9th Feb - Discovery of oncogenes Flashcards
What are the key oncogenes?
Src Ras Erb-b BRAF BCR-ABL Myc
Who identified oncogenes in 1984 and how?
Downward and Waterfield 1984
They generated amino acid sequences for proteins thought to be important in growth regulation, and then compared the sequences of the DNA sequences with retroviral oncogenes
How was the src gene identified?
In 1958 Rubin and Termin altered chicken fibroblasts morphologically by RSV transfection. 2 years later Termin determined it was due to a genetic property of RSV (later determined to be src)
What are NIH 3T3 cells?
Murine fibroblasts that have a contact inhibition response
How was Ras detected?
Using the 3T3 transformation assay
What is the 3T3 Transformation assay?
Transfect DNA into NIH 3T3 cells
Foci form
Inject foci into mice or transfect new culture with foci cells DNA
See if tumour develops
What is the key limit of the 3T3 transformation assay?
It only works for very strong oncogenes as most cause cancer in 1 mutation
How is the transforming DNA sequence identified?
Sequence hybridisation
Molecular cloning
Sequencing
What are double minute chromosomes?
Small circular fragments of extrachromosomal DNA composed of chromatin
Why are double minute chromosomes important in cancer?
They are a manifestation of gene amplification thus accumulate during tumour development
They frequently contain amplified oncogenes
What are homogenously staining regions?
Chromosomal segments with various lengths and uniform staining intensity after G banding
What do homogenously stained regions indicate?
That a gene is amplified many times
What is a prognostics factor for Neuroblastoma?
N-Myc
Give some examples of some frequently amplified chromosomal regions and the cancer they are present in
erbB1 in glioblastoma
k-sam in gastric and breast cancer
k-RAS in lung, ovarian and bladder cancer
What are the 4 forms of chromosomal rearrangements?
Deletions
Translocations
Duplications
Inversions
How can oncogenes be activated by chromosomal translocation?
The breakpoint can occur within introns of 2 genes –> a chimeric protein with novel properties e.g. BCR-ABL
Translocation may place a proto-oncogene sownstream of a strong constitutive promoter from another gene e.g. Myc in burkitt’s lymphoma
What is the Philadelphia Chromosome?
A common chromosomal translocation in CML, it is a translocation of Abl from Chr 9 –> Chr 22,
How does the Philadelphia Chromosome Cause Cancer?
It creates a 3’ fused Bcr/Abl gene –> a chimeric BCR-ABL protein which causes deregulated tyrosine kinase activity. This activates Grb2, Paxillin, SFK and Actin leading to proliferation and inhibition of apoptosis and cell adhesion
Describe the common chromosomal translocation in burkitt’s lymphoma
A reciprocal translocation occurs between the tip of chromosome 14 to 8, translocating Myc from chromosome 8 to chromosome 14 putting Myc under the control of an IgH enhancer.
How can one view chromosomal rearrangements?
FISH
Multiplex FISH
The CGH method
What is FISH?
Flourescent DNA label labels target sequence through hybridisation.
What is multiplex FISH?
FISH but every chromosome is visualised with a different colour to avoid false positives
What is the CGH method?
Comparitive Genomic Hybridisation which allows one to compare the chromosomes of a normal cell with a cancerous cell.
Each cell’s DNA is labelled a different colour.
Both cells DNA is added to another normal cells metaphase spread of chromosomes.
Flourescently coloured signals are then compared. A higher intensity of the test sample colour in a particular region indicates a gain of material and a lower intensity indicates a loss
What is the most modern method of finding oncogenes?
DNA sequencing
How was BRAF identified as an oncogene?
Genomic DNA from 15 cancer cell lines and the corresponding lymphoblastoid cell lines were screened for variants using sequencing –> identification of 2 BRAF mutations in exon 15
Screen an additional 530 cancer cell lines - BRAF mutation identified in 43 cell lines
What form of cancer has the highest frequency of BRAF mutations?
Malignant melanoma
How does the V600E mutation in BRAF cause cancer?
It makes BRAF constitutively active, with 500x more activity than WT BRAF. Leading to constant MAPK activation transforming fibroblasts and melanocytes
How was Vemurafenib discovered?
Used a structure guided discovery approach looking for selective inhibitors of active B-Raf –> PLX 4720 which creates a DFG-out conformation making B-raf inactive. This blocked tumour growth in malignant astrocytes in mice and became FDA approved in 2011 as Vermurafenib
