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 ROSHypoxiaIR,UVOncogenes mdm2 p53 Organismal Aging?? TransientTerminal Cell Cycle Arrest Apoptosis Senescence Telomere loss ? ? SirT1 p66Shc ?

AGE (YEARS) % SURVIVORS p53+/+ n = 56 p53+/- n = 217 p53-/- n = 72

14 ABERRANT GENE TARGETING INTRODUCES A p53 DELETION Aberrant Gene Targeting * * deletion Pr 1-6 Wild Type p53 Allele “m” p53 Allele AAA AUG “m” mRNA p53 a.a “m” protein Truncated C-terminal p53 fragment Missing: Transactivation domain DNA binding domain

M allele mRNA is translated into a C-terminal truncated p53 protein in vitro and in vivo WTm 53kd 24kD 7 C In vitro translation of m allele message. IP with p53 Ab PAb kD GST GST-p53 m protein Interacts with Wt p53 in vitro 24 kD + m + m - m Spleen Kidney Heart Liver Saos2 m protein present in tissues p53+/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 +/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 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

Muscle Mass (mg) 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 mice24M mice +/+ +/m

Aging Phenotypes of p53 +/m Mice Phenotypep53+/+p53+/m Median Life Span118 weeks96 weeks Maximum Life Span164 weeks136 weeks Cancer Incidence>45%<6% Body WeightReduced by 30mReduced by 18m Organ Weights (24m)Minimal loss 25-40% loss Lymphoid AtrophyModeratePronounced LordokyphosisModestPronounced OsteoporosisMinimalPronounced Blood ChemistryNormalNormal Peripheral WBC, RBC CountsNormal Normal Male FecundityNormal Normal Hair Graying and AlopeciaMinimalMinimal Hair RegrowthModestly Reduced Greatly Reduced Dermal ThicknessModerately Reduced Greatly Reduced Subcutaneous AdiposeModerately Reduced Greatly Reduced Wound HealingNormal Re-epithelializationReduced Re-ep. Muscle AtrophyModeratePronounced Tolerance Anesthetic StressGoodPoor 5-FU MyeloablationRobust WBCReduced 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 3 month 21 month 3 month 21 month

3 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 Damage Oncogenes Latent WT p53 Activated WT p53 P Latent WT p53 C-Term TA DNA Bind m Cell cycle arrest Apoptosis Senescence 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 Lab p53+/m Cancer Resistance Early Aging Phenotypes Serrano Super p53 Cancer Resistance Normal Aging Scrable Truncated p53 Transgenic Runted Mice Early Aging Phenotypes 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.

Young HSC proliferation 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 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.

Reduced Numbers of p53+/m Sca-1+ SP Stem Cells in Marrow 18 month old mice

Stem Cell Functional Capacity Homeostasis p53 +/+ p53+/m p53+/- TUMOR Death DOES p53 DOSAGE AFFECT STEM CELL FUNCTION? Reduced Cellularity Reduced Function Reduced Stress Tolerance

Acknowledgements nDonehower Lab nStuart Tyner nJene Choi nNader Ghebranious nSundaresan Venkatachalam nXiongbin Lu nHerbert Igelmann nMelissa Dumble nBradley Lab nAllan Bradley nSteve Jones n Baylor Cory Brayton Gerard Karsenty Dennis Roop Peggy Goodell Monica Justice Andy Salinger Kentucky Gary Van Zant National Cancer Institute National Institute on Aging