oncogenes and tumour suppressors

AMES test

1. homogenised rat liver - source of metabolic enzymes

2. add to test compound to activate metabolism

3. add to his negative salmonella

4. addition of metabolically active compound should provide his for salmonella to grow

effect of rsv on cell monolayer

no longer contact inhibited

can alter morphology of cells

anchorage independent

immortal

reduced requirement for GFs

high saturation density

increased glucose metabolism

experiment showing src causes cell transformaton

temp sensitive src mut

transform culture with rsv = morphology change

increase to sensitive temperature = return to normal

back to normal temp = rsv morphology

origin of viral oncogenes

virus infected host and co opted src gene

evidence - probe restriction digested host DNA with viral DNA = homology

mechanisms of activation of proto oncogenes

deregulated activity, deregulated expression

cellular processes normally subverted in cancer

GFRs, Myc, proliferative, checkpoints, apoptosis

genetic changes in tumorigenesis

small changes - indels e.g. FAP

structural changes - deletion, inversion, duplication, translocation

growth factor signalling changes

overexpressed GFRs, production of own GF ligands, mutated GFRs

oncogenic src activation

no pTyr in C terminus which is normally inhibitory of active src structure

increases pTyr of substrates

elevated expression - myc

retroviral insertion

mutation of control genes

genomic amplification

chromosomal translocation - EBV e.g

stimulation by upstream oncogenic activity

types of tumour suppressors

caretakers, gatekeepers, stress sensors, landscapers

caretakers

maintain genetic integrity

loss of genome integrity arises as a consequence of DNA damage and erosion of repair machinery

BRCA1/2 - caretakers

BRCA1 = DSB recognition

BRCA 2 = homologous recombination

BRCA1 forms dimers with BARD1 - binds damaged DNA and interacts with machinery

gatekeepers

cell cycle checkpoints and signal attenuators

APC - gatekeeper

FAP = 1 inactive APC gene predisposes to second mtuation

activation of wnt pathway in absence of ligand = increased myc

RTKs - gatekeepers

NF1, PTEN

Rb - gatekeeper

mitogenic signals activate G1/S cyclin-CDK complexes to inhibit Rb by phosphorylation = removal of E2F inhibition

E2Fs dimerise with DP1/2 partners to activate S phase and repress differentiation/quiescence

stress sensors - p53

monitor dysfunction and coordinate repair

activated by stress and DNA damage (ARF, ATM, ATR, chk)

trigger cytostatic and apoptotic factors

inactive in 85% cancer

landscapers

stromal remodelling, angiogenesis

restrain precocious clonal expansion through restrictive impact on environment