Mod 3 Oncogenes

Proto-oncogene conversion to an ongene by a retrovirus

** expression of the proto=oncogene is controlled by retroviral transcriptional promoter 1) Avian Leukosis Virus (ALV) infects a cell. 2) Reverse transcriptase transcribes RNA to DNA 3) Viral dsDNA integrates into the host cell DNA. 4) dsDNA becomes incorporated next to c-src proto-oncogene in an infected chicken cell 5) ALV provirus and adjacent c-src proto-oncogene are co-transcribed into a single hybrid RNA transcript. 6) The hybrid viral RNA is packaged into a virus particle that became the ancestor of RSV. 7) After insertion into the retrovirus genome, the expression of c-src is now controlled by a retroviral transcription of a promoter that activates the c-src proto-oncogene into an oncogene. The cell proliferation ability of an oncogene causes cancer.

Mechanisms leading to oncogene activation

**proto-oncogenes can be activated by genetic changes affecting either protein EXPRESSION or STRUCTURE 1) Chromosomal Translocation: fusing of a region of a chromosome to a second unrelated chromosome 2) Amplification: replication of a limited region of chromosome DNA; observed at metaphase. The dark region are concentrated and called homogeneously staining regions (HSRs) 3) Provirus Integration/Insertion: deregulation of myc. Insertional mutagenesis causes expression of c-myc proto-oncogene to be placed under the transcriptional control of ALV provirus that has integrated into nearby chromosomal DNA; overexpression of c-myc

Huamn Epidermal Growth Factor Receptor 2 (HER2)

- HER2 gene amplification present in 30% of breast cancers SOUTHERN BLOTTING - Southern blotting: molecular biology technique used for the detection of a specific DNA sequence; detects the levels of gene duplication in cancers - Experiment: Southern blotting of 5 human breast carcinomas TAKEAWAYs: 1) gel imaging indicated that some breast canrcinomas carried extra copies of HER2 genes (lanes 1,2,3) 2) some tumors overexpressed the mRNA without gene amplification (lane 5 ) GRAPH: Disease-free survival(y) months after diagnosis(x) - HER2 gene amplification is correlated with a poor prognosis (>5 genes is poor prognosis) - breast cancers with high levels of HER2 are more likely to spread and less likely to respond to treatment, and there is relapse in the first 18 months after dx and tx - targeted treatment abrogates signaling from the HER2 receptor inhibiting tumor growth - targeting HER2 improves prognosis for patients with HER2 overexpressing breast cancer - Pts whose cancers expressed normal levels of this protein showed a median survival of 6-7 years after diagnosis - Pts with elevated levels, had immediate survival of 3 years Conclusion: Data proves that HER2 is involved in malignant growth tx: Targeted tx reduces HER2 signaling and inhibits growth

Gene amplification

-increased number of copies of that gene -increased RNA and protein from that gene - growth in cancer cells caused by the amplified gene - resistance to anti-cancer drugs

Lecture Notes Mod 3: Oncogenes

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A conflict of theories

1) Cancer is caused by the activation of endogenous retroviruses (they become activated cells and multiply by cell to cell infection) 2) Cancer us caused by chemical agents ****Chemical agents cause mutations in normal genes, making them function abnormally (i.e become cellular oncogenes), resulting in cancer formation.

6. What two theories existed about what caused cancer? Which theory was proved to be correct?

1) Cancer is caused by the activation of endogenous retroviruses. 2) Cancer is caused by chemical agents therefore carcinogens function as mutagens. The second theory was proved to be correct because it was difficult to verify infectious retroviral particles in human tumors, and RT-containing virus particles were difficult to pinpoint.

Activating endogenous Retroviruses

1) Transmission of infectious virus particles (inserted into germline) 2) Infection of germ cells (in testes) which then fertilizes an egg to form an infected zygote that carries the provirus 3) activation of cancer in one cells leads to spreading into the offspring

15. Several retrovirus-associated oncogenes have been discovered altered in cancer. List four. [raf, abl, erbB, myc, cbl, akt, ras, myb]

1. Rous sarcoma: src 2. Abelson leukemia: abl 3. Avian erythroblastosis: arbB 4. McDonough feline sarcoma: fms

4. What mutation occurred in human bladder carcinomas that made them come cancerous?

A mutation to the normal human H-ras proto-oncogene led to formation of H-ras oncogene, resulting in human bladder carcinoma. The point mutation occurred in a 350base pair region by a single base substitution in which a G (guanosine nucleotide) in the proto-oncogene was replaced by a T (thymidine) nucleotide in the oncogene. Therefore, the change in nucleotide was able to alter the amino acid coded from glycine to valine in the 12th codon.

Oncogen causing Bladder carcinoma

A mutation to the normal human H-ras proto-oncogene led to formation of H-ras oncogene, resulting in human bladder carcinoma. The point mutation occurred in a 350base pair region by a single base substitution in which a G (guanosine nucleotide) in the proto-oncogene was replaced by a T (thymidine) nucleotide in the oncogene. Therefore, the change in nucleotide was able to alter the amino acid coded from glycine to valine in the 12th codon.

17. Proto-oncogene:

A proto-oncogene is a normal gene that when altered by a mutation it becomes an oncogene that can develop into cancer.

3. What activation mechanisms collaborate to activate oncogenes and how do those mechanisms work?

Both activation mechanisms, regulatory and structural, collaborate to create an oncogene. A structural mechanism can be a point mutation to a codon region that causes a change in nucleotide serious enough to alter the amino acid for that codon. This structural change results in a disruption to the function of the protein. A regulatory mechanism includes that of WT mice exposed to chemical carcinogen that develop lung tumor carrying K-ras oncogenes while Knock-in mice will develop H-ras oncogenes following this treatment; expression of the ras proto-oncogene activated depends on the regulatory sequences. An example of the collaboration of both regulatory and structural aspects is that of avian myelocytomatosis virus that carries the myc oncogene. While there is deregulation of gene expression by viral transcription factor (regulatory), there was enhancement of transformative activity by the alterations (structural) to the reading frame of myc oncogene.

2. What cancer arises from the event in Question 1?

Burkitt lymphoma due to potent oncogene that drives proliferation of lymphoid cells.

2. What experiments were done to demonstrate the role endogenous retroviruses play in cancer?

Experiment 1: 1) Transmission of infectious virus particles (retroviruses) inserted into the germline (i.e sperm). 2) fertilization of the ovum by infected virus leads to a zygote carrying a provirus. 3) proviruses are then transmitted like Mendelian alelles from one generation to the next, activating latent endogenous proviruses which have the ability to encode infectious retrovirus particles that cause mutations leading to cancer. Experiment 2: 1) Avian Leukosis Virus (ALV) infects a cell. 2) Reverse transcriptase transcribes RNA to DNA 3) Viral dsDNA integrates into the host cell DNA. 4) dsDNA becomes incorporated next to c-src proto-oncogene in an infected chicken cell 5) ALV provirus and adjacent c-src proto-oncogene are co-transcribed into a single hybrid RNA transcript. 6) The hybrid viral RNA is packaged into a virus particle that became the ancestor of RSV. 7) After insertion into the retrovirus genome, the expression of c-src is now controlled by a retroviral transcription of a promoter that activates the c-src proto-oncogene into an oncogene. The cell proliferation ability of an oncogene causes cancer. Experiment 3: 1) Culturing of endogenous retroviral (ERV) genome in fibroblasts in the presence of thymidine analog bromodeoxyuridine (BrdU). 2) connective tissue cells (fibroblasts) begin to release retrovirus particles (by endogenous provirueses). 3) latent endogenous proviruses are activated in vivo in a small number of cells in a mouse. 4) Once infectious virus particles are released from these few cells, they can multiple by cell-to-cell infection, spreading and causing leukemia.

13. T/F: Inhibition of the HER2 gene is always associated with increased protein levels.

FALSE (The observed amplification of erbB2/HER2 gene was often but not always correlated with an increased expression of its encoded protein)

11. T/F: Gene amplification always results in greater over-expression of a gene than chromosomal translocation.

FALSE (only 40-60% of the genes that are found to be amplified in cancer cell genomes show corresponding increases in their RNA transcripts (and thus proteins. So there must be negative feedback mechanisms that control too many copies and expression from forming)

4. What makes HER2 an effective cancer target?

HER2 when mutated will lead to increased signaling, indefinite division of cells, and protection from apoptosis. These three factors make it an essential target due to efficient machinery and high survival. What makes this gene unique is that when activated, it will also induce the expression of closely linked genes mapping on both sides of itself. Therefore, the amplification activity of this gene must be made up of a stretch of chromosomal DNA that extends past the HER2 gene region (past both ends). Genes surrounding HER2 gene possess proliferation and survival qualities and interact with apoptosis machinery. Together, these grouped genes will offer a more forceful tactic to generate the cancer phenotype.

What causes cancer?

In the 1970s, DNA and RNA tumor viruses provided cancer biologists with a theory about how human tumors developed. But, the passage of time made it clear that human cancer did not spread from one individual to another as infectious disease Therefore, 1. Some researchers clung to tumor viruses as the cause of cancers and 2. Other researchers started to look at the cellular genome as a potential source of oncogenes This led to the studies of viruses in association w cancer

N-Myc Amplification

Kaplan Meier plots the status of the patient EFS of some other clinical parameter as a function of the time elapsed following the initial diagnosis/treatment Here it illustrates that tumors with minimal N-myc amplification have a very good prognosis while tumors with high N-myc amplification have a poorer prognosis sna d decreased survival times

List some types of Oncogenes

PIK3CA(kinase protein): lung ,ovarian, breast r-myc (transcription factor): leukemia, carcinoma cdk6(transcription factor): gliomas

10. T/F: Non-viral oncogenes exist in chemically transformed cells. TRUE

TRUE

12. T/F: Similar homology exists between transfected oncogenes and retroviral oncogenes.

TRUE

9. T/F: Activating an endogenous retrovirus can cause cancer.

TRUE

14. T/F: Chromosomal translocations that involve protein encoding genes affect cell behavior.

TRUE (both chromosomal translocation of 1) protein encoding and 2) nonprotein encoding genes affect cell behavior.)

21. What is the hybrid protein that results from from the fusion of chromosomes 9 and 22?

The Bcr-abl hybrid gene (Philadelphia chromosome), a truncated gene, results from the reciprocal translocation between chromosomes 9 and 22. Moreover, this translocation results in the fusion of two protein-coding sequences, which is associated to Chronic Myelogenous Leukemia.

BCR-ABL: Chromosomal translocation

The Bcr-abl hybrid gene (Philadelphia chromosome), a truncated gene, results from the reciprocal translocation between chromosomes 9 and 22. Moreover, this translocation results in the fusion of two protein-coding sequences, which is associated to Chronic Myelogenous Leukemia. - Novel hybrid proteins account for >95% of cases of chronic mylegenous leukemia - the abl gene was originally discovered as acquired oncogene in Abelson murine leukemia virus, a rapidly tumorigenic retrovirus - diffusion of Abl with the with the breakup cluster region (bcr) results in deregulation of normal abl protein causing it to emit promoting signals

1. You've read about the two conflicting theories about how cancer arises. Which theory was proven to be correct and why?

The first theory is that cancer is caused by the activation of endogenous retroviruses. The second theory is that cancer is caused by chemical agents therefore carcinogens function as mutagens. To prove the first theory, there needed to be verification reports of infectious retroviral particles in human tumors, which was not met. Moreover, it was difficult to pinpoint human tumors with revealed reverse transcriptase-containing virus particles. What was concluded is that the endogenous retroviral genomes that were found in human genomes were actually the demarcations of ancient germ-line infections, which was too long ago to give any indication whatsoever on infectious retroviruses. In fact, a very minimal insignificant amount of the human genome is made retrovirus-derived segments. This raises collective consensus on supporting the second theory as the origin of cancer.

1.Name the process pictured in the image and describe how it occurs.

The process is a reciprocal chromosomal translocation occurring between two unrelated chromosomes( 8 and 14), which is the most common. The myc proto-oncogene is found in the region on human chromosome 8, which is associated with three translocations involving chromosomes 2,14,22.While myc was on one side, the other side were the transcription-promoting sequences from any one of three distinct immunoglobulin (antibody) genes. Specifically on Chromosome 14 shown in the image, there is a heavy chain cluster (IgH). During translocation, part of the antibody gene is fused together with the myc proto-oncogene. Importantly, the c-myc is removed from its normal transcriptional promoter (in chrmx 8) and moved below IgH (in chrmx 14) which means it is under the control of the transcription controlling enhancer sequences of immunoglobulin heavy chain genes. Once it its expression is subjected by the antibody gene promoters, the result is overexpression of c-myc, a potent oncogene. Therefore, there is proliferation of lymphoid cells in which these transcriptional promoters are highly active.

C-MYC: Cancer from chromosomal translocation

The process is a reciprocal chromosomal translocation occurring between two unrelated chromosomes( 8 and 14), which is the most common. The myc proto-oncogene is found in the region on human chromosome 8, which is associated with three translocations involving chromosomes 2,14,22.While myc was on one side, the other side were the transcription-promoting sequences from any one of three distinct immunoglobulin (antibody) genes. Specifically on Chromosome 14 shown in the image, there is a heavy chain cluster (IgH). During translocation, part of the antibody gene is fused together with the myc proto-oncogene. Importantly, the c-myc is removed from its normal transcriptional promoter (in chrmx 8) and moved below IgH (in chrmx 14) which means it is under the control of the transcription controlling enhancer sequences of immunoglobulin heavy chain genes. Once it its expression is subjected by the antibody gene promoters, the result is overexpression of c-myc, a potent oncogene. Therefore, there is proliferation of lymphoid cells in which these transcriptional promoters are highly active. C-myc expression leads to non-Hodgkins lymphoma (affected B cells); fastest growing tumor Burkitt Lymphoma: associated with impaired immunity and is fatal if left untreated

Oncogene Activation: Myc and HER2

These genes are normal until they suffer some change Myc -activated by multiple mechanisms - proteins of the myc family possess potent growth-promoting capacity -plays a role in cell cycle progression, apoptosis, and cellular transformation - the copy # of this gene is elevated (more than 2x copies) - Translocation involving this gene are associated with Burkitt Lymphoma and multiple myeloma in human patients - in some human tumors, myc gene expression is driven by its own natural transcription or promoter - Types: C-myc, N-myc, L-myc HER2 - a member of the Epidermal Growth Factor (EGF) receptor family of receptor tyrosine kinases - Normally, HER2 receptors help control how healthy breast cells. - Amplification of gene results in transformation - Amplifications involving this gene are associated with breast and ovarian tumors.

Nonviral oncogenes and chemically transformed cells (via transfection)

Transfection: [Gene transfer procedure enabling the introduction of DNA into mammalian cells]; Transfection is the process of gene transfer into cells or animals to induce them with a specific reaction (i.e disease, tumor growth). This procedure can be used to detect oncogenes in the DNA of cancer cells. The steps include: 1) DNA is extracted from cancer cells on petri dish. 2) calcium ions are added via transfection using calcium phosphate co-precipitation procedure. 3) crystals that form are added to a monolayer of cells. 4) Transformed cells proliferate, and form a clump of cells (focus). 5) Injection or morphologically transformed cells into mouse host. 6) Tumor formation. Conclusion: CaPO4 was found to facilitate the uptake of DNA fragments by cells thus demonstrating that if a transforming gene or oncogene was present in donor DNA then it became incorporated into the genome of one of the recipient cells and could transform that recipient cell

5. You can experimentally fuse the myc gene with the one for the estrogen receptor. What happens to the cells with this fused gene when estrogen is present versus when it is absent?

When the myc gene is fused with the one for estrogen receptor, there is a likelihood that any time that estrogen is present, the myc gene will be transcribed and there will be expression of the gene. This occurs because estrogen will bind the estrogen receptor that is neighboring the myc gene, which induces transcription factors associated with activating myc gene or simply the promoter of the estrogen receptor will transcribe the fused myc gene on its way. It is the close physical association of the receptor and myc gene creates a functional link between these two genetic elements. The normal mechanisms that control myc gene expression have been overtaken by the estrogen receptor. In women, there are higher levels of estrogen naturally or by estrogen supplements. As a result, myc gene function will be enhanced such that it causes cell cycle progression, cellular transformation, and apoptosis. In fact, estrogen-induced myc expression may develop into breast cancer (Wang). In contrast, absence of estrogen will not trigger expression of myc gene because there will be no estrogen available to bind the adjacent estrogen receptor. Wang, Chunyu et al. "Estrogen induces c-myc gene expression via an upstream enhancer activated by the estrogen receptor and the AP-1 transcription factor." Molecular endocrinology (Baltimore, Md.) vol. 25,9 (2011): 1527-38. doi:10.1210/me.2011-1037

5. Which of the methods below is not a method of activating the myc proto-oncogene?

Yes: Chromosomal translocation, Gene amplification, Proviral Insertion No: DNA retroviral transduction

18. Gene amplification:

[Duplication of genes, such as those for transcription factors, that can cause cancer]; occurs when there is a mutation in a gene that causes an increased number of copies of that gene. There is also increased RNA and protein from that gene, growth in cancer cells caused by the amplified gene, and resistance to anti-cancer drugs.

19. Transfection:

[Gene transfer procedure enabling the introduction of DNA into mammalian cells]; Transfection is the process of gene transfer into cells or animals to induce them with a specific reaction (i.e disease, tumor growth). This procedure can be used to detect oncogenes in the DNA of cancer cells. The steps include: 1) DNA is extracted from cancer cells on petri dish. 2) calcium ions are added via transfection using calcium phosphate co-precipitation procedure. 3) crystals that form are added to a monolayer of cells. 4) Transformed cells proliferate, and form a clump of cells (focus). 5) Injection or morphologically transformed cells into mouse host. 6) Tumor formation.

16. What are two viruses that are implicated in human cancer causation?

[HPV, HCV, HBV, HTLV, HIV, HHV8, EBV] 1. Human Papilloma Virus (HPV): cervical cancer 2. Epstein Barr Virus (EBV): nasopharyngeal cancer

3. How many genes were necessary in the NIH 3T3 experiment to create an oncoprotein?

[Only a single gene was statistically likely to have developed an oncoprotein.] The NIH 3T3 cell line cells, derived from mouse embryo fibroblasts, were especially useful for taking up and integrating into their genomes the foreign DNA by transfection. DNA was taken from several such 3-MC transformed mouse cell lines. After several weeks of transfection into NIH 3T3 cell, these led to foci, resulting in anchorage-independence and tumorigenecity. Additionally, exposure of 3-MC carcinogen converted a previously normal C3H10T1/2 gene(s) into a mutant allele that now could function as a tranforming oncogene when introduced into NIH 3T3 cells. Researchers investigated if a single or many oncognes transformed recipient cells. They discovered that only anout 0.1% of a cell's genome's worth of donor DNA became established in the genome of each transfected recipient cell. Therefore, only a single gene was responsible for the transformation of NIH 3T3 cells after transfection of donor tumor cell DNA. It was determined that exposure of normal C3H10T1/2 mouse cells to the 3-MC carcinogen years earlier had caused the formation of a single mutant oncogenic allele, which could transform C3H10T1/2 cells and also the recipient NIH 3T3 cells.

20. Exon:

[Portion of the gene dedicated to encoding amino acid sequence]; An exon is a region of RNA transcript that is retained after splicing (process to remove noncoding intron regions) occurs. Human genomic composition is mostly introns and very little coding exon region.

7. Describe the experiments performed to investigate whether activating an endogenous retrovirus cause cancer.

[Retroviruses can be inserted into the germ-line resulting in the production of a sperm and then a zygote carrying a provirus. An animal carrying the provirus in all of its cells can pass the provirus onto its descendants, resulting in the transcriptional activation of the virus in one cell which infects all of the cells throughout the body.] Experiment 1: 1) Transmission of infectious virus particles (retroviruses) inserted into the germline (i.e sperm). 2) fertilization of the ovum by infected virus leads to a zygote carrying a provirus. 3) proviruses are then transmitted like Mendelian alelles from one generation to the next, activating latent endogenous proviruses which have the ability to encode infectious retrovirus particles that cause mutations leading to cancer. Experiment 2: 1) Avian Leukosis Virus (ALV) infects a cell. 2) Reverse transcriptase transcribes RNA to DNA 3) Viral dsDNA integrates into the host cell DNA. 4) dsDNA becomes incorporated next to c-src proto-oncogene in an infected chicken cell 5) ALV provirus and adjacent c-src proto-oncogene are co-transcribed into a single hybrid RNA transcript. 6) The hybrid viral RNA is packaged into a virus particle that became the ancestor of RSV. 7) After insertion into the retrovirus genome, the expression of c-src is now controlled by a retroviral transcription of a promoter that activates the c-src proto-oncogene into an oncogene. The cell proliferation ability of an oncogene causes cancer. Experiment 3: 1) Culturing of endogenous retroviral (ERV) genome in fibroblasts in the presence of thymidine analog bromodeoxyuridine (BrdU). 2) connective tissue cells (fibroblasts) begin to release retrovirus particles (by endogenous provirueses). 3) latent endogenous proviruses are activated in vivo in a small number of cells in a mouse. 4) Once infectious virus particles are released from these few cells, they can multiple by cell-to-cell infection, spreading and causing leukemia.

8. What were the conclusions about homology from the experiments investigating whether the same group of proto-oncogenes become activated into oncogenes by infectious as well as non-infectious agent?

[There is homology between transfected oncogenes and retroviral oncogenes, which indicates that the same group of proto-oncogenes activated by both infectious and non-infectious agents are the same.] In other words, this is investigating whether there was an association between retrovirus-associated / retroviral oncogenes (infectious) and transfected oncogenes by tumor cell DNA (non-infectious), which both result in cancer phenotype. A southern blot test was done on mouse embryo fibroblast cells. This test detects DNA concentrations/sizes on a gel. A DNA probe made from the H-ras oncogene in Harvery rat sarcoma virus binded to oncogene detected by transfection in the DNA of a human bladder carcinoma cell. This formed a hybrid that was visualized in the darker bands on Southern imaging ( lanes a and k share similar bands to tumor cell DNA transfected oncogenes( G, H, I, J) with a same molecular weight of 6.6kb). In conclusion, there is homology between the transfected oncogenes and the retroviral oncogenes since H-ras oncogene was able to form hybrids with the fragments from transfected cells.

Homology between transfected oncogenes and retroviral oncogenes

[There is homology between transfected oncogenes and retroviral oncogenes, which indicates that the same group of proto-oncogenes activated by both infectious and non-infectious agents are the same.] In other words, this is investigating whether there was an association between retrovirus-associated / retroviral oncogenes (infectious) and transfected oncogenes by tumor cell DNA (non-infectious), which both result in cancer phenotype. A southern blot test was done on mouse embryo fibroblast cells. This test detects DNA concentrations/sizes on a gel. A DNA probe made from the H-ras oncogene in Harvery rat sarcoma virus binded to oncogene detected by transfection in the DNA of a human bladder carcinoma cell. This formed a hybrid that was visualized in the darker bands on Southern imaging ( lanes a and k share similar bands to tumor cell DNA transfected oncogenes( G, H, I, J) with a same molecular weight of 6.6kb). In conclusion, there is homology between the transfected oncogenes and the retroviral oncogenes since H-ras oncogene was able to form hybrids with the fragments from transfected cells.

Oncogenes vs proto-oncogenes

proto-oncogenes: a normal gene which when altered by mutation becomes an oncogene that can contribute to cancer Oncogenes: overactive growth-promoting genes; transforms a normal cell into a cancer