The Cell Cycle

Introduction

• DNA duplication• cell division• the phases have been

known 40 years but last 3-4 years - the genes involved in control of cycling process

• The cell cycle divided into four phases– the first gap phase (G1)– DNA replication (S)

– the second gap phase (G2) -- interphase

– mitosis (M)

M

G2 G1

S

G2 DNA

damage

checkpoint

DNA synthesiscompletion

G1/S DNA damagecheckpoint

START or restriction point

Correct spindleformation & metaphase plate

Initiation ofmitosis The length of the

cell cyclearound 16-24 h.

The cell cycle showing checkpoints at which DNA is monitored before the next stage of

the cycle is entered

• G1 preparing to synthesize DNA, biosynthesis of RNA & proteins

• S phase, DNA is replicated & histones are synthesized. At the end of S phase DNA doubled & Chromosomes replicated

• G2, cells are preparing for cell division, replicated DNA complexes with proteins, biosynthesis continues

• nucleus & cytoplasm divide during mitosis

• two daughter cells are produced - can begin interphase of new cell cycle

• cells can also enter a resting phase (G0)

• External stimuli: growth factors (to enter into G1): GF -induced signals and GF inhibitors (TGF beta)

• The progression of cycle from one stage to next is controlled by cyclin-dependent kinases (CDKs) & CDKInhibitors

• -> Signal transduction

• Number of recognized checkpoints:

• START or restriction (R) point (late in G1)

• during S phase • at the G2 to M

transition to monitor the completion of DNA synthesis

• prevent chromosome segregation if not intact

• the degradation of various cyclin

Cell CycleControl Genes

Introduction

• Two further groups of genes play a major role in the development of cancer :

1. genes those intimately involved in the positive and negative control of the cell cycle

2. genes those involved in the repair of DNA mismatches (initially identified in colorectal cancer)

Cyclins and Cyclin-Dependent Kinases

• Transition from one stage to the next in the cell cycle :

regulated at a number of checkpoints at which the integrity of the DNA are checked which prevent entry into subsequent stages with damaged DNA

carefully controlled by the sequential : activation and degradation of the cyclins activation of the cyclin-dependent kinases

(CDKs)

Cyclins and Cyclin-Dependent Kinases

• Thirteen mammalian cyclins have been identified, each one of which is required at a different stage of the cell cycle

• Six mammalian CDKs have been identified

• Activation of the CDKs occurs by :

phosphorylation of a conserved threonine residue (at position 160)

binding of the cyclin

AB 1-3CD 1-4EFGH

Cyclin

MitoticMitoticG1

G1

G1

???

Family

CDC2, CDK2CDC2?CDK2,4,5,6CDK2,4,5,6??p40 mo15

CDK

S, G1, MM?G1

G1/S???

Stage

Mammalian Cyclins and Cyclin-Dependent Kinases

The Stages of the Cell Cycle and Expression of the Cyclins and CDKs

M

G2 G1

S

Cyclin D and CDK2,4,5,6

Cyclin E + CDK2

Cyclin A and CDC2Cyclin B and CDC2

Cyclin A1CDK2

Cyclin-Dependent Kinase Inhibitors• Control of cyclins and CDKs is now also known to

occur via a group of inhibitor proteins known as cyclin-dependent kinase inhibitor (CDKIs)

• There are seven different CDKIs in mammalian cells which belong to two different classes

p15p16p18p19p21p27

p57

Inhibitor

CDK4,6CDK4,6CDK4,6CDK4,6CDK2,3,4,6CDK2,4,6

CDK2,3,4

Target

9p219p211p3219p136p2112p12-13

11p15

Chromosomal location

TGFβ???p53, TGFβRapamycincAMP?

Regulator

Control of the Cell Cycle• Each of the cyclin-CDK complexes, together with

the CDKIs : responsible for controlling different stages of

the cell cycle by preventing progression through checkpoints

in the presence of DNA damage deregulation of these processes has been

implicated in tumorigenesis

1. START

• The cyclin-CDK complexes linked to the regulation of START are the D type cyclins

• There are four cyclin Ds : D1, D2, D3 and D4

expressed in a cell lineage-specific manner

synthesized in response to growth factors

very short lived, rapidly degraded when growth stimuli are withdrawn regardless of the position of the cell cycle

if removed during G1, cells will not enter S phase

• Deregulation of cyclin D synthesis :

make cells less dependent on growth stimuli

likely to contribute to tumorigenesis

• Overexpression of cyclin D1 is associated with esophageal, breast and gastric cancers

• CDK4 : has a potential role in tumorigenesis

a target for TGFβ in some cells

• CDKIs : involved in tumor development at this stage of the cell cycle

p16, encoded by the CDKN2 or MTS1 gene, is an inhibitor of CDK4

2. G1 to S Phase

• Cells which have suffered DNA damage : prevented from entering S phase

blocked at G1

• this process is dependent on : the tumor suppressor gene, p53 the cyclin-dependent kinase inhibitor, p21

DNA damage

Activation of p53

Increased p21 Bind to a number of cyclin-CDK complexes :• cyclin D-CDK4• cyclin E-CDK2• cyclin A-CDK2

Prevents phosphorylation of RB

Cell cycle arrest in G1

E2F DP-1

RB

E2F DP-1

RB

E2F DP-1

RB

RB

RBP

P

P

RBP

P

P

E2F DP-1

TTTCGCGC

No transcription

No transcription

Transcription

No transcription

Dephosphorylation

Cyclin A/CDK2

Cyclin E/CDK2

Cyclin D1/CDK4-6

G0

G1

S

G2

M

G0 G1

Mode of Action of RB

During G0 and G1 RB is underphosphorylated and is bound to the E2F-1 transcription factor complexed with DP-1.

During G1, cyclin D1/CDK4-6 and cyclin E/CDK2 phosphorylate RB and E2F-1 is released to interact with and promote transcription from genes necessary for S phase.

Phosphorylation of RB is maintained by cyclin A/CDK2 until mitosis when it is dephosphorylated ready either to re-enter G1 or to go into the stationary phase.

3. S Phase

• Cyclin A :

expressed from S phase through G2 and M

binds to two different CDKs : complexed to CDK2 (during S phase) complexed to CDC2 (during G2 and M)

has a role in both transcriptional regulation and replication

binds to the E2F transcription factor one of the first cyclins to be implicated in

tumor development

• The cyclin A gene was the unique insertion site for the hepatitis B virus in one clonal tumor

• The virus integrate into the second intron of the gene → production of a chimeric protein (in which the region, N terminal to the cyclin box, was replaced with viral sequences) → removal of the ‘destruction’ box necessary for the degradation of the cyclin in mitosis

4. Mitosis

• Entry to mitosis is signaled by the activation of the cyclin B-CDC2 complex

• This complex accumulates during S and G2 but is kept inactive by phosphorylation of tyrosine 15 and threonine 14 residues

Mismatch Repair Genes

Mismatch Repair Genes• Both sporadic and hereditary colorectal cancers

shows defect in these genes

• Hereditary nonpolyposis colon cancer (HNPCC) accounts for around 10-15% of all colorectal cancer

• The causative gene was mapped to chromosome 2p21 by linkage studies

• When DNA from tumors was compared with DNA from normal tissues :

tumor DNA showed widespread alterations in short repeated sequences distributed throughout the genome

seen as additional bands over and above the usual one or two alleles identified in the normal tissue DNA

replication errors, caused by slippage of DNA polymerase, had occurred during tumor development and had not been repaired

MutS

MutLMutLMutL

Bacteria

MSH2

MLH1PMS1PMS2

Yeast

hMSH2GTBPhMLH1hPMS1hPMS2

Human

Mismatch Repair Genes

STOP

G0

DNA repair

APOPTOSISTumor

Suppressor genes

DNA lesion

Apoptosis

Apoptosis (Kerr et al., 1972)

• Program Cell Death (PCD)

• Genetically controlled unwanted cell

- during morphogenesis

- during proliferation and differentiation

• critical point of cellular control

• modulated physiologically (itself & its environment)

• normal regulation

• self destruction mechanism

Characteristic of Apoptosis

•Apoptotic cells are recognized & phagocytosis

•Differs from necrosis / accidental cell death

- active process

- no surrounding tissue damage or

induction of inflammatory responses

• Characteristic morphology :

- condensation of nuclear heterochromatin

- cell shrinkage

- loss of positional organization of organellas in

cytoplasm

- ladder phenomen

• Controlled by protooncogenes & tumor suppressor genes

Apoptosis

Intrinsic

Extrinsic • TNFα• TGFβ

• p53• MYC• Interleukin-1β converting enzyme• BAX/BCLXS

• BCL2/BCLXL

• A20

Many, for example : • erythropoietin• PDGF/IGF1• sex hormones

Promoting Inhibiting

Protein Controlling Apoptosis

Therapeutic induction of apoptosis

• Haemopoietic malignancies (responsive therapy) - Apoptosis >>

• high expression bcl-2 - inhibit the cell - apoptotic responsiveness to dexamethazone, methotrexate, etoposide, vincristine, cisplastin & Cyclophosphamide

• Mechanism of drug resistance ?

APOPTOSIS NECROSIS

Physiological or pathological

Always pathological

Single cell Sheets of cells

Energy dependent Energy independent

Cell shrinkage Cell swelling

Membrane integrity maintained

Membrane integrity lost

APOPTOSISAPOPTOSIS NECROSISNECROSIS

Role of mitochondria & cytochrome c

No role for mitochondria

No leak of lysosomal enzymes

Leak of lysosomal enzymes

Characteristic nuclear changes

Nuclei lost

Apoptotic bodies form Do not form

DNA cleavage No DNA cleavage

Activation of specific proteases

No activation

Regulatable Not regulated

APOPTOSIS NECROSIS

Evolutionarily conserved

Not conserved

Dead cells ingested by neighboring cells

Dead cells ingested by neutrophils &macrophages

START S PHASE G2 PHASE MITOSIS

DNA synthesis

E2F

serum RB

Cyclin DCDK4

Cyclin ECDK2

Cyclin ACDK2

Cyclin ACDC2

Cyclin BCDC2

p16p21 p27

Cyclin D

degradation

Cyclin Eproteolysis

Cyclin A & B proteolysis

p53

DNA damage

TGF beta

cdc25A CDC25c

Interaction of the cyclins