INTERNAL CONTROLS OF CELL CYCLE - “CHECKPOINTS”

1. G1/S checkpoint : checks for DNA damage ,prevents replication of cells that have defects in DNA ; cell cycle arrest mediated through p53

2. G2/M checkpoint : monitors completion of DNA replication ; cell cycle arrest by p53- dependent & p53-independent mechanisms

Defects in cell cycle checkpoints are major cause of genetic instability

in cancer cells

Products of tumor suppressor genes apply “brakes” to cell proliferation – network of check points

Tumor suppressors like RB & p53 , are part of regulatory network that recognize genotoxic stress and respond by shutting down prolif

Another set of tumor suppressors involved in cell differentiation causing cells to enter postmitotic differentiated pool without replicative potential

RB - The 1st and prototypic tumor suppressor gene

60% retinoblastomas are sporadic Familial Retinoblastoma inherited as

an autosomal dominant trait. Patients with familial retinoblastoma

are also at greatly increased risk of developing osteosarcoma and other soft-tissue sarcomas.

“two-hit” hypothesis of oncogenesis proposed by Knudson

Knudson's hypothesis can be stated as follows :

 Two mutations (hits) involving both alleles of RB at chromosome locus 13q14, are required to produce retinoblastoma

In familial cases, children inherit one defective copy of the RB gene in the germ line (one hit ) ; the other copy is normal.

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Knudson's hypothesis :

 Retinoblastoma develops when the normal RB allele is mutated in retinoblasts as a result of spontaneous somatic mutation (second hit).

In sporadic cases both normal RB alleles must undergo somatic mutation in the same retinoblast (two hits). A retinal cell that has completely lost RB function becomes cancerous.

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RB (RB protein) – product of Rb gene is nuclear phosphoprotein.

Active form – hypophosphorylated in quiescent cells

Inactive form – hyperphosphrylated in G1/S transition

Key role in G1/S checkpoint ; if RB is absent as a result of gene mutation the “molecular brakes” on cell cycle are released

Initiation of DNA replication requires activity of cyclinE – CDK2 ; expression of cyclin E is dependent on E2F family of transcription factors

In its active form RB binds to E2F & blocks E2F mediated transcription of cyclin E

RB couples control of cell cycle progression at G1 with diferentiation ; diff is associated with exit from cell cycle

Role of RB in checking G 1- S checkpoint of cell cycle

p53 gene is located on chromosome 17p

Most common target for genetic alteration over 50% of human tumors contain mutations in this gene

Homozygous loss of p53 occurs in virtually every type of cancer, including CA lung, colon breast—the three leading causes of CA death

P53 protein functions as a transcription factor

In response to DNA damage, p53 is phosphorylated by genes that sense the damage & are involved in DNA repair.

p53 links cell damage with DNA repair, cell cycle arrest, and apoptosis.

Assists in DNA repair by causing G1 arrest and inducing DNA-repair genes.

A cell with DNA damage beyond repair is directed to undergo apoptosis.

In view of these activities, p53 has

been rightfully called a “guardian of the genome.”

With loss of function of p53, DNA damage goes unrepaired, mutations accumulate in dividing cells, and the cell marches along a one-way street leading to malignant transformation.

The role of p53 in maintaining the integrity of the genome.

p53 prevents neoplastic transformation by three interlocking mechanisms: activation of temporary cell

cycle arrest (quiescence) induction of permanent cell

cycle arrest (senescence) or triggering of programmed cell

death (apoptosis)

Li – Fraumeni syndrome : individuals who inherit one

mutant allele of p53 have a 25 fold greater chance

of developing malignant tumor by age 50 than general population

APC/β – CATENIN PATHWAY

Adenomatous polyposis coli genes (APC) is a tumor suppressor ; down-regulate growth-promoting signals.

Germ-line mutations at the APC (5q21) loci are associated with familial adenomatous polyposis

Almost invariably, one or more of these polyps undergoes malignant transfor- mation, giving rise to colon cancer.

APC /β –C ATENIN PATHWAY Both copies of the APC gene must be lost /

mutated for tumor to arise APC (& β-catenin ) are component of the

WNT signaling pathway, which has a major role in controlling cell fate, adhesion, and cell polarity during embryonic development

An important function of the APC protein is to down-regulate β-catenin

Cells with loss of APC behave as if they are under continuous WNT signaling

A, The role of APC in regulating the stability and function of β-catenin. APC and β-catenin are components of the WNT signaling pathway. In resting cells (not exposed to WNT), β-catenin forms a macromolecular complex containing the APC protein. This complex leads to the destruction of β-catenin, and intracellular levels of β-catenin are low.

B, When cells are stimulated by WNT molecules, the destruction complex is deactivated, β-catenin degradation does not occur, and cytoplasmic levels increase. β-catenin translocates to the nucleus, where it binds to TCF, a transcription factor that activates genes involved in cell cycle progression.

C, When APC is mutated or absent, the destruction of β-catenin cannot occur. β-catenin translocates to the nucleus and coactivates genes that promote entry into the cell cycle, and cells behave as if they are under constant stimulation by the WNT pathway.

Accumulation of neoplastic cells may also result from mutations in the genes that regulate apoptosis.

Apoptosis represents a barrier that must be surmounted for cancer to occur.

In the adult, cell death by apoptosis is a physiologic response to several pathologic conditions that might contribute to malignancy if the cells remained viable.

CD95 receptor- induced (extrinsic pathway) and DNA damage–triggered (intrinsic) pathways of apoptosis & mechanisms used by tumor cells to evade cell death.

1. Reduced CD95/Fas (death receptors) level.

2. Inactivation of death-induced signaling complex by FLICE protein (caspase 8)

3. Reduced egress of cytochrome c from mitochondrion as a result of up-regulation of BCL2.

4. Reduced levels of pro-apoptotic BAX resulting from loss of p53.

5. Loss of apoptotic peptidase activating factor 1 (APAF1).

6. Up-regulation of inhibitors of apoptosis (IAP).

Most normal human cells have a capacity of 60 to 70 doublings only, then become senescent due to progressive shortening of telomeres

Short telomeres are recognized by the DNA-repair machinery as double - stranded DNA breaks, and this leads to cell cycle arrest mediated by p53 and RB.

In cells in which the checkpoints are disabled by p53 or RB1 mutations, the nonhomologous end-joining pathway is activated to save the cell, joining the shortened ends of two chromosomes

At mitosis the dicentric chromosomes are pulled apart, generating random double-stranded breaks, activating DNA-repair pathways, leading to the random association of double-stranded ends and the formation, again, of dicentric chromosomes

Genomic instability from the repeated “bridge-fusion-breakage cycles” eventually produces mitotic catastrophe, characterized by massive cell death.

For tumors to grow indefinitely tumor cells must

Reactivate / Re-expression of telomerase

Re-expression of telomerase allows the cells to

escape the bridge-fusion-breakage cycle

Sequence of events in the development of limitless replicative potential.

• Replication of somatic cells, which do not express telomerase, leads to shortened telomeres.

• In the presence of competent checkpoints, cells undergo arrest and enter nonreplicative senescence.

• In the absence of checkpoints, DNA-repair pathways are inappropriately activated, leading to the formation of dicentric chromosomes.

Solid tumors cannot enlarge beyond 1 to 2 mm in diameter unless they are vascularized

Cancer cells can stimulate neoangiogenesis or in some cases vasculogenesis

Tumor angiogenesis is controlled by the balance between angiogenesis promoters and inhibitors (e.g p53)

Tumor vasculature is abnormal, vessels are leaky , dilated and have a haphazard pattern of connection

Most human tumors do not induce

angiogenesis early remain small /in situ,

possibly for years, until the angiogenic

switch terminates this stage

The molecular basis of the angiogenic switch

involves increased production of angiogenic

factors and/or loss of angiogenic inhibitors

These factors may be produced directly by

the tumor cells ,or by inflammatory cells

(e.g., macrophages) or stromal cells

Neovascularization has a dual effect on

tumor growth:

1. Perfusion - supplies needed nutrients

& O2

2. Endothelial cells secrete growth factors

(IGFs, PDGF, GMCSF) promoting growth

of adjacent tumor cells Angiogenesis is required also for access

to the vasculature and hence for metastasis.

Angiogenesis is thus a necessary biologic correlate of malignancy