Transcript No Slide Title - Weizmann Institute of Science

Cancer results from mutations in
genes regulating cell growth
• Two classes of genes are involved:
• 1) Oncogenes - positive regulators
promote cancer by hyperactivity (one allele
is enough)
• 2) Suppressor genes - negative regulators,
promote cancer by loss of activity (two
allele must be mutated)
• The first tumor suppressor- Rb (retinoblastoma)
• The most frequent mutated tumor suppressor - p53
• p53 was first cloned at the weizmann institute and
is mutated in > 50% of all cancer.
• 25,000 papers published on p53 in 22 years
How p53 functions as a tumor suppressor?
Evidence that p53 is a tumor suppressor (II)
•
•
•
Transformation of primary rat embryo fibroblasts (REF) with a combination of
two different oncogenes (e.g. myc+ras) gives rise to transformed foci.
Wild type p53 (wtp53) can suppress the transformation of REF by oncogenes.
M utant forms of p53, found in tumors,can not suppress transformation(in fact,
•
they enhance tra
nsformation).
C onclusion: p53 is a tum or suppre ssor.
myc +ras
myc +ras+ wtp53
myc +ras+ mutp53
Additional evidence that p53 is a tumor suppressor
•
1. Li-Fraumeni syndrome. An inherited syndrome of familial
•
2 Re-introduction of wt p53 into human cancer-derived, p53-negative cells. Parental (non-
cancer. Affected fami ly members
tend to develop cancer at early age (below 40). The most common tumors are br east cancer and soft
tissue sarcoma, but other types of cancer are al so frequent. In the majority of families (about 80%),
affected individuals carry one mutated germline allele of p53 (in all somatic cells of their body). In the
tumors of the LFS pat ients, the normal allele is either lost or mutated, and only the mutant allele is
retained. As a result, the tumor cells do not express any wt p53.
corrected) cells form tumor s in nude mice. Stable cel l lines can be obtained which express low levels of
wt p53. In some cases, the cells grow more slowly in culture and are non-tumorigenic in nude mice. In
other cases the cells grow normally in culture, but still are non-tumorigenic in mice. Sim ilarly, reintroduction ofwt p53 in p53-negative mouse ly mphoma cells causes a reduct ion of tumorigenicity in
syngeneic mice.
•
3. p53 knock-out mice. The mice appear healthy upon birth (with some specific exceptions- a
percentage of femal e embryos die because of a defect in neural tube closure). Mice develop more or less
normally, with no gross developmental defects after birth. However, within very few months, all mice
develop cancer and die. The predomi nant type of cancer is lymphoma of T cell or igin.
Induction of cell death by active p53
Parental M1
M1-p53Val135
clones
at 32oC
Activation of temperature sensitive p53 prevents
transformation
Myc + Ras +
no p53
temperature sensitive p53
ts p53 in mutant
conformation
ts p53 in wild type
conformation
100
1
N
I
200
II
Transactivation
Proline rich
III
300
IV
Specific DNA binding
Protein-protein interactions,
Transcriptional repression
apotosis
V
393
C
Negative regulation
NLS1
of DNA binding
Tetramerisation
Most of p53 mutations are found in the conserved regions
of the central DNA binding domain
(Ko LJ & Prives C, Genes & Dev. 10:1054-1072, 1996)
(Cho Y et al., Science 265:346-55, 1994)
Activated
p53
Transactivation
p21/Waf1
Other genes
Other activities
(C terminal = TFIIH binding?)
(N terminal = SH3 binding?)
Bax, IGF-BP3, Fas,
killer/DR5, Noxa, PIG3,
p53AIP1, PIDD, Puma
etc, etc, etc
Apoptosis
Growth arrest
Growth arrest, apoptosis
Functional p53
DNA damage, oncogene activation
Loss of p53
function
Three Experimental Systems
1. Primary and Secondary Targets of p53
2. Target genes related to apoptosis
3. Comparison of p53 and p73
A chip of 10,000 genes is more than ten
thousand northern blots
Filter hybridization
MDM2
LIG1
PCNA
p21
Representation of 7000 genes on 1 cm2 chip
DIRECT AND INDIRECT
TARGETS
CHX INHIBITS PROTEIN SYNTHESIS AND PREVENTS
ACTIVATION OF SECONDARY TARGETS
Effect of Cycloheximide on H1299 val135 Cells
Only ˜10% of the genes changed expression in all
3 repeats
et al. 2000)
Primary p53 target genes in presence of CHX
38
24
(Coller
Clustering of 259 genes upregulated without CHX
at least three times > 2.5 fold
a. 9 genes (false?)
b. All 38 primary
c. 87% of primary
p53 - Upregulated Genes in H1299-Val135 system
GENE/ PROTEIN NAME
p21waf1
MIC1 - member of TGF-b family
MDM2
PCNA
GADD45
Mitochondrial Stress 70 (Mortalin2)
p57KIP2-CDK inhibitor 1C
RATIO FUNCTION
PIG3-p53 induced gene 3
FAS/APO1
BAX-Bcl2 associated X protein
BAK1- Bcl2 antagonist/killer 1
11.0
3.8
3.7
1.5
38.0
10.0
8.3
CELL CYCLE
3.9
3.0
1.5
APOPTOSIS
P53 - Upregulated Genes in H1299-Val135 system
GENE/ PROTEIN NAME
DDB2-Nucleotide Excision Repair
LIG1 - DNA ligase 1
ERCC5 - DNA Excision Repair related
TDG - G/T mismatch DNA Glycosylase
RPA1 - Replication Factor A Protein 1
RATIO FUNCTION
6.5
DNA REPAIR
2.3
1.9
1.8
1.6
MAPK14
MAP4K5 Activaes Jun N-term Kinase
MAP2K1 - MEK1
3.8
1.6
1.5
KINASE
MYD88 - Myeloid differentiation
Retinoic Acid Receptor Beta
FKBP4
HOXD3 - Homeobox protein
CSPG2 - Chondroitin sulfate
proteoglycan 2
5.4
4.6
3.1
1.8
2.0
RECEPTOR
DEVELOP.
IMMUNOPHIL.
DEVELOP.
ECM
p53- DRIVEN APOPTOSIS
A different cell line (M1) that
undergoes apoptosis by p53 at 32
APOPTOSIS :
- Apoptosis is a genetically controlled program of cell death, also referred to
as cell suicide or Programmed Cell Death (PCD).
- It is an evolutionary conserved mechanism.
- It ultimately leads to elimination of undesired cells
•either superfluous
•or potentially harmful when damaged
- It plays an essential role during developmental as well as adult stages by
allowing tissue remodeling, tissue renewal and maintenance of tissue
homeostasis
Pro apop
Anti apop
APOPTOSIS IN LTR6 CELLS AT 32C.
Sub G1
SCATTER PLOTS OF 404 GENES THAT WERE REGULATED BY
P53 IN LTR6 CELLS
C
B
2h M1
2h M1
12h LTR6
9h LTR6
A
2h M1
D
2h M1
Clustering of 404 genes based on expression kinetics at 32C
Control
Cell type
Time (hr)
M1 2hr
M1
12
M1 12hr
LTR6
2
6
M1
9 12 2
LTR6
2
LTR6 2hr
M1
6 9 12 12 2
LTR6
6 9 12
A
B
C
D
E
F
Kinetics of transcriptional activation
Relative ratio
120
100
Cluster E
Cluster D
Cluster C
80
60
40
20
0
M1
2
6
9
12
M1 2
6
9 12
M1 2 6
9
12
Apoptosis related genes upregulated by p53
H1299 Val Cells
ACCESSION NO.
X63717
U82987
U00115
U16811
MOUSE
ACCESSION NO.
AB021961
M83649
U82532
J04953
Z16410
AW060710
X74504
AF064071
RATIO OF
EXPRESSION (12h)
Fas/APO-1 cell surface antigen
9.8
Bcl-2 binding component 3 (bbc3) PUMA
30.5
Bcl-6
6.8
Bak
7.4
LTR 6 Cells
p53
Fas antigen/TNFR6
TNFR18
Gelsolin
Btg1
EST=PIG8 (Etoposide induced)
T10 mRNA/human sentrin/SUMO-1
Apaf-1
HUMAN
57.8
54.5
11.3
2.9
9.0
6.2
4.3
7.2
ACCESSION NO.
A1909620
X89101
A1923712
X04412
X61123
R11732
U83117
AL135220
Northern Analysis of LTR6 Cells
Apaf-1 mRNA induced at 32 C by
ts-p53
APAF-1 promoter contains p53 target at -604
bp
800
400
RRRCWWGYYY N{0-13 bp}
RRRCWWGYYY
APAF-1 AGACATGTCT GGAGACCCTAGGA cGACAAGCCC
BAX
tcACAAGTTa G
AGACAAGCCT
GADD45 GAACATGTCT
AAGCATGCTg
MDM2
GGACAcGTCC
GGtCAAGTTg
p53 Binding to APAF-1 Target by Gel Shift Analysis
Others
Oligo
p53
Oligo
Oligo
Oligo
Oligo
A
A:
B:
C:
D:
A
+
Ab
A
+
x’sX x’sA
A
A
A
+
+
mut
B C D
+
+
+
AGACATGTCTGGAGACCCTAGGACGACAAGCCC
AGACATGTCT
CGACAAGCCC
AGACATGTCTGGAGAC
CGACAAGCCC
AGAaATGTCTGGAGACCCTAGGACGAaAAGCCC
Apoptotic stimuli
p53
Bax
PUMA
Bcl-2
Smac
Cyt c
/dATP
Apaf-1
Caspase-9
IAPs
Caspase-3
Cellular targets
Apoptosis
p53
p53 family members
Comparison of p73 and p53
induced genes
Scatter plot
Activated
p53
p21/Waf1 /Cip1
cyclin E, D,
cdk 2,4,6
1 4 .3 .3 
GADD4 5
p21
cdk inactivation
Rb fam ily
(active)
cyclin B
cdc 2
p21
Rb
E2F
(inactive)
G1 arrest
G2 arrest
Melanoma
p16
p16
CDK4
cy cD
CDK4
Breast
cy cD
Retina, Lung
RB
P
RB
E2 F
X
E2 F
G1
Active
S