Tumour suppressor genes

Question 1

using the brake and accelerator analogy - describe how dominant oncogenes and tumour suppressor genes work

Answer 1

Activation of +ve regulators = gain of function caused by dominnat oncogenes (accelerator)
Inactivation of -ve regulators = loss of function by tumour suppressor genes (brake)

Question 2

Explain how the RAS- G protein family activates and deactivates

Answer 2

In-active p21ras is bound to GDP, stimulation by coupled receptor casue active p21ras to be bound to GTP by displacing the GDP
- Active RAS sends growth signal
Deactivation occurs when GAP proteins use GTPase to turn the bound GTP to GDP (the inactive form)

Question 3

how can ras proteins lead to cancer

Answer 3

• when RAS is mutated there may be increased or prolonged growth signals
• either the protein is no longer recognised by GAP proteins or there is a deletion/mutation in the GAP genes

Question 4

Describe how cell hydrid studies work and what they accomplish

Answer 4

○ Normal cells can suppress the transformed and tumourigenic phenotype, i.e. transformation and malignancy can be recessive
○ Cells that revert to a transformed phenotype show loss of specific chromosomes (e.g. HeLa and Ch11)
○ Fusion of different tumour cell lines can also produce normal cell hybrids, implying different tumour cells have different recessive mutations
○ The pattern of chromosome loss in revertants can be used to assign putative tumour suppressor genes to particular chromosomes

Question 5

What is the evidence behind what we know of tumour suppressor genes

Answer 5

• cell hybrid studies
• familial cancer
• non-random chromosomes losses in tumours

Question 6

Explain the retinoblastoma paradigm and what type of evidence investigating TSG is it

Answer 6

• familial cancer
• Retinoblastomas is a childhood tumour of the eye and has two forms hereditary and sporadic

Question 7

What do heriditary childhood cancers show

Answer 7

pattern of inheritance suggest a two hit activation process

Question 8

Explain the difference between the familial & sporadic forms of retinoblastoma

Answer 8

Familial forms
-Occurs bilateral (both eyes)
- Mean age on set 14 months
-High penetrance = 95% of tumour developing by age 8
Sporadic
- Unilateral (one eye)
- Mean age of onset = 30 months
- 1/30000
- no family history !

Question 9

Based on analysis of the epidemiological evidence what did Knudson proposes ? Explain

Answer 9

• Two hit hypothesis
  Familial retinoblastomas patients predisposed by the inheritance of a single mutated allele form one parent
  a second mutation in the normal allele during eye development results in a tumour
  sporadic tumours arise from two independent somatic mutation of the target gene in the same cell hence rare

Question 10

how was cloning of the RB gene done ? and how was this linked to retinoblastomas

Answer 10

Linkage analysis location to Ch13q14
Homozygous deletion of Ch13q14 markers found in two retinoblastomas (Friend et al; 1986)
Marker sequence used for sequential isolation of overlapping sequences from a genomic DNA library (chromosome walking)
Sequences sought which were conserved in evolution and expressed in retinoblasts, but absent or altered in retinoblastoma cells

Question 11

During RB gene cloning, genome mapping occurer
how ?

Answer 11

linkage to esterase D gene polymorphism

Question 12

What is the characteristics of the RB gene and products

Answer 12

• 4.7kb mRNA
• 928 amino acid (105-110kD) nuclear phosphoprotein –p105Rb/p110Rb
• Found associated with DNA, but not directly bound
• Expressed abundantly in most normal tissues

Question 13

Give examples of DNA tumour viruses which express transforming protein which bind and inactivates RB

Answer 13

Adenovirus (E1A)
SV40 (large T antigen)
Human papilloma virus (E7 protein)

Question 14

What are the functions of RB protein

Answer 14

Transcriptional regulation
Cell cycle control

Question 15

how does RB protein effect transcriptional regulation

Answer 15

• Binds and regulates a range of transcription factors and cell cycle control proteins
• E2F transcription factor first identified as essential for transcription of the adenovirus E2 gene

Question 16

How does RB effect cell cycle control

Answer 16

◊ Cell cycle regulatory function of RB is controlled through its phosphorylation status
◊ During Go/G1 phase of the cell cycle the RB is hypo-phosphorylated when it binds to and inactivates the E2F transcription factor
◊ On cell cycle progression RB phosphorylation leads to dissociation of the E2F/RB complex, releasing the transcriptional activity of E2F

Question 17

what is E2F

Answer 17

transcription factor
E2F drives expression of genes required for entry into S phase, including enzymes required for the synthesis of DNA and DNA precursors
(DNA polymerase-α, thymidylate synthase, ribonucleotide reductase and dihydrofolate reductase)

Question 18

describe the involvement of RB defects in other tumours other than retinoblastomas

Answer 18

• RB patients and relatives show an increased risk of non-occular tumours - (particularly osteosarcomas and soft-tissue sarcomas
  Structural abnormalities of the RB gene found in these tumours)
• RB gene structural abnormalities found in common sporadic tumours (Bladder, Small cell lung carcinoma, Breast cancer)
• Loss of heterozygosity (LOH) for RB-linked polymorphic markers on Ch13q (reported for breast and bladder cancers coupled with more subtle point mutations of the remaining allele)
• RB protein expression studies in breast and bladder cancer indicate that low levels of RB are associated with invasive lesions and poor prognosis

Question 19

Tumour cell karotypes are U_. The , and_ are not r_

Answer 19

unstable
gains, losses and rearrangments
random

Question 20

what deletion is common in chromsomses

Answer 20

17p

Question 21

the incidence of 17p deletion in colon cancer is what

Answer 21

75%

Question 22

17p deletion mapping idenified what - How

Answer 22

p53
- Detailed mapping of Chromosome 17p deletions in colorectal cancer pointed to 17p12-17p13.3 as the critical region
- The gene (TP53) for the p53 nuclear phosphoprotein (mapped to this region) had a history that made it a strong candidate as the relevant tumour suppressor
- p53 was originally discovered in cells transformed by the SV40 tumour virus, as a host protein that was bound to the large-T antigen transforming protein encoded by the virus
- In tumours showing 17p loss, sequencing of the remaining TP53 allele revealed a high proportion to be mutated, consistent with the Knudson hypothesis and a tumour suppressor role for p53

Question 23

what is the link between p53 and cancer

Answer 23

gene is mutated in up to 50% of cancer
- typically a missence mutation in one allele coupled with loss of the 2nd allele
- germline mutation of TP53 found in the Li-Fraumeni familial cancer predisposition syndrome

Question 24

Describe the characteristics and products of p53

Answer 24

• Maps to 17p13.1 region deleted in a wide range of common cancers
• TP53 occupies ~20kB of DNA and comprises 11 exons - Exons 2-10 code for the p53 protein
• p53 is a nuclear phosphoprotein
• An acidic transcriptional transactivation domain is located at the N-terminal end
  -□ The central region comprises a DNA binding domain
• The basic C-terminal end is involved in binding to other p53 molecules to form oligomers and includes sequences required for localisation to the nucleus

Question 25

how does p53 interact with DNA

Answer 25

Binds to DNA in a sequence specific manner and acts as a transcription factor for a range of genes

Question 26

Explain the pattern of p53 mutations

Answer 26

• most frequent genetic alteration in human cancer
• mutation mainly within the central DNA binding domain (consistent with loss of function)
  [mutational hotspot (5/6 hotspots involve an arginine residue)]

Question 27

The turnover and activity of p53 is regulated by _. Describe this process

Answer 27

Post-translational modification, including phosphorylation.
- The principal sites of phosphorylation show clustering in the N-terminal transcriptional transactivation domain, which also turns out to be the a binding site for the MDM2 oncoprotein, which acts as a negative regulator of p53

Question 28

X-ray crystallography structure of the p53 core domain showed _

Answer 28

The significance of the mutational hotspot apparent when the 3-D structre of p53 bound to DNA was obtained
Mutational hotspots correspond to AA residues critical for the structure and bind to DNA

Question 29

what are some of the AA residues which are muattional hotspots in p53

Answer 29

R175,R245,R249,R248,R273,R282

Question 30

How does germline mutation of TP53 link to Li-Fraumeni syndrome

Answer 30

Transgenic mice overexpressing mutant alleles of p53 produce offspring with a high incidence of osteosarcoma, lung adenocarcinoma and lymphoma
Too rare for linkage studies – p53 investigated as a candidate gene
5 families: germ line mutations at codons 248, 258, 245, 248 & 252

Question 31

What is li-fraumeni syndrome

Answer 31

Li-fraumeni syndrome is a rare heritary disorder where the patient has a high susceptibility to a range of malignant tumours (developing before the age of 30)
High incidence of premenopausal breast cancer, childhood soft tissue sarcomas and other unusually early-onset cancers

Question 32

what altered properties due mutated p53 have

Answer 32

• Mutated p53 undergoes conformational changes detectable by antibodies
• In normal cells p53 is rapidly degraded, but some mutant forms of p53 are stabilised and accumulate to levels readily detectable by immunohistochemical (IHC) staining with antibodies
  (IHC staining for p53 alone is insufficient for assessing TP53 mutational status)
• The mutated protein binds less strongly to SV40largeT antigen and loses the ability to inhibit SV40 replication
  = may imply that p53 mutant tumours are more susceptible to the lytic effects of SV40

Question 33

What is p53 in response to DNA damage, how does mutation effect this function

Answer 33

Cell cycle arrest
□ Normal p53 accumulates in response to DNA damage and induces cell cycle arrest
□ Cells expressing mutant p53 fail to undergo cell cycle arrest after DNA damage, resulting in replication on a damaged template
□ Led to the “Molecular Policeman” or “Guardian of the Genome” hypothesis (David Lane)
Apoptosis
□P53 promotes programmed cell death

Question 34

how does p53 acts as a transcriptional regulator

Answer 34

Non-specific
□ suppresses a variety of promoters containing TATA elements
Specific
□ Binds directly to specific DNA promoter elements consisting of a 20bp consensus binding site in which two copies of the 10bp sequence:
5’-PuPuPuC(A/T)(T/A)GPyPyPy-3’ are separated by up to 13bp
□ Reporter gene constructs have shown this sequence is required for stimulation of gene transcription by p53

Question 35

What is MDM2 and WAF1

Answer 35

DNA damage induces cell cycle arrest via induction of the WAF1 gene
MDM2 and WAF1(CDKN1A) are two genes induced by P53 in response to DNA damage
MDM2 forms an autoregulatory feedback loop, by binding to p53, blocking its transcriptional activity and targeting it for degradation
p21WAF1 is a cyclin-dependent kinase inhibitor that stops RB being phosphorylated and results in cell cycle arrest