The Cell Cycle, Chromosome segregation,
Aneuploidy and Cancer
Susanne Lens
Dept. of Medical Oncology
[email protected]
Conly Rieder
The cell cycle and cyclin/cdk activity
The wonderful world of mitosis (cell division)
short break
The mitotic checkpoint
aneuploidy and cancer
Conly Rieder
The mammalian cell cycle
mitosis
(nuclear division)
cytokinesis
(cytoplasmic division)
46
G1
M
DNA replication (S phase)
G2
G1
2 x 46
G2
mitosis and cytokinesis
(M phase)
S
DNA
replication
46
G1
46
G1
Checkpoints ensure that each daughter cell ends up
with an equal number of undamaged chromosomes
chromosomes improperly
attached to mitotic spindle
damaged or
incompletely
replicated DNA
M
mitosis
(nuclear division)
G2
incompletely
replicated
DNA
unfavorable
extracellular
environment
cytokinesis
(cytoplasmic division)
S
DNA
replication
G1
DNA damage
(p53)
The cell cycle control system:
Order and timing of cell cycle events
DNA
replication
Chromosome
segregation
The cell cycle control system:
Oscillation of protein kinase activities
divide
replicate
divide
activity
replicate
time
replicate
divide
Lessons from cell fusion (1)
G1 phase
Mitosis
M-phase
Promoting
Factor
Lessons from cell fusion (2)
S phase
Mitosis
M-phase
Promoting
Factor
G1 phase
S-phase
S-phase
Promoting
Factor
Lessons from cell fusion (3)
G1 phase
S-phase
S-phase
Promoting
Factor
S phase
G2-phase
DNA
Re-replication
Inhibitor
G1 phase
G2-phase
M-phase
Delaying
Factor
Lessons from cell fusion: Interdependence
DNA
replication
Chromosome
segregation
Cyclins (1)
cyclins
mitosis
MPF
interphase
mitosis
MPF
cyclins
Cyclin/cdks
G1 & G1/S cyclins
M
cdk
cyclin
G2
mitotic cyclin
G1
S
cdk
cyclin
Cyclin/cdks in mammals
Cdk4
Cyclin B
Cdk1
M
M
G1
G2
G2
S
SS
Cyclin D
Cyclin E
Cdk2
Cdk2
Cyclin A
Cdk6
CyclinB-cdk1
CyclinA-cdk2
CyclinE-cdk2
G1
Cyclin B
S
G2
M
Protein level
Kinase activity
Cyclin B /cdk1 (1)
CyclinB-cdk1
CyclinA-cdk2
CyclinE-cdk2
APC
NF-Y
B-Myb
FoxM1
cdk2
G1
S
CycA
CycA
E2F
Ub
Ub
Cyclin B
CycB
G2
Ub
Ub
Ub
M
APC
CycB
CycE
Spindle Checkpoint
Protein level
Kinase activity
Cyclin B /cdk1 (2)
Cdk activation (1)
Wee1
Myt1
CAK
P
Cdc25
P
P
Cdk1
Cyclin B
Cdk activation: positive feedback
Wee1
Myt1
CAK
-
+
+
Cdc25
P
P
P
Cdk1
CyclinB
kinase
activity
Cdk1 levels
Cyclin B1-Cdk1 activity
Positive feedback leads to a bistable
system
Cyclin B1 levels
Cyclin B/Cdk1: Mitosis Promoting Factor
•
•
mitotic entry and
mitotic exit
Prepares cells for nuclear division (mitosis)
From: The cell cycle: Principles of Control by David O Morgan
Mitosis in living cells
Newt Lung Cell
Mitosis in living cells
African Blood Lily
Walther Flemming (1882)
Salamander embryo
Mitosis in the 21st century
interphase
DNA
condensation
prophase
centrosome
separation
late telophase
prometaphase
NEB
formation
mitotic spindle
cytokinesis
cell membrane
ingression
chromosome spindle
attachments
telophase
anaphase B
DNA
decondensation
reformation NE
metaphase
anaphase A
chromosome segregation
spindle elongation
prometaphase
chromosome
alignment
(biorientation)
pictures Geert Kops
The Mitosis ‘to do’ list
 Preparing the chromosomes for segregation
 Centrosome separation
 Mitotic spindle formation
 Connect chromosomes to mitotic spindle
 Check
 Segregation (Anaphase)
 Exit and cytokinesis
The Mitosis ‘to do’ list
 Preparing the chromosomes for segregation
 Centrosome separation
 Mitotic spindle formation
 Connect chromosomes to mitotic spindle
 Check
 Segregation (Anaphase)
 Exit and cytokinesis
Chromosomes are dramatically reorganized in
mitosis
condensation
resolution
(DNA decatenation)
resolution
(cohesin
dissociation)
separation
(cohesin cleavage)
From: The cell cycle: Principles of Control by David O Morgan
Chromatin and chromosome condensation
interphase
mitosis
nucleosome
30nm fiber of interphase
chromatin is folded into
larger and larger coils
fiber formation:
histone tails
and additional
proteins
From: The cell cycle: Principles of Control by David O Morgan
Condensin
Cdk1 substrates
From: Nasmyth K and Hearing CH, Ann. Rev. Biochem, 2005, 74:595-648
Condensin and chromosome condensation
Condensin acts in a
cooperative manner forming
filaments along the
longitudinal axis of the
chromatid
Individual condensin rings may
trap different regions one
chromatid to confer
intrachromatid cohesion
From: Nasmyth K and Hearing CH, Ann. Rev. Biochem, 2005, 74:595-648
Sister-chromatid resolution
Step 1: DNA decatenation
catenation/decatenation/ topoisomerase II
association of sister-chromatids is
now fully dependent on cohesin
From: The cell cycle: Principles of Control by David O Morgan
Cohesin
hinge
ATP
cleaved in
anaphase
From: Nasmyth K and Hearing CH, Ann. Rev. Biochem, 2005, 74:595-648
Cohesin embraces the two sisterchromatids (ring model)
From: Nasmyth K and Hearing CH, Ann. Rev. Biochem, 2005, 74:595-648
Sister-chromatid resolution and separation
Step 2: Removal of cohesin
resolution
Cohesin
(arm)
From: The cell cycle: Principles of Control by David O Morgan
separation
Cohesin
(centromere)
Removal of cohesin: the prophase pathway
and protection of centromeric cohesin
resolution
Cohesin
(arm)
phosphorylation of
Scc3 (SA2) by
Plk1
separation
PP2A
Sgo1
Cohesin
(centromere)
cleavage
of Scc1 by
separase
The Mitosis ‘to do’ list
 Preparing the chromosomes for segregation
 Centrosome separation
 Mitotic spindle formation
 Connect chromosomes to mitotic spindle
 Check
 Segregation
 Exit and cytokinesis
Centrosomes are the microtubule organizing
centers of the cell
centrosomes consist of two centrioles
pericentriolar matrix
contains many γ-tubulin ring complexes (γ-TuRC)
>>> nucleation of microtubuli
nine triplet MT
The centrosome cycle parallels the
chromosome cycle
Chromosome
Centrosome cycle
cycle
M
G1
G2
separation
S
duplication
maturation
Cdk2/Cyclin E and A
Plk4
The Mitosis ‘to do’ list
 Preparing the chromosomes for segregation
 Centrosome separation
 Mitotic spindle formation
 Connect chromosomes to mitotic spindle
 Check
 Segregation
 Exit and cytokinesis
The mitotic spindle: a bipolar array of
microtubules
From: The cell cycle: Principles of Control by David O Morgan
Connecting chromosomes to the mitotic spindle
Aim: obtain bipolar attachments!
Conly Rieder
The Centromere and Kinetochore
Kinetochores are the attachment sites
for the mitotic spindle
CENP-A
Bruce McEwen
Search and capture of chromosomes by
the centrosomal spindle
lateral attachments
and sliding to pole
conversion into
end-on attachment
bi-orientation
tension > stabilization
From: The cell cycle: Principles of Control by David O Morgan
capture or sliding
along existing K-fiber
Some microtubules originate at kinetochore and
contribute to bi-orientation
Maiato and Kodjakov, JCB, 2004
The Mitosis ‘to do’ list
 Preparing the chromosomes for segregation
 Centrosome separation
 Mitotic spindle formation
 Connect chromosomes to mitotic spindle
 Check
 Segregation (anaphase)
 Exit and cytokinesis
short break
•
The mitotic checkpoint
•
Aneuploidy and cancer
Checkpoints ensure that each daughter cell
ends up with an equal number of undamaged
chromosomes
chromosomes improperly
attached to mitotic spindle
damaged or incompletely
replicated DNA
46
2 x 46
incompletely
replicated DNA
46
DNA damage (p53)
46
Cancer and Chromosomal INstability (CIN)
structural and numerical CIN
normal cell
cancer cell
cancer cells have lost the ability to maintain a stable
diploid genome
Chromosome missegregation can result in
aneuploidy
DIC
cell membrane
H2B-GFP
chromosomes
47
45
How can aneuploidy (CIN) contribute to
tumor formation?
- Increased chance of LOH
- Amplification of oncogene
- Haploinsufficiency
- Adaptation to changing environment:
Resistance to anti-cancer drugs
Cell division errors and aneuploidy
normal cell division
chromosome missegregation
46
45
46
cytokinesis failure
92
47
centrosome
defective chromosomespindle attachments
cohesion defects
mitotic checkpoint
dysfunction
The Spindle Checkpoint “Live”
cell 1
cell 2
U2OS cells
Mitosis in living cells
Newt Lung Cell
The Anaphase Promoting Complex/Cyclosome
is an E3 ubiquitin ligase
(Pro)Metaphase
APC/C
Separase
Anaphase
Ub
Ub
Ub
Securin
Cdc20
APC/C
Securin
Cyclin B
cdk1
Ub
Ub
Ub
Cyclin B
cdk1
The Mitotic Checkpoint: Discovering the players
(spindle poison)
chrom. segr. errors
From: Hardwick et al., JCB 2000
Van Oosthuyse et al., MCB 2004
The Mitotic Checkpoint Proteins
Vertebrate
Bub1
1
BubR1
1
Bub3
1
Mps1
1
Mad1
1
Mad2
1
kinase
1086
1050
328
858
718
205
Conserved region
Bub3-binding
Yeast
Fly
Worm
Bub1p
Bub1
BUB1
Mad3p/Bub1p
BubR1
MAD3
Bub3p
Bub3
BUB3
Mps1p
Mps1
MPS1
Mad1p
Mad1
MAD1
Mad2p
Mad2
MAD2
Cdc20-binding
Mad2-binding
Mad1-binding
WD40 repeat
The Mitotic Checkpoint: Mad2
PtK1 cells injected with anti-hMad2 antibodies
From: Canman et al., Curr Biol 2000
Mad2 localizes to unattached kinetochores
prior to metaphase
GFP-hMad2 in PtK1 cells
From: Howell et al., JCB 2000
Mad2 preferentially localizes to unattached
kinetochores
Cell treated with the spindle
depolymerzing drug nocodazole
>> all chromosomes unattached
From: The cell cycle: Principles of Control by David O Morgan
1 mono-oriented
chromosome: 2
kinetochores (1 pair)
…So does Mad1...but BubR1 leaves once
attachments have generated tension
From: Howell et al., Curr Biol 2004
Mitosis in living cells
Newt Lung Cell
A single unattached kinetochore is sufficient to
prevent anaphase onset
Rieder et al (1995) JCB 130: 941
How is the cell cycle inhibited by a lone
unattached kinetochore?
APC/C
APC/C
APC/C
APC/C
APC/C
APC/C
Mad2 shows very fast kinetics at the
unattached kinetochore
From: Shah et al., Curr Biol 2004
‘Activation’ of checkpoint proteins at
unattached kinetochores
inhibition of
APC/Cdc20
amplification
Musacchio, 2005
Yu, 2005
The Mitotic Checkpoint: Activation
prometaphase
Separase
Securin
APC/C
Cyclin B
Mad2-C
BubR1
Bub3
Cdc20
cdk1
Mps1
Bub3
BubR1
R M
Z da Mad2-C
Z 1
Bub1
Mad2-O
Mad2-C
The Mitotic Checkpoint: Inactivation
metaphase
Mps1
Kinetochore MT
Bub3
BubR1
R
Z
Z
Bub1
Mad2-O
M
a Mad2-C
d
1
The Mitotic Checkpoint: Inactivation
anaphase
Ub
Ub
Ub
p31comet/
CMT2
Securin
Mad2-C
Cdc20
Ub
Ub
Ub
Cyclin B
APC/C
BubR1
Bub3
Mitotic Checkpoint defects and anueploidy
Premature
Anaphase
APC/C
Mad2
BubR1
Cdc20
Mps1
Aneuploid Progeny
Bub3 BubR1
M
a Mad2
d
1
Zw10 complex
Bub1
Mad2
Mad2
Bub3
Partial Checkpoint
Inactive Checkpoint
Single
Chromosome
Loss or Gain
Massive
Chromosome
Loss or Gain
Cancer
Death
Mutations in Mitotic Checkpoint Genes
Mutation
Change in Protein
1
2
3
4
5
6
7
8
D76-141+fs
A130S
H151D
G250D
Y259C
H265N
S492Y
S950G
colon
colon
lung
lymphoid
pancreatic
pancreatic
colon
rectal
1
2
3
4
5
1
2
3
4
5
6
7
8
9
T40M
E180D
D537-552
Fs 1023
L875Q
S29L
R59C
T299A
D318-end
R359Q
E515K
R556C
E569K
DY649-end
colon
lymphoid
lymphoid
colon
lymphoid
lymphoid
prostate
lung
prostate
prostate
breast
prostate
brast
lymphoid
1
2
I190V
D192-end
bladder
breast
1
2
N123T
S623G
colon
colon
1
DV67-end
colon
1
E2199D
colon
1
Bub1
1
1086
23 456
BubR1
1050
2
3
45
718
1
12
Mad2
8
1
1
Mad1
7
3 45
678 9
205
1
12
ZW10
1
Zwilch
1
Rod
1
kinase
779
1
2
591
1
2209
1
Conserved region
Bub3-binding
Cdc20-binding
Cancer
Mad2-binding
Mad1-binding
Further Study
The Cell Cycle: Principles of Control
David O Morgan (2007, Oxford Univ. Press)
The Kinetochore: From Molecular Discoveries
to Cancer Therapy
De Wulff and Earnshaw (2009, Springer)
Ways to Tweak Checkpoint Strength
p53mut RBmut BRCA1mut
mutations
BCSG1
Bub1
Tax
E2F
BubR1
Mad1
Mad2
M
a
ZW10
Mad2
BubR1
d
Rod
1
Zwilch
Altered Checkpoint Function
ANEUPLOIDY/CIN