High incidence of lung, bone, and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene.

We have investigated the role of the p53 gene in oncogenesis in vivo by generating transgenic mice carrying murine p53 genomic fragments isolated from a mouse Friend erythroleukemia cell line or BALB/c mouse liver DNA. Elevated levels of p53 mRNA were detected in several tissues of two transgenic lines tested. Increased levels of p53 protein were also detected in most of the tissues analyzed by Western blotting (immunoblotting). Because both transgenes encoded p53 proteins that were antigenically distinct from wild-type p53, it was possible to demonstrate that overexpression of the p53 protein was mostly, if not entirely, due to the expression of the transgenes. Neoplasms developed in 20% of the transgenic mice, with a high incidence of lung adenocarcinomas, osteosarcomas, and lymphomas. Tissues such as ovaries that expressed the transgene at high levels were not at higher risk of malignant transformation than tissues expressing p53 protein at much lower levels. The long latent period and low penetrance suggest that overexpression of p53 alone is not sufficient to induce malignancies and that additional events are required. These observations provide direct evidence that mutant alleles of the p53 oncogene have oncogenic potential in vivo and that different cell types show intrinsic differences in susceptibility to malignant transformation by p53. Since recent data suggest that p53 may be a recessive oncogene, it is possible that the elevated tumor incidence results from functional inactivation of endogenous p53 by overexpression of the mutant transgene. The high incidence of lung and bone tumors suggests that p53 transgenic mice may provide a useful model to investigate the molecular events that underlie these malignancies in humans.     

The haploinsufficient tumor suppressor p18 upregulates p53 via interactions with ATM/ATR

p18 was first identified as a factor associated with a macromolecular tRNA synthetase complex. Here we describe the mouse p18 loss-of-function phenotype and a role for p18 in the DNA damage response. Inactivation of both p18 alleles caused embryonic lethality, while heterozygous mice showed high susceptibility to spontaneous tumors. p18 was induced and translocated to the nucleus in response to DNA damage. Expression of p18 resulted in elevated p53 levels, while p18 depletion blocked p53 induction. p18 directly interacted with ATM/ATR in response to DNA damage. The activity of ATM was dependent on the level of p18, suggesting the requirement of p18 for the activation of ATM. Low p18 expression was frequently observed in different human cancer cell lines and tissues. These results suggest that p18 is a haploinsufficient tumor suppressor and a key factor for ATM/ATR-mediated p53 activation.     

Loss of Borealin/DasraB leads to defective cell proliferation, p53 accumulation and early embryonic lethality

Borealin/DasraB is a member of the chromosomal passenger protein complex (CPC) required for proper segregation of chromosomes during mitosis. In Drosophila melanogaster, inactivation of Borealin/DasraB results in polyploidy, delayed mitosis and abnormal tissue development, indicating its critical role for cell proliferation. However, the in vivo role of mammalian Borealin/DasraB remains unclear. Here, we analyzed the expression of Borealin/DasraB and found that borealin is widely expressed in embryonic tissues and later restricted to adult tissues which relies on rapid cell proliferation. To determine the role of borealin during mouse development, we generated borealin-null mice through targeted disruption. While heterozygous mice developed normally, disruption of both borealin alleles resulted in early embryonic lethality by 5.5 dpc (days postcoitus) due to mitotic defects and apoptosis in blastocyst cells that showed microtubule disorganization and no CPC enrichment. At 5.5 dpc, borealin-null embryos exhibited excessive apoptosis and elevated expression of p53. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that Borealin/DasraB inactivation triggered impaired mitosis and apoptosis though p53-independent mechanisms. Our data show that Borealin/DasraB is essential for cell proliferation during early embryonic development, and its early embryonic lethality cannot be rescued by the loss of p53.     

Suppression of p53: a new approach to overcome side effects of antitumor therapy

The p53 protein is traditionally believed to be a tumor suppressor. Activation of p53-dependent apoptosis in response to damage to cell DNA provides for the elimination of possible tumor cell precursors. However, in some cases the activity of p53 can be dangerous for the organism. Thus, p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of antitumor therapy and, therefore, limits its efficiency. This review analyzes experimental data on the role of p53 in the primary and late tissue response to DNA-damaging exposures. Comparison of normal and p53-deficient mice indicated that the apoptosis in radiosensitive tissues during the first hours after irradiation is really caused by the activity of p53 which, in turn, is determined by a high level of expression of mRNA of p53. We supposed that a temporary suppression of p53 can decrease the damage to sensitive tissues and accelerate their recovery after the antitumor radio- and chemotherapy. To test this hypothesis, we have isolated a chemical inhibitor of p53 and determined its activity in vitro and in vivo. This compound, called pifithrin-alpha, protects wild-type mice against lethal doses of radiation, has no effect on p53-deficient animals, and does not induce visible tumors. These results show that the suppression of p53 is a promising approach in the prevention of side effects of antitumor therapy.     

Epithelial cells of different organs exhibit distinct patterns of p53-dependent and p53-independent apoptosis following DNA insult.

The present study shows that DNA damage induces different patterns of p53-dependent and p53-independent apoptosis in epithelial cells of various organs of adult mice. Genotoxic stress induced a biphasic apoptotic response in the small intestine and tongue. While the first immediate apoptotic wave was p53-dependent, the second was slower in rate and was p53-independent. Under the same experimental conditions a single rapid, but a more extended, p53-independent response was evident in the skin of the tail. Indeed, exposure of p53+/+ mice to 400 R induced in epithelium of the small intestine and tongue an immediate rapid response that was followed by a second delayed p53-independent apoptotic wave. p53-/- mice exhibited in these organs the second wave only. However, epithelium of the tail derived from the same mice showed a single rapid apoptotic response that lasted much longer than the p53-dependent response and was similar in the p53-/- and the p53+/+ mice. Variations in apoptotic patterns observed in epithelial cells derived of the different tissues may point to differences in the physiological pathways expressed.     

DNA damage-induced neural precursor cell apoptosis requires p53 and caspase 9 but neither Bax nor caspase 3

Programmed cell death (apoptosis) is critical for normal brain morphogenesis and may be triggered by neurotrophic factor deprivation or irreparable DNA damage. Members of the Bcl2 and caspase families regulate neuronal responsiveness to trophic factor withdrawal; however, their involvement in DNA damage-induced neuronal apoptosis is less clear. To define the molecular pathway regulating DNA damage-induced neural precursor cell apoptosis, we have examined the effects of drug and gamma-irradiation-induced DNA damage on telencephalic neural precursor cells derived from wild-type embryos and mice with targeted disruptions of apoptosis-associated genes. We found that DNA damage-induced neural precursor cell apoptosis, both in vitro and in vivo, was critically dependent on p53 and caspase 9, but neither Bax nor caspase 3 expression. Neural precursor cell apoptosis was also unaffected by targeted disruptions of Bclx and Bcl2, and unlike neurotrophic factor-deprivation-induced neuronal apoptosis, was not associated with a detectable loss of cytochrome c from mitochondria. The apoptotic pathway regulating DNA damage-induced neural precursor cell death is different from that required for normal brain morphogenesis, which involves both caspase 9 and caspase 3 but not p53, indicating that additional apoptotic stimuli regulate neural precursor cell numbers during telencephalic development.     

Cooperation between p53 mutation and high telomerase transgenic expression in spontaneous cancer development

Telomerase reintroduction in adult somatic tissues is envisioned as a way to extend their proliferative capacity. It is still a question, however, whether constitutive telomerase expression in adult tissues impacts the normal aging and spontaneous cancer incidence of an organism. Here, we studied the aging and spontaneous cancer incidence of mice with transgenic telomerase expression in a wide range of adult tissues, K5-Tert mice. For this, we maintained large colonies of K5-Tert mice for more than 2 years. K5-Tert mice showed a decreased life span compared to wild-type cohorts associated with a higher incidence of preneoplastic and neoplastic lesions in various tissue types. Neoplasias in K5-Tert mice were coincident with transgene expression in the affected tissues. These observations suggest that high telomerase activity may cooperate with genetic alterations that occur with age to promote tumorigenesis. Indeed, we demonstrate here that increased cancer incidence and the reduced viability of K5-Tert mice are aggravated in a p53(+/-) genetic background, indicating that telomerase cooperates with loss of p53 function in inducing tumorigenesis. Altogether, these results demonstrate that constitutive high levels of telomerase activity result in a decreased life span associated with an increased incidence of neoplasias as the organism ages.     

ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis

Cisplatin is one of the most effective anti-cancer drugs; however, the use of cisplatin is limited by its toxicity in normal tissues, particularly injury of the kidneys. The mechanisms underlying the therapeutic effects of cisplatin in cancers and side effects in normal tissues are largely unclear. Recent work has suggested a role for p53 in cisplatin-induced renal cell apoptosis and kidney injury; however, the signaling pathway leading to p53 activation and renal apoptosis is unknown. Here we demonstrate an early DNA damage response during cisplatin treatment of renal cells and tissues. Importantly, in the DNA damage response, we demonstrate a critical role for ATR, but not ATM (ataxia telangiectasia mutated) or DNA-PK (DNA-dependent protein kinase), in cisplatin-induced p53 activation and apoptosis. We show that ATR is specifically activated during cisplatin treatment and co-localizes with H2AX, forming nuclear foci at the site of DNA damage. Blockade of ATR with a dominant-negative mutant inhibits cisplatin-induced p53 activation and renal cell apoptosis. Consistently, cisplatin-induced p53 activation and apoptosis are suppressed in ATR-deficient fibroblasts. Downstream of ATR, both Chk1 and Chk2 are phosphorylated during cisplatin treatment in an ATR-dependent manner. Interestingly, following phosphorylation, Chk1 is degraded via the proteosomal pathway, whereas Chk2 is activated. Inhibition of Chk2 by a dominant-negative mutant or gene deficiency attenuates cisplatin-induced p53 activation and apoptosis. In vivo in C57BL/6 mice, ATR and Chk2 are activated in renal tissues following cisplatin treatment. Together, the results suggest an important role for the DNA damage response mediated by ATR-Chk2 in p53 activation and renal cell apoptosis during cisplatin nephrotoxicity.     

The murine Cyp1a1 gene is expressed in a restricted spatial and temporal pattern during embryonic development

In adult mice the cytochrome P450 Cyp1a1 gene is not constitutively expressed but is highly inducible by foreign compounds acting through the aryl hydrocarbon (Ah) receptor. However, the expression profile of the Cyp1a1 gene in the developing embryo is not well under-stood. Using established transgenic mouse lines where 8.5 kb of the rat CYP1A1 promoter is cloned upstream of the lacZ reporter gene (1), we describe the expression of the CYP1A1-driven reporter gene in all tissues through-out stages E7-E14 of embryonic development. In contrast to the absence of constitutive Cyp1a1 and lacZ transgene expression in tissues of the adult mouse, a constitutive cell-specific and time-dependent pattern of CYP1A1 promoter activity was observed in the embryo. This expression pattern was confirmed as reflecting the endogenous gene by measuring Cyp1a1 mRNA levels and protein expression by immunohistochemistry. The number of cells displaying endogenous CYP1A1 activity could be increased in the embryo upon xenobiotic challenge, but only within areas where the CYP1A1 promotor was already active. When reporter mice were bred onto a genetic background expressing a lower affinity form of the Ah receptor (DBA allele), transgene and murine Cyp1a1 protein expression were both attenuated in the adult mouse liver upon xenobiotic challenge. By comparison, constitutive CYP1A1 promoter activity in the embryo was identical in the presence of either the high or low affinity Ah receptor. These novel data suggest that the Cyp1a1 protein may play a role in murine development and that regulation of the Cyp1a1 gene during this period is either through the action of a high affinity Ah receptor ligand or by an alternative regulatory pathway.