Molecular Cell Biology Bio 5068

1
Molecular Cell Biology (Bio 5068) Cell Cycle
Lectures
Dr. Helen Piwnica-Worms November 18, 20
25, 2008
2
Suggested Reading Molecular Biology of The Cell
(Alberts). Chapter 17 Chapter 24
Harper, J.W. and P. D. Adams. 2001. Cyclin
Dependent Kinases. Chem. Rev. 1012511-2526.
Morgan, D. O. 1997. Cyclin Dependent Kinases
Engines, Clocks and Microprocessors. Ann. Rev.
Cell Dev. Biol. 13261-91.

Harper, J.W. and SJ. Elledge. 2007. The DNA
Damage Response Ten Years After. Mol Cell 28
739-745.

Paulsen, R.D. and K.A. Cimprich. 2007. The ATR
Pathway Fine Tuning the fork. DNA Repair
6953-966

3
CELL DIVISION CYCLE
MITOSIS
4
Mitosis 1880's Fleming and Strasburger first
describe mitosis Mitos Greek for 'thread'
mitosis 'action of threads in
division' Observed elongated chromosome
threads forming within the nucleus, watched
them shorten and thicken during mitosis
5
Mitosis 3 stages in terms of regulationProphas
e chromosomes condense centrosomes move to
opposite sides of nucleus (centrosome in higher
eukaryotes spindle pole body in yeast) ends
when nuclear envelope breaks down
(NEBD).Metaphase commences upon
NEBD chromosomes align on the metaphase
plate microtubules form mitotic spindle that
attach to chromosomes (at kinetochores).Anapha
se Linkage between sister chromatids is broken
sister chromatids separate and migrate towards
centrosomes.
6
Basic Properties of the Cell Cycle
7
1950's

De
fi
ne

s
t
age
s
o
f

c
e
ll
cyc
l
e
.

P
r
i
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o

1950
i
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pha
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e

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a
s "unde
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ib
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t"


I
n
t
erph
a
se
pa
rt
o
f

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e
ll
cyc
le

t
ha
t

el
apse
s
b
e
t
w
een

on
e

mit
os
is
and
t
he n
e
xt
.
Prior to 1950s see cell cycle like this
M
I
T
O
S
I
S
M
8
Interphase mitosis Interphase G1, S, G2
(centrosomes duplicate during interphase DNA
content doubles during a discrete period termed S
phase
9
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10
Cell Cycles of Model Organisms
11
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12
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13
Experiments demonstrating that mitotic cells
contain a dominant inducer of mitosis
Stage VI
progesterone
(G2 arrest)
(Metaphase arrest)
14
1971 Masui Marker1971 Smith Ecker
DISCOVERY AND NAMING OF MPF (MATURATION
PROMOTING FACTOR)

• By repeated passage you demonstrate
• that you are not carrying along
• progesterone. Therefore, whatever
• promotes maturation is a component
• of egg.
• Universality of MPF
• 1976 starfish oocytes eggs.
• Wasserman/Smith demonstrated
• activity is present in cleaving frog embryos
• therefore present in mitotic cell cycles.
• mouse oocytes
• 1979 mammalian tissue culture cells
• 1982 budding yeast

15
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16
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17
Final Purification Activity co-purified with a
32 45kDa proteins
18
DISCOVERY AND NAMING OF CYCLINS
19
Clam no maturation prior to fertilization
Fertilization releases from G2 arrest--undergo
meiosis I and II then mitotic divisions begin.
20
CYCLIN IS LINKED TO MPF
F
i
r
s
t

indi
cat
i
o
n
t
h
a
t
cy
cl
i
n

i
s

a co
mp
o
n
e
n
t

of

M
P
F
S
wen
s
on

R
ude
rm
an 1986

(
c
l
a
m

c
yc
l
in

A
)
Pi
nes

Hun
t
1987
(
sea

u
r
ch
i
n cy
cl
in
)
M
ic
ro
i
n
j
ec
t
m
RNA

fo
r
c
la
m

cyc
li
n

A o
r

s
ea ur
c
hin cyc
li
n

B
i
n
t
o

s
t
ag
e
V
I
oocy
te
o
f
Xenopu
s
.

Ob
s
e
r
ve
i
nduc
ti
on

of

M
-
pha
s
e

(
GV
B
D)
i
n ab
s
ence o
f
p
r
oges
t
e
r
one
MPF activity that promotes M-phase in presence
of cycloheximide. Cyclin A injection experiments
were done in the absence of cycloheximide, thus
cyclin A could have induced expression of
something else which, in turn, promoted M-phase
entry.
21
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22
Add
m
RNA

fo
r

s
ea ur
c
hin cyc
li
n

B
23
Determination that cyclin destruction required
for mitotic exit
CYC
L
IN DE
S
TR
U
CTI
O
N

U
B
I
Q
U
I
TIN P
A
T
H
WAY


u
s
e
d s
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a

u
rc
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cy
cl
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Ub
i
qu
iti
n
is
7kDa

p
r
ot
ei
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e
s to
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ys
i
ne

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e
s
i
due
i
n
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ys
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a
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equ
ir
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i
qu
i
na
ti
on
24
Exit from Mitosis Requires Degradation of mitotic
cyclins
cytoplasm from
Wt cyclin stable

EGTA-treated
frog eggs
(high
MPF
)
2

-
Ca
High
MPF
extracts remain arrested
35
in metaphase
S-methionine
o
cycloheximide
23
wt cyclin
40 min.
2

Extracts have high

Ca
levels of histone
H1
kinase activity
(high
MPF
)
25
Determination that cyclin destruction required
for mitotic exit
CYC
L
IN DE
S
TR
U
CTI
O
N

U
B
I
Q
U
I
TIN P
A
T
H
WAY


u
s
e
d s
e
a

u
rc
hin
cy
cl
i
n

B
Ub
i
qu
iti
n
is
7kDa

p
r
ot
ei
n
a
tt
ach
e
s to
l
ys
i
ne

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e
s
i
due
i
n
t
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o
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e
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ys
i
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t

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equ
ir
es

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i
qu
i
na
ti
on
26
Proteolytic degradation of
cyclin
is required for
MPF-
inactivation and cytokinesis BUT not for anaphase
cytoplasm from
Wt cyclin stable

EGTA-treated
90
stable
frog eggs
D
(high
MPF
)
2

-
Ca
High
MPF
extracts remain arrested
in metaphase
o
cycloheximide
23
40 min.
2

Extracts have high

Ca
Wt cyclin degraded
levels of histone
H1
90
stable
D
kinase activity
(high
MPF
)
High
MPF
extracts arrest
in anaphase
27
CELL DIVISION CYCLE
MITOSIS
28
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29
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30

APC

A
naphase
P
romoting
C
omplex
31
Components F Box adapter Brings substrate to
E3 ligase. F-box binds to Skp1 Additional protein
interaction domains (PID WD repeat,
leucine-rich repeat) binds to substrate E2
UBIQ. Conjugating enzyme (transfers UB to
substrate) Skp1 Bridges F-box to
cullin Cullin Organizes and activates E3
complex Recruits E2-UBIQ conjugating enzyme
Ring finger protein Participates in E2
binding and catalysis
32
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33
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34

• 1. What are the protein components of MPF?
• 2. Where was the catalytic subunit of MPF
  identified?
• 3. What distinguishing characteristics of
  cyclins made them stand out?
• 4. How was it demonstrated that cyclin is
  necessary and sufficient for driving frog
  interphase extracts into mitosis?
• 5. How is cyclin normally destroyed?
• 6. What stage of mitosis is cyclin B normally
  destroyed in?
• 7. Where do mitotic extracts expressing a
  non-degradable form of cyclin arrest?
• 8. What E3 ligase is responsible for cyclin B
  degradation?

35
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36
D. Immunoblot Cdc2 IP with Antibody specific
for phosphotyrosine
37
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38
CELL CYCLE REGULATION OF Cdc2
Inhibitory kinase(s)
Phosphatase(s)
T161
T14 Y15 T161
P
P
P
P
Cdc2
Cdc2
Cdc2
Cdc2
Cdc2
Cyclin B
Cyclin B
Cyclin B
INACTIVE
INACTIVE
INACTIVE
INACTIVE
ACTIVE
Cyclin B
Activating Kinase(s)
39

• Questions
• Why is monomeric Cdk inactive as a protein
  kinase?
• How does cyclin binding activate Cdk and promote
  phosphorylation of Thr 14, Tyr 15 and Thr 161?
• Why is the Thr 160 phosphorylation necessary for
  full activity of the Cdk2 kinase?
• How does Thr 14 Tyr 15 phosphorylation keep
  Cdc2/cyclin B complex inactive?

40
Review
CELL DIVISION CYCLE
MITOSIS
41
ENTRY INTO MITOSIS
Cdc2 Cdk1


Cyclin
B
Cyclin
B
M
M
G2
G2
G1
G1
S
S
42
CELL CYCLE REGULATION OF Cdc2
Inhibitory kinase(s)
Phosphatase(s)
T161
T14 Y15 T161
P
P
P
P
Cdc2
Cdc2
Cdc2
Cdc2
Cdc2
Cyclin B
Cyclin B
Cyclin B
INACTIVE
INACTIVE
INACTIVE
INACTIVE
ACTIVE
Cyclin B
Activating Kinase(s)
43

• Questions
• Why is monomeric Cdk inactive as a protein
  kinase?
• How does cyclin binding activate Cdk and promote
  phosphorylation of Thr 14, Tyr 15 and Thr 161?
• Why is the Thr 160 phosphorylation necessary for
  full activity of the Cdk2 kinase?
• How does Thr 14 Tyr 15 phosphorylation keep
  Cdc2/cyclin B complex inactive?

44
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45
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46
Structure of Cdk2 (inactive)
Structure of PKA (active)
47
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48
t
o
f

a
c
ti
ve
sit
e,

hydroxy
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group
s
wou
l
d

be

expo
s
ed

a
nd

ac
c
es
si
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o

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.
Cyc
lin

A
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ot
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as

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er

a
nd

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co
mpl
ex

w
i
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h C
d
k
2
.
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wo

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ur
e
s ar
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ti
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ll
y
t
he
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e
,

i
nd
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ca
ti
ng
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h
a
t Cdk2 b
i
nd
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ng doe
s
no
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gre
at
ly
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f
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in

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confo
rm
a
ti
on.
49
Cyclin Binding Induces Conformational Changes in
Cdk2
Green monomeric Cdk2 Red/Yellow Cyclin bound
Cdk2
50
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51
Genetics in Fission yeast identified key Cdc2
regulators
Inhibitory kinase(s)
Phosphatase(s)
T161
T14 Y15 T161
P
P
P
P
Cdc2
Cdc2
Cdc2
Cdc2
Cdc2
Cyclin B
Cyclin B
Cyclin B
INACTIVE
INACTIVE
INACTIVE
INACTIVE
ACTIVE
Cyclin B
Activating Kinase(s)
52
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53
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54
CELL CYCLE REGULATION OF Cdc2
Wee1 Mik1
Fission Yeast (Tyr. only)
Wee1 Myt1
Cdc25
Higher eukaryotes (Tyr and Thr)
T161
T14 Y15 T161
P
P
P
P
Cdc2
Cdc2
Cdc2
Cdc2
Cdc2
Cyclin B
Cyclin B
Cyclin B
INACTIVE
INACTIVE
INACTIVE
INACTIVE
ACTIVE
Cyclin B
CAK
55
Mitotic Regulators cdc2 Encodes
serine/threonine protein kinase cdc13 Encodes
a B-type cyclin wee1 First gene to be
identified in screen as an inhibitor of
mitosis. Cells mutant in wee1 initiate mitosis
at a size significantly smaller than that of
wild-type cells (wee phenotype). wee1 encodes
a protein kinase that autophosphorylates on
serine, threonine and tyrosine residues. Wee1
phosphorylates Cdc2 on Tyr15. mik1 Encodes a
protein kinase (Mik1) that has 48 sequence
identity to Wee1. Phosphorylates Cdc2 on Tyr
15. cdc25 Isolated in genetic screen as a
dosage-dependent inducer of mitosis. Encodes a
dual-specificity protein phosphatase.
Dephosphorylates Cdc2 on Tyr15 (yeast) and on
both Thr 14 and Tyr 15 (higher eukaryotic
organisms). In higher Eukaryotic organisms there
are three family members (Cdc25A, -B and -C)
56
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57
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58
Additional Levels of Regulation
59
Human Cdc25C INTERPHASE
Impairs nuclear import
14-3-3
NES
Catalytic Domain
S216 NLS
182
199
240 244
473
1
282
MITOSIS
Blocks phosphorylation of serine 216
ACTIVATES PHOSPHATASE
NES
Catalytic Domain
NLS
T48T67 S122 T130 S167
S214
1
177
204
240 244
473
282
60
INTERPHASE
MITOSIS
14-3-3
14-3-3
P
P
P
P
P
P
Low activity,
WEE1
WEE1
low protein levels
WEE1
WEE1
Active
P
P
P
P
P
P
Cyclin B1/Cdk1
Reduced interactions


DOCKING SITE
DOCKING SITE
MYT1
MYT1
Cyclin
B1.
with Cdk1/
Active
MYT1
MYT1
Enhanced activity,
P
P
P
P
Low activity,
14-3-3
14-3-3
nuclear, phosphorylation
P
P
CDC25C
CDC25C
of S216 blocked.
Cytoplasmic
P
P
CDC25C
CDC25C
CyclinB1/Cdk1


DOCKING SITE
Low protein levels,


14-3-3
14-3-3
P
P
interactions with
P
P
P
P
P
P
CDC25A
P
CDC25A
P
P
CyclinB1/Cdk1
P
P
CDC25A
CDC25A
blocked.
P
P
P
P
T14 Y15
T14 Y15
T14 Y15
T14 Y15
Inactive
Cytoplasmic
Cdc2
Cyclin
B1
Cdc2
Cyclin
B1
Cdc2
Cdc2
Cyclin
Cyclin
B1
B1
NES
NES
P
P
P
P
61
GENETIC ANALYSIS OF THE CELL CYCLE Model
Organisms (budding yeast) One CDK, multiple
cyclins. CDK Cyclin-Dependent protein
Kinase Budding yeast Saccharomyces cerevisiae
G1 control Fission yeast Schizosaccharomyce
s pombe G2 control Nomenclature S.
pombe S. cerevisiae wild type
gene cdc2 CDC28 Mutant (recessive) cdc2 or
cdc2- cdc28 (dominant) cdc2D Cdc28 protein
Cdc2 Cdc28
62
Budding yeast- model for G1
control Advantage morphology stage of cell
cycle easily distinguishable in microscope (shape
of cell where you are in cell cycle). With
mammalian cell---cannot tell by shape where you
are (ie G1 vs. G2). Fission yeast cell length
indicates where you are in cell cycle.
Size of bud relative to mother gives rough
estimate of cells position in cell cycle.
63
Hartwell (late 60's early 70's) postulated that
one should be able to apply general mutagenesis
strategies that had been developed
for bacteriophage assembly to identify genes that
regulate the cell cycle. Applied conditional
mutagenesis strategies-looked for ts
mutations. Looked for mutants (at NP temp) that
stopped at particular stages in cell cycle (as
opposed to all over)- called these cdc
mutants (cell division cycle mutants). A cdc
mutant arrests all cells in the population at
the same point in the cell cycle.
32 isolates --now over 60. Lay down genetic
basis of cell cycle control one mutant (cdc28)
particularly important.
64
In addition, CDK is activity is turned ON. APC is
turned OFF.
65
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66
DEFINES START transition in the cell cycle that
initiates the processes that lead ultimately to
cell division. Start was defined as the point
in the cell cycle at which budding, DNA
replication and spindle pole body duplication
become insensitive to loss of CDC28
function. START a point in late G1 where cell
becomes irrevocably committed to entering
S-phase and traversing the cell cycle.
67

• START CONTROL IN BUDDING YEAST
• CDC28 cloned wild-type CDC28 isolated by its
  ability to
• complement mutant cdc28ts cells at the
  nonpermissive temperature.
• CDC28 encodes for a 34 kDa serine/threonine
  protein kinase.
• In 1982 Beach Nurse show that CDC28 complements
  cdc2 in
• S. pombe. Cdc28p requires cyclin binding for
  activity. There are
• Three G1-cyclins in yeast called CLN's CLN 1, 2,
  3.
• CLN 1 2 were isolated as high copy suppressers
  of a ts CDC28
• mutant (cdc28-4 mutation) This mutants is blocked
  at START
• at nonpermissive temperature Screen was set up
  to identify yeast
• genes that could overcome the cell cycle block.
  Using a high copy
• plasmid carrying yeast library they pulled out
  CLN 1 and CLN 2.
• HOW do you explain this rescue?

68
CLN 3 (originally called DAF1/ WHI1) was isolated
prior to CLN1 and 2. Isolated as two dominant
alleles that cause resistance to the G1-arrest
caused by mating pheromone and cause small cell
size (even in absence of mating factor).
WHAT types of mutations in a G1 cyclin might
explain this phenotype??
69
Cln1 and Cln2 levels rise and fall, like other
cyclins. Levels of Cln3 do not oscillate
greatly but its translation is regulated by
availability of nutrients. Cells can grow if
they contain any one of the three CLN's but
a knockout of all three is lethal (if two are
knocked-out and the third is made ts, then cells
arrest at G1 at nonpermissive temperature).
Overproduction of one Cln increases fraction of
cells in S and G2, indicating that high levels of
Cdc28/G1 cyclin complex drives cells through
START. Sequence of CLN's showed homology to
cyclins isolated in other systems-it was already
known that cyclins bound Cdc2. Looked for
complex formation with Cdc28 Looked for cell
cycle regulation Antibodies against cyclins
pulled out Cdc28 Cyclin/Cdc28 complexes had
kinase activity
70
Various Cdc28/cyclin heterodimers regulate
progress through the cell cycle in S. Cerevisiae
Cdc28 Clb1 Cdc28 Clb2
M
Cdc28 Cln3
G2
G1
Cdc28 Clb3 Cdc28 Clb4
S
Cdc28 Cln1 Cdc28 Cln2
Cdc28-Clb5 Cdc28-Clb6
71
G1 Control in Higher Eukaryotic Cells Yeast
one Cdk (fission yeast Cdc2 budding yeast
Cdc28) multiple cyclins. Higher Eukaryotic
Cells multiple Cdks multiple cyclins. New
Mode of Cdk regulation Cdk inhibitors
72

• Four experiments indicate that Cdc2 does not
  control
• G1/S in higher eukaryotic cells
• Xenopus cell free system Depletion of Xe Cdc2
  DNA
• synthesis fine. Extracts fail to enter
  into mitosis. Depletion of
• Cdk2 blocks DNA replication Fang and
  Newport.(1991).
• Cell 66, 731-742.
• mouse cell line (FT210) contains ts Cdc2 At
  nonpermissive
• temperature DNA synthesis fine but cells
  arrest in late G2.
• Th'ng et al (1990) Cell 63, 313-324.
• III. microinject antibodies to Cdc2--cells
  arrest in late G2--no
• G1 arrest Pagano et al. (1993) J. Cell
  Biol. 121, 101-111.

73

• Dominant negative change Asp (146 in Cdc2 and
  145 in Cdk2)
• to Asn corresponding Asp in PKA
  involved in chelating Mg2
• and orienting ? and ? phosphates of MgATP
  in catalytic cleft.
• Assay Cotransfection of plasmids expressing
  dominant negative
• kinase with plasmid encoding a cell surface
  marker (CD20 from B cells). Monitor cell cycle
  distribution of CD20 cells.

Result DN-Cdc2 shifts cell cycle distribution
to increased G2/M population. DN-Cdk2 shifted
cell cycle distribution to increased G1
population (transfection of wild-type Cdk2 or
Cdc2 had no effect). see van den Heuvel and
Harlow (1993) Science 262, 2050-2054.
74

• Identification of other Cdk's
• PCR strategies or hybridization--indicated that a
  large family
• of Cdk's existed.
• Genetic screen (rescue experiments)--isolated
  human Cdc2
• (Lee Nurse) human Cdk2 (Elledge/
  Matsumoto)
• a). Cdk cyclin dependent protein
  kinase--require cyclin binding for activity
  family members are numbered by their order of
  discovery ie Cdc2Cdk

75
Cdk Cyclin Rescue H1
phos. Cdc2Cdk1(p34) B1, B2, B3 yes
Cdk2 (p33) A1, A2 yes E1,
E2 Complementation (Elledge Matsumoto) PCR
(Tsai/Harlow) Cdk4 (p36) D1, 2 3 no
- H1 Cdk4/D most prevalent in fibroblasts
Rb and macrophages
Cdk5 p35 post-mitotic neurons Does
not require CAK (its activating
partner is only in
post-mitotic neurons Cdk6 most similar to
Cdk4 D1, 2 3 - H1 Cdk6/D most
prevalent
Rb in lymphocytes Cdk7p40MO15CAK H
(p37) assoc. with TFIIH Kinase for Thr160
(Cdk2)
Kin 28 budding yeast
Thr161(Cdc2), Thr 172 (Cdk4) homolog.

76
To determine which Cdk is involved in G1
control Do they rescue yeast CDC28 mutant?
Yes Cdc2, Cdk2 Do they contain expected
properties of a G1 Cdk? Antibodies to Cdk2 were
used to characterize kinase throughout cell
cycle. Cdk2 present throughout
cell cycle, its kinase activity fluctuates,
peaking just prior to S-phase. Cdk2
associates with cyclins E A
Functional tests that argue Cdk2 activity
required for S-phase (Cdk2 G1
Cdk) microinjection of antibodies specific for
Cdk2 blocks entry into S-phase dominant
negative mutations in Cdk2 block G1/S
transition depletion of egg extracts of Cdk2
block S-phase no effect on mitosis
77
Q How do dominant negative mutants work?
Dominant negative experiments van den Heuvel
and Harlow (1993) Science 262,
2050-2054. Cdk arrest rescue (cdk) rescue
(cyclin) Cdc2 G2/M Cdc2 B Cdk2 G1 Cdk2 A,
E No effects of Cdk4, Cdk5, Cdk6 and PCTAIRE-1
78

• Identification of G1 cyclins in higher eukaryotic
  organisms
• Eddie Arnold--mechanism that causes adenoma of
  parathyroid--characteristic
• translocation--parathyroid promoter translocates
  to one region of chrom. 11.
• Cloned gene turned on at high levels named PRAD.
  Sequence indicated it was a
• cyclin-like molecule (turned out to be cyclin
  D1). First evidence cyclin deregulated
• in human disease.
• Budding yeast rescue experiment three mammalian
  G1 cyclins were isolated
• because they were able to functional
  complement a strain lacking
• CLN1CLN2CLN3 (delete CLN1, CLN2, put CLN3 on
  regulatable promoter
• (GAL1)(Roberts Beach) or delete CLN1, CLN3,
  put CLN2 on regulatable
• promoter (GAL1) (Reed).
• Beach D1
• Roberts E
• Reed C, D, E
• O'Farrell Drosophila C

79

• Sherr Isolated CLY1 (murine equivalent of human
  cyclin D1) as a CSF-1 inducible
• gene (delayed early gene) in murine
  macrophages.
• D2 (CLY2) and D3 (CLY3) were isolated by low
  stringency hybridization
• (using CLY) and PCR techniques Sherr
  Beach).
• D1 not expressed in mature IL-2 dependent T
  cells
• D2 ubiquitous
• D3 not expressed in proliferating CSF-1
  dependent macrophages
• 4. Cyclin F (Elledge-complementation experiment
  in yeast)
• 5. Cyclin H Morgan biochemically purified CAK
  human p40M015 (Cdk7)
• cyclin H

80
Cyclin Periodicity
81

• Regulation of CDK's
• 1. subunit rearrangement (cyclin binding and
  destruction)
• 2. posttranslational modification
  stimulatory and inhibitory phosphorylations
• transcriptional regulation Cdc2 has E2F-1
  binding sites in its promoter region
• its transcription is repressed in G1 and
  goes up in S and G2 (protein synthesis up
• in S and G1 as well).
• 4. subcellular compartmentalization
• 5. small regulatory proteins p21 and p16
  families
• Small molecular weight inhibitors
• (CKI's cyclin-dependent kinase inhibitors)
• Beach's lab first described them as proteins
  that co-precipitate with Cdk complexes
• (saw them via 35S-methionine labeling) in normal
  human diploid fibroblasts but not in
• transformed cells.

82

• Two families of small molecular weight
  inhibitors
• CIP/KIP family (p21, p27Kip1, p57Kip2) Binds
  to cyclin/Cdk
• complexes and inhibits activity. Will bind to
  Cdk4, Cdk6 and Cdk2
• complexes.
• INK4 family (p16, p15, p18, p19). Specific for
  Cdk4 and Cdk6.
• Binds Cdk subunit alone and prevents cyclin D
  binding or can bind
• to Cdk4/6-Cyclin D heterodimer and inhibit
  activity.
• p21
• Beach laboratory isolated p21 as a protein that
  co-IPs with cyclin D1
• Harper isolated it in a two hybrid screen with
  Cdk2
• Vogelstein isolated it from a subtraction library
  looking for p53 inducible genes
• Morgan purified it as a Cdk2-associated activity
  that inhibited Cdk2 complexes
• Smith isolated it as mRNA that increased 10 to 20
  fold in senescent cells (relative
• to early passage cells).

83
p27 Isolated in two hybrid screen using cyclin
D1/Cdk4 as bait purified using a cyclin E/Cdk2
affinity column. Got protein sequence and then
cloned cDNA. p16INK4a(MTS1, CDKN2, CDK4I)
Beach isolated it in a two hybrid screen using
Cdk4 as bait. Also called MTS1 (multiple tumor
suppressor) maps to chromosome 9p21 locus
(disrupted in variety of human cancers). Other
family members P15INK4b, p18INK4c, p19INK4d
84
How do the small molecular weight inhibitors
inactivate cyclin/Cdk complexes? Structure of
phosphorylated Cdk2/Cyclin A in complex with p27
has been solved (includes 69 amino acid segment
of p27 that is known to be sufficient for
inhibitory activity) Reference Nature (1996),
382325-331. p27 binding disrupts the
conformation of the active site. The p27
peptide stretches across the top of Cdk/cyclin
complex in an extended conformation.
Makes contacts with both Cdk and cyclin. It also
binds within the active site of Cdk2 to
block ATP binding. p27 has an RXL motif,
binds to a region of cyclin A that is used to
target cyclin/cdk complexes to
RXL-containing substrates
85
(No Transcript)
86
G1 Control
M
Cdk 4 6 Cyclin D1, 2, 3
INK4a proteins (p15,16, 18, 19)
G2
Assembly Sequestration
G1
S
Cdk2 Cyclin E
Cip/Kip proteins (p21, p27, p57)
87
CELL CYCLE REGULATION OF Cdc2
Wee1 Mik1
Fission Yeast (Tyr. only)
Wee1 Myt1
Cdc25
Higher eukaryotes (Tyr and Thr)
T161
T14 Y15 T161
P
P
P
P
Cdc2
Cdc2
Cdc2
Cdc2
Cdc2
Cyclin B
Cyclin B
Cyclin B
INACTIVE
INACTIVE
INACTIVE
INACTIVE
ACTIVE
Cyclin B
CAK
88
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89
Various Cdc28/cyclin heterodimers regulate
progress through the cell cycle in S. Cerevisiae
Cdc28 Clb1 Cdc28 Clb2
M
Cdc28 Cln3
G2
G1
Cdc28 Clb3 Cdc28 Clb4
S
Cdc28 Cln1 Cdc28 Cln2
Cdc28-Clb5 Cdc28-Clb6
90
G1 Control in Higher Eukaryotic Organisms
M
Cdk 4 6 Cyclin D1, 2, 3
INK4a proteins (p15,16, 18, 19)
G2
Assembly Sequestration
G1
S
Cdk2 Cyclin E
Cip/Kip proteins (p21, p27, p57)
91

• Regulation of CDK's
• 1. subunit rearrangement (examples )
• Reversible phosphorylation (examples )
• Ubiquitin-mediated proteolysis (examples)
• 4. subcellular compartmentalization
  (examples)

92
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93
G1 Control
RB E2F
MITOGENIC SIGNALS
CYCLIN D STABILITY CYCLIN D-DEPENDENT
KINASES (Cdk4/Cdk6)
94
(No Transcript)
95
G1 Control
CYCLIN E CDK2
RB E2F
CYCLIN E
S-Phase
CYCLIN D STABILITY CYCLIN D-DEPENDENT
KINASES (Cdk4/Cdk6)
MITOGENIC SIGNALS
E2F
CYCLIN A S-PHASE GENES
Relief of Rb- mediated transcriptional
repression
S-Phase
S-PHASE GENES
96
G1 Control
p27KIP1-Phosphorylation Ubiq-Mediated proteolysis
p27KIP1
Assembly Sequestration
CYCLIN E CDK2
RB E2F
CYCLIN E
S-Phase
CYCLIN D STABILITY CYCLIN D-DEPENDENT
KINASES (Cdk4/Cdk6)
MITOGENIC SIGNALS
E2F
CYCLIN A S-PHASE GENES
Relief of Rb- mediated transcriptional
repression
S-Phase
S-PHASE GENES
97
Checkpoints What are they? How were they
defined? How does their derailment contribute to
cancer?
98
Checkpoints intracellular signaling pathways
that determine if previous steps are complete
before proceeding onto the next stage (complete
DNA synthesis before entering mitosis spindles
must be assembled before exiting metaphase and
entering into anaphase) and whether there has
been any damage to the DNA.
DNA damage checkpoint integrity of DNA (DNA
damage is repaired before entering S-phase,
completing S-phase or entering mitosis). DNA
replication Checkpoint replication state of DNA
(must complete DNA synthesis before
mitosis). Spindle assembly checkpoint
integrity of spindle (spindles must be
assembled before exiting metaphase into
anaphase).
99

CHECKPOINTS
IMPROPER SPINDLE
ASSEMBLY
M
M
DNA DAMAGE
UNREPLICATED DNA
STOP!
G2
G2
G1
G1
S
S
DNA DAMAGE
100
IR-induced DNA Damage Checkpoint

• Irradiation of cells produces single stranded
  nicks and gaps and double stranded breaks
• in chromosomal DNA.
• If cells are irradiated in G1 they arrest in G1
  to try and correct any DNA damage before
• replicating the DNA.
• If cells are in S-phase when irradiated they
  delay in S-phase and try to correct damage
• before replicating.
• If cells are in G2 at the time of irradiation
  they delay in G2 (damaged DNA keeps them
• from entering mitosis until cells have repaired
  the damage).

First clue this was due to feedback or checkpoint
control came in 1974 irradiated tissue culture
cells (p53 deficient cells) delay in G2 if
aminopurine (AP), staurosporin or caffeine were
added cells advanced into mitosis
prematurely. Thus, these agents somehow abrogate
the signals that detect damaged DNA and tell the
cell to delay entry into mitosis.
101
(A). To identify genes involved in
sensing/repairing damaged DNA Genetic studies in
yeast irradiate yeast isolate yeast mutants
that are radiation-sensitive identify genes
that detect or respond to damaged DNA(
checkpoint genes) EXAMPLE rad9- this mutant
goes through mitosis without waiting to repair
damaged DNA (but repair machinery is fine in a
rad9 mutant). Thus, rad9 checkpoint gene.
Susan Forsberg
102
(B). To identify genes that detect unreplicated
DNA mutagenize yeast replica plate in
presence or absence of HU hydroxyurea. screen
(visual) for mutants that are particularly
sensitive to hydroxyurea Fission yeast called
hus mutants.
(C). To identify genes whose protein products
monitor the integrity of spindle before exiting
mitosis. In most cells, treatment with
microtubule depolymerizing drugs induces a
mitotic arrest by activating a feedback control
that detects improperly assembled spindles.
Genetic screens have been used to isolate
mutants that lack feedback control mad-
mitotic arrest defect bub- budding
uninhibited by benomyl (microtubule
polymerization inhibitor)
103
CELLULAR RESPONSES TO IONIZING RADIATION
104
Ionizing Radiation Exposure to ionizing radiation
(IR) comes from both natural (background
radiation) man made sources (imaging and
therapy procedures in medicine). Background
radiation Naturally occurring isotopes are in
soil, rocks, and as a result in building
materials. In addition, we are exposed to cosmic
rays from the sun.
UV light and certain forms of ionizing radiation,
such as X-rays and gamma rays, are all part of
the electromagentic spectrum.
Short wavelengths
Ionizing radiation induces DNA double strand
breaks
105
IR- INDUCED DNA DAMAGE CHECKPOINT
M
IR-INDUCED G2-PHASE CHECKPOINT
STOP!
G2
G1
S
IR-INDUCED G1- PHASE CHECKPOINT
IR-INDUCED S-PHASE CHECKPOINT
106
GENOTOXIC STRESS
DNA DS Breaks
ssDNA RPA
ATM-MRN Mre11, Rad50, Nbs1 Brca1, 53BP1,
MDC1 (contain BRCT domains)
ATR-ATRIP Rad17 Rfc2-5 911 (Rad 9, Rad1,
Hus1)
Claspin, Brca1, TopBP1
Chk1 Chk2
p53 Cdc25 phosphatases
MRN complex, BRCA1
CELL CYCLE DELAYS

DNA REPAIR
APOPTOSIS
Genomic Instability Cancer
107
IR- Induced Nuclear Foci
In response to IR, ATM phosphorylates histone
H2AX. Nuclear foci form within seconds after IR
and this is coincident with histone H2AX
phosphorylation ( ?-H2AX). MDC1 (Mediator of
DNA Damage Checkpoint) binds to
phosphorylated H2AX. This is followed by
recruitment of 53BP1, Brca1, Mre11/Rad50/NBS1 to
foci. MDC1, 53BP1 and Brca1 all have BRCT
domains (pSer/pThr binding domains).
108
Ionizing Radiation (IR)
DNA Double Strand Breaks
Foci
pChk2 pChk1
p53 pMdm2
p53
Cdc25A
P phosphorylated by ATM
109
ATM
MRN COMPLEX Mre11/Rad50/NBS1
MDC1Mediator of DNA Damage Checkpoint
FHA Domain binds pTxx(acidic/aliphatic) motif or
pTxx(Y/F) in case of RNF8
BRCT Domain binds p(S/T)xxF motifs
UIM Ubiquitin-interacting domain
AIM Abraxas interacting domain
RING Domain E3 ligase UBC13 E2 ubiquitin
conjugating enzyme
Both RAP80 ABRAXAS are phosphorylated on SQ/TQ
sites in response to IR
110
How do we assess the integrity of the IR-
induced DNA damage checkpoint in the laboratory?
111
IR-INDUCED G1 PHASE CHECKPOINT
IR-INDUCED G1- phase checkpoint
Cells arrest at the restriction point (2 to 3
hours prior to S-phase). Meaure as failure of
cells to enter S-phase several hours after
IR-treatment. Arrest can be hours to days.
112
IONIZING RADIATION-INDUCED G1 CHECKPOINT
Chk2 Chk1
Cdc25A
S
G1
G2
M
IR-INDUCED G1- CHECKPOINT
DEATH
113
IR-INDUCED S-PHASE CHECKPOINT
Lasts 2 to 3 hours. Replicon initiation but not
elongation is blocked. Measure reduction in 3H
thymidine incorporation at early time points
after IR. Express results as of control - see
50 decrease.
IR-INDUCED S-phase checkpoint
114
ABROGATION OF THE IR-INDUCED S-PHASE
CHECKPOINT
mock
IR-Induced S-phase checkpoint
125
Chk1-siRNA
100
DNA Synthesis ( of control)
Meaure reduction in 3H-thymidine incorporation
at early time points after IR. Express results
as of control - see 50 decrease.
75
50
25
0
5
10
20
Dose (Gy)
115
IONIZING RADIATION-INDUCED S-PHASE CHECKPOINT
IR
ATM
ATR
Brca1(S1387) Nbs1 Smc1
Transcriptional regulation DNA Repair
Chk1
Cdc25A
S
G1
G2
M
IR-INDUCED G1- CHECKPOINT
IR-INDUCED S-CHECKPOINT
IR-INDUCED G2-CHECKPOINT
DEATH
116
IR-INDUCED G2-PHASE CHECKPOINT
IR-INDUCED G2-phase checkpoint
G2 Checkpoint
Occurs 30 min. prior to entry into mitosis (cells
enter mitosis for first 30 min. after IR).
Dose-independent (1 to 10 Gy), requires ATM,
Brca1, Rad17. Measure failure of cells to
enter mitosis beginning 30 min. after IR and for
next 3 to 4 h.
117
Premature Entry of G2-phase cells into mitosis
Control - IR
Control IR
0.5
1.9
74 DECREASE
Luciferase RNAi -IR
Luciferase RNAi IR
0.8
2.7
DNA Content
70 DECREASE
Chk1 siRNA -IR
Chk1 siRNA IR
2.3
1.6
30 DECREASE
FITC-Histone-H3 -p
118
IONIZING RADIATION-INDUCED G2-PHASE CHECKPOINT
IR
ATM
BRCA1(S1423) Rad 17
ATR
Chk1
Cdc25A
Transcription DNA REPAIR
S
G1
G2
M
IR-INDUCED G1- CHECKPOINT
IR-INDUCED S-CHECKPOINT
IR-INDUCED G2-CHECKPOINT
DEATH
119
CYTOPLASM NUCLEUS
120
A HYPOPHOSPHORYLATED STABLE FORM OF Cdc25A
ACCUMULATES IN CHK1-DEFICIENT CELLS
IR
121
Mammalian Tissue Culture Cells How do you
establish them? How do you grow them? What is
serum? What is an immortal cell? What is a
transformed cell? What is a senescent
cell? What is the role of telomerase in the
immortalization process?
122
(No Transcript)
123
Incubator (37oC, humidity, CO2) Treated
plastic dishes (negatively charged), Cells
secrete ECM components which adhere to this
surface- allows cells to attach to a
substratum. Media (supplies 9 essential amino
acids (histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan,
and valine) as well as cysteine, glutamine,
tyrosine and vitamins, salts, fatty acids,
glucose Serum (fluid remaining after after
noncellular part of blood (plasma) has been
allowed to clot). Serum contains protein factors
required for proliferation of mammalian cells-
insulin, transferrin (provides iron), growth
factors. Some cells require factors not in serum
i.e. T lymphocytes require IL-2
124

• Establishing human vs mouse tissue culture cells
• Mouse Embryo Fibroblasts Human cells
• 15-30 generations 60-80 doublings before they
  stop dividing
• telomeres are 60kb telomeres are 12 kb
• telomerase widely expressed telomerase not
  normally expressed (germ cells/stem
• cells
• Mouse cells (easier to immortalize than human
  cells) telomerase
• on therefore hypothesis is that cells
  experience culture shock due
• to culture conditions, activate p53 and growth
  arrest (senesce). Rare
• cells emerge that have mutated either p53 or
  p19ARFthese cells are immortal.
• MEFs derived from p53-/- or ARF-/- mice never
  experience senescence,
• these cells divide forever in culture.
• Role of Rb in senescence of mouse MEFs not
  thought to play a role because
• if you overproduce cyclin D, or lose Rb or p16
  cells still arrest.

125
Human cells much more difficult to establish
than mouse cultures. SV40 TAg (inactivates both
p53 and Rb) extends lifespan but does not
immortalize cells Idea culture shock
activates p53 and Rb dependent checkpoints
cells senesce. Can bypass this with SV40 but
cells undergo CRISIS (due to telomere erosion
and subsequent chromosome end fusions) Rare
survivors emerge because they reactivate TERT or
activate ALTnow they are established.
Establish TERT in normal diploid
fibroblasts-cells remain diploid and genetically
stable and do not undergo CRISIS. Conclusion
Human cells rely on p53/Rb checkpoints telomere
maintenance to limit establishment in culture.
126
Human Cells Must inactivate Rb and P53 to get
past senescence but telomeres continue to
shorten. Crisis telomeres so short that you
get chromosome end fusion, aneuploidy and most
cells die.
Human telomerase ribonucleotide enzyme
complex hTERT reverse transcriptase (not
present in adult cells) hTR template RNA TP1
telomerase associated protein 1 (KO had no
phenotype) p23/hsp90 hTERT associated proteins
(chaperones). Required for assembly but not
activity.
127
TRANSFORMED IMMORTAL
128
Normal vs Immortal vs Transformed
Cell PROPERTIES Normal Immortal Transformed C
ontact Inhibition
- Growth Factor Requirements
reduced Anchorage Independence -
- Ability to grow as tumors
in nude mice - -
Proliferative Lifespan finite
infinite infinite Telomerase
activity or ALT absent
present present (except germ
cells, stem cells)
129
Cancer is a Genetic Disease
Cancer arises not from a single mutation, but
from the accumulation of several mutations.
130

• Types of Cancer
• Sporadic isolated cases, no or only distant
  relatives affected
• Familial multiple cases in immediate family
  (BRCA1, 2)

131
Naming of tumors Carcinomas derive from
endoderm (gut epithelium) or ectoderm (skin and
neural epithelia) Sarcomas derive from mesoderm
(muscle, blood, and connective tissue
precursors) Leukemias (class of sarcomas) grow
as individual cells in blood, whereas most other
tumors are solid masses)
132
Acquired Capabilities of Cancer Cells
Cell 100, 57-70 (2000)
133
FACTORS THAT KEEP CANCER AT BAY
Cancer arises not from a single mutation, but
from the
accumulation of several mutations within one cell.
Cells are equipped with enzymes that can repair
errors.
Damaged cells can be eliminated through apoptosis.
Some mutations occur in genes that are not

cancer-promoting

.

134
Cancer-Promoting Mutations

• Genes encoding proteins that positively regulate
• cellular proliferation (proto-oncogenes- ras,
  HER2/neu,
• cell cycle genes- cyclin D1, cyclin E, Cdc25A)
• Genes encoding proteins that negatively regulate
  cell
• proliferation (tumor suppressors-Rb, p16, ARF)
• Genes encoding proteins that regulate programmed
• cell death (Bcl 2, Bcl-xL)
• Genes encoding proteins involved in DNA repair
• (FA, NBS1, MRE11, BRCA1, BRCA2)
• Genes encoding checkpoint proteins
• (ATM, Chk2, p53)

135
Gain of function mutations convert
proto-oncogenes to oncogenes Point mutations
change in a single base pair in a proto-oncogene
that results in a constitutively active protein
product. i.e. ras mutations that substitute
glycine 12 with another aa.- reduces Ras GTPase
activity -keeps it in active form.
Chromosomal translocations fuses two genes
together to produce a hybrid gene encoding a
chimeric protein, whose activity, unlike that of
the parent proteins, often is constitutive.
Bcr-Abl (exists in tetramer and exhibits
constitutive Abl kinase activity. Gleevac
inhibits Abl -used to treat patients with chronic
myelogenous leukemia (CML).
136
Chromosomal translocations brings a growth
regulatory gene under the control of a different
promoter that causes inappropriate or
over-expression of the gene. Burkitts
lymphoma c-myc translocated close to an
antibody gene, leading to its overproduction. Amp
lification abnormal DNA replication of a DNA
segment including proto-oncogenes so that
numerous copies exist, leads to overproduction
of the encoded protein. Overproduction of Her2
RTK due to gene amplification observed in a
subset of Breast cancers. Monoclonal Ab specific
for Her2 causes internalization of the receptor
and is used to treat these breast cancers.
137
Transformation of human cells in culture can be
accomplished by

• Inactivating p53 pathway
• Inactivating Rb pathway
• Expressing hTERT (to maintain telomere length)
• Expressing constitutively-active Ras (oncogene)
• Inhibiting PP2A phosphatase

138
(No Transcript)
139
p53 PATHWAY AND CANCER
Overproduced in certain cancers.
Inactivated in certain cancers.
140
The p53/Rb PATHWAY