Transcript Document

Lectures 7 -10
Cell Cycle Regulation and
Cancer
Introduction to the Cell Cycle
The Eukaryotic Cell Cycle and Chromosome Dynamics
Identification and Experimental Basis of
Cell Cycle Regulatiory Factors
Major players:
• Cyclins , cdk’s (cyclin – dependant kinases)
• MPF (Maturation Promoting Factor) = cyclin Bcdc2 (yeast) or cdk1 kinase (“mitotic cdk”)
• CAK (Cdk Activating Kinases) regulators of cdks
• CKI – cdk inhibitory subunits
• APC (Anaphase Promoting Complex) and
cdc34/SCF ubiquitination pathways which activate
proteolysis via proteosomes.
Introduction to the Regulation of Cell Cycle
Also termed SCF
pathway
Identification and Experimental Basis of
Cell Cycle Regulatory Factors contd…
Mutant studies in Saccharomyces cerevisiae (budding yeast) and
Schizosaccharomyces pombe (fission yeast)
Complementation studies of the cdc (cell division cycle) temperature
sensitive mutants using a plasmid library of wild type yeast DNA have
enabled identification of numerous proteins involved in cell cycle
regulation.
Identification and Experimental Basis of
Cell Cycle Regulatory Factors contd…
Biochemical studies with oocytes:
• Oocytes from Xenopus laevis, the South African clawed
toad, are a store house for factors required for cell
cycle progression & cell division from the oocyte stage
to the fertilized egg through gastrulation during
embryogenesis.
• In vitro studies using this model system have led to the
identification of various components and events in the
cell cycle. This began with the identification of “MPF”
(maturation promoting factor) which controls entrance
into meiosis and mitosis. It was later shown to be the
equivalent of the cyclin b-cdc2/cdk1 complex
Functions of MPF (cyclin b-cdc2/cdk1)
• Reversible breakdown of the NE & role of nuclear lamin
proteins via DNA transfection experiments.
• Genetic studies on S.cerevisiae identified a family of
proteins that are required for normal chromosome
segregation and are termed SMC ( Structural
Maintenance Complex) proteins. They are found in
organisms up to humans and are typically called
“condensins”.
• Phosphorylation of condensins by MPF or another
protein kinase regulated by MPF is necessary for
maintaining the condensed state of the chromosomes via
DNA binding.
• Phosphorylation of APC results in initiation of anaphase.
Identification of MPF (Maturation Promoting Factor)
and Cyclins in Xenopus Oocytes
• Oocyte maturation to the meiotic II metaphase stage is studied by microinjection of
the female steroid hormone, progesterone, into G2 arrested oocytes.
• This can be mimicked by the microinjection of cytoplasm from the metaphase II stage.
• The “factor” that mediates this oocyte maturation was purified and termed MPF.
• MPF oscillates in coordination with meiosis in the oocyte and mitosis during early
cleavage stages following fertilization.
• This oscillation was due to the cyclic degradation and biosynthesis of cyclin B.
• It was subsequently demonstrated that MPF is mitotic cyclin b-cdc2 or -cdk1 complex.
G2-arrested oocyte Meiosis I
Meiosis II
Experimental Demonstration that the Synthesis &
Degradation of Cyclin B are Required for the Cycling of
MPF and Mitotic Events in Xenopus Egg Extracts
Sperm
Chromatin
(mutation in destruction box sequence)
Sperm
Chromatin
Polyubiquitination of Mitotic Cyclins
• Regulated degradation of mitotic cyclins occurs in late anaphase via
the APC-ubiquitin pathway which makes use of a “destruction box”
sequence common to the N-T of mitotic cyclins.
• This ubiquitin pathway involves E1 (ubiquitin activating enzyme) , E2
(ubiquitin conjugating enzyme) & E3 (ubiquitin ligase or APC).
• Mutant genes that code for non-degradable cyclins have been
produced by site directed mutagenic deletion of the destruction box.
Regulation of Mitotic Cyclin Levels in Cycling Cells
• Regulated degradation of mitotic cyclins is a result of a
corresponding activation of APC activity via
phosphorylation by mitotic cdk and Cdh-1 binding.
• This is an example of two protein complexes which
regulate each other in concert with cell cycle regulation.
Cdh-1
cyclin B degrades
Mechanism for the Role of APC in Initiating Anaphase
• Anaphase is induced by degradation of the “anaphase inhibitor”
(“securin”) via the APC (anaphase promoting complex) pathway
activated by MPF (P) (cyclin B-cdk1) and Cdc20 addition to APC.
• This protein is involved in the stabilization of a multicomplex of
proteins called cohesins. Cohesins act as “linchpins” to tie together
sister chromatids at the centromeric attachments and other sites
along the chromatid pairs and maintain this association despite the
pulling of the kinetochore attached spindle fibers.
• Some of the cohesins are members of the SMC protein family and
thus related to the condensin proteins which maintain
chromosomes in a condensed state.
• Degradation of anaphase inhibitor (securin) results in inactivation
of cohesin function allowing the poleward force exerted on
kinetochore to move sister chromatids towards opposite poles.
• Destruction of the mitotic cyclin B via the APC pathway occurs in
late anaphase when APC complexes with cdh-1.
Model for Induction of Anaphase by
Regulation of Cohesin Complexes
Regulation of Sister
Chromatid Pairing by
the Cohesin Complex
• Sister chromatid pairing is
mediated by the cohesin complex
in which Smc1/Smc3 together with
Scc1/Scc3 form a bridge between
the two chromatids.
• Scc1 is the critical protein that
serves as a “linker” between
cohesin complexes on the two
sister chromatids
• APC/Cdc20 mediates release of the
‘anaphase inhibitor’ (“securin”) by
activating securin degradation. This
results in activation of separase and
cleavage of Scc1 linkages between
cohesin complexes
Cdc20
Summary of the Dual Effect of APC on Mitosis
P
+ Cdh-1
[Securin] + Cdc20
Active Separase breaks
Cohesin links
Cell Cycle Regulatory Components
• S. pombe genetic studies especially complementation
and recombination studies of the cdc temperature
sensitive mutants led to the identification of cdc2 and
cdc 13 as cdc2 kinase and cyclin b.
• Both of them are linked in a complex as MPF or cyclin
b-cdc2 kinase complex.
• It also identified other regulatory components such as
wee 1, cdc 25 and CAK (cdc2 activating enzyme).
• Multiple pathways are involved in regulating the
mitotic-cdk complexes which are cdc2 in yeast and
cdk1 in mammals.
• All these cell cycle regulatory proteins are typically
well conserved from yeast to humans.
Regulation Wheel of CDK’s
(Morgan, Nature 374 (1995) 131-134)
Tyr-15
P-Thr-161
The Regulatory Aspects of DNA Replication
Genetic studies with S.cerevisiae have dissected the
regulatory aspects of DNA replication:
(a) Control of S-phase by regulated proteolysis of sic 1:
(i) Sic 1 is a CKI specific for the S-phase cdk-cyclin complex.
(ii) This proteolysis is initiated by the G1 phase cdk-cyclin which
phosphorylates sic1 making it a target for cdc34 ubiquitin conjugating
enzyme and a trimeric ubiquitin ligase called SCF.
The Regulatory Aspects of DNA Replication contd…
(b) Regulation of pre
replication complexes:
cdk’s simultaneously activate
initiation of replication and
prevent re-initiation at origin
sites by phosphorylation &
disassociation of McM’s from
the ORCs (origin recognition
complex) which are
permanently associated with
replication origins.
For the McM’s to reassemble
at the origins they need to be
dephosphorylated which does
not occur until mitosis.
P
P
Cell Cycle Control in Mammalian Cells
Multiple cdks and cyclins regulate passage of
mammalian cells through the cell cycle.
Cyclin D is Required for Passage Through the Restriction Point in
the Mammalian Cell: An In Vivo Depletion Experiment
Cells microinjected anti-cyclin D
8 h after growth factors
Control
DAPI
An in vivo
Depletion exp
BrdU
anti-Cyclin D
microinjection
Early and Delayed Genes and Regulation at the
“Restriction Point”
• Activation of Go cells into the cell cycle of growth and proliferation
requires growth factor stimulation and involves transcriptional
induction of early response and delayed response genes (DRG).
• Induction of early response genes is not blocked by inhibitors of
protein synthesis (e.g. cycloheximide) because the transcriptional
factors that control these genes are already present in the Go cells
and are activated by post transcriptional modifications e.g.,
phosphorylation via protein kinases activated by signal transduction
systems activated by growth factors.
• Many of the early response genes code for general transcription
factors (TF) e.g. (c-Fos and c-Jun) that stimulate transcription of the
delayed response genes (DRG). Some of the DRG encode additional
components of cell cycle regulation, e.g., the cyclins and G1cdks.
EF2 is a TF that regulates transcription of cyclins A & E and cdk2
genes.
Mechanism for Restriction Point Regulation
• If growth factors are withdrawn before the passage
through the “Restriction point” (RP), transcription of the
DRGs (e.g., G1 cyclins and cdks) rapidly decreases.
• Since these proteins and the mRNAs encoding them are
unstable , their concentrations in the cell fall rapidly. Thus
the cells do not pass the RP and do not enter S-phase and
thus do not proliferate.
Role of E2F and Rb Protein in Regulation of RP Passage
• The E2F transcription factor regulates the cyclin A & D and cdk2 genes.
• Rb (retinoblastoma) protein inhibits E2F transcription factor.
• Rb is the protein product of the tumor supressor gene RB which if mutated
enhances the probability of retinoblastoma and other cancers.
• Normally this protein is not active until mid/late G1 phase when the
buildup of cdk2-cyclin E and cdk4/6-cyclin D leads to phosphorylation of
Rb and release from supression of the E2F transcription factor.
• After mitosis Rb is dephosphorylated due to the low concentrations of the
G1 cdks and cyclins. Rb will undergo phosphorylation as the cyclin levels
rise in G1. If not, the cell cycle goes into Go (i.e., it does not pass RP).
Mid-G1
Late-G1
A
+ Cyclin
Cdk2
release from RP
Checkpoints in Cell Cycle Regulation
To ensure orderly passage of the cells through cell cycle there are four
defined “checkpoints” where certain events need to occur before the
cell will proceed in the cell cycle; otherwise the cell will be “arrested”
at that checkpoint: G1 arrest, S arrest, G2 arrest and M arrest.
There are specific checkpoint protein kinases (chks) that are directly
involved in arresting the cells.
Role of p53 in G1 Checkpoint Arrest
• Most commonly mutated tumor suppressor gene
associated with human cancers is p53 which encodes
the transcription factor (TF) p53 protein.
Cell sorting
Wild-type cells
• This protein is rapidly degraded in normal cells so its
concentration is very low.
Cell sorting
cells
p53- mutant
• In response to DNA damage in G1, the protein level of
p53 rises dramatically due to the activation of a
checkpoint protein kinase (chk2) that phosphorylate
p53 and make it less susceptible to degradation by the
ubiquitination/proteasome pathway
• This enables p53 to activate transcription of the cdk
inhibitor (CKI) p21 which binds to all cdks and inhibits
their action and thus “arrests” the cells until the DNA
damage can be repaired. Then the p53 fall back to
normal levels and the cell is “rescued “ from checkpoint
arrest.
P
P P
(which activates a checkpoint kinase (phosphorylate p53)
• Under severe DNA damage p53 activates genes that lead to apoptosis (cell death).
Cancer and Growth Related Factors: ProtoOncogenes and Tumor Supressor Genes
• Cancer is characterized by the ability to grow in
absence of growth promoting factors and
resistance to signals that stop growth or program
cells for apoptosis.
• Cancer is a disease of: unregulated growth,
proliferation and cell to organ function.
• Genes coding for these regulatory factors can be
divided according to their cancer related
properties into either: (a) Oncogenes or (b)
Tumor Suppressor Genes
Oncogenes
• Oncogenes produce proteins that have the capacity to
stimulate growth and proliferation.
• They are dominant or “gain of function” mutations.
• First discovered through the ability of Rous sarcoma
virus (RSV) to cause cancer in chickens.
• Mutant studies of RSV: the src gene causes cancer!
• Transfection of cells with src or other oncogenes. e.g.,
ras or jun leads to neoplastic transformation.
• There is a normal cell equivalent of this so-called
“oncogene” and it codes for a protein that has been
associated with tyrosine kinase activity, that
stimulated growth and proliferation via protein
phosphorylation in signal transduction pathways.
Oncogenes contd…
• The activity of the normal protein is regulated by
another tyrosine kinase that phosphorylates src at
T-527 (p-527 src is inactive).
• v-src has a deletion in 18 aa at the C-T that
includes T-527.
• Therefore , v-src or appropriate cellular mutants (csrc) have constitutive tyrosine kinase activity,
therefore unregulated cell growth and proliferation.
• All oncogenes have been found in normal cell
equivalent genes/proteins and are termed “protooncogenes”.
Oncogene proteins
• Growth factors – rare but an
example is sis which codes for a
mutant PDGF (platelet derived
growth factor) and aberrantly auto
stimulates proliferation of cells
containing PDGF receptors.
• Growth factor receptors- erb
b is a mutant form of epidermal
growth factor. This receptor
functions as a tyrosine protein
kinase (CT of protein) located on the
cytoplamic side of the membrane
with the ligand binding region (NT of
protein) facing the cell exterior.
In erb b the receptor protein is
missing the N-terminal ligand
binding domain on the cell surface
exterior and the C-terminal tyrosine
protein kinase is in a permanently
turned on state.
•Intracellular transducers e.g., ras (a mutant
form of an inner cell surface GTPase) , whose
function activates a number of other regulatory
factors including another oncogene termed raf (a
protein kinase) that work in concert to regulate cell
proliferation via the MAP Kinase Pathway which
activates transcription factors
•Nuclear transcription factors like myc. The
myc protein is a key factor involved in activation of
gene transcription in cells going from a non –
proliferating state. If myc gene expression is
blocked using anti-sense oligonucleotides, cell cycle
progression is stopped. Over expression of c-myc is
characteristic of many cancers.
Oncogenes Contribute To Cancer By
Over Expression Of Normal
Regulatory Components
(1) Point mutations that lead to constitutively
acting protein products.
(2) Amplification of the gene or change in
chromosomal location of the gene that puts
the gene under the regulation of a different
promoter or activator.
Tumor Supressor Genes
• Tumor supressor genes produce proteins that
suppresses growth and proliferation.
• These are “loss of function” or recessive mutations.
• Being heterozygous enhances the probability of
cancer but this will require a mutation in the
corresponding other allele. e.g., it need to be
homozygous for the gene.
• Tumor supressor genes include: (a) Proteins that
regulate or inhibit progression through a cell cycle
stage like CKI’s: p16, p21; (b) Receptors for secreted
hormones/growth factors that function to inhibit
proliferation: e.g., TGF-beta; (c) Restriction point (Rb)
& check point (p53) control proteins.
Human Colorectal Cancer:
Multi-Tumor Suppressors and
Oncogenes
• APC –tumor supressor gene
inhibits ability of wnt protein to
activate expression of the myc
oncogene.
• Absence of functional APC thus
leads to an uncontrolled
expression of myc oncogene
protein that activates genes
required for the G1-S-phase
progression.
• This leads to polyp (small tumors)
formation along the colon wall.
• Cancer progression can then
proceed towards malignancy if
subsequent mutations occur: ras
oncogene, DCC-tumor suppressor
gene, p53 tumor suppressor gene.
The RB Gene and Heredity Retinoblastoma
Loss of Heterozygosity (LOH) of Tumor Suppressor Genes
Why do patients who inherit one mutant allele of RB have a high
probability of developing retinal tumors in childhood?
This is characteristic of cancers related to the loss of tumor suppressor
genes and a likely explanation is the predisposition of such genes to a
“lose of heterozygosity” or (LOH).
Conclusion
Cancer is characterized by many abnormalities
in chromosome numbers (aneuploidy) and
organization (translocations, inversions
deletions, and amplifications). This is a
direct result of the lack of checkpoint control
during the cell cycle.
CANCER CELLS ARE CHARACTERIZED BY GLOBAL
ABERRATIONS IN THE NUMBER SIZE AND BANDING
PATTERNS OF CHROMOSOMES GIVING RISE TO THE IDEA
OF A “CHAOTIC GENOME” IN CANCER CELLS.
Chromosome
painting of an
osteosarcoma
cell line (MG-63)
Chromosome
painting of
normal diploid
human cells
PLEASE DO NOT SMOKE