L12 - Oncogenes Flashcards
What quantity of tumours are induced by viruses
Minority
How many genes do viral genomes have
Few genes
What can be said about the few genes that viruses do contain
They are enough to drive the tumour phenotype
- Extremely potent genes which perturb the complex regulatory circuit of the host cells that they infext
RSV
Rous Sarcoma Virus
How was the carcinogenic agent in RSV discovered
Chicken with sarcoma in breast
Sarcoma removed and ground with sand
Collect filtrate that had passed through a fine-pore filter
Inject filtrate into a young chicken
Observe the formation of a sarcoma in the injected chicken
What are foci
Clusters of tumour cells which appear after infection
Describe the morphology of the foci and how they grow
Rounded cells that grow one on top of the other
What characteristics of the foci is similar to the cells of the tumour
Have similar metabolism
Define cell transformation
Converstion of a normal cell into a cancer cell
Describe the transmission of the cancer between the cells of the foci
Transformed phenotype was transmitted from the initially infected cells to its descendents
What is a TS RSV mutant
RSV virus which carries a temperature sensisitve mutation
Describe how the TS RSV mutant functions
Proteins are able to function at the permissive temperature (37) if raised about this the proteins don’t have TW function
What can be said of the dependence of the transformed phenotype on the virus
Viral transforming gene is required to initiate and maintain the transformed phenotype of the infected cells
Properties of transformed cells
Altered morphology Loss of contact inhibition Ability to grow w/o attachment Ability to proliferate indefintely Reduced requirement for MGFs High saturation density Inability to halt proliferation in the absence of GFs Increased uptake of glucose Tumorigenicity
How many genes in the RSV virus
4
What are the four genes in RSV
Gag, Pol, Env, C
What is the effect of the removal of Gag, Pol and Env
Virus is unable to replicate but is still able to transform other cells
What is the effect of the removal of X
Virus is able to replicate but is unable to induce cell transformation
The single gene required for transformation is known as
Src
How many genes are required for replication of the RSV virus
3,
Gag, pol and env
What is the derivations behind the name Src
Ability to induce a sarcoma in the chicken
What can be used to understand the origin and functions of Src especially useful to be able to follow it in infected cells
Src specific DNA probe
Src when it is normally found in organisms is known as
C-Src
What type of Src is found in viruses
What can it act as
V-Src
Potent oncogene
What is an oncogene
Gene which is able to transform a normal cell into a tumour one
What type of gene is C-src
What is it a precurrsor to
Proto-oncogene
Precursor of an active oncogene
What are the two hypothesies to describe how oncogenes become activated
Through endogenous retroviruses
Induction through mutants
Describe how you would be able to identify wether chemicall transformed cells are able to carry mutated genes
TRANSFECTION STUDIES
Chemically transform mouse fibroblasts
Isolate DNA - transfect using calcium phosphate
Inject into normal mouse fibroblasts
Formation of a focus of morphologically transformed cells
Injection of morphologically transformed cells into mouse host
Formation of a tumour
What was the overall conclusion of the transfection studies
Donor tumour DNA carried by one or several genetic elements is able to convert a normal cells into a tumorigenic one
Oncogenes detected by transfection were derived from ____
Pre-existing normal cellualr genes which lack previously lacked oncogenic function - similar scenario to the viral oncogenes
Give an example of an oncgoene detected in human bladder carcinoma
H-Ras
Is H-Ras expressed through gene amplification
No - mutation present in only the one copy
What is the mutation which must occur in order for h-Ras to become oncogenic
Point mutation at residue 12
Glycine –> Valine
Describe the method which is used to identify the sequence which has changed
Creation of hybrids - 1/2 and 1/2 of the known cancer and normal
If the hybride can transform you know thar THE CANCER HALF CONTAINS THE MUTATED SEQUENCE
Perform again
What are the 3 Ras proteins
What do they have in common
H, K and N Ras
Point mutations are able to convert from protooncogenes to oncogenes
Describe the cycling seen in small GTPases
cycle between the active form - with GTP bound
AND
The inactive form with GDP bound
What is required to activated Ras
GEF
What is required to cause a cessation in the signalling of Ras
GAPS
What is significant about Ras G12V (Glycine –> Valine at position 12)
It is unable to hydrolyse GTP
Protein is consituitively active
In the presence of no growth factor describe the activity of the Ras G12V
ONCOGENIC RAS
No growth factor but mutant RAS is always ON
Leading to cell division and proliferation
What are the four immediate downstream targets of Ras
RAF
PI3K
RALGDS
PLC
How many mechanisms are there which would lead to the activation of the myc oncogene
3
What are the three mechanisms leading to oncogenic transformation of myc
1) Expression driven by normal promoter but gene is amplifyed
2) Chromosomal translocation
3) Pro-virus integration causing an insertional mutagenesis
How does Expression driven by normal promoter but gene is amplifyed arise?
More than 2 copies => high levels of gene product
What can be said about the prognosis of myc at the various copy levels
With a higer myc copy number the prognosis for patients is worse
How many chromosomal translocation cause oncogenic activation of Mtc
C-myc would be under the control of a foreign promoter
**Most common to have Myc expressed under a Ig (immunoglobin) promoter
During a chromosomal translocation is there any change to the strucutre of myc
NO
Strucutall normal but is expressed at high ammounts
Wht type of lymphoma is caused by a chromosomal translocation affecting myc
Burkitt lymphoma
What is ALV
Avian leukosis virus
Describe how pro-virus integration may lead to elevated myc expression
Virus integrates immeidatly adjacent to the myc gene so expression of myc is now driven by the active viral promoter leads to an INCREASE IN GENE PRODUCT
Describe how changes in the EGF receptor may form ocogenees
Trucation of the EC ligand bidning domain results in a consituitively active receptor
What is the normal structure and mechanism of activation of the EGF receptor
EC ligand binding domain - when a growth factor binds here the IC domain is able to signal
What are 4 ways in which EGF signalling could be manipulated to oncogenic levels
Over expression of EGF ligand
Overexpression of the receptor
Ligand independent dimerisation (dimerisation in the absence of ligand biinding)
Mutations resulting in the formation of a constituitvelt active receptor
