The role of p53 in vivo during skeletal muscle post-natal development and regeneration: studies in p53 knockout mice

The tumour suppressor gene p53 is recognised as a central regulator of the cell cycle and apoptosis. Post-natally, p53 mutations are associated with many cancers and mice lacking p53 are prone to spontaneous tumour formation. The present study examines skeletal muscle formation in post-natal mice lacking p53 using two different models of skeletal muscle regeneration. The level of endogenous myogenic cell proliferation in mature skeletal muscle was examined and the time course of muscle regeneration after whole muscle transplantation or crush injury were compared in p53 (-/-) and control C57Bl/6J adult mice, using desmin and proliferating cell nuclear antigen (PCNA) immunohistochemistry and histological analysis. The pattern of inflammation, myoblast proliferation and myotube formation in regenerating p53 (-/-) skeletal muscles appears normal and similar to those in control C57Bl/6J muscle. These data indicate that p53 is not required for the regulation of myoblast proliferation, differentiation and myotube formation in vivo during myogenesis of adult skeletal muscle.     

p19ARF determines the balance between normal cell proliferation rate and apoptosis during mammary gland development

This study demonstrated, for the first time, the following events related to p19(ARF) involvement in mammary gland development: 1) Progesterone appears to regulate p19(ARF) in normal mammary gland during pregnancy. 2) p19(ARF) expression levels increased sixfold during pregnancy, and the protein level plateaus during lactation. 3) During involution, p19(ARF) protein level remained at high levels at 2 and 8 days of involution and then, declined sharply at day 15. Absence of p19(ARF) in mammary epithelial cells leads to two major changes, 1) a delay in the early phase of involution concomitant with downregulation of p21(Cip1) and decrease in apoptosis, and 2) p19(ARF) null cells are immortal in vivo measured by serial transplantion, which is partly attributed to complete absence of p21(Cip1) compared with WT cells. Although, p19(ARF) is dispensable in mammary alveologenesis, as evidenced by normal differentiation in the mammary gland of pregnant p19(ARF) null mice, the upregulation of p19(ARF) by progesterone in the WT cells and the weakness of p21(Cip1) in mammary epithelial cells lacking p19(ARF) strongly suggest that the functional role(s) of p19(ARF) in mammary gland development is critical to sustain normal cell proliferation rate during pregnancy and normal apoptosis in involution possibly through the p53-dependent pathway.     

Methylation of p53 by Set7/9 mediates p53 acetylation and activity in vivo

The protein methyltransferase Set7/9 was recently shown to regulate p53 activity in cancer cells. However, the impact of Set7/9 on p53 function in vivo is unclear. To explore these issues, we created a null allele of Set7/9 in mice. Cells from Set7/9 mutant mice fail to methylate p53 K369, are unable to induce p53 downstream targets upon DNA damage, and are predisposed to oncogenic transformation. Importantly, we find that methylation of p53 by Set7/9 is required for the binding of the acetyltransferase Tip60 to p53 and for the subsequent acetylation of p53. We provide the first genetic evidence demonstrating that lysine methylation of p53 by Set7/9 is important for p53 activation in vivo and suggest a mechanistic link between methylation and acetylation of p53 through Tip60.     

Accelerated development and aging of the immune system in p53-deficient mice.

Development and aging of the immune system lead to an accumulation of memory T cells over the long term. The predominance of T cells of the memory phenotype in the T cell population induces an age-related decline in protective immune responses. We found that development and aging of the immune system were accelerated in p53-deficient (p53-/-) mice; the accumulation of memory T cells was spontaneously accelerated, and a strong T cell-dependent Ab response and Th2 cytokine expression (IL-4, IL-6, and IL-10) were induced by Ag stimulation in young p53-/- mice in the developmental stage. The high T cell proliferative response in the young mice rapidly progressed to a depressed proliferative response in adult mice. It was suggested that the loss of regulation of the cell cycle, DNA repair, and apoptosis by p53 deficiency potentially leads to immunosenescence with the accumulation of memory T cells.     

Telomere shortening is associated with malformation in p53-deficient mice after irradiation during specific stages of development

The natural ends of linear chromosomes, the telomeres, recruit specific proteins in the formation of protective caps that preserve the integrity of the genome. Unprotected chromosomes induce DNA damage checkpoint cascades and ultimately lead to senescence both in mouse and man in a p53 dependent manner and initial telomere length setting therefore determines the proliferative capacity of each cell. Yet, only little information is available on telomere biology during embryonic development. We have previously shown that the p53 gene plays a crucial role in the development of malformations (exencephaly, gastroschisis, polydactyly, cleft palate and dwarfism) in control and irradiated mouse embryos. Here, we investigated telomere biology and the outcome of radiation exposure in wild type (p53+/+) and p53-mutant (p53+/-- and--/--) C57BL mouse foetuses irradiated at three different developmental stages. We show that telomeres are significantly shorter in malformed foetuses as compared to normal counterparts. In addition, our results indicate that the observed telomere attrition is primarily associated with p53-deficiency but is also modulated by irradiation, more specifically during the gastrulation and organogenesis stages. In conclusion, we formulate a hypothesis in which telomere shortening is linked to the absence of p53 in mouse foetuses and that when, in the presence of shorter telomeres, these foetuses are irradiated, the chance for the occurrence of developmental defects increases substantially.     

Microwave-accelerated fixation and lacZ activity staining of testicular cells in transgenic mice.

Microwave energy has been used in conjunction with glutaraldehyde to rapidly fix testicular samples of transgenic mice (whole tubules, individual cells, and cryosections) as a preparation for histochemical bacterial beta-galactosidase activity staining. The results demonstrate that the microwave-enhanced aldehyde fixation step is a convenient and simple adaptation for routine analyses, with almost no artifactual consequences or gross distortions in morphology at the microscopic level. The entire procedure (from sacrificing the animal to microscopic observation of the blue spermatogenic cells) can be completed in 1 h.     

p53 mutations associated with increased sensitivity to ionizing radiation in human head and neck cancer cell lines

The p53 tumour suppressor gene is activated following cellular exposure to DNA-damaging agents. The functions of wild-type p53 protein include transient blocking of cell cycle progression, direct or indirect stimulation of DNA repair machinery and triggering of apoptosis if DNA repair fails. Therefore, the status of p53 protein may be critically associated with tumour cell radiosensitivity.
In the present study we examine the intrinsic radiosensitivity of 20 human carcinoma cell lines derived from 15 patients with different types of head and neck tumour. Radiosensitivities were measured in a 96-well plate clonogenic assay in terms of the mean inactivation dose, surviving fraction at 2 Gy, and constants α and β in the linear quadratic survival curve. The p53 allele status was determined by amplifying exons 4–10 by the polymerase chain reaction (PCR), screening for mutations using single-strand conformation polymorphism (SSCP) analysis and determining the exact type and location of a mutation by direct sequencing. The results showed that prevalence of p53 mutations in squamous cell carcinoma (SCC) cell lines is high (80%), and that deletion of one or both wild-type alleles is common (75%). Intrinsic radiosensitivity of the cell lines varied greatly in terms of mean inactivation dose, from 1.4±0.1 to 2.6±0.2 Gy. Radiosensitivity correlated well with the p53 allele status so that cell lines carrying a wild-type p53 allele were significantly ( P <0.01) more radioresistant (mean inactivation dose 2.23±0.15 Gy) than cell lines which lacked a wild-type gene (1.82±0.24 Gy).
Evaluation of our own results and those published in the literature lead us to conclude that absence of the wild-type p53 allele in human head and neck cancer cell lines is associated with increased radiosensitivity. However, the sensitivity is also strongly dependent on the exact type and location of the p53 mutation.     

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.     

Variations of chromosome radiation sensitivity in fetal and adult mice during gestation

Mice were exposed to 2 Gy of γ-rays at various days of pregnancy, and just before and after gestation. Chromosomes were analyzed 4 h after irradiation in spontaneously dividing hematopoietic cells from liver for fetuses and bone marrow for mothers. On average, there was significantly less chromosome damage in fetuses than in mothers. A very strong increase of chromosome breakage was observed in mothers at days 16–19 of gestation. This increase parallels that of gestation hormones, suggesting a direct relationship. The differences between fetuses and mothers in relation to gestation age result from the increase in the rate of chromatid and chromosome breaks but not of chromatid exchanges, which remained stable. This suggests that a DNA repair step involved in joining broken extremities is the cause. More experiments are needed to understand the origin of these variations of radiation sensitivity and the possible extrapolation of these observations to other species.     

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.     

p53 facilitates pRb cleavage in IL-3-deprived cells: novel pro-apoptotic activity of p53.

In the interleukin-3 (IL-3)-dependent lymphoid cell line DA-1, functional p53 is required for efficient apoptosis in response to IL-3 withdrawal. Activation of p53 in these cells, by either DNA damage or p53 overexpression, results in a vital growth arrest in the presence of IL-3 and in accelerated apoptosis in its absence. Thus, IL-3 can control the choice between p53-dependent cell-cycle arrest and apoptosis. Here we report that the cross-talk between p53 and IL-3 involves joint control of pRb cleavage and degradation. Depletion of IL-3 results in caspase-mediated pRb cleavage, occurring preferentially within cells which express functional p53. Moreover, pRb can be cleaved efficiently by extracts prepared from DA-1 cells but not from their derivatives which lack p53 function. Inactivation of pRb through expression of the human papillomavirus (HPV) E7 oncogene overrides the effect of IL-3 in a p53-dependent manner. Our data suggest a novel role for p53 in the regulation of cell death and a novel mechanism for the cooperation between p53 and survival factor deprivation. Thus, p53 makes cells permissive to pRb cleavage, probably by controlling the potential activity of a pRb-cleaving caspase, whereas IL-3 withdrawal provides signals that turn on this potential activity and lead to the actual cleavage and subsequent degradation of pRb. Elimination of a presumptive anti-apoptotic effect of pRb may then facilitate conversion of p53-mediated growth arrest into apoptosis.     

Genotoxic effects of radiation and hyperthermia in linear mice with different radiation sensitivity

The features of the repair processes of DNA damage and their implementation at the chromosome level in marrow cells of experimental animals (mices of the CBA and C3H lines) with different radiation sensitivity were studied. It is shown that the radiomodifying efficiency of moderate hyperthermia (HT) is higher for animals of the radioresyst line. At the same time, it was possible to observe intensive elimination of chromosomal damages, the level of which in later observations almost was not distinguished from the control values. For mices of more radiosensitive lines, a long-term potentiation damaging action of irradiation at the chromosomal level was observed as an additional HT effect.     

p53 restoration kills primitive leukemia cells in vivo and increases survival of leukemic mice

Loss of p53 function is a common feature of human cancers and it is required for differentiated tumor cell maintenance; however, it is not known whether sustained inactivation of the p53 pathway is needed for cancer stem cell persistence. Chronic myeloid leukemia (CML) is caused by a chromosome translocation that generates the BCRABL oncogene encoding a constitutively active protein tyrosine kinase. The disease originates in a hematopoietic stem cell and is maintained by leukemic stem cells (LSCs). Treatment with specific tyrosine kinase inhibitors does not eliminate LSCs because they do not depend on the oncogene for survival. We have combined a switchable p53 knock-in mouse model, p53^KI/KI, with the well-characterized Sca1-BCRABLp210 CML transgenic model, to show that transient restoration of p53 slows disease progression and significantly extends the survival of leukemic animals, being the mechanism responsible for this effect, apoptotic death of primitive leukemia cells. In agreement with these in vivo findings, in vitro assays show that restoring p53 reduces hematopoietic colony formation by cells of leukemic animals. These results suggest that reestablishing p53 function may be a therapeutic strategy for the eradication of leukemic stem cells and to prevent disease progression.     

neu/ERBB2 cooperates with p53-172H during mammary tumorigenesis in transgenic mice.

Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mutations in p53. The most common p53 mutation in human breast cancers is an Arg-to-His mutation at codon 175, an allele that functions in a dominant oncogenic manner in tumorigenesis assays and is thus distinct from loss of p53. Transgenic mice expressing mouse mammary tumor virus-driven neu transgene (MMTV-neu) develop clonal mammary tumors with a latency of 234 days, suggesting that other events are necessary for tumor development. We have examined the role of mutations in p53 in tumor development in these mice. We have found that 37% of tumors arising in these mice have a missense mutations in p53. We have directly tested for cooperativity between neu and mutant p53 in mammary tumorigenesis by creating bitransgenic mice carrying MMTV-neu and 172Arg-to-His p53 mutant (p53-172H). In these bitransgenic mice, tumor latency is shortened to 154 days, indicating strong cooperativity. None of the nontransgenic mice or the p53-172H transgenic mice developed tumors within this time period. Tumors arising in the p53-172H/neu bitransgenic mice were anaplastic and aneuploid and exhibited increased apoptosis, in distinction to tumors arising in p53-null mice, in which apoptosis is diminished. Further experiments address potential mechanisms of cooperativity between the two transgenes. In these bitransgenic mice, we have recapitulated two common genetic lesions that occur in human breast cancer and have shown that p53 mutation is an important cooperating event in neu-mediated oncogenesis.     

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)