Transcript Cancer and the Cell Cycle

Cancer and the
Cell Cycle
Outline of the lecture
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What is cancer?
Review of the cell cycle and regulation of cell
growth
Which types of genes when mutated can 
cancer?
Roles for screening for mutations in specific
genes
Tumor suppressor p53
Have you figured it out?
What is cancer?
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Cancer = uncontrolled proliferation of cells within
the body  tumor.
Tumor = clone of cells resulting from series of
sequential genetic mutations  loss of growth
control.
Cancer is also known as malignancy.
Development of cancer = oncogenesis
Study or treatment of cancer = oncology
Cancer is a multistep process.
How does this happen?
In the following slides:
= a non- dividing cell
1, 2, 3 = successive
mutations, each contributing
in some way to an increased
rate of cell division or
decreased rate of cell death.
1
Non-dividing
cells
1
1
Non-dividing
cells
2
1
1
1
1
2
2
1
32
2
Non-dividing
cells
Non-dividing cells
1
1
12
12
1
123
12
12
123
123
123
123
123
123
123
123
This process continues, with each successive mutation
leading to a faster rate of cell division, slower rate of cell
death, and eventually loss of cell adhesion.
Review of the
Eukaryotic Cell Cycle
The cell cycle
• The cell cycle has four phases: M, during
which the cell divides; G1, during which
the cell grows larger; S , during which
DNA synthesis occurs; and G2, during
which the cell continues to grow and
prepare for mitosis. The cycle is regulated
at several points.
• The restrictive point in late G1 phase is a
time when the decision is made whether
to continue the cycle or to to exit the cycle
in a nondividing state called G0.
• Cells in G0 may differentiate and assume
specialized functions.
• A cell can remain in G0 indefinitely, or it
may re-enter the cell cycle in response to
signals from a variety of growth factors.
The cell cycle
• Once the cell passes the restriction point
in G1, the cycle will continue until it is
arrested at one of several checkpoints in
response to some problem that needs to
be corrected.
• Progression is halted in late G1 and late
G2 if DNA damage has occurred. The
checkpoints allow time for the damaged
DNA to be repaired before the cycle
resumes.
• The checkpoint in G2 also responds to the
presence of unreplicated DNA and
prevents mitosis from occurring until all of
the DNA has been copied.
• A checkpoint in late M phase halts the cell
cycle until all of the chromosomes are
properly aligned.
The Cell Cycle
• Proteins from several different families
interact to regulate progression through
the cell cycle.
• Cyclins, cyclin-dependent kinases (Cdks),
and Cdk inhibitors (CKIs) all interact either
to block or unblock phases of the cycle.
• Cyclins, and Cdks act together as a dimer,
functioning as the regulatory and catalytic
subunits, respectively.
• Cyclins are degraded at the end of their
functional period, thus inactivating their
Cdk partner in the dimer.
• The assembly of the dimers is regulated
by other proteins.
The cell cycle
• In the event that a cell enters an S
phase with damaged DNA, apoptosis
may be triggered to prevent the
mutant cell from reproducing itself.
Seven levels of
regulation of
cell growth
An unrepaired
mutation in a gene
for a DNA-repair
protein, a cell-cycle
control protein, or
an anti-apoptosis
protein can
increase the
likelihood of a
cancer developing.
An Example of Cell Cycle
Regulation by a Serum Growth
Factor
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Cyclin D is made following the
binding of the serum growth factor
to its receptor and the ensuing
cascade of phosphorylations.
An Example of Cell Cycle
Regulation by a Serum Growth
Factor
An Example of Cell Cycle
Regulation by a Serum Growth
Factor
Cyclin D associates with either Cdk4 or
Cdk6.
 P16 may block the assembly.
 After assembly the Cdk becomes
phosphorylated.
 This may be blocked by either p21 or p27
 The target of the active dimer is Rb which
is bound to a transcription factor called
E2F.
 The Rb/E2F dimer blocks transcription of
genes needed to enter the S phase.
 Phosphorylation of Rb results in its
dissociation from E2F.
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An Example of Cell Cycle
Regulation by a Serum Growth
Factor
 This
results in activation of S
phase genes.
 In addition to its ability to block the
association of cyclin D with a Cdk,
P16 can also directly block the
phosphorylation of Rb.
Phosphorylation of Rb
An Example of Cell Cycle
Regulation by a Serum Growth
Factor
 P16,
p21, and p27 are regulated
by p53 (more on this later) which
blocks the cell cycle in the G1
phase if there is DNA damage.
 P53, Rb, p21, p16, and p27 are
called tumor supressors because
their normal function is to prevent
the growth of cells with damaged
DNA.
An Example of Cell Cycle
Regulation by a Serum Growth
Factor
P53 also responds to unrepaired DNA
damage by triggering apoptosis of the
injured cell.
 It interacts with a member of the Bcl-2
family of proteins which, in turn activate
special enzymes called caspases.
 Caspases initiate a protease cascade
that results in digestion of the DNA.
 This ultimately leads to cell death.
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Apoptosis
Which types of genes when mutated
can  cancer?
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Oncogenes = genes whose products turn DNA
synthesis ON
Tumor suppressors/anti-oncogenes = genes whose
products turn DNA synthesis OFF
Genes whose products contribute to genomic
stability
Genes whose products contribute to cell longevity
Which types of genes when mutated
can  cancer?
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Oncogenes (turn DNA synthesis ON)
 In
progression towards cancer, a gene for a protein
that normally stimulates DNA synthesis (protooncogene) is either
 consitutively
expressed at high levels or
 mutated such that protein product is constitutively
active, i.e., can not be inactivated
 Classes
I-IV from Slide # 15 generally give rise to
dominantly active oncogenes.
 Examples: see next slide.
Oncogenes
Which types of genes when mutated
can  cancer?
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Tumor suppressors/anti-oncogenes (turn DNA
synthesis OFF)
 In
the progression towards cancer, a gene for a
protein that normally inhibits DNA synthesis is either
 permanently
inactivated or
 mutated such that the protein product is inactive
 Mutations
in Class VI, cell-cycle control proteins, from
Slide #15.
 Examples:
 APC
inhibits Wnt gene product from activating myc
 Rb inhibits activation of transcription of DNA synthesis
genes
Which types of genes when mutated
can  cancer?
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Contributors to genomic stability
Some tumor suppressors turn DNA synthesis off when DNA is
damaged.
 The progression toward cancer occurs when a gene for a
protein which contributes to DNA repair is
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permanently inactivated or
 mutated such that protein product is inactive
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Mutations in repair genes increase likelihood of mutations in
proto-oncogenes and tumor suppressors.
 Examples:
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p53 gene product induces genes for DNA repair
 MDM2 gene product destabilizes p53
 MutS and MutL gene products repair UV or chemically damaged
DNA
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Which types of genes when mutated
can  cancer?
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Contributors to cell longevity (anti-apoptosis genes)
 Progression
toward cancer can occur when an antiapoptosis gene is
 constitutively
expressed or
 mutated such that protein product is constitutively
active
 Allows
survival of cells with oncogenic mutations
 Example: Bcl2
Roles for screening for mutations
in specific genes
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To determine
 Type
of cancer
 Familial predispositions
 Progression of the cancer
What is p53?
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A protein of ~53 kilodaltons
A nuclear phosphoprotein
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What is p53?
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Transcriptional regulator
 Binds
to 12 bp recognition sequence in the
promoters (regulatory regions) of the genes it
regulates
 Activates transcription by interacting with RNA
polymerase complex
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What is p53?
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Acts as a tetramer
 Individual
molecules associate at tetramerization
region
 Oligomerization of mutated p53 with wt p53 
inactive p53 complex
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What is p53?
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Binding of damaged DNA fragments to p53 causes
p53 to be stabilized and accumulate in the cell.
When damaged DNA is not present, p53 is turned
over rapidly and does not accumulate because
 the
protein MDM2 binds to the transcriptionactivation region of p53 and targets p53 for
degradation by a proteosome.
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(TAD)
 Note: MDM2 binds when the TAD is NOT phosphorylated.
p53 as a transcriptional regulator
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If DNA damage is detected by binding of DNA fragments to the
non-specific DNA binding region of p53, p53 stops DNA
synthesis until the damage is repaired.
If DNA damage is detected, then
p53 is phosphorylated by a protein known as ATM
 MDM2 is released from being bound to the transcriptional
activation domain of p53 and
 p53 is able to act as a transcriptional activator and turn on genes
for
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cyclin dependent kinase inhibitor p21, which
• stops or prevents DNA synthesis
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DNA repair
• Example: GADD45
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If DNA damage is extensive and can not be repaired, p53
induces genes for apoptosis (programmed cell death).
p53 as a transcriptional regulator
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p53 activates the gene for MDM2
 MDM2
 targets
p53 for degradation and prevents inappropriate
build up
 prevents transcriptional activation by p53
 So,
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it’s a negative feedback loop!
p53 also turns expression of some genes off.
How does p53 inhibit DNA
synthesis? Let’s work backwards.
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E2F transcription factor turns on transcription of
genes for DNA synthesis.
E2F can’t turn on genes if it is bound to Rb, a
tumor suppressor.
Rb can’t bind E2F if it is heavily phosphorylated.
Rb is phosphorylated by cylin-dependent kinases
(CDKs).
How does p53 inhibit DNA
synthesis?
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Cylin dependent kinases can be inhibited by cyclin
dependent kinase inhibitors (CDKIs). If CDKs are
inhibited
 Rb
won’t be phosphorylated
 E2F will be bound by Rb
 DNA synthesis gene will not be transcribed
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And remember . . . .
P53 induces expression of CDKI p21, a cyclin
dependent kinase inhibitor!
Check out the next slide for a visual of these
pathways.
Phosphorylation of Rb
Figure legend on next slide.
p53 Mutations - where are they?
This magnification of mutations in the DNA binding region of
p53 gives more information regarding how the mutation
affects p53. Note particularly that some mutations cause p53
to be misfolded (denatured) and others do not.
Have you figured it out?
For our assay, the samples are cell extracts from two mouse
cell lines, BC3H1 and C2C12.
One line is wild type for p53; one is mutant.
One accumulates detectable levels of p53; one
doesn’t.
Based on this lecture and your assay results, have
you figured out which cell line does what?