Regulation of gene expression by mutant and
oncogenic forms of the p53 tumor suppressor
“The Cell Cycle”
DNA Damage Leads to p53Mediated Arrest of Cell Cycle
p53 Regulatory Pathways
DNA Repair
Phosphorylation
Acetylation
Methylation
Oncogene Expression
G2 Arrest
c-Myc and Ras
(14-3-3s, Gadd45)
(-)
p19ARF
DNA Damage
UV, Irradiation,
Genotoxic
Compounds
p53
MDM2
ATM
G1 Arrest
(p21Waf1/Cip1)
DNA Repair
Apoptosis
(Bax)
DNA Damage
UV, Irradiation,
Genotoxic
Compounds
Carcinogens can Damage the p53
Gene
p53 Mutations in Human Cancers
p53 mutations in human cancer
p53 mutations in colon cancer
p53 mutations in lung cancer
p53 mutations in liver cancer
Can we identify genes
regulated by mutant p53?
Expression of mutant p53
_
p53-null cells 10(3) expressing p53
mutant(248) from an expression vector
Growth Rates of cells +/- mutant p53
8
Number of cells (in Millions)
7
CM248.2
6
CM248.6
5
4
10(3)
3
2
1
0
0
24
48
72
Hours
96
120
144
600 Gene Array
Gene
Expression
Array
p53 null
Mutant p53
Genes demonstrating altered expression in
the presence of R248W p53
• Induced
_
_
_
_
_
_
_
_
Repressed
Multidrug resistance gene (MDR1)
Tissue inhibitor of metalloproteinases-3 (TIMP-3)
Connexin-32
Monocyte chemotactic protein-3 (MCP-3)
Ribosomal protein gene L37
Ribosomal protein gene RPP-1
Ribosomal protein gene S1
Transcription/Termination Factor I (TTF-1)
PW29, Calcium-binding protein
Heatshock 86Kd protein
Some Properties of TIMP-3
_
_
_
_
_
Inhibitor of matrix metalloproteinases
Required for proper tissue remodeling, vascularization and
matrix turnover
Loss of expression in a mouse tumor progression model potential tumor suppressor
Reduced expression observed in metastatic human colon
cancer
Reconstituted/elevated expression of TIMP-3 leads to
reduced metastasis and reduced angiogenesis
TIMP-3 Reporter Construct
TIMP-3
Promoter
Inhibition of TIMP promoter by
mutant p53
Deletions in the TIMP-3 promoter
-2500
-2000
-1000
-600
-2900
AP-1
2xNFkB
4xAP-1
p53 AP-1
2xSp1
TATA
-500
-400
-300
-200
-100
Deletions eliminate mutant
but not wild type p53 repression
7
Relative Fold Repressio n
6
5
4
wt p53
mut p53
3
2
1
0
TIMP-3
1
-600bp
2-1000bp
-2000bp
3
-2500bp4
5
Deletions in the TIMP-3 promoter
-2500
-2000
-1000
-600
-2900
AP-1
2xNFkB
4xAP-1
p53 AP-1
2xSp1
TATA
-500
-400
-300
-200
-100
Loss of p53-mediated repression
Relative Fold Repression
12
10
8
6
4
2
0
TIMP-3
-100
-200
-300
-400
-500
Inhibition of TIMP-3 in cells expressing
mutant p53
Relative Luciferase Activity
60000
50000
40000
2900bp
30000
-100bp
20000
10000
0
10(3)
CMV 248
Cl.2
Colo 320
SW 837
Deletions in the TIMP-3 promoter
-2500
-2000
-1000
-600
-2900
AP-1
2xNFkB
4xAP-1
p53 AP-1
2xSp1
TATA
-500
-400
-300
-200
-100
Two Nuclear Factors Bind to TIMP-3 in the
Presence of Mutant p53
TIMP3(1)
mutant p53
-2800
-2900
-2800
*
*
-
+
+
TIMP3(4)
+
-
+
+
+
A 25-bp element causes repression
by p53
Relative Luciferase Activity
50000
45000
40000
35000
2900bp
30000
-100bp
25000
20000
-100 + (1)
-100 + (4)
15000
10000
5000
0
CMV 248 Cl.2
SW 837
10(3)
RNAi-mediated inhibition of mutant p53
expression
_
_
_
_
_
RNA-guided regulation of gene expression
conserved in most eukaryotic organisms.
involves dsRNA inhibiting the expression of genes with complementary nucleotide
sequences.
thought to have evolved as a form of innate immunity in defense against viruses,
plays major regulatory roles in development and genome maintenance.
RNAi inhibition of p53
CMV 248 Cl.2
1
2
Colo 320
3
4
1
2
3
SW 837
1
2
3
4
4
5
6
7
T98G
5
6
1
2
3
4
5
6
7
8
p53 mRNA levels in Colo 320 siRNA
expressing clones
Cl.4
1
Cl.5
2
Cl.7
3
Cl.12
4
Cl.10
5
Cl.11
6
Cl.17
7
Cl.18
8
9
Inhibition of p53 leads to
increase in TIMP-3 expression
untransfected
siRNA expressing
Cl. 4
Cl.5
6
7
Cl.7
Cl.12
GAPDH
1
2
3
4
5
8
9
Cancer results from numerous genetic
mutations
Model for role of mutant p53 expression in
progression to metastasis
Future Goals
1. Characterize factors that bind to the TIMP promoter
2. Determine role of mutant p53 in regulating these factors
3. Determine whether inhibition of mutant p53 can result in
elevated TIMP and inhibition of tumor progression
4. Assess contribution of the pathway to cancer progression
ACKNOWLEDGEMENTS
Troy Loging
Sondra Spiegl
Tricia Solito
Shana Thomas