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)
What are the kinetics for the different types of retinoblastoma?
Unilateral - Two-hit kinetics curve
Bilateral - One-hit kinetics linear line
Familial Retinoblastoma
Tumour formation requires only one “event” in any cell of somatic tissues
Tumours develop from many progenitors during development
Patients present with multiple tumours in both eyes
Sporadic Retinoblastoma
Tumours are rare because two “events” are required in the same cell of somatic tissues
Patients present with a single tumour
How is the retinoblastoma gene cloned?
13q14 deletion in non-tumour cells of the patient
The Rb gene was cloned using a probe derived from a chromosome 13 library
How is the Rb gene authenticated?
Structural alterations observed in both copies of the Rb gene in tumours of patients
Studies of Rb gene structure in tumours and lymphocytes verified Knudson’s hypothesis at the molecular level
Antisera raised against the Rb protein (synthetic fusion protein using expression vector)
This showed that tumour cell lines lacked an intact form of the Rb protein
The wild-type Rb gene was cloned and reintroduced into retinoblastoma and osteosarcoma cell lines
This resulted in cell cycle arrest at G1 phase and suppression of the tumorigenic phenotype (mutant Rb alleles had no effect)
What are teh biological functions of tumour suppressor genes?
TSGs restrain/antagonise cell proliferation
Many interact directly or indirectly with an oncogene product/pathway
Cellular properties controlled by TSGs - cell cycle (Rb), apoptosis (p53) or cell migration/adhesion (APC colon)
Biochemical activities of TSGs - cell signalling (eg neurofibromin interacts in ras pathway) and transcription (eg p53, Rb)
Give an overview of p53 and cancer?
It is a tumour suppressor gene
Identified as a 53K protein found in complexes with SV40 T antigen
Both alleles are inactivated in many human tumours
Associated with hereditary cancers (Li-Fraumeni syndrome; wide range of cancers at early age)
P53 mutations in human cancer - 26,597 somatic mutations and 535 germline mutations
Describe the normal role of p53?
It is involved in co-ordinating a variety of stress signals
E.g. Lack of nucleotides, UV radiation, ionising radiation, oncogene signalling, hypoxia or blockage of transcription
Induction of the p53 protein occurs via the inhibition of its degradation
p53 is a transcription factor that activates genes involved in diverse processes
It is involved in cell cycle arrest, DNA repair, block of angiogenesis or apoptosis
What are the mechanisms of p53 inactivation in human cancer?
p53 germline and somatic mutations
Interaction with protein partners
products of oncogenic viruses - adenovirus E1B; SV40 T antigen
MDM2 (mouse double minute 2)
cellular protein that leads to p53 degradation
oncogene that is amplified in human sarcomas
Loss of ARF
AFR inactivates MDM2 (it seems to ‘mop it up’)
MDM2 is an oncogene and ARF is a TSG
What is the nature of p53 mutations?
There are a lot of mutations in the sequence-specific DNA binding domain
This inhibits the ability of p53 to bind to target DNA - therefore can’t control to downstream effects
Mutant p53 functions as a dominant-negative allele - often missense
The presence of a mutant in the mixed tetramers = non-functional
= p53 is very vulnerable
