Lecture 2 - Oncogenes

Where were oncogenes first identified?

Inside retroviruses.

• Found they have transforming properties due to an extra gene (oncogene)

Why did the discovery of viral oncogenes (v-onc) lead to the discovery of cellular oncogenes (c-onc)?

Researchers found that the genes present inside retroviruses were extremely similar structurally to mammalian genes.

• This allowed they to conclude that the mammalian genome contains oncogenes

What were some of the experiments done to discover cellular oncogenes?

• Analysis of transduced cellular gene sequences in acutely transforming retroviruses

• Identification of preferred integration sites of retroviruses

• Characterisation of chromosomal translocations

• Characterisation of amplified DNA sequences

Which cell line was typically used for detection of oncogene’s functon?

NIH 3T3 cells.

• Immortalised fibroblast cell line

What is special about NIH 3T3 cells?

They are immortal but are not cancerous.

• Allows oncogenes to be transduced in to see the effects. 

Using NIH 3T3 cells, what were oncogenes found to cause?

Oncogenes cause:

• Increased growth rate

• Reduced serum requirement 

• Cells lift off plate and grow in solution 

  • Loss of anchorage-dependence 

• Foci forming (multi-layering) due to loss of contact inhibition. 

• Tumour formation in nude mice

What is special about ‘nude’ mice models?

• Lack a thymus due to a mutation in FOXN1

  • No T-cell production so can’t mount a response against foreign cells 

  • Allows tumours to be grown without the immune system fighting it

What is Foci forming?

Cells grow in multiple layers on the plate

• Due to loss of contact inhibition 

Quantitative activation of oncogenes means what?

Increase in production of an unaltered product 

Qualitative activation of oncogenes means what?

Production of modified or novel product 

Oncogene activation needs to affect both alleles for there to be an effect.

• Mutation of both copies of gene

True or False.

False.

Activation only needs to affect one allele

• A mutation only needs to affect one copy of the gene

What are the three main mechanisms of oncogene activation?

• Point mutations

  • Hyperactive protein made in normal amounts

• Amplification

  • Normal protein greatly overproduced 

• Translocation/rearrangement 

  • Nearby strong enhancer causes normal protein to be overproduced

  • Fusion with an actively transcribed gene

What is an example of a point mutation in cancer?

RAS oncogenes

How many RAS oncogenes are there? What are they called?

Three genes

• HRAS - Found in mice Harvey sarcoma virus

• KRAS - Found in mice Kiersten sarcoma virus

• NRAS - Found in Neuroblastoma (not virus)

What role do the RAS oncogenes play?

Mediate G-protein coupled receptor signalling

• RAS oncogene found in common tumour type

  • Colon, lung, breast, etc

What is the function of wild-type RAS?

• Ligand binds to receptor

• Causes GTP to bind to RAS which induces signal transmission

• RAS has GTPase activity and converts GTP to GDP

• RAS then becomes inactive and signal transmission stops

How does a point mutation in RAS alter its function?

• Ligand binds to receptor

• Causes GTP to bind to RAS which induces signal transmission

• RAS loses its GTPase activity so GTP is not converted to GDP

• RAS doesn’t become inactivated and signal transmission doesn’t stop

What does the RAS oncogene cause?

• Increased proliferation

• Expression of anti-apoptotic genes

• Increased metastatic gene expression 

What is gene amplification?

When one structurally normal gene is copies 10s-100s of times.

• This causes the overexpression of the protein it encodes

What are homogenously staining regions in gene amplification?

Insertions of the oncogene within the normal chromosome.

• Represent hundreds of copies of amplified DNA

What are double minutes (DMs) in gene amplification?

Small paired chromatin bodies which are separate from the chromosomes

• These can contain many copies of the oncogene

What is an amplicon?

A segment of genetic material which is the product of gene amplification

May contain multiple genes 

Following gene amplification, homogenously staining regions can contain multiple genes derived from different chromosomes. 

True or False?

False.

This can only occur in double minutes (DMs)

How can chromosomal translocation result in novel chimeric gene formation?

Following chromosomal translocation fusion of an oncogene and another gene can occur

• This creates a chimeric gene which escapes normal regulation and has abnormal transforming properties 

What is an example of a chimeric gene formed from chromosomal translocation?

BCR-ABL

Which chromosomes are ABL and BCR normally found on?

ABL found on chromosome 9

BCR found on chromosome 22

What kind of protein is BCR-ABL fusion protein?

Novel tyrosine kinase protein

How does BCR-ABL form?

Reciprocal translocation between chromosomes 9 and 22

• They break and re-join together

• 5’ region of ABL and 3’ region of BCR join 

What is the name of the chromosome which is created following reciprocal translocation between chromosomes 9 and 22?

‘Philadelphia’ chromosome

• This contains the novel BCR-ABL fusion protein

STI-571 (Imatinib) can be used to treat what tumorigenic fusion protein?

BCR-ABL

• Binds to BCR-ABL and prevents ATP binding

• This prevents the negative effects of BCR-ABL tyrosine kinase activity

What is a key gene activation which occurs in Burkitt’s Lymphoma?

MYC activation

• Translocation of MYC to an immunoglobulin locus

How does MYC become overexpressed in Burkitt’s Lymphoma?

MYC is placed close to an immunoglobulin locus due to chromosomal translocation. 

• The immunoglobulin locus is highly expressed in antibody-producing B-cells which results in MYC overexpression 

In Burkitt’s Lymphoma, exon1 of the MYC gene is often lost. Why is this important?

Exon1 is thought to play a role in transcriptional regulation of MYC and in mRNA stability.

• Loss of exon1 can result in increased expression of MYC and an extended half-life of MYC mRNA

Overexpression of MYC in antibody-producing B-cells is likely caused by what process?

Likely caused by errors in immunoglobulin V-D-J gene rearrangements.