9th Feb - Discovery of oncogenes

Question 1

What are the key oncogenes?

Answer 1

Src
Ras
Erb-b
BRAF
BCR-ABL
Myc

Question 2

Who identified oncogenes in 1984 and how?

Answer 2

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

Question 3

How was the src gene identified?

Answer 3

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)

Question 4

What are NIH 3T3 cells?

Answer 4

Murine fibroblasts that have a contact inhibition response

Question 5

How was Ras detected?

Answer 5

Using the 3T3 transformation assay

Question 6

What is the 3T3 Transformation assay?

Answer 6

Transfect DNA into NIH 3T3 cells
Foci form
Inject foci into mice or transfect new culture with foci cells DNA
See if tumour develops

Question 7

What is the key limit of the 3T3 transformation assay?

Answer 7

It only works for very strong oncogenes as most cause cancer in 1 mutation

Question 8

How is the transforming DNA sequence identified?

Answer 8

Sequence hybridisation
Molecular cloning
Sequencing

Question 9

What are double minute chromosomes?

Answer 9

Small circular fragments of extrachromosomal DNA composed of chromatin

Question 10

Why are double minute chromosomes important in cancer?

Answer 10

They are a manifestation of gene amplification thus accumulate during tumour development
They frequently contain amplified oncogenes

Question 11

What are homogenously staining regions?

Answer 11

Chromosomal segments with various lengths and uniform staining intensity after G banding

Question 12

What do homogenously stained regions indicate?

Answer 12

That a gene is amplified many times

Question 13

What is a prognostics factor for Neuroblastoma?

Answer 13

N-Myc

Question 14

Give some examples of some frequently amplified chromosomal regions and the cancer they are present in

Answer 14

erbB1 in glioblastoma
k-sam in gastric and breast cancer
k-RAS in lung, ovarian and bladder cancer

Question 15

What are the 4 forms of chromosomal rearrangements?

Answer 15

Deletions
Translocations
Duplications
Inversions

Question 16

How can oncogenes be activated by chromosomal translocation?

Answer 16

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

Question 17

What is the Philadelphia Chromosome?

Answer 17

A common chromosomal translocation in CML, it is a translocation of Abl from Chr 9 –> Chr 22,

Question 18

How does the Philadelphia Chromosome Cause Cancer?

Answer 18

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

Question 19

Describe the common chromosomal translocation in burkitt’s lymphoma

Answer 19

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.

Question 20

How can one view chromosomal rearrangements?

Answer 20

FISH
Multiplex FISH
The CGH method

Question 21

What is FISH?

Answer 21

Flourescent DNA label labels target sequence through hybridisation.

Question 22

What is multiplex FISH?

Answer 22

FISH but every chromosome is visualised with a different colour to avoid false positives

Question 23

What is the CGH method?

Answer 23

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

Question 24

What is the most modern method of finding oncogenes?

Answer 24

DNA sequencing

Question 25

How was BRAF identified as an oncogene?

Answer 25

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

Question 26

What form of cancer has the highest frequency of BRAF mutations?

Answer 26

Malignant melanoma

Question 27

How does the V600E mutation in BRAF cause cancer?

Answer 27

It makes BRAF constitutively active, with 500x more activity than WT BRAF. Leading to constant MAPK activation transforming fibroblasts and melanocytes

Question 28

How was Vemurafenib discovered?

Answer 28

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