1. BB20023/0110: Cell cycle and cancer
Cell-Cycle Regulation and the Genetics of Cancer
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2. Outline The control of cell division Normal cell cycle
Yeast as a model organism
Cell cycle control: Molecular Mechanisms
Regulation of cyclin-CDK activity
Checkpoints that regulate passage through cell cycle

3. The normal cell division
BB20023/0110: Cell cycle and cancer
The normal cell division
Cyclin-dependent kinases (CDKs) collaborate with cyclins to ensure the proper timing and sequence of cell-cycle events
Figure 18.2
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4. Experiments with yeast helped identify genes that control cell division
Properties of yeast
Grow as haploid or diploid organisms
Can identify recessive mutations in haploids
Complementation analysis in diploids
Budding – daughter cell arises on surface of mother cell and grows in size during cell cycle. Helps determine stage of cell cycle.

5. Isolation of temperature-sensitive mutants in yeast
BB20023/0110: Cell cycle and cancer
Isolation of temperature-sensitive mutants in yeast
Mutants grow normally at permissive temperature
Mutants loses gene function at restrictive temperature
Thousands of cell cycle mutants have been identified
Figure 18.3
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6. A cell-cycle mutant in yeast
BB20023/0110: Cell cycle and cancer
A cell-cycle mutant in yeast
(a) growth at permissive temperature displays buds of all sizes
(b) growth at restrictive temperature shows cells have finished first cell cycle and arrested in the second
Figure 18.4
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7. A double mutant reveals which mutation is needed
BB20023/0110: Cell cycle and cancer
A double mutant reveals which mutation is needed
Figure 18.5
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8. BB20023/0110: Cell cycle and cancer
Human CDKs and cyclins can function in yeast in place of native proteins
Figure 18.8
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9. 70 cell-cycle genes identified through temperature-sensitive mutation screens

10. BB20023/0110: Cell cycle and cancer
Cell Cycle Control: Cyclin-dependent kinases (CDK) and their regulatory subunits, the cyclins
Figure 18.7a
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11. BB20023/0110: Cell cycle and cancer
Cyclins
50-90kDa proteins with conserved ‘cyclin box’ region
Forms 5 alpha helices.
Conservation between Rb and TFIIB suggests that cyclin box domain regulates protein interactions related to cdk regulation and transcription
Cyclins so called because their levels rise and fall during the cell cycle
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12. Cyclin – CDK interactions
BB20023/0110: Cell cycle and cancer
Cyclin – CDK interactions
Cdk – blue; cyclin - purpleActivation occurs through
phosphorylation of the T loop (green) and the binding of cyclin (purple) at the PSTAIRE helix (red). These events lead to a conformational change that
produces a functional active site (yellow,
Cdks ( blue) by themselves are inactive.
Activation occurs through phosphorylation of the T loop (green) and the binding of cyclin (purple) at the PSTAIRE helix (red). These events lead to a conformational change that produces a functional active site (yellow)
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13. How enzymes select their substrate
BB20023/0110: Cell cycle and cancer
How enzymes select their substrate
a, b, In general, enzymes recognize their targets through structural complementarity between the substrate and the enzyme's active site (indicated here by the shape of the 'pocket'). Small substrates (a) and relatively small modification sites on proteins (b) can be recognized by this mechanism.
c, Some enzymes make additional, specific contacts with the substrate that enable them to distinguish between proteins that have identical or related sites of modification.
d, cyclin-dependent protein kinases (CDKs) have relegated that function to the exchangeable cyclin subunit, enabling a single CDK catalytic subunit to exist in numerous forms with different specificities.
Cdk – blue; cyclin - purpleActivation occurs through
phosphorylation of the T loop (green) and the binding of cyclin (purple) at the PSTAIRE helix (red). These events lead to a conformational change that
produces a functional active site (yellow,
Cell cycle: cyclin guides the way by C Wittenberg
Nature 434, (3 March 2005)
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14. BB20023/0110: Cell cycle and cancer
Higher eukaryotes have more forms of both cyclins and CDKs compared to lower eukaryotes.
Figure 18.7a
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15. BB20023/0110: Cell cycle and cancer
Cyclin-dependent kinases (CDKs) control the cell cycle by phosphorylating specific serine and threonine residues of select proteins during different phases of the cell cycle other proteins
Figure 18.7a
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16. BB20023/0110: Cell cycle and cancer
E.g. Nuclear lamins
CDK substrates
Underlie inner surface of the nuclear membrane
Probably provide structural support for nucleus
May also be site for assembly of DNA replication, transcription, RNA transport, and chromosome structure proteins
Dissolution of nuclear membrane during mitosis is triggered by CDK phosphorylation of nuclear lamins
Figure 18.7b
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17. REGULATON OF CYCLIN–CDK ACTIVITY
1. Cyclin availability
Association with a cyclin is absolutely required for Cdk activity.
Cyclin levels can be changed by transcriptional regulation and/or by ubiquitin-dependent proteolysis.
E.g. cyclins D and E contain a PEST sequence [segment rich in proline(P), glutamic acid (E), serine (S) and threonine (T) residues]: which are required for efficient ubiquitin-mediated cyclin proteolysis at the end of a cell cycle

18. REGULATON OF CYCLIN–CDK ACTIVITY
2. Inhibitory phosphorylation
Cyclin–Cdk complexes can also be inactivated by phosphorylation of tyrosine and threonine residues close to the active site of the Cdk subunit.
This phosphorylation is mediated by Wee1-type protein kinases, and the inhibitory phosphate groups are removed by Cdc25-type phosphatases

19. REGULATON OF CYCLIN–CDK ACTIVITY
3. Stoichiometric inhibition
CDK activity can be regulated by stoichiometric inhibitors (cyclin kinase inhibitors-CKIs), which bind to CDK alone or to the CDK-cyclin complex and regulate CDK activity.
Lower eukaryotes possess a single CycB–Cdk1 specific inhibitor, whereas in higher eukaryotes 2 distinct families exist; the INK4 family and Cip/Kip family

20. REGULATON OF CYCLIN–CDK ACTIVITY
3. Stoichiometric inhibition…
CKIs are regulated both by internal and external signals:
E.g. expression of p21 is under transcriptional control of the p53 tumour suppressor gene (internal), whereas the expression and activation of p15 and p27 increases in response to transforming growth factor b (TGF-b), contributing to growth arrest (external)

21. REGULATON OF CYCLIN–CDK ACTIVITY
4. Intracellular localisation
Intracellular localization of different cell cycle-regulating proteins also contributes to CDK regulation

22. BB20023/0110: Cell cycle and cancer
Checkpoints integrate repair of chromosome damage with events of cell cycle
G1-S checkpoint
p53 – transcription factor that induces expression of DNA repair genes and CDK inhibitor p21
p53 pathway activated by ionizing radiation or UV light (causing DNA damage) during G1 phase delays entry into S phase
DNA is repaired before cell cycle continues
If DNA is badly damaged cells commit suicide (programmed cell death or apoptosis)
Figure 18.11a
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23. BB20023/0110: Cell cycle and cancer
G1-S phase transition
Figure 18.9
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24. Mutations in p53 disrupt G1-S transition
BB20023/0110: Cell cycle and cancer
Mutations in p53 disrupt G1-S transition
Figure 18.11a
Gene amplification in tumour cells that appear as homogenously staining regions (HSR)
Small chromosome-like bodies (called minutes) in tumour cells that lack centromeres and telomeres
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25. BB20023/0110: Cell cycle and cancer
p53 mutants do not induce p21 and cell cycle is not arrested
Cells replicate damaged DNA
Cells die or DNA is degraded and cell is engulfed and digested by neighboring cells (apoptosis, or programmed cell death)
Figure c,d
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26. BB20023/0110: Cell cycle and cancer
S phase checkpoint
codes for a protein kinase with homology to the catalytic domain of
phosphatidylinositol 3-kinase (PI-3 kinase) and serves as a checkpoint gene in response to DNA damage.
Individuals affected by ataxia telangiectasia (AT), an autosomal recessive disorder, are unable to slow down DNA replication after exposure to radiation. The AT gene (ATM) codes for a protein kinase with homology to the catalytic domain of phosphatidylinositol 3-kinase (PI-3 kinase) and serves as a checkpoint gene in response to DNA damage. ATM is central to dsb responses.
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27. G2-M transition is controlled by phosphorylation and dephosphorylation
BB20023/0110: Cell cycle and cancer
G2-M transition is controlled by phosphorylation and dephosphorylation
Figure 18.10
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28. Two checkpoints act at the G2-M transition double strand breaks
BB20023/0110: Cell cycle and cancer
Two checkpoints act at the G2-M transition double strand breaks
Figure 18.12a
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29. Checkpoint in M spindle damage
BB20023/0110: Cell cycle and cancer
Checkpoint in M spindle damage
Figure 18.12b
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30. Checkpoints ensure genomic stability
Defective checkpoints
Chromosome aberrations
Aneuploidy
Changes in ploidy
Single-stranded nicks – normally repaired in G1 phase
Chromosome loss or gain – normally corrected in G2-M checkpoint

31. Three classes of error lead to aneuploidy in tumor cells
BB20023/0110: Cell cycle and cancer
Three classes of error lead to aneuploidy in tumor cells
Figure 18.13a
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32. BB20023/0110: Cell cycle and cancer
Normal cells
Cancerous cells
Figure b
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33. BB20023/0110: Cell cycle and cancer
General reading:
MBoC by Alberts et al (4th ed): pgs OR
Cancer Biology by RJB King : pgs ……..OR
Chapter 9 Mol & Cell Biol of Cancer by Knowles and Selby
Optional reading:
The cell cycle: a review…. targets in cancer by K Vermeulen, DR. Van Bockstaele and ZN. Berneman Cell Proliferation (June 2003) 36(3) pp
Cell cycle by Gary S Stein et al
Figure b
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