Transcript Cancer and Aging Phenotypes in p53 Mutant Mice

p53 Mutant Mice with Altered Cancer and Aging
Phenotypes
Larry Donehower
Baylor College of Medicine
Houston, Texas 77030
p53 - A Major Node in the Cellular Stress Response
ROS
Hypoxia
Oncogenes
p53
mdm2
Cell Cycle
Arrest
Transient
IR,UV
Apoptosis
?
?
Senescence
SirT1
p66Shc
Terminal
Telomere loss
?
Organismal
Aging??
100
p53+/+
n = 56
% SURVIVORS
80
p53+/60
n = 217
40
p53-/n = 72
20
0
0
0.5
1.0
1.5
AGE (YEARS)
2.0
2.5
3.0
ABERRANT GENE TARGETING INTRODUCES A p53 DELETION
Wild Type p53 Allele
1
*
4
5
11
8
7
Aberrant Gene Targeting
deletion 1-6
“m” p53 Allele
Pr
*
8
7
“m” mRNA
11
AAA
AUG
Truncated C-terminal p53 fragment
Missing: Transactivation domain
DNA binding domain
p53 a.a. 240-390
“m” protein
M allele mRNA is translated into a C-terminal
truncated p53 protein in vitro and in vivo
In vitro translation
of m allele message.
IP with p53 Ab PAb421
WT C
m protein
Interacts with
Wt p53 in vitro
m protein present in tissues
p53+/m mice
GST GST-p53
m
+ m + m + m + m -
m
53 kd
24 kD
24 kD
24 kD
7 kD
Spleen Kidney Heart Liver Saos2
Possible Effects of m Protein on Tumorigenesis
No Effect (Null Allele)
m
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
1
Cumulative Survival
.8
p53+/+
N= 56
45% TUM
.6
p53+/m
N= 35
6% TUM
.4
p53+/N= 217
> 80% TUM
.2
0
0
20
40
60
80
100
120
140
Age (Weeks)
+/m medium lifespan = 96 weeks
+/+ median lifespan = 118 weeks
160
p53 +/m mouse phenotype
p53 +/+
p53 +/m
•
•
•
•
•
•
•
•
Tumor resistance
•
Reduced longevity
•
Reduced body weight
Osteoporosis
Lordokyphosis
Organ atrophy
Decreased regeneration &
stress tolerance
Mice appear normal until 12
months, overt phenotype by
16-18 months.
Age-associated organ atrophy in p53 +/m mice and humans
Humans
p53 +/+
p53 +/m
Mice
Body
Spleen
Liver
Kidney
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
Muscle atrophy in 24 month p53+/m mice
Muscle Mass (mg)
150
100
50
0
p53+/+
p53+/m
p53 +/m mouse exhibits a decreased
regenerative response
Wound healing:
+/m
+/+
3M mice
24M mice
• Re-epithelization of skin following 3mm biopsy punch.
• 24 month p53 +/m mice show reduced ability to close wound.
Aging Phenotypes of p53 +/m Mice
Phenotype
Median Life Span
Maximum Life Span
Cancer Incidence
Body Weight
Organ Weights (24m)
Lymphoid Atrophy
Lordokyphosis
Osteoporosis
Blood Chemistry
Peripheral WBC, RBC Counts
Male Fecundity
Hair Graying and Alopecia
Hair Regrowth
Dermal Thickness
Subcutaneous Adipose
Wound Healing
Muscle Atrophy
Tolerance Anesthetic Stress
5-FU Myeloablation
p53+/+
118 weeks
164 weeks
>45%
Reduced by 30m
Minimal loss
Moderate
Modest
Minimal
Normal
Normal
Normal
Minimal
Modestly Reduced
Moderately Reduced
Moderately Reduced
Normal Re-epithelialization
Moderate
Good
Robust WBC
p53+/m
96 weeks
136 weeks
<6%
Reduced by 18m
25-40% loss
Pronounced
Pronounced
Pronounced
Normal
Normal
Normal
Minimal
Greatly Reduced
Greatly Reduced
Greatly Reduced
Reduced Re-ep.
Pronounced
Poor
Reduced WBC
Senescence-associated beta galactosidase assay
1.
Assay, developed by Campisi and colleagues, is specific for senescent
cells. 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
Senesence Associated B-galactosidase Assay: Liver
12
% Senescent Cells
10
8
6
4
2
0
p53 +/+
p53 +/+
p53 +/m
p53 +/m
3 month
21 month
3 month
21 month
Old p53+/m mice show higher percentages of senescent spleen cells
Senescence Associated B-galactosidase Assay: Spleen
3
% Senescence
2.5
2
1.5
1
0.5
0
p53 +/+
p53 +/+
3 month
21 month
p53 +/m
p53 +/m
3 month
21 month
Proposed model of m function
C-Term
TA
DNA Damage
Oncogenes
TA
DNA Bind
Latent WT p53
P Ac
P
Removal of
stressors
C-Term
DNA Bind
Activated WT p53
Cell cycle arrest
Apoptosis
Senescence
m
TA
C-Term
P
DNA Bind
TA
Latent WT p53
P Ac
DNA Bind
Activated WT p53
C-Term
Elevated p53 levels and stability in the
presence of the m protein
• Before and after 5G g-IR
Mice with Increased p53 Activity
Our Lab
Cancer Resistance
p53+/m Early Aging Phenotypes
Mut p53
WT p53
WT p53 TG
Serrano
Super p53
Cancer Resistance
Normal Aging
WT p53
WT p53
Scrable
Runted Mice
Truncated
p53 Transgenic Early Aging Phenotypes
Mut p53 TG
WT p53
WT p53
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 agerelated 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.
Cobblestone Frequency
Frequency (per
000
Cobblestone
(per100,
100,
000
cells)
cells)
Young HSC numbers
35
35
30
30
25
25
WT
WT
20
20
+/+/+/m
+/m
15
15
-/-/-
10
10
5
5
0
0
D7
D7
D14
D14
D21
D21
Days of Culture
D28
D28
D35
D35
Days of Culture
•
•
p53 +/m mice appear to have reduced numbers of progenitor HSC compared
to p53 +/+, +/- and -/- mice.
No significant difference between the p53 +/+, +/- and -/- mice.
60
60
50
Cells (%)
Percentage
of Proliferating
Percentage
of Proliferating
Cells (%)
Young HSC proliferation
40
50
40
30 30
20 20
10 10
0
0
WT
WT
+/-
+/-
-/-
-/-
+/m
+/m
p53 Genotype
p53 Genotypes
• p53 -/- and +/- HSC proliferative index approximately two fold
more than +/+ and +/m counterparts.
• These results comparable to the MSC proliferation profile.
Analysis of SP-HSC in p53 +/+ and +/m mice
p53 +/+
Sca-1+= 36.62%
p53 +/m
Sca-1+= 18.97%
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.
Reduced Numbers of p53+/m Sca-1+ SP Stem Cells in Marrow
18 month old mice
Percentage
of Sca+
SP SP
cells
in in
total
bone
Percentage
of Sca+
cells
total
bone
marrow
(%)
marrow
(%)
0.09
0.09
0.08
0.08
0.07
0.07
0.06
0.06
0.05
0.05
0.04
0.04
0.03
0.02
0.03
0.01
0.02
0
0.01
WT
+/-
+/m
p53 Genotypes
0
WT
+/p53 Genotypes
+/m
DOES p53 DOSAGE AFFECT STEM CELL FUNCTION?
TUMOR
p53+/-
Stem Cell
Functional
Capacity
Homeostasis
p53+/+
p53+/m
Reduced Cellularity
Reduced Function
Reduced Stress Tolerance
Death
Acknowledgements
 Donehower Lab
Baylor






Gerard Karsenty
Dennis Roop
Stuart Tyner
Jene Choi
Nader Ghebranious
Sundaresan Venkatachalam
Xiongbin Lu
Herbert Igelmann
Cory Brayton
Peggy Goodell
 Melissa Dumble
Monica Justice
Andy Salinger
 Bradley Lab
Kentucky
 Allan Bradley
 Steve Jones
Gary Van Zant

National Cancer Institute
National Institute on Aging