"Super p53" mice exhibit enhanced DNA damage response,are tumor resistant and age normally

The tumor suppressor p53 is critical in preventing cancer due to its ability to trigger proliferation arrest and cell death upon the occurrence of a variety of stresses, most notably, DNA damage and oncogenic stress. Here, we report the generation and characterization of mice carrying supernumerary copies of the p53 gene in the form of large genomic transgenes. Prior to this, we demonstrate that the p53 transgenic allele (p53-tg), when present in a p53-null genetic background, behaves as a functional replica of the endogenous gene. "Super p53" mice, carrying p53-tg alleles in addition to the two endogenous alleles, exhibit an enhanced response to DNA damage. Importantly, "super p53" mice are significantly protected from cancer when compared with normal mice. Finally, in contrast to previously reported mice with constitutively active p53, "super p53" mice do not show any indication of premature aging, probably reflecting the fact that p53 is under normal regulatory control. Together, our results prove that cancer resistance can be enhanced by a simple genetic modification and in the absence of undesirable effects.     

The roles of p53 and p21 in normal development and hyperthermia‐induced malformations

BACKGROUND : Hyperthermia (HS) is a well‐studied teratogen that induces serious malformations, including neural tube defects. Our previous studies have shown that HS induces apoptosis by activating the mitochondrial apoptotic pathway. Prior to activation of the mitochondrial apoptotic pathway, HS also activates p53 and its target genes. In the present study, we determine whether p53 and/or p21 play a role as teratogen suppressors or inducers of HS‐induced malformations. METHODS : Pregnant mice carrying all three p53 or p21 genotype embryos were exposed to HS on day 8.5. Subsequently, fetuses were collected on day 15.5, and genotyped. In addition to genotype, we also determined the number of resorptions and dead fetuses as well as the number and types of external malformations. RESULTS : In the absence of HS exposure, fetuses exhibiting exencephaly and spina bifida were observed in approximately 11% of p53 ?/? fetuses, whereas no malformations were observed among p21 ?/? fetuses. Exposure to HS resulted in an increase in exencephaly and polydactyly in fetuses of all three p53 genotypes. However, the incidence of these malformations was statistically significantly higher in p53 ?/? compared to p53 +/? and p53 +/+ fetuses. Exencephaly was the only malformation observed in p21 fetuses exposed to HS, with an approximately 2‐fold increase among p21 +/? and a 3‐fold increase among p21 ?/? compared to p21 +/+ fetuses. CONCLUSIONS : Our study confirms that p53 plays a role in normal development and has shown, for the first time that p53 and p21 function to suppress HS‐induced malformations. Birth Defects Res (Part B) 86:40‐47, 2009. © 2009 Wiley‐Liss, Inc.     

Uncoupling p53 functions in radiation-induced intestinal damage via PUMA and p21

The role of p53 in tissue protection is not well understood. Loss of p53 blocks apoptosis in the intestinal crypts following irradiation but paradoxically accelerates gastrointestinal (GI) damage and death. PUMA and p21 are the major mediators of p53-dependent apoptosis and cell-cycle checkpoints, respectively. To better understand these two arms of p53 response in radiation-induced GI damage, we compared animal survival, as well as apoptosis, proliferation, cell-cycle progression, DNA damage, and regeneration in the crypts of WT, p53 knockout (KO), PUMA KO, p21 KO, and p21/PUMA double KO (DKO) mice in a whole body irradiation model. Deficiency in p53 or p21 led to shortened survival but accelerated crypt regeneration associated with massive nonapoptotic cell death. Nonapoptotic cell death is characterized by aberrant cell-cycle progression, persistent DNA damage, rampant replication stress, and genome instability. PUMA deficiency alone enhanced survival and crypt regeneration by blocking apoptosis but failed to rescue delayed nonapoptotic crypt death or shortened survival in p21 KO mice. These studies help to better understand p53 functions in tissue injury and regeneration and to potentially improve strategies to protect or mitigate intestinal damage induced by radiation.     

p53 involvement in the pathogenesis of fatty liver disease

Obesity is a major health problem in industrialized societies, and fatty liver disease (hepatic steatosis) is common in obese individuals. Oxidative stress originating from increased intracellular levels of fatty acids has been implicated as a cause of hepatocellular injury in steatosis, although the precise mechanisms remain to be elucidated. p53, widely known as a tumor suppressor, has been shown often to be activated in stressed cells, inducing cell cycle arrest or death. Here we demonstrate that p53 is involved in the molecular mechanisms of hepatocellular injury associated with steatosis. We found that p53 in the nucleus is induced in the liver from two mouse models of fatty liver disease, ob/ob and a transgenic mouse model that overexpresses an active form of sterol regulatory element-binding protein-1 in the liver (TgSREBP-1), the one with obesity and the other without obesity. This activation of the p53 pathway leads to the elevation of p21 mRNA expression, which can be considered an indicator of p53 activity, because ob/ob mice lacking p53 generated by targeting gene disruption exhibited the complete restoration of the p21 elevation to wild type levels. Consistent with these results, the amelioration of hepatic steatosis caused by Srebp-1 gene disruption in ob/ob mice lowered the p21 expression in a triglyceride content-dependent manner. Moreover, p53 deficiency in ob/ob mice resulted in a marked improvement of plasma alanine aminotransferase levels, demonstrating that p53 is involved in the mechanisms of hepatocellular injury. In conclusion, we revealed that p53 plays an important role in the pathogenesis of fatty liver disease.     

Loss of p53 compensates for alpha v-integrin function in retinal neovascularization

alpha(v)-Integrin antagonists block neovascularization in various species, whereas 20% of alpha(v)-integrin null mice are born with many normal looking blood vessels. Given that blockade of alpha(v)-integrins during angiogenesis induces p53 activity, we utilized p53 null mice to elucidate whether loss of p53 can compensate for alpha(v)-integrin function in neovascularization of the retina. Murine retinal vascularization was inhibited by systemic administration of an alpha(v)-integrin antagonist. In contrast, mice lacking p53 were refractory to this treatment, indicating that neovascularization in normal mice depends on alpha(v)-integrin-mediated suppression of p53. Blockade of alpha(v)-integrins during neovascularization resulted in an induction of p21(CIP1) in wild type and, surprisingly, in p53 null retinas, indicating that alpha(v)-integrin ligation regulates p21(CIP1) levels in a p53-independent manner. In conclusion, we demonstrate for the first time an in vivo intracellular mechanism for compensation of integrin function and that p53 and alpha(v)-integrins act in concert during retinal neovascularization.     

Loss of poly(ADP-ribose) polymerase-1 causes increased tumour latency in p53-deficient mice.

PARP-1-deficient mice display a severe defect in the base excision repair pathway leading to radiosensitivity and genomic instability. They are protected against necrosis induced by massive oxidative stress in various inflammatory processes. Mice lacking p53 are highly predisposed to malignancy resulting from defective cell cycle checkpoints, resistance to DNA damage-induced apoptosis as well as from upregulation of the iNOS gene resulting in chronic oxidative stress. Here, we report the generation of doubly null mutant mice. We found that tumour-free survival of parp-1(-/-)p53(-/-) mice increased by 50% compared with that of parp- 1(+/+)p53(-/-) mice. Tumour formation in nude mice injected with oncogenic parp-1(-/-)p53(-/-) fibroblasts was significantly delayed compared with parp-1(+/+)p53(-/-) cells. Upon gamma-irradiation, a partial restoration of S-phase radiosensitivity was found in parp-1(-/-)p53(-/-) primary fibroblasts compared with parp-1(+/+)p53(-/-) cells. In addition, iNOS expression and nitrite release were dramatically reduced in the parp-1(-/-)p53(-/-) mice compared with parp-1(+/+)p53(-/-) mice. The abrogation of the oxydated status of p53(-/-) cells, due to the absence of parp-1, may be the cause of the delay in the onset of tumorigenesis in parp-1(-/-)p53(-/-) mice.     

Gene and microRNA expression in p53‐deficient day 8.5 mouse embryos

BACKGROUND: Neural tube defects (NTDs) are one of the most common human birth defects, with a prevalence of approximately 1 in 1000 live births in the United States. In animal studies, deletion of p53 leads to a significant increase in embryos that exhibit exencephaly. Whereas several studies have closely investigated the morphologic changes of p53‐deficient embryos, no study has reported the molecular‐level alteration in p53‐deficient embryos. Here we attempt to identify genes and microRNAs (miRNAs) modified by deletion of p53 in day 8.5 mouse embryos. METHODS: Mouse embryos from p53 heterozygous crosses were collected, genotyped, and embryos of similar genotype (+/+; +/?; ?/?) were pooled. RNA from the pooled samples was isolated to determine mRNA and miRNA expression levels using Whole Genome Bioarrays and Low Density Arrays, respectively. RESULTS: In p53 ?/? embryos, 388 genes showed statistically significant alteration in gene expression of more than twofold compared to p53 +/+ embryos. Expression of p53 and well known p53 target genes, such as p21 and cyclin G1, were significantly down‐regulated in p53 ?/? embryos. In contrast, expression of other p53 target genes, such as Mdm2, Noxa, and Puma, were unchanged. We also identified six genes (Csk, Itga3, Jarid2, Prkaca, Rarg, and Sall4), known to cause NTDs when deleted, that are also down‐regulated in p53 ?/? embryos. Finally, five miRNAs (mir‐1, mir‐30e‐3p, mir‐142‐3p, mir‐301, and mir‐331) also showed statistically significant alterations in expression levels in p53 ?/? embryos compared to p53 +/+ embryos. Combined analysis of the experimental data using stepwise regression model and two publicly available algorithms identified putative target genes of these miRNAs. CONCLUSIONS: Our data have identified genes and miRNAs that may be involved in the mechanisms underlining NTDs and begin to define the developmental role of p53 in the etiology of NTDs. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

PUMA inactivation protects against oxidative stress through p21/Bcl-XL inhibition of bax death

The tumor suppressor protein p53 activates growth arrest and proapoptotic genes in response to DNA damage. It is known that negative feedback by p21(Cip1/Waf1/Sdi1) represses p53-dependent transactivation of PUMA. The current study investigates PUMA feedback on p53 during oxidative stress from hyperoxia and the subsequent effects on cell survival mediated through p21 and Bcl-X(L). Deletion of PUMA in HCT116 colon carcinoma cells increased levels of p53 and p21, resulting in a larger G(1) population during hyperoxia. P21-dependent increase in Bcl-X(L) levels protected PUMA-deficient cells against hyperoxic cell death. Bax and Bak were both able to promote hyperoxic cell death. Bcl-X(L) protection against hyperoxic death was lost in cells lacking Bax, not PUMA, suggesting that Bcl-X(L) acts to inhibit Bax-dependent death. These results indicate that PUMA exerts a negative feedback on p53 and p21, leading to p21-dependent growth suppressive and survival changes. Enhanced survival was associated with increased Bcl-X(L) to block Bax activated cell death during oxidative stress.     

Inactivation of the p19(ARF) tumor suppressor affects intestinal epithelial cell proliferation and integrity

p19(ARF) is a tumor suppressor that is frequently deleted in human cancer. It lies at chromosome 9p21 and shares exons 2 and 3 with p16(ink4a), which is also inactivated by these cancer-associated deletions. The "canonical pathway" by which p19(ARF) is thought to suppress tumorigenesis through activation of the p53 tumor suppressor. In response to hyperproliferative signals, such as expression of oncogenes, p19(ARF) is induced and binds to the MDM2 ubiquitin ligase, sequestering it in the nucleolus to allow the accumulation of p53. However, p19(ARF) also has MDM2 and p53 independent functions. In human colon cancer, p19(ARF) is only rarely deleted, but it is more frequently silenced by DNA promoter methylation. Here we show that inactivation of p19(ARF) in mice increases the number of cycling cells in the crypts of the colonic epithelium. Moreover, inactivation of p19(ARF) exacerbated the ulceration of the colonic epithelium caused by dextran sodium sulfate (DSS). These effects were similar to those observed in mice lacking myeloid translocation gene-related-1 (Mtgr1), and mice lacking both of these genes showed an even greater sensitivity to DSS. Surprisingly, inactivation of p19(ARF) restored the loss of the secretory lineage in mice deficient in Mtgr1, suggesting an additional role for p19(ARF) in the small intestinal epithelium.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian, and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G2/M "arrest," showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis, and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL.p53^-/- mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^-/- mice show chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfb/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfb/Smad pathway.     

Glucose and lipid metabolism alterations in liver and adipose tissue pre-dispose p47phox knockout mice to systemic insulin resistance

Oxidative stress due to enhanced production or reduced scavenging of reactive oxygen species (ROS) has been associated with diet (dyslipidemia) induced obesity and insulin resistance (IR). The present study was undertaken to assess the role of p47^phox in IR using wild type (WT) and p47^phox?/? mice, fed with different diets (HFD, LFD or Chow). Augmented body weight, glucose intolerance and reduced insulin sensitivity were observed in p47^phox?/? mice fed with 45% HFD and 10% LFD. Further, body fat and circulating lipids were increased significantly with 5 weeks LFD feeding in p47^phox?/? mice, while parameters of energy homeostasis were reduced as compared with WT mice. LFD fed knockout (KO) mice showed an enhanced hepatic glycogenolysis, and reduced insulin signalling in liver and adipose tissue, while skeletal muscle tissue remained unaffected. A significant increase in hepatic lipids, adiposity, as well as expression of genes regulating lipid synthesis, breakdown and efflux were observed in LFD fed p47^phox?/? mice after 5 weeks. On the other hand, mice lacking p47^phox demonstrated altered glucose tolerance and tissue insulin sensitivity after 5 weeks chow feeding, while changes in body weight, respiratory exchange ratio (RER) and heat production are non-significant. Our data demonstrate that lack of p47^phox is sufficient to induce IR through altered glucose and lipid utilization by the liver and adipose tissue.     

p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.     

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Mdm2 and Mdm4 loss regulates distinct p53 activities

Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or Mdm4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2(-/-) and Mdm4(-/-) embryos display divergent phenotypes, suggesting that Mdm2 and Mdm4 exert distinct control over p53. To explore the interaction between Mdm2 and Mdm4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2, and Mdm4. These mice had identical survival curves and tumor spectrum as p53(-/-) mice, substantiating the principal role of Mdm2 and Mdm4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, Mdm4, or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking Mdm4. Cells lacking both Mdm2 and Mdm4 had a stable p53 that initiated apoptosis similar to Mdm2-null cells. Additionally, stabilization of p53 in cells lacking Mdm4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and Mdm4 in the regulation of p53 activities.     

MdmX regulates transformation and chromosomal stability in p53-deficient cells

The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.     

Epimorphic regeneration in mice is p53-independent

The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M “arrest,” showed that p21^Cip1/Waf1 was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53^−/− mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21^−/− mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cyclerelated mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.Key words: mouse, regeneration, p53, p21, MRL, ear-hole, Tgfβ