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Regulation of gene expression by mutant and oncogenic forms of the p53 tumor suppressor. “The Cell Cycle”. DNA Damage Leads to p53-Mediated Arrest of Cell Cycle. p53 Regulatory Pathways. DNA Repair. Phosphorylation Acetylation Methylation. Oncogene Expression c-Myc and Ras. G 2 Arrest - PowerPoint PPT Presentation
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Regulation of gene expression by mutant andRegulation of gene expression by mutant andoncogenic forms of the p53 tumor suppressoroncogenic forms of the p53 tumor suppressor
““The Cell Cycle”The Cell Cycle”
DNA Damage Leads to p53-DNA Damage Leads to p53-Mediated Arrest of Cell CycleMediated Arrest of Cell Cycle
p53 Regulatory Pathways
MDM2
Apoptosis (Bax)
Oncogene Expressionc-Myc and Ras
DNA DamageUV, Irradiation,
Genotoxic Compounds
p19ARF
(-)
(p21Waf1/Cip1)G1 Arrest
DNA Repair
DNA DamageUV, Irradiation,
Genotoxic Compounds
PhosphorylationAcetylationMethylation
G2 Arrest (14-3-3, Gadd45)
DNA Repair
ATM
p53
Carcinogens can Damage the p53 Carcinogens can Damage the p53 GeneGene
p53 Mutations in Human Cancersp53 Mutations in Human Cancers
p53 mutations in human cancerp53 mutations in human cancer
p53 mutations in colon cancerp53 mutations in colon cancer
p53 mutations in lung cancerp53 mutations in lung cancer
p53 mutations in liver cancerp53 mutations in liver cancer
Can we identify genes Can we identify genes regulated by mutant p53?regulated by mutant p53?
Expression of mutant p53Expression of mutant p53
_ p53-null cells 10(3) expressing p53 mutant(248) from an expression vector
Growth Rates of cells +/- mutant p53Growth Rates of cells +/- mutant p53
1441209672482400
1
2
3
4
5
6
7
8
Hours
Nu
mb
er
of c
ells
(in
Mill
ion
s)
10(3)
CM248.2
CM248.6
600 Gene Array600 Gene Array
Gene Gene Expression Expression
Array 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-3Some 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-3Promoter
TIMP-3 Reporter Construct
Inhibition of TIMP promoter by Inhibition of TIMP promoter by mutant p53mutant p53
Deletions in the TIMP-3 promoterDeletions in the TIMP-3 promoter
p53 TATAAP-1AP-1-2900 2xNFkB 4xAP-1 2xSp1
-2500-2000-1000 -600
-500-400-300-200-100
0
1
2
3
4
5
6
7
1 2 3 4 5
Rel
ati
ve
Fo
ld R
epre
ssi
on
wt p53
mut p53
TIMP-3 -600bp -1000bp -2000bp -2500bp
Deletions eliminate mutant but not wild type p53 repression
Deletions in the TIMP-3 promoterDeletions in the TIMP-3 promoter
p53 TATAAP-1AP-1-2900 2xNFkB 4xAP-1 2xSp1
-2500-2000-1000 -600
-500-400-300-200-100
Loss of p53-mediated repressionLoss of p53-mediated repression
0
2
4
6
8
10
12
TIMP-3 -100 -200 -300 -400 -500
Rel
ativ
e F
old
Rep
ress
ion
0
10000
20000
30000
40000
50000
60000
10(3) CMV 248Cl.2
Colo 320 SW 837
Rela
tive L
ucif
era
se A
cti
vit
y
2900bp
-100bp
Inhibition of TIMP-3 in cells expressingInhibition of TIMP-3 in cells expressing mutant p53mutant p53
Deletions in the TIMP-3 promoterDeletions in the TIMP-3 promoter
p53 TATAAP-1AP-1-2900 2xNFkB 4xAP-1 2xSp1
-2500-2000-1000 -600
-500-400-300-200-100
Two Nuclear Factors Bind to TIMP-3 in the Two Nuclear Factors Bind to TIMP-3 in the Presence of Mutant p53Presence of Mutant p53
-2900
-2800
-2800
* *
mutant p53 - + + +
- + + +
TIMP3(1)
TIMP3(4)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
CMV 248 Cl.2 SW 837 10(3)
Rel
ativ
e Lu
cife
rase
Act
ivit
y
2900bp
-100bp
-100 + (1)
-100 + (4)
A 25-bp element causes repressionA 25-bp element causes repression by p53by p53
RNAi-mediated inhibition of mutant p53 RNAi-mediated inhibition of mutant p53 expressionexpression
_ 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 p53RNAi inhibition of p53
1 2 3 4 1 2 3 4 5 6 7
1 2 3 4 5 6 1 2 3 4 5 6 7 8
SW 837 T98G
CMV 248 Cl.2 Colo 320
p53 mRNA levels in Colo 320 siRNA p53 mRNA levels in Colo 320 siRNA expressing clonesexpressing clones
1 2 3 4 5 6 7 8 9
Cl.4 Cl.5 Cl.7 Cl.12 Cl.10 Cl.11 Cl.17 Cl.18
Inhibition of p53 leads to Inhibition of p53 leads to increase in TIMP-3 expressionincrease in TIMP-3 expression
1 2 3 4 5 6 7 8 9
GAPDH
untransfected siRNA expressing
Cl. 4 Cl.5 Cl.7 Cl.12
Cancer results from numerous genetic Cancer results from numerous genetic mutationsmutations
Model for role of mutant p53 expression in Model for role of mutant p53 expression in progression to metastasisprogression to metastasis
Future GoalsFuture Goals
1.1. Characterize factors that bind to the TIMP promoterCharacterize factors that bind to the TIMP promoter2.2. Determine role of mutant p53 in regulating these factorsDetermine role of mutant p53 in regulating these factors3.3. Determine whether inhibition of mutant p53 can result inDetermine whether inhibition of mutant p53 can result in
elevated TIMP and inhibition of tumor progressionelevated TIMP and inhibition of tumor progression4. 4. Assess contribution of the pathway to cancer progressionAssess contribution of the pathway to cancer progression
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
Troy LogingTroy LogingSondra SpieglSondra SpieglTricia SolitoTricia SolitoShana ThomasShana Thomas