THE CELL CYCLE

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THE CELL CYCLE

• Basic Cell Biology
• GENETICS and BIOCHEMISTRY
• cdks and CYCLINS
• CELL CYCLE CHECKPOINTS
• Mechanisms of Carcinogenesis
• TUMOR SUPPRESSOR GENES
• ONCOGENES
• ? Exploitable Targets in Cancer Therapy

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THE CELL CYCLE
S
G1
G2
M
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CELL CYCLE - Genetics

• MUTANTS OF YEAST
• STUCK AS BIG CELL AND CANNOT DIVIDE
• CLASSICAL GENETICS
• FIND COMPLEMENTING GENE AND ISOLATE
• PAUL NURSE ISOLATED cdc2
• COMPLEMENTATION FROM YEAST TO MAN

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THE CELL CYCLE 3 basic components

• CYCLIN DEPENDENT KINASES (cdk)
• CYCLINS
• Regulators of CYCLIN/cdk
• Activating Phosphatases
• Inhibitory Kinases
• Non-kinase inhibitors

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CYCLIN DEPENDENT KINASES
tyr15-P
thr14-P
P-thr161
- protein kinase - binds to cyclin - kinase
domain - regulatory domain - present throughout
cell cycle
e.g. cdk1 ( cdc2)
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CYCLINS
- no intrinsic enzymatic activity - binds cdk -
synthesized and degraded each cycle - essential
component for cdk activity
e.g. Cyclin B
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CYCLIN/CDK
tyr15-P
thr14-P

• Regulated by
• -tyr15 phosphorylation
• inhibitory kinases
• activating phosphatases
• -direct interaction
• inhibitory proteins
• e.g. p21, p27, p57
• p16, p15, p18,p19

cdk1 (cdc2)
P-thr161
cyclin B
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FROM YEAST TO MAN cdksconservation of sequence
across evolution
cdk1 G2/M transition

• budding yeast (S cerevisiae) cdc28
• fission yeast (S pombe) cdc2
• man cdc2 homolog p34

cdk2 G1/S transition

• budding yeast (S cerevisiae) cdc28
• fission yeast (S pombe) cdc2 (S form)
• man cdk2 p33 - PSALRE motif

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CYCLINS cell cycle phase specific?FUNCTION IN
CELL CYCLE

• CYCLIN B G2/M with cdc2 cdc13 CLB1-4
• CYCLIN A S G2 with cdk2 ?cig2 CLB5,6
• CYCLIN E G1/S with cdk2 ?cig1 ?puc1
  CLN1,2,3
• CYCLIN D G1 with cdk4,5,6 ? in yeast (early
  vs late G1)
• CYCLIN C present through cycle CTD of RNA pol
  II
• CYCLIN F peaks like A ?cyclin stability and
  proteolysis
• CYCLIN G induced 3 hr growth stim and by p53
  ?function
• CYCLIN H part of CAK (with cdk7)
• CYCLIN J sorry, it exists

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CELL CYCLE CHECKPOINTS
CYCLIN E / cdk2
CYCLIN A/ cdk2
S
G1
G2
CYCLIN D / cdk4,5,6
M
CYCLIN B/ cdc2
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CELL CYCLE CHECKPOINTS

• MITOSIS ENTRY (G2/M)
• Replication Complete
• Growth/ Protein Synthesis adequate
• No DNA Damage
• S-PHASE ENTRY (G1/S)
• Mitosis Complete ?signal - cyclin degradation
• Growth/ Protein Synthesis (G1 CYCLINS)
• No DNA Damage
• OTHERS
• MITOSIS EXIT ?coupling to S-phase
• S PHASE coupling to mitosis
• also in response to DNA damage
• G1 sequence of events
• signaling from cell surface

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DNA Damage - Cell Cycle Arrestdamage dependent
checkpoints
G1 - S - G2
G1 - S - G2
CELL No.
wild-type
DNA content
DNA content
X-ray treated G1/S block G2/M block (6-9 hours)
asynchronous
loss of G1/S in p53 deficient cells
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G2/M CHECKPOINT IN RESPONSE TO DAMAGE

• MAMMALIAN CELLS
• chk1/chk2 kinases involved
• ?upstream pathway uncertain ?ATM ?ATR
• many other proteins e.g. 9-1-1 complex
• YEAST
• RAD9 radiation sensitive (resistant with MBC
  G2 hold)
• cf RAD52 radiation sensitive /- MBC
• RAD17,24 MEC3 late S signal (RAD9)
• MEC1,2 mitosis entry (late S or pre-S)

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S PHASE CHECKPOINT IN RESPONSE TO DAMAGE

• MAMMALIAN CELLS
• involves Rad50/mre11/NBS1
• ?upstream pathway uncertain ?ATM ?ATR
• many other proteins ?BRCA1? 9-1-1 complex
• YEAST (pombe)
• RAD9/ RAD1/ Hus1
• cdc18
• Rad17, Rad24

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G1/S CHECKPOINTRESPONSE TO DNA DAMAGE

• In Mammalian Cells
• A p53 DEPENDENT PATHWAY
• Upstream activators of p53 ATM/DNA-PK?/
• activity of protein to transactivate
• levels of protein
• Effector Pathway p21 (WAF1/cip1/sdi1)
  transactivated
• p21 inhibits CYCLIN E or A / cdk2 CYCLIN D /
  cdk4
• N-terminal of p21 inhibitory domain
  interaction with cyclin has been demonstrated by
  X-ray crystallography

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G1/S CHECKPOINTIN RESPONSE TO DAMAGE
X-rays
P-tyr15
cdk2
strand break
p53
p21
ATM
cyclin E
p21 CKI class (cyclin dependent kinase
inhibitors) N-terminal of p21 forms complex with
cyclin / cdk - inhibit kinase
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Cell Cycle Regulators and Cancer
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STABLE STATES IN CELL CYCLEtimes when the cell
can pause in the cell cycle

• G0 (EARLY G1)
• G0 NUTRIENT DEPLETED
• G0 GROWTH ARRESTED eg cell contact
• POST-REPLICATION
• FUNCTION UNKNOWN
• METAPHASE ARREST
• MITOSIS ENTRY BUT NOT EXIT
• cyclin protease
• cyclin destruction
• allow chromosome separation

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Spindles and Chromosomesmore checkpoints!
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CONCLUSIONS

• CDKs, CYCLINs and REGULATORS of Cyclin/cdks
• CELL CYCLE EVENTS ARE COUPLED TO MAINTAIN THE
  CORRECT SEQUENCE
• G0/G1, G1/S and Exit from Mitosis TRANSITIONS
  THAT ARE OFTEN ALTERED IN CELL TRANSFORMATION/
  ONCOGENESIS
• DNA DAMAGE LEADS TO RAPID CESSATION OF DNA
  SYNTHESIS, and CYCLE ARREST AT G1/S and G2/M
• STABLE STATES ARE G0, S exit, M exit
• G2/M TRANSITION HAS GREATER IMPACT ON CELL
  SURVIVAL AFTER DNA DAMAGE, RELATIVE TO G1/S