Tumour Supressor Genes Flashcards
How does the karyotype of a cancer change with clonal evolution?
Human tumors with minimal chromosome change (diploid acute leukemia, chronic granulocytic leukemia) are considered to be early in clonal evolution; human solid cancers, typically highly aneu- ploid, are viewed as late in the developmental process.
What sort of genes are mutated in cancer to allow for uncontrolled proliferation?
Regulation of cell proliferation can either be positive or negative; the acquisition of tumourgenicity results from genetic changes that affect these controls:
- Interference with controls that restrain cell multiplication-loss of function mutations
- Gain of function mutations resulting in constitutive activation of signals to proliferate
What evidence suggests that LOF mutation of tumour supressors plays a role in carcinogenesis?
1) Cell hybrids, 2), familial cancer, 3) loss of heterozygosity, 4) downregulation of gene expression.
The first insight into the activity of tumor suppressor genes came from somatic cell hybridization experiments, carried out by Henry Harris and colleagues in 1969.
The fusion of normal cells with tumour cells yielded hybrid cells containing chromosomes from both parents. In most cases they were unable to form tumors in animals. It appeared that genes derived from the normal cell parent acted to inhibit or suppress tumour development.
The observations seen here can now be described through our knowledge of loss of function mutations in tumour supressor genes, which were subsequently replaced by the somatic cell.
What is the difference between tumour supressor genes and oncogenes?
Oncogenes are genes that, when mutated to produce a gain/increase of function, promote the formation of tumours. Hence the oncogene products – oncoproteins – are responsible for promoting proliferation, growth and survival etc.
Tumour suppressor genes, also called anti-oncogenes, are genes that, when mutated to cause a loss/reduction of function, lead to increased cancerogenesis. Hence they are genes whose products supress cancer trends such as proliferation growth and survival.
What was the first tumour supressor gene identified?
The first tumor suppressor gene was identified by studies of retinoblastoma, a rare childhood eye tumor. 50% of children from the parents of those affected would go on to develop RB, consistent with Mendelian transmission of a single recessive gene that confers susceptibility to tumor development.
In hereditary cases of retinoblastoma (40% of cases) an earlier age of tumor development is inflicted, suggesting tumor suppressor genes to have already been lost from inheritance.
What hypothesis did the age of onset for hereditary Rb lead to?
That the children were not born with retinoblastoma led to the proposal of the two hit hypothesis.
In 1971 Alfred Knudson proposed that the development of RB requires two mutations, which are now known to correspond to the loss of both the functional copies of the rb gene. With only one defective copy being inherited due to heterozygosity, another mutation in the functional RB is required for tumor formation.
This Loss of Heterozygosity (LOH) is more frequent than a random event which would induce heterozygosity.
What is Rb?
Is a nuclear phosphoprotein of 105 kDa and is a member of a family of pocket proteins that interact with E2F promoter regions. There are three members, p105, p107, p130, which are inactivated in cells via sequestration.
How is Rb regulated in G0?
The pocket domain described directly interacts with proteins holding an LXCXE motif, the phosphorylation of which regulates its activity. In GO it is dephosphorylated, which allows the RB protein to bind to promoter regions of E2F shielding from transcription factors.
How is Rb regulated at the restriction point?
The restriction point RB undergoes phosphorylation by CDK4/6 and cyclin D complexes, through a transphopshorlyation reaction in which phosphates of the threonine 160/161 residues of CDK4/6 are transferred to RB proteins, leading to its inactivation by preventing binding of LXCXE motifs in the pocket domain.
Other than the obvious, what cancer is Rb involved in in humans and mice? What do Rb null mouse models show about its redundancy?
It contributes to small cell lung carcinoma, sarcomas and bladder carcinoma.
RB-/- mice- embryonic lethal- die day 13.5 to 15.5 due to defects in fetal liver erythropoiesis, neurogenesis and lens development/ problem with placenta
Chimeric Rb (-/-) and Rb (+/+) mice do not develop retinoblastoma but acquire pituitary tumours with age. This implies a functional redudancy in Rb, with other pathways being able to compensate.
How do viral proteins interact with Rb?
SV40 & polyoma virus large T antigens, Ad E1A and HPV16/18 E7 viral proteins bind the de/hypophosphorylated form, restricting entry into late G1 to immortalise cells by competetively inhibiting Rb.
How do the levels of the different pRb family members change through G1 into S?
p105 (pRb) only slowly and slightly increases consistently during this time, but its activity is highly regulated.
p107 is highly repressed during G0, but its expression is consistently increased until its peak when the cell passes the R-point when it plateaus.
p130 is highly expressed during G0, being important for its regulation, and its level is consistently decreased during G1 to it’s lowest point at the beginning of S-phase after which it gets a slight uptick.
How does Rb suppress E2F stimulated genes?
Not only does it sequester free E2F to prevent it acting as a TF, it can bind to DNA bound E2F and recruit HDACs and HMTs to epigenetically silence the genes.
What are the varying roles of the E2F TFs?
E2F1, 2 and 3a activate transcription of genes essential for S phase. Expression of these proteins is regulated by cell growth via RB.
E2F3b, 4 and 5 (found in quiescent cells) and 6-8 function as transcriptional repressors of S phase genes as well as the E2F1, 2 and 3a proteins
E2F4 and 5 are not regulated in response to position within the cell cycle.
Where do E2F TFs bind to DNA?
E2Fs as TFs bind to the TTTCCCGC (or slight variations of this sequence) consensus binding site in the target promoter sequence. They often bind when dimerised with dimerisation proteins (DP1 & 2).
This is found in genes such as c-myc, B-myc, cdc2, DHFR, TK, cyclinA, PCNA, cyclinE and E2F1.
