L12 - Oncogenes Flashcards
What is the suspected cause of human cancers?
Tumour viruses
In reality only a minority of tumours are virally-induced
What are the characteristics of viral genomes?
Very simple – only a few genes
Enough to drive tumour phenotype
Potent genes to perturb complex regulatory circuitry of the host cell they infect
What experiment did Peyton Rous undertake?
- Chicken with sarcoma in breast muscle
- Removed sarcoma and broke up tissue into small chunks
- Ground up sarcoma with sand
- Collected filtrate that passed through fine pore filter
- Injected filtrate into young chicken and observe sarcoma formation
What did Peyton Rous experiment show?
Showed the carcinogenic agent was very small as it could pass through filter
RSV - Rous Sarcoma Virus
- Caused sarcoma formation in injected chicken – tumour always appeared after a set
What provided the opportunity to induce cancer?
RSV capable of multiplying within chicken tissues virus can be recovered in big amounts
How can RSV transform infected cells in culture?
Chicken embryo fibroblasts infected with RSV show traits associated with cancer cells
The cells showed cell transformation - conversion of a normal cell into cancer cell
Showed cancer was a disease of malfunctioning cells
What traits do chicken embryo fibroblasts show when infected with RSV?
Foci (clusters) appear after infection
Similar metabolism to cells isolated from tumours
What are the two explanations of why the transformation phenotype is transmitted from infected cells to descendants?
RVS particles transformed the progenitor cells and their descendants
- Virus must be present all the time to maintain transformed phenotype
RVS only needed to transform progenitors, which then transmit phenotype to descendants
- Virus only needs to be present at the beginning
What experiment was undertaken to decide which explanation for how the transformation phenotype is transmitted from infected cells to descendants was correct?
Temperature sensitive RSV mutant - function at permissive temperature
Transformed state lost when cells kept at non-permissive temperature
Transformed state regained when permissive temperature reached
- Continued actions of viral temperature sensitive protein required to maintain transformed phenotype
What were the conclusions from the temperature sensitive RSV mutant experiments?
Viral transforming gene required to both initiate and maintain transformed phenotype
What are the properties of transformed cells?
Altered morphology - rounded shape
Loss of contact inhibition – can grow over one another
Ability to grow without attachment - anchorage independence
Ability to proliferate indefinitely – immortalisation
Reduced requirement for mitogenic growth factors
High saturation density in culture dish
Inability to halt proliferation in the absence of growth factors
Increased uptake of glucose
Tumorigenicity
How was it worked out which gene was responsible for RSV induced transformation?
RSV viral constituents – gag, pol, env and unknown
When either one of the genes removed the virus was
- Unable to replicate
- Able to transform cells
When the unknown gene was removed the virus
- Able to replicate
- Unable to transform cells
Shows a single gene (unknown) is required for transformation (not for viral replication)
- Unknown – src
What are the RSV viral constituents?
gag, pol, env and src
What was used to understand origins, functions and follow src in infected cells?
Src specific DNA probe
What are the two types of src?
C-src – cellular src - present in genome of normal organisms, acts as a proto-oncogene
- Genomes carry a agene that has the potential, under certain circumstances to induce cell transformations and thus cancer
V-src - viral src - acts as an oncogene
What are the two hypothesis for the causes of cancer?
Activation of endogenous retrovirus
Induced by mutagens – physical or chemical agents that can mutate growth controlling genes
Do non-viral oncogenes exist in chemically transformed cells?
Chemically transformed cells carry mutated genes –> responsible for aberrant growth of these cells
Donor tumour DNA carried one or several genetic elements able to convert a normal cell into a tumorigenic one
How do you identify the mutant genes responsible for aberrant growth of transformed cells?
Transfection
- Chemically transformed mouse fibroblasts
- DNA transfection using calcium phosphate co-precipitation procedure
- Injected into normal mouse fibroblasts
- Formation of a focus of transformed cells
- Injection of transformed cells into mouse host leading to tumour
Does fibroblast transformation involve one or more genes?
Only 0.1% of donor mouse DNA became established in recipient cell genome
Low probability of 2 independent genes meaning only 1 gene responsible for transformation
- Similar experiments done with human tumour cells
Oncogenes detected by transfection were derived from?
Pre-existing normal cellular genes lacking oncogenic function
- Were converted by the chemical transformation
- Same scenario that happens with viral oncogenes
Could the same group of proto-oncogenes be activated by viruses or mutagens?
Yes
DNA probes for retroviral associated oncogenes used on chemically transformed cells
- Many proto-oncogenes found in activated, oncogenic form in human tumour genomes
How can proto-oncogenes be converted to active oncogenes?
Activated by genetic changes
How was H-ras detected?
By transfection of human bladder carcinoma DNA
No gene amplification as only present in 1 copy
What method was used to find the difference between an oncogene and proto-oncogene?
Ras oncogene isolated from transformed cells – by molecular cloning
Ras proto-oncogene isolated from normal cells
A similar size fragment from both types of cells
- To find out the difference in size – fragment recombination
- Looked to see if hybrid gene was able to transform the cells
– Narrowed down the section responsible for the difference in behaviour
What was the difference between oncogene and proto-oncogene for H-ras?
One single change in amino acid – glycine swapped for valine
Many human tumours were found to carry point mutations in one of 3?
Ras genes
- H-ras, K-ras and N-ras
What is the Ras signalling cycle?
Small GTPases
Cycle between active GTP-bound and inactive GDP-bound
What is the Ras signalling cycle mediated by?
GEF - guanine nucleotide exchange factors
GAP - GTPase-activating proteins
When Ras G12V mutant is present what happens?
Unable to hydrolyse GTP to GDP
Ras is constitutively active
Leads to activation of downstream pathways resulting in more proliferative cells
Myc is over expressed in?
70% of human tumours
What are the 3 members of the myc family?
C-myc, N-myc, L-myc
Conversion of myc protooncogene to oncogene occurs via which 3 mechanisms?
Gene amplification
Chromosomal translocation
Pro-virus integration
Myc proteins in the nucleus leads to?
Growth promoting transcription factors
What is the chromosomal translocation method of myc oncogene formation?
C-myc under control of foreign transcriptional promoter –> Burkitt lymphoma
Structurally normal myc protein but abnormally high amounts
What is the gene amplification method of myc oncogene formation?
N-myc amplification –> 30% childhood neuroblastoma
Expression driven by normal promoter
Increased levels of gene product
What is the pro-virus integration method of myc oncogene formation?
Insertional mutagenesis
Viral transcriptional promoter disrupts mechanisms controlling myc expression –> Avian leukosis virus
- Increased expression and high levels of myc protein
80% ALV-induced leukaemia –> integration of ALV pro-virus adjacent to myc proto-oncogene
Structural changes in what protein can lead to oncogenes?
De-regulation of the epidermal growth factor receptor
What structural changes in EGF receptor can lead to cancer?
Over expression of EGF receptor
- Can lead to ligand independent or ligand dependent signalling
Mutations in EGF receptor
- Extracellular function of receptor lost
- Receptor constitutively active
What 3 mechanisms can proto-oncogenes be activated by?
Ras: point mutations –> G12V substitution –> constitutively active protein
Myc: gene amplification, chromosomal translocation, insertional mutagenesis
EGFR: structural changes
