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