resisting cell death and cellular immortilisation

what confers resistance to apoptosis and chemo/radiotherapy

• loss of p53

• gain of function Bcl-2 anit apoptopic molecules

what limits replicative life span

• senescence

• telomere erosion

how is sensescence bypassed

• inactivation of p53 and pRb

how is crisis bypassed

• reactivation of telomerase activity

bcl2 proteins general structure

• shared domain structure

• pro survival - bcl2

• pro apoptopic - bax and bh3

• transmembrane domain - allows MOMP

How do BH3 proteins interact with pro and anti apoptopic multidomain Bcl-2 proteins?

• via BH3 domain

• can either activate pro apoptopic

• or inhibit anti apoptopic

BH3 protein function

promote Bax and Bak to directly permeabilise the OMM

What are BH3 proteins triggered by?

• hypoxia

• dna damaging agents

• withdraw of growth factor

• osmotic stress

• UV radiation

tBid function

• proapoptotic

• activates Bax and Bak leading to MOMP

• release of cytochrome c, caspase activation

Bim activation

• sequestered to microtubules of cytoskeleton (inactive)

• held by DLC-1

• when signal is received Bim is released

Bmf activation

• sequestered to actin cytoskeleton (inactive)

• held by DCL-2

• signal causes Bmf to be released

Bad inactivation

• phosphorylated

• binding site at 14-3-3

• Bad sequestered to cytoplasm

• cannot bind and neutralise Bcl-2

activation of Bad

• dephosphorylation

• release from 14-3-3

• induces apoptosis

Bid activation

• inactive full length molecule

• cleaved by caspases

• produces truncated tBid which promotes release of pro apoptopic factors

direct activation model for apoptosis

• Bax and Bak are activated from binding to Bim and Bid (BH3 only proteins)

• cytochrome c release

• apoptosis

indirect activation of apoptosis

• Bak is released from complexes with BCL-xL and MCL-1

• binds to bid and bim

• allows cytochrome c release

• apoptosis

PI3K-Akt survival pathway

• receptor tyrosine kinase activates PI3

• phosphorylation to form PIP3

• Akt activation

• phosphorylation of pro apoptopic protein Bad at serine residues, 14-3-3 complex, leads to inactivation

what leads to hyperactivation of PI3-Akt pathway

• mutations or loss of inhibitory enzyme PTEN

• seen in many cancer cells

what happens when p53 is induced

• p21 Ras is inhibited

• inhibition of CDKs

• blocking of the cell cycle

regulation of p53

• oncoprotein MDM2

• E3 ligase responsible for conjugating poly ubiquitin to p53

• requires inhibition of ubiquitattion

How do the various inputs create a pause in p53 degradation?

Ser/thr phosphorylation of the N-terminus of p53 by damage sensors e.g. Chk2, ATM, ATR blocks association with Mdm2 and thereby spares p53 from proteasomal degradation.

Why does p53 dominate the regulation of apoptosis?

Because it transduces a plethora of stress/danger signals and promotes extrinsic and intrinsic apoptosis signalling and suppresses survival signalling.

therapeutics to target resisting cell death hallmark

• ABT-263

• Nutlin-2

ABT-263

small molecule BH3 mimetic which competes with BH3 proteins for binding to certain anti-apoptotic Bcl2 proteins

Nutlin-2

• binds the p53-binding pocket of MDM2

• inhibiting its interaction with p53

• prevents p53 degradation and indues apoptosis in cancer cells

• only works with wild type p53 (not oncogenic mutation_

senescence phase cells

• large, stop proliferating, irreversible

• fried egg morphology

• express a senescence-associated acidic β-galactosidase

• inflammatory factors

do somatic cells express telomerase

no

what is the big driver of cell crisis

• loss of telomeres cause fusion of chromatids or chromosomes

• scrambling of the genome

what causes scrambling of chromosomes and the genome?

• crisis

• erosion of telomeres

How is telomerase reactivated?

• TERT promoter mutations

• seen in human cancers

Validation of telomerase as a therapeutic agent

Suppression of telomerase inhibits cancer cell growth

• use of hTERT kills cancer cells by inducing crisis

• the shorter the telomres the quicker the onset of crisis

where do most of the point mutations found in naturally occuring cancers occur within the p53 gene?

central dna binding domain

MDM2

• ubiquitin ligase

• responsible for limiting the levels of p53

what will inhibit MDM2

• ARF

• oncogene induced

how does metabolic stress activate p53

• through AMPK

• may allow cells to proliferate in nutrient poor conditions, allows advantage to tumour cells

how are low levels of dna damage dealt with?

• p53 lowers ROS levels

• promotes the survival of slightly damaged cell to allow repair

how are high levels of dna damage dealt with?

• p53 from oncogene activate will switch to induction of apoptosis

• unable to induce apoptosis in absence of puma, puma knock out mice show tumorigenesis accelerated with activated oncogene cells

what are the 2 major approaches to killing telomerase pos tumour cells?

• direct telomerase inhibitor

• vaccines directed against TERT