Lecture 2 - cellular oncogenes

Question 1

Give 5 examples of viruses associated with human cancers and encode viral oncoproteins that manipulate DNA repair and cell cycle pathways as part of their oncogenic effects?

Answer 1

Epstein-Barr Virus (EBV), Human Papillomavirus (HPV), Human T-cell leukemia virus (HTLV-1), Kaposi-sarcoma herpesvirus (KSHV), Hepatitus C virus (HCV)

Question 2

Do oncogenic viruses develop cancers in patients immediately after infection

Answer 2

No, only a small fraction of individuals who harbour these viruses develop cancer, and this usually does not occur until 20 - 40 years after infection.

Question 3

Are Simian virus 40 (SV40) and Adenovirus (Ad) capable of causing human cancers?

Answer 3

No, but they are able to immortalise and transform cells in culture via the same mechanisms employed by other oncogenic viruses. They are therefore a simple and powerful model system for the molecular mechanisms that govern transformation by oncogenic viruses.

Question 4

How was RSV discovered

Answer 4

Discovered by Peyton Rous

He removed sarcomas from chicken breast muscle, broke the sarcoma up in sand and passed the resultant filter through a fine pour filter. The filtrate was then injected into the wing of a young chicken.

This led to the development of a sarcoma weeks later. This process was repeated and the next chicken also developed a sarcoma.

Question 5

What was the importance of the fine pore filtering used in the protocol for inducing sarcomas in chickens

Answer 5

Made sure that the resultant filtrate had no bacteria present in it, and the sarcoma that developed was due to a viral oncogene.

Question 6

Is the interaction of RSV with host cells virulent or temperate?

Answer 6

Temperate - The host cell survives for extended periods, all the while harbouring the viral genome and releasing the progeny virus particles to surrounding cells.

Question 7

What did Harry Rubin discover when stocks of RSV were added into petri dishes carrying cultures of chicken embryo fibroblasts.

Answer 7

RSV infected cells survived, seemingly indefinitely. Suggested that RSV infects cells, forcing them to produce a steady stream of viral progeny. (Temperate relationship).

Question 8

What characteristics did RSV infected fibroblasts display?

Answer 8

Displayed similar traits of cancerous cells.

1. Altered morphology - increased thickness of cell layer, loss of contact inhibition (the cells grow over one another) and a more rounded morphology.
2. Anchorage independent growth - Unlike most cells, RSV infected fibroblasts grow and aggregate together when put into a semisolid medium.

Question 9

What are the uses of SCID mice

Answer 9

Lack a thymus so produce few immune cells. Capable of accepting a human derived xenograph. If cells injected develop tumuors its a good indicator of the tumorigenic capacity of the cells. Can also see if treatments are capable of reducing the effects of cancer in these models.

Question 10

What is the structure of the virion of RSV

Answer 10

Three major types of viral protein:

1. The glycoprotein spikes encoded by the viral env gene. These aid in the adsorption of the virion to the surface of a cell.
2. A complex internal protein coat formed by the several core proteins encoded by the viral gag gene.
3. Within the protein shell are two identical copies of the viral genomic RNA and a number of reverse transcriptase molecules specified by the viral pol gene.

Question 11

How was it shown that RSV transforming gene was required to both initiate and maintain the transformed phenotype of virus infected cells.

Answer 11

By creating a temperature sensitive mutant version of RSV. RSV ceased to function at 41 degrees but did at 37. Cells were infected with RSV and lest at the permissive temp leading to the formation of foci.

When the temperature was shifted up to the restrictive temp cells were reverted to their original morphology. This shows that the genome of RSV persisted in the infected cells long after infection as the temp sensitive proteins were continueing to be made.

Question 12

What cancer is EBV virus associated with

Answer 12

Lymphoma and nasopharyngeal cancers.

Question 13

What cancer is HCMV associated with

Answer 13

Malignant glioma, colon and prostate cancer

Question 14

How was SV40 investigated to discover that viran genomes can integrate into host cell genomes?

Answer 14

Viral DNA in SV40-transformed cells is tightly associated with the chromosomal DNA. SV40 DNA co-sedimented following centrifugation with the high molecular weight chromosomal DNA of the host cell. SV40 DNA had become covalently linked to host cell DNA. This integration means transmission of viral DNA from mother cells to their progeny is guarunteed. (Viral DNA gets replicated during S phase)

Question 15

Which cancer has the presence of HPV genomes integrated into the host cells DNA? And was the entire viral genome present?

Answer 15

Cervical carcinomas (almost 99.7% of tumours contain elements of the viral DNA)

Only part of the viral DNA that persisted in host cells was the oncogenic element - portions that enable the virus to replicate and construct progeny viruses was almost always absent of fragmented.

Question 16

Outline the life cycle of an RNA tumour virus like RSV

Answer 16

• Infecting virion introduces ssRNA genome into the cytoplasm of a cell together with the enzyme reverse transcriptase.
• RT makes a dsDNA copy of the viral RNA
• RTDNA moves into the nucleus where it becomes integrated into the host cellular chromosomes.
• The integrated provirus is transcribed into ssRNA by RNA polymerase of the host cell.
• Progeny RNA molecules are exported into the cytoplasm where they serve as mRNAs to make viral proteins or are packaged into progeny virus particles that leave the cell and initiate a new round of infection.

Question 17

Why was the theory that chemicals and radiation activate endogenous retroviruses and subsequent transformation of cells flawed?

Answer 17

If it were correct, we would expect to see clusters of outbreaks and we would be able to isolate virus particles from human tumour cells, neither of which are seen.

Question 18

What is mutagenesis and what are they a common factor in

Answer 18

Common factor in chemical, viral and physical causes of cancer. Cpable of inducing mutations into a cell and inducing genomic instability.

Question 19

What happens as a result of adding benzopyrene to a plate of cells.

Answer 19

It causes mutations, resulting in aberrant growth programs in cells, which can be seen as the development of foci.

Question 20

How was 3 methylcholanthrene used to investigate mutagenesis.

Answer 20

It was added to cells that a free from tumour virsus and cells and the DNA was extracted out and transfected into a host cell. The host cells are incubated in a dish and changes in morphology are looked out for. If transformation occurs this is soley due to the exposure to a mutagen. This experiment showed it was unlikely that more than 0.1% of the genome was taken up per cell and that more than 1 mutated gene was transfected into each cell. This suggested that we might only need one genetic mutation to cause transformation of cells.

Question 21

Outline the process of trasfection using calcium phosphate

Answer 21

DNA is extracted from cancerous cells (which dont posess any viral oncogenes)

DNA is then introduced into a phosphate buffer. When calcium ions are added, a co-precipitate of DNA and calcium phosphate crystals is formed. Phosphate crystals facilitate the uptake of DNA fragments by cells. If an oncogene is present in the donor DNA, it may become incorporated into the genome of recipient cells. This transformed cell will now proliferate and form clumps of cells.
Injection of these cells into a host mouse model and results in tumour formation which confirms the trasformed state of the transfected cells.

Question 22

How was c-Ras discovered?

Answer 22

DNA extracted out of cells treated with mutagens and attached a bacterial gene essential for bacterial cell growth. Every fragment of DNA from the cells has a bacterial gene attached.
Modified DNA was then added back into normal fibroblasts. Cells that form a focus have their DNA removed and introduced into bacteria that lack the essential bacterial gene.
Only the bacteria that live will contain the transfected DNA
The growing bacteria are taken and the process is repeated, eventually purifying to the one strand with the oncogene.

Question 23

What technique was used to discover c-Ras

Answer 23

Southern blotting - looks at the expression of genes withing cells. Southern blotting determines the relatedness of DNA sequences

Question 24

How was southern blotting used to discover c-Ras

Answer 24

The DNA probe they used was known and identified from RSV. The DNA sequence of a gene identified from RSV was pretty much identical to a gene from human cells that had been mutated with a chemical.

The gene is oncogenic ras and one single mutation from G TO T is responsible for forming the oncogene.

Question 25

How do structural changes (particularly in RTKs) create oncogenes

Answer 25

RTKs and other receptors have an intraceullar signalling domain that is only active when the extraceullular domain is bound by a growth factor and inhibits the inhibition the extracellular domain exhibits on the intracellular. If the extracellular domain of the receptor is lost then the receptor signals intracllularly even in the absence of its ligand (usually a growth factor)

Question 26

How does N-myc expression change in childhood neuroblastomas? And is this due to translocation of the gene.

Answer 26

The gene is still driven by its own natural promoter.

But the copy number of the gene is found to be elevated to levels many times more than the two copies normally present in the human genome. both c-myc and N-myc (a close relitive) have increased number of gene copies. Resulting in a larger amount of gene product (myc and N-myc protein) which possess potent growth promoting powers.

Question 27

How does gene amplification of c/N-myc occur

Answer 27

Preferential replication of a number of a limited region of chromosomal DNA. Leaving more distantly located chromosomal DNA unaffected. The amplicon usually contains DNA far greater than the gene encoding c-myc. Therefore the amlified chromosomal regions can often be seen during metaphase through a light microscope.

Question 28

How is Burkitts lymphoma caused

Answer 28

Translocation of the c-myc gene to foreign transcriptional promoters.
The enzyme responsible for rearranging the antibody gene sequences during the development of the immune system occassionally loses specificity and instead of creating a rearranged antibody gene - inadvertantly fuses part of the antibody gene with the c-myc proto-oncogene. IgH expression is constitutively high creating a large amount of c-myc expression.

Question 29

Which specific chromosome translocation is seen for burkitt’s lymphoma?

Answer 29

IgH is on chromosome 14

c-myc is on chromosome 8

c-myc undergoes reciprocal translocation downstream of IgH promoter on Chr. 14

Question 30

How is fluorescent in situ hybridisation (FISH) used to probe for amplification changes

Answer 30

DNA probe unique to a DNA sequence of a particualr gene used. The fluorescently tagged label reveals the location of the gene within a cell.

There should normally only be one or two copies of a gene. However when amplification of a gene occurs (like in c/N-myc) you can visualise many more copies of the gene.

Question 31

How is data analysis of the nature of the tumour used to predict outcomes

Answer 31

Expression of proto-oncogenes can be amplified or not amplified. In the case of HER-2 (breast cancer) when HER-2 is amplified there is a much lower survival rate compared to patients with low HER-2 amplification.