Cell cycle I

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BISC666
Targeting Cell Cycle Regulators in Medicine
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Overview

• Mechanisms of cell cycle control
• Mechanisms of checkpoints
• Targets for cancer therapies
• basis, examples, problems

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(No Transcript)
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The stages outside M phase are collectively
known as interphase. The cell cycle is
completed with the separation of the two daughter
cells (cytokinesis). Most non-dividing cells
exit the cell cycle at G1 into G0 (quiescence).
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Mitosis
Prophase
Metaphase
Prometaphase
Anaphase
Telophase
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Mitosis
Molecular Biology of the Cell Vol. 9, 1603-1607,
July 1998
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From http//www.sciencemag.org/cgi/content/full/3
00/5616/91/DC1
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Cell cycle phase can be observed by DNA staining
followed by flow cytometry
DNA is stained with a fluorescent dye
(propidium iodide). The intensity of staining
is proportional to the amount of DNA in
individual cells. The DNA content of a large
number of cells is analyzed.
Methods
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No. of cells
G1
G2
S
DNA content
Methods
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BrdU (bromodeoxyuridine) labeling followed by
flow cytometry is more accurate. (BrdU is a dNTP
analogue)
Methods
Source Poon Lab
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Although both the duration and the morphology
of the cell cycle vary greatly between different
types of cells, but the underlying successive
processes of growth, DNA replication, mitosis,
and cytokinesis are the same. At the molecular
level, the components and the mechanisms that
control the cell cycle are conserved throughout
evolution, from yeast to insects to frogs to
human.
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The cell cycle turns out to be driven by a cell
cycle engine composed of protein kinases
(cyclin-dependent kinases) and their regulatory
subunits (cyclins). Proteolysis also turns out to
be a key mechanism that regulate the cell
cycle. It was an important revelation that
likes so many other processes in the cell, the
cell cycle is regulated by phosphorylation and
proteolysis.
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Cyclin A
CDK2
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Different cyclins and CDKs control different
points in the cell cycle
Cyclin B-CDC2
Cyclin A-CDC2
Cyclin D-CDK4 Cyclin D-CDK6
Cyclin E-CDK2
Cyclin A-CDK2
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Cyclin-dependent kinases (CDKs) are defined as
protein kinases whose kinase activities are
depended on binding to a cyclin subunit.
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Cyclins are defined as protein with sequence
homology to the originally discovered mitotic
cyclins (A and B).
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Cyclins and CDKs Cell Cycle functions
Modified from Murray and Marks (2001) Nature 409,
844-846
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Cyclins and CDKs Transcription functions
Modified from Murray and Marks (2001) Nature 409,
844-846
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Cyclins and CDKs Other/Unknown functions
Modified from Murray and Marks (2001) Nature 409,
844-846
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Evidence of the functions of different cyclin-CDK
in the cell cycle
G2/M cyclin-CDK see MPF as described above.
G1/S cyclin-CDK were studied by -
overexpression of the protein shorten G1 phase
- micro-injection of specific antibodies or
antisense oligonucleotides lengthen G1
Cyclin B-CDC2
Cyclin D-CDK4 Cyclin D-CDK6
Cyclin A-CDC2
Cyclin E-CDK2
Cyclin A-CDK2
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G2 - mitosis
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M-phase promoting factor (MPF) is composed of
cyclin B and CDC2
MPF was first purified from Xenopus eggs and
starfish. Purified MPF contains two polypeptides
CDC2 and cyclin B
Oocyte (G2)
Oocyte (G2)
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This activity is termed Maturation-Promoting
Factor (MPF)
MPF can be found in mitotic cells in organisms
ranging from yeast to human. MPF can also drive
G2-arrested cells into mitosis, and is
interchangeable between different organisms. -
indicate that MPF is not only able to induce
certain special meiotic G2-arrested cells into M
phase, but represents a universal M-phase
activator in all eukaryotic cells (hence also
called M phase-Promoting Factor).
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Substrates of MPF
Very little is known about the substrates of MPF,
but its phosphorylation of lamins and breakdown
of nuclear lamina is understood, and provides
some insight into how other mitotic events may be
regulated by MPF.
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Substrates of MPF
lamins (A, B and C) ? dimers ? intermediate
filaments ? nuclear lamina ? nuclear envelope
(DNA)
Source Poon Lab
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Substrates of MPF
MPF phosphorylates a serine residue in lamins ?
depolymerization of intermediate filaments.
Containing phosphorylated lamins
Nuclear envelope
Chromatin
Phosphatase
MPF
Mitosis
Interphase
Interphase
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Substrates of MPF
Evidence lamins satisfied all the requirements
as substrates for MPF (see above) when mutant
lamins (non-phosphorylatable mutant changing Ser
to Ala) were put into cells, the lamins no longer
depolymerized and the nuclear envelop did not
break down during mitosis.
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Dephosphorylation trigger exit from mitosis
Inactivation of MPF coincides with the late
stages of mitosis (anaphase and telophase)
sister chromatids separate and move to opposite
poles chromosomes decondense nuclear envelope
re-forms endoplasmic reticulum and Golgi are
remodeled cytokinesis Dephosphorylation is
carried out by various phosphatases including
PP2A and CDC14
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Dephosphorylation trigger exit from mitosis
Dephosphorylation of lamins leads to formation
of nuclear lamina around each daughter nucleus.
Containing phosphorylated lamins
Phosphatase
Mitosis
Interphase
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Degradation of mitotic cyclins
Mitotic cyclins are degraded rapidly at the
onset of anaphase.
Metaphase
Anaphase
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Prophase
Metaphase
Prometaphase
Anaphase
Telophase
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Mitotic cyclins are degraded by ubiquitination
Cyclin degradation requires the destruction box
sequence
Destruction box
Cyclin box
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Mitotic cyclins are degraded by ubiquitination
Evidence cyclin mutants that lack the
destruction box are not degraded transfer of
the destruction box to another protein leads to
the degradation of the fusion protein during
anaphase
Degraded
Stable
Degraded
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Mitotic cyclins are degraded by ubiquitination

• Cyclin degradation involves ubiquitination.
• involves a recognition protein that binds
  specially to the cyclin destruction box, and
  directs ubiquitin ligase to covalently attach
  multiple copies a small protein called ubiquitin
  to lysine residues in cyclin.
• - polyubiquitinated proteins are then degraded by
  multiprotein complexes of proteolytic enzymes
  called proteasomes.

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The enzymology of ubiquitination
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Mitotic cyclins are degraded by ubiquitination
Cyclin
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Mitotic cyclins are degraded by ubiquitination

• The ubiquitin ligase for mitotic cyclins is a
  large complex called the anaphase-promoting
  complex / cyclosome (APC/C).
• APC/C is a large multi-subunit complex. It
  contains a targeting subunit called CDC20 that
  targets cyclin (and some other proteins) for
  ubiquitination.

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Mitotic cyclins are degraded by ubiquitination

• The ubiquitin ligase for mitotic cyclins is a
  large complex called the anaphase-promoting
  complex / cyclosome (APC/C).
• APC/C is a large multi-subunit complex. It
  contains a targeting subunit called CDC20 that
  targets cyclin (and some other proteins) for
  ubiquitination.

CDC2
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Mitotic cyclins are degraded by ubiquitination
APC/C-CDC20 is activated by cyclin B-CDC2
itself.
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Mitotic cyclins are degraded by ubiquitination
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Mitotic cyclins are degraded by ubiquitination

• APC/C-CDC20 is activated at the onset of
  anaphase

another APC/C complex, APC/C-CDH1, is activated
after mitosis. unlike APC/C-CDC20, APC/C-CDH1
is inhibited by cyclin B-CDC2.
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Mitotic cyclins are degraded by ubiquitination
Mitosis
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Mitotic cyclins are degraded by ubiquitination
G1
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Mitotic cyclins are degraded by ubiquitination
G1
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Mitotic cyclins are degraded by ubiquitination
G1
Prevents activation of CDC2 in G1
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APC/C also triggers sister chromatids separation
Securin also contains a similar D-box as cyclin
Securin
Separase
Cohesin
Cohesin
Cohesin
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APC/C also triggers sister chromatids separation
Separase
Cohesin
Cohesin
Cohesin
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APC/C also triggers sister chromatids separation
Separase
Cohesin
Cohesin
Cohesin
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APC/C also triggers sister chromatids separation
Separase
Sister chromatids separation
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APC/C also triggers sister chromatids separation
Prophase
Metaphase
Prometaphase
Anaphase
Telophase
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SCF Complexes
Another major type of ubiquitin ligase in cell
cycle control is the SCF complexes.
Hermand Cell Division 2006 130
doi10.1186/1747-1028-1-30
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The activity of CDC2 is also regulated by
phosphorylation
What is the underlying reason for this lag in
the activation of MPF?
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The activity of CDC2 is also regulated by
phosphorylation

• Effects on CDC2
• Cyclins
• Phosphorylation on Thr14 and Tyr15 -
• Phosphorylation on Thr161

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The activity of CDC2 is also regulated by
phosphorylation
Cyclin B1
OFF
CDC2
T161
T14/Y15
WEE1 MYT1
CAK
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The activity of CDC2 is also regulated by
phosphorylation
Cyclin B1
OFF
CDC2
CDC25C
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The activity of CDC2 is also regulated by
phosphorylation
Cyclin B1
ON
CDC2
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Cyclin-CDK complexes act autocatalytically to
stimulate its own activation (positive / negative
feedback loops).
Cyclin B1
WEE1 MYT1
CDC25
CDC2
Phosphorylation of CDC25 by active cyclin
B-CDC2 activates more CDC25 Phosphorylation of
WEE1/MYT1 by active cyclin B-CDC2 inactivates
WEE1/MYT1
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However, it is not clear how the first
cyclin-CDC2 is activated. One mechanism may
involve activation of CDC25 by another protein
kinase Polo-like kinase (PLK1) partial
activation of CDC2 by PLK1 leads to activation of
the feedback loop and complete activation of CDC2.
Cyclin B1
WEE1 MYT1
CDC2
CDC25
PLK1
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Events during G2-M
Cyclin B1
CDC2
WEE1 MYT1
CAK
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Cyclin B1
CDC2
CDC25
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Cyclin B1
CDC2
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APC/C
Cyclin B1
CDC2
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APC/C
CDC2
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G1 - S phase
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One major substrate of G1 cyclin-CDK complexes is
the retinoblastoma gene product (pRb)
pRb was first found to be mutated in
retinoblastoma (and later in many cancers)
(Image from Wikipedia)
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One major substrate of G1 cyclin-CDK complexes is
the retinoblastoma gene product (pRb)
pRb is hypophosphorylated for most part of the
cell cycle. Hypophosphorylated pRb sequesters
the transcription factor E2F. When pRb is
hyperphosphorylated by G1 cyclin-CDK complexes
(cyclin D-CDK4/6 and cyclin E-CDK2) ? E2F is
released ? E2F is allowed to activate
transcription of genes essential for G1/S
transition.
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Events during G1-S
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HDAC
pRb
E2F
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HDAC
pRb
E2F
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HDAC
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One major substrate of G1 cyclin-CDK complexes is
the retinoblastoma gene product (pRb)
Histone deacetylase (HDAC) binding to Rb is
important for Rbs inhibition of E2F
transcriptional activity because deacetylation
of histones is believed to turn off gene
transcription.
Rb
P
HDAC
P
Rb
P
HDAC
E2F
E2F
Ac
Ac
OFF
ON
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Degradation of G1 and S phase cyclins
Like the mitotic cyclins, other cyclins are
also degraded by ubiquitination-dependent
proteolysis. The ubiquitin ligase for these
cyclins SCF complexes.
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Cyclin-CDK and DBF4-CDC7 control DNA replication
Major questions How DNA replication is
coordinated in the cell cycle? How replication
is initiated once and once only (how to prevent
re-replication of the same DNA)?
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ORC protein complex binds DNA all the time
ORC
Origin of replication
G1 phase
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Pre-replicative complex
MCM2-7
CDC6
CDT1
MCM10
ORC
G1 phase
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MCM2-7
CDC6
CDT1
MCM10
ORC
G1 phase
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Phosphorylation of MCM proteins stimulates
binding of CDC45
MCM2-7
CDC6
CDT1
MCM10
ORC
CDC45
S phase
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MCM2-7
CDT1
MCM10
CDC6
RPA stabilizes ssDNA
ORC
DNA pol
CDC45
Unwinding of origin
Initiation of DNA replication
S phase
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Preventiion of re-replication
MCM2-7
CDT1
MCM10
CDC6
ORC
DNA pol
CDC45
(1) The disassembly of pre-replicative complex
prevents the re-firing from the same origin.
S phase
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MCM2-7
CDT1
MCM10
CDC6
ORC
DNA pol
CDC45
(2) S phase cyclin-CDK also phosphorylates CDC6,
leading to its degradation.
S phase
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MCM2-7
CDT1
MCM10
ORC
DNA pol
CDC45
(2) S phase cyclin-CDK also phosphorylates CDC6,
leading to its degradation.
S phase
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MCM2-7
CDT1
Geminin
MCM10
ORC
DNA pol
CDC45
S phase
(3) CDT1 is inhibited by Geminin.
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Reset for next cycle
APC/C
MCM2-7
CDT1
Geminin
MCM10
ORC
M phase
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Reset for next cycle
CDK2
APC/C
MCM2-7
CDT1
MCM10
ORC
M phase
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Reset for next cycle
MCM2-7
CDT1
MCM10
CDC6
ORC
G1 phase
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Reset for next cycle
Pre-replicative complex
MCM2-7
CDC6
CDT1
MCM10
ORC
G1 phase
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Cancer Therapies Approaches

• Selectively kill / inhibit cancer cells while
  sparing normal cells.
• Agents that inhibits the cell cycle engine

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CDK inhibitors
Inhibition of CDKs by small inhibitors will
stop the cell cycle. But is it possible to
selectively interrupt the cell cycle regulation
in cancer cells ? The idea is that cancer
cells may be more sensitive to killing after cell
cycle inhibition than normal cells. Since
cancer cells divide much faster than
non-cancerous cells, they are far more
susceptible to agents that targets the cell
cycle.
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CDK inhibitors
Example Flavopiridol a flavone
synthetically derived from an alkaloid isolated
from leaves and stems of Amoora rohituka and
Dysoxylum binectariferum (plants indigenous to
India). a pan-CDK inhibitor induced cell
cycle arrest. the first CDK inhibitor tested in
clinical trials (Senderowicz et al., 1998).
phase I clinical trials 200-400 nM inhibits
CDKs. phase II clinical trials poor results.
likely to have many targets other than CDKs, e.g.
potent inhibitor of global transcription by
inhibiting the transcriptional elongation factor
P-TERb. Review Cell Cycle. 2004
Dec3(12)1537-42.
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CDK inhibitors
Example R-roscovitine (Seliciclib /CYC202)
a purine analog inhibits several CDKs (CDK2,
CDK7, CDK1, CDK9) induces apoptosis
Currently undergoing Phase II clinical trial of
non-small cell lung cancer (NSCLC).
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CDK inhibitors
Crystal Structure of human CDK2 complexed with
roscovitine From European J. Biochem. 243 518
(1997)
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Checkpoints
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Concepts
It is essential to regulate the cell cycle to
ensure that each stage of the cell cycle is
completed before the next stage is initiated.
Important checkpoints in the cell cycle sense
DNA damage Incomplete DNA replication
Incomplete spindles assembly
To ensure that the cell does not enter the next
phase of the cell cycle with damaged DNA,
incompletely replicated DNA, or incompletely
assembled spindles.
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Concepts
Most checkpoints are mechanisms that inhibits
the normal cell cycle machinery. Mutation of
checkpoint mechanisms allow cells to proceed to
the next stage of the cell cycle without
completing the preceding stage or with damaged
DNA. These lead to genome instability and
cancer.
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Concepts

• Defects in these checkpoints may result in
  genome instability and have been linked with
  diseases like ataxia telangiectasia (AT, see
  later) and cancer.
• Genome instability
• at the DNA base levels
• at the chromosomal levels

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Spectral karyotype (SKY)
A visualization of all of an organism's
chromosomes together, each labeled with a
different color. This technique is useful for
identifying chromosome abnormalities.
Methods
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Spectral karyotype (SKY)
A breast cancer cell line (MCF7)
Methods
From Cancer Genomics Program,University of
Cambridge, Departments of Pathology and Oncology
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Spectral karyotype (SKY)
By using a series of specific probes (chromosome
paint) each with varying amounts of the dyes,
different chromosomes have unique spectral
characteristics. Slight variations in color,
undetectable by the human eye, are detected by a
computer program.
Methods
From The Biology Project, University of Arizona
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Many checkpoint genes are first identified in
yeast
Defects in checkpoints usually sensitize the
cells to the checkpoint-inducing agents

• Discuss about yeast screens
• DNA damage checkpoint mutants
• DNA replication checkpoint mutants
• Spindle-assembly checkpoint mutants

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Spindle-Assembly Checkpoint
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Abnormal spindle mitosis
Normal mitosis
Missing chromosome
Extra chromosome
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Abnormal checkpoint
Normal mitosis
Normal checkpoint
nocodazole (disrupts spindles)
Block in mitosis
Exit mitosis without division
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Source Poon Lab
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movie
Poon Lab
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Spindle
Kinetochore
Spindle pole
Cohesin
Cohesin
Cohesin
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When not all kinetochores are attached
Cohesin
Cohesin
Cohesin
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Activates MAD2 mechanism unknown Involves
several proteins, Including MAD1, BUB1 (a protein
kinase). MAD2 is activated by a conformational
change.
MAD2
Cohesin
Cohesin
Cohesin
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MAD2
MAD2 inhibits APC/C-CDC20
MAD2
Cohesin
Cohesin
Cohesin
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MAD2
Securin
MAD2
Separase
Cell stay in prometaphase
Cohesin
Cohesin
Cohesin
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MAD2
Securin
When all the kinetochores are attached to the
spindles (i.e. metaphase)
Separin
Cohesin
Cohesin
Cohesin
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Securin
MAD2 signal disappear
Separase
Cohesin
Cohesin
Cohesin
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Separase
Cohesin
Cohesin
Cohesin
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Separase
Cohesin
Cohesin
Cohesin
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Separase
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Pre-replication complex
Separase
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Cancer Therapies Approaches

• Selectively kill / inhibit cancer cells while
  sparing normal cells.
• Agents that inhibits the cell cycle engine
• Agents that activates the checkpoints

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Checkpoint activator Taxol
Example Taxol (Paclitaxel) isolated from
the bark of the Pacific yew tree (Taxus
brevifolia). (now obtained by total syhthesis)
Image from http//en.wikipedia.org/wiki/Taxus_brev
ifolia
Image from http//en.wikipedia.org/wiki/Taxol
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Checkpoint activator Taxol
Clinically, Taxol is now used to treat patients
with cancer of the lung, ovarian, breast, head
and neck cancer, and advanced forms of Kaposi
sarcoma. Taxol inhibits depolymerization of
microtubules, locking the cells in mitosis,
leading to apoptosis. One possible mechanism
may be through phosphorylation of BCL2 (T69, S70,
S87) - inactivates BCL2 (Yamamoto et al., 1999).
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DNA Damage Checkpoints
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DNA damage induced by e.g. radiation
G1 Checkpoint
Entry into S phase will duplicate the damaged DNA
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G1 Checkpoint
Cell Cycle Arrest DNA Repair
(Option I)
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G1 Checkpoint
Apoptosis (see elsewhere)
(Option II)
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DNA damage induced by e.g. radiation
G2 Checkpoint
Entry into M phase will propagate the damaged
DNA to the daughter cells
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Flow cytometry reveals DNA damage checkpoints in
both G1 and G2
Wild type cells
No. of cells
No irradiation
G1
S
G2
No. of cells
After irradiation (8 hr)
G1
S
G2
Flow cytometry
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Summary
Responses after DNA damage

• Arrest in G1, repair DNA, before DNA replication
  (S).
• (2) Arrest in G2, repair DNA, before cell
  division (M).
• (3) Apoptosis.

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Normal G2-M
Cyclin B1
CDC2
WEE1 MYT1
CAK
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Cyclin B1
CDC25C
CDC2
CDC25A
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Cyclin B1
CDC2
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APC/C
Cyclin B1
CDC2
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APC/C
CDC2
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DNA Damage
Everything is normal up to the buildup of
inactive MPF
Cyclin B1
CDC2
WEE1 MYT1
CAK
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DNA Damage
14-3-3
ATM / ATR
Cyclin B1
CDC25C
CDC2
CHK1 / CHK2
CDC25A

• Phosphorylation of CDC25CSer216
• Inactivates phosphatase activity directly.
• Creates 14-3-3 binding site, masks a proximal NLS
  and sequesters CDC25C in the cytoplasm (cannot
  access nuclear cyclin B1-CDC2).

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DNA Damage
14-3-3
ATM / ATR
Cyclin B1
CDC25C
CDC2
CHK1 / CHK2
CDC25A
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DNA Damage
14-3-3
ATM / ATR
Cyclin B1
CDC25C
CDC2
CHK1 / CHK2
Degradation SCF?-TrCP-dependent
ubiquitination. Phosphorylation of CDC25ASer76 by
CHK1 is required for the phosphorylation of a
phosphodegron centered at Ser82 (by an
as-yet-unidentified kinase), creating a binding
site for ?-TrCP.
Cell stay in G2.
136
Ataxia-telangiectasia

• Ataxia-telangiectasia mutated (ATM) is mutated
  in the human genetic disorder ataxia-telangiectasi
  a (AT).
• AT
• Ataxia- Telangiectasia
• Immune System Problems
• Predisposition to Cancer 1,000 times more
  frequently than the general population mainly
  lymphoma and leukemia.

http//www.neuro.wustl.edu/neuromuscular/ataxia/dn
arep.html
137
ATM and ATR
AT cells exhibit an enhanced radiosensitivity,
radioresistant DNA synthesis, and chromosomal
instability. ATM encodes a product related to
phosphatidyl-inositol-3 kinases. ATR is a
protein kinase related to ATM. Mainly activated
after stall of replication (see Replication
checkpoint), UV, and hypoxia.
138
ATM and ATR
The upstream sensors that initiate the activation
of ATM/ATR consist of an intricate network of
large protein complexes, with many components
being BRCT domain-containing proteins
RAD9-HUS1-RAD1 (9-1-1) clamp RAD17-containing
clamp loader BRCA1, BLM, and the MRN
(MRE11-RAD50-NBS1) complex form part of a large
complex, termed BRCA1-associated genome
surveillance complex (BASC), and localize to DNA
lesion-associated nuclear foci also participate
in ATM/ATR activation
139
ATM and ATR
Histone H2AX is also phosphorylated at Ser139
(called gamma-H2AX) by ATM at DNA damage foci.
Confocal immunofluorescent analysis of NIH/3T3
cells, untreated (left) or etoposide-treated
(right), double-labeled with Phospho-Histone
H2A.X (Ser139) (20E3) Rabbit mAb (Alexa Fluor 488
Conjugate) (green) and beta-Tubulin Antibody
2146 (red). From Cell Signaling Technology
140
Cancer Therapies Approaches

• Selectively kill / inhibit cancer cells while
  sparing normal cells.
• Agents that inhibits the cell cycle engine
• Agents that activates the checkpoints
• Agents that bypass the checkpoints

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Checkpoint activator Uncoupling of checkpoints
Caffeine sensitized cells to UV (Rauth 1967),
and subsequently to many DNA damaging agents.
Targets of caffeine are ATM and ATR. Caffeine
cannot be used clinically because µM range is
required.
142
DNA Damage
Caffeine
Premature entry into mitosis
X
14-3-3
ATM / ATR
Cyclin B1
CDC25C
X
CDC2
CHK1 / CHK2
CDC25A
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Checkpoint activator Uncoupling of checkpoints
UCN-01 was 100,00 times more potent than
caffeine in disrupting checkpoint (Bunch and
Eastman , 1996) UCN-01 was found to bind
strongly to ?1 acid glycoprotein in human plasma,
leading to plasma concentration gt 30 µM (Fuse et
al., 1998 Sausville et al., 2001) - i.e.
probably difficult to obtain adequate amount of
free drug in the body. Many clinical trials in
combination with DNA damaging agents.
144
Checkpoint activator Uncoupling of checkpoints
Known to be a PKC inhibitor in the
beginning. UCN-01 target CHK1 (Busby et al.,
2000 Graves et al. 2000), and also CHK2 (Yu et
al., 2002) Probably many more targets.
145
CDK inhibitors
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HDAC
pRb
E2F
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HDAC
pRb
E2F
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HDAC
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Inhibits transcription
Transports to cytoplasm
Ubiquitination
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Inhibits transcription
Transports to cytoplasm
Ubiquitination
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DNA Damage
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DNA Damage
Ubiquitin-mediated degradation.
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DNA Damage
Redistribution of CDK inhibitors.
154
CDK inhibitors
Apart from cyclin binding and phosphorylation,
CDKs can also be turned off by binding to CDK
inhibitors Two families
155

• Effects on CDK
• Cyclins
• Phosphorylation on Thr14 and Tyr15 -
• Phosphorylation on Thr161
• CDK inhibitors -

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p27
Cyclin A
CDK2
157
DNA Damage
ATM, ATR CHK1, CHK2 p38
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DNA Damage
ATM, ATR DNA-PK CHK1, CHK2
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p53
A tumor suppressor - one of the best
characterized. Functions causes cell cycle
arrest / apoptosis after DNA damage and other
stresses (has been called Guardian of the
genome).
p53
DNA
160
p53

• Evidence
• (1) Mutated in more than 50 of all cancers.
• p53 KO mice viable, but develop tumor
  spontaneously.
• (3) Li-Fraumeni syndrome (germline mutation in
  p53 gene) cancer predisposition.

161
p53
A transcriptional factor. Function as a
tetramer. The majority of mutations of p53 in
cancers are in the DNA binding domain.
Transcriptional targets includes p21 (for cell
cycle checkpoint) BAX (for apoptosis - see later)
162
p53
Inhibited by binding to MDM2. MDM2 binds to
the N-terminal region of p53 (transcriptional
activation domain) and (1) Inhibits
transcription directly (2)Targets p53 for
ubiquitination (MDM2 itself is a ubiquitin
ligase) (3) Transport p53 out to the cytoplasm
(to be degraded by the proteasome)
163
p53
Phosphorylation of the N-terminal region of p53
after DNA damage - by ATM/ATR (Ser15) or
CHK1/CHK2 (Ser20) - prevents binding of MDM2 to
p53.
P
MDM2
ATM also activates p53 indirectly by
phosphorylating MDM2 (Ser395 in human MDM2),
which reduces the p53-inhibitory potential of
MDM2 by disrupting the nucleo-cytoplasmic
shuttling.
164
Wild type cells
p53-negative cells
No. of cells
No irradiation
No. of cells
G1
S
G2
G1
S
G2
After irradiation (8 hr)
No. of cells
No. of cells
G1
S
G2
G1
S
G2
165
pRb mutation
E2F drives cells into S phase prematurely
HDAC
X
166
p16 and ARF mutated in melanoma and many other
cancers. ARF is read from alternative reading
frame (!). p16 CDK inhibitor (specific for
CDK4 CDK6). ARF MDM2 inhibitor (sequester
MDM2 to the nucleolus).
167
p16 gene
Normal cells
p16
ARF
168
p16 gene
Normal cells
p16
ARF
169
X
p16 gene
p16 gene mutation
X
X
p16
ARF
170
X
p16 gene
p16 gene mutation
X
X
X
p16
ARF
No inhibition of CDK4/6, pRb phosphorylated
inappropriately
171
X
p16 gene
p16 gene mutation
X
X
X
p16
ARF
172
X
p16 gene
p16 gene mutation
X
X
X
p16
ARF
X
X
X
173
Viruses and Cancer
DNA viruses
Human Papillomaviruses (HPV) Cervical Cancer
With few exceptions, papillomaviruses are
exclusively epitheliotropic and cause a
self-limiting infection characterized by
abnormal maturation and differentiation of
epithelial cells. But HPV can also cause
cervical cancer and skin cancer. More than 70
different HPV types. 1/3 of known HPV types
preferentially infect epithelium of the genital
tract - probably one of the most frequently
acquired sexually transmitted diseases. Produce
benign genital warts, squamous intraepithelial
neoplasia, or latent infections.
174
Viruses and Cancer
DNA viruses
Human Papillomaviruses (HPV) Cervical Cancer
(Depending on HPV subtype) may later develop
squamous intraepithelial lesions of the cervix
and invasive cervical cancer. Cervical cancers
frequently contain HPV sequence integrated into
the host genome - integration appears to be
random. E6 and E7 proteins of HPV are expressed
at high level in cervical cancers. E6 binds and
destabilizes p53 E7 binds to pRb
175
HDAC
E7
pRb
E2F
176
E7 displaces E2F from pRb (even in the absence of
phosphorylation by cyclin-CDK). Cell enters S
phase inappropriately.
HDAC
E7
pRb
177
The cell responses by activation of p53 to induce
apoptosis.
HDAC
BAX
E7
pRb
Apoptosis
178
The virus counters by E6, which binds and
degrades p53
E6
HDAC
BAX
E7
pRb
Apoptosis
179
The virus counters by E6, which binds and
degrades p53
E6
X
HDAC
BAX
E7
pRb
X
Apoptosis
180
Cancer Therapies
Adenoviruses ONYX-015 (dl1520) is an
engineered adenovirus that lacks E1B-55K.

181
HDAC
E1A
pRb
E2F
182
E1A displaces E2F from pRb (similar to HPV
E7). Cell enters S phase inappropriately. Virus
can replicate.
HDAC
E1A
pRb
183
The cell responses by activation of ARF to
inactivate MDM2.
p16 gene
ARF
HDAC
E1A
pRb
184
p16 gene
Activation of p53 induces cell cycle arrest or
apoptosis. Limits virus infection.
ARF
HDAC
BAX
p21
E1A
pRb
Apoptosis
/ARREST
185
The virus counters by E1B-55K, which binds and
degrades p53 (like HPV E6)
E1B-55K
HDAC
BAX
p21
E1A
pRb
Apoptosis
/ARREST
186
Virus can continue to replicate.
E1B-55K
X
X
HDAC
BAX
p21
E1A
pRb
X
Apoptosis
/ARREST
187
ONYX-015 contains E1A but lacks E1B-55K. Virus
cannot replicate in normal cells.
p16 gene
ARF
HDAC
BAX
p21
E1A
pRb
Apoptosis
/ARREST
188
Many cancer cells have defective ARF-p53
pathway. ONYX-015 can replicate in these cancer
cells. Replication of virus subsequently lyse
the cell (leaving the surrounding normal cells)
X
p16 gene
X
ARF
X
X
HDAC
BAX
p21
E1A
pRb
X
Apoptosis
/ARREST
189
Cancer Therapy (ONYX-015)
X
E1B-55K
p53
p21
Cell cycle
E1A
190
Cancer Therapy (ONYX-015)
Normal cells
X
p53
Cell cycle
p21
E1A
191
Cancer Therapy (ONYX-015)
Cancer cells
X
X
X
X
p53
Cell cycle
p21
E1A
Cell lysis
192
Nat Med. 2000 Aug6(8)879-85. a controlled
trial of intratumoral ONYX-015, a
selectively-replicating adenovirus, in
combination with cisplatin and 5-fluorouracil in
patients with recurrent head and neck cancer.
Khuri FR, Nemunaitis J, Ganly I, Arseneau J,
Tannock IF, Romel L, Gore M, Ironside J,
MacDougall RH, Heise C, Randlev B, Gillenwater
AM, Bruso P, Kaye SB, Hong WK, Kirn DH. ONYX-015
is an adenovirus with the E1B 55-kDa gene
deleted, engineered to selectively replicate in
and lyse p53-deficient cancer cells while sparing
normal cells. Although ONYX-015 and chemotherapy
have demonstrated anti-tumoral activity in
patients with recurrent head and neck cancer,
disease recurs rapidly with either therapy alone.
We undertook a phase II trial of a combination of
intratumoral ONYX-015 injection with cisplatin
and 5-fluorouracil in patients with recurrent
squamous cell cancer of the head and neck. There
were substantial objective responses, including a
high proportion of complete responses. By 6
months, none of the responding tumors had
progressed, whereas all non-injected tumors
treated with chemotherapy alone had progressed.
The toxic effects that occurred were acceptable.
Tumor biopsies obtained after treatment showed
tumor-selective viral replication and necrosis
induction.
193
(No Transcript)
194
Cancer Therapies Approaches

• Selectively kill / inhibit cancer cells while
  sparing normal cells.
• Agents that inhibits the cell cycle engine
• Agents that activates the checkpoints
• Agents that bypass the checkpoints
• Agents that increase / restore p53 function

195
Cancer Therapies Reactivating p53

• Idea reintroduction of p53 into p53-negative
  tumors.
• The idea is promising because restoring p53
  alone is sufficient to cause regression of
  several different tumor types in mouse models
  (Ventura et al., 2007 Xue et al., 2007).
• Approaches
• Reintroducing exogenous p53 gene
• Blocking association of p53 with MDM2
• Reverting mutant p53 to wild-type conformation

196
Cancer Therapies Reactivating p53
Compounds that inhibit the interaction between
MDM2 and p53 (such as nutlin and RITA) (Vassilev
et al., 2004 Issaeva et al., 2004). Mimic the
binding of p53 to the p53-binding pocket of MDM2.
197
Cancer Therapies Reactivating p53
Cannot simply express wild-type p53 (e.g. mice
with higher activity of p53 exhibit enhanced
resistance to spontaneous tumors, but aged
earlier). Compounds that stabilize the DNA
binding domain of p53 in the active
conformation e.g. CP-31398 restores mutant p53
proteins to their normal function (Science 286
2507 (1999)). CP-31398 reduced UV-induced skin
cancer in mouse model (J. Clin. Invest. 117,
3753-3764 (2007))
198
References
Morgan, D.O. The Cell Cycle Principles and
Control New Science Press (2006) Fung, T.K. and
Poon, R.Y.C. A roller coaster ride with the
mitotic cyclins. Seminars in Cell and
Developmental Biology 16 335-342 (2005) Woo,
R.A. and Poon, R.Y.C.Cyclin-dependent kinases and
S phase control in mammalian cells. Cell Cycle
2 316-324 (2003). Yam, C.H., Fung, T.K., and
Poon, R.Y.C. Cyclin A in cell cycle control and
cancer. Cellular and Molecular Life Sciences 59
1317-1326 (2002). Poon, R.Y.C. Cell cycle
control. Encyclopedia of Cancer 2nd Edition? Ed.
Bertino, J.R. Academic Press, San Diego 393-404
(2002).
199
Randy Y.C. Poon Department of Biochemistry Hong
Kong University of Science and Technology Clear
Water Bay Hong Kong bcrandy_at_ust.hk