Cancer Biology II Flashcards
How can we identifiy oncogenes?
By DNA transfection
This approach is relevant when the genetic change that “activates” the proto-oncogene does not result in obvious “large-scale” chromosome abnormalities
eg. activation of oncogenes by point mutation
Describe identification of oncogenes by DNA transfection?
- Chemical transformed mouse fibroblasts - with 3-methylcholanthrene
- The DNA was extracted and transfected using calcium phosphate co-precipitation procedure, into normal mouse fibroblast that form a focus of morphologically transformed cells
Two options from here
Inject the morphologically transformed cells into mouse host = tumour
Or clone the transfected oncogene from the focus of transformed cells or analyse by southern blotting
Transfected oncogenes isolated from human tumours are homologous to retroviral oncogenes - mainly the ras gene family (K-Ras, H-Ras and N-Ras)
Describe how the H-Ras proto-oncogene becomes ‘activated’ to an oncogene?
The H-Ras gene is present as a single copy in bladder cancer cells (Southern blotting)
gene amplification not involved in activation
The H-Ras bladder carcinoma oncogene was isolated by molecular cloning and shown to have the same overall structure as the H-ras gene from normal cells
chromosome translocation/deletions not involved
What experiement can be done to determine the mutation of the Ras proto-oncogene?
Localise mutation by successive restriction cleavage and recombination, then testing for transforming activity
Found the oncogenic end is found in the 5’ end and a 350 np fragment
Mutations were found usually in codon 12 or (less frequently) 13 or 61
E.g. G12V
Describe gene amplification?
This can be detected by fluorescent in situ hybridisation (FISH)
This can identify parts of chromosomes that
Examples:
Breast cancer cells with amplified copies of the HER2/Neu oncogene borne on double-minute (DM) chromosomes
HER2 encodes a growth factor receptor (epidermal growth factor receptor 2)
Human neuroendocrinal tumour showing amplification of the myc oncogene (yellow) - double minutes and homogenously staining region (HSR)
myc encodes a transcription factor that activates genes that control cell proliferation
However, gene amplification is a poor prognostic indicator in human cancer
Describe deregulation of receptor firing in tumour cells?
When there is overexpression of the receptor - likely to get spontaneous dimerisation without a ligand
When there are mutations affecting structure this can lead to ligand independent firing
Truncation of the extracellular domain can also cause spontaneous dimerisation
Describe chromosomal translocations?
Chromosome painting - use chromosome-specific DNA probes - meaning each chromosome is “painted” a different colour
Chromosome translocations may lead to the over-expression of an oncogene OR to the production of an abnormal protein product
Describe the mechanism of oncogene activation by chromosome translocation?
Example - Overexpression of the c-Myc gene in Burkitt’s lymphoma (BL)
The c-myc oncogene is overexpressed because it is placed under control of the strong immunoglobulin gene enhancers/promoter
There is a breakpoint outside the coding region - leading to a translocation of c-myc gene (chr 8) downstream of the Ig gene enhancer (chr 14)
The c-myc oncogene can be “activated” by different mechanisms in different human tumours gene amplification (eg neuroendocrinal tumours) or chromosome translocation (eg BL)
How else can chromosome translocation lead to oncogene activation?
Example - Chronic myelogenous Leukaemia
Translocation between chromosomes 9 and 22 leads to production of a BCR-ABL fusion protein
The breakpoint is within the coding region of the bcr and abl (bcr = breakpoint cluster region)
It forms a chimeric protein with increased biological activity
This is relevant for target therapies for CML
=oncogenic fusion proteins
Describe the the Src genes?
C-Src encodes for 3 domains
Catalytic domain
TK domain
And SH2/SH3 are regulatory domains
In the v-Src gene:
There is a truncation of a phosphorylated Tyrosine from the CTD to position 416
With it still being phosphorylated it is active
How is Src regulated?
Auto inhibited:
SH2 domain of c-Src binds phosphotyrosine (Tyr 527) at C-terminus (intramolecular)
SH3 domain of Src binds polyproline helix in linker region between SH2 and kinase domains (intramolecular)
SH2 and SH3 domains are coupled through a rigid linker
Activated
Phosphorylated PDGF-R interacts with SH2 domain of Src
PolyPro domain of PDGF-R interacts with SH3 domain of Src
Catalytic cleft of Src is opened up
Phosphorylation of Tyr 416 leads to moving of the obstructing activation loop (dark green) out of the catalytic cleft to yield full tyrosine kinase activity
Signalling downstream from Src - contributes to control of many pathways
Describe tumour suppressor genes?
Tumour Suppressor Genes (TSGs) regulate cell proliferation and apoptosis:
Normal function is to restrain cell proliferation or to promote apoptosis
Altered/loss of TSG activity in tumours leads to uncontrolled proliferation or loss of apoptotic potential = uncontrolled increase in cell numbers
Mutations in TSGs in cancer are loss-of-function leading to:
Absence of protein or
Normal levels of mutant protein that has lost its activity
Mutant TSG allele is recessive to wild-type
Development of cancer requires loss of both alleles of a TSG
Inheritance of one mutant allele of a TSG results in hereditary predisposition to cancer
How were tumour suppressor genes discovered?
Via somatic cell fusion studies - using Sendai virus or polyethylene glycol (PEG) to fuse the cell membranes together
If the hybrid cell is tumorigenic - cancer alleles are dominant
If the hybrid cell is non-tumorigenic - cancer alleles are recessive (=tumour repressor)
The normal cell contributed “tumour suppressing” activity that imposed normal growth behaviour on the hybrid cell
Describe reversion to malignancy?
This correlates with loss of specific chromosomes
The loss of a chromosome goes from non-malignant to malignant revertant
The gain of a chromosome goes from Carcinoma to non-malignant
Give evidence of tumour suppressor genes?
Retinoblastoma - eye tumours in the retina
Two forms:
Sporadic Form (60% of cases; unilateral retinoblastoma)
Unilateral eye involvement; single tumour
No family history
No increased risk of other malignancies in later life
Heritable Form (40% of cases; bilateral retinoblastoma)
Multiple independent tumours in both eyes
Presents early in life (<5 years)
Positive family history
Autosomal dominant inheritance of the risk of developing disease - each offspring of an affected parent has a 50% chance of developing the disease
Increased risk of other malignancies in later life (osteosarcoma)
