A role for Xrcc2 in the early stages of mouse development

Xrcc2 is one of a family of five Rad51-like genes with important roles in the repair of DNA damage by homologous recombination (HR) in mammals. We have shown previously that loss of Xrcc2 in mice results in severe but variable developmental defects and embryonic lethality, potentially linked to excessive apoptosis. To look at the causes of lethality, and possibly to allow Xrcc2-/- mice to survive to birth, we have produced double knockout mice deficient in either the p53 oncoprotein or Ataxia telangiectasia mutated (Atm). Overall we show that the excessive apoptosis observed in Xrcc2-/- embryos is p53-dependent, and that loss of p53 can restore growth capacity to Xrcc2-/- fibroblasts in culture, but that it cannot rescue the embryonic lethality. Additionally, although the Xrcc2-/- Trp53-/- embryos show a near-normal morphology they remain relatively small in size. Loss of Atm in an Xrcc2-/- embryo has little effect, suggesting that response to loss of HR capacity is not mediated through the Atm kinase in the early stages of mouse development. Further, as seen by reduced expression of the early developmental marker, Delta-like1, the normal developmental programme is perturbed in Xrcc2-/- embryonic tissues, particularly during neurogenesis and somitogenesis. Taken together our data suggest that the accumulation of spontaneous damage in HR-deficient embryos has severe consequences for the development and survival of mammals due to the unregulated loss of cells important to the developmental programme.     

Prenatal radiation-induced limb defects mediated by Trp53-dependent apoptosis in mice

We reported previously that in utero radiation-induced apoptosis in the predigital regions of embryonic limb buds was responsible for digital defects in mice. To investigate the possible involvement of the Trp53 gene, the present study was conducted using embryonic C57BL/6J mice with different Trp53 status. Susceptibility to radiation-induced apoptosis in the predigital regions and digital defects depended on both Trp53 status and the radiation dose; i.e., Trp53 wild-type (Trp53(+/+)) mice appeared to be the most sensitive, Trp53 heterozygous (Trp53(+/-)) mice were intermediate, and Trp53 knockout (Trp53(-/-)) mice were the most resistant. These results indicate that induction of apoptosis and digital defects by prenatal irradiation in the later period of organogenesis are mediated by the Trp53 gene. These findings suggest that the wild-type Trp53 gene may be an intrinsic genetic susceptibility factor that is responsible for certain congenital defects induced by prenatal irradiation.     

p53-dependent and -independent pathways of apoptotic cell death in sepsis

Sepsis induces extensive apoptosis of lymphocytes, which may be responsible for the profound immune suppression of the disorder. Two potential pathways of sepsis-induced lymphocyte apoptosis, Fas and p53, were investigated. Lymphocyte apoptosis was evaluated 20-22 h after sepsis by annexin V or DNA nick-end labeling. Fas receptor-deficient mice had no protection against sepsis-induced apoptosis in thymocytes or splenocytes. p53 knockout mice (p53-/-) had complete protection against thymocyte apoptosis but, surprisingly, had no protection in splenocytes. p53-/- mice had no improvement in sepsis survival compared with appropriately matched control mice with sepsis. We conclude that both p53-dependent and p53-independent pathways of cell death exist in sepsis. This differential apoptotic response of thymocytes vs splenocytes in p53-/- mice suggests that either the cellular response or the death-inducing signal is cell-type specific in sepsis. The fact that p53-/- lymphocytes of an identical subtype (CD8-CD4+) were protected in thymi but not in spleens indicates that cell susceptibility to apoptosis differs depending upon other unidentified factors.     

E2F1-dependent pathways are involved in amonafide analogue 7-d-induced DNA damage, G2/M arrest, and apoptosis in p53-deficient K562 cells

The E2F1 gene well known is its pivotal role in regulating the entry from G1 to S phase, while the salvage antitumoral pathway which implicates it, especially in the absence of p53, is not fully characterized. We therefore attempted to identify the up‐ and down‐stream events involved in the activation of the E2F1‐dependent pro‐apoptotic pathway. For this purpose, a amonafide analogue, 7‐d (2‐(3‐(2‐(Dimethylamino)ethylamino)propyl)‐6‐(dodecylamino)‐1H‐benzo[de]isoquinoline‐1,3(2H)‐dione) was screened, which exhibited high antitumor activity against p53‐deficient human Chronic Myelogenous Leukemia (CML) K562 cells. Analysis of flow cytometry and western blots of K562 cells treated with 7‐d revealed an appreciable G2/M cycle arrest and apoptosis in a dose and time‐dependent manner via p53‐independent pathway. A striking increase in “Comet tail” formation and γ‐H2AX expression showed that DNA double strand breaks (DSB) were caused by 7‐d treatment. ATM/ATR signaling was reported to connect E2F1 induction with apoptosis in response to DNA damage. Indeed, 7‐d‐induced G2/M arrest and apoptosis were antagonized by ATM/ATR signaling inhibitor, Caffeine, which suggested that ATM/ATR signaling was activated by 7‐d treatment. Furthermore, the increased expression of E2F1, p73, and Apaf‐1 and p73 dissociation from HDM2 was induced by 7‐d treatment, however, knockout of E2F1 expression reversed p73, Apaf‐1, and p21^Cip1/WAF1 expression, reactivated cell cycle progression, and inhibited 7‐d‐induced apoptosis. Altogether our results for the first time indicate that 7‐d mediates its growth inhibitory effects on CML p53‐deficient cells via the activation of an E2F1‐dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets p73, Apaf‐1, and p21^Cip1/WAF1. J. Cell. Biochem. 113: 3165–3177, 2012. © 2012 Wiley Periodicals, Inc.     

Inhibition of mutant p53 phosphorylation at serine 15 or serine 315 partially restores the function of wild-type p53.

The tumor suppressor protein p53 is a phosphoprotein and has growth and transformation suppression functions. Phosphorylation of wild-type p53 is known to modulate its function. To investigate the role of phosphorylation in modulating the functions of mutant p53, we constructed a series of phosphorylation site mutants based on mutant p53 Ala143 (p53-143) and p53 His175 (p53-175). When transfected into p53-negative Saos-2 cells, parental mutant p53-143 and p53-175 abolished both growth suppression and induction of apoptosis. However, DNA-activated protein kinase (DNA-PK) or cyclin-dependent kinase (cdks) phosphorylation site double mutants partially restored the growth suppression and induction of apoptosis and recovered the p53-specific DNA binding activity. We also observed a difference in sensitivity to calpain from parental mutants p53-175 and p53-175/15 or p53-175/315. These results suggest that the lack of phosphorylation at either the DNA-PK or cdks site in p53 mutants partially restores the wild-type functions by altering their conformation.     

Simultaneous haploinsufficiency of Pten and Trp53 tumor suppressor genes accelerates tumorigenesis in a mouse model of prostate cancer

Tumor suppressor gene PTEN is important in the initiation and progression of human prostate carcinoma, whereas the role of TP53 remains controversial. Since Pten/Trp53 double conditional knockout mice show earlier onset and fast progression of prostate cancer when compared to Pten knockout mice, we asked whether heterozygosity of these two tumor suppressor genes was sufficient to accelerate prostatic tumorigenesis. To answer this question we examined prostatic lesion progression of Pten/Trp53 double heterozygous mice and a series of controls such as Pten heterozygous, Pten conditional knockout, Trp53 heterozygous and Trp53 knockout mice. Tissue recombination of adult prostatic epithelium coupled with embryonic rat seminal vesicle mesenchyme was used as a tool to stimulate prostatic epithelial proliferation. In our study, high-grade prostatic intraepithelial neoplasia (PIN) was found with high frequency at 8 weeks post-tissue recombination transplantation. PIN lesions in Pten/Trp53 double heterozygous mice were more severe than those seen in Pten heterozygous alone. Furthermore, morphologic features attributable to Pten or Trp53 loss appeared to be enhanced in double heterozygous tissues. LOH analysis of Pten and Trp53 in genomic DNA collected from high-grade PIN lesions in Pten heterozygous and Pten/Trp53 double heterozygous mice showed an intact wild-type allele for both genes in all samples examined. In conclusion, simultaneous heterozygosity of Pten and Trp53 accelerates prostatic tumorigenesis in this mouse model of prostate cancer independently of loss of heterozygosity of either gene.     

E2F and p53 induce apoptosis independently during Drosophila development but intersect in the context of DNA damage

In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression of pro-apoptotic genes generates an additional sensitivity to apoptosis that requires both dE2F/dDP and dp53 activity. This sensitivity differs from DNA damage-induced apoptosis in wild-type discs (and from dE2F/dDP-induced apoptosis in un-irradiated rbf1 mutant eye discs) by being dependent on both hid and reaper. These results show that pro-apoptotic activities of dE2F1 and dp53 are surprisingly separable: dp53 is required for dE2F-dependent apoptosis in the response to DNA damage, but it is not required for dE2F-dependent apoptosis caused simply by the inactivation of rbf1.