Chapter 8: pRb and Control of the Cell Cycle Clock

Chapter 8:
pRb and Control of the
Cell Cycle Clock
From: Biology of Cancer: Robert Weinberg: Copyright © Garland Science 2007
pRb and the Cell cycle
• Cell-cell signaling decides cell fate
• Normal cells will not divide without growth
• TGF-B can stop growth in presence of growth
• Cells can divide OR
• Cells can become quiescent (G0 phase)
• Cells can become post-mitotic – an irreversible
state of non-division
Signaling and Growth
• Surface receptors collect signals and funnel
them into the cell
• Integration of signals by proteins help cells
decide to divide/not-divide
• There must be a master cellular program
deciding cell fate (quiescence, division,
• Master Program Called Cell Cycle Clock
Figure 8.1 The Biology of Cancer (© Garland Science 2007)
Cancer Cells
• Always proliferate
• Oncogenes keep cell cycle on, regardless of
lack of growth signals
• Tumor suppressor genes can be hijacked by
cancer cells to change circuitry and keep
cell cycle on
Normal Cell Cycle
• Cells in culture with growth medium will keep
growing and dividing
• In vivo, a newly divided cell has to decide whether
to divide again or not
• Cell division requires circuits for
– Growth in size
– Duplication of chromosomes
– Correct chromosome complements in divided cell
Normal Cell Cycle
• After a new cell is made, RNA and protein
synthesis begins immediately
• But chromosome duplication is delayed by
12-15 hours (G1 or Gap 1 phase)
• In G1, cell is deciding what to do
• G1 is followed by S phase (6-8 hours)
• Then by G2 phase (Gap 2) of 3-5 hours
• Then by M phase (~1-2 hours)  division
• Prophase – chromosomes condense, become
visible, mitotic spindle forms
• Metaphase – chromosomes align along plane that
bisects cell, nuclear membrane disappears, mitotic
spindle migrates to poles
• Anaphase – chromosome halves pulled apart to
poles by mitotic splindle
• Telophase – Chromatids decondense, nuclear wall
forms around them
• Cell splits into 2 daughter cells – mitosis complete
• Mitosis Demo
Figure 8.3b The Biology of Cancer (© Garland Science 2007)
Checkpoints MUST exist
Necessary for fidelity of cell division
Decision on when to enter cycle phases
Accurate duplication of chromosomes
Division of correct complement to daughter
Where are these checkpoints?
• G1  S phase checkpoint
– Integrate growth signals and decide its time to divide
– Check that genome is undamaged
• S  G2 phase checkpoint
– Accurate DNA replication (no DNA damage)
• G2 M phase checkpoint
– DNA replication completed
• Anaphase checkpoint in M
– Chromosomes attached to splindle
• Other checkpoints must exist
– Decatenation checkpoint in late G phase (no tangles in Chr)
Figure 8.4 The Biology of Cancer (© Garland Science 2007)
R (Restriction) checkpoint
• Cells make growth decision during specific
interval in G1 phase
– In culture, if cells are < 80-90% in G1 phase,
inhibition of growth serum will restrict entry
into S phase
– In last 10% of G1 phase, restriction of serum
has no effect. Cell committed to S phase entry
– TGF-B is able to halt cell cycle only in early G1
Figure 8.6 The Biology of Cancer (© Garland Science 2007)
R Checkpoint
• This is a GO/NO-GO decision made by the
cell in late G1 phase
• More important than other checkpoints
– Once in S phase, cells seem oblivious to
external signaling
– They activate fixed schedule SG2M
• Cancers must subvert R checkpoint
Cyclin Dependent Kinases
• Embryonic frog and sea urchin cells have a
cyclical expression of Cyclin-B correlated
with cell cycle
Cyklin Dependent Kinases
• Play a major role in R checkpoint
• Act only in the presence of cyclins which are
subunits (form a dimer-complex)
• Phosphorylated at Serine/threonine instead of
tyrosine (as in receptors – eg EGF)
• 40% identity amongst themselves (evolved by
gene duplication from progenitor gene)
• 430 distinct known CDKs in humans
• Enormous enzymatic potential
– Cyclin A + CDK2 increases activity by 400,000 X
When are they active?
• G1 phase
– Kinase CDK4/CDK6
– Cyclin D1, D2, D3
• Early S phase
– Kinase CDK2
– Cyclin E1, E2
• Late S phase
– Kinase CDC2 (CDK1)
– Cyclin A1, A2
• G2 phase
– Kinase CDC2 (CDK1)
– Cyclin B1, B2
Figure 8.10 The Biology of Cancer (© Garland Science 2007)
What dergrades them so fast?
• Ubiquitylation followed by degradation in
Cyclin D’s are different
• Controlled by extracellular signaling (mitogens)
• They inform cell about external conditions
• Three different types D1, D2, D3 for flexibility of
response and refinement of input sensing
• Most often/easily targeted by carcinogens, viruses,
• Probably newer than other CDKs (since the
evolution of complexity) and so more error prone
Factors affecting Cyclin
D1 levels
Figure 8.11b The Biology of Cancer (© Garland Science 2007)
Each cyclin pairs with a specific Cyclin Dependent Kinase
Figure 8.12 The Biology of Cancer (© Garland Science 2007)
CDK inhibitors p21, p27
Early G1: Cyclin D-CDK4/6 complexes accumulate, bind and
sequester p27 and p21 including those associated with
E-CDK2 complexes
Late G1: Sufficient numbers of p21, p27 are bound to D-CDK4/6 to
liberate enough E-CDK2 complexes to transit R point
How does TGF-B work?
• Increases p15 levels which block formation
of D-CDK4/6 complexes and inhibits those
already formed
• Cell cannot advance through R checkpoint
• If Cell is already past R, TGF-B has no
effect since D-CDK4/6 complex is
pRb gene controls R Checkpoint
• Defective pRb gene isolated in 1986 in
• Encodes a nuclear phospho-protein ~ 105 kD
• Called pRb or RB
• pRb is unphosphorylated in G0
• pRb is hypo-phosphorylated in early G1
• pRb is hyper-phosphorylated in late G1 and rest of
cell cycle
• After exit through mitosis, phosphate groups on
pRb are stripped off by PP1 (protein phosphatase 1)
How was it identified as important?
• Several DNA virus (eg. SV40) oncoproteins
target pRb by binding to it to sequester it or
inactivate it
• They only bind to (seem to care only about)
hypo-phosphorylated state of pRb
• This means that only the hypo-P state of
pRb is relevant for tumors
How does pRb get Phosphorylated?
• D-CDK4/6 initiate phosphorylation of pRb
• E-CDC2 complex complete the
phosphorylation of pRb
• Puzzle: pRb has homologues p107 and p130
which also have similar phosphorylation
states and histories. Why are they not
involved in tumorogenesis?
Figure 8.22 The Biology of Cancer (© Garland Science 2007)
Figure 8.19 The Biology of Cancer (© Garland Science 2007)
So why is hypo-P of pRb important?
• Unphosphorylated pRb
• Binds and sequesters E2F
• Hypo-phosphorylated pRb
• Releases some E2F
• Hyper-phosphorylated pRb
• Releases a lot of E2F
Figure 8.23a The Biology of Cancer (© Garland Science 2007)
What does E2F do?
• E2F is bound to promotor region of genes that
initiate transcription
• When E2F is bound to pRB, it attracts histonedeacytylases – which remove acetyl groups from
histones and make them inaccessible to
transcription machinery (inhibits transcription)
• When E2F is not bound to pRB, it attracts
histone-acytelases which add acetyl groups to
histones and make them accessible to transcription
machinery (promotes transcription)
Transcription control by
chromatin modification
induced by by Pocket
Proteins pRb, p107, p130
Figure 8.24a The Biology of Cancer (© Garland Science 2007)
Many cancers disable pRb
• Retinoblastomas, osteosarcomas, small cell
lung carcinomas: pRb lost by mutation
• Cervical CA: HPV protein E7 binds to pRb
and makes it inactive
• Breast Cancer : Overexpression of Cyclin
D1 by gene amplification
• Melanoma : methylation of p16 gene
promotor etc etc……
Myc and Subtle Dis-Regulation of
• Myc overexpression
– increases Cyclin D2  phosphorylation of pRb
– Overexpression of CDK4  same effect
– Sequesters p27, drives E-CDK2 complexes and makes
cell enter S phase
– Degrades Cull protein which degrades p27, induces
expression of genes which encode E2Fwhich drives
• Myc Disregulation Resets Cell-Cycle
Regulatory Dials  Causes proliferation !!
Eukaryotic Cell Cycle and Pathway
Table 8.3 The Biology of Cancer (© Garland Science 2007)
Table 8.4 The Biology of Cancer (© Garland Science 2007)
Cyclin E levels and Breast Cancer Progression: Plot shows survival
in women presenting with stage III disease (large primary tumors
and LNs but no distant metastasis)
Figure 8.38 The Biology of Cancer (© Garland Science 2007)
Next week
• Read Chapter 9
• We will talk about the p53 gene, guardian
and master executioner !