Transcript The Cell Cycle

The Cell Cycle and its
implications in diseases
Hansjörg Hauser
Dept. of Gene Regulation and Differentiation
Molecular Biotechnology
Helmholtz Centre for Infection Research,
Braunschweig
Cell division is a prerequisite
for life
•Microorganisms reproduce by cell division
•Mammals need cell division during embryogenesis and for
tissue homeostasis
Example:
Adult humans produce several milions of new cells per
second (more than 1011 per day – about 100 grams)
Cell division can be fast or slow
•Microorganisms > 20 min per division
•Multicellular organisms: 8 min and several weeks per
division
•All species can halt cell division
The Cell Cycle
M
G2
G1
S
S
Start, G1 Checkpoint
Point of no return
Methods to measure cell division
•Counting
•Amount of DNA
•Enzymatic activities
•Incorporation of labeled DNA precursors
•Cell cycle analysis (FACS)
•Dilution of dyes
•Time lapse microscopy
While cdks are constitutively expressed
the appearance of cyclins in the cell cycle
is transient – they cycle
The presence of cyclins regulates the
activity of the cdks
Cyclic activity of Cyclin kinases
Temporal control of the animal cell cycle. The cyclin-E-, cyclin-A- and
cyclin-B-dependent kinases are active at different times in the cell cycle. On
this basis, cyclin E–Cdk2 appears to have a role in promoting S phase, cyclin
A–Cdk2 in S phase and at G2-to-M phase, and cyclin B–Cdk1 during mitosis.
The kinase activity of cdc-cyclin compexes
is regulated by phosphorylation and
dephosphorylation
Examples
MO16 is an activating kinase
Wee1 is an inhibitory kinase
cdc25 is a phosphatase that removes the
inhibitory phosphate from the cdk
Regulation of cyclin-dependent kinases.
Arrowheads represent activating events and perpendicular ends
represent inhibitory events. Genes known to perform the indicated
functions are listed below. Both cyclins and some CKIs (Cdk inhibitors)
are regulated by synthesis and ubiquitin-mediated proteolysis. Checkpoint
pathways could act to promote inhibitory pathways or inhibit activating
pathways to cause cell cycle arrest
Example: The CKI (cdk inhibitor) p27
p27 inhibits cdk2 and thereby the relvant
complexes CyclinE/cdk2 and CyclinA/cdk2
p27 underlies multiple regulations:
transcriptional,
translational,
degradation,
localisation (cytoplasmic versus nuclear),
phosphorylation,
sequestering by binding to CyclinD1/cdk4
(without becoming inactivated)
The progression through the cell cycle
underlies many controls: Example DNA
replication
A re-replication block ensures that no
segment of DNA is replicated more than
once
Passage through mitosis removes the rereplication block
Feedback controls generally depend on
inhibitory signals
Checkpoint pathways
(A) A genetic pathway illustrating intrinsic and extrinsic checkpoint mechanisms.
Letters represent cell cycle processes. The pathway shown as red symbols indicates
an intrinsic checkpoint mechanism that operates to ensure that event C is completed
before event E. After event B is completed, an inhibitory signal is activated that
blocks completion of event E. After event C is completed, a signal is sent to turn off
the inhibitory signal from B, thereby allowing completion of E. The blue symbols
represent an extrinsic mechanism that is activated when defects such as DNA damage
or spindle errors are detected. It is arbitrarily located on the D to E pathway but
could also function by inhibiting a later step in the B to C pathway. In that case, the
extrinsic pathway would utilize the intrinsic mechanism for cell cycle arrest.
Mutations in any of the red or blue symbols would result in a checkpoint-effective
phenotype.
DNA damage leads to a block in cell cycle
progression
Replication of damaged DNA would fix
mutations for all daughter cells
Possible biochemical function of the Rad24 group of checkpoint proteins.
Rad24, together with the four small subunits of RFC, is a component of a
pentameric complex. By analogy with RFC, this complex might recognise the
transition between ssDNA and dsDNA. Such a structure is produced by many
repair pathways but the Rad24 complex may only efficiently recognise it in the
context of repair complexes (not shown here). Once the Rad24 complex is
bound, it then functions to recruit the ‘PCNA-like’ Rad17/Mec3/Ddc1 complex
to the DNA, followed by additional recruitment of checkpoint proteins involved
in signal transduction (e.g. Mec1 and Rad53)
Gene expression in G1
G1
G0
Activation of
early response
genes:
fos, jun,..
delayed response
genes:
E2F
Cyclins E, D
S
DNA synthesis
genes
Resting cells:
The retinoblastoma protein Rb blocks cell
cycle progression in G1 by binding to and
sequestering E2F
Rb-P
Rb
+ E2F
Phosphorylation causes
Inactivation of Rb
Rb : E2F
Target of the CyclinE/cdk2 and
CyclinD/cdk4(6) complexes
Phosphorylation of pRB
Cell cycle progression by growth factors
Phosphorylation causes
Inactivation of Rb
Rb-P
Proliferation
Rb
E2F
CyclinD.cdk4
CyclinD
MAPK pathway
Ras
EGF
Rb: E2F
Rb captures E2F:
E2F cannot activate
proproliferative genes
Proliferation
Cell cycle progression
Growth block
Phosphorylation causes
Inactivation of Rb
Rb-P
Rb captures E2F, so that
it cannot activate
proproliferative genes
Rb
CyclinD.cdk4
+ E2F
CyclinD
Rb: E2F
MAPK pathway
Ras
P16 Ink4A
Regulators the cell cycle progression
CDK1
CycB
M
CDK4/6
CycD
pRB
E2F
P
G2
INK4
p15
p16
p18
p19
P
pRB
G1
E2F
CDK2
CycE
CDK1
CycA
S
CDK2
CycA
Kip/Cip
p21
p27
p57
Mammalian cells:
The protein p53 is sensing DNA damage
p53 becomes phosphorylated and stabilized
P53 is a general gatekeeper for the G1
checkpoint
p53 is a transcriptional activator:
One of the genes induced by p53 is p21,
an inhibitor of the cdk4 kinase activity
p27
Rb captures E2F, so that
it cannot activate
proproliferative genes
CyclinE.cdk2
Rb-P
p53
Rb
CyclinD.cdk4
+ E2F
p21
CyclinD
MAPK pathway
Ras
Rb: E2F
p16 Ink4A
Cellular products influencing the cell cycle
Viral and cellular proteins influencing p53
activity
Cell cycle control in mono- versus
multicellular systems
•Monocellular systems:
Unlimited proliferation
Control by size, nutrients and sex
•Multicellular systems:
Proliferation is limited to specific regions
and circumstances: Growth factors,
cell:cell-interactions,
In mammals growth and proliferation are
independently regulated
Influence of cellular and viral proteins in
the cell cycle machinery
Growth factor stimulation through membrane receptors
Extracellular
ligand binding
domain
Transmembrane
domain
Tyrosine
kinase
domain
EGF
receptor
FGF
receptor
Growth factor stimulation through a membrane receptor
EGFreceptor
Tyrosine
kinase
Cell growth inhibitors that act through a membrane receptor
Anti-GrowthFactors e.g.
TGFß
p16
Cycl D:CDK4
RB
E2Fs
Changes in
Gene Expression
p15
Smads
p27
Cycl E:CDK4 -
Cell Proliferation
(Cell Cycle)
p21
Rb regulates the cell cycle by
binding nuclear transcription
factors E2F/myc
Rb-P
Rb
+ E2F
Phosphorylation causes
Inactivation of Rb
TGFβ
E2F
Rb myc
Cancer and the cell cycle
Introduction
Current view:
• accumulation of multiple mutations within genes of a
single cell
• mutations confer a competitive advantage for cell growth
and (de-) differentiation
• mutations lead to initiation and progression of
malignancies
Proto-oncogenes
• control cell proliferation and differentiation
• are expressed in all subcellular compartments (nucleus,
cytoplasm, cell surface)
• act as protein kinases, growth factors, growth factor
receptors, or membrane associated signal transducers
Oncogenes
• Mutations in proto-oncogenes alter the
normal structure and/or expression
pattern
• Act in a dominant fashion
 gain of function
Mechanisms of oncogene
action
Biochemically, there are three known
mechanisms by which these genes act:
• phosphorylation of proteins, with serine, threonine
and tyrosine as substrates
• signal transmission by GTPases
• regulation of DNA transcription
Tumor Suppressor Genes
• Have normal, diverse functions to regulate cell
growth in a negative fashion (restrain neoplastic
growth; act as cellular “brakes”)
• physical or functional loss of both alleles frees
the cell from constraints imposed by their
protein products
 loss of function
What causes cancer?
• Chemical Carcinogenes
– Aflatoxin B1,, Vinylchloride, β-Propiolacton
Dimethylsulfate ...
• Radiation:
– UV, X-Ray, α,-β,-γ-radiation
• Viruses
– RNA-viruses, DNA-viruses
• Spontaneoud mutations Loss of DNA-repair
machinery  p53
„... manifestation of six essential alterations in cell physiology
that collectively dictate malignant growth.“
Cell, Vol. 100, 2000
1. Self-Sufficiency in Growth Signals
2. Insensitivity to Antigrowth Signals
3. Evading Apoptosis
4. Limitless Replicative Potential
5. Sustained Angiogenesis
6. Tissue Invasion and Metastasis
Summary
„... manifestation of six essential
alterations in cell physiology that
collectively dictate malignant growth.“
Cell, Vol. 100, 2000
1. Self-Sufficiency in Growth Signals
growth signals (PDGF, TGFα) --> autocrine stimulation
overexpression or mutation of receptors (EGF-R, HER2)
disruption of intracellular circuits
(SOS-Ras-Raf -Map-Kinase)
2. Insensitivity to Antigrowth Signals
Disruption of Rb-pathway,
downregulation of death receptors
3. Evading Apoptosis
Loss of proapoptotic regulators (p53),
nonsignaling deathreceptors (FAS)
4. Limitless Replicative Potential
Telomerase activation
5. Sustained Angiogenesis
Increased expression of angiogen. inducers (VEGF, bFGF)
loss of p53 -->downregulation of inhibitors
(thrombospondin-1)
6. Tissue Invasion and Metastasis
„Out-of-order“ CAMs (E-cadherin),
changing integrin expression pattern,
overexpression of extracellular proteases,
downregulation of protease inhibitor genes