Transcript Thightly Controlled Reversible Immortalization with a

The Cell Cycle and its
implications in diseases
Hansjörg Hauser
Dept. of Gene Regulation and Differentiation
Molecular Biotechnology
HZI, 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
Neben einem zum Vergleich dargestellten Zellkern in der Interphase sind verschiedene
Stadien der Mitose gezeigt (entsprechend der deutschen Literatur, daher ohne
Prometaphase).
Schematische Darstellung des Zellzyklus. Zur besseren Veranschaulichung sind
Chromosomen hier auch in Interphase so gezeichnet, wie sie in der Mitose aussehen.
Dies entspricht jedoch nicht der Wirklichkeit.
The Cell Cycle
Schematic of the cell
cycle.
Outer ring:
I=Interphase,
M=Mitosis;
Inner ring: M=Mitosis,
G1=Gap 1, G2=Gap 2,
S=Synthesis;
Not in ring: G0=Gap
0/Resting.
Cell cycle control was studied in early
embryogenesis of frogs, yeasts and
mammalian cells.
The mechanisms and involved molecules are
highly conserved
The names are sometimes confusing!
Cell division and duplication of
cellular constituents
•DNA
•Proteins, Lipids, Carbohydrates
•Organelles, Membranes...
Standard versus early embryonic
cell cycle
•in early embryonic cells DNA replication is
uncoupled from other synthesis
•The eggs contain more cytoplasm than normal
cells: Stocks
•Some organisms have eggs with 100.000times
more cytoplasm than normal cells
•16 –17 divisions are possible without significant
protein production.
•These divisions are running without feedback
control
Methods to measure cell division
•Counting
•Amount of DNA
•Enzymatic activities
•Incorporation of labeled DNA precursors
•Cell cycle analysis (FACS)
•Dilution of dyes (CFSE)
•Time lapse microscopy
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent cell staining dye.
CFSE is commonly confused with CFDA-SE, although they are not strictly the same
molecule; CFDA-SE is cell permeable, while CFSE is not. As CFDA-SE enters the
cell cytoplasm, intracellular esterases convert the molecule to the fluorescent ester,
CFSE, which is retained within cells.
CFSE is a simple and sensitive technique for analysis of multiple parameters of cells.
This method allows us to examine specific populations of proliferating cells and
identify 7–10 successive cell generations, which has first been employed to detect
proliferation of T cells in experimental animals.[1] CFSE consists of a fluorescent
molecule containing a succinimydyl ester functional group and two acetate moieties.
CFSE diffuses freely inside the cells and intracellular esterases cleave the acetate
groups converting it to a fluorescent, membrane impermeable dye. This dye is not
transferred to adjacent cells. CFSE is retained by the cell in the cytoplasm and does
not adversely affect cellular function. During each round of cell division, relative
fluorescence intensity of the dye is decreased by half. In addition, unlike other
methods, CFSE-labeled viable cells can be recovered for further analysis.
FACS profiles of resting and growing cells
G1 82%
S 12%
G2 6%
G1 33%
S 47%
G2 19%
Kinases control the progression through the
cell cycle
Example MPF (maturation promoting factor)
MPF is composed of a cyclin and a cyclin
dependent protein kinase (cdk)
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
Yeasts only have one Cyclin kinase (cdk1)
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.
Cyclin kinases
Targets of cyclin kinases:
the G2 Kinase complex (MPF)
The kinase activity of cdc-cyclin compexes
is regulated by phosphorylation and
dephosphorylation
Example
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
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.
Checkpoint pathways
(B) Schematic representation of several cell cycle checkpoints. The colored
arrows depict complex signaling pathways that operate in G1 to transmit
information regarding cell proliferation. The red lines connecting particular
events and cell cycle transitions represent the inhibitory signals generated by
checkpoint pathways in response to those events. The points of contact of the
negative growth factor and contact inhibition pathways with the cell cycle are
arbitrary and meant to indicate arrest in G1.
http://www.1lec.com/Genetics/Cell%20Growth
/index.html
Activation of DNA replication
through 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
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
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
P53 is a general gatekeeper for the G1
checkpoint
P53 is a DNA binding protein
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)
Organisation of the DNA-damage-dependent checkpoint pathways of budding yeast. Two distinct
types of DNA damage, in the context of repair complexes, are represented in the schematic. Some
of the components of the NER complex, specific for UV photoproducts, are indicated. The pointers
indicate the incisions generated by the structure-specific endonucleases ERCC1/XPF and XPG. The
Rad50/Mre11/Xrs2 complex is involved in DSB repair. RAD9 and RAD24 define upstream ‘sensor’
branches of the pathway and seem to respond, primarily outside of S phase, to multiple types of
DNA damage. Currently, no other members of the RAD9 branch have been identified but the RAD24
Mammalian cells:
The protein p53 is sensing DNA damage
P53 has a very high turn-over
DNA damage: p53 becomes phosphorylated
and stabilized
The Events in p53 Activation DNA damage (indicated by the break in
the double line at the top) is recognized by a "sensor" molecule that
identifies a specific type of lesion and possibly by the p53 protein,
using its C-terminal domain. The sensor modifies p53 (by
phosphorylation) when both molecules correctly determine that there
is damage. A modified p53 is more stable (enhanced half-life), and a
steric or allosteric change in p53 permits DNA binding to a specific
DNA sequence regulating several downstream genes (p21, MDM2,
GADD45, Bax, IGF-BP, and cyclin G). Two modes of signaling for
cellular apoptosis are possible: one requiring transcription and one
involving direct signaling with no transcription of downstream genes
required.
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
Phosphorylation causes
Inactivation of Rb
Rb-P
Rb
E2F
CyclinD.cdk4
CyclinD
MAPK pathway
Ras
EGF
Rb: E2F
Rb captures E2F:
E2F cannot activate
proproliferative genes
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
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
1. Self-Sufficiency in Growth Signals
• Cancer cell strategies:
– Alteration of extracellular growth signals
– Alteration of transcellular transducers of
growth signals
– Alteration of intracellular circuits that
translate growth signals
– Synthesis of „own“ GS  autocrine stimulation
e.g. production of PDGF, TGFα by glioblastomas
and sarcomas
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)
1. Self-Sufficiency in Growth Signals
EGFreceptor
Tyrosine
kinase
2. Insensitivity to Antigrowth Signals
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
2. Insensitivity to Antigrowth Signals
Disruption of Rb-pathway,
downregulation of death receptors
3. Evading Apoptosis
Apoptotic machinery: sensors and effectors
Survival signals:
IGF-1/IGF-2  Receptor: IGF-1R
IL-3
 Receptor: IL-3R
Death signals:
FAS
 Receptor: FAS-R
TNFα
 Receptor: TNF-R1
3. Evading Apoptosis
Intracellular signals:
• There are both proapoptotic genes (cell death agonists
such as Bax, Bak, Bid, Bim) and antiapoptotic genes (cell
death antagonists such as Bcl-2, Bcl-xL)
• The prototypic gene in this category is Bcl-2
3. Evading Apoptosis
Loss of proapoptotic regulators (p53),
nonsignaling deathreceptors (FAS)
3. Evading Apoptosis
Death Factor
CELL DEATH
Caspase 9
Cytochrom C
Caspase 8
FADD
Bid
MITO
Bax
Bim, etc.
p53
Abnormality
sensor
DNA damage
sensor
4.Limitless Replicative Potential
• With each cell division, there is a shortening of
specific tracts of DNA at the ends of
chromosomes (50 – 100 bp)
• These tracts are called telomeres and are
composed of repetitive DNA sequences
• Once shortened beyond a certain point, cells die
• Telomere shortening, therefore, acts as a clock
that counts cell divisions
4.Limitless Replicative Potential
• In germ cells, telomere shortening is prevented by
the enzyme complex telomerase
• Telomerase adds back any repetitive telomere
sequences lost after a cell division
• Most somatic cells lack telomerase
• For a cell to divide indefinitely, it must prevent
telomere shortening
• Tumor cells do this by activating telomerase
5. Sustained Angiogenesis
• Angiogenesis (growth of new blood vessels)
• Cells reside within 100 µm of a blood
vessel (nutrients/oxygen supply)
• Regulating signals may stimulate/block
angiogenesis
• Initiating signals: e.g. VEGF/ bFGF
• Inhibitor signals: e.g. Thrombospondin-1
5. Sustained Angiogenesis
Increased expression of angiogen. inducers (VEGF, bFGF)
loss of p53 -->downregulation of inhibitors
(thrombospondin-1)
5. Sustained Angiogenesis
• Tumor cells change the Inducer/Inhibitor balance
• Possibilities:
– Increased expression of VEGF/FGF
– Downregulation of Thrombospondine-1,
or ß-interferon
• Loss of p53  Thrombospondine-1
• Activation of ras Increased expression of VEGF
• Proteases release bFGF stored in the ECM
6. Tissue Invasion and Metastasis
6. Tissue Invasion and Metastasis
• Affected proteins:
– cell-cell-adhesion molecules (CAMs)  e.g.
Ca-dependent cadherin families
– Integrins  responsible for linking cells to
extra cellular matrix (ECM)
6. Tissue Invasion and Metastasis
Actin,
Myosin II,
Tropomyosin
Α-Actinin,
Vinculin
α β
Integrin
Fibronectin
6. Tissue Invasion and Metastasis
Signal
transmission
E-cadherin
Ca2+
Cell-to-Cell coupling
by E-cadherin
Signal
transmission
6. Tissue Invasion and Metastasis
• Cancer cells use extracellular proteases to
facilitate invasion into: nearby stroma, across
blood vessel walls, „normal“ epithelian cells
• Upregulation of protease genes, downregulation of
inhibitor genes
• Inactive zymogen forms of proteases are
converted into active enzymes
• Often cancer cell contacted stromal and
inflammatory cells deliver the proteases
6. Tissue Invasion and Metastasis
„Out-of-order“ CAMs (E-cadherin),
changing integrin expression pattern,
overexpression of extracellular proteases,
downregulation of protease inhibitor genes
Summary
„... manifestation of six essential
alterations in cell physiology that
collectively dictate malignant growth.“
Cell, Vol. 100, 2000