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Week 7: Cell Cycle Control and Apoptosis (Other regulation (p53…
Week 7: Cell Cycle Control and Apoptosis
Apoptosis
Cell death overview
Normal
Asymmetric mitosis
Stem cells
proliferate and differentiate to replace dead cells, which are constantly being turned over at low levels
Abnormal
Dividing too rapidly, irreparable DNA damage, viral infection, etc.
Apoptotic
mechanisms survey for abnormalities and execute self-destruct mechanisms
Unproblematic given
homeostasis
works normally to replenish cells
Sequential cell destruction
Organelle disruption
.
Driven by
caspases
: Cysteine-containing, aspartate-specific proteases
State
Inactive
Zymogen
: harmless to cell, activated by proteolysis
Active
Flag other proteins for destruction by cleaving target proteins at
aspartate residues
that initiate apoptosis
Types
Initiator
Cleaved in response to activation signals
Cleaves executioner caspase, chain of cleaving activity until all are active
Executioner
Activate other caspases and cleave target proteins in damage cell
E.g. target:
sequestering protein
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E.g. target:
actin
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Chr. fragmentation
Cell fragmentation
1) Fragmentation, disruption, loss of shape
2) Cell breaks up into apoptotic bodies
3) Phagocytosis by scavenger cells
Signalling
Intercellular
Neg control: maintain system in 'off' mode
Pos control: rapid response
Possibility that self-destruction is default state, and cells receive constant signals to promote living (e.g. survival factors incl.
mitogens
); advantage is able to eliminate abnormal cells rapidly
Intracellular
Cell-cell communication, secreted molecules, direct cell-cell contact
Fas system
FasL
(membrane-bound protein) protrudes from cell surface and signals adjacent cell to self-destruct
FasL binds receptor on adjacent cell, inducing
trimerisation
of cytoplasmic domains activating
Apaf
(initiator caspase)
Cell cycle
Overview
Definition
A cyclic process of duplication of cell contents then division to produce two daughter cells
Highly regulated and controlled by ordered series of
biochemical switches
Requirements
Cell growth
aspect coordination
Macromolecule biosynthesis
DNA synthesis
Organelle biosynthesis
Events
Newly synthesised DNA
Reformed nucleus
Increase in cell content/membranes
Divided cell
Increase in cell size
Studies
DNA content
Flow cytometry
Stain cells with dye that fluoresces when bound to DNA
2n to 4n to 2n
Morphological
Interventional
Add growth factors to G0 cell (arrested)
Microinject anti-cyclin D antibody to stop DNA synthesis
Add BrdU (thymidine analogue)
BrdU+ cell
: incorporates thymidine analogue and hence has synthesised DNA
BrdU- cells:
cyclin D bound to antibody
Overview (M phase & Interphase)
_
G2
M
G0
Terminally-differentiated
cells withdraw from cycle indefinitely (quiescence)
Interphase cells
Nuclear envelope intact
Individual chromosomes undistinguishable
DNA replicated and centrosome duplicated
Mitosis
2 daughter cells
Cytokinesis
APC/C induces anaphase to allow daughter chromosome separation
Drop in mitotic cyclin-CDK and Cdc14 results in late mitosis steps
Mitotic cells
Nuclear envelope absent
Individual chr. distinguishable
Arrest: chr. improper attachment to mitotic spindle/kinteochores to microtubules; stops degradation of anaphase inhibitor
.
No DNA synthesis
RNA/protein synthesis continue
Increasing Cyclin B
Inactive MPF
Arrest: damaged or incompletely replicated DNA
Late: M cyclin-CDK activates early mitosis steps
S
G1 Restriction point
G1
Re-entry point
:
Cell returning from G0 enters early G1 phase
RNA/protein synthesis
G phases provide time for cell to monitor
internal and external environment
, ensuring conditions are suitable and preparations are complete before S/M phases
Length varies considerably depending on external conditions and extracellular signals from other cells
Under unfavourable conditions, cells delay progress through G1
Arrest: damaged DNA
Cyclin B degradation
G1 cyclin-CDK activates transcription genes for DNA replication
A cell that passes this point is committed to S phase
Late G1:
CDK2-Cyclin A
acts on Rb
.
DNA synthesis (doubles DNA content)
RNA/protein synthesis
Arrest: damaged or incompletely replicated DNA
S-Cdk triggers degradation of Cdc6; activates DNA replication origins
Initiated by SCF ubiquitin protein ligase
DNA tumour viruses
target cell cycle
Proteins of
Human papilloma virus
E7 binds and inhibits RB; E6 binds and inhibits p53
Sufficient to induce loss of cell cycle control even in absence of mutations
E5 causes sustained activation of growth factor receptor PDGF-R
Proteins of
monkey SV40 papova virus
Larget T binds and inhibits Rb and p53
15% cancer linked to infection
Intercellular signalling
Negative
Protein ligands inhibit cell division when growth is unnecessary
TGF-beta
secreted under growth-inhibitory conditions, binds to receptor, initiates signalling cascade, blocks phosphorylation and inactivation of Rb (i.e. ultimately switches off cell cycle)
Positive
Signals from neighbouring cells
Immune system cells: small percentage of cells allowed to become functional; the rest eliminated by apoptosis through
Fas system
and other
death receptors
Heterodimeric protein kinases
Regulate passage through cell cycle via
phosphorylation
and
degradation
Cyclins
Regulatory subunit
Family:
A, B, D, E
Levels increase and decrease during cell cycle
Mammals: D1-D3
Cyclin A
Required for DNA synthesis (S Phase)
Similarities to yeast suggest triggers
pre-replication complexes
(PRC) assembled during G1, and inhibits formation of new PRCs
PRC =
ORC
(origin recognition complex) +
Cdc6
@ origin of replication
G1 to S
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Positive & negative effects limit DNA replication to once per cycle
Cyclin B
G2:
Accumulates through interphase; near end, active MPF reaches critical level causing entry into mitosis (M Phase)
M:
Degraded and causes active MPF level to drop; cell re-enters interphase
Interphase (G1/S/G2:
Level increases; combines with CDK to produce more inactive MPF
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_
Cyclins bind to determine substrate phosphorylation and activate CDK kinases forming
complexes
S-Cdk
Cyclin A + Cdk2
.
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G1/S-Cdk
Cyclin E + Cdk2
M-Cdk
Cyclin B + Cdk1
G1-Cdk
Cyclin D + Cdk4/6
Sequential activation
controls CC progression
Turn on
TFs
that activate genes, the
protein products
of which are necessary for next CC phase
G1 CDK-cyclin
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CDK
Catalytic subunit
Serine/threonine
kinase activity when bound to appropriate cyclin
Family:
CDK1, CDK2, CDK4, CDK6
CDK2
Free, inactive until bound by cyclin
Active site inaccessible to protein substrates (obscured by yellow regions in model)
CDK1 originally known as CDC2 in vertebrates
Other regulation
Species
Have cell cycle regulatory mechanisms similar to somatic cells of humans
Clam
_
Well-conserved mechanisms including complex
network of signalling pathways
that control
cell division
Yeast
Worms
Sea urchins
Xenopus
Role of mammalian CDK/cyclin confirmed by complementarity to yeast mutants with
defective cell cycle controls
Restriction Point
Cells in culture
Growth factor or mitogen
Cells pass through restriction point
Committed to entering & completing cell cycle within 24h
Cell cycle continues even if extracellular signals removed
No growth factors
Cells stop at restriction point
Enter G0: quiescent, non-dividing state
Critical timepoint
Inhibitor (anti-cyclin D in BrdU assay) works well at 10-12h, but not 14-16h
Retinoblastoma protein
Binds and inhibits
E2F
(TF)
Binding activity inhibited by phosphorylation
Cyclin D/E + CDK
regulates phosphorylation
Cdk2-Cyclin A
(Late G1) phosphorylates Rb
Changes shape of Rb and frees
E2F
to promote transcription of genes vital for DNA synthesis /chromosome replication/next cyclin (S phase)
Rb is unphosphorylated in non-proliferating cells
Rb-E2F
complex present in late M Phase to middle of G1 does not promote transcription
CDKs
Mitotic CDK
Synthesised in S/G2 but activity held in check until DNA synthesis complete
Activates chr. condensation, nuclear envelope breakdown, spindle assembly, alignment of chr. at metaphase plate
Activates
APC
(
Anaphase Promoting Complex
) after assembly of mitotic spindle and chromosomes
APC/C in late anaphase
A multi protein complex that directs the
ubiquitin
-mediated proteolysis of mitotic cyclins
Controls degradation of mitotic cyclins by proteasome and exit from mitosis
MPF kinase activity drops and telophase is triggered
Regulated by
Cdh1
Phosphorylated Cdh1 is inactivated by G1 cyclin-CDKs
Phosphatase (
Cdc14
) removes phosphate from Cdh1; once inhibited in G1, mitotic cyclins and hence mitosis increases
CDK regulation
CDK Inhibitors (CKIs)
INK4:
inhibitors of kinase 4
CIP
: CDK inhibitory proteins
Cyclin degradation, e.g. ubiquitination
Phosphorylation levels (phosphates inhibit cyclin-CDK complex)
S-phase onset
Regulated by proteolysis of
Sic1
(S-phase inhibitor)
Sic1 inhibits
S-phase cyclin-CDK complexes
accumulated in G1, preventing DNA replication until cell is prepared
G1 cyclin-CDK
phosphorylates Sic1 at multiple steps, marking it for
polyubiquitination
and degradation
Active S-phase cyclin-CDK triggers DNA synthesis by phosphorylation of components of pre-initiation complexes assembled at DNA replication origins
DNA
damage
UV radiation, gamma irradiation
Must be repaired; hence arrested in cell cycle
p53
Transcription factor/ 53 kDa protein
First identified as
tumour suppressor protein
, as cells lacking p53 do not arrest in G1 (p53 also contributes to G2 arrest)
Mutant p53 found in 50% of human tumours
Found in low levels in cells as it is unstable
Targets gene that encodes
p21
, an inhibitor of G1 CDKs (allows time for DNA damage repair during G1/2 arrest)
Stabilisation
Phosphorylated by protein kinases activated during 'stress'
#
p53 acts as TF; binds to specific
response elements
, and up-regulates expression of specific proteins
In extensive DNA damage, stabilised p53 drives expression of pro-apoptotic proteins
Mutations
abolish ability to bind DNA and activate transcription/expression
Cannot properly act as transcription factor
Checkpoint control does not operate properly
Damaged DNA is replicated, mutations are formed; cells with
homozygous mutated p53 alleles
do not delay
S-phase entry
and do not
apoptose
, leading to tumour formation
Changes to interacting partners of p53
E6/E1b
(sequence-specific DNA-binding domain)
(Tetramerization domain)
MDM2
(transcription-activation domain)
Binding to
N-terminus of p53
represses its transcriptional activity (preventing increase of p21 levels) and mediates p53 degradation; inhibits p53 activity and keeps levels low
60% human lung cancers have G-T transvehrsion at 174, 548, and 273 on p53