Transcript powerpoint

Cell-Cycle
Regulation and the
Genetics of Cancer
Outline
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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
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The normal cell division
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Cyclin-dependent kinases
(CDKs) collaborate with
cyclins to ensure the proper
timing and sequence of cellcycle events
Experiments with yeast helped
identify genes that control cell
division
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Properties of yeast
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Grow as haploid or diploid organisms
Can identify recessive mutations in haploids
 Complementation analysis in diploids
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Budding – daughter cell arises on surface
of mother cell and grows in size during cell
cycle. Helps determine stage of cell cycle.
Isolation of temperaturesensitive mutants in yeast
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Mutants grow
normally at
permissive
temperature
Mutants loses gene
function at
restrictive
temperature
Thousands of cell
cycle mutants have
been identified
A cell-cycle mutant in yeast
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(a) growth at
permissive
temperature displays
buds of all sizes
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(b) growth at
restrictive
temperature shows
cells have finished first
cell cycle and arrested
in the second
A double mutant reveals which
mutation is needed
Human CDKs and cyclins can function in
yeast in place of native proteins
70 cell-cycle genes identified through
temperature-sensitive mutation screens
Cell Cycle Control:
Cyclin-dependent kinases (CDK) and their
regulatory subunits, the cyclins
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
Cyclin – CDK interactions
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)
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.
Cell cycle: cyclin guides the way by C Wittenberg
Nature 434, 34-35 (3 March 2005)
Higher eukaryotes have more forms of both
cyclins and CDKs compared to lower
eukaryotes.
CDK1
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
E.g. Nuclear
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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
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
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
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
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)
REGULATON OF CYCLIN–CDK ACTIVITY
4. Intracellular localisation
Intracellular localization of different cell cycleregulating proteins also contributes to CDK
regulation
Checkpoints integrate repair of chromosome
damage with events of cell cycle
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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)
G1-S phase transition
Mutations in p53 disrupt G1-S transition
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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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)
S phase checkpoint
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 3kinase (PI-3 kinase) and serves as a checkpoint gene in response to
DNA damage. ATM is central to dsb responses.
G2-M transition is controlled by
phosphorylation and dephosphorylation
Two checkpoints act at the G2-M transition
double strand breaks
Checkpoint in M
spindle damage
Checkpoints ensure genomic
stability
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Defective checkpoints
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Chromosome aberrations
Aneuploidy
 Changes in ploidy
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Single-stranded nicks – normally repaired
in G1 phase
 Chromosome loss or gain – normally
corrected in G2-M checkpoint
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Three classes of error lead to
aneuploidy in tumor cells
Normal cells
Cancerous cells
General reading:
MBoC by Alberts et al (4th ed): pgs 863-906 OR
Cancer Biology by RJB King : pgs 148 - 158……..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) pp131-149.
Cell cycle by Gary S Stein et al www.els.net