Testicular germ cell sensitivity to TRAIL-induced apoptosis is dependent upon p53 expression and is synergistically enhanced by DR5 agonistic antibody treatment

The ability of the TRAIL/DR5 signaling pathway to induce apoptosis has generally been limited to tumor cells. Here we report that in primary testis explants, addition of TRAIL (0.5 μg/ml) caused a three-fold increase in germ cell apoptosis. Furthermore, exposure of C57BL/6 mice to the testicular toxicant, mono-(2-ethylhexyl) phthalate (MEHP), caused an increased p53 stability and elevated DR5 mRNA levels coincident with increases in the levels of apoptosis in spermatocytes. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo TRAIL/DR5-mediated apoptosis, we used the germ cell lines GC-1spg and GC-2spd(ts) (a temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32°C but not 37°C). Addition of TRAIL and the anti-DR5 monoclonal antibody, MD5-1, triggered a robust synergistic increase of apoptosis in p53 permissive GC-2 cells (32°C) but not in GC-1 cells. In addition, DR5 levels on the plasma membrane of permissive cells were considerably enhanced concomitant with p53 expression and after MD5-1 treatment. These data represent the first indication that testicular germ cells, specifically spermatocytes, can undergo TRAIL-mediated apoptosis and the clinically relevant observation that pretreatment with a DR5 monoclonal antibody can greatly sensitize their apoptotic response to TRAIL. This work was supported, in part, by grants from the National Institute of Environmental Health Sciences/NIH (ES09145, JHR), Toxicology Training grant (ES T32 ES007247, CM), NIH Center Grant (P30 ES07784^, JHR) and the Center for Molecular and Cellular Toxicology (CMCT).     

Influence of TRP53 status on FAS membrane localization, CFLAR (c-FLIP) ubiquitinylation, and sensitivity of GC-2spd (ts) cells to undergo FAS-mediated apoptosis

Previously we reported that testicular germ cells undergo FAS-mediated apoptosis after exposure of mice to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP) and that this process is partially dependent on the TRP53 protein (p53). Recent reports have suggested that TRP53 may influence the ubiquitinylation and consequent proteosomal degradation of a negative regulator of FAS, CFLAR (L) (c-FLIP [L]), in human colon cancer cells. To further characterize the relationship between CFLAR and TRP53, we used the transformed germ cell line GC-2spd (ts), which harbors a temperature-sensitive Trp53 mutation that allows for TRP53 activation at 32 degrees C. We report here that GC-2 cells expressed a 10-fold increase in basal cell membrane FAS levels and an increased sensitivity to FAS agonistic antibody (JO2)-triggered apoptosis only when they were maintained at the permissive TRP53 temperature. After JO2 exposure, CFLAR (L) protein levels were enhanced only at the nonpermissive TRP53 temperature (37 degrees C) while real-time PCR results indicated an absence of Cflar (L) mRNA changes in GC-2 cells regardless of the temperature. Furthermore, transfection of GC-2 cells at 37 degrees C with siRNA against Cflar resulted in reduction of CFLAR (L) protein levels and increased sensitivity to JO2-mediated apoptosis. The CFLAR (L) protein was also more strongly ubiquitinylated in response to JO2 treatment at the permissive TRP53 temperature. Taken together, these data suggest that the TRP53 protein influences the sensitivity of GC-2 cells to undergo FAS-mediated apoptosis by modulating the expression of FAS on their cell membranes and subsequently influencing the degradation of the antiapoptotic protein CFLAR (L).     

Fas is involved in the p53-dependent apoptotic response to ionizing radiation in mouse testis

Apoptosis induced in male germ cells following ionizing radiation is dependent on functional p53 (Trp53) being present. We sought to determine whether Fas (Tnfrsf6/CD95/APO-1), an apoptotic factor, is involved in this p53-dependent germ cell death. In p53 knock-out mice exposed to 5 Gy of x-radiation, germ cells were protected from cell death, as assessed by counting apoptotic seminiferous tubules 12 h following radiation. Similarly, spermatid head counts in p53 knock-out mice remained near normal 29 days after exposure to 0.5 Gy of radiation, whereas wild-type animals had a more than twofold reduction in spermatid head counts. Fas mRNA expression remained at pretreatment levels in p53 knock-out mice; however, Fas increased in a time-dependent manner in wild-type mice following exposure to 5 Gy of radiation, indicating that radiation-induced Fas expression is p53-dependent. The functional significance of Fas involvement was demonstrated when lpr(cg) mice, having a nonfunctional Fas receptor, were exposed to 5 Gy of radiation; the number of apoptotic seminiferous tubules 12 h following radiation was significantly reduced compared to that of wild-type mice. Additionally, lpr(cg) mice exposed to 0.5 Gy of radiation had increased spermatid head counts 29 days following radiation compared to wild-type mice. Interestingly, gld mice with a non-functional Fas ligand (Tnfsf6/FasL/CD95L) were as sensitive to radiation as wild-type animals, and levels of FasL mRNA were not affected by radiation treatment. These results indicate that apoptosis and up-regulation of Fas following radiation are both p53-dependent events. Although Fas is necessary, in part, for radiation-induced p53-dependent apoptosis, FasL is not.     

p53 and Fas ligand are required for psoralen and UVA-induced apoptosis in mouse epidermal cells

A combination of 8-methoxypsoralen (8-MOP) and ultraviolet-A (UVA) radiation (320-400 nm) (PUVA) is widely used in the treatment of psoriasis and other skin diseases. PUVA is highly effective in eliminating hyperproliferative cells in the epidermis, but its mechanism of action has not been fully elucidated. In this study, we used immortalized JB6 mouse epidermal cells, p53(-/-), and Fas ligand deficient (gld) mice to investigate the molecular mechanism by which PUVA induces cell death. The results indicate that PUVA treatment induces apoptosis in JB6 cells. In addition, PUVA treatment of JB6 cells results in p53 stabilization, phosphorylation, and nuclear localization as well as induction of p21(Waf/Cip1) and caspase-3 activity. In vivo studies reveal that PUVA treatment induces significantly less apoptosis in the epidermis of p53(-/-) mice compared to p53(+/+) mice. Furthermore, FasL-deficient (gld) mice are completely resistant to PUVA-induced apoptosis compared to wild-type mice. These results indicate that PUVA treatment induces apoptosis in mouse epidermal cells in vitro and in vivo and that p53 and Fas/Fas ligand interactions are required for this process, at least in vivo. This implies that similar mechanisms may be involved in the elimination of psoriatic keratinocytes from human skin following PUVA therapy.     

Involvement of death receptor Fas in germ cell degeneration in gonads of Kit-deficient Wv/Wv mutant mice

Kit and its ligand stem cell factor (SCF) play a fundamental role in hematopoiesis, melanogenesis and gametogenesis. Homozygous W(v) mutant mice with a mutation in kit show abnormalities in these cell lineages. Fas is a member of the death receptor family inducing apoptosis. In this study, we generated double-mutant mice (W(v)/W(v):Fas(-/-)) and analyzed histologically their reproductive organs. In testes and ovaries of the double-mutant mice, testicular germ cells and oocytes were detected, respectively, whereas the same-aged W(v)/W(v) mice contained neither cells. In addition, inhibition of Kit signals by administration of anti-Kit mAb, which induces degeneration of testicular germ cells in vivo in wild-type mice, did not cause degeneration in Fas-deficient mice. In testicular germ cells of W(v)/W(v) mutant mice, an increase of Fas expression was observed in spermatogonia. Further, in vitro treatment with SCF was shown to downregulate Fas on fibroblasts expressing exogenous Kit through activation of PI3-kinase/Akt. All the results clearly indicate that Fas-mediated apoptosis is involved in germ cell degeneration accompanied by defects in Kit-mediated signals, and Kit signaling negatively regulates Fas-mediated apoptosis in vivo.     

Deficiency of Trp53 rescues the male fertility defects of Kit(W-v) mice but has no effect on the survival of melanocytes and mast cells.

Mutations of the receptor tyrosine kinase, Kit, or its ligand, mast growth factor (Mgf), affect three unrelated cell populations: melanocytes, germ cells, and mast cells. Kit signaling is required initially to prevent cell death in these lineages both in vitro and in vivo. Mgf appears to play a role in the survival of some hematopoietic cells in vitro by modulating the activity of p53. Signaling by Mgf inhibits p53-induced apoptosis of erythroleukemia cell lines and suppresses p53-dependent radiation-induced apoptosis of bone marrow cells. We tested the hypothesis that cell survival in Kit mutant mice would be enhanced by p53 deficiency in vivo. Double-mutant mice, which have greatly reduced Kit receptor tyrosine kinase activity and also lack Trp53, were generated and the affected cell lineages examined. Mast cell, melanoblast, and melanocyte survival in the double Kit(W-v/W-v):Trp53(-/-) mutants was not increased compared to the single Kit(W-v/W-v):Trp53(+/+) mutants. However, double-mutant males showed an increase in sperm viability and could father litters, in contrast to their homozygous Kit mutant, wild-type p53 littermates. This germ cell rescue appears to be male specific, as female ovaries were similar in mice homozygous for the Kit mutant allele with or without p53. We conclude that defective Kit signaling in vivo results in apoptosis by a p53-independent pathway in melanocyte and mast cell lineages but that in male germ cells apoptosis in the absence of Kit is p53-dependent.     

Physiological and pathological cell deaths in the reproductive organs

Apoptosis of testicular germ cells and oocytes and their supporting cells in the gonads occurs at physiological and normal conditions or after exposure to pathological stimuli. Cell-death regulators, including Bcl-2 family members, caspases, Fas and p53 are thought to be involved in these processes. This article reviews the details of the apoptotic machinery in the reproductive organs by describing briefly the abnormal phenotypes observed in transgenic and gene-ablated mice.     

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germcells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flowcytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes ofepithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphologywas normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.     

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.     

Differential effects of phthalates on the testis and the liver

Phthalates have been shown to elicit contrasting effects on the testis and the liver, causing testicular degeneration and promoting abnormal hepatocyte proliferation and carcinogenesis. In the present study, we compared the effects of phthalates on testicular and liver cells to better understand the mechanisms by which phthalates cause testicular degeneration. In vivo treatment of rats with di-(2-ethylhexyl) phthalate (DEHP) caused a threefold increase of germ cell apoptosis in the testis, whereas apoptosis was not changed significantly in livers from the same animals. Western blot analyses revealed that peroxisome proliferator-activated receptor (PPAR) alpha is equally abundant in the liver and the testis, whereas PPAR gamma and retinoic acid receptor (RAR) alpha are expressed more in the testis. To determine whether the principal metabolite of DEHP, mono-(2-ethylhexyl) phthalate (MEHP), or a strong peroxisome proliferator, 4-chloro-6(2,3-xylindino)-2-pyrimidinylthioacetic acid (Wy-14,643), have a differential effect in Sertoli and liver cells by altering the function of RAR alpha and PPARs, their nuclear trafficking patterns were compared in Sertoli and liver cells after treatment. Both MEHP and Wy-14,643 increased the nuclear localization of PPAR alpha and PPAR gamma in Sertoli cells, but they decreased the nuclear localization of RAR alpha, as previously shown. Both PPAR alpha and PPAR gamma were in the nucleus and cytoplasm of liver cells, but RAR alpha was predominant in the cytoplasm, regardless of the treatment. At the molecular level, MEHP and Wy-14,643 reduced the amount of phosphorylated mitogen-activated protein kinase (activated MAPK) in Sertoli cells. In comparison, both MEHP and Wy-14,643 increased phosphorylated MAPK in liver cells. These results suggest that phthalates may cause contrasting effects on the testis and the liver by differential activation of the MAPK pathway, RAR alpha, PPAR alpha, and PPAR gamma in these organs.     

Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.     

Spontaneous mutagenesis is enhanced in Apex heterozygous mice

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.     

Disruption of the MDM2�Cp53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2–p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.     

ApoE-/-Fas-/- C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia

To establish a mouse model of accelerated atherosclerosis in lupus, we generated apolipoprotein E-deficient (apoE(-/-)) and Fas(lpr/lpr) (Fas(-/-)) C57BL/6 mice. On a normal chow diet, 5 month old apoE(-/-)Fas(-/-) mice had enlarged glomerular tuft areas, severe proteinuria, increased circulating autoantibody levels, and increased apoptotic cells in renal and vascular lesions compared with either single knockout mice. Also, double knockout mice developed increased atherosclerotic lesions but decreased serum levels of total and non-HDL cholesterol compared with apoE(-/-)Fas(+/+) littermates. Moreover, female apoE(-/-)Fas(-/-) mice had lower vertebral bone mineral density (BMD) and bone volume density (BV/TV) than age-matched female apoE(-/-)Fas(+/+) mice. Compared with apoE(-/-)Fas(+/+) and apoE(+/+)Fas(-/-) mice, apoE(-/-)Fas(-/-) mice had decreased circulating oxidized phospholipid (OxPL) content on apoB-100 containing lipoprotein particles and increased serum IgG antibodies to OxPL, which were significantly correlated with aortic lesion areas (r = 0.58), glomerular tuft areas (r = 0.87), BMD (r = -0.57), and BV/TV (r = -0.72). These results suggest that the apoE(-/-)Fas(-/-) mouse model might be used to study atherosclerosis and osteopenia in lupus. Correlations of IgG anti-OxPL with lupus-like disease, atherosclerosis, and bone loss suggested a shared pathway of these disease processes.