p53 Mutant Mice with Altered Cancer and Aging Phenotypes Larry Donehower Baylor College of Medicine Houston, Texas 77030
Jan 11, 2016
p53 Mutant Mice with Altered Cancer and Aging Phenotypes
Larry DonehowerBaylor College of Medicine
Houston, Texas 77030
p53 - A Major Node in the Cellular Stress Response
ROS Hypoxia IR,UV Oncogenes
mdm2 p53
Organismal Aging??
Transient Terminal
Cell CycleArrest Apoptosis
Senescence
Telomere loss
?
?
SirT1
p66Shc
?
0
20
40
60
80
100
AGE (YEARS)
% S
UR
VIV
OR
S
0.50 1.0 1.5 2.0 2.5 3.0
p53+/+n = 56
p53+/-
n = 217
p53-/-n = 72
1 4
ABERRANT GENE TARGETING INTRODUCES A p53 DELETION
Aberrant Gene Targeting
5 11
11
7
*
*
78
8
deletion
Pr
1-6
Wild Type p53 Allele
“m” p53 Allele
AAA
AUG
“m” mRNA
p53 a.a. 240-390 “m” proteinTruncated C-terminal p53 fragment
Missing: Transactivation domainDNA binding domain
M allele mRNA is translated into a C-terminaltruncated p53 protein in vitro and in vivo
WT m
53 kd
24 kD
7 kD
C
In vitro translationof m allele message.
IP with p53 Ab PAb421
24 kD
GST GST-p53
m proteinInteracts withWt p53 in vitro
24 kD
+ m + m + m + m - m
Spleen Kidney Heart Liver Saos2
m protein present in tissuesp53+/m mice
Possible Effects of m Protein on Tumorigenesis
m
No Effect (Null Allele)
Oncogenic Effect
Tumor Suppressor Effect
p53+/m --> tumors same as p53+/-
p53+/m --> tumors before p53+/-
p53+/m --> tumors later than p53+/-
p53 +/m mice have reduced longevity
0
.2
.4
.6
.8
1
0 20 40 60 80 100 120 140 160
Age (Weeks)
p53+/-N= 217> 80% TUM
p53+/mN= 356% TUM
p53+/+N= 5645% TUM
+/m medium lifespan = 96 weeks+/+ median lifespan = 118 weeks
p53 +/m mouse phenotype
• • Tumor resistance• • Reduced longevity• • Reduced body weight• Osteoporosis• Lordokyphosis • Organ atrophy• Decreased regeneration &
stress tolerance
p53 +/+
p53 +/m
Mice appear normal until 12 months, overt phenotype by 16-18 months.
Age-associated organ atrophy in p53 +/m mice and humans
Body Spleen
Liver Kidney
p53 +/+
p53 +/m Mice
Humans
Osteoporosis in the p53 +/m mouse
p53 +/+
p53 +/m
Skin Atrophy in p53+/m Mice
p53+/+ 3 mo
p53+/m 3 mo
p53+/+ 24 mo
p53+/m 24 mo
0
50
100
150
Mus
cle
Mas
s (m
g)
p53+/+ p53+/m
Muscle atrophy in 24 month p53+/m mice
p53 +/m mouse exhibits a decreased regenerative response
• Re-epithelization of skin following 3mm biopsy punch.• 24 month p53 +/m mice show reduced ability to close wound.
Wound healing:
3M mice 24M mice
+/+
+/m
Aging Phenotypes of p53 +/m Mice
Phenotype p53+/+ p53+/m
Median Life Span 118 weeks 96 weeksMaximum Life Span 164 weeks 136 weeksCancer Incidence >45% <6%Body Weight Reduced by 30m Reduced by 18mOrgan Weights (24m) Minimal loss 25-40% lossLymphoid Atrophy Moderate PronouncedLordokyphosis Modest PronouncedOsteoporosis Minimal PronouncedBlood Chemistry Normal NormalPeripheral WBC, RBC Counts Normal NormalMale Fecundity Normal NormalHair Graying and Alopecia Minimal MinimalHair Regrowth Modestly Reduced Greatly ReducedDermal Thickness Moderately Reduced Greatly ReducedSubcutaneous Adipose Moderately Reduced Greatly ReducedWound Healing Normal Re-epithelialization Reduced Re-ep. Muscle Atrophy Moderate PronouncedTolerance Anesthetic Stress Good Poor5-FU Myeloablation Robust WBC Reduced WBC
Senescence-associated beta galactosidase assay
1. Assay, developed by Campisi and colleagues, is specific for senescentcells. Quiescent, presenescent, or immortal cells not stained.
2. Senescent cells stain blue when incubated at pH 6.0 with X-gal.
3. Recently, we have tested this assay in fixed tissues in situ (below is a 20 month p53+/m liver section with blue stained senescent cells).
21 month +/m Liver
Old p53+/m mice show higher percentages of senescent liver cells
3 month 21 month
Senesence Associated B-galactosidase Assay: Liver
0
2
4
6
8
10
12
p53 +/+ p53 +/+ p53 +/m p53 +/m
% Senescent Cells
3 month 21 month
Senescence Associated B-galactosidase Assay: Spleen
0
0.5
1
1.5
2
2.5
3
p53 +/+ p53 +/+ p53 +/m p53 +/m
% Senescence
3 month 3 month21 month 21 month
Old p53+/m mice show higher percentages of senescent spleen cells
Proposed model of m function
C-Term
TA DNA Bind TA DNA Bind
DNA DamageOncogenes
Latent WT p53 Activated WT p53
P
Latent WT p53
C-Term
TA DNA Bind
m
Cell cycle arrestApoptosisSenescence
TA DNA Bind C-Term
Activated WT p53
P P Ac
C-Term
P Ac
Removal of stressors
Elevated p53 levels and stability in the presence of the m protein
• Before and after 5G -IR
Mice with Increased p53 Activity
Mut p53 Our Labp53+/m
Cancer ResistanceEarly Aging Phenotypes
SerranoSuper p53
Cancer ResistanceNormal Aging
ScrableTruncated
p53 Transgenic
Runted MiceEarly Aging Phenotypes
WT p53
WT p53
WT p53
WT p53
WT p53
Mut p53 TG
WT p53 TG
Tissue stem cells and aging
• Adult tissue stem cells are critical for maintaining organ cellularity and function (homeostasis).
• Multiple adult stem cells have been shown to exhibit age- related decline in functionality.
• Relative reduction of HSC functionality may vary with mouse strain longevity (Van Zant).
• p53+/m mice exhibit early organ atrophies and reduced regenerative responses, suggesting earlier failures in maintaining organ homeostasis.
• These p53+/m phenotypes suggest an earlier age-associated reduction in stem cell functionality.
Young HSC numbers
• p53 +/m mice appear to have reduced numbers of progenitor HSC compared to p53 +/+, +/- and -/- mice.
• No significant difference between the p53 +/+, +/- and -/- mice.
0
5
10
15
20
25
30
35
D7 D14 D21 D28 D35
Days of Culture
Cobblestone Frequency (per 100, 000
cells)
WT
+/-
+/m
-/-
0
5
10
15
20
25
30
35
D7 D14 D21 D28 D35
Days of Culture
Cobblestone Frequency (per 100, 000
cells)
WT+/-+/m-/-
Young HSC proliferation
0
10
20
30
40
50
60
WT +/- -/- +/m
p53 Genotype
Percentage of Proliferating Cells (%)
• p53 -/- and +/- HSC proliferative index approximately two fold more than +/+ and +/m counterparts.
• These results comparable to the MSC proliferation profile.
0
10
20
30
40
50
60
WT +/- -/- +/m
p53 Genotypes
Percentage of Proliferating
Cells (%)
Analysis of SP-HSC in p53 +/+ and +/m mice
Sca-1+= 36.62% Sca-1+= 18.97%
p53 +/+ p53 +/m
SP cells are selected from total bone marrow by Hoechst/PI sorting.Pure SP cells are identified by selection of Sca-1+ and GR-1- cells.
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
WT +/- +/m
p53 Genotypes
Percentage of Sca+ SP cells in total bone
marrow (%)
Reduced Numbers of p53+/m Sca-1+ SP Stem Cells in Marrow
18 month old mice
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
WT +/- +/m
p53 Genotypes
Percentage of Sca+ SP cells in total bone
marrow (%)
Stem CellFunctionalCapacity Homeostasis
p53+/+p53+/m
p53+/-
TUMOR
Death
DOES p53 DOSAGE AFFECT STEM CELL FUNCTION?
Reduced CellularityReduced FunctionReduced Stress Tolerance
Acknowledgements Donehower Lab Stuart Tyner Jene Choi Nader Ghebranious Sundaresan Venkatachalam Xiongbin Lu Herbert Igelmann Melissa Dumble
Bradley Lab Allan Bradley Steve Jones
BaylorCory Brayton
Gerard KarsentyDennis Roop
Peggy Goodell
Monica JusticeAndy Salinger
KentuckyGary Van Zant
National Cancer InstituteNational Institute on Aging