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22. What happened to the lac operon in the absence of lactose?

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22. What happened to the lac operon in the absence of lactose?

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When lactose is absent the lac operon is switched off. This is becasue a repressor protein is produced which binds to the operator region. This prevents RNA polymerase from binding to the operon and therefore prevents transcription of the structual genes.

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According to our findings based on this article it states ," As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme β-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis in studies assessing new anticancer therapies. "

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. 2012 Aug 21;(66):e4162.

doi: 10.3791/4162.

Affiliation

¹ Laboratory for Orthopedic Research, Balgrist University Hospital, Zurich. marlt@research.balgrist.ch

Free PMC article

Matthias J E Arlt et al. J Vis Exp.

Free PMC article

. 2012 Aug 21;(66):e4162.

doi: 10.3791/4162.

Affiliation

¹ Laboratory for Orthopedic Research, Balgrist University Hospital, Zurich. marlt@research.balgrist.ch

Abstract

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited. While animal models have proven to be invaluable tools for cancer research, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR, FACS or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme β-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis in studies assessing new anticancer therapies. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al. who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model, which was originally described to be non-metastatic.

Figures

Similar articles

Bacterial lacZ gene as a highly sensitive marker to detect micrometastasis formation during tumor progression.

Lin WC, Pretlow TP, Pretlow TG 2nd, Culp LA. Lin WC, et al. Cancer Res. 1990 May 1;50(9):2808-17. Cancer Res. 1990. PMID: 2183931

Histochemical staining following LacZ gene transfer underestimates transfection efficiency.

Couffinhal T, Kearney M, Sullivan A, Silver M, Tsurumi Y, Isner JM. Couffinhal T, et al. Hum Gene Ther. 1997 May 20;8(8):929-34. doi: 10.1089/hum.1997.8.8-929. Hum Gene Ther. 1997. PMID: 9195215

LacZ transgene expression in the subcutaneous Dunn/LM8 osteosarcoma mouse model allows for the identification of micrometastasis.

Arlt MJ, Banke IJ, Walters DK, Puskas GJ, Steinmann P, Muff R, Born W, Fuchs B. Arlt MJ, et al. J Orthop Res. 2011 Jun;29(6):938-46. doi: 10.1002/jor.21304. Epub 2011 Jan 31. J Orthop Res. 2011. PMID: 21284029

Overview and assessment of the histochemical methods and reagents for the detection of β-galactosidase activity in transgenic animals.

Trifonov S, Yamashita Y, Kase M, Maruyama M, Sugimoto T. Trifonov S, et al. Anat Sci Int. 2016 Jan;91(1):56-67. doi: 10.1007/s12565-015-0300-3. Epub 2015 Sep 22. Anat Sci Int. 2016. PMID: 26394634 Free PMC article. Review.

References

Frese KK, Tuveson DA. Maximizing mouse cancer models. Nature. 2007;7:645-658. - PubMed

Coussens LM, Fingleton B, Matrisian LM. Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science (New York, N.Y.) 2002;295:2387-2392. - PubMed

Fass L. Imaging and cancer: a review. Mol Oncol. 2008;2:115-152. - PMC - PubMed

Puaux AL. A comparison of imaging techniques to monitor tumor growth and cancer progression in living animals. Int. J. Mol. Imaging. 2011;2011:321538. - PMC - PubMed

Malek A, Catapano CV, Czubayko F, Aigner A. A sensitive polymerase chain reaction-based method for detection and quantification of metastasis in human xenograft mouse models. Clinical & experimental metastasis. 2010;27:261-271. - PubMed

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national center for biotechnology information

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Improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and X-Gal staining of lacZ-tagged tumor cells - PubMed

# data[].attributes.attachment_link.description
Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited. While animal models have proven to be invaluable to ...

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Q21 What is the function of each translated enzyme encoded by structural genes?

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Q21 What is the function of each translated enzyme encoded by structural genes?

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Lac Z: encodes for β-galactosidase
Lac Y: encodes for permease
Lac A: encodes for transacetylase

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Q20 What is the function of the enzyme mention in Q (19)?

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Q20 What is the function of the enzyme mention in Q (19)?

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The binding of the activator and the release of the repressor – allow RNA polymerase to bind strongly to the promoter and give it a clear path for transcription. They lead to strong transcription of the lac operon and production of enzymes needed for lactose utilization.

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Q19 Name the enzyme that binds to promoter site?

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Q19 Name the enzyme that binds to promoter site?

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RNA polymerase is the main transcription enzyme. Transcription begins when RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through helper proteins).

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Q18 What happen to repressor protein after steps that occurs in Q (17)?

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Q18 What happen to repressor protein after steps that occurs in Q (17)?

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Change the shape of repressor protein.

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LAC OPERON PART 2: POSITIVE AND NEGATIVE CONTROL

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Similarly, mutations in the lac promoter are cis-acting, since they alter the binding site for RNA polymerase. When RNA polymerase cannot initiate transcription of the lac operon, none of the genes in the operon can be expressed irrespective of the function of the repressor. In general, trans-acting genes that regulate expression of genes on other DNA molecules encode diffusible products. In most cases these are proteins, but in some cases RNA molecules can act in trans to regulate gene expression.

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Chains of alpha glucose compose starch. Since the foundation of starch is alpha glucose, it can be easily broken down into simple sugars. Meanwhile, chains of beta glucose compose cellulose. Unlike starch, cellulose is not easy to break down; hence it is a perfect, building material. The tasty parts of plants are composed of starch while the hard parts of plants are made of cellulose.

Since plants are our main sources for glucose, which comes in the form of starch and cellulose, we depend greatly on them. For plants to store sugar, they need chains of alpha glucose to build starch. For plants to build structural material, they need chains of beta glucose to create cellulose. Humans have the capacity to break down starch while we cannot break down cellulose. Though this is the situation, cellulose is still important in our body system because cellulose is otherwise known as fiber. Fiber plays an important role in our digestive system. There are animals which can digest cellulose, specifically livestock animals such as horses, and cows. Termites can also break down the strong, structural form of cellulose.

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(Most mutations in the operator, the binding site for repressor,) lead to lower affinity for the repressor and hence less binding.  Thus these mutations allow continued transcription (and thus expression) of the lac operon even in the absence of inducer; this is referred to constitutive expression.

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Q5. who proposed the operon model in bacteria?

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Q5. who proposed the operon model in bacteria?

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The operon theory was first proposed by the French microbiologists François Jacob and Jacques Monod in the early 1960s. In their classic paper they described the regulatory mechanism of the lac operon of Escherichia coli, a system that allows the bacterium to repress the production of enzymes involved in lactose metabolism when lactose is not available.

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Q6. what part in the operon that code enzymes?

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Q6. what part in the operon that code enzymes?

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A typical operon consists of a group of structural genes that code for enzymes involved in a metabolic pathway, such as the biosynthesis of an amino acid. These genes are located contiguously on a stretch of DNA and are under the control of one promoter (a short segment of DNA to which the RNA polymerase binds to initiate transcription). A single unit of messenger RNA(mRNA) is transcribed from the operon and is subsequently translated into separate proteins

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Operon Definition

An operon is a cluster of functionally-related genes that are controlled by a shared operator. Operons consist of multiple genes grouped together with a promoter and an operator. Operons are present in prokaryotes ( bacteria and archaea), but are absent in eukaryotes. In some situations multiple operons are controlled by the same regulatory protein; in these cases the operons form a regulon. Operons were first identified as a mode of gene expression control in 1961 by François Jacob and Jacques Monod.

Operon Structure

Operons are regions of DNA that contain clusters of related genes. They are made up of a promoter region, an operator, and multiple related genes. The operator can be located either within the promoter or between the promoter and the genes. RNA polymerase initiates transcription by binding to the promoter region. The location of the operator is important as its regulation either allows or prevents transcription of the genes into mRNA.

Operon Function

An operon is a complete package for gene expression and synthesis of polypeptides. By combining the related genes, all polypeptides required for a specific function are synthesized in response to a single stimulus. For example, the bacterium Escherichia coli contains a number of genes clustered into operons and regulons: the Lac operon which is involved in lactose degradation, the Trp operon which is involved in tryptophan biosynthesis, and the His operon which is involved in histidine biosynthesis. These operons are turned on when the gene products are needed.

Operons can be under negative or positive control. Negative control involves turning off the operon in the presence of a repressor; this can be either repressible or inducible. A repressible operon is one that is usually on but which can be repressed in the presence of a repressor molecule. The repressor binds to the operator in such a way that the movement or binding of RNA polymerase is blocked and transcription cannot proceed. An inducible operon is one that is usually off. In the absence of an inducer the operator is blocked by a repressor molecule. When the inducer is present it interacts with the repressor protein, releasing it from the operator and allowing transcription to proceed. Repressible operons are generally involved in anabolic pathways, or the synthesis of an essential component, while inducible operons are generally involved in catabolic pathways, or the breakdown of a nutrient. Positive control of an operon is when gene expression is stimulated by the presence of a regulatory protein.

Lac Operon

The Lac operon is the classic operon example, and is responsible for the degradation of the milk protein lactose. The Lac operon is an inducible operon; in the absence of lactose the operator is blocked by a repressor protein. The operon is made up of a promoter with operator, and three genes (lacZ, lacY, and lacA) which encode β-galactosidase, permease, and transacetylase. The three genes are involved in the breakdown of lactose into its metabolites: β-galactosidase breaks lactose down into glucose and galactose, while the other two proteins aid in the metabolic process. The expression of the Lac operon is controlled by the regulatory gene lacI, located immediately adjacent to the promoter region. LacI encodes an allosteric repressor protein that keep the Lac operon "off".

In order for the Lac operon to be turned on, an inducer molecule must inactivate the repressor protein. The inducer molecule in this system is allolactose, an isomer of lactose. When lactose and its isomer are present in the cell, allolactose will bind to allosteric sites on the repressor protein, changing its conformation and rendering it inactive. As the repressor protein detaches from the operator, RNA polymerase can bind to the promoter, transcription can occur, and the three lactose degradation genes can be synthesized.

The figure shows the structure of the Lac operon and the adjacent lacR repressor gene.

The Lac operon is also under positive gene regulation. While the removal of the repressor protein in the presence of lactose is required for synthesis of the lacZ, lacY, and lacA genes, the gene expression will remain low. The level of gene expression is controlled by the amount of the preferred energy source, glucose, in the cell. This control is regulated by an allosteric regulatory protein, catabolite activator protein (CAP). When glucose levels in the cell are low, the organic molecule cyclic AMP is in high concentration. Cyclic AMP activates CAP by binding to the allosteric sites, causing CAP to attach to the Lac operon promoter. Unlike the repressor proteins, binding of CAP to the Lac operon stimulates gene expression. When the cell glucose levels increase, the cyclic AMP levels in the cell decrease, and the activator protein will disassociate from the promoter. Transcription will return to low levels, or will turn off if the repressor protein reattaches.

The figure depicts the Lac operon and how its gene expression is under both positive and negative control.

Trp Operon

The Trp operon is responsible for synthesis of the amino acid trytophan when it is not available in the environment. The Trp operon is made up of a promoter with an operator, and five genes that encode enzymes for tryptophan synthesis. The Trp operon is regulated by the regulatory gene trpR, a gene that is located at a distance from the Trp operon.

The Trp operon is an example of a repressible operon; it is on unless turned off by a repressor protein. The repressor protein is synthesized by trpR. While the repressor protein is always present in the cell, it is synthesized in an inactive form. When a corepressor is present, in this case tryptophan, it binds to the repressor protein in an allosteric site. This changes the conformation of the protein such that it can bind to the operator and block transcription by preventing the binding of RNA polymerase to the promoter. In this way the cell saves energy by not producing tryptophan when it is already present.

The figure depicts the Trp operon with the repressor gene, the promoter, operator, and five tryptophan-synthesizing genes.

Quiz

3. Are repressible operons under positive or negative control?
A. positive
B. negative

References

Campbell, N. A., & Reece, J. B. (2005).Biology, 7th. ed. Ch. 18. San Francisco, CA: Benjamin Cummings. ISBN: 0-8053-7171-0.

Madigan, M. T., & Martinko, J. M. (2006).Brock biology of microorganisms, 11th. ed. Chs. 8 and 10. Upper Saddle River, NJ: Pearson Prentice Hall. ISBN: 0-13-144329-1.

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Operon - Definition, Structure and Function | Biology Dictionary

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An operon is a cluster of functionally-related genes that are controlled by a shared operator. Operons consist of multiple genes grouped together with a promoter and an operator. Operons are present in prokaryotes ( bacteria and archaea), but are absent in eukaryotes.

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Q7. what are the components of lac operon

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Q7. what are the components of lac operon

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Chapter 6 EP 12 / Sem 1 :Lac Operon and Its Function(Matriculation Biology)

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Q7. what are the components of lac operon

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Q7. what are the components of lac operon

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An operon consists of
i. an operator
ii. promoter
iii. regulator
iv. structural genes.

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Lac Operon (Basic) Animation || Gene regulation in Prokaryotes

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Q8. what is the gene that is not part of the operon system?

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Q8. what is the gene that is not part of the operon system?

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The structural genes in the lac operon activate specific enzymes for the metabolism of lactose. From the above information, we have found that operon does not contain an enhancer in their structure. An enhancer has no specific role in the functioning of the operon. Enhancer sequences are regulatory DNA sequences. These sequences are bound by specific proteins called transcription factors. The binding of these proteins will enhance the transcription of an associated gene. They are not part of the operon structure.

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Q23. give the significance of the presence of lactose in E.coli.

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Q23. give the significance of the presence of lactose in E.coli.

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The lactose operon (lac operon) is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. ... The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose.

Lactose, being large and highly hydrophilic cannot pass through the E. coli cell membrane. Lactose permease is a membrane protein that allows lactose to enter the cell, moving down its concentration gradient.

Therefore, when E. coli grows on sources other than lactose, such as carbon-based sources, these genes are not transcribed, leading to a very low concentration of these enzymes in the cell. In contrast, the presence of lactose considerably increases the synthesis of these enzymes

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An inducer (allolactose or an analog) binds to the repressor and prevents its binding to the operator, thereby releasing the repression and allowing transcription of the lac operon.

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RNA polymerase bind at promoter

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NURIZZATUL AQMAR BINTI ZULKURNAIN KM-Pelajar

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Stages of the Transcription Initiation Process

Transcription initiation is the phase during which the first nucleotides in the RNA chain are synthesized. It is a multistep process that starts when the RNAP holoenzyme binds to the DNA template and ends when the core polymerase escapes from the promoter after the synthesis of approximately the first nine nucleotides.

-The stages of the transcription initiation process, which are summarized in Figure 2, can be described in terms of the types of interaction between the RNAP and the nucleic acids that are involved

-The first stage in transcription initiation is the formation of a complex between the holoenzyme and the DNA sequence at the promoter, which is in the form of a double-stranded DNA.

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4.1.2 Use of quantitative reverse transcriptase PCR to analyze induction of genes indicative of activation of individual branches of the UPR

Transcriptional activation of a small number of genes has been reported to be nearly exclusively dependent on one of the three branches of the mammalian UPR (Adachi Zhang Bustin, 2002; Bustin et al., 2008; Harding et al., 2003; Lee et al., 2003; Okada et al., 2002; Wu et al., 2006). Induction of these two genes may be indicative of activation of the ATF6 branch of the UPR. Measuring mRNA levels for these genes using Northern blotting or quantitative reverse transcriptase PCR techniques ( et al., 2009; Wilhelm and Pingoud, 2003) will indicate which pathways of the UPR are activated in the experimental system under investigation. et al., 2007; Yamamoto et al., 2007). Genes nearly exclusively regulated by the IRE1-XBP1 and the PERK pathway can be readily identified in the literature (Table 15.3). However, transcriptional activation of many chaperone genes by ATF6α (Fig. 15.3) overlaps with IRE1 and PERK signaling activities. A CREB-H·ATF6α heterodimer induces inflammatory genes encoding C reactive protein ( CRP) and serum amyloid P-component (SAP;

Where available, oligonucleotide sequences for mouse sequences are provided. Human or other species will need alternative primers. Detailed PCR protocols can be found in the references in the footnote to the table.

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Stages of the Transcription Initiation Process

Transcription initiation is the phase during which the first nucleotides in the RNA chain are synthesized. It is a multistep process that starts when the RNAP holoenzyme binds to the DNA template and ends when the core polymerase escapes from the promoter after the synthesis of approximately the first nine nucleotides.

The stages of the transcription initiation process, which are summarized in Figure 2, can be described in terms of the types of interaction between the RNAP and the nucleic acids that are involved. The first stage in transcription initiation is the formation of a complex between the holoenzyme and the DNA sequence at the promoter, which is in the form of a double-stranded DNA. This complex is termed a closed binary complex or closed complex. The second stage is the unwinding of a short region of DNA within the sequence that is bound to the RNAP. The complex between the polymerase and the partially melted DNA is termed an open binary complex or open complex. The conversion of the closed complex into the open complex leads to the establishment of tight binding between the RNAP and the promoter sequence. For strong promoters, the conversion into an open complex is irreversible. The third stage is the incorporation of two ribonucleotides and the formation of a phosphodiester bond between them. Because the complex at that stage contains an RNA as well as DNA, it is called an initiation ternary complex. Up to seven additional ribonucleotides can be added to the RNA chain without any movement of the polymerase. After the addition of each base, there is a certain probability that the enzyme will release the short (up to nine bases long) RNA chain. Such an unsuccessful initiation event is termed an 'abortive initiation'. Following an abortive initiation, RNAP begins again to incorporate the first base. Several rounds of abortive initiations usually occur and the result is the formation of short RNA chains that are 2-7 bases long. When initiation succeeds, that is, a nine-base-long RNA chain is formed and is not released, the last stage in transcription initiation occurs. At that stage the σ factor is released from the polymerase. As a consequence, a complex containing core polymerase, DNA, and RNA is formed. This complex is called an elongation ternary complex. The departure of the polymerase from the promoter to resume elongation is termed promoter escape or promoter clearance.

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Catabolite Activator Protein

Transcription activation is a process carried out by a combination of a complex set of gene activators. The catabolite activator protein (CAP, also known as cAMP receptor protein, CRP) is a transcriptional activator, present as homodimer in solution, each subunit including a ligand-binding domain at the N-terminus and a DNA-binding domain at the C-terminus. Two cAMP molecules bind dimeric CAP and function as allosteric effectors by increasing the affinity for DNA. Components of the CAP superfamily have been linked to several biological functions, including immune defense. Many components contain a CAP domain, whereas others contain additional C-terminus extensions related to biological roles. A differential screening between LPS-treated and naïve C. intestinalis allowed the isolation of a full-length cDNA. Analysis of the deduced amino acid sequence showed that the protein (CiCAP) displays a modular structure with similarities to the vertebrate CAP superfamily as well as to a collagen-binding adhesin of Streptococcus mutans. Quantitative mRNA expression, performed by real-time PCR analysis, showed that the gene transcription is promptly activated in the pharynx after LPS inoculation. Moreover, in situ hybridization assay disclosed that CiCAP mRNA is highly produced by hemocytes with large granules that are contained inside the pharynx vessels. Thus CiCAP represents a protein with novel structural domains, involved in ascidian immune responses, probably as a component of the transcription activation complex.

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Franklyn F. BolanderJr., in Molecular Endocrinology (Third Edition) , 2004

Motifs and Molecular Mechanisms

Transcription activation domains have been mapped in several factors; they are often characterized by an abundance of a particular amino acid rather than a specific sequence. The most studied motif is one enriched in acidic amino acids, known as an acid blob, negative noodle, or acid noodle; the names refer to both the charge and the original belief that these sequences formed random structures. Later, they were postulated to form amphipathetic α-helices that interacted with other transcription factors having basic helices. Most recently, they have been predicted to form a hydrophobic β-sheet, and the hydrophobic amino acids have been shown to be critical for transcription. The negative charges may only be necessary to make the lipophilic sheet more accessible. Whatever the secondary structure, acidic domains are known to stimulate RNA polymerase II. They also bind to the TATA-box factors TFIIB and TFIID; to SWI/SNF (switch and sucrose nonfermentors gene products), a chromatin-remodeling complex; and to the replication protein A. The latter protein is involved with DNA unwinding. In addition to direct contacts with the transcriptional machinery, acidic domains can bind coactivators, which form bridges between the TAD and general transcription factors.

Other domains rich in glutamine, proline, isoleucine, or serine-threonine have also been documented. Like the acidic motif, the activity of the glutamine TADs is dependent on the hydrophobic amino acids and not the glutamine. The glutamine domain can bind the TBP. The proline-rich regions probably form polyproline helices (see Chapter 7); they can bind TFIIB and several transcriptional coactivators. With the exception of the S-T TAD, these TADs are all hydrophobic; this property makes them "sticky" and allows them to recruit other proteins. The S-T domains probably act as regulatory sites through phosphorylation and/or glycosylation.

From this discussion, several general mechanisms of action can be envisioned for TADs. First, TADs can recruit general transcription factors either directly or through coactivators; for example, binding to TBP, TFIIB, TFIID, TFIIF, and TFIIH have all been documented. Second, TADs may alter the conformation of either the general transcription factor and/or the DNA. Finally, TADs may stimulate the covalent modification of proteins; they have been shown to recruit kinases, acetyltransferases, and methyltransferases. These mechanisms are discussed further in the following section on mechanisms for transcription effects.

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1.1.1.1.2.1 Location in the Epigenome

Transcriptional activation or inactivation of specific miRNA genes is largely influenced by epigenetics. Such epigenetic regulation includes the proximity of the miRNA gene promoter to a CpG island, various histone modifications to the chromatin, and availability of factors that maintain and regulate expression from the epigenome. The expression of mir-127, a miRNA located near a CpG island is dependent on the methylation status of the promoter, implying epigenetic control on the expression of miRNAs (Lujambio, Ropero, Ballestar, et al., 2007; Saito, Liang, Egger, et al., 2006). MiRNAs also undergo massive upregulation when the DNA methylatransferases 1 and 3b (DNMT1, DNMT3) are downregulated (Lujambio et al., 2007), lending further support to the role of DNA methylation in regulating miRNA expression (reviews: Calin & Fabbri, 2010; Sato et al., 2011).

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G Histone Modifications Modulate MPC Fate

Transcriptional activation and repression involves characteristic changes in the chromatin structure of regulated genes. Because key developmental regulators often bind to thousands of genes (e.g., Refs. 76,77), broad changes in chromatin structure might be expected. Histone deacetylases (HDAC) are central players in the control of chromatin structure through their regulated activity that removes covalent acetyl-modifications from histones and other chromosomal proteins including DNA-binding TFs. Chemical inhibitors of HDACs, for example, can induce widespread gene activation. Surprisingly, in some developmental contexts, nonspecific inhibition of HDAC activity can favor one developmental lineage over another. In particular, treatment of embryonic pancreatic explants in long-term culture with HDAC inhibitors suppresses acinar development. In explants treated with the inhibitors, the number of cells that express the proendocrine TF Ngn3 increases several-fold and persists many days longer than normal. This observation suggests that HDAC activity normally biases the cell-fate options toward the acinar program by suppressing the opportunity to enter the bipotent intermediate toward islet or ductal fates (Fig. 3). A thorough analysis of histone marks and DNA methylation in acinar and nonacinar cells will provide a road map for the pancreatic epigenome.

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Alternative Splicing

Transcription initiation is certainly the first point that comes to mind when chromatin structure and gene expression are related. However, control of splicing might be at least as important. Splicing is executed by the trans-acting splicing factors that remove introns from pre-mRNA and fuse the remaining exons. A genome-wide survey of nucleosome positioning data in human T cells discovered that nucleosome density is higher over exons than over introns (Schones et al., 2008). Presumably, pausing of polymerase II (Pol II) at exons allows more time for the splicing machinery to recognize exons and splice out introns. Later, H3K36me3, H3K27me, H3K79me1/2, H4K20me1, and H2BK5me1 were found to be enriched in exons (reviewed in Zhou et al., 2014). Alternate splicing is one of the "hallmarks" of cancer progression (Oltean and Bates, 2014) and the existence of specific cancer isoforms is of diagnostic and eventually therapeutic interest. Mutations and dysregulation of expression of splice factors in cancer (Sveen et al., 2016) are confounding with a potential impact of cis-acting epigenetic modifications. However, both can and probably will contribute to aberrant splicing in tumor cells.

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URL: https://www.sciencedirect.com/science/article/pii/B9780128043103000065

Initiation

Transcription initiation begins with formation of the pre-initiation complex (PIC) on promoter DNA. The PIC contains Pol II and the general transcription factors TFIIB, -D, -E, -F, and -H. The general factors are involved in sequence-specific promoter recognition (TFIIB and TFIID), prevention of nonspecific DNA binding (TFIIF), and DNA melting and CTD phosphorylation (TFIIE and TFIIH). Recently, the structure of RNA Pol II in complex with TFIIB was presented. The structure shows that to initiate gene transcription, promoter DNA is positioned over the Pol II active center cleft with the B-core domain that binds the wall at the end of the cleft. DNA is then opened with the help of the B-linker that binds the Pol II rudder and clamp coiled coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the B-reader that approaches the active site. RNA synthesis initiates within the bubble. The early transcribing complex is functionally unstable. Short RNAs are frequently released and Pol II has to restart transcription (abortive cycling).

Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger TFIIB release and EC formation. The elongating RNA extends over the dock domain, preventing reassociation of the TFIIB ribbon with Pol II during elongation.

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URL: https://www.sciencedirect.com/science/article/pii/B9780123786302002644

Abstract

Transcriptional activation of proapoptotic genes play an important role to initiate cell death during animal development or in response to cytotoxic stress. In particular, P53-mediated induction of proapoptotic genes serves as a major tumor suppression mechanism in eliminating genetically compromised cells. During animal development, the sensitivity of cells to intra- and extracellular stress-induced cell death varies greatly depending on tissue types and differentiation status. Emerging evidences from Drosophila, as well as mammalian systems, indicated that targeted epigenetic regulation of proapoptotic genes, especially those that are transcriptional targets of P53, controls cellular sensitivity to stress-induced apoptosis during animal development. Epigenetic regulation of proapoptotic genes distinguishes itself from canonical epigenetic regulation of homeotic genes in three major aspects. First, it is not strictly determined by cell lineage or position. Second, it is often reversible in response to changes of the extracellular and intracellular environment. Last, but probably most importantly, epigenetic regulation of P53 target loci introduces differential sensitivity to stress among adjacent cells in the same tissue compartment. This phenotypic plasticity generated by the epigenetic mechanism may play an import role in maintaining appropriate number of cells during animal development. The detailed mechanism of how stress-responsive proapoptotic genes is specifically targeted for epigenetic suppression remains to be explored. However, abundant evidence suggests that such a mechanism can be hijacked by oncogenic viruses or dysregulated by oncogene overexpression to promote tumorigenesis. Comprehensive understanding of epigenetic regulation of proapoptotic genes holds the promise of improving the efficacy of cancer therapy.

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URL: https://www.sciencedirect.com/science/article/pii/B9780127999586000160

3.2 Epigenetic Pathways

Transcription activation by ER is known to involve several coactivators. These coactivators have acetyltransferase activity and are capable of acetylating histones, which destabilizes nucleosomes and promotes transcription. Acetylation is regulated in a steady state by the enzymes histone acetyltransferase (HAT) and histone deacetylases (HDACs) activity. Expression of ER can be lost by epigenetic modifıcations such as histone deacetylation, which can cause endocrine resistance.

Pfister et al. demonstrated a correlation between histone modification and tumor phenotypes as well as patient outcomes. Higher levels of acetylation and methylation were associated with better outcomes compared with moderate or low levels.

There is also some evidence of partial restoration of functional ER in cells that have lost ER expression as a result of acquired resistance to endocrine therapy.

HDAC inhibitors (HDACi) were shown to induce growth arrest, differentiation, and cell death in breast cancer cells. HDACi inhibit ERα expression and cell proliferation. In breast cancer cells, TSA, which is a potent and reversible HDACi, led to a significant reduction in ERα accumulation independent of ER ligands. In human cancer cells, inhibition of HDACs regulates the expression of the ERα gene and the transcriptional activity in response to partial antiestrogens. Inhibition of HDAC enzymatic activity and modulation of ERα levels affect the relative agonist activity of partial antiestrogens on a stably integrated reporter transgene.

Clinically the combination of vorinostat, a HDACi, and tamoxifen was shown to reverse hormone resistance, with a response rate of 19% and a clinical benefit rate of 40%.

Epigenetic pathways therefore play a significant role in endocrine resistance and provide a potential therapeutic target.

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URL: https://www.sciencedirect.com/science/article/pii/S1877117316300783

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Transcriptional activation of a small number of genes has been reported to be nearly exclusively dependent on one of the three branches of the mammalian UPR (Adachi Zhang Bustin, 2002; Bustin et al., 2008; Harding et al., 2003; Lee et al., 2003; Okada et al., 2002; Wu et al., 2006).

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Transcription: an overview of DNA transcription (article) | Khan Academy

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In transcription, the DNA sequence of a gene is transcribed (copied out) to make an RNA molecule.

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Related research article Denard B, Lee C, Ye J. 2012. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1. eLife :e00090. doi: 10.7554/eLife.00090

Image Cleavage of the transcription factor CREB3L1

Many clinically available drugs are used without a clear understanding of how they work. This is particularly true in oncology, where powerful cytotoxic drugs are given to patients despite the fact that we do not fully understand their mechanism of action. Even targeted therapies such as rapamycins and receptor tyrosine kinase inhibitors are active in only a subset of the tumours for which they were originally developed, which has led to calls for better methods to identify tumours that will respond to specific drugs. This issue is even more pressing for conventional cytotoxic agents because, in addition to being toxic to cancer cells, they can also be toxic to healthy cells. Now, writing in eLife, Brey Denard, Ching Lee and Jin Ye provide evidence that doxorubicin, a widely used cancer drug, induces cellular toxicity via a novel mechanism that involves the synthesis of ceramide followed by activation of a transcription factor called CREB3L1 ( Denard et al., 2012).

This is a story that began 40 years ago with the introduction of doxorubicin and daunorubicin into clinical practice. Both of these compounds belong to a class of drugs called anthracyclines that are derived from bacteria belonging to the genus Streptomyces. Extensive clinical studies have demonstrated that they are active against a wide variety of tumours ( Minotti et al., 2004). However, despite this, the clinical use of anthracyclines has been limited because of a significant risk for cardiac damage. The chances of this life-threatening side effect depend on cumulative dosage, and can occur decades after exposure ( Kremer et al., 2001). Over the years, many mechanisms of action have been proposed for these drugs-including topoisomerase II inhibition, DNA intercalation, and free radical generation-but there has until recently been a lack of definitive evidence for all three of these mechanisms ( Gewirtz, 1999).

Denard, Lee and Ye, who are based at the University of Texas Southwestern (UTSW) Medical Center, propose that doxorubicin can combat tumours via a mechanism called regulated intramembrane proteolysis. In this process, a membrane-bound protein is cleaved, liberating a soluble messaging molecule that can play a role in a variety of cellular processes, including apoptosis, lipid metabolism and the response to viral infection ( Lal and Caplan, 2011).

In previous work, the UTSW group showed that CREB3L1-a membrane protein with a carboxy-domain inside the lumen of the endoplasmic reticulum, and an amino-terminal domain that is in the cytoplasm of the cell-undergoes proteolytic cleavage during viral infection to release the amino-terminal domain, which then translocates to the nucleus and drives the transcription of genes that inhibit cellular proliferation ( Denard et al., 2011). In the present study, Denard, Lee and Ye extend these results by treating multiple cell lines with doxorubicin and showing that the drug produces lipid molecules called ceramides that trigger the cleavage of CREB3L1 (see ). This is again followed by translocation of the resulting amino-terminal fragment to the nucleus and the increased expression of several genes ( Denard et al., 2012). Importantly, the down-regulation of CREB3L1 diminishes doxorubicin-induced effects on cell proliferation, whereas overexpression of CREB3L1 makes tumour cells more sensitive to doxorubicin. Collectively, these observations provide the first evidence for a model in which the anthracycline-induced synthesis of ceramides leads to the cleavage of CREB3L1, resulting in altered expression of genes that might contribute to the effect of the drug.

While intriguing, these results represent only a first step in testing this model. In particular, as discussed by the authors, there are several open questions: Does ceramide alter CREB3L1 trafficking to the Golgi apparatus, where the intramembrane proteases are located? Does CDKN1A, one of the genes that is overexpressed when cells are treated with doxorubicin, have a critical role in the action of the drug? And does expression or activation of CREB3L1 correlate with doxorubicin sensitivity across a panel of cancer cell lines, such as the Cancer Cell Line Encyclopedia ( Barretina et al., 2012), or clinical specimens? In addition, it will be important to establish whether other chemo-therapeutic agents also induce proteolysis of CREB3L1 or other transmembrane proteins.

It will also be interesting to see whether the ideas put forward by the UTSW group can be integrated into existing models of doxorubicin action. Emerging data suggest that the enzyme topoisomerase IIβ has a critical role in the toxic effects of doxorubicin on cardiac tissue (see ), and it has been reported that the targeted deletion of the Top2b gene abolishes doxorubicin-induced effects on cardiac function ( Zhang et al., 2012). By contrast, it has been suggested that the Top2a gene contributes to the anticancer effects of doxorubicin and other anthracyclines ( Mao et al., 1999). This work, and also the work of the UTSW group, needs to be rigorously extended by studying in detail how doxorubicin kills cancer cells in appropriate cancer models and clinical samples.

The fact that doxorubicin remains the subject of intense study four decades after its introduction into the clinic is a reflection of its complex biology and its importance as a cancer drug. Hopefully, these recent studies will ultimately allow clinicians to better understand this important agent and to utilize it in a more targeted fashion.

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Related research article Denard B, Lee C, Ye J. 2012. Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1. eLife :e00090. doi: 10.7554/eLife.00090 Image Cleavage of the transcription factor CREB3L1 Many clinically available drugs are used without a clear understanding of how they work.

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Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter. This signals the DNA to unwind so the enzyme can ‘‘read’’ the bases in one of the DNA strands. The enzyme is now ready to make a strand of mRNA with a complementary sequence of bases.

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Learning Outcomes

Understand the basic steps in the transcription of DNA into RNA

Transcription takes place in the nucleus. It uses DNA as a template to make an RNA (mRNA) molecule. During transcription, a strand of mRNA is made that is complementary to a strand of DNA. Figure 1 shows how this occurs.

Transcription takes place in three steps: initiation, elongation, and termination. The steps are illustrated in Figure 2.

Step 1: Initiation

Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter. This signals the DNA to unwind so the enzyme can ''read'' the bases in one of the DNA strands. The enzyme is now ready to make a strand of mRNA with a complementary sequence of bases.

Step 2: Elongation

Elongation is the addition of nucleotides to the mRNA strand. RNA polymerase reads the unwound DNA strand and builds the mRNA molecule, using complementary base pairs. There is a brief time during this process when the newly formed RNA is bound to the unwound DNA. During this process, an adenine (A) in the DNA binds to an uracil (U) in the RNA.

Step 3: Termination

Termination is the ending of transcription, and occurs when RNA polymerase crosses a stop (termination) sequence in the gene. The mRNA strand is complete, and it detaches from DNA.

This video provides a review of these steps. You can stop watching the video at 5:35. (After this point, it discusses translation, which we'll discuss in the next outcome.)

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Understand the basic steps in the transcription of DNA into RNA Transcription takes place in the nucleus. It uses DNA as a template to make an RNA (mRNA) molecule. During transcription, a strand of mRNA is made that is complementary to a strand of DNA. Figure 1 shows how this occurs.

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Chemotherapy is one of the most common treatments for cancer. It uses certain drugs to kill cancer cells or to stop them from growing and spreading to other parts of your body. Your doctor might prescribe chemo by itself or with surgery or radiation therapy. You might also take newer kinds of cancer-fighting drugs along with chemotherapy.

You can take chemo as pills or shots. You might go to a clinic or hospital so you can get the drugs through an IV, what doctors call an infusion.

To help your body regain strength and grow new, healthy cells, you might take the drugs over a few weeks. You might take doses every day, every week, or every month. It depends on the type of cancer you have and how severe it is.

Your cancer doctor, called an oncologist, may prescribe one chemo drug or a mix of different ones, depending on:

Your type of cancer

Whether you've had cancer before

If you have other health problems like diabetes or heart, kidney, or liver disease

Why You Need Chemotherapy

Even after surgery to remove a tumor, your body might still have cancer cells. These cells can grow new tumors or spread the cancer to other parts of your body.

Chemotherapy drugs help destroy, shrink, or control those cells. It might also treat symptoms the cancer causes, like pain. You might also get chemo to shrink a tumor before your doctor removes it in surgery.

How It Works

Chemotherapy drugs work in a few different ways. They can:

Kill both cancerous and healthy cells

Fight only cancer cells

Keep tumors from growing blood vessels, which help them thrive

Attack the cancer cells' genes so the cells die and can't grow into new tumors

Common Chemotherapy Drugs

There are dozens of chemotherapy drugs that doctors can prescribe. They're often divided into groups based on how they work and what they're made of. Each group of drugs destroys or shrinks cancer cells in a different way.

Some drugs damage the DNA of cancer cells to keep them from making more copies of themselves. They are called alkylating agents, the oldest type of chemotherapy. They treat many different types of cancer, such as leukemia, lymphoma, Hodgkin's disease, multiple myeloma, and sarcoma, as well as breast, lung, and ovarian cancers. Some examples of alkylating agents are cyclophosphamide, melphalan, and temozolomide. As they kill bad cells, though, they can also destroy your bone marrow in the process, which can cause leukemia years later. To lower this risk, you can take the drugs in small doses. One type of alkylating agent -- platinum drugs like carboplatin, cisplatin, or oxaliplatin -- has a lower risk of causing leukemia.

One type of chemo drug interferes with the normal metabolism of cells, which makes them stop growing. These drugs are called antimetabolites. Doctors often use them to treat leukemia and cancer in the breasts, ovaries, and intestines. Drugs in this group include 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine, and methotrexate, among many others.

Anthracycline chemotherapy attacks the enzymes inside cancer cells' DNA that help them divide and grow. They work for many types of cancer. Some of these drugs are actinomycin-D, bleomycin, daunorubicin, and doxorubicin, among others. High doses of anti-tumor antibiotics can damage your heart or lungs. So your doctor will have you take them for a short time.

Drugs called mitotic inhibitors stop cancer cells from making more copies of themselves. They can also stop your body from making the proteins that cancer cells need to grow. Doctors might prescribe them for breast and lung cancers and types of myeloma, leukemia, and lymphoma. Mitotic inhibitors include docetaxel, estramustine, paclitaxel, and vinblastine.

Another type of medicine, called topoisomerase inhibitors, also attacks enzymes that help cancer cells divide and grow. They treat some types of leukemia and cancer of the lung, ovaries, and intestines, among other types. This group of medicine includes etoposide, irinotecan, teniposide, and topotecan. Some of them, though, may raise your odds of getting a second cancer a few years later.

Steroids are drugs that act like your body's own hormones. They are useful in treating many types of cancer, and they can keep you from having nausea and vomiting after a round of chemo. They can also prevent allergic reactions to some of the drugs. Some of the steroids your doctor might prescribe are prednisone, methylprednisolone, and dexamethasone.

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Chemotherapy is one of the most common treatments for cancer. It uses certain drugs to kill cancer cells or to stop them from growing and spreading to other parts of your body. Your doctor might prescribe chemo by itself or with surgery or radiation therapy.

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Lactose occurs in two isomeric forms, α-lactose and β-lactose
α and β, differing only in the configuration of the hydroxyl group at the anomeric hemiacetal carbon. α has an axial hydroxyl whereas β hydroxyl is equatorial

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15. What happen to the lac operon in the presence of lactose?

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15. What happen to the lac operon in the presence of lactose?

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If lactose is available, the lac repressor will be released from the operator (by binding of allolactose). This allows RNA polymerase to move forward on the DNA and transcribe the operon.

These two events in combination – the binding of the activator and the release of the repressor – allow RNA polymerase to bind strongly to the promoter and give it a clear path for transcription. They lead to strong transcription of the lac operon and production of enzymes needed for lactose utilization.

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Chapter 6 EP 14 / Sem 1 : Lac Operon in the absence & presence of lactose  (Matriculation Biology)

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14. If glucose is present, is the lac operon activated?

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14. If glucose is present, is the lac operon activated?

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No, The lac operon of E. coli contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent.
The lac operon has an added level of control so that the operon remains inactive in the presence of glucose even if lactose also is present. High concentrations of glucose catabolites produce low concentrations of cAMP, which must form a complex with CAP to permit the induction of the lac operon.

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13. What is the function of lac operon in E. coli?

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13. What is the function of lac operon in E. coli?

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The lac operon is an operon, or group of genes with a single promoter (transcribed as a single mRNA). The genes in the operon encode proteins that allow the bacteria to use lactose as an energy source.
E. coli bacteria can break down lactose, but it's not their favorite fuel. If glucose is around, they would much rather use that.

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Elongation
- is a subsequent addition of RNA nucleotides complementary to the DNA template strands.

1. Direction of RNA polymerase elongating is 3' to 5'. RNA polymerase is moves along the 3' to 5' DNA template, free nucleotides are added in the 5' to 3' direction complementary to DNA template.
2. RNA nucleotide is being added at the 3' end of the RNA transcript/strand.
3. Product of this process is RNA.
4. Bond that links between these nucleotides is ester bond. A phosphodiester bond is formed between sugar base of one nucleotide and phosphate group of the adjacent nucleotide .
5. Type of nucleotide that makes the product is adenine (A), guanine (G), cytosine (C), and uracil (U). During elongation, nucleotides are added to the growing RNA strands according to the same base pairing rules as for DNA.

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Chapter 6 EP 7 / Sem 1 : Transcription (Matriculation Biology)

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An operon is a cluster of functionally-related genes that are controlled by a shared operator. Operons consist of multiple genes grouped together with a promoter and an operator.

There are three basic components to an operon

Promoter

Operator

Structural genes

what is an operator?
-An operator is a genetic sequence which allows proteins responsible for transcription to attach to the DNA sequence.
-The gene, or genes, which get transcribed when the operator is bound are known as the operon.

what is a promoter?
-A type of DNA sequence that activates the expression of the lactose genes by acting as an RNA polymerase binding site for the lac operon.

what is a structural gene?
-Structural genes are those genes that control the production of a protein that performs a structural role in cell.

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Operon Definition

An operon is a cluster of functionally-related genes that are controlled by a shared operator. Operons consist of multiple genes grouped together with a promoter and an operator. Operons are present in prokaryotes ( bacteria and archaea), but are absent in eukaryotes. In some situations multiple operons are controlled by the same regulatory protein; in these cases the operons form a regulon. Operons were first identified as a mode of gene expression control in 1961 by François Jacob and Jacques Monod.

Operon Structure

Operons are regions of DNA that contain clusters of related genes. They are made up of a promoter region, an operator, and multiple related genes. The operator can be located either within the promoter or between the promoter and the genes. RNA polymerase initiates transcription by binding to the promoter region. The location of the operator is important as its regulation either allows or prevents transcription of the genes into mRNA.

Operon Function

An operon is a complete package for gene expression and synthesis of polypeptides. By combining the related genes, all polypeptides required for a specific function are synthesized in response to a single stimulus. For example, the bacterium Escherichia coli contains a number of genes clustered into operons and regulons: the Lac operon which is involved in lactose degradation, the Trp operon which is involved in tryptophan biosynthesis, and the His operon which is involved in histidine biosynthesis. These operons are turned on when the gene products are needed.

Operons can be under negative or positive control. Negative control involves turning off the operon in the presence of a repressor; this can be either repressible or inducible. A repressible operon is one that is usually on but which can be repressed in the presence of a repressor molecule. The repressor binds to the operator in such a way that the movement or binding of RNA polymerase is blocked and transcription cannot proceed. An inducible operon is one that is usually off. In the absence of an inducer the operator is blocked by a repressor molecule. When the inducer is present it interacts with the repressor protein, releasing it from the operator and allowing transcription to proceed. Repressible operons are generally involved in anabolic pathways, or the synthesis of an essential component, while inducible operons are generally involved in catabolic pathways, or the breakdown of a nutrient. Positive control of an operon is when gene expression is stimulated by the presence of a regulatory protein.

Lac Operon

The Lac operon is the classic operon example, and is responsible for the degradation of the milk protein lactose. The Lac operon is an inducible operon; in the absence of lactose the operator is blocked by a repressor protein. The operon is made up of a promoter with operator, and three genes (lacZ, lacY, and lacA) which encode β-galactosidase, permease, and transacetylase. The three genes are involved in the breakdown of lactose into its metabolites: β-galactosidase breaks lactose down into glucose and galactose, while the other two proteins aid in the metabolic process. The expression of the Lac operon is controlled by the regulatory gene lacI, located immediately adjacent to the promoter region. LacI encodes an allosteric repressor protein that keep the Lac operon "off".

In order for the Lac operon to be turned on, an inducer molecule must inactivate the repressor protein. The inducer molecule in this system is allolactose, an isomer of lactose. When lactose and its isomer are present in the cell, allolactose will bind to allosteric sites on the repressor protein, changing its conformation and rendering it inactive. As the repressor protein detaches from the operator, RNA polymerase can bind to the promoter, transcription can occur, and the three lactose degradation genes can be synthesized.

The figure shows the structure of the Lac operon and the adjacent lacR repressor gene.

The Lac operon is also under positive gene regulation. While the removal of the repressor protein in the presence of lactose is required for synthesis of the lacZ, lacY, and lacA genes, the gene expression will remain low. The level of gene expression is controlled by the amount of the preferred energy source, glucose, in the cell. This control is regulated by an allosteric regulatory protein, catabolite activator protein (CAP). When glucose levels in the cell are low, the organic molecule cyclic AMP is in high concentration. Cyclic AMP activates CAP by binding to the allosteric sites, causing CAP to attach to the Lac operon promoter. Unlike the repressor proteins, binding of CAP to the Lac operon stimulates gene expression. When the cell glucose levels increase, the cyclic AMP levels in the cell decrease, and the activator protein will disassociate from the promoter. Transcription will return to low levels, or will turn off if the repressor protein reattaches.

The figure depicts the Lac operon and how its gene expression is under both positive and negative control.

Trp Operon

The Trp operon is responsible for synthesis of the amino acid trytophan when it is not available in the environment. The Trp operon is made up of a promoter with an operator, and five genes that encode enzymes for tryptophan synthesis. The Trp operon is regulated by the regulatory gene trpR, a gene that is located at a distance from the Trp operon.

The Trp operon is an example of a repressible operon; it is on unless turned off by a repressor protein. The repressor protein is synthesized by trpR. While the repressor protein is always present in the cell, it is synthesized in an inactive form. When a corepressor is present, in this case tryptophan, it binds to the repressor protein in an allosteric site. This changes the conformation of the protein such that it can bind to the operator and block transcription by preventing the binding of RNA polymerase to the promoter. In this way the cell saves energy by not producing tryptophan when it is already present.

The figure depicts the Trp operon with the repressor gene, the promoter, operator, and five tryptophan-synthesizing genes.

Quiz

3. Are repressible operons under positive or negative control?
A. positive
B. negative

References

Campbell, N. A., & Reece, J. B. (2005).Biology, 7th. ed. Ch. 18. San Francisco, CA: Benjamin Cummings. ISBN: 0-8053-7171-0.

Madigan, M. T., & Martinko, J. M. (2006).Brock biology of microorganisms, 11th. ed. Chs. 8 and 10. Upper Saddle River, NJ: Pearson Prentice Hall. ISBN: 0-13-144329-1.

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Operon - Definition, Structure and Function | Biology Dictionary

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An operon is a cluster of functionally-related genes that are controlled by a shared operator. Operons consist of multiple genes grouped together with a promoter and an operator. Operons are present in prokaryotes ( bacteria and archaea), but are absent in eukaryotes.

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2.In what type of cell operon system can be found?                                                                                                       

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2.In what type of cell operon system can be found?                                                                                                       

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-Operons occur primarily in prokaryotes but also in some eukaryotes.
-A prokaryotic cell is a type of cell that does not have a true nucleus or membrane-bound organelles.

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In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.

Originally, operons were thought to exist solely in prokaryotes (which includes organelles like plastids that are derived from bacteria), but since the discovery of the first operons in eukaryotes in the early 1990s, more evidence has arisen to suggest they are more common than previously assumed. In general, expression of prokaryotic operons leads to the generation of polycistronic mRNAs, while eukaryotic operons lead to monocistronic mRNAs.

Operons are also found in viruses such as bacteriophages. For example, T7 phages have two operons. The first operon codes for various products, including a special T7 RNA polymerase which can bind to and transcribe the second operon. The second operon includes a lysis gene meant to cause the host cell to burst.

The term "operon" was first proposed in a short paper in the Proceedings of the French Academy of Science in 1960. From this paper, the so-called general theory of the operon was developed. This theory suggested that in all cases, genes within an operon are negatively controlled by a repressor acting at a single operator located before the first gene. Later, it was discovered that genes could be positively regulated and also regulated at steps that follow transcription initiation. Therefore, it is not possible to talk of a general regulatory mechanism, because different operons have different mechanisms. Today, the operon is simply defined as a cluster of genes transcribed into a single mRNA molecule. Nevertheless, the development of the concept is considered a landmark event in the history of molecular biology. The first operon to be described was the lac operon in . The 1965 Nobel Prize in Physiology and Medicine was awarded to François Jacob, André Michel Lwoff and Jacques Monod for their discoveries concerning the operon and virus synthesis.

Operons occur primarily in prokaryotes but also in some eukaryotes, including nematodes such as C. elegans and the fruit fly, Drosophila melanogaster. rRNA genes often exist in operons that have been found in a range of eukaryotes including chordates. An operon is made up of several structural genes arranged under a common promoter and regulated by a common operator. It is defined as a set of adjacent structural genes, plus the adjacent regulatory signals that affect transcription of the structural genes. ⁵ The regulators of a given operon, including repressors, corepressors, and activators, are not necessarily coded for by that operon. The location and condition of the regulators, promoter, operator and structural DNA sequences can determine the effects of common mutations.

Operons are related to regulons, stimulons and modulons; whereas operons contain a set of genes regulated by the same operator, regulons contain a set of genes under regulation by a single regulatory protein, and stimulons contain a set of genes under regulation by a single cell stimulus. According to its authors, the term "operon" is derived from the verb "to operate".

An operon contains one or more structural genes which are generally transcribed into one polycistronic mRNA (a single mRNA molecule that codes for more than one protein). However, the definition of an operon does not require the mRNA to be polycistronic, though in practice, it usually is. Upstream of the structural genes lies a promoter sequence which provides a site for RNA polymerase to bind and initiate transcription. Close to the promoter lies a section of DNA called an operator.

All the structural genes of an operon are turned ON or OFF together, due to a single promoter and operator upstream to them, but sometimes more control over the gene expression is needed. To achieve this aspect, some bacterial genes are located near together, but there is a specific promoter for each of them; this is called gene clustering. Usually these genes encode proteins which will work together in the same pathway, such as a metabolic pathway. Gene clustering helps a prokaryotic cell to produce metabolic enzymes in a correct order.

An operon is made up of 3 basic DNA components:

Promoter - a nucleotide sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. In RNA synthesis, promoters indicate which genes should be used for messenger RNA creation - and, by extension, control which proteins the cell produces.

Operator - a segment of DNA to which a repressor binds. It is classically defined in the lac operon as a segment between the promoter and the genes of the operon. The main operator (O1) in the lac operon is located slightly downstream of the promoter; two additional operators, O2 and O3 are located at -82 and +412, respectively. In the case of a repressor, the repressor protein physically obstructs the RNA polymerase from transcribing the genes.

Structural genes - the genes that are co-regulated by the operon.

Not always included within the operon, but important in its function is a regulatory gene, a constantly expressed gene which codes for repressor proteins. The regulatory gene does not need to be in, adjacent to, or even near the operon to control it.

An inducer (small molecule) can displace a repressor (protein) from the operator site (DNA), resulting in an uninhibited operon.

Alternatively, a corepressor can bind to the repressor to allow its binding to the operator site. A good example of this type of regulation is seen for the trp operon.

Control of an operon is a type of gene regulation that enables organisms to regulate the expression of various genes depending on environmental conditions. Operon regulation can be either negative or positive by induction or repression.

Negative control involves the binding of a repressor to the operator to prevent transcription.

In negative inducible operons, a regulatory repressor protein is normally bound to the operator, which prevents the transcription of the genes on the operon. If an inducer molecule is present, it binds to the repressor and changes its conformation so that it is unable to bind to the operator. This allows for expression of the operon. The lac operon is a negatively controlled inducible operon, where the inducer molecule is allolactose.

In negative repressible operons, transcription of the operon normally takes place. Repressor proteins are produced by a regulator gene, but they are unable to bind to the operator in their normal conformation. However, certain molecules called corepressors are bound by the repressor protein, causing a conformational change to the active site. The activated repressor protein binds to the operator and prevents transcription. The trp operon, involved in the synthesis of tryptophan (which itself acts as the corepressor), is a negatively controlled repressible operon.

Operons can also be positively controlled. With positive control, an activator protein stimulates transcription by binding to DNA (usually at a site other than the operator).

In positive inducible operons, activator proteins are normally unable to bind to the pertinent DNA. When an inducer is bound by the activator protein, it undergoes a change in conformation so that it can bind to the DNA and activate transcription.

In positive repressible operons, the activator proteins are normally bound to the pertinent DNA segment. However, when an inhibitor is bound by the activator, it is prevented from binding the DNA. This stops activation and transcription of the system.

The lac operon of the model bacterium Escherichia coli was the first operon to be discovered and provides a typical example of operon function. It consists of three adjacent structural genes, a promoter, a terminator, and an operator. The lac operon is regulated by several factors including the availability of glucose and lactose. It can be activated by allolactose. Lactose binds to the repressor protein and prevents it from repressing gene transcription. This is an example of the derepressible (from above: negative inducible) model. So it is a negative inducible operon induced by presence of lactose or allolactose.

Discovered in 1953 by Jacques Monod and colleagues, the trp operon in E. coli was the first repressible operon to be discovered. While the lac operon can be activated by a chemical ( allolactose), the tryptophan (Trp) operon is inhibited by a chemical (tryptophan). This operon contains five structural genes: trp E, trp D, trp C, trp B, and trp A, which encodes tryptophan synthetase. It also contains a promoter which binds to RNA polymerase and an operator which blocks transcription when bound to the protein synthesized by the repressor gene (trp R) that binds to the operator. In the lac operon, lactose binds to the repressor protein and prevents it from repressing gene transcription, while in the trp operon, tryptophan binds to the repressor protein and enables it to repress gene transcription. Also unlike the lac operon, the trp operon contains a leader peptide and an attenuator sequence which allows for graded regulation. This is an example of the corepressible model.

The number and organization of operons has been studied most critically in E. coli. As a result, predictions can be made based on an organism's genomic sequence.

One prediction method uses the intergenic distance between reading frames as a primary predictor of the number of operons in the genome. The separation merely changes the frame and guarantees that the read through is efficient. Longer stretches exist where operons start and stop, often up to 40-50 bases.

An alternative method to predict operons is based on finding gene clusters where gene order and orientation is conserved in two or more genomes.

Operon prediction is even more accurate if the functional class of the molecules is considered. Bacteria have clustered their reading frames into units, sequestered by co-involvement in protein complexes, common pathways, or shared substrates and transporters. Thus, accurate prediction would involve all of these data, a difficult task indeed.

Pascale Cossart's laboratory was the first to experimentally identify all operons of a microorganism, Listeria monocytogenes. The 517 polycistronic operons are listed in a 2009 study describing the global changes in transcription that occur in L. monocytogenes under different conditions.

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In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e.

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3. What are the example of organism in prokaryotic cells?

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3. What are the example of organism in prokaryotic cells?

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-Example of Prokaryotes-

bacteria -They constitute a large domain of prokaryotic microorganisms and  are small single-celled organisms like the bacterium Escherichia coli (E. coli)

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Biology Articles, Tutorials & Dictionary Online

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Prokaryote
n., plural: prokaryotes
[prəʊˈkærɪɒt]
Definition: An organism lacking a distinct membrane-bound nucleus

Prokaryote refers to any of the group of organisms primarily characterized by the lack of true nucleus and other membrane-bound cell compartments: such as mitochondria and chloroplasts, and by the possession of a single loop of stable chromosomal DNA in the nucleoid region and cytoplasmic structures, such as plasma membrane, vacuoles, primitive cytoskeleton, and ribosomes. Examples of prokaryotes are bacteria and archaea.

Prokaryote Definition

A prokaryote is defined as any organism that is chiefly characterized by a cell devoid of a well-defined nucleus as opposed to a eukaryote that has a nucleus. Instead of a nucleus, the prokaryotes have a nucleoid region where the genetic materials are located.

Etymology

The term prokaryote (plural: prokaryotes) came from the Latin pro, meaning " in favour of" or " on behalf of" and káry(on), meaning " nut", "kernel". The term prokaryotic is a derived word and used to refer to a prokaryote. Compare: eukaryote. Variant: procaryote.

Prokaryotic Cell

Prokaryotes are unicellular organisms that lack a well-defined nucleus. They have instead a nucleoid region in their cytoplasm where their genetic material occurs in most instances as a single, circular molecule of DNA. They generally reproduce asexually, which is by binary fission or by budding. Most of them are unicellular, others are capable of forming stable aggregate communities. Conjugation, apparently, is the counterpart of sexual reproduction in eukaryotes where two cells exchange genetic materials via a conjugation tube.

Although prokaryotes lack the true organelles found in a eukaryotic cell, they possess certain cytoplasmic structures. For instance, they have a primitive cytoskeleton. Flagellin is the cytoskeletal protein that confers structural support to bacteria that is essential during chemotaxis. Other cytoplasmic structures are ribosomes, carboxysomes, chlorosomes, magnetosomes, and thylakoid systems. Some references regard them as prokaryotic organelles. However, they are not bounded by lipids; rather, they are proteinaceous. For example, carboxysomes are protein-shell compartments that are involved in carbon fixation in certain bacteria. Chlorosomes are light-harvesting complex in green sulfur bacteria. Magnetosomes are present in magnetotactic bacteria. Thylakoids are present in photosynthetic bacteria, such as cyanobacteria. Some prokaryotes have a cell wall that surrounds the cell membrane. Bacterial cell walls are composed chiefly of peptidoglycan. Its thickness can be used to determine if the bacterial cell is Gram-positive (thicker cell wall) or Gram-negative (thinner cell wall). As for the archaea, their cell wall is made up of glycoprotein S-layers, pseudopeptidoglycan, or polysaccharides rather than peptidoglycan (except for a group of methanogens).

Eukaryote vs. Prokaryote

Eukaryotes are organisms in which at the cellular level most of their genetic material is located inside a double-membraned nucleus. Other genetic materials outside the nucleus are found in the mitochondria and the chloroplasts (if present). The chromosomes of eukaryotes are linear strands of DNA. Both eukaryotes and prokaryotes store their genetic information in their genes. The main source of metabolic energy is ATP.

In prokaryotes, the chromosome is mostly circular. Prokaryotes are smaller in size than eukaryotes. Therefore, prokaryotes have a large surface area to volume ratio. And because of this, they have a high metabolic rate and high growth rate.

Both eukaryotes and prokaryotes have ribosomes that serve as the site of protein synthesis. However, the composition of their ribosomes differs. The prokaryotic ribosome is 70S and it is made up of 50S (large subunit) and 30S (small subunit). The eukaryotic ribosome is 80S and it consists of 60S (large subunit) and 40S (small subunit). [N.B. the S units do not add up since they represent measures of sedimentation rate, not mass.]

Examples of prokaryotes are eubacteria and archaea.

Examples

Prokaryotes include the domains, Eubacteria and Archaea. Examples of prokaryotes are bacteria, archaea, and cyanobacteria (blue-green algae).

Bacteria

Bacteria are microscopic, single-celled organisms that belong to Domain Eubacteria (true bacteria). Their cells lack lipid-bound subcellular compartments and their DNA is found in the cytoplasm (nucleoid region) rather than inside a nucleus. They reproduce by fission or by forming spores. They can inhabit all kinds of environments, such as in soil, acidic hot springs, radioactive waste, seawater, deep in the Earths crust, in the stratosphere, and even in the bodies of other organisms. Bacteria include the bacilli, the cocci, the spirochetes, and the vibrios.

Archaea

Archaea belong to the Domain Archaea. They are unicellular microorganisms that are genetically distinct from bacteria and eukaryotes. Similar to prokaryotes, they lack a nucleus. However, the genes of archaebacterial are more similar to eukaryotes. Both of them produce certain enzymes that are used in transcription, translation, and other metabolic pathways. Many archaebacterial are found thriving in extreme habitats. Archaebacteria include the halophiles (those inhabiting extremely salty environments), the methanogens (archaea species producing methane), and the thermophiles (those that can thrive in extremely hot habitats).

Cyanobacteria

Cyanobacteria, also called blue-green algae, are microorganisms that are formerly considered as protists for being single-celled and photosynthetic. However, they now belong to a group or phylum of photosynthetic bacteria that inhabit aquatic habitats and moist soils. Cyanobacteria are ecologically significant because a huge percentage of gaseous oxygen comes from their photosynthetic activity. They may lack a nucleus but they possess microcompartments (e.g. thylakoids and carboxysomes). They also have photosynthetic pigments (particularly, phycobiliproteins) that account for the bluish-green color of their cells. Cyanobacteria include Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, and
Stigonematales.

Read:

References:

1. Prokaryotic vs. Eukaryotic. (2019). Retrieved from Nku.edu website: https://www.nku.edu/~whitsonma/Bio150LSite/Lab%205%20Cells/Bio150LRCellTypes.htm

2. Long, B. M., Badger, M. R., Whitney, S. M., & Price, G. D. (October 2007). "Analysis of carboxysomes from Synechococcus PCC7942 reveals multiple Rubisco complexes with carboxysomal proteins CcmM and CcaA". The Journal of Biological

3. Chemistry. 282 (40): 29323-35. doi:10.1074/jbc.M703896200.

4. Armstronf, J. E. (2015). How the Earth Turned Green: A Brief 3.8-Billion-Year History of Plants. The University of Chicago Press. Retrieved from https://www.press.uchicago.edu/ucp/books/book/chicago/H/bo16465693.html

© Biology Online. Content provided and moderated by Biology Online Editors

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Prokaryote n., plural: prokaryotes [prəʊˈkærɪɒt] Definition: An organism lacking a distinct membrane-bound nucleus Prokaryote refers to any of the group of organisms primarily characterized by the lack of true nucleus and other membrane-bound cell compartments: such as mitochondria and chloroplasts, and by the possession of a single loop of stable chromosomal DNA in the nucleoid region and cytoplasmic structures, such as plasma membrane, vacuoles, primitive cytoskeleton, and ribosomes.

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4. Example of operon system that operate in E.coli.

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4. Example of operon system that operate in E.coli.

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The lactose metabolism system (lac operon)

The lac operon is an operon, or group of genes with a single promoter (transcribed as a single mRNA). The genes in the operon encode proteins that allow the bacteria to use lactose as an energy source.

The lac operon of E. coli contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent.

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Regulation of gene expression and cell specialization

Current time:0:00Total duration:11:05

Regulation of gene expression and cell specialization

Video transcript

we're now going to talk about one of the most famous operons and this is the lac operon and it is part of eco lies genome and it is involved and the lac right over here is referring to lactose and so you can imagine that it codes for genes involved in the metabolism of lactose and the word lactose might already be familiar to you it is a sugar found in milk some of us including myself are lactose intolerant I have trouble digesting lactose so I have mixed feelings regarding this but in general for a cell to make use of it it needs to be able to absorb the lactose it needs to be able to split it up into simpler sugars that it can actually use for fuel and that is what the genes in the lac in the lac operon actually do code for so just as an example the lac z gene right over here this codes for an enzyme that helps cleave the lactose into simple sugars the lac y gene codes for an enzyme that allows for the absorption of lactose through cellular membranes Lac a is a little bit more interesting a little less understood but the general idea here is all three of these are involved in the metabolism and the absorption of lactose and it is an operon so we have our promoter here where our RNA polymerase would attach and I've also drawn some other sites I've drawn this the operator right over here where you can imagine a repressor and it is indeed the lac repressor will could bind and over here this cap site or CA p site cap stands for Khattab a lighter protein catabolized catabolize activator whoops acti activator protein and so this is you can imagine where a protein called the Khattab elect evader protein can bind and perhaps be an activator so with that out of the way let's think about different scenarios let's think about a scenario let's think about a scenario where the e coli is an environment where there is no lactose so what do you think should happen over here and a lot of these things are very logical if you just assume that a lot of biological organisms are quite stingy they don't want to just waste resources well if there's no lactose well why transcribe why transcribe the genes that can be translated into enzymes for the metabolism of lactose so there's no lactose you can almost view this as a default state right over here you actually have the lac repressor protein being bound to the operator so this is the lac repressor lac repressor right over there and so you won't be able to transcribe these things the RNA polymerase the RNA polymerase won't won't be able to get anything done it's not no transcription is going to occur so no lactose no transcription transcription which makes a lot of sense the bacteria or the bacterium singular that doesn't want to waste resources so what do you think should happen if there is lactose so I'll keep this up here so you can see it so lactose present lactose lactose present well you can imagine well you don't want that repressor around anymore and that is indeed what happens is that you have an isomer of lactose called a low lactose so if lactose is present you're going to have also a low lactose present right over here and so that is a low a low lactose which can act as an inducer of transcription and the way that it acts as an inducer is if it binds to the Lac repressor the lac repressor can no longer bind to the operator site so when this is when the a low lactose is present it will bind to the repressor and then the repressor is going to leave the operator site it's not going to be able to bind as well and so let me draw that so in this case the operator sorry the repressor I should say the operators where the repressor binds so this is the repressor right over here you have some allolactose let me do that in white you have some aloe lactose that has bound to it and because of that it's not going to bind to the operator and since it's not bound to the operator well now the RNA polymerase can actually transcribe these genes and that's valuable because by transcribing these genes we are going to be able to metabolize this lactose so lactose present you have transcription transcription transcription occurs now that's a very high-level simple view of the lac operon but there is more involved because there's other sugars in particular glucose which is preferred by the cell so whoops I'm moving the wrong part there you go so let's think about what will happen in the presence of glucose and not in the presence of glucose so let me write here so glucose glucose and no and no glucose actually let me do it let me do it well let me I'll do no glucose first let's say we have no glucose and remember glucose is preferred to lactose simpler sugar if you have glucose around why worry about the lactose and then here we have we have glucose we have glucose around and we could talk about both of these both of these situations in the presence of lactose or not in the presence of lactose but if we don't have if we don't have any lactose around then we're not going to have the a low lactose around and then you're just going to have the repressor sit on the operator and you're not going to have any transcription so you can and that's going to be whether or not we have glucose so I'm going to think about no glucose but we do have lactose plus lactose and in here you have glucose plus glucose plus lactose well the lactose part if we have lactose around then we're going to have the a low lactose around and we just covered this scenario the a low lactose binds binds to the lac repressor keeps the lac repressor from binding to the operator and so you have you have your RNA polymerase is able to actually perform the transcription but that's not it URF you in a situation with no glucose you actually are going to also involve the cap side you're going to have an activator that's going to make this happen even more because if you don't have glucose around man you really need that lactose and so what you have is something called so let me draw this the Khattab alight activator protein right over here the Khattab alight activator protein and in the presence of cyclic ANP Edina side ICD design monophosphate it's a derivative of ATP and so this is that right over there cyclic a MP you'll see that come up a lot in biology so this is the Khattab alight activator protein in the presence of CA MP and we'll talk about how cyclic a MP relates to glucose in a second in that presence it is going to bind to this the cap site and it is going to further activate the transcription so in this situation no glucose plus lactose you're going to have even more transcription so let me write this down lots of transcription lots of trans transcription lots of transcription now I know you're probably asking this is what I first asked myself when people told me about cyclic a and B well how does cyclical A&P relate to glucose well I'm not going to go into a huge amount of detail here but what you need to know here and it makes sense is that if you have glucose glue so let me write it this way if you have high glucose high glucose I'm having trouble writing high glucose then that's going to inhibit the production of cyclic a and P so low cyclic adenosine monophosphate and if you have low glucose or no glucose it's like a tongue twister if you have low glucose well you're not going to inhibit the creation of cyclic a and P and so you're going to have high cyclic a and P so if you have no glucose or low glucose we are in this scenario right over here you're going to have high constant or higher concentrations of cyclic a and P which can bind to the catabolized of a toe protein which then acts as an activator to allow even more transcription of the lac operon which once again why is it important well if there's no glucose or low glucose you're really going to need that lactose so you really want to transcribe these genes as much as possible now what about the situation where there is glucose and lactose well once again if there is lactose then you're going to have a low lactose which is going to be able to bind to the lac repressor and by it binding to the lac repressor the lac repressor is not going to be able to bind to the operator and so you do have once again the RNA polymerase is going to be able to transcribe but because you have glucose present because you have glucose present you're going to have low or I'll just write no all right low cyclic a MP and since you have low or no cyclic A&P around well that cyclic a and P isn't going to be able to bind to the Khattab 'light activator protein and so the Khattab later the Khattab light activator protein isn't going to be able to act as an activator I know this is I'm using a lot of words multiple times and so it's not going to bond to the to the activator side right here to the to the cap site and so you're going to have less transcription less transcription transcription which once again makes sense you've got glucose and lactose around the cell would prefer to use glucose simpler sugar why waste resources you have plenty of energy around just go straight to the glucose but if you don't have glucose around well then use more resources so that you can digest the lactose

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Overview of gene regulation in the Lac operon. Discussion of CAP, cAMP, lac repressor and allolactose in regulation of lac operon.

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The Role of Translation Control in Tumorigenesis and Its Therapeutic Implications

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The Role of Translation Control in Tumorigenesis and Its Therapeutic Implications

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TRANSLATION INHIBITORS IN CANCER TREATMENT 

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TRANSLATION INHIBITORS IN CANCER TREATMENT 

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A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland

International Clinical Research Center, St. Anne University Hospital, Brno, Czechia

Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czechia

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

University Hospital Tübingen, Department of Internal Medicine VIII, University of Tübingen, Tübingen, Germany

Transcription and translation are fundamental cellular processes that govern the protein production of cells. These processes are generally up regulated in cancer cells, to maintain the enhanced metabolism and proliferative state of these cells. As such cancerous cells can be susceptible to transcription and translation inhibitors. There are numerous druggable proteins involved in transcription and translation which make lucrative targets for cancer drug development. In addition to proteins, recent years have shown that the "undruggable" transcription factors and RNA molecules can also be targeted to hamper the transcription or translation in cancer. In this review, we summarize the properties and function of the transcription and translation inhibitors that have been tested and developed, focusing on the advances of the last 5 years. To complement this, we also discuss some of the recent advances in targeting oncogenes tightly controlling transcription including transcription factors and KRAS. In addition to natural and synthetic compounds, we review DNA and RNA based approaches to develop cancer drugs. Finally, we conclude with the outlook to the future of the development of transcription and translation inhibitors.

Introduction

Cancer refers to a large group of diseases of uncontrolled cell growth and division where a general cure or containment is nowhere to be seen. As a testament to this, the research in cancer is ongoing vigorously and journals specifically tailored for cancer research are now more than 50, ranging from the general to the specific types of cancer. According to the World Health Organization, the various types of cancer accounted for nearly 10 million deaths in 2018, which made it the second most common cause of death. The common factor in cancers is the malignant transformation of cells due to acquired genetic mutations. These are often many and can include both driver and passenger mutations, that confer a growth advantage ( Pon and Marra, 2015). These mutations can lead to activation (gain of function) or deactivation (loss of function,) in some of the biological processes that lead to cancer. When transcriptional or translational processes are disrupted a tumor might be formed.

Chemotherapy is a general term used for all chemicals to treat cancer. It includes for example hormone therapy, which is used to slow down hormone-reactive cancer growth, and cisplatin which prevents the replication of DNA. Also, the focus of this review, targeted therapies are included under chemotherapy. Targeted therapy differs from general chemotherapy by taking a more specialized approach which can be compared to a sniper rifle (targeted therapy) vs. a shotgun (chemotherapy). In other words, the probability of killing healthy cells is lower with targeted therapies than with general chemotherapeutics. While chemotherapy is directed to the inhibition of cell mitosis or inducing autophagy, targeted small molecules inhibitors act on the transcription and translation processes.

Targeting Transcription

Most small molecules used in targeted therapy affect transcription, at three of the four different steps of this process. On the pre-initiation complex, bromodomain, and extra-terminal motif inhibitors reversibly inhibit the ability of the bromodomain to bind to acetylated histones ( Alqahtani et al., 2019), effectively slowing down the formation of the PIC ( Figure 1). Still, at this first step, cyclin-dependent kinase (CDK) inhibitors have been researched and widely tested due to their role in the cell cycle ( Blachly and Byrd, 2013). Their importance is not limited to cancer diseases ( Malumbres and Barbacid, 2001, 2009) but also other diseases such as in HIV ( Galons et al., 2013). At the second step of the transcription process, Pol II inhibitors block the formation of the Pol II-transcription factor complexes. The study of these inhibitors dates back to the late 80's and is still a subject of study today ( Logan et al., 1989; Sharma et al., 2019) ( Figure 1). The last step that can be acted upon at transcription level is the elongation step. There are two main methods of inhibiting this step: inhibition of transcription factors and usage of premature transcription chain terminators ( Figure 1). After being considered undruggable for a long time, recent years have seen a growing number of transcription factor inhibitors and although the sheer number of known TF's makes it difficult to find a general acting drug, it also allows for a greater range of inhibitors to be developed ( Bushweller, 2019). The transcription chain termination is naturally processed by Pols and to terminate it prematurely nucleoside analogs and/or inhibitors, such as fludarabine, are used.

Inhibitors of the Epigenetic Machinery

RNA Polymerase Inhibitors

RNA polymerases produce various RNA molecules. In humans, RNA polymerase I (Pol I) synthesizes the precursors of ribosomal (r)RNA, the main component of ribosomes; Pol II synthesizes precursors of mRNA and most of the snore (sn)RNA and micro (mi)RNAs; while RNA polymerase III synthesizes transfer (t)RNAs and other small RNAs. Cancer drugs or drug candidates have been developed to target all three human RNA polymerases. Some potent natural toxins, such as α-amatinin, inhibit RNA polymerases ( Seifart and Sekeris, 1969). There have been efforts to convert α-amatinin into a cancer drug by combining it with antibodies for specificity. In addition to these conjugates, there are synthesized inhibitors of RNA polymerases, some of which have made it into clinical trials but unfortunately none of these RNA polymerase inhibitors have been approved for therapy as of yet (3/2020). Structures of the discussed RNA polymerase inhibitors are shown in Figure 2.

α-amatinin Conjugates

CX-5461

BMH-21

Small molecule BMH-21 ( Figure 2) is a DNA intercalator which also inhibits Pol I in a manner which is not dependent on DNA damage ( Peltonen et al., 2010; Colis et al., 2014). BMH-21 both inhibits Pol I and induces the degradation of the largest subunit of Pol I ( Peltonen et al., 2014). The inhibition of Pol I by BMH-21 targets the transcription elongation phase and induces pausing in the transcription process ( Wei et al., 2018). Despite the promising in vitro results, no clinical trials have been started with BMH-21.

TAS-106

Metarrestin

Transcriptional Complex Disruptors

Different complexes of proteins can be disrupted during transcription. The first complex to form is the pre-initiation complex which can be disturbed by bromodomain inhibitors. It is also possible to disturb the elongation process by preventing the binding of elongation factors to RNA polymerase, which is the mechanism of action of triptolide. The compounds included in this section are protein-protein interaction inhibitors, and their structures are shown in Figure 3.

Bromodomain Inhibitors

A short overview of the topic will be given here, since there are thorough reviews about bromodomain inhibitors from recent years ( Pérez-Salvia and Esteller, 2016; Alqahtani et al., 2019; Letson and Padron, 2019). Bromodomain (BRD) inhibitors or BET (bromodomain and extraterminal domain) inhibitors prevent the interaction of bromodomain family proteins (BRD2, BRD3, BRD4, and BRDT) with acetylated histones and transcription factors ( Filippakopoulos et al., 2010; Junwei and Vakoc, 2014; Fu et al., 2015). Since the bromodomain-containing proteins regulate gene expression through various processes including histone recognition and modification, chromatin remodeling, and regulation of the transcriptional machinery, BRD and BET inhibitors can be potent transcriptional inhibitors ( Fujisawa and Filippakopoulos, 2017). In cancer, the acetylation state of histones and other proteins is altered, and BRDs promote the expression of many oncogenes, such as c-Myc and Bcl-2, and thus their inhibition provides a way to inhibit cancer cell growth (see also section Inhibitors of Transcription Factor Gene Expression) ( Alqahtani et al., 2019). Most of the bromodomain inhibitors, such as JQ-1, compete for the acetylated lysine binding site and are thus competitive inhibitors ( Alqahtani et al., 2019). However, some of its analogs have made it into the clinical trials (such as birabresib in leukemia and glioblastoma) ( Alqahtani et al., 2019). The results of BET inhibitors as monotherapy have been suffering from resistance, lack of response and toxicity issues ( Bolden et al., 2014; Letson and Padron, 2019), even though they were relatively effective in preclinical models of various cancers ( Kharenko et al., 2016; Waring et al., 2016; Letson and Padron, 2019). This has sparked an interest in using BRD inhibitors in combination with other chemotherapeutic agents, which has shown promising results in animal models so far ( Alqahtani et al., 2019). There are still dozens of on-going clinical trials for various BRD inhibitors in multiple different cancers with or without other chemotherapeutic agents. With more than 600 unique interaction partners in the cells, this family of proteins will continue to spark the curiosity of researchers for a long time.

Triptolide

Triptolide ( Figure 3) is a diterpene triepoxide produced by thunder god vine, a plant regularly used in Chinese traditional medicine for rheumatoid arthritis ( Su et al., 1990). In addition to inhibiting heat shock protein 70, it is an inhibitor of Pol I and Pol II which functions by blocking the transcription elongation process while binding to transcription factor TFIIH ( Titov et al., 2011). It also facilitates degradation of the largest subunit of Pol II in a CDK7-dependent manner ( Vispé et al., 2009; Manzo et al., 2012). Triptolide kills colorectal cancer cells in vitro and inhibits the growth of colorectal xenografts in a mouse model ( Wang et al., 2009; Oliveira et al., 2015). Recently, Liang et al. tested triptolide in adenomatous polyposis coli (Apc) mutated mice where it effectively inhibited colorectal cancer proliferation ( Liang et al., 2019b). Interestingly, they noted that triptolide also reduced Pol III mediated transcription by inhibiting TFIIIB formation at Pol III target genes. Specifically, it did so by blocking the interaction of TBP and Brf1 at Pol III promoters, thereby reducing tRNAs and 5S rRNA transcription. The inhibition of cancer growth both in vitro and in vivo makes triptolide a potential drug candidate for colorectal cancer. However, due to the toxicity of triptolide, only the prodrug disodium salt form of it, minnelide is being studied in human trials for pancreatic and liver cancers ( Banerjee and Saluja, 2015).

Premature Transcription Chain Terminators

Transcription is terminated when specific ending codons are reached on the DNA and after it the mRNA transcript unbinds from the RNA polymerase. The termination is coupled with the 3′-end processing which includes cleavage and addition of a poly-adenosine (A) chain ( Cramer, 2004). This process can be disturbed by mimetics of adenosine or fludarabine ( Figure 4).

Adenosine Analogs

Since transcription generally terminates when a poly-A chain is added to the mRNA transcript, modified adenosine analogs have been the focus of research as premature transcription chain terminators. 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-NH ₂-Ado) ( Figure 4) can be incorporated to both the body and the poly(A) tail of the transcript, where they inhibit further synthesis of it ( Gandhi et al., 2001). In addition to incorporation to mRNA, 8-Cl-Ado, and 8-NH ₂-Ado can be phosphorylated by adenosine kinase into corresponding ATP analogs which decreases the amount of available ATP in the cell ( Frey and Gandhi, 2010). Interestingly, these compounds are not cytotoxic for non-transformed cells ( Balakrishnan et al., 2005; Dennison et al., 2010). 8-Cl-Ado is in phase I clinical trials for acute myeloid leukemia and chronic lymphocytic leukemia ( Stellrecht et al., 2017). Recently, 8-Cl-Ado showed a positive synergistic effect with another cancer drug in a mice xenograft model of acute myeloid leukemia ( Buettner et al., 2019).

Fludarabine

CDK Inhibitors

Alvocidib (Flavopiridol)

Alvocidib (formerly flavopiridol, Figure 5) is a semisynthetic flavonoid resembling rohitukine and it inhibits CDK1, CDK2, CDK4, CDK6, and CDK9. It was the first CDK inhibitor which reached clinical trials in 1998 ( Senderowicz, 1999; Kelland, 2000). Since then more than 60 phase I and phase II clinical trials in various cancers have been conducted using it ( Asghar et al., 2015). The broad target spectrum lead to promising in vitro results, but unfortunately the clinical tests showed only a little activity ( Asghar et al., 2015). There are few positive results for leukemia and lymphoma, and new phase I and II clinical trials are continuously started for alvocidib ( Byrd et al., 2007; Blum et al., 2011). Despite all the investments and thorough studies, alvocidib has not made it into phase III clinical trials, as of 2020. The main issues with alvocidib and other non-selective CDK inhibitors are the uncertainty of their mechanism of action, the problems in patient selection for clinical trials and the lack of a therapeutic window as a result of CDK inhibition in healthy cells ( Asghar et al., 2015).

Palbociclib

Palbociclib ( Figure 5) is a selective inhibitor of CDK4 and CDK6 and it was the first inhibitor of CDKs that was approved as a cancer therapy in combination with letrozole, an aromatase inhibitor ( Lu, 2015; Turner et al., 2015; Finn et al., 2016). Targeting only these CDKs in tumors that have CDK4/CDK6 dysregulation, causes arrest of the cell cycle mediated by retinoblastoma 1 ( Malumbres et al., 2004; Toogood et al., 2005). This results in lower levels of cyclins, nucleotide biosynthesis, DNA replication machinery and mitotic regulatory genes ( Dean et al., 2010; Rivadeneira et al., 2010). Palbociclib is a product of multiple cycles of chemical screening and optimization which started from a set of pyrido[2,3-d]pyrimidin-7-one compounds with a 2-amino pyridine side chain at the C2 position that were showing specificity for CDK4/CDK6 over other CDKs ( VanderWel et al., 2005). During the extensive clinical trials of palbociclib, its cytotoxic effect have been proven, and the main adverse effect has been neutropenia ( Asghar et al., 2015). Neutropenia is a common adverse effect of chemotherapies, but in the case of palbociclib it is a rapidly reversible condition which can be avoided by intermittent dosage. The clinical tests also revealed that palbociclib has a beneficial effect in combination with hormone therapy in estrogen receptor (ER) positive breast cancer cell lines ( Finn et al., 2009). Importantly, the inhibition of CDK4 and CDK6 showed activity in multiple ER positive cell lines that had developed resistance to ER antagonists ( Miller et al., 2011; Thangavel et al., 2011). This lead to many phase II studies which confirmed the significant improvement in the median progression-free survival and granted palbociclib the Breakthrough Therapy designated from the FDA in 2013( Asghar et al., 2015).

Ribociclib

Abemaciclib

Targeting Translation

In the translation initiation phase, the 80S ribosome binds to the start of the mRNA after which tRNA carrying a methionine are able to bind to the starting codon AUG ( Schuller and Green, 2018). The initiation phase is assisted by a wide variety of eukaryotic translation initiation factors (eIFs). For drug design, the most important eIF is eIF4G ( Figure 6). During the elongation phase, the 80S ribosome moves along the mRNA template, binding new tRNA molecules with corresponding amino acids to synthesize the polypeptide chain. This process is coordinated by the eukaryotic elongation factors (eEFs). Once the 80S ribosome encounters a termination codon which is recognized by eukaryotic peptide chain release factors (eRFs), it releases from the mRNA and the polypeptide chain. Finally, the 80S ribosome complex separates into subunits 40 and 60S to begin a new round of translation. At the translation level, it has been shown that many signaling pathways are dysregulated in cancers ( Wolfe et al., 2014; Faller et al., 2015). This association has been bringing translational control into the foreground of targeted cancer therapies ( Vogel and Marcotte, 2012), where the spotlight was previously reserved for transcription level inhibitors. Here, the first step, initiation, is the most targeted by inhibitors, with the mammalian target of rapamycin (mTOR) inhibitors being in recent years one of the main targets of study ( Figure 6) ( Hua et al., 2019). Bioengineered non-coding RNA agents (BERAs) and antisense oligonucleotides, single-chain DNA that prevent translation by interacting with mRNA are also targeting this initial step ( Duan and Yu, 2016; Jian et al., 2017). On the second step of translation, tRNA is targeted for inhibition, thus blocking the protein synthesis process. These inhibitors act by binding the free ribosome, interfering with the normal tRNA binding and thus blocking the elongation step ( Figure 6) ( Gandhi et al., 2014).

mTOR Related Inhibitors

The mammalian (or mechanistic) target of rapamycin (mTOR) is a protein kinase which is the main component of mTOR complexes 1 and 2 (mTORC1 and mTORC2) which are one of the most important regulators of translation ( Sabers et al., 1995). It functions as a serine/threonine protein kinase regulating many cellular processes, such as protein, lipid and nucleotide synthesis as well as nutrient sensing ( Hsieh et al., 2012; Kim and Guan, 2019). The mTOR complexes affect translation by phosphorylation of multiple translation factors, including eIF4G ( Iadevaia et al., 2012). The MTOR gene itself is often mutated in cancer ( Grabiner et al., 2014). In addition, mTORC1 and mTORC2 affect oncogenic pathways, such as phosphatidylinositol-3-OH kinase (PI3K)-PKB/Akt pathway, and thus their signaling is frequently activated in cancer, for example in glioblastoma and follicular lymphoma ( Kim and Guan, 2019). The readers interested in a more detailed overview of mTOR and its biology are referred to the recent and comprehensive review written by ( Kim and Guan, 2019).

The inhibitors of mTOR complexes can be divided into three generations, first of which consist of rapamycin and its analogs termed rapalogs, which affect only specific parts of the mTOR complexes ( Kim and Guan, 2019; Tian et al., 2019). The latter generation is termed ATP-competitive catalytic inhibitors or mTOR kinase inhibitors, and they target the catalytic activity of mTOR. The newest additions to the mTOR related drugs are called RapaLinks which consist of rapamycin linked with an mTOR kinase inhibitor, a combination of the previous two generations.

Rapamycin

Rapamycin (sirolimus, Figure 7) is an antifungal molecule produced by Streptomyces hygroscopicus and it was first discovered from a soil sample from the Easter Island (Rapa Nui) ( Vézina et al., 1975). Structurally, rapamycin resembles the immunosuppressant tacrolimus, and it has similar immunosuppressive effect and mechanism of action via the inhibition of T- and B-cells ( Sehgal, 2003). Because of these immunosuppressive properties rapamycin is one of approved drugs for the prophylaxis of renal transplantation ( Pidala et al., 2012). It was later discovered that in addition to its antifungal and immunosuppressive properties, it is a potent inhibitor of many mammalian kinases ( Chung et al., 1992; Kuo et al., 1992; Price et al., 1992; Sehgal, 2003). About 20 years after the discovery of rapamycin, its target was identified and aptly named, the mammalian target of rapamycin, mTOR ( Sabers et al., 1995; Wiederrecht et al., 1995). Rapamycin functions by binding to 12-kDa FK506-binding protein (FKBP12) forming a complex which then allosterically inhibits mTORC1, but not mTORC2 ( Li et al., 2014). Long-term rapamycin treatment can also affect mTORC2 signaling but the mechanism of this is not clear ( Kim and Guan, 2019). Since mTORC1 complex is activated in numerous human cancers to keep the cancer cells proliferative and increase their nutrient uptake and energy metabolism, rapamycin impairs cancer metabolism and it has been thoroughly studied as a cancer drug ( Li et al., 2014). However, the unmodified rapamycin has poor water solubility which leads to pharmacokinetic issues and facilitated the development of multiple analogs called rapalogs, the first generation mTOR inhibitors.

Rapalogs

Two water-soluble derivatives of rapamycin, temsirolimus, and everolimus ( Figure 7), have been approved for the treatment of renal cancer carcinoma ( Li et al., 2014). Everolimus is also approved for the treatment of progressive neuroendocrine tumors of pancreatic origin, and refractory mantle cell lymphoma in the EU. Even though many water soluble rapalogs were tested in cell and animal models, their effect in the clinic is generally only modest or weak ( Kim and Guan, 2019). In some patients, rapalog usage inadvertently promotes cell survival by enhancing the PKB/Akt activity or by activating autophagy which promotes survival in oxygen-deprived microenvironments ( Tabernero et al., 2008; Palm et al., 2015; Johnson and Tee, 2017). In some cases, these effects can be countered by combining rapalogs with an autophagy inhibitor hydroxychloroquine ( Rangwala et al., 2014). Rapalogs function as allosteric inhibitors of mTORC1, and as such only prevent the phosphorylation of some of the mTORC1 substrates ( Choo et al., 2008; Kang et al., 2013). This, in connection with the negative-feedback loop of PKB/Akt activation associated with the mTOR pathways is possibly the cause for the relatively poor clinical success of rapalogs.

mTOR Kinase Inhibitors

Torin1 and PP242 ( Figure 7) were the first identified ATP-competitive mTOR inhibitors which block the kinase function of mTOR ( Feldman et al., 2009; Thoreen et al., 2009). Later, Torin2 was characterized and tested alone and in combination with other kinase inhibitors against various cancer cell lines ( Liu et al., 2013). Unlike rapamycin and rapalogs, mTOR kinase inhibitors directly inhibit the catalytic activity of both mTOR complexes. The mTOR kinase inhibitors display stronger inhibition of cancer cell proliferation than rapamycin due to their increased efficacy toward mTORC2. However, since mTOR signaling is essential for cell viability, blocking both mTOR complexes causes mTOR kinase inhibitors to have more severe side effects than rapamycin or rapalogs ( Xie et al., 2016). The most advanced mTOR kinase inhibitor in clinical trials is a derivative of PP242, AZD2014, which is currently in phase II clinical trials for ER positive breast cancer ( Guichard et al., 2015).

RapaLinks

The newest members of the mTOR targeting drugs consist of rapamycin linked with an mTOR kinase inhibitor, and they are aptly named RapaLinks ( Rodrik-Outmezguine et al., 2016; Fan et al., 2017). In addition to having activity against many cancer cell lines, RapaLinks are effective against cancerous cells that are resistant to first or second generation mTOR inhibitors ( Rodrik-Outmezguine et al., 2016). All published RapaLinks (1-3) have MLN0128 as the mTOR kinase inhibitor part, and they differ by the linker connecting the two-parts ( Figure 7) ( Rodrik-Outmezguine et al., 2016; Fan et al., 2017). MLN0128 by itself is not very effective in vivo due to its short residence time and its usage is limited by toxicity ( Graham et al., 2018). Even though RapaLinks are large and contain a poorly water-soluble rapamycin, they can pass the blood-brain barrier and their efficacy has been shown in animal models of glioblastoma ( Fan et al., 2017). If the in vivo results imply anything about clinical usability, we expect to see RapaLinks in clinical trials within the next few years.

Silvestrol

Silvestrol ( Figure 8) is a rocaglate derivative that can be isolated from the fruits and twigs of Aglaia foveolate ( Pan et al., 2014). It is cytotoxic toward multiple cancer cell lines in vitro and it displays similar potency to paclitaxel or camptothecin ( Hwang et al., 2004; Pan et al., 2014; Chen et al., 2016b). Silvestrol inhibits the translation initiation by binding to the initiation factor eIF-4A which prevents the ribosome loading onto the mRNA template ( Cencic et al., 2009). This kills cells by inducing early autophagy and caspase-mediated apoptosis ( Chen et al., 2016b). Silvestrol exhibits cytotoxic effects against different human cancer cell lines, such as melanoma, acute myelogenous leukemia, cervical cancer and oral carcinoma ( Hwang et al., 2004; Lucas et al., 2009; Rodrigo et al., 2012; Kogure et al., 2013). Even though silvestrol is effectively cytotoxic against multiple cancer cell lines in vitro, only partial protein synthesis inhibition was observed in mice models of lymphoma ( Bordeleau et al., 2008). The main issue with silvestrol and its analogs is that they upregulate multi-drug-resistant gene ABCB1 and that they are substrates of p-glycoprotein, a well-known resistance-causing efflux transporter ( Gupta et al., 2011). Despite the decade long research, silvestrol and its analogs remain at the preclinical drug research stage, and none of them has made it into clinical trials ( Peters et al., 2018).

Omacetaxine

Omacetaxine, formerly known as homoharringtone ( Figure 8), is a plant alkaloid from Cephalotoxus fortune. It was identified in 1970s as the inhibitor of the initial elongation step of translation ( Huang, 1975; Fresno et al., 1977). Omacetaxine binds to the A-site cleft in the peptidyl-transferase site of the ribosome where it prevents the correct positioning of the incoming aminoacyl-tRNAs, thus functioning as a competitive inhibitor ( Gürel et al., 2009). Because omacetaxine affects the elongation step, it is a more general translation inhibitor than other molecules that target translation initiation which inhibit only the translation of specific sequences ( Wetzler and Segal, 2011). Treatment with omacetaxine leads to a rapid decrease in the number of proteins with short half-lives, including the oncogenic cyclin D1 and c-Myc ( Robert et al., 2009). Omacetaxine was intensely studied after its discovery both in vitro and in vivo against chronic myeloid leukemia but after the approval of imatinib and other tyrosine kinase inhibitors, the scientific interest toward it dwindled ( Wetzler and Segal, 2011). Recently, new clinical studies around omacetaxine have been started due to its synergistic effect with tyrosine kinase inhibitors, especially in the treatment of cancers with mutations in the tyrosine kinase genes ( Marin et al., 2005; Cortes et al., 2012; Rosshandler et al., 2016; Maiti et al., 2017). Omacetaxine is approved by the FDA for the treatment of chronic myeloid leukemia if the disease does not respond to two or more tyrosine kinase inhibitors ( Cortes et al., 2012, 2013; Rosshandler et al., 2016). This way, omacetaxine can help patients who suffer from lack of effect of those drugs, intolerance or drug-drug interactions. The most common adverse effects of omacetaxine are myelosuppression and thrombocytopenia which are observed in almost all patients but they can be managed with supportive care, dose delays and reduction in the number of days that omacetaxine is administered ( Rosshandler et al., 2016).

Antisense Oligonucleotides

ASOs have different mechanisms of actions including enzyme-mediated target RNA degradation, steric-hindrance of translation, as well as modulation of splicing and transcription ( MacLeod and Crooke, 2017). Historically, the first mechanism of ASOs identified was RNase H-dependent degradation, which entails the hydrolysis of the RNA strand in a RNA/DNA duplex ( Crooke, 2017). This is efficiently mediated by the ubiquitous RNase H and has the advantage that the oligonucleotide can be targeted to any part of the RNA molecule. However, problems of specificity due to activation following partial hybridization have been observed and pose a concern. Modified oligomers deviate from RNase H-induced cleavage and can inhibit protein expression via other mRNA quality control decay pathways. These include the non-sense-mediated decay (NMD) ( Ward et al., 2014; Nomakuchi et al., 2016) and the no-go decay (NGD) ( Liang et al., 2019a) which is triggered by ribosome stalling due to obstacles and collision of multiple ribosomes on the mRNA ( Harigaya and Parker, 2010). Oligonucleotides targeted to either the 5′ or 3′ splice sites can interfere with splicing ( Havens and Hastings, 2016; Singh et al., 2018). Interestingly, this can be used either to block mature protein expression or to correct aberrant splicing thereby restoring the protein function. Furthermore, oligonucleotides can result in steric hindrance of translation by preventing ribosome binding when targeted near the translation initiation codon ( Chery, 2016; Goyal and Narayanaswami, 2018). Alternatively, ASOs targeted to the 5′UTR can inhibit translation by preventing 5′ cap formation.

Trabedersen (AP 12009)

AZD4785

RNA Interference-Based Inhibitors

Following the seminal discovery that double stranded RNAs (dsRNAs) could cause post-transcriptional gene silencing (PTGS) in C. elegans ( Fire et al., 1998), it did not last many years until RNA-based therapeutic inhibitors were in clinical trials ( Whelan, 2005) and in 2018 patirisan received FDA approval as the first RNA based drug for the treatment of polyneuropathy. RNA interference (RNAi) as an inherent cellular process of RNA-mediated suppression of gene expression; it achieves this by inhibition of mRNA translation or targeting mRNA for degradation or sequestration (reviewed in detail in Jinek and Doudna, 2009; Setten et al., 2019). The short interfering (si)RNA required for activation of the RNAi pathway, can be derived exogenously from synthetic dsRNA or small hairpin (sh)RNA, in parallel endogenous miRNA can be generated following processing of transcribed pri-micro (mi)RNA. Whereas, the miRNA biogenesis pathway involves cleavage of pri-miRNA in the nuclear microprocessor complex and then with Dicer complexed to TAR RNA binding protein (TRBP) in the cytoplasm, shorter dsRNA can bypass this and is similarly but directly incorporated into the RNA-induced silencing complex (RISC) ( Ha and Kim, 2014; Setten et al., 2019). In this complex, Argonaut 2 (AGO2) removes the passenger (sense) strand and the guide (antisense) strand is maintained, normally due to higher 5′ stability, in the mature and active RISC. In the case of siRNA, the guide RNA is then able to bind with perfect complementarity to target mRNA within the coding region, and in so doing results in mRNA cleavage by the AGO endonucleases. In contrast, miRNA that can bind to several mRNAs and often have their target site in the 3′UTR of mRNA, tend to do so with only partial complementarity. The partial base pairing compromises the AGO slicer catalytic activity and instead results in either translation repression or degradation of mRNA. In both cases small RNA result in suppression of mRNA and subsequently the matching protein.

G12D KRAS -Targeted siRNA

EphA2-Targeted siRNA

miRNA-34a Prodrugs and Mimics

BERAs

Although different chemical modification of synthetic RNA molecules intended for RNAi therapeutics have increased stability and demonstrated favorable pharmacokinetics properties, these Chemo-engineered RNAs are different from naturally transcribed RNA molecules in living cells, which are largely unmodified. This difference affects the structure, properties, and possibly the activity and immunogenicity of these molecules (reviewed in Yu et al., 2019). Also, effort has been made to bioengineer RNA molecules in living cells, including in bacteria and yeast ( Huang et al., 2013; Chen et al., 2015; Pereira et al., 2017b; Ho et al., 2018; Kaur et al., 2018; Duman-Scheel, 2019). These bioengineered RNA agents (BERA) can be produced in large scale and carry no or minimal posttranscriptional modifications ( Li et al., 2015; Wang et al., 2015). Different strategies for producing BERA have been tested, including strategies using RNA-binding proteins such as viral p19, inclusion into tRNA or 5S rRNA scaffolds and tRNA/pre-miRNA chimeras. The last of these has proven to be the most versatile platform as siRNA, miRNAs, and RNA aptamers of different sizes and forms have been produced at large scale with high yields ( Duan and Yu, 2016; Yu et al., 2019).

Vulnerability of naked BERAs to RNAse-degradation in blood necessitates additional formulation for BERA to protect the RNA molecules. This can be done by cationic lipids, polymers, and peptides ( Kim et al., 2016). Specifically, polyethylenimine (PEI)-based polyplexes (complexes of nucleotides and polycations) have facilitated efficient delivery in tumor models ( Zhao et al., 2016; Tu et al., 2019). However due to potential toxicity of polyplexes ( Lv et al., 2006), formulation based on lipidation of these has been recently tested for delivery of tumor associated miRNA ( Zhang et al., 2018; Jilek et al., 2019). Increased serum stability of these BERAs as well as improved delivery, therapeutic effectiveness and survival of tumor bearing mice were observed. These positive results encourage the further development of BERA for tumor therapy.

Targeting Oncogenes

Oncogenes are genes that can cause cancer once mutated or when expressed at high levels ( Croce, 2008). Many oncogenic pathways lead to altered transcription or translation of various proteins. In order to keep the topic of this review we will focus on two oncogenic targets that are involved with transcription and translation; transcription factors and KRAS. Both of these oncogenes were previously thought to be undruggable but nevertheless, a few inhibitors for both of them have been published in the last few years. The readers interested in the drugs designed for oncogenic kinases or other oncogenic pathways are referred to other reviews ( Bhullar et al., 2018; Solassol et al., 2019; Tang and Zhao, 2019; Wang et al., 2019; Zhang et al., 2019b).

Transcription Factors

Protein-Protein Interaction Inhibitors

Inhibitors of Transcription Factor Gene Expression

There is one CDK-inhibitor (SY-1365) in phase I clinical trial for ovarian and breast cancer ( Hu et al., 2019). The inhibition with SY-1365 leads to decreased levels of multiple oncogenic transcription factors and it exhibits the inhibitory effects on multiple cancer cell lines at nanomolar level. In addition, mouse xenograft studies showed modest antitumor activity in both AML as well as ovarian cancer, and a synergistic effect with venetoclax ( Hu et al., 2019).

Two BET inhibitors are in clinical trials that target specifically the transcription of transcription factors, INCB057643 and BMS-986158 ( Forero-Torres et al., 2017; Gavai et al., 2018). Both inhibitors lower the expression levels of the c-Myc oncogene and the proliferation rates of multiple cancer cell lines, and in animal models they display suitable properties for oral dosing in humans. The clinical trials are ongoing for advanced cancers, both solid tumors and leukemias.

RAS Inhibitors

Drugs affecting transcription and translation are difficult to develop. This statement has been a harsh reality for those who have aimed to design oncogene RAS inhibitors, especially inhibitors of KRAS. The RAS GTPase family includes HRAS (Harvey rat sarcoma viral oncogene homolog), KRAS (Kristen rat sarcoma viral oncogene homolog), KRAS split variant KRASB (KRASA is same as KRAS) and NRAS (neuroblastoma RAS viral oncogene homolog). As RAS proteins are the most commonly mutated proteins in cancer and at the same time are part of the signaling cascade from the EGFR receptor, these have been a natural target for drug discovery. After more than 30 years of research, we now have the first compounds in clinical trials targeting mutated RAS protein, namely G12C KRAS. The difficulty of this process has been aptly stated in a recent editorial in the British Journal of Cancer "To put this development into context, KRAS was first described in 1983 and it has taken 35 years to reach this point, whereas identification of oncogenic BRAF mutations in 2002 was followed by an effective targeted drug in 2009" ( Lindsay and Blackhall, 2019).

RAS proteins are small GTPases, thus removing the gamma-phosphate of GTP to produce GDP. The biological function is related to conformational cycle between GTP-bound active RAS and GDP-bound inactive RAS. The GTP-bound RAS can adopt a conformation able to bind with downstream signaling kinases (like PI3K or RAF) and this allows further activation of kinase pathways. Those interested to know more about RAS protein structure and function are advised to look at recently published review ( Pantsar, 2020) and another review about early drug discovery work on RAS by Ostrem and Shokat (2016). In this part of our review we will concentrate on the recent works of KRAS G12C targeting covalent inhibitors as those are the most promising KRAS inhibitors currently known. The KRAS -targeting siRNA was discussed in section G12D KRAS -Targeted siRNA.

As stated KRAS is cycling between inactive GDT-bound and active GTP-bound stated. Direct targeting of GTP-binding pocket is not a realistic option as GTP has a high 0.5 mM concentration near the intracellular site where RAS proteins are located ( Traut, 1994) and at the same time low femtomolar binding affinity ( John et al., 1993; Ford et al., 2009). As there are no other clear druggable pockets in RAS direct targeting seemed to be an impossible mission. The problem was partially solved by the seminal work of Shokat lab which demonstrated that covalent interaction targeting mutated G12C residues is able to deliver in vivo relevant inhibition of RAS activation ( Ostrem et al., 2013). The initial compounds presented were developed by disulphide-fragment based screening using tethering compounds. After early hit-optimization guided by X-ray crystallography an optimized G12C targeting covalent inhibitor was presented. In the optimized compounds disulfides were replaced by different electrophilic warheads and especially acrylamides were used. Upon covalent interaction G12C position compounds were binding previously unknown allosteric pocket. Even more important is the finding that these G12C inhibitors are RAS-GDP specific. This effect will undoubtedly explain the positive clinical outcome of two later-developed G12C inhibitors, namely AMG 520 and MRTX849 ( Canon et al., 2019; Hallin et al., 2020). Both AMG 520 and MRTX849 seem to give better results with combination of either upstream or downstream kinase inhibitors. This is not surprising as such, but more surprising is a very recent report concerning the rapid non-uniform adaptation to KRAS G12C inhibition ( Xue et al., 2020). According to Xue et al. (2020) G12C inhibitors targeting the RAS-GDP state promote a feedback mechanism in which KRAS G12C is maintained in active state by EGFR and Aurora kinase A (AURKA) signaling. If this finding is validated it would indicate that EGFR and/or AURKA inhibition are mandatory to support and maintain KRAS G12C inhibition. Partially this effect might be specific for the currently used G12C inhibitor (ARS1620) but due to similar binding mode as with AMG 520 it seems that this conclusion will be a general one ( Janes et al., 2018). Besides of AMG 520 and MRTX849 there are also two other KRAS G12C targeting inhibitors, namely JNJ-74699157 (NCT04006301) and LY349946 (NCT04165031), in the clinical trials.

Conclusions and Future Remarks

History has shown us that it is indeed not straight-forward to develop transcription or translation inhibitors. Even more difficult is to target these inhibitors only toward malignant cells. There have been a few clinical successes in their development, such as the CDK inhibitors, especially in combination with other chemotherapeutics. Targeting these central cellular processes has advantages to be more directed to cancer cells than non-specific chemotherapeutic agents such as cisplatin. On the side of disadvantages, targeting transcription and translation may affect multiple pathways hence circumventing the targeted pathway, and there are inevitable side effects arising from the fact that all cells require transcription and translation for their proper function. Transcription and translation are fundamental processes that targeting them is bound to result in cell death, as such cancer treatment based on these processes needs to be done in a manner that is safe for healthy cells. To achieve the specific and effective treatment, the myriad of proteins involved will continue to offer drug design possibilities far into the future. In addition, RNA was previously considered to be undruggable and not suitable as a drug itself, but now RNA-targeting and RNA-based drugs can be used as very precise methods to target some cancers. The recently discovered RNA activation of transcription offers uncharted possibilities in the treatment of cancer. Furthermore, transcription factors and oncogenes were also thought to be undruggable, but within the last few years we have seen some molecules targeting them entering clinical trials. By widening the scope of drug targets from traditional proteins with specified binding sites to transcription factors, oncogenes and RNA molecules, we are discovering new and specific ways to target cancer cells. So far, we have produced some highly specific, safe and efficacious cancer therapies which inhibit transcription and translation, and the newly discovered targets and our ever-increasing knowledge about the biological basics of these processes is bound to keep this field of inhibitor development ongoing far into the future.

Author Contributions

NL-K, GP, AP, and PK wrote sections and participated in the creation of the graphics of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

Funding

The authors thank Maire Taponen Foundation (NL-K personal grant) and competitive funding to strengthen university research profiles, 5th call, funding to University of Eastern Finland, funded by the Academy of Finland, funding decision 325022. This work was supported by the Technology Agency of Czech Republic (TN0100013).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors apologize for colleagues whose work could not be referenced due to word count limits.

Abbreviations

ASO, Antisense oligonucleotide; AURKA, Aurora kinase A; BERA, Bioengineered non-coding RNA agent; BET, Bromodomain and extraterminal domain; CDK, Cyclin-dependent kinase; EGFR, Epidermal growth factor receptor; Pol, RNA polymerase; RAS, Rat sarcoma viral oncogene homolog; RISC, RNA-induced silencing complex; RNAi, RNA interference.

References

Friday, B., Lassere, Y., Meyers, C. A., Mita, A., Abbruzzese, J. L., and Thomas, M. B. (2012). A phase I study to determine the safety and pharmacokinetics of intravenous administration of TAS-106 once per week for three consecutive weeks every 28 days in patients with solid tumors. Anticancer Res. 32, 1689-1696. Available online at: http://ar.iiarjournals.org/content/32/5/1689.long

PubMed Abstract | Google Scholar

Gandhi, V., Ayres, M., Halgren, R. G., Krett, N. L., Newman, R. A., and Rosen, S. T. (2001). 8-chloro-cAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma cells. Cancer Res. 61, 5474-5479. Available online at: https://cancerres.aacrjournals.org/content/61/14/5474.long

PubMed Abstract | Google Scholar

Hortobagyi, G. N., Stemmer, S. M., Burris, H. A., Yap, Y.-S., Sonke, G. S., Paluch-Shimon, S., et al. (2016). Ribociclib as first-line therapy for HR-positive, advanced breast cancer. 375, 1738-1748. doi: 10.1056/NEJMoa1609709

Lucas, D. M., Edwards, R. B., Lozanski, G., West, D. A., Shin, J. D., Vargo, M. A., et al. (2009). The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo. Blood 113, 4656-4666. doi: 10.1182/blood-2008-09-175430

Pereira, P., Pedro, A. Q., Queiroz, J. A., Figueiras, A. R., and Sousa, F. (2017b). New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli. Bioengineered 8, 670-677. doi: 10.1080/21655979.2017.1284710

Watson, J., Baker, T., Bell, S., Gann, A., Levine, M., and Losick, R. (2013). "Part 5: regulation," in Molecular Biology of the Gene, ed B. Wilbur (New York, NY: Pearson), 609-792.

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Transcription and Translation Inhibitors in Cancer Treatment

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Transcription and translation are fundamental cellular processes that govern the protein production of cells. These processes are generally up regulated in cancer cells, to maintain the enhanced metabolism and proliferative state of these cells. As such cancerous cells can be susceptible to transcription and translation inhibitors.

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Life Science - Protein synthesis (Translation)

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Enhancers are short regulatory elements of accessible DNA that help establish the transcriptional program of cells by increasing transcription of target genes.

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- the process of copying and duplicating DNA molecule
-DNA replication occurs in the cytoplasm of prokaryotes and in the nucleus of eukaryotes
-During cell division, DNA replication occur during interphase

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The 3 proposed models of DNA replication

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The 3 proposed models of DNA replication

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In 1957, Matthew s. Meselson and Franklin W. Stahl confirmed semiconservative replication of DNA experimentally

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In 1957, Matthew s. Meselson and Franklin W. Stahl confirmed semiconservative replication of DNA experimentally

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THE MOST BEAUTIFUL EXPERIMENT IN BIOLOGY: Meselson & Stahl, The Semi-Conservative Replication of DNA

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Bacterial RNA polymerase (RNAP) is a proven target for broad-spectrum antibacterial therapy. The suitability of bacterial RNAP as a target for broad-spectrum antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP-subunit sequences are highly conserved (providing a basis for broad-spectrum activity), and the fact that bacterial RNAP-subunit sequences are not highly conserved in human RNAPI, RNAPII, and RNAPIII (providing a basis for therapeutic selectivity).
The rifamycin antibacterial agents--rifampin, rifapentine, rifabutin, and rifamixin--bind to and inhibit bacterial RNAP. The rifamycins bind to a site on bacterial RNAP adjacent to the RNAP active center and prevent extension of RNA chains beyond a length of 2-3 nucleotides. The rifamycins are in current clinical use in treatment of Gram-positive and Gram-negative bacterial infections. The rifamycins are of particular importance in treatment of tuberculosis; the rifamycins are first-line antituberculosis agents and are among the only antituberculosis agents able to clear infection and prevent relapse. The clinical utility of the rifamycin antibacterial agents is threatened by the existence of bacterial strains resistant to rifamycins. Resistance to rifamycins typically involves substitution of residues in or adjacent to the rifamycin-binding site on bacterial RNAP--i.e., substitutions that directly interfere with rifamycin binding.
In view of the public health threat posed by drug-resistant and multidrug-resistant bacterial infections, there is an urgent need for new classes of broad-spectrum antibacterial agents that (1) target bacterial RNAP (and thus have the same biochemical effects as rifamycins), but that (2) target sites within bacterial RNAP that do not overlap the rifamycin-binding site (and thus do not show cross-resistance with rifamycins).
We have identified new drug targets within the structure of bacterial RNAP. Each of these new targets can serve as a potential binding site for compounds that inhibit bacterial RNAP and thereby kill bacteria. Each of these new targets is present in most or all bacterial species, and thus compounds that bind to these new targets are active against a broad spectrum of bacterial species. Each of these new targets is different from targets of current antibiotics, and thus compounds that bind to these new targets are not cross-resistant with current antibiotics. For each of these new targets, we have identified at least one lead compound that binds to the target, and we have synthesized analogs of the lead compound comprising optimized lead compounds. Several of the lead compounds and optimized lead compounds are extremely promising: they exhibit potent activity against a broad spectrum of bacterial pathogens (including Staphylococcus aureus MSSA, Staphylococcus aureus MRSA, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile, Mycobacterium tuberculosis, Bacillus anthracis, Francisella tularensis, Burkholderia mallei, and Burkholderia pseudomallei) and exhibit no cross-resistance with current antibiotics.

In support of this work, we are identifying new small-molecule inhibitors of bacterial RNAP by analysis of microbial and plant natural products, by high-throughput screening, and by virtual screening. We are also using genetic, biochemical, biophysical, and crystallographic approaches to define the mechanism of action of each known, and each newly identified, small-molecule inhibitor of bacterial RNAP, and we are using microbiological approaches to define antibacterial efficacies, resistance spectra, and spontaneous resistance frequencies of known and new small-molecule inhibitors of bacterial RNAP.

We seek to address the following objectives: to develop new classes of antituberculosis agents and broad-spectrum antibacterial agents, to develop antibacterial agents effective against pathogens resistant to current antibiotics, to develop antibacterial agents effective against pathogens of high relevance to public health, and to develop antibacterial agents effective against pathogens of high relevance to biodefense.

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Inhibitors of Transcription: Antibacterial Drug Discovery

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Bacterial RNA polymerase (RNAP) is a proven target for broad-spectrum antibacterial therapy. The suitability of bacterial RNAP as a target for broad-spectrum antibacterial therapy follows from the fact that bacterial RNAP is an essential enzyme (permitting efficacy), the fact that bacterial RNAP-subunit sequences are highly conserved (providing a basis for broad-spectrum activity), and the fact that bacterial RNAP-subunit sequences are not highly conserved in human RNAPI, RNAPII, and RNAPIII (providing a basis for therapeutic selectivity).

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Stages of transcription: initiation, elongation & termination (article) | Khan Academy

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An in-depth looks at how transcription works. Initiation (promoters), elongation, and termination.

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Elongation is the addition of nucleotides to the mRNA strand. RNA polymerase reads the unwound DNA strand and builds the mRNA molecule, using complementary base

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Okay, so an anticodon is a three-letter sequence that's complementary, or matches up, to the codon sequence found in the RNA. And the RNA, typically a messenger RNA, codes for proteins. Proteins are made up of amino acids, and these three-letter codons in the RNA are what code for the amino acids to match up with those three-letter codon sequences you need an anticodon sequence to match up with the complimentary bases. And you'll find that anticodon sequence in the transfer RNA, which is carrying the correct amino acid that it's going to attach to the protein that is being generated by that messenger RNA

Elliott Margulies, Ph.D.

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Okay, so an anticodon is a three-letter sequence that's complementary, or matches up, to the codon sequence found in the RNA. And the RNA, typically a messenger RNA, codes for proteins.

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VCAC: Molecular Processes: Translation: Advanced Look

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Questions? Comments? Please contact Dr. Phillip McClean or Christina Johnson.

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Cisplatin is chemotherapy medicine used to treat a variety of cancers, including bladder cancer,cervical cancer, head and neck cancer, lung cancer, 🤬 cancer and cancer of the ovaries.

Cisplatin works by stopping the cancer cells from multiplying. It does this by binding together the strands of the cells' genetic material, DNA. DNA is needed for growth and multiplication of cells. Cisplatin damages the DNA or RNA inside the cancer cells that tells the cell how to copy itself in division and so prevents them from multiplying. If the cells are unable to divide, they die. The faster the cells are dividing, the more likely it is that chemotherapy will kill the cells, causing the tumor to shrink.

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Cisplatin is a platinum-based chemotherapy medicine used to treat a variety of cancers, including bladder cancer, cervical cancer, head and neck cancer, lung cancer, testicular cancer and cancer of the ovaries.

Cisplatin is often used in combination with other chemotherapy medicines.

Here's everything you need to know about cisplatin, including how it works, who shouldn't have it and the potential side effects:

How does cisplatin work?

Cancers form when some cells within the body multiply uncontrollably and abnormally. These cells spread, destroying nearby tissues. Cisplatin works by stopping the cancer cells from multiplying. It does this by binding together the strands of the cells' genetic material, DNA. DNA is needed for growth and multiplication of cells. Cisplatin damages the DNA inside the cancer cells and so prevents them from multiplying.

Unfortunately, cisplatin can also affect normal, healthy cells, particularly those that multiply quickly, such as blood cells and hair cells. The most important side effect is on the bone marrow where blood cells are made. Cisplatin can decrease the production of blood cells, leaving people susceptible to infection. Regular blood tests are therefore needed to monitor the levels of blood cells.

In most chemotherapy regimens, doses are administered in courses at various intervals to allow normal cells to recover from the adverse effects of the anticancer medicines between doses. However, during this period, cancer cells will also recover and start to replicate again. Successful treatment depends on the administration of the next course of therapy before the cancer has regrown to its previous size and the net effect is to decrease the amount of cancer with each successive course.

How cisplatin treatment given?

Cisplatin infusion is given via a drip into a vein (intravenous infusion). The infusion can take up to eight hours.

Treatment with cisplatin is given in several cycles of treatment. The length of your treatment and the number of cycles you have will depend on the type of cancer you are being treated for, how well it responds and how well your body copes with the treatment.

Cisplatin important information

Your doctor will want to monitor your kidney function while you are receiving treatment with this medicine, as it can affect your kidneys. You will be given fluid through a drip before and after your cisplatin treatment to help keep your kidneys working normally and your doctor may also want you to drink extra fluids before and after the treatment. Follow the instructions you are given by the hospital. You may be asked to keep a record of how much you drink and how much urine you produce. If you notice that you are producing less urine than normal you should let your doctor or nurse know straight away.

Chemotherapy medicines can decrease the number of blood cells in your blood. A low white blood cell count can increase your susceptibility to infections; a low red blood cell count causes anaemia and a low platelet count can cause problems with blood clotting. For this reason, you will need regular blood tests to monitor your blood cells during treatment with this medicine. Tell your doctor immediately if you experience any of the following symptoms during your treatment, as they may indicate problems with your blood cells: unexplained bruising or bleeding, purple spots, sore mouth or throat, mouth ulcers, high temperature (fever) or other signs of infection, or suddenly feeling tired, breathless, or generally unwell.

Your doctor will want to monitor your kidney function while you are receiving treatment .

This medicine can affect the nerves (this is called peripheral neuropathy). This may cause feelings of numbness or tingling, especially in the hands, feet, neck or throat. You may also have difficulty in performing fiddly tasks, such as buttoning clothes. It is important to report your symptoms to your doctor, as they may be controlled by slightly lowering the dose of this medicine. Although in the majority of cases these symptoms disappear completely, there is a possibility that symptoms of weakness or numbness because of nerve damage may continue after the end of the treatment.

Tell your doctor if you notice any problems with your hearing, or sensation of ringing in your ears (tinnitus) while you are having treatment with this medicine.

This medicine may be harmful to an unborn baby, and for this reason you should use effective contraception to avoid getting pregnant or fathering a child during treatment. You should continue to use contraception to prevent pregnancy for at least a few months after stopping this medicine. Discuss this with your doctor. Women should consult their doctor immediately if they get pregnant during treatment.

Your ability to get pregnant or father a child may be affected by treatment with this medicine. It is important to discuss fertility with your doctor before starting treatment.

Who shouldn't use cisplatin?

● People with a history of allergic reaction to any other platinum compounds, for example carboplatin or oxaliplatin.

● People with severely decreased production of blood cells by the bone marrow (myelosuppression), resulting in very low levels of white blood cells or platelets in the blood (for example due to radiotherapy or previous courses of chemotherapy).

● Severely decreased kidney function.

This medicine should not be used if you are allergic to any of its ingredients. Please inform your doctor or pharmacist if you have previously experienced such an allergy.

If you feel you have experienced an allergic reaction, stop using this medicine and inform your doctor or pharmacist immediately.

Meanwhile, certain people should use cisplatin with caution. They include:

People with decreased kidney function.

People taking other medicines that can affect the kidneys (see end of factsheet for examples).

People with hearing problems.

People with low levels of red blood cells, white blood cells or platelets in their blood.

Cisplatin and pregnancy and breastfeeding

Certain medicines should not be used during pregnancy or breastfeeding. However, other medicines may be safely used in pregnancy or breastfeeding providing the benefits to the mother outweigh the risks to the unborn baby. Always inform your doctor if you are pregnant or planning a pregnancy, before using any medicine.

This medicine is likely to be harmful if used during pregnancy. It should not be used during pregnancy unless considered essential by your doctor to treat life-threatening disease.

Women who could get pregnant must use a highly effective method of contraception to prevent pregnancy, and men should use effective contraception to avoid fathering a child, both during treatment, and for at least a few months after treatment is finished. Seek further medical advice from your doctor.

It is not known if this medicine passes into breast milk. Mothers who need treatment with this medicine should not breastfeed. Seek medical advice from your doctor.

Cisplatin side effects

Medicines and their possible side effects can affect individual people in different ways. The following are some of the side effects that are known to be associated with this medicine. Just because a side effect is stated here, it does not mean that all people using this medicine will experience that or any side effect.

Nausea and vomiting which may last for up to a week after treatment. You will given medicines to help with this.

Loss of appetite.

Weakness or loss of strength (asthenia).

Decreased kidney function (see warning section above).

Disturbances in the levels of electrolytes such as calcium, magnesium, sodium and potassium in the blood.

Hearing problems such as ringing in the ear or loss of hearing (see warning section above).

Disorder of the peripheral nerves causing weakness and numbness (peripheral neuropathy - see warning section above).

Decrease in the normal numbers of white blood cells, red blood cells or platelets in the blood (see warning section above).

Increased susceptibility to infections.

Diarrhoea.

Changes in taste.

Skin reactions such as rash, itching.

Hair loss.

Problems with vision such as loss of vision, blurring of vision and changes in the colour vision.

Increased heart rate or irregular heart beats.

Alteration in results of liver function tests.

High blood uric acid level (hyperuricaemia) which can cause kidney problems and gout.

Seizures.

The side effects listed above may not include all of the side effects reported by the medicine's manufacturer.

For more information about any other possible risks associated with this medicine, please read the information provided with the medicine or consult your doctor or pharmacist.

Cisplatin and other medicines

It is important to tell your doctor or pharmacist what medicines you are already taking, including those bought without a prescription and herbal medicines, before you start treatment with this medicine. Similarly, check with your doctor or pharmacist before taking any new medicines during treatment this one, to make sure that the combination is safe.

Vaccines may be less effective in people receiving chemotherapy. This is because chemotherapy medicines reduce the activity of the immune system and can prevent the body forming adequate antibodies. Live vaccines should be postponed until at least six months after finishing chemotherapy because they may cause infection. Live vaccines include the following: oral polio; rubella; measles, mumps and rubella (MMR); BCG; chickenpox; yellow fever and oral typhoid vaccines.

There may be an increased risk of side effects on the kidneys and ears if this medicine is used in combination with the following medicines:

aminoglycoside antibiotics such as gentamicin

amphotericin B

capreomycin

diuretics such as furosemide

polymixin antibiotics such as colistin or polymixin B

vancomycin.

Cisplatin may reduce the ability of the kidneys to excrete the medicines bleomycin and methotrexate and so may increase the risk of side effects from these medicines.

Cisplatin may lower the blood level of the anticonvulsant medicine phenytoin and so could make it less effective.

There may be an increased risk of side effects if this medicine is used in combination with other medicines that can affect blood cell counts, for example other chemotherapy medicines, or the antipsychotic clozapine.

Other medicines containing the same active ingredient: Cisplatin infusion is only available generically (ie without a brand name).
Last updated 5.7.2020
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Cisplatin is a platinum-based chemotherapy medicine used to treat a variety of cancers, including bladder cancer, cervical cancer, head and neck cancer, lung cancer, testicular cancer and cancer of the ovaries. Cisplatin is often used in combination with other chemotherapy medicines.

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A term derived from the lac operon, structural genes are typically viewed as those containing sequences of DNA corresponding to the amino acids of a protein that will be produced, as long as said protein does not function to regulate gene expression. Structural gene products include enzymes and structural proteins.
(nabilah)

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In RNA splicing, specific parts of the pre-mRNA, called introns are recognized and removed by a protein-and-RNA complex called the spliceosome. Introns can be viewed as "junk" sequences that must be cut out so the "good parts version" of the RNA molecule can be assembled.

The pieces of the RNA that are not chopped out are called exons. The exons are pasted together by the spliceosome to make the final, mature mRNA that is shipped out of the nucleus.

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Translation of an mRNA molecule by the ribosome occurs in three stages

initiation

elongation

termination

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Translation is the process by which a protein is synthesized from the information contained in a molecule of messenger RNA (mRNA). During translation, an mRNA sequence is read using the genetic code, which is a set of rules that defines how an mRNA sequence is to be translated into the 20-letter code of amino acids, which are the building blocks of proteins. The genetic code is a set of three-letter combinations of nucleotides called codons, each of which corresponds with a specific amino acid or stop signal. Translation occurs in a structure called the ribosome, which is a factory for the synthesis of proteins. The ribosome has a small and a large subunit and is a complex molecule composed of several ribosomal RNA molecules and a number of proteins. Translation of an mRNA molecule by the ribosome occurs in three stages: initiation, elongation, and termination. During initiation, the small ribosomal subunit binds to the start of the mRNA sequence. Then a transfer RNA (tRNA) molecule carrying the amino acid methionine binds to what is called the start codon of the mRNA sequence. The start codon in all mRNA molecules has the sequence AUG and codes for methionine. Next, the large ribosomal subunit binds to form the complete initiation complex. During the elongation stage, the ribosome continues to translate each codon in turn. Each corresponding amino acid is added to the growing chain and linked via a bond called a peptide bond. Elongation continues until all of the codons are read. Lastly, termination occurs when the ribosome reaches a stop codon (UAA, UAG, and UGA). Since there are no tRNA molecules that can recognize these codons, the ribosome recognizes that translation is complete. The new protein is then released, and the translation complex comes apart.

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Translation is the process by which a protein is synthesized from the information contained in a molecule of messenger RNA (mRNA).

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Introduction

Ever wonder how antibiotics kill bacteria-for instance, when you have a sinus infection? Different antibiotics work in different ways, but some attack a very basic process in bacterial cells: they knock out the ability to make new proteins.

To use a little molecular biology vocab, these antibiotics block translation. In the process of translation, a cell reads information from a molecule called a messenger RNA (mRNA) and uses this information to build a protein. Translation is happening constantly in a normal bacterial cell, just like it is in most of the cells of your body, and it's key to keeping you (and your bacterial "visitors") alive.

When you take certain antibiotics (e.g., erythromycin), the antibiotic molecule will latch onto key translation molecules inside of bacterial cells and basically "stall" them. With no way to make proteins, the bacteria will stop functioning and, eventually, die. That's why infections clear up when they're treated with the antibiotic.

Cells need translation to stay alive, and understanding how it works (so we can shut it down with antibiotics) can save us from bacterial infections. Let's take a closer look at how translation happens, from the first step to the final product.

Translation: The big picture

Translation involves "decoding" a messenger RNA (mRNA) and using its information to build a polypeptide, or chain of amino acids. For most purposes, a polypeptide is basically just a protein (with the technical difference being that some large proteins are made up of several polypeptide chains).

The genetic code

In an mRNA, the instructions for building a polypeptide come in groups of three nucleotides called codons. Here are some key features of codons to keep in mind as we move forward:

These relationships between mRNA codons and amino acids are known as the genetic code (which you can explore further in the genetic code article).

Codons to amino acids

In translation, the codons of an mRNA are read in order (from the 5' end to the 3' end) by molecules called transfer RNAs, or tRNAs.

Each tRNA has an anticodon, a set of three nucleotides that binds to a matching mRNA codon through base pairing. The other end of the tRNA carries the amino acid that's specified by the codon.

tRNAs bind to mRNAs inside of a protein-and-RNA structure called the ribosome. As tRNAs enter slots in the ribosome and bind to codons, their amino acids are linked to the growing polypeptide chain in a chemical reaction. The end result is a polypeptide whose amino acid sequence mirrors the sequence of codons in the mRNA.

That's the big picture of translation. But what about the nitty gritty of how translation begins, proceeds, and finishes? Let's take a look.

Translation: Beginning, middle, and end

A book or movie has three basic parts: a beginning, middle, and end. Translation has pretty much the same three parts, but they have fancier names: initiation, elongation, and termination.

Initiation ("beginning"): in this stage, the ribosome gets together with the mRNA and the first tRNA so translation can begin.

Elongation ("middle"): in this stage, amino acids are brought to the ribosome by tRNAs and linked together to form a chain.

Termination ("end"): in the last stage, the finished polypeptide is released to go and do its job in the cell.

Let's take a closer look at how each stage works.

Initiation

In order for translation to start, we need a few key ingredients. These include:

A ribosome (which comes in two pieces, large and small)

An mRNA with instructions for the protein we'll build

An "initiator" tRNA carrying the first amino acid in the protein, which is almost always methionine (Met)

During initiation, these pieces must come together in just the right way. Together, they form the initiation complex, the molecular setup needed to start making a new protein.

Inside your cells (and the cells of other eukaryotes), translation initiation goes like this: first, the tRNA carrying methionine attaches to the small ribosomal subunit. Together, they bind to the 5' end of the mRNA by recognizing the 5' GTP cap (added during processing in the nucleus). Then, they "walk" along the mRNA in the 3' direction, stopping when they reach the start codon (often, but not always, the first AUG).

In bacteria, the situation is a little different. Here, the small ribosomal subunit doesn't start at the 5' end of the mRNA and travel toward the 3' end. Instead, it attaches directly to certain sequences in the mRNA. These Shine-Dalgarno sequences come just before start codons and "point them out" to the ribosome.

Why use Shine-Dalgarno sequences? Bacterial genes are often transcribed in groups (called operons), so one bacterial mRNA can contain the coding sequences for several genes. A Shine-Dalgarno sequence marks the start of each coding sequence, letting the ribosome find the right start codon for each gene.

Elongation

I like to remember what happens in this "middle" stage of translation by its handy name: e longation is when the polypeptide chain gets long er.

But how does the chain actually grow? To find out, let's take a look at the first round of elongation-after the initiation complex has formed, but before any amino acids have been linked to make a chain.

Our first, methionine-carrying tRNA starts out in the middle slot of the ribosome, called the P site. Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be the "landing site" for the next tRNA, one whose anticodon is a perfect (complementary) match for the exposed codon.

Once the matching tRNA has landed in the A site, it's time for the action: that is, the formation of the peptide bond that connects one amino acid to another. This step transfers the methionine from the first tRNA onto the amino acid of the second tRNA in the A site.

Not bad-we now have two amino acids, a (very tiny) polypeptide! The methionine forms the N-terminus of the polypeptide, and the other amino acid is the C-terminus.

But...odds are we may want a longer polypeptide than two amino acids. How does the chain continue to grow? Once the peptide bond is formed, the mRNA is pulled onward through the ribosome by exactly one codon. This shift allows the first, empty tRNA to drift out via the E ("exit") site. It also exposes a new codon in the A site, so the whole cycle can repeat.

And repeat it does...from a few times up to a mind-boggling times! The protein titin, which is found in your muscles and is the longest known polypeptide, can have up to amino acids.

Termination

Polypeptides, like all good things, must eventually come to an end. Translation ends in a process called termination. Termination happens when a stop codon in the mRNA (UAA, UAG, or UGA) enters the A site.

Stop codons are recognized by proteins called release factors, which fit neatly into the P site (though they aren't tRNAs). Release factors mess with the enzyme that normally forms peptide bonds: they make it add a water molecule to the last amino acid of the chain. This reaction separates the chain from the tRNA, and the newly made protein is released.

What next? Luckily, translation "equipment" is very reusable. After the small and large ribosomal subunits separate from the mRNA and from each other, each element can (and usually quickly does) take part in another round of translation.

Epilogue: Processing

Our polypeptide now has all its amino acids-does that mean it's ready to do its job in the cell?

Not necessarily. Polypeptides often need some "edits." During and after translation, amino acids may be chemically altered or removed. The new polypeptide will also fold into a distinct 3D structure, and may join with other polypeptides to make a multi-part protein.

Many proteins are good at folding on their own, but some need helpers ("chaperones") to keep them from sticking together incorrectly during the complex process of folding.

Some proteins also contain special amino acid sequences that direct them to certain parts of the cell. These sequences, often found close to the N- or C-terminus, can be thought of as the protein's "train ticket" to its final destination. For more about how this works, see the article on protein targeting.

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Stages of translation (article) | Khan Academy

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Learn for free about math, art, computer programming, economics, physics, chemistry, biology, medicine, finance, history, and more. Khan Academy is a nonprofit with the mission of providing a free, world-class education for anyone, anywhere.

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In bacteria, RNA transcripts are ready to act as messenger RNAs and get translated into proteins right away. In eukaryotes, things are a little more complex, though in an pretty interesting way. The molecule that's directly made by transcription in one of your (eukaryotic) cells is called a pre-mRNA, reflecting that it needs to go through a few more steps to become an actual messenger RNA (mRNA). These are:

Addition of a 5' cap to the beginning of the RNA

Addition of a poly-A tail (tail of A nucleotides) to the end of the RNA

Chopping out of introns, or "junk" sequences, and pasting together of the remaining, good sequences (exons)

Once it's completed these steps, the RNA is a mature mRNA. It can travel out of the nucleus and be used to make a protein.

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Nobel laureates Francois Jacob and Jacques Monod conducted much of the seminal work describing the lac operon's structure and control mechanisms. By examining mutant strains of E. coli that exhibited defects in lactose metabolism, Jacob and Monod were able to learn how the lac operon is regulated to metabolize lactose (Jacob & Monod, 1962). The duo noted that the lac operon contains three genes that encode proteins involved in lactose metabolism. These are referred to as lac z, lac y, and lac a. The lac z gene encodes beta-galactosidase, the lac y gene encodes a permease, and the lac a gene encodes the transacetylase enzyme. Together, these gene products act to import lactose into cells and break it down for use as a food source. As in other operons, the genes of the lac operon lie along a contiguous stretch of DNA such that their expression can be easily coregulated. In addition to these so-called structural genes, the lac operon also contains other sequences that direct the bacterial gene expression machinery.

The organization of genes into an operon allows for simultaneous expression of all the genes that are located (i.e., on the same contiguous piece of DNA) in the operon. Several features contribute to this characteristic of operons (Figure 1). First, all of the operon's genes are downstream of a single promoter. This promoter serves as a recognition site for the transcriptional machinery of the RNA polymerase complex. Second, all genes in an operon actually become part of a single messenger RNA molecule, which is subsequently translated into individual protein gene products.

Several other regulatory sequences also ensure coordinated regulation of the lac operon. These include the operator and the terminator. The operator is a special DNA sequence located between the promoter sequence and the structural genes that enables repression of the entire lac operon, following binding by the inhibitor ( lac i) protein. Expression of the lac operon is, in fact, regulated by the presence of lactose itself. The ability to turn lactose-metabolizing genes on or off as a group therefore provides an efficient way to quickly adapt to environmental changes. The terminator, on the other hand, instructs the transcriptional machinery to terminate transcription. As such, the promoter serves as a transcriptional start site, the terminator serves as a stop site, and the operator helps determine whether transcription will occur.

Because of the common control mechanism for all of the genes in the lac operon, mutations in this operon can have multiple, or pleiotropic, effects. For example, mutations affecting the promoter can prevent all of the operon's genes from being expressed, because RNA polymerase will be unable to bind and commence transcription. Other mutations may affect expression of only some of the genes in the operon. For instance, nonsense mutations in the lac z gene encoding beta-galactosidase might interfere with expression of downstream permease and transacetylase genes by causing the ribosome to fall off prior to their translation. This kind of mutation is said to have a "polar" effect on the pathway in that it affects downstream genes but not upstream genes.

Thus, although bacteria may be considered simpler organisms than humans, it is clear that bacterial gene regulation is extremely efficient and that the bacterial genome is highly organized. Bacteria appear to be perfectly adapted to a variety of environments, and they are ready to respond to whatever environmental changes they encounter by employing elegant and complex regulatory mechanisms.

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bacterial genes are organized in operons

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jacob and monod operons  lactose operon  bacterial gene organization  repressor protein

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Bacterial Genes Are Organized in Operons

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Nobel laureates Francois Jacob and Jacques Monod conducted much of the seminal work describing the lac operon's structure and control mechanisms. By examining mutant strains of E. coli that exhibited defects in lactose metabolism, Jacob and Monod were able to learn how the lac operon is regulated to metabolize lactose (Jacob & Monod, 1962).

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A codon is a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis. DNA and RNA molecules are written in a language of four nucleotides; meanwhile, the language of proteins includes 20 amino acids. Codons provide the key that allows these two languages to be translated into each other. Each codon corresponds to a single amino acid (or stop signal), and the full set of codons is called the genetic code. The genetic code includes 64 possible permutations, or combinations, of three-letter nucleotide sequences that can be made from the four nucleotides. Of the 64 codons, 61 represent amino acids, and three are stop signals. For example, the codon CAG represents the amino acid glutamine, and TAA is a stop codon. The genetic code is described as degenerate, or redundant, because a single amino acid may be coded for by more than one codon. When codons are read from the nucleotide sequence, they are read in succession and do not overlap with one another.

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A codon is a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis. DNA and RNA molecules are written in a language of four nucleotides; meanwhile, the language of proteins includes 20 amino acids.

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The Transcriptional Inhibitors, Actinomycin D and α-Amanitin, Activate the HIV-1 Promoter and Favor Phosphorylation of the RNA Polymerase II C-terminal Domain *

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Actinomycin D and α-amanitin are commonly used to inhibit transcription. Unexpectedly, however, the transcription of the human immunodeficiency virus (HIV-1) long terminal repeats (LTR) is shown to be activated at the level of elongation, in human and murine cells exposed to these drugs, whereas the Rous sarcoma virus LTR, the human cytomegalovirus immediate early gene (CMV), and the HSP70 promoters are repressed.

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The ribosome usually moves forward from the start to the stop codon on the mRNA in exact steps of one codon at a time

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The ribosome usually moves forward from the start to the stop codon on the mRNA in exact steps of one codon at a time

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Proteins are synthesized from mRNA templates by a process that has been highly conserved throughout evolution . All mRNAs are read in the 5´ to 3´ direction,

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Max Planck researchers unravel how the ribosome slides along non-coding mRNA
When synthesizing proteins, ribosomes move in exact small steps over long distances along a messenger RNA (mRNA). At certain sites, ribosomes stumble and start sliding before they land at a defined site and resume their accurate work. Marina Rodnina and her team at the Max Planck Institute (MPI) for Biophysical Chemistry in Göttingen have now solved the question what makes the ribosome slide.
Ribosomes are the macromolecular machines that synthesize proteins according to the genetic blueprint stored in the DNA. Copies of the DNA, the mRNAs, deliver the information blueprints to the ribosome. Three nucleotides of the mRNA, called a codon, encode one amino acid, which are the building blocks of proteins. Each codon is decoded with the help of a transfer RNA (tRNA), which delivers the respective amino acid to the ribosome. The region of an mRNA between a start codon and a stop codon encodes for a protein and is called open reading frame (ORF).

Steps of three bases at a time

The ribosome usually moves forward from the start to the stop codon on the mRNA in exact steps of one codon at a time. For each cycle of protein synthesis, a tRNA delivers an amino acid to the ribosome and the peptide grows by one amino acid. Then, the ribosome moves one codon further on the mRNA with the help of a protein called elongation factor G (EF-G) in order to read the next codon. For each step, EF-G has to hydrolyze an energy-rich molecule called GTP to promote fast forward movement. To ensure that the protein is correctly produced, it is crucial that the ribosome moves by exactly one codon at each step.

When the ribosome stumbles

Remarkably, during the translation of particular mRNAs ribosomes slide over a non-coding region within an ORF to synthesize a single protein in a process called translational bypassing. The best-characterized example of bypassing is the gene 60 mRNA of bacteriophage T4. The ribosome reads the first 46 mRNA codons of the ORF up to the triplet coding for the amino acid glycine. The subsequent codon is a stop codon, but instead of terminating protein synthesis, the ribosome takes off and slides over a 50 nucleotides-long non-coding gap. It lands again at a distal glycine codon, and resumes translation to the end of the ORF.

Model of translational bypassing. The ribosome (grey and yellow) is forced into a hyper-rotated state by the growing protein (green) and stem-loop in the messenger RNA (red). Elongation factor G (cyan) promotes take off and sliding. At the landing site the ribosome resumes translation.

What trips and shoves the ribosome

Combining biochemical and single-molecule approaches, Max Planck Director Marina Rodnina and her team have now unraveled how bypassing is initiated and driven. Residues of the synthesized peptide together with an mRNA stem-loop at the take-off site induce an unusual, hyper-rotated state of the ribosome. In this situation EF-G triggers ribosome take-off by shoving the stem-loop, akin of a tRNA in regular translocation. During bypassing EF-G hydrolyzes close to two molecules of GTP per nucleotide of the non-coding gap. The hyper-rotated conformation of the ribosome is also observed with ribosomes stalled by other mRNA sequences, suggesting common ribosome dynamics during translation stalling. These results demonstrate a new function of EF-G in promoting ribosome sliding along the mRNA, in contrast to a codon-wise ribosome movement during normal translation, and suggest a mechanism by which ribosomes could traverse untranslated parts of mRNAs. (Frank Peske)

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How the ribosome starts sliding instead of walking

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Max Planck researchers unravel how the ribosome slides along non-coding mRNA When synthesizing proteins, ribosomes move in exact small steps over long distances along a messenger RNA (mRNA). At certain sites, ribosomes stumble and start sliding before they land at a defined site and resume their accurate work.

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A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland

International Clinical Research Center, St. Anne University Hospital, Brno, Czechia

Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czechia

School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland

University Hospital Tübingen, Department of Internal Medicine VIII, University of Tübingen, Tübingen, Germany

Transcription and translation are fundamental cellular processes that govern the protein production of cells. These processes are generally up regulated in cancer cells, to maintain the enhanced metabolism and proliferative state of these cells. As such cancerous cells can be susceptible to transcription and translation inhibitors. There are numerous druggable proteins involved in transcription and translation which make lucrative targets for cancer drug development. In addition to proteins, recent years have shown that the "undruggable" transcription factors and RNA molecules can also be targeted to hamper the transcription or translation in cancer. In this review, we summarize the properties and function of the transcription and translation inhibitors that have been tested and developed, focusing on the advances of the last 5 years. To complement this, we also discuss some of the recent advances in targeting oncogenes tightly controlling transcription including transcription factors and KRAS. In addition to natural and synthetic compounds, we review DNA and RNA based approaches to develop cancer drugs. Finally, we conclude with the outlook to the future of the development of transcription and translation inhibitors.

Introduction

Cancer refers to a large group of diseases of uncontrolled cell growth and division where a general cure or containment is nowhere to be seen. As a testament to this, the research in cancer is ongoing vigorously and journals specifically tailored for cancer research are now more than 50, ranging from the general to the specific types of cancer. According to the World Health Organization, the various types of cancer accounted for nearly 10 million deaths in 2018, which made it the second most common cause of death. The common factor in cancers is the malignant transformation of cells due to acquired genetic mutations. These are often many and can include both driver and passenger mutations, that confer a growth advantage ( Pon and Marra, 2015). These mutations can lead to activation (gain of function) or deactivation (loss of function,) in some of the biological processes that lead to cancer. When transcriptional or translational processes are disrupted a tumor might be formed.

Chemotherapy is a general term used for all chemicals to treat cancer. It includes for example hormone therapy, which is used to slow down hormone-reactive cancer growth, and cisplatin which prevents the replication of DNA. Also, the focus of this review, targeted therapies are included under chemotherapy. Targeted therapy differs from general chemotherapy by taking a more specialized approach which can be compared to a sniper rifle (targeted therapy) vs. a shotgun (chemotherapy). In other words, the probability of killing healthy cells is lower with targeted therapies than with general chemotherapeutics. While chemotherapy is directed to the inhibition of cell mitosis or inducing autophagy, targeted small molecules inhibitors act on the transcription and translation processes.

Targeting Transcription

Most small molecules used in targeted therapy affect transcription, at three of the four different steps of this process. On the pre-initiation complex, bromodomain, and extra-terminal motif inhibitors reversibly inhibit the ability of the bromodomain to bind to acetylated histones ( Alqahtani et al., 2019), effectively slowing down the formation of the PIC ( Figure 1). Still, at this first step, cyclin-dependent kinase (CDK) inhibitors have been researched and widely tested due to their role in the cell cycle ( Blachly and Byrd, 2013). Their importance is not limited to cancer diseases ( Malumbres and Barbacid, 2001, 2009) but also other diseases such as in HIV ( Galons et al., 2013). At the second step of the transcription process, Pol II inhibitors block the formation of the Pol II-transcription factor complexes. The study of these inhibitors dates back to the late 80's and is still a subject of study today ( Logan et al., 1989; Sharma et al., 2019) ( Figure 1). The last step that can be acted upon at transcription level is the elongation step. There are two main methods of inhibiting this step: inhibition of transcription factors and usage of premature transcription chain terminators ( Figure 1). After being considered undruggable for a long time, recent years have seen a growing number of transcription factor inhibitors and although the sheer number of known TF's makes it difficult to find a general acting drug, it also allows for a greater range of inhibitors to be developed ( Bushweller, 2019). The transcription chain termination is naturally processed by Pols and to terminate it prematurely nucleoside analogs and/or inhibitors, such as fludarabine, are used.

Inhibitors of the Epigenetic Machinery

RNA Polymerase Inhibitors

RNA polymerases produce various RNA molecules. In humans, RNA polymerase I (Pol I) synthesizes the precursors of ribosomal (r)RNA, the main component of ribosomes; Pol II synthesizes precursors of mRNA and most of the snore (sn)RNA and micro (mi)RNAs; while RNA polymerase III synthesizes transfer (t)RNAs and other small RNAs. Cancer drugs or drug candidates have been developed to target all three human RNA polymerases. Some potent natural toxins, such as α-amatinin, inhibit RNA polymerases ( Seifart and Sekeris, 1969). There have been efforts to convert α-amatinin into a cancer drug by combining it with antibodies for specificity. In addition to these conjugates, there are synthesized inhibitors of RNA polymerases, some of which have made it into clinical trials but unfortunately none of these RNA polymerase inhibitors have been approved for therapy as of yet (3/2020). Structures of the discussed RNA polymerase inhibitors are shown in Figure 2.

α-amatinin Conjugates

CX-5461

BMH-21

Small molecule BMH-21 ( Figure 2) is a DNA intercalator which also inhibits Pol I in a manner which is not dependent on DNA damage ( Peltonen et al., 2010; Colis et al., 2014). BMH-21 both inhibits Pol I and induces the degradation of the largest subunit of Pol I ( Peltonen et al., 2014). The inhibition of Pol I by BMH-21 targets the transcription elongation phase and induces pausing in the transcription process ( Wei et al., 2018). Despite the promising in vitro results, no clinical trials have been started with BMH-21.

TAS-106

Metarrestin

Transcriptional Complex Disruptors

Different complexes of proteins can be disrupted during transcription. The first complex to form is the pre-initiation complex which can be disturbed by bromodomain inhibitors. It is also possible to disturb the elongation process by preventing the binding of elongation factors to RNA polymerase, which is the mechanism of action of triptolide. The compounds included in this section are protein-protein interaction inhibitors, and their structures are shown in Figure 3.

Bromodomain Inhibitors

A short overview of the topic will be given here, since there are thorough reviews about bromodomain inhibitors from recent years ( Pérez-Salvia and Esteller, 2016; Alqahtani et al., 2019; Letson and Padron, 2019). Bromodomain (BRD) inhibitors or BET (bromodomain and extraterminal domain) inhibitors prevent the interaction of bromodomain family proteins (BRD2, BRD3, BRD4, and BRDT) with acetylated histones and transcription factors ( Filippakopoulos et al., 2010; Junwei and Vakoc, 2014; Fu et al., 2015). Since the bromodomain-containing proteins regulate gene expression through various processes including histone recognition and modification, chromatin remodeling, and regulation of the transcriptional machinery, BRD and BET inhibitors can be potent transcriptional inhibitors ( Fujisawa and Filippakopoulos, 2017). In cancer, the acetylation state of histones and other proteins is altered, and BRDs promote the expression of many oncogenes, such as c-Myc and Bcl-2, and thus their inhibition provides a way to inhibit cancer cell growth (see also section Inhibitors of Transcription Factor Gene Expression) ( Alqahtani et al., 2019). Most of the bromodomain inhibitors, such as JQ-1, compete for the acetylated lysine binding site and are thus competitive inhibitors ( Alqahtani et al., 2019). However, some of its analogs have made it into the clinical trials (such as birabresib in leukemia and glioblastoma) ( Alqahtani et al., 2019). The results of BET inhibitors as monotherapy have been suffering from resistance, lack of response and toxicity issues ( Bolden et al., 2014; Letson and Padron, 2019), even though they were relatively effective in preclinical models of various cancers ( Kharenko et al., 2016; Waring et al., 2016; Letson and Padron, 2019). This has sparked an interest in using BRD inhibitors in combination with other chemotherapeutic agents, which has shown promising results in animal models so far ( Alqahtani et al., 2019). There are still dozens of on-going clinical trials for various BRD inhibitors in multiple different cancers with or without other chemotherapeutic agents. With more than 600 unique interaction partners in the cells, this family of proteins will continue to spark the curiosity of researchers for a long time.

Triptolide

Triptolide ( Figure 3) is a diterpene triepoxide produced by thunder god vine, a plant regularly used in Chinese traditional medicine for rheumatoid arthritis ( Su et al., 1990). In addition to inhibiting heat shock protein 70, it is an inhibitor of Pol I and Pol II which functions by blocking the transcription elongation process while binding to transcription factor TFIIH ( Titov et al., 2011). It also facilitates degradation of the largest subunit of Pol II in a CDK7-dependent manner ( Vispé et al., 2009; Manzo et al., 2012). Triptolide kills colorectal cancer cells in vitro and inhibits the growth of colorectal xenografts in a mouse model ( Wang et al., 2009; Oliveira et al., 2015). Recently, Liang et al. tested triptolide in adenomatous polyposis coli (Apc) mutated mice where it effectively inhibited colorectal cancer proliferation ( Liang et al., 2019b). Interestingly, they noted that triptolide also reduced Pol III mediated transcription by inhibiting TFIIIB formation at Pol III target genes. Specifically, it did so by blocking the interaction of TBP and Brf1 at Pol III promoters, thereby reducing tRNAs and 5S rRNA transcription. The inhibition of cancer growth both in vitro and in vivo makes triptolide a potential drug candidate for colorectal cancer. However, due to the toxicity of triptolide, only the prodrug disodium salt form of it, minnelide is being studied in human trials for pancreatic and liver cancers ( Banerjee and Saluja, 2015).

Premature Transcription Chain Terminators

Transcription is terminated when specific ending codons are reached on the DNA and after it the mRNA transcript unbinds from the RNA polymerase. The termination is coupled with the 3′-end processing which includes cleavage and addition of a poly-adenosine (A) chain ( Cramer, 2004). This process can be disturbed by mimetics of adenosine or fludarabine ( Figure 4).

Adenosine Analogs

Since transcription generally terminates when a poly-A chain is added to the mRNA transcript, modified adenosine analogs have been the focus of research as premature transcription chain terminators. 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-NH ₂-Ado) ( Figure 4) can be incorporated to both the body and the poly(A) tail of the transcript, where they inhibit further synthesis of it ( Gandhi et al., 2001). In addition to incorporation to mRNA, 8-Cl-Ado, and 8-NH ₂-Ado can be phosphorylated by adenosine kinase into corresponding ATP analogs which decreases the amount of available ATP in the cell ( Frey and Gandhi, 2010). Interestingly, these compounds are not cytotoxic for non-transformed cells ( Balakrishnan et al., 2005; Dennison et al., 2010). 8-Cl-Ado is in phase I clinical trials for acute myeloid leukemia and chronic lymphocytic leukemia ( Stellrecht et al., 2017). Recently, 8-Cl-Ado showed a positive synergistic effect with another cancer drug in a mice xenograft model of acute myeloid leukemia ( Buettner et al., 2019).

Fludarabine

CDK Inhibitors

Alvocidib (Flavopiridol)

Alvocidib (formerly flavopiridol, Figure 5) is a semisynthetic flavonoid resembling rohitukine and it inhibits CDK1, CDK2, CDK4, CDK6, and CDK9. It was the first CDK inhibitor which reached clinical trials in 1998 ( Senderowicz, 1999; Kelland, 2000). Since then more than 60 phase I and phase II clinical trials in various cancers have been conducted using it ( Asghar et al., 2015). The broad target spectrum lead to promising in vitro results, but unfortunately the clinical tests showed only a little activity ( Asghar et al., 2015). There are few positive results for leukemia and lymphoma, and new phase I and II clinical trials are continuously started for alvocidib ( Byrd et al., 2007; Blum et al., 2011). Despite all the investments and thorough studies, alvocidib has not made it into phase III clinical trials, as of 2020. The main issues with alvocidib and other non-selective CDK inhibitors are the uncertainty of their mechanism of action, the problems in patient selection for clinical trials and the lack of a therapeutic window as a result of CDK inhibition in healthy cells ( Asghar et al., 2015).

Palbociclib

Palbociclib ( Figure 5) is a selective inhibitor of CDK4 and CDK6 and it was the first inhibitor of CDKs that was approved as a cancer therapy in combination with letrozole, an aromatase inhibitor ( Lu, 2015; Turner et al., 2015; Finn et al., 2016). Targeting only these CDKs in tumors that have CDK4/CDK6 dysregulation, causes arrest of the cell cycle mediated by retinoblastoma 1 ( Malumbres et al., 2004; Toogood et al., 2005). This results in lower levels of cyclins, nucleotide biosynthesis, DNA replication machinery and mitotic regulatory genes ( Dean et al., 2010; Rivadeneira et al., 2010). Palbociclib is a product of multiple cycles of chemical screening and optimization which started from a set of pyrido[2,3-d]pyrimidin-7-one compounds with a 2-amino pyridine side chain at the C2 position that were showing specificity for CDK4/CDK6 over other CDKs ( VanderWel et al., 2005). During the extensive clinical trials of palbociclib, its cytotoxic effect have been proven, and the main adverse effect has been neutropenia ( Asghar et al., 2015). Neutropenia is a common adverse effect of chemotherapies, but in the case of palbociclib it is a rapidly reversible condition which can be avoided by intermittent dosage. The clinical tests also revealed that palbociclib has a beneficial effect in combination with hormone therapy in estrogen receptor (ER) positive breast cancer cell lines ( Finn et al., 2009). Importantly, the inhibition of CDK4 and CDK6 showed activity in multiple ER positive cell lines that had developed resistance to ER antagonists ( Miller et al., 2011; Thangavel et al., 2011). This lead to many phase II studies which confirmed the significant improvement in the median progression-free survival and granted palbociclib the Breakthrough Therapy designated from the FDA in 2013( Asghar et al., 2015).

Ribociclib

Abemaciclib

Targeting Translation

In the translation initiation phase, the 80S ribosome binds to the start of the mRNA after which tRNA carrying a methionine are able to bind to the starting codon AUG ( Schuller and Green, 2018). The initiation phase is assisted by a wide variety of eukaryotic translation initiation factors (eIFs). For drug design, the most important eIF is eIF4G ( Figure 6). During the elongation phase, the 80S ribosome moves along the mRNA template, binding new tRNA molecules with corresponding amino acids to synthesize the polypeptide chain. This process is coordinated by the eukaryotic elongation factors (eEFs). Once the 80S ribosome encounters a termination codon which is recognized by eukaryotic peptide chain release factors (eRFs), it releases from the mRNA and the polypeptide chain. Finally, the 80S ribosome complex separates into subunits 40 and 60S to begin a new round of translation. At the translation level, it has been shown that many signaling pathways are dysregulated in cancers ( Wolfe et al., 2014; Faller et al., 2015). This association has been bringing translational control into the foreground of targeted cancer therapies ( Vogel and Marcotte, 2012), where the spotlight was previously reserved for transcription level inhibitors. Here, the first step, initiation, is the most targeted by inhibitors, with the mammalian target of rapamycin (mTOR) inhibitors being in recent years one of the main targets of study ( Figure 6) ( Hua et al., 2019). Bioengineered non-coding RNA agents (BERAs) and antisense oligonucleotides, single-chain DNA that prevent translation by interacting with mRNA are also targeting this initial step ( Duan and Yu, 2016; Jian et al., 2017). On the second step of translation, tRNA is targeted for inhibition, thus blocking the protein synthesis process. These inhibitors act by binding the free ribosome, interfering with the normal tRNA binding and thus blocking the elongation step ( Figure 6) ( Gandhi et al., 2014).

mTOR Related Inhibitors

The mammalian (or mechanistic) target of rapamycin (mTOR) is a protein kinase which is the main component of mTOR complexes 1 and 2 (mTORC1 and mTORC2) which are one of the most important regulators of translation ( Sabers et al., 1995). It functions as a serine/threonine protein kinase regulating many cellular processes, such as protein, lipid and nucleotide synthesis as well as nutrient sensing ( Hsieh et al., 2012; Kim and Guan, 2019). The mTOR complexes affect translation by phosphorylation of multiple translation factors, including eIF4G ( Iadevaia et al., 2012). The MTOR gene itself is often mutated in cancer ( Grabiner et al., 2014). In addition, mTORC1 and mTORC2 affect oncogenic pathways, such as phosphatidylinositol-3-OH kinase (PI3K)-PKB/Akt pathway, and thus their signaling is frequently activated in cancer, for example in glioblastoma and follicular lymphoma ( Kim and Guan, 2019). The readers interested in a more detailed overview of mTOR and its biology are referred to the recent and comprehensive review written by ( Kim and Guan, 2019).

The inhibitors of mTOR complexes can be divided into three generations, first of which consist of rapamycin and its analogs termed rapalogs, which affect only specific parts of the mTOR complexes ( Kim and Guan, 2019; Tian et al., 2019). The latter generation is termed ATP-competitive catalytic inhibitors or mTOR kinase inhibitors, and they target the catalytic activity of mTOR. The newest additions to the mTOR related drugs are called RapaLinks which consist of rapamycin linked with an mTOR kinase inhibitor, a combination of the previous two generations.

Rapamycin

Rapamycin (sirolimus, Figure 7) is an antifungal molecule produced by Streptomyces hygroscopicus and it was first discovered from a soil sample from the Easter Island (Rapa Nui) ( Vézina et al., 1975). Structurally, rapamycin resembles the immunosuppressant tacrolimus, and it has similar immunosuppressive effect and mechanism of action via the inhibition of T- and B-cells ( Sehgal, 2003). Because of these immunosuppressive properties rapamycin is one of approved drugs for the prophylaxis of renal transplantation ( Pidala et al., 2012). It was later discovered that in addition to its antifungal and immunosuppressive properties, it is a potent inhibitor of many mammalian kinases ( Chung et al., 1992; Kuo et al., 1992; Price et al., 1992; Sehgal, 2003). About 20 years after the discovery of rapamycin, its target was identified and aptly named, the mammalian target of rapamycin, mTOR ( Sabers et al., 1995; Wiederrecht et al., 1995). Rapamycin functions by binding to 12-kDa FK506-binding protein (FKBP12) forming a complex which then allosterically inhibits mTORC1, but not mTORC2 ( Li et al., 2014). Long-term rapamycin treatment can also affect mTORC2 signaling but the mechanism of this is not clear ( Kim and Guan, 2019). Since mTORC1 complex is activated in numerous human cancers to keep the cancer cells proliferative and increase their nutrient uptake and energy metabolism, rapamycin impairs cancer metabolism and it has been thoroughly studied as a cancer drug ( Li et al., 2014). However, the unmodified rapamycin has poor water solubility which leads to pharmacokinetic issues and facilitated the development of multiple analogs called rapalogs, the first generation mTOR inhibitors.

Rapalogs

Two water-soluble derivatives of rapamycin, temsirolimus, and everolimus ( Figure 7), have been approved for the treatment of renal cancer carcinoma ( Li et al., 2014). Everolimus is also approved for the treatment of progressive neuroendocrine tumors of pancreatic origin, and refractory mantle cell lymphoma in the EU. Even though many water soluble rapalogs were tested in cell and animal models, their effect in the clinic is generally only modest or weak ( Kim and Guan, 2019). In some patients, rapalog usage inadvertently promotes cell survival by enhancing the PKB/Akt activity or by activating autophagy which promotes survival in oxygen-deprived microenvironments ( Tabernero et al., 2008; Palm et al., 2015; Johnson and Tee, 2017). In some cases, these effects can be countered by combining rapalogs with an autophagy inhibitor hydroxychloroquine ( Rangwala et al., 2014). Rapalogs function as allosteric inhibitors of mTORC1, and as such only prevent the phosphorylation of some of the mTORC1 substrates ( Choo et al., 2008; Kang et al., 2013). This, in connection with the negative-feedback loop of PKB/Akt activation associated with the mTOR pathways is possibly the cause for the relatively poor clinical success of rapalogs.

mTOR Kinase Inhibitors

Torin1 and PP242 ( Figure 7) were the first identified ATP-competitive mTOR inhibitors which block the kinase function of mTOR ( Feldman et al., 2009; Thoreen et al., 2009). Later, Torin2 was characterized and tested alone and in combination with other kinase inhibitors against various cancer cell lines ( Liu et al., 2013). Unlike rapamycin and rapalogs, mTOR kinase inhibitors directly inhibit the catalytic activity of both mTOR complexes. The mTOR kinase inhibitors display stronger inhibition of cancer cell proliferation than rapamycin due to their increased efficacy toward mTORC2. However, since mTOR signaling is essential for cell viability, blocking both mTOR complexes causes mTOR kinase inhibitors to have more severe side effects than rapamycin or rapalogs ( Xie et al., 2016). The most advanced mTOR kinase inhibitor in clinical trials is a derivative of PP242, AZD2014, which is currently in phase II clinical trials for ER positive breast cancer ( Guichard et al., 2015).

RapaLinks

The newest members of the mTOR targeting drugs consist of rapamycin linked with an mTOR kinase inhibitor, and they are aptly named RapaLinks ( Rodrik-Outmezguine et al., 2016; Fan et al., 2017). In addition to having activity against many cancer cell lines, RapaLinks are effective against cancerous cells that are resistant to first or second generation mTOR inhibitors ( Rodrik-Outmezguine et al., 2016). All published RapaLinks (1-3) have MLN0128 as the mTOR kinase inhibitor part, and they differ by the linker connecting the two-parts ( Figure 7) ( Rodrik-Outmezguine et al., 2016; Fan et al., 2017). MLN0128 by itself is not very effective in vivo due to its short residence time and its usage is limited by toxicity ( Graham et al., 2018). Even though RapaLinks are large and contain a poorly water-soluble rapamycin, they can pass the blood-brain barrier and their efficacy has been shown in animal models of glioblastoma ( Fan et al., 2017). If the in vivo results imply anything about clinical usability, we expect to see RapaLinks in clinical trials within the next few years.

Silvestrol

Silvestrol ( Figure 8) is a rocaglate derivative that can be isolated from the fruits and twigs of Aglaia foveolate ( Pan et al., 2014). It is cytotoxic toward multiple cancer cell lines in vitro and it displays similar potency to paclitaxel or camptothecin ( Hwang et al., 2004; Pan et al., 2014; Chen et al., 2016b). Silvestrol inhibits the translation initiation by binding to the initiation factor eIF-4A which prevents the ribosome loading onto the mRNA template ( Cencic et al., 2009). This kills cells by inducing early autophagy and caspase-mediated apoptosis ( Chen et al., 2016b). Silvestrol exhibits cytotoxic effects against different human cancer cell lines, such as melanoma, acute myelogenous leukemia, cervical cancer and oral carcinoma ( Hwang et al., 2004; Lucas et al., 2009; Rodrigo et al., 2012; Kogure et al., 2013). Even though silvestrol is effectively cytotoxic against multiple cancer cell lines in vitro, only partial protein synthesis inhibition was observed in mice models of lymphoma ( Bordeleau et al., 2008). The main issue with silvestrol and its analogs is that they upregulate multi-drug-resistant gene ABCB1 and that they are substrates of p-glycoprotein, a well-known resistance-causing efflux transporter ( Gupta et al., 2011). Despite the decade long research, silvestrol and its analogs remain at the preclinical drug research stage, and none of them has made it into clinical trials ( Peters et al., 2018).

Omacetaxine

Omacetaxine, formerly known as homoharringtone ( Figure 8), is a plant alkaloid from Cephalotoxus fortune. It was identified in 1970s as the inhibitor of the initial elongation step of translation ( Huang, 1975; Fresno et al., 1977). Omacetaxine binds to the A-site cleft in the peptidyl-transferase site of the ribosome where it prevents the correct positioning of the incoming aminoacyl-tRNAs, thus functioning as a competitive inhibitor ( Gürel et al., 2009). Because omacetaxine affects the elongation step, it is a more general translation inhibitor than other molecules that target translation initiation which inhibit only the translation of specific sequences ( Wetzler and Segal, 2011). Treatment with omacetaxine leads to a rapid decrease in the number of proteins with short half-lives, including the oncogenic cyclin D1 and c-Myc ( Robert et al., 2009). Omacetaxine was intensely studied after its discovery both in vitro and in vivo against chronic myeloid leukemia but after the approval of imatinib and other tyrosine kinase inhibitors, the scientific interest toward it dwindled ( Wetzler and Segal, 2011). Recently, new clinical studies around omacetaxine have been started due to its synergistic effect with tyrosine kinase inhibitors, especially in the treatment of cancers with mutations in the tyrosine kinase genes ( Marin et al., 2005; Cortes et al., 2012; Rosshandler et al., 2016; Maiti et al., 2017). Omacetaxine is approved by the FDA for the treatment of chronic myeloid leukemia if the disease does not respond to two or more tyrosine kinase inhibitors ( Cortes et al., 2012, 2013; Rosshandler et al., 2016). This way, omacetaxine can help patients who suffer from lack of effect of those drugs, intolerance or drug-drug interactions. The most common adverse effects of omacetaxine are myelosuppression and thrombocytopenia which are observed in almost all patients but they can be managed with supportive care, dose delays and reduction in the number of days that omacetaxine is administered ( Rosshandler et al., 2016).

Antisense Oligonucleotides

ASOs have different mechanisms of actions including enzyme-mediated target RNA degradation, steric-hindrance of translation, as well as modulation of splicing and transcription ( MacLeod and Crooke, 2017). Historically, the first mechanism of ASOs identified was RNase H-dependent degradation, which entails the hydrolysis of the RNA strand in a RNA/DNA duplex ( Crooke, 2017). This is efficiently mediated by the ubiquitous RNase H and has the advantage that the oligonucleotide can be targeted to any part of the RNA molecule. However, problems of specificity due to activation following partial hybridization have been observed and pose a concern. Modified oligomers deviate from RNase H-induced cleavage and can inhibit protein expression via other mRNA quality control decay pathways. These include the non-sense-mediated decay (NMD) ( Ward et al., 2014; Nomakuchi et al., 2016) and the no-go decay (NGD) ( Liang et al., 2019a) which is triggered by ribosome stalling due to obstacles and collision of multiple ribosomes on the mRNA ( Harigaya and Parker, 2010). Oligonucleotides targeted to either the 5′ or 3′ splice sites can interfere with splicing ( Havens and Hastings, 2016; Singh et al., 2018). Interestingly, this can be used either to block mature protein expression or to correct aberrant splicing thereby restoring the protein function. Furthermore, oligonucleotides can result in steric hindrance of translation by preventing ribosome binding when targeted near the translation initiation codon ( Chery, 2016; Goyal and Narayanaswami, 2018). Alternatively, ASOs targeted to the 5′UTR can inhibit translation by preventing 5′ cap formation.

Trabedersen (AP 12009)

AZD4785

RNA Interference-Based Inhibitors

Following the seminal discovery that double stranded RNAs (dsRNAs) could cause post-transcriptional gene silencing (PTGS) in C. elegans ( Fire et al., 1998), it did not last many years until RNA-based therapeutic inhibitors were in clinical trials ( Whelan, 2005) and in 2018 patirisan received FDA approval as the first RNA based drug for the treatment of polyneuropathy. RNA interference (RNAi) as an inherent cellular process of RNA-mediated suppression of gene expression; it achieves this by inhibition of mRNA translation or targeting mRNA for degradation or sequestration (reviewed in detail in Jinek and Doudna, 2009; Setten et al., 2019). The short interfering (si)RNA required for activation of the RNAi pathway, can be derived exogenously from synthetic dsRNA or small hairpin (sh)RNA, in parallel endogenous miRNA can be generated following processing of transcribed pri-micro (mi)RNA. Whereas, the miRNA biogenesis pathway involves cleavage of pri-miRNA in the nuclear microprocessor complex and then with Dicer complexed to TAR RNA binding protein (TRBP) in the cytoplasm, shorter dsRNA can bypass this and is similarly but directly incorporated into the RNA-induced silencing complex (RISC) ( Ha and Kim, 2014; Setten et al., 2019). In this complex, Argonaut 2 (AGO2) removes the passenger (sense) strand and the guide (antisense) strand is maintained, normally due to higher 5′ stability, in the mature and active RISC. In the case of siRNA, the guide RNA is then able to bind with perfect complementarity to target mRNA within the coding region, and in so doing results in mRNA cleavage by the AGO endonucleases. In contrast, miRNA that can bind to several mRNAs and often have their target site in the 3′UTR of mRNA, tend to do so with only partial complementarity. The partial base pairing compromises the AGO slicer catalytic activity and instead results in either translation repression or degradation of mRNA. In both cases small RNA result in suppression of mRNA and subsequently the matching protein.

G12D KRAS -Targeted siRNA

EphA2-Targeted siRNA

miRNA-34a Prodrugs and Mimics

BERAs

Although different chemical modification of synthetic RNA molecules intended for RNAi therapeutics have increased stability and demonstrated favorable pharmacokinetics properties, these Chemo-engineered RNAs are different from naturally transcribed RNA molecules in living cells, which are largely unmodified. This difference affects the structure, properties, and possibly the activity and immunogenicity of these molecules (reviewed in Yu et al., 2019). Also, effort has been made to bioengineer RNA molecules in living cells, including in bacteria and yeast ( Huang et al., 2013; Chen et al., 2015; Pereira et al., 2017b; Ho et al., 2018; Kaur et al., 2018; Duman-Scheel, 2019). These bioengineered RNA agents (BERA) can be produced in large scale and carry no or minimal posttranscriptional modifications ( Li et al., 2015; Wang et al., 2015). Different strategies for producing BERA have been tested, including strategies using RNA-binding proteins such as viral p19, inclusion into tRNA or 5S rRNA scaffolds and tRNA/pre-miRNA chimeras. The last of these has proven to be the most versatile platform as siRNA, miRNAs, and RNA aptamers of different sizes and forms have been produced at large scale with high yields ( Duan and Yu, 2016; Yu et al., 2019).

Vulnerability of naked BERAs to RNAse-degradation in blood necessitates additional formulation for BERA to protect the RNA molecules. This can be done by cationic lipids, polymers, and peptides ( Kim et al., 2016). Specifically, polyethylenimine (PEI)-based polyplexes (complexes of nucleotides and polycations) have facilitated efficient delivery in tumor models ( Zhao et al., 2016; Tu et al., 2019). However due to potential toxicity of polyplexes ( Lv et al., 2006), formulation based on lipidation of these has been recently tested for delivery of tumor associated miRNA ( Zhang et al., 2018; Jilek et al., 2019). Increased serum stability of these BERAs as well as improved delivery, therapeutic effectiveness and survival of tumor bearing mice were observed. These positive results encourage the further development of BERA for tumor therapy.

Targeting Oncogenes

Oncogenes are genes that can cause cancer once mutated or when expressed at high levels ( Croce, 2008). Many oncogenic pathways lead to altered transcription or translation of various proteins. In order to keep the topic of this review we will focus on two oncogenic targets that are involved with transcription and translation; transcription factors and KRAS. Both of these oncogenes were previously thought to be undruggable but nevertheless, a few inhibitors for both of them have been published in the last few years. The readers interested in the drugs designed for oncogenic kinases or other oncogenic pathways are referred to other reviews ( Bhullar et al., 2018; Solassol et al., 2019; Tang and Zhao, 2019; Wang et al., 2019; Zhang et al., 2019b).

Transcription Factors

Protein-Protein Interaction Inhibitors

Inhibitors of Transcription Factor Gene Expression

There is one CDK-inhibitor (SY-1365) in phase I clinical trial for ovarian and breast cancer ( Hu et al., 2019). The inhibition with SY-1365 leads to decreased levels of multiple oncogenic transcription factors and it exhibits the inhibitory effects on multiple cancer cell lines at nanomolar level. In addition, mouse xenograft studies showed modest antitumor activity in both AML as well as ovarian cancer, and a synergistic effect with venetoclax ( Hu et al., 2019).

Two BET inhibitors are in clinical trials that target specifically the transcription of transcription factors, INCB057643 and BMS-986158 ( Forero-Torres et al., 2017; Gavai et al., 2018). Both inhibitors lower the expression levels of the c-Myc oncogene and the proliferation rates of multiple cancer cell lines, and in animal models they display suitable properties for oral dosing in humans. The clinical trials are ongoing for advanced cancers, both solid tumors and leukemias.

RAS Inhibitors

Drugs affecting transcription and translation are difficult to develop. This statement has been a harsh reality for those who have aimed to design oncogene RAS inhibitors, especially inhibitors of KRAS. The RAS GTPase family includes HRAS (Harvey rat sarcoma viral oncogene homolog), KRAS (Kristen rat sarcoma viral oncogene homolog), KRAS split variant KRASB (KRASA is same as KRAS) and NRAS (neuroblastoma RAS viral oncogene homolog). As RAS proteins are the most commonly mutated proteins in cancer and at the same time are part of the signaling cascade from the EGFR receptor, these have been a natural target for drug discovery. After more than 30 years of research, we now have the first compounds in clinical trials targeting mutated RAS protein, namely G12C KRAS. The difficulty of this process has been aptly stated in a recent editorial in the British Journal of Cancer "To put this development into context, KRAS was first described in 1983 and it has taken 35 years to reach this point, whereas identification of oncogenic BRAF mutations in 2002 was followed by an effective targeted drug in 2009" ( Lindsay and Blackhall, 2019).

RAS proteins are small GTPases, thus removing the gamma-phosphate of GTP to produce GDP. The biological function is related to conformational cycle between GTP-bound active RAS and GDP-bound inactive RAS. The GTP-bound RAS can adopt a conformation able to bind with downstream signaling kinases (like PI3K or RAF) and this allows further activation of kinase pathways. Those interested to know more about RAS protein structure and function are advised to look at recently published review ( Pantsar, 2020) and another review about early drug discovery work on RAS by Ostrem and Shokat (2016). In this part of our review we will concentrate on the recent works of KRAS G12C targeting covalent inhibitors as those are the most promising KRAS inhibitors currently known. The KRAS -targeting siRNA was discussed in section G12D KRAS -Targeted siRNA.

As stated KRAS is cycling between inactive GDT-bound and active GTP-bound stated. Direct targeting of GTP-binding pocket is not a realistic option as GTP has a high 0.5 mM concentration near the intracellular site where RAS proteins are located ( Traut, 1994) and at the same time low femtomolar binding affinity ( John et al., 1993; Ford et al., 2009). As there are no other clear druggable pockets in RAS direct targeting seemed to be an impossible mission. The problem was partially solved by the seminal work of Shokat lab which demonstrated that covalent interaction targeting mutated G12C residues is able to deliver in vivo relevant inhibition of RAS activation ( Ostrem et al., 2013). The initial compounds presented were developed by disulphide-fragment based screening using tethering compounds. After early hit-optimization guided by X-ray crystallography an optimized G12C targeting covalent inhibitor was presented. In the optimized compounds disulfides were replaced by different electrophilic warheads and especially acrylamides were used. Upon covalent interaction G12C position compounds were binding previously unknown allosteric pocket. Even more important is the finding that these G12C inhibitors are RAS-GDP specific. This effect will undoubtedly explain the positive clinical outcome of two later-developed G12C inhibitors, namely AMG 520 and MRTX849 ( Canon et al., 2019; Hallin et al., 2020). Both AMG 520 and MRTX849 seem to give better results with combination of either upstream or downstream kinase inhibitors. This is not surprising as such, but more surprising is a very recent report concerning the rapid non-uniform adaptation to KRAS G12C inhibition ( Xue et al., 2020). According to Xue et al. (2020) G12C inhibitors targeting the RAS-GDP state promote a feedback mechanism in which KRAS G12C is maintained in active state by EGFR and Aurora kinase A (AURKA) signaling. If this finding is validated it would indicate that EGFR and/or AURKA inhibition are mandatory to support and maintain KRAS G12C inhibition. Partially this effect might be specific for the currently used G12C inhibitor (ARS1620) but due to similar binding mode as with AMG 520 it seems that this conclusion will be a general one ( Janes et al., 2018). Besides of AMG 520 and MRTX849 there are also two other KRAS G12C targeting inhibitors, namely JNJ-74699157 (NCT04006301) and LY349946 (NCT04165031), in the clinical trials.

Conclusions and Future Remarks

History has shown us that it is indeed not straight-forward to develop transcription or translation inhibitors. Even more difficult is to target these inhibitors only toward malignant cells. There have been a few clinical successes in their development, such as the CDK inhibitors, especially in combination with other chemotherapeutics. Targeting these central cellular processes has advantages to be more directed to cancer cells than non-specific chemotherapeutic agents such as cisplatin. On the side of disadvantages, targeting transcription and translation may affect multiple pathways hence circumventing the targeted pathway, and there are inevitable side effects arising from the fact that all cells require transcription and translation for their proper function. Transcription and translation are fundamental processes that targeting them is bound to result in cell death, as such cancer treatment based on these processes needs to be done in a manner that is safe for healthy cells. To achieve the specific and effective treatment, the myriad of proteins involved will continue to offer drug design possibilities far into the future. In addition, RNA was previously considered to be undruggable and not suitable as a drug itself, but now RNA-targeting and RNA-based drugs can be used as very precise methods to target some cancers. The recently discovered RNA activation of transcription offers uncharted possibilities in the treatment of cancer. Furthermore, transcription factors and oncogenes were also thought to be undruggable, but within the last few years we have seen some molecules targeting them entering clinical trials. By widening the scope of drug targets from traditional proteins with specified binding sites to transcription factors, oncogenes and RNA molecules, we are discovering new and specific ways to target cancer cells. So far, we have produced some highly specific, safe and efficacious cancer therapies which inhibit transcription and translation, and the newly discovered targets and our ever-increasing knowledge about the biological basics of these processes is bound to keep this field of inhibitor development ongoing far into the future.

Author Contributions

NL-K, GP, AP, and PK wrote sections and participated in the creation of the graphics of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

Funding

The authors thank Maire Taponen Foundation (NL-K personal grant) and competitive funding to strengthen university research profiles, 5th call, funding to University of Eastern Finland, funded by the Academy of Finland, funding decision 325022. This work was supported by the Technology Agency of Czech Republic (TN0100013).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

The authors apologize for colleagues whose work could not be referenced due to word count limits.

Abbreviations

ASO, Antisense oligonucleotide; AURKA, Aurora kinase A; BERA, Bioengineered non-coding RNA agent; BET, Bromodomain and extraterminal domain; CDK, Cyclin-dependent kinase; EGFR, Epidermal growth factor receptor; Pol, RNA polymerase; RAS, Rat sarcoma viral oncogene homolog; RISC, RNA-induced silencing complex; RNAi, RNA interference.

References

Friday, B., Lassere, Y., Meyers, C. A., Mita, A., Abbruzzese, J. L., and Thomas, M. B. (2012). A phase I study to determine the safety and pharmacokinetics of intravenous administration of TAS-106 once per week for three consecutive weeks every 28 days in patients with solid tumors. Anticancer Res. 32, 1689-1696. Available online at: http://ar.iiarjournals.org/content/32/5/1689.long

PubMed Abstract | Google Scholar

Gandhi, V., Ayres, M., Halgren, R. G., Krett, N. L., Newman, R. A., and Rosen, S. T. (2001). 8-chloro-cAMP and 8-chloro-adenosine act by the same mechanism in multiple myeloma cells. Cancer Res. 61, 5474-5479. Available online at: https://cancerres.aacrjournals.org/content/61/14/5474.long

PubMed Abstract | Google Scholar

Hortobagyi, G. N., Stemmer, S. M., Burris, H. A., Yap, Y.-S., Sonke, G. S., Paluch-Shimon, S., et al. (2016). Ribociclib as first-line therapy for HR-positive, advanced breast cancer. 375, 1738-1748. doi: 10.1056/NEJMoa1609709

Lucas, D. M., Edwards, R. B., Lozanski, G., West, D. A., Shin, J. D., Vargo, M. A., et al. (2009). The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo. Blood 113, 4656-4666. doi: 10.1182/blood-2008-09-175430

Pereira, P., Pedro, A. Q., Queiroz, J. A., Figueiras, A. R., and Sousa, F. (2017b). New insights for therapeutic recombinant human miRNAs heterologous production: Rhodovolum sulfidophilum vs Escherichia coli. Bioengineered 8, 670-677. doi: 10.1080/21655979.2017.1284710

Watson, J., Baker, T., Bell, S., Gann, A., Levine, M., and Losick, R. (2013). "Part 5: regulation," in Molecular Biology of the Gene, ed B. Wilbur (New York, NY: Pearson), 609-792.

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Transcription and Translation Inhibitors in Cancer Treatment

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Transcription and translation are fundamental cellular processes that govern the protein production of cells. These processes are generally up regulated in cancer cells, to maintain the enhanced metabolism and proliferative state of these cells. As such cancerous cells can be susceptible to transcription and translation inhibitors.

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The lac operon of E. coli contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent.

Two regulators turn the operon "on" and "off" in response to lactose and glucose levels: the lac repressor and catabolite activator protein (CAP).

The lac repressor acts as a lactose sensor. It normally blocks transcription of the operon, but stops acting as a repressor when lactose is present. The lac repressor senses lactose indirectly, through its isomer allolactose.

Catabolite activator protein ( CAP) acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the "hunger signal" molecule cAMP.

Introduction

Lactose: it's what's for dinner! While that may not sound delicious to us (lactose is the main sugar in milk, and you probably don't want to eat it plain), lactose can be an excellent meal for E. coli bacteria. However, they'll only gobble up lactose when other, better sugars - like glucose - are unavailable.

With that for context, what exactly is the lac operon? The lac operon is an operon, or group of genes with a single promoter (transcribed as a single mRNA). The genes in the operon encode proteins that allow the bacteria to use lactose as an energy source.

What makes the lac operon turn on?

E. coli bacteria can break down lactose, but it's not their favorite fuel. If glucose is around, they would much rather use that. Glucose requires fewer steps and less energy to break down than lactose. However, if lactose is the only sugar available, the E. coli will go right ahead and use it as an energy source.

To use lactose, the bacteria must express the lac operon genes, which encode key enzymes for lactose uptake and metabolism. To be as efficient as possible, E. coli should express the lac operon only when two conditions are met:

How are levels of lactose and glucose detected, and how how do changes in levels affect lac operon transcription? Two regulatory proteins are involved:

One, the lac repressor, acts as a lactose sensor.

The other, catabolite activator protein (CAP), acts as a glucose sensor.

These proteins bind to the DNA of the lac operon and regulate its transcription based on lactose and glucose levels. Let's take a look at how this works.

Structure of the lac operon

The lac operon contains three genes: lacZ, lacY, and lacA. These genes are transcribed as a single mRNA, under control of one promoter.

Genes in the lac operon specify proteins that help the cell utilize lactose. lacZ encodes an enzyme that splits lactose into monosaccharides (single-unit sugars) that can be fed into glycolysis. Similarly, lacY encodes a membrane-embedded transporter that helps bring lactose into the cell.

In addition to the three genes, the lac operon also contains a number of regulatory DNA sequences. These are regions of DNA to which particular regulatory proteins can bind, controlling transcription of the operon.

The promoter is the binding site for RNA polymerase, the enzyme that performs transcription.

The operator is a negative regulatory site bound by the lac repressor protein. The operator overlaps with the promoter, and when the lac repressor is bound, RNA polymerase cannot bind to the promoter and start transcription.

The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter.

Let's take a closer look at the lac repressor and CAP and their roles in regulation of the lac operon.

The lac repressor is a protein that represses (inhibits) transcription of the lac operon. It does this by binding to the operator, which partially overlaps with the promoter. When bound, the lac repressor gets in RNA polymerase's way and keeps it from transcribing the operon.

When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon.

This change in the lac repressor is caused by the small molecule allolactose, an isomer (rearranged version) of lactose. When lactose is available, some molecules will be converted to allolactose inside the cell. Allolactose binds to the lac repressor and makes it change shape so it can no longer bind DNA.

Allolactose is an example of an inducer, a small molecule that triggers expression of a gene or operon. The lac operon is considered an inducible operon because it is usually turned off (repressed), but can be turned on in the presence of the inducer allolactose.

Catabolite activator protein (CAP)

When lactose is present, the lac repressor loses its DNA-binding ability. This clears the way for RNA polymerase to bind to the promoter and transcribe the lac operon. That sounds like the end of the story, right?

Well...not quite. As it turns out, RNA polymerase alone does not bind very well to the lac operon promoter. It might make a few transcripts, but it won't do much more unless it gets extra help from catabolite activator protein ( CAP). CAP binds to a region of DNA just before the lac operon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription.

CAP isn't always active (able to bind DNA). Instead, it's regulated by a small molecule called cyclic AMP ( cAMP). cAMP is a "hunger signal" made by E. coli when glucose levels are low. cAMP binds to CAP, changing its shape and making it able to bind DNA and promote transcription. Without cAMP, CAP cannot bind DNA and is inactive.

CAP is only active when glucose levels are low (cAMP levels are high). Thus, the lac operon can only be transcribed at high levels when glucose is absent. This strategy ensures that bacteria only turn on the lac operon and start using lactose after they have used up all of the preferred energy source (glucose).

So, when does the lac operon really turn on?

The lac operon will be expressed at high levels if two conditions are met:

Glucose must be unavailable: When glucose is unavailable, cAMP binds to CAP, making CAP able to bind DNA. Bound CAP helps RNA polymerase attach to the lac operon promoter.

Lactose must be available: If lactose is available, the lac repressor will be released from the operator (by binding of allolactose). This allows RNA polymerase to move forward on the DNA and transcribe the operon.

These two events in combination - the binding of the activator and the release of the repressor - allow RNA polymerase to bind strongly to the promoter and give it a clear path for transcription. They lead to strong transcription of the lac operon and production of enzymes needed for lactose utilization.

Putting it all together

Now that we've seen all the moving parts of the lac operon, let's put what we've learned together to see how the operon reacts to a variety of different conditions (presence or absence of glucose and lactose).

Summary of lac operon responses

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The lac operon of E. coli contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent. Two regulators turn the operon "on" and "off" in response to lactose and glucose levels: the lac repressor and catabolite activator protein (CAP). The lac repressor acts as a lactose sensor.

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Genetic code. The story of the genetic code is the story of biology and genetics in the 19th, 20th, and 21st centuries, as well as its promises and its perils. Oswald Avery in 1944, for example, proved that the genetic code - that DNA -was indeed the carrier of hereditary information, ending more than 80 years of productive speculation. But as important as DNA was to the so-called heroic era of molecular biology, spanning the generation of scientific discovery after the Second World War, and as important as DNA is to the revolutionary sciences of genetics and genomics, neither genes nor DNA determine who you are or what you shall do.

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The instructions in a gene that tell the cell how to make a specific protein.

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Transcription and mRNA processing | Biomolecules | MCAT | Khan Academy

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Abstract

Over the past forty years, anthracyclines and ellipticines have attracted attention as promising cytostatics. In this review, we focus on their mechanisms of cytoxicity, DNA-damaging effects and adverse side-effects. We also summarize ways to enhance the therapeutic effects of these drugs together with a decrease in their adverse effects. Current drug design strategies are focused on drug bioavailability and their tissue targeting, whereas drug delivery to specific intracellular compartments is rarely addressed. Therefore, therapies utilizing the antineoplastic activities of anthracyclines and ellipticines combined with novel strategies such as nanotechnologies for safer drug delivery, as well as strategies based on gene therapy, could significantly contribute to medical practice.

View full text

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Anthracyclines and ellipticines as DNA-damaging anticancer drugs: Recent advances

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Over the past forty years, anthracyclines and ellipticines have attracted attention as promising cytostatics. In this review, we focus on their mechanisms of cytoxicity, DNA-damaging effects and adverse side-effects. We also summarize ways to enhance the therapeutic effects of these drugs together with a decrease in their adverse effects.

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Translation is the process by which a protein is synthesized from the information contained in a molecule of messenger RNA (mRNA). During translation, an mRNA sequence is read using the genetic code, which is a set of rules that defines how an mRNA sequence is to be translated into the 20-letter code of amino acids, which are the building blocks of proteins. The genetic code is a set of three-letter combinations of nucleotides called codons, each of which corresponds with a specific amino acid or stop signal. Translation occurs in a structure called the ribosome, which is a factory for the synthesis of proteins. The ribosome has a small and a large subunit and is a complex molecule composed of several ribosomal RNA molecules and a number of proteins. Translation of an mRNA molecule by the ribosome occurs in three stages: initiation, elongation, and termination. During initiation, the small ribosomal subunit binds to the start of the mRNA sequence. Then a transfer RNA (tRNA) molecule carrying the amino acid methionine binds to what is called the start codon of the mRNA sequence. The start codon in all mRNA molecules has the sequence AUG and codes for methionine. Next, the large ribosomal subunit binds to form the complete initiation complex. During the elongation stage, the ribosome continues to translate each codon in turn. Each corresponding amino acid is added to the growing chain and linked via a bond called a peptide bond. Elongation continues until all of the codons are read. Lastly, termination occurs when the ribosome reaches a stop codon (UAA, UAG, and UGA). Since there are no tRNA molecules that can recognize these codons, the ribosome recognizes that translation is complete. The new protein is then released, and the translation complex comes apart.

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Translation is the process by which a protein is synthesized from the information contained in a molecule of messenger RNA (mRNA).

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Abstract

Transcription by RNA polymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC(50) in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity. We have shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis.

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pol1 transcription initiation complex proteins / metabolism

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national center for biotechnology information

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Old drug, new target: ellipticines selectively inhibit RNA polymerase I transcription - PubMed

# data[].attributes.attachment_link.description
Transcription by RNA polymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linke ...

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ribosome is a non membrane organelle  that acts as the site for protein systhesis

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ribosome is a non membrane organelle  that acts as the site for protein systhesis

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Organelles that are not fluid-filled don't need to be separated from the rest of the cell in the same way, so they don't have a membrane. These are the non-membrane bound organelles

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Abstract

The bacterial lacZ gene has been used to genetically tag tumor cells. This has been of value, specifically in metastasis research where it allowed to visualize micrometastases and single tumor cells in tissues. lacZ tagging has also provided the feasibility of re-isolating metastatic tumor cell populations for ex vivo molecular analyses. This review summarizes studies that have utilized lacZ tagged tumor cells to understand aspects of metastasis including dormancy, tumor-host interactions, gene modulation and anti-tumor immunity. lacZ tagging is also a valuable tool for evaluating cancer therapies.

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Oncogenic Signaling Pathways Perturb Specific Translational Components

There is a growing body of evidence supporting the idea that deregulation of translational control serves as a common mechanism by which diverse oncogenic pathways (e.g., PI3K, Myc, and Ras) promote cellular transformation and tumor development ( Silvera et al. 2010; van Riggelen et al. 2010; Hannan et al. 2011). In a normal cell, these pathways act as sensors of energy, stress, and nutrient availability, as well as growth factor signals, and integrate these inputs to direct control of ribosome production and gene expression at the translational level. One of the primary reasons for this cross talk is to couple these external stimuli with the execution of cell growth, division, and survival, which are directly coupled to protein synthesis (see below). Importantly, all these signals become oncogenic when hyperactivated. How do these signaling pathways alter translational control when mutated in cancer? This question has been answered, at least in part, by the discovery that the oncogenic signals directly modulate the activity and expression of specific translational components (). Notable examples include the PI3K-AKT-mTOR and Ras-MAPK signal transduction pathways, as well as transcriptional programs regulated by oncogenic Myc ( Frederickson et al. 1992; Waskiewicz et al. 1999; Schuhmacher et al. 2001; Zeller et al. 2006; Furic et al. 2010; Silvera et al. 2010; van Riggelen et al. 2010; Hannan et al. 2011). I discuss the role of Myc in protein synthesis and cell growth in the next section.

One of the best-studied examples of oncogenic signaling impinging on translational control is the PI3K-AKT-mTOR pathway, which directly modulates translation initiation largely through activation of the kinase mammalian target of rapamycin complex 1 (mTORC1) ( Zoncu et al. 2011). The PI3K-AKT-mTOR pathway is one of the most commonly mutated pathways in cancer ( Vogt et al. 2011). mTORC1 phosphorylates ribosomal protein S6 kinase 1/2 (S6K1/2) and the eIF4E-binding proteins (4E-BPs), which negatively regulate the major cap-binding protein eIF4E ( Dennis et al. 1996; Hara et al. 1997; Gingras et al. 1999). Phosphorylation of 4E-BPs leads to a conformational change that frees eIF4E, which, with the help of other eIFs, ultimately recruits the 40S ribosomal subunit to the 5′ cap of mRNAs during translation initiation ( Gingras et al. 1999; Ruggero and Sonenberg 2005). Initiation is thought to be the rate-limiting step of cap-dependent translation and eIF4E is considered the key factor in controlling this step ( Duncan et al. 1987). This deduction is in part based on the fact that eIF4E activity is highly regulated at both the mRNA and protein level. Indeed, eIF4E is up-regulated at the mRNA level by a number of transcription factors including the oncogene Myc ( Jones et al. 1996; De Benedetti and Graff 2004; Ruggero and Sonenberg 2005). At the protein level, eIF4E activity is also controlled through phosphorylation at serine 209 by the MAP kinase targets MNK1/2, in addition to inhibitory interactions with 4E-BPs (see above and ) ( Lachance et al. 2002; Ueda et al. 2004, 2010; Wendel et al. 2007; Furic et al. 2010). This tight regulation of eIF4E activity provides a rapid mechanism for cells to modulate translation initiation in response to numerous stimuli, including growth factor and oncogenic signaling.

The 4E-BPs/eIF4E axis is among the most well-characterized nodes in translation control and cancer. For example, eIF4E gene amplification has been reported in human breast, head, and neck cancer specimens, and eIF4E is also found overexpressed in a variety of tumors ( Sorrells et al. 1998, 1999; Crew et al. 2000; Berkel et al. 2001; Rosenwald et al. 2001; Salehi and Mashayekhi 2006; Wang et al. 2009a; Yoshizawa et al. 2010). The oncogenic potential of eIF4E hyperactivity has been faithfully recapitulated both in vitro and in vivo. Overexpression of eIF4E is sufficient to induce transformation of immortalized murine fibroblasts and human epithelial cells ( Lazaris-Karatzas et al. 1990; Avdulov et al. 2004). Constitutive overexpression of eIF4E in a mouse model that mimics the human oncogenic lesion leads to increased cancer susceptibility; eIF4E transgenic mice develop lymphomas, angiosarcomas, lung carcinomas, and hepatomas ( Ruggero et al. 2004). Furthermore, in vivo overexpression of eIF4E cooperates with c-Myc to drive lymphomagenesis in part via a mechanism by which eIF4E overcomes Myc-induced apoptosis, a cellular barrier to tumor formation ( Ruggero et al. 2004; Wendel et al. 2004). Phosphorylation of eIF4E also plays an important role in cancer formation. In fact, by using an adoptive transfer method in vivo, it has been shown that phosphorylated eIF4E contributes to Myc-induced tumorigenesis mainly by suppressing apoptosis ( Wendel et al. 2007). In addition, whole body expression of a knockin mutant of eIF4E, which can no longer be phosphorylated at the serine 209 residue, was found to decrease the incidence and grade of prostatic intraepithelial neoplasia in a mouse prostate cancer model driven by PTEN loss ( Furic et al. 2010).

Although these findings support the notion that eIF4E is a bona fide oncogene, they do not address the extent to which specific oncogenic signaling (i.e., PI3K-AKT-mTOR pathway) relies on eIF4E translational activity for tumor development. Some of the first evidence for such a connection came from a study showing that pharmacological inhibition of oncogenic Ras and AKT in glioblastoma cells caused a rapid and profound change in mRNA translation that far outweighed transcriptional changes. These translational changes were associated with loss of mTORC1-dependent phosphorylation of 4E-BPs ( Rajasekhar et al. 2003). In addition, transfection of a 4E-BP1 phosphorylation site mutant into breast carcinoma cells suppressed their tumorigenicity by inducing apoptosis ( Avdulov et al. 2004). It has recently been shown in vitro and in vivo that the 4E-BPs/eIF4E axis exerts significant control over cap-dependent translation, cell growth, cancer initiation, and progression downstream of mTOR hyperactivation ( Dowling et al. 2010; Hsieh et al. 2010). Most importantly, restoring eIF4E oncogenic activity to normal levels downstream of AKT-mTOR hyperactivation results in blockage of tumor progression in a mouse model for lymphomagenesis associated with a drastic increase in overall survival ( Hsieh et al. 2010).

Do these findings indicate that cells transformed by mTOR hyperactivation become "addicted" to oncogenic eIF4E-mediated translation control? Oncogenic addiction describes a tumor's dependence on specific oncogenic lesions for its survival and growth. This represents a potential "Achilles heel" in tumor formation by which inactivation of a specific oncogene can lead to apoptosis, senescence, and tumor regression ( Weinstein 2000, 2002; Weinstein and Joe 2006). The importance of this concept resides in the fact that although cancer cells harbor many oncogenic lesions, targeting the mediators of oncogenic addiction would herald a landmark improvement in cancer therapy. Although the mechanisms underlying this "Achilles heel" are poorly understood, it is tempting to speculate that the 4E-BPs/eIF4E axis may represent a novel node of oncogenic addiction to target for therapeutic intervention (see below). At the mechanistic level, targeting eIF4E activity in cancer cells can be a key to initiate a cell death program resulting in tumor regression. In support of this hypothesis, it has been shown that eIF4E hyperactivation is able to enhance the translation of select mRNAs ( De Benedetti and Graff 2004), including some encoding antiapoptotic factors ( Mamane et al. 2007). The 5′ UTR of these mRNAs are believed to harbor regulatory elements, such as complex secondary structures, that impart this selectivity (). One important example is Mcl-1, an antiapoptotic factor containing a complex 5′ UTR that is translationally up-regulated specifically upon eIF4E hyperactivation, leading to enhanced survival of cancer-initiating cells ( Mills et al. 2008; Hsieh et al. 2010). Therefore, cancer cells may be addicted to eIF4E-promoted survival, which would serve as a promising target for therapeutic intervention.

Regulation of eIF4E is not the only node in which information from signaling pathways is received by the translational machinery. It is now clear that an entire repertoire of translational components may be co-opted to promote cancer initiation. For example, AKT hyperactivation also modulates translation elongation (; ) ( Wang et al. 2001). Additional regulated translational components include eIF2α, which is part of the ternary complex required to chaperone the initiator transfer RNA (tRNA) to the ribosome. Phosphorylation of eIF2α by several kinases including the double-stranded RNA-dependent protein kinase (PKR) and the endoplasmic reticulum kinase PERK leads to consequent inhibition of protein synthesis and this is a major cell growth checkpoint ( Ron and Harding 2007). Disruption of the eIF2α checkpoint can lead to the transformation of immortalized rodent and human cells ( Koromilas et al. 1992b; Meurs et al. 1993; Barber et al. 1995; Donze et al. 1995). However, prevention of eIF2α phosphorylation is not tumorigenic in vivo ( Yang et al. 1995; Abraham et al. 1999; Scheuner et al. 2001). In addition, it has been shown that the PERK-eIF2α axis represents a survival pathway for cancer cells that need to adapt and overcome hypoxic stress during tumor progression ( Fels and Koumenis 2006). Transformed cells with inactivating PERK or eIF2α mutations form smaller tumors that grow more slowly in nude mice that grow more slowly, and show higher levels of apoptosis in hypoxic areas compared to control cells with intact eIF2α phosphorylation ( Bi et al. 2005). eIF2α is also commonly overexpressed in cancers and may thereby provide an uncontrolled stimulus leading to increased rates of protein synthesis ( Wang et al. 1999). Interestingly, overexpression of the initiator tRNA itself is able to drive cellular transformation ( Marshall et al. 2008). Another translation factor that promotes cellular transformation is eIF6, which regulates the joining of the 60S ribosomal subunit to the 48S preinitiation complex to begin translation ( Gandin et al. 2008). Importantly, eIF6 has been shown to be rate limiting for translation, cell growth, and transformation ( Gandin et al. 2008). This initiation factor also interacts with RACK1, a ribosome-associated scaffolding protein that coordinates signaling by PKC and src kinases ( Ceci et al. 2003). Therefore, signaling through RACK1 to eIF6 may be another key node of oncogenic regulation.

Although a wealth of research (described above), including elegant genetic studies, has shown that eIF4E hyperactivation and other nodes of translational control play a key role in cancer development, the repertoire of mRNAs that are specifically sensitive to translational perturbations is not completely understood. There are now emerging technologies that may facilitate their identification. In particular, the ability to deep-sequence ribosome-protected mRNAs will enable codon-by-codon resolution of ribosome occupancy on specific mRNAs ( Ingolia et al. 2009). Furthermore, through deep sequencing, it is now possible to determine the secondary structures of mRNAs by using a novel strategy termed parallel analysis of RNA structures (PARS) ( Kertesz et al. 2010). The combination of these two technologies may provide a very accurate portrait of how mRNA secondary structures control the translation of the cancer genome. Furthermore, techniques such as high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) and affinity RNA purification followed by mass spectrometry have successfully identified cis-regulatory elements and specific regulatory factors associated with RNAs ( Ji et al. 2004; Darnell et al. 2011; Tsai et al. 2011; Darnell and Richter 2012). Such technologies could be used to characterize the mRNAs and the translational complex directly bound to eIFs such as eIF3, eIF4G, eIF5B, and eIF4A. Undertaking these approaches in a time course manner during the different steps in tumorigenesis would be valuable in identifying novel translational regulatory networks and might help to reveal the interplay between these networks during the stages of tumor development. Fluorescence-based living cell approaches such as fluorescence resonance energy transfer (FRET) could also enable the analysis of molecular interactions of a specific translational complex in real time during cellular transformation ( Huranova et al. 2009; Lorenz 2009). Simply analyzing a static complex paints an incomplete picture. By studying the dynamic assembly and disassembly of these regulatory nodes, we could gain crucial insight into their ability to promote distinct translation initiation modes, ultimately contributing to cancer development.

Why Has the Translational Control of Specific mRNAs Been Selected for Cancer Development?

To consider this question, it is perhaps important to consider mechanisms that drive cancer evolution, whereby altering specific cellular processes may lead to uncontrolled growth. In this respect, perturbing translational control could provide a rapid change in gene expression upon environmental cues that allows the cancer cell to adapt and survive even under unfavorable conditions.

But how does the cancer program control specific mRNAs to support the growth of cancer cells? A central principle emerging from detailed molecular studies is that important regulatory elements (e.g., IRES, complex 5′ and 3′ UTRs, RNA-binding proteins, and microRNA sites) may render mRNAs encoding key tumor suppressors and protooncogenes (including cell-cycle and survival factors) exquisitely vulnerable to any perturbations in translational control (). Many of these mRNAs possess long 5′ UTRs that contain upstream ORFs and/or stable secondary structures ( Stoneley and Willis 2003; Pickering and Willis 2005; Sonenberg and Hinnebusch 2009). These specific cis features act as inhibitory elements that interfere with the activity of the eIF4F initiation complex and ribosome scanning, thereby maintaining the translational efficiency of these mRNAs at a level that does not exceed a threshold necessary for cellular homeostasis. Importantly, these mRNAs are aberrantly translated as a consequence of overexpressed eIF4E or the other components of the eIF4F complex, which overcome the translational inhibitory effects imposed by their 5′ UTR cis elements () ( Koromilas et al. 1992a; Stoneley and Willis 2003; Pickering and Willis 2005; Ruggero and Sonenberg 2005; Mills et al. 2008; Hsieh et al. 2010). In addition, overexpression of RNA-binding proteins such as CPEB4 has been shown to increase the translation of specific oncogenic mRNAs by binding to cis elements present in their 3′ UTRs ( Ortiz-Zapater et al. 2011; for further review, please see van Kouwenhove et al. 2011). Furthermore, the sequence and structural integrity of the 5′ and 3′ UTRs in the p53 tumor suppressor mRNA are required for its translation () ( Chen and Kastan 2010).

Do genetic perturbations in mRNA regulatory elements directly lead to cancer formation? This is an intriguing line of research that remains to be fully explored. Alterations in the 5′ UTR of MDM2, δ-catenin, or Myc oncogenes increase the translational level of their mRNAs in multiple myeloma, Burkitt's lymphoma, and prostate cancer ( Paulin et al. 1996; Landers et al. 1997; Capoulade et al. 1998; Stoneley et al. 1998; Brown et al. 1999; Jin et al. 2003; Wang et al. 2009b). A mutation in the 5′ UTR of the CDKN2a tumor suppressor (also known as p16 INK4A) is associated with increased susceptibility to melanoma. This mutation creates an aberrant upstream translation initiation codon impairing translation initiation efficiency from the wild-type AUG ( Liu et al. 1999). Additionally, cancer-derived mutations in the p53 gene may alter an IRES element present in the 5′ UTR of its mRNA decreasing p53 protein expression ( Grover et al. 2011).

Altogether, the remarkable repertoire of deregulated translational components, whose activity is directly controlled downstream of specific oncogenic signals, strongly supports their critical and causal role in cancer initiation and progression. What also emerges from these studies is that perturbations in translational control provide highly specific outcomes for gene expression that impinge on distinct steps along the pathway toward cancer development.

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Translational Control in Cancer Etiology

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There is a growing body of evidence supporting the idea that deregulation of translational control serves as a common mechanism by which diverse oncogenic pathways (e.g., PI3K, Myc, and Ras) promote cellular transformation and tumor development ( Silvera et al. 2010; van Riggelen et al. 2010; Hannan et al. 2011).

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Abstract

Cisplatin and other platinating agents are some of the most widely used chemotherapy agents. These drugs exert their antiproliferative effects by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The cross-links mobilize signaling and repair pathways, including the Rad9-Hus1-Rad1-ATR-Chk1 pathway, a pathway that helps tumor cells survive the DNA damage inflicted by many chemotherapy agents. Here we show that Rad9 and ATR play critical roles in helping tumor cells survive cisplatin treatment. However, depleting Chk1 with small interfering RNA or inhibiting Chk1 with 3-(carbamoylamino)-5-(3-fluorophenyl)- N-(3-piperidyl)thiophene-2-carboxamide (AZD7762) did not sensitize these cells to cisplatin, oxaliplatin, or carboplatin. Moreover, when Rad18, Rad51, BRCA1, BRCA2, or FancD2 was disabled, Chk1 depletion did not further sensitize the cells to cisplatin. In fact, Chk1 depletion reversed the sensitivity seen when Rad18 was disabled. Collectively, these studies suggest that the pharmacological manipulation of Chk1 may not be an effective strategy to sensitize tumors to platinating agents.

The platinum-based chemotherapy drugs cisplatin, carboplatin, and oxaliplatin are among the most active and widely used agents for the treatment of malignancies, including testicular, head and neck, ovarian, lung, colorectal, and bladder cancers ( Kelland, 2007). It is generally accepted that these agents kill tumor cells primarily by creating DNA lesions, which are most cytotoxic during S-phase, probably because the lesions are potent inhibitors of DNA replication ( Donaldson et al., 1994).

When a replication fork is stalled by either intra- or interstrand cross-links, sophisticated repair and signaling pathways are called into action. In the case of bulky adducts such as intrastrand cross-links (which comprise the majority of platin-induced lesions), the stalled replication fork triggers the monoubiquitylation of proliferating cell nuclear antigen ( Lehmann, 2006). Ubiquitylated proliferating cell nuclear antigen then recruits one or more translesion synthesis (TLS) polymerases, which have active sites that can accommodate bulky lesions, thereby allowing error-prone bypass of the lesion. In contrast, interstrand cross-links, which account for a few percentage of cisplatin-induced DNA lesions but are far more cytotoxic, cannot be simply bypassed. Instead, their repair involves a complex interplay between a series of DNA repair pathways, including the TLS, Fanconi's anemia (FA), and homologous repair (HR) pathways ( Dronkert and Kanaar, 2001). Although the complete mechanistic details of how these pathways accomplish this repair remain unknown, it is clear that defects in these pathways dramatically sensitize cells to agents that cause interstrand cross-links, including the platinating agents ( Dronkert and Kanaar, 2001). It is noteworthy that defects in these pathways are frequently found in tumor cells, raising the possibility that these repair deficiencies contribute to enhanced sensitivity of tumor cells to platinating agents ( Gudmundsdottir and Ashworth, 2006; Lyakhovich and Surralles, 2006; Gossage and Madhusudan, 2007; Miyagawa, 2008).

In addition to triggering repair pathways, stalled replication forks also activate the Rad9-Hus1-Rad1 (9-1-1)-ATR-Chk1 signaling pathway ( Cimprich and Cortez, 2008). The pathway is initiated when the replicative helicase that unwinds the double-stranded DNA continues advancing in front of the stalled DNA polymerase. This creates extensive regions of single-stranded DNA that are coated with the replication protein A complex. The replication protein A-coated single-stranded DNA then triggers the Rad17-mediated loading of the 9-1-1 clamp complex and the binding of the ATM and Rad3-related-ATR-interacting protein complex. The chromatin-bound 9-1-1 clamp, which associates with the ATR activator TopBp1, then triggers ATR activation ( Burrows and Elledge, 2008). Activated ATR phosphorylates multiple substrates that regulate DNA repair and cell cycle arrest, including Chk1, which helps cells survive replication stress by preventing the firing of origins of replication, delaying G ₂ exit, stabilizing the stalled replication forks, and regulating DNA repair ( Cimprich and Cortez, 2008).

Consistent with the multiple roles of the 9-1-1-ATR-Chk1 pathway in regulating cell cycle arrest, DNA repair, and replication fork stability, much work has now shown that the pathway plays a pivotal role in helping cells survive a wide range of genotoxic stresses, including radio- and chemotherapies. These findings have provoked intense interest in pharmacologically targeting this pathway as a means to increase the cytotoxicity of genotoxic cancer therapies, with most of these efforts focused on identifying small-molecule inhibitors of Chk1, the most "druggable" component in the signaling pathway. Consistent with that prediction, recent work has shown that Chk1 inhibitors potentiate the activity of nucleoside analogs and topoisomerase I inhibitors in cell lines and xenografts, and these inhibitors are now in early-stage clinical trials in combination with gemcitabine and irinotecan ( Ashwell and Zabludoff, 2008).

Although platinating agents are among the most widely used chemotherapy agents, little is known about what checkpoint signaling pathways are activated by these agents or how these pathways affect the survival of tumor cells treated with these agents. To that end, we performed a stepwise analysis and examined the role the 9-1-1-ATR-Chk1 pathway in cells treated with platinating agents to gain insight into which aspects of this signaling pathway are important for tumor cell survival and to assess whether Chk1 plays an important role in facilitating tumor cell survival after treatment with platinating agents.

Materials and Methods

Reagents. Cisplatin and carboplatin were from NovaPlus (Novation, Irving, TX). Oxaliplatin was from Sigma-Aldrich (St. Louis, MO). Gemcitabine was obtained from Eli Lilly & Co. (Indianapolis, IN). Antibodies that recognize the indicated proteins were obtained as follows: Chk1 and ATM, from Santa Cruz Biotechnology (Santa Cruz, CA); Rad18 from Novus Biologicals (Littleton, CO); Rad51 from Thermo Fisher Scientific (Waltham, MA); Cdc25A from Neomarkers (Fremont, CA); phospho-Ser ³⁴⁵-Chk1 and BRCA1 from Cell Signaling Technology (Danvers, MA); Rad9 from Volkmer and Karnitz ( 1999); ATR and BRCA2 from Calbiochem (San Diego, CA); FancD2 from GeneTex (Irvine, CA); heat shock protein 90 from David Toft (Mayo Clinic, Rochester, MN); and β-actin from Sigma. The Chk1/Chk2 inhibitor AZD7762 was purchased from Axon Medchem BV (Groningen, The Netherlands).

Cell Culture, siRNA Transfections, Clonogenic Assays, and Drug Treatment. HeLa, HCT-116, and U2OS cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum. Stable clones of Rad9(-/-) mouse embryonic stem (ES) cells ( Hopkins et al., 2004) transfected with empty vector or expressing wild-type Rad9 were derived and cultured as described previously ( Roos-Mattjus et al., 2003). The following siRNAs (synthesized by Thermo Fisher Scientific) were used: luciferase, CUUACGCUGAGUACUUCGA ( Elbashir et al., 2001); ATR, CCUCCGUGAUGUUGCUUGA ( Casper et al., 2002); Chk1, GCGUGCCGUAGACUGUCCA ( Zhao et al., 2002); BRCA1, GUGGGUGUUGGACAGUGUA ( Bartz et al., 2006); BRCA2, GACUCUAGGUCAAGAUUUA ( Bartz et al., 2006); Rad51, CUCCUAGAGGUGAAACCCU ( Lio et al., 2004); ATM, GCACCAGUCCAGUAUUGGC ( Wang and Qin, 2003); Rad9, ACUUGAAUCUUAGCAUUCA; Rad18, GCUCUCUGAUCGUGAUUUA; and FancD2, GGUCAGAGCUGUAUUAUUC. On day 1, siRNA (900 ng) was combined with 12 μl of HiPerFect reagent (Qiagen, Valencia, CA), incubated at room temperature for 5 min, and added to cells in the well for a final siRNA concentration of 30 nM. Transfections were repeated on day 2. On day 3, cells were replated in 100-mm tissue culture dishes. On day 4, cells were trypsinized, used to set up clonogenic assays, and lysed for immunoblotting. Clonogenic assays were performed as described previously ( Karnitz et al., 2005) using 24-h drug treatments. Cell lysis and immunoblotting were performed as described previously ( Volkmer and Karnitz, 1999), and blots were developed with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).

Cell Cycle Analysis. Trypsinized cells were permeabilized with ice-cold 70% ethanol in phosphate-buffered saline, stored at -20°C for 1 h, centrifuged, resuspended in phosphate-buffered saline containing 50 μg/ml propidium iodide and 100 μg/ml RNase, incubated at 30°C for 30 min, and analyzed by flow microfluorometry (FACS-can; BD Biosciences, San Jose, CA).

Results

Cells Lacking Rad9 Are Sensitive to the Antiproliferative Effects of Cisplatin. To begin a stepwise assessment of the role(s) of 9-1-1-ATR-Chk1 pathway in tumor cells treated with cisplatin, initial experiments focused on Rad9, a key participant in DNA repair and Chk1 signaling. Using a previously described model system of mouse Rad9(-/-) ES cells stably transfected to express wild-type Rad9 or transfected with empty vector ( Roos-Mattjus et al., 2003; Hopkins et al., 2004), we assessed the impact of Rad9 status on the ability of these cells to form colonies after a 24-h treatment with graded concentrations of cisplatin. As shown in , cells lacking Rad9 were exceptionally sensitive to the antiproliferative effects of this cross-linking agent.

Rad9 and ATR Depletion Sensitizes HeLa Cells to Cisplatin. To further evaluate the role of Rad9 and ATR in resistance to cisplatin (and to demonstrate that the results of a Rad9 deficiency were not specific to mouse ES cells), we analyzed the effects of depleting Rad9 and ATR from HeLa cells using siRNAs (). Because we had shown previously that Rad9 and ATR play critical roles in helping tumor cells (including HeLa cells) survive treatment with gemcitabine, a nucleoside analog that disrupts DNA replication ( Karnitz et al., 2005), we also treated the cells with gemcitabine. Depletion of either Rad9 or ATR sensitized HeLa cells to cisplatin () and gemcitabine (), thus demonstrating that these checkpoint proteins play critical roles in facilitating the survival of cisplatin-treated tumor cells.

Disrupting Chk1 Signaling Does Not Sensitize HeLa Cells to Platinating Agents. An important target substrate for activated ATR is Chk1, a protein kinase that participates in blocking cell cycle progression and regulating DNA repair after DNA damage or replication stress ( Ashwell and Zabludoff, 2008). Given the central role of Chk1 in ATR signaling and the fact that Chk1 inhibition sensitizes many tumor cell lines to genotoxic chemotherapies, including gemcitabine () ( Karnitz et al., 2005), we asked whether Chk1 depletion () affected HeLa cell clonogenicity after treatment with cisplatin, oxaliplatin, or carboplatin ( Fig. 1, C, E, and F , respectively). It is surprising that even though Chk1 depletion sensitized cells to gemcitabine (), Chk1 depletion did not sensitize HeLa cells to any of the platinating agents.

To further probe the role of Chk1 in cisplatin cytotoxicity, we used AZD7762, a small molecule that inhibits both Chk1 and Chk2 with similar potency ( Zabludoff et al., 2008). Although this agent dramatically sensitized HeLa cells to gemcitabine (), it did not sensitize the cells to cisplatin (). This result suggests that neither Chk1 nor Chk2 plays an important role in helping cells survive cisplatin treatment. Consistent with this finding, codepletion of Chk1 and Chk2 with siRNAs () did not sensitize HeLa cells to cisplatin (). Taken together, these results demonstrate that although ATR is important for tumor cell survival after treatment with platinating agents, Chk1 is not, even when Chk2 is also inhibited.

Cisplatin Activates Chk1. In view of the unexpected finding that Chk1 depletion did not sensitize HeLa cells to platinating agents, we asked whether the DNA damage induced by cisplatin could activate Chk1. HeLa cells were treated with cisplatin concentrations that reduced clonogenicity by 10% (250 nM) and 90% (600 nM), and Chk1 phosphorylation on Ser ³⁴⁵, a site phosphorylated by ATR and required for Chk1 activation ( Liu et al., 2000), was assessed (). In addition, to demonstrate that the phosphorylated Chk1 was relaying signals to downstream targets, we analyzed Cdc25A, a Chk1 substrate that is targeted for proteasomal degradation after Chk1-mediated phosphorylation. Consistent with previous results, cisplatin induced Chk1 phosphorylation under all conditions tested, and there was a corresponding decrease in the levels of Cdc25A (, lanes 2-5). As a control for this experiment, we initially treated cells with concentrations of gemcitabine that also reduced clonogenicity by 10% (30 nM) and 90% (100 nM), but we observed nearly undetectable Chk1 phosphorylation (, lanes 6 and 8, and data not shown); notably, however, a high concentration of gemcitabine (1000 nM) induced robust Chk1 phosphorylation and Cdc25A degradation (, lane 7). Taken together, these results suggest that cisplatin-at isotoxic concentrations-is a better inducer of Chk1 phosphorylation than gemcitabine; however, Chk1 only plays a role in helping cells survive gemcitabine but not cisplatin treatment.

Depleting Chk1 Disrupts the Cisplatin-Induced S-Phase Arrest. A major function of Chk1 after genotoxic stress is to block the origin firing and S-phase progression. To assess whether the Chk1 activated in cisplatin-treated HeLa cells was indeed promoting an S-phase arrest, we examined the cisplatin-induced cell cycle arrest in control and Chk1-depleted HeLa cells. For these assays, cells were treated for 20 h with 1 and 4 μM cisplatin. (These comparatively high concentrations of cisplatin were used because we did not observe robust S-phase accumulation in the control cells at lower concentrations.) Consistent with previously published results, 1 μM cisplatin induced mid-S-phase accumulation in control cells, with the higher concentration of cisplatin causing an early S-phase accumulation (). In contrast, in Chk1-depleted cells, this S-phase arrest was partially disrupted and the cells accumulated in late S phase or G ₂/M. Taken together, these results suggest that Chk1-mediated inhibition of S-phase progression does not play an important role in helping HeLa cells survive cisplatin treatment.

Multiple Tumor Cell Lines Are Not Sensitized to Cisplatin by Chk1 Depletion. To further explore the surprising finding that Chk1 depletion does not sensitize HeLa cells to cisplatin, we examined the effect of depleting Chk1 in additional cell lines. HCT-116 and U2OS cells, which were derived from a colorectal carcinoma and an osteosarcoma, respectively, were selected for these studies because patients with these tumors are often treated with platinating agents. Consistent with the results for HeLa cells (), Chk1 depletion did not sensitize either HCT-116 or U2OS cells to cisplatin, whereas both cells lines were sensitized to gemcitabine (). Likewise, Chk1 depletion did not sensitize HCT-116 cells to oxaliplatin, an agent that is often used to treat colon cancer, or the lung cancer cell line A549 to cisplatin (data not shown). Collectively, these results show that Chk1 does not play a rate-limiting role in preventing the antiproliferative effects of platinating agents in multiple cell types, including cell lines derived from tumors that are routinely treated with these drugs.

Disabling DNA Repair Pathways Does Not Make Cisplatin-Treated Tumor Cells Reliant on Chk1. We reasoned that Chk1 signaling pathways might assume increased importance if the pathways that repair platinum-induced lesions were disabled. Many of the tumors that are treated with cisplatin harbor defects in repair pathways for cisplatin-induced lesions. Thus, if Chk1 depletion sensitized a tumor cell with a defect in a specific repair pathway, then Chk1 inhibitors might be useful to sensitize these tumors to platinating agents. To test this idea, we first depleted HeLa cells of Rad51, BRCA1, Rad18, FancD2, or BRCA2 (Supplemental Fig. 1), all of which participate in the repair of cisplatin-induced lesions. In all cases, knockdown of any single repair protein increased the sensitivity of the cells to cisplatin (). When the effects of simultaneously depleting Chk1 with each individual repair protein were examined, we observed that in no case did codepletion of Chk1 and the repair protein further sensitize the cells to cisplatin. To the contrary, simultaneous depletion of Rad18 or FancD2 with Chk1 rendered cells less sensitive to cisplatin than depletion of Rad18 or FancD2 alone ().

Discussion

In the present study, we examined the role of the 9-1-1-ATR-Chk1 pathway in protecting a series of tumor cell lines from the antiproliferative effects of cisplatin and other platinating agents. Previously published studies, using small-molecule Chk1 inhibitors and RNA interference approaches, demonstrated variable sensitization of some tumor cell lines to platinating agents when Chk1 is disabled ( Gao et al., 2006; Blasina et al., 2008; Ganzinelli et al., 2008). However, none of these studies addressed the role of the entire 9-1-1-ATR-Chk1 pathway, nor did they examine the effects of disabling specific DNA repair pathways in the context of Chk1 inhibition. Our studies demonstrate that cells lacking Rad9 and ATR are exquisitely sensitive to platinating agents. In stark contrast, however, Chk1 depletion did not enhance the antiproliferative effects of cisplatin in multiple cell lines, even though Chk1 was activated and relayed a checkpoint signal that caused Cdc25A degradation and slowed S-phase progression in cisplatin-treated cells. In addition, we showed that depleting key repair proteins, which are part of DNA repair pathways that are frequently disabled in a variety of tumor cells, did not render cells more dependent on Chk1. In fact, in some cases, depleting Chk1 from cells lacking specific repair proteins reversed the sensitivity caused by the deficiency of the repair protein.

Multiple studies have shown that Chk1 depletion and Chk1 inhibitors potently sensitize tumor cells to the damage induced by S-phase-active agents such as gemcitabine, hydroxyurea, or 5-fluorouracil ( Cho et al., 2005; Karnitz et al., 2005; Morgan et al., 2006; Robinson et al., 2006; Matthews et al., 2007; Blasina et al., 2008). During S-phase, Chk1 contributes to cell survival by blocking the firing of unfired origins of replication, preventing cells from exiting G ₂, stabilizing stalled replication forks, and regulating DNA repair ( Enders, 2008). Because the intrastrand and interstrand cross-links caused by cisplatin are also potent inhibitors of DNA replication, we expected that Chk1 would also facilitate tumor cell survival after cisplatin treatment. Surprisingly, however, even though cisplatin provoked robust Chk1 activation and this activation was important in blocking progression through S-phase, Chk1 depletion did not sensitize these tumor cell lines to platinating agents.

Such results strongly suggest that not all stalled replication forks require Chk1 to maintain their stability. Moreover, they also indicate that the Chk1-mediated block of origin firing does not contribute to increased cell survival. One possible explanation is that the Chk1-mediated suppression of origin firing is most important when continued replication would actually create additional DNA damage, such as when additional gemcitabine is incorporated into the genome. In contrast, when the damage is pre-existing, as with cisplatin, additional origin firing would not incorporate further damage into the genome. This latter point is of particular interest because a recent study has shown that the repair of interstrand cross-links is initiated only when two opposing replication forks converge on the lesion ( Räschle et al., 2008), thus raising the possibility that the repair of these lesions might depend on the activation of additional replication origins.

Chk1, in addition to regulating origin firing and replication of fork stability, also positively regulates DNA repair pathways that are important for the repair of interstrand cross-links in at least two ways. First, Chk1 promotes HR, in part by phosphorylating Rad51 ( Sørensen et al., 2005; Huang et al., 2008). Second, Chk1 phosphorylates FancE ( Wang et al., 2007), which stimulates the repair of interstrand cross-links through the FA pathway ( Wang, 2007). Because our results clearly demonstrate that the HR and FA pathways are important in HeLa cells treated with cisplatin, the lack of an effect on cell survival when Chk1 is depleted suggests that Chk1 does not play a major regulatory role in these repair pathways in the cell lines examined.

We also explored the possibility that Chk1 might only become important in cisplatin-treated cells when specific DNA repair pathways were disrupted. This is of particular relevance because tumors often have defective DNA repair pathways, and the defects in these pathways probably contribute to the sensitivity of the tumor to chemotherapy regimens ( García and Benitez, 2008; Powell and Kachnic, 2008). For example, patients with defects in BRCA1 and BRCA2 have better overall responses (including longer survival) to platinum-based therapies ( Foulkes, 2006; Sakai et al., 2008), probably because BRCA1 and BRCA2 play critical roles in repairing the cisplatin-induced damage. If Chk1 was important in such cells, then tumors that harbor these defects might be good candidates for clinical trials that combine cisplatin and a Chk1 inhibitor. We did not observe such an outcome. Instead, we found that Chk1 depletion actually reduced the sensitivity of cells with disabled FA (i.e., FancD2) and TLS (i.e., Rad18) pathways. Not only do these results further suggest that Chk1 inhibitors might not be useful agents to sensitize tumors to platinating agents, they also suggest that the addition of a Chk1 inhibitor to combination therapies containing cisplatin should be undertaken with great caution.

The present findings suggest that Chk1 inhibitors may be of limited use to sensitize tumor cells to platinum-induced damage. In fact, given that Chk1 depletion actually reversed the sensitivity of cells with defects in repair pathways that are often defective in tumors treated with cisplatin, the use of such inhibitors may be counterproductive in some patients. In contrast, because both Rad9 and ATR depletion cause profound sensitization to cisplatin, the identification of small-molecule inhibitors that disrupt this portion of the pathway may be effective agents to sensitize tumors to platinating agents.

Notes

This work was supported by the National Institutes of Health National Cancer Institute [Grant CA084321] and the Mayo Foundation.

ABBREVIATIONS: TLS, translesion synthesis; 9-1-1, Rad9-Hus1-Rad1; FA, Fanconi's anemia; HR, homologous recombination; siRNA, small interfering RNA; AZD7762, 3-(carbamoylamino)-5-(3-fluorophenyl)- N-(3-piperidyl)thiophene-2-carboxamide; ES, embryonic stem.

S⃞The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

References

Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, et al. (2000) Chk1 is an essential kinase that is regulated by ATR and required for the G[[₂/M DNA damage checkpoint. Genes Dev 14 1448-1459.PubMed] PMC free article] [Google Scholar]

Volkmer E and Karnitz LM (1999) Human homologs of [Schizosaccharomyces pombe Rad1, Hus1, and Rad9 form a DNA damage-responsive protein complex. J Biol Chem 274 567-570. [PubMed] Google Scholar]

Zhao H, Watkins JL, and Piwnica-Worms H (2002) Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G[[₂ checkpoints. Proc Natl Acad Sci U S A 99 14795-14800.PubMed] PMC free article] [Google Scholar]

# data[].attributes.attachment_link.title
Cisplatin-Induced DNA Damage Activates Replication Checkpoint Signaling Components that Differentially Affect Tumor Cell Survival

# data[].attributes.attachment_link.description
Cisplatin and other platinating agents are some of the most widely used chemotherapy agents. These drugs exert their antiproliferative effects by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The cross-links mobilize signaling and repair pathways, including the Rad9-Hus1-Rad1-ATR-Chk1 pathway, a pathway that helps tumor cells survive the DNA damage inflicted by many chemotherapy agents.

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PROJECT BASED LEARNING- TASK : The realization that the increase in rRNA expression has an active role in cancer progression, not only through increased protein synthesis and thus proliferative capacity but also through control of cellular check points and chromatin structure, has opened up new therapeutic avenues for the treatment of cancer through direct targeting of Pol I transcription.  As the group of research scientist appointed by  Cancer Research Malaysia, SJMC  you are required to do research  which  drugs can be used to inhibit targeting of Pol I transcription in term as alternative cancer treatment?

OBJECTIVE:
1) Applying the concept of transcription process in eukaryotic
2) How to inhibit transcription process by using drugs

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PROJECT BASED LEARNING- GENETIC ENGINEERING

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PROJECT BASED LEARNING- GENETIC ENGINEERING

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When genes work properly, they tell cells when it is the right time to grow and divide. When cells divide, they make exact copies of themselves. One cell divides into 2 identical cells, then 2 cells divide into 4, and so on. In adults, cells normally grow and divide to make more cells only when the body needs them, such as to replace aging or damaged cells.

Existing cells in our body divides through the process of meiosis and mitosis to produce new cells. The division of the cells would slowly halve the size of the genome until each cell would die if there is no new copies of DNA is made before undergoes cell division process, which probably would not take long. Therefore, S phase of interphase in cell cycle of a cell is important in keeping the cells alive. What is the process involved in copying DNA in S phase?

A cancer cell doesn’t act like a normal cell. It starts to grow and divide out of control instead of dying when it should. They also don’t mature as much as normal cells so they stay immature. Although there are many different types of cancer, they all start because of cells that are growing abnormally and out of control. Cancer can start in any cell in the body.  Cisplatin is one type of drugs used in cancer treatment. Discover how this drugs overcome the growing of cancerous cell.

OBJECTIVE:
1) Applying the concept of replication process in eukaryotic
2) How to inhibit replication process by using drugs

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The link between perturbations in translational control and cancer etiology is becoming a primary focus in cancer research. It has now been established that genetic alterations in several components of the translational apparatus underlie spontaneous cancers as well as an entire class of inherited syndromes known as “ribosomopathies” associated with increased cancer susceptibility.  As the group of research scientist appointed by  Cancer Research Malaysia, SJMC  you are required to do research  how to control translation in cancer Etiology in term as alternative cancer treatment?

OBJECTIVE:
1) Applying the concept of translation process in eukaryotic
2) How to inhibit translation process to Control in Cancer Etiology.

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Assume that you have discovered two new strains of E.coli that has a mutation in the lac operon structures. One type of strain has mutation in operator region and another type in promoter region. Explain how this phenomenon would affect the metabolism of lactose.

The bacterial lacZ gene: an important tool for metastasis research and evaluation of new cancer therapies. As the group of research scientist appointed by  Cancer Research Malaysia, SJMC  you are required to do research how this therapies will help  in term as alternative cancer treatment?

Project objective :
1. Applying the concept of lac operon process in prokaryotic cell
2. How to use LacZ in therapy