Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.

Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

Cell autonomous apoptosis defects in acid sphingomyelinase knockout fibroblasts.

A body of evidence suggests that stress-induced sphingomyelin hydrolysis to the second messenger ceramide initiates apoptosis in some cells. Although studies using lymphoblasts from Niemann-Pick disease patients or acid sphingomyelinase (ASMase)-deficient mice have provided genetic support for this hypothesis, these models have not been universally accepted as definitive. Here, we show that mouse embryonic fibroblasts (MEFs) prepared from asmase mice manifest cell autonomous defects in apoptosis in response to several stresses. In particular, asmase(-/-) MEFs failed to generate ceramide and were totally resistant to radiation-induced apoptosis but remained sensitive to staurosporine, which did not induce ceramide. asmase(-/-) MEFs were also partially resistant to tumor necrosis factor alpha/ actinomycin D and serum withdrawal. Thus, resistance to apoptosis in asmase(-/-) MEFs was not global but rather stress type specific. Most importantly, the sensitivity to stress could be restored in the asmase(-/-) MEFs by administration of natural ceramide. Overcoming apoptosis resistance by natural ceramide is evidence that it is the lack of ceramide, not ASMase, that determines apoptosis sensitivity. The ability to rescue the apoptotic phenotype without reversing the genotype by the product of the enzymatic deficiency provides proof that ceramide is obligate for apoptosis induction in response to some stresses.

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase.

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.

Nitroxides tempol and tempo induce divergent signal transduction pathways in MDA-MB 231 breast cancer cells.

Tempol and tempo are stable free radical nitroxides that possess antioxidant properties. In this study, we examined the effects of these compounds on components of the mitogen-activated protein kinase signal transduction cascade. Tempo treatment (15 min) of MDA-MB 231 human breast cancer cells resulted in significant levels of tyrosine phosphorylation of several as yet unidentified proteins compared with equimolar concentration of tempol (10 mM). Both compounds caused tyrosine phosphorylation and activation of Raf-1 protein kinase (30 min, 2-3-fold). Interestingly, however, only tempol caused increased extracellular signal-regulated kinase 1 activity (2 h, approximately 3-fold). On the other hand, tempo, but not tempol, potently activated stress-activated protein kinase (2 h, >3-fold). Consistent with these data, tempol was found to be noncytotoxic, whereas tempo induced apoptotic cell death (2 h, >50%). Tempo treatment also resulted in significant elevation of ceramide levels at 30 min (54% over control) and 1 h (71% over control) posttreatment, preceding stress-activated protein kinase activation and apoptosis. These data suggest that in the absence of an environmental oxidative stress, tempol and tempo elicit distinct cellular signaling pathways. The recognition of the molecular mechanisms of nitroxide action may have important implications for biological effectiveness of these compounds.

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis in LNCaP cells is mediated through ceramide synthase.

Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKC alpha by 12-O-tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that de novo ceramide generation through the enzyme ceramide synthase is required. TPA induced rapid ceramide generation, which was detectable by 1 h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in ceramide synthase activity that persisted for at least 16 h. Treatment with fumonisin B1, a specific natural inhibitor of ceramide synthase, abrogated both ceramide production and TPA-induced apoptosis. Ceramide analogues bypassed fumonisin B1 inhibition to initiate apoptosis directly. Thus, ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.

Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells.

BACKGROUND: Protein kinase C (PKC) is a family of enzymes that function in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dihydrosphingosine, is a specific inhibitor of PKC and might represent a novel agent for anticancer therapy. Preclinical animal studies show that safingol alone has a minimal effect on tumor cell growth, but combining this compound with conventional chemotherapy agents dramatically potentiates their antitumor effects. It has been suggested that many chemotherapeutic agents exert their antitumor effects by inducing apoptosis. PURPOSE: We wanted to determine the extent to which safingol, alone or in combination with a standard chemotherapeutic agent (mitomycin C [MMC]), would promote apoptosis in gastric cancer cells in vitro. Furthermore, we investigated whether the induction of apoptosis in the treated cells was affected by their p53 tumor suppressor status or their drug-resistance status. METHODS: SK-GT-5 (p53-deficient and MMC-resistant) and MKN-74 (p53 wild-type and MMC-sensitive) gastric cancer cells were exposed to either no drug, safingol (50 microM) alone, MMC (5 micrograms/mL) alone, or a combination of safingol (50 microM) and MMC (5 micrograms/mL). In some experiments, cells were exposed simultaneously to safingol and the PKC activator, 3-phorbol 12-myristate 13-acetate (PMA), prior to treatment with MMC. Apoptosis was measured by two methods: 1) quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzamide trihydrochloride (Hoechst-33258), and 2) terminal deoxynucleotidyl transferase (TdT) labeling of the 3'-OH ends of DNA fragments produced in apoptotic cells. RESULTS: As determined by quantitative fluorescence microscopy, exposure of SK-GT-5 cells to safingol alone induced apoptosis in 2% +/- 1% (mean +/- SD) of the cells, and MMC alone increased that level to 18% +/- 1%. However, the combination of safingol and MMC induced apoptosis in 39% +/- 1% of the cells (P < .001, for the drug combination versus MMC alone). With MKN-74 cells, safingol alone induced apoptosis in 8% +/- 3% of the cells, whereas MMC alone induced apoptosis in 40% +/- 4% of treated cells and the combination of safingol and MMC induced apoptosis in 83% +/- 4% of the cells. Similar results were obtained with the TdT assay. Simultaneous exposure of cells to safingol and PMA abrogated the safingol-mediated enhancement of MMC-induced apoptosis. CONCLUSIONS: The PKC inhibitor safingol enhances the cytotoxic effect of the chemotherapeutic agent MMC in gastric cancer cells by promoting drug-induced apoptosis. The induction of apoptosis occurs regardless of the p53 status or the drug-resistance status of the cells.

Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis.

Recent investigations provided evidence that the sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor alpha (TNF-alpha) in several hematopoietic and nonhematopoietic cells. In this pathway, TNF-receptor interaction initiates sphingomyelin hydrolysis to ceramide by a sphingomyelinase. Ceramide acts as a second messenger stimulating a ceramide-activated serine/threonine protein kinase. The present studies show that ionizing radiation, like TNF, induces rapid sphingomyelin hydrolysis to ceramide and apoptosis in bovine aortic endothelial cells. Elevation of ceramide with exogenous ceramide analogues was sufficient for induction of apoptosis. Protein kinase C activation blocked both radiation-induced sphingomyelin hydrolysis and apoptosis, and apoptosis was restored by ceramide analogues added exogenously. Ionizing radiation acted directly on membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis enzymatically through a neutral sphingomyelinase. These studies provide the first conclusive evidence that apoptotic signaling can be generated by interaction of ionizing radiation with cellular membranes and suggest an alternative to the hypothesis that direct DNA damage mediates radiation-induced cell kill.

Basic fibroblast growth factor protects endothelial cells against radiation-induced programmed cell death in vitro and in vivo.

Apoptosis (programmed cell death) serves as a common mechanism of interphase cell death after radiation exposure in thymic, lymphoid, and hematopoietic cells but has infrequently been documented in other adult mammalian cell types. The present study demonstrates that apoptotic interphase cell death occurs in endothelial cells after exposure to clinically relevant radiation doses and that basic fibroblast growth factor (bFGF) protects endothelial cells against this mode of the lethal effects of radiation. Radiation exposure produced heterologous double-stranded DNA breaks in endothelial cells, but the cells exhibited a similar competence for repair of this damage in the presence or absence of bFGF. However, subsequent to the completion of this repair process, a second process of DNA fragmentation became apparent, which was detected only in the absence of bFGF and was associated with a DNA ladder of oligonucleosomal fragments characteristic of apoptosis. The apoptotic DNA degradation occurred mainly in G0-G1 phase cells and was inhibited by bFGF stimulation. C3H/HeJ mice exposed to lethal doses of whole lung irradiation exhibited similar apoptotic changes in the endothelial cell lining of the pulmonary microvasculature within 6-8 h after radiation exposure. bFGF given i.v. immediately before and after irradiation inhibited the development of apoptosis in these cells and protected mice against the development of lethal radiation pneumonitis. These findings suggest that interphase apoptosis may represent a biologically relevant mechanism of radiation-induced cell kill in nonlymphoid mammalian cells both in vitro and in vivo and that natural protection mechanisms against this effect may be associated with the level of radiation resistance in normal and malignant tissues in vivo.

Protein kinase C mediates basic fibroblast growth factor protection of endothelial cells against radiation-induced apoptosis.

Basic fibroblast growth factor (bFGF) was found to protect bovine aortic endothelial cells against the lethal effects of ionizing radiation by inhibiting the programmed cell death (apoptosis) induced in these cells by radiation exposure. The involvement of the bFGF receptor tyrosine kinase in this function was demonstrated by abrogation of the radioprotective effect of bFGF by a specific inhibitor of the bFGF receptor tyrosine kinase, the tyrphostin AG213. The downstream signaling after stimulation of the bFGF receptor tyrosine kinase in bovine aortic endothelial cells involved translocation of the alpha isotype of cytoplasmic protein kinase C (PKC) into the membrane and its activation within 30 s after bFGF stimulation. The involvement of PKC in the radioprotective effect conferred by bFGF was suggested by the demonstration that nonspecific PKC activation by short-term exposure (30 min) to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA; 30 ng/ml) mimicked the radioprotective effect of bFGF. Furthermore, treatment of the cells with the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (20 microM) abrogated the radioprotective effect of bFGF, as was observed after the depletion of cellular PKC by overnight preincubation with high-dose TPA (200 nM). Agarose gel electrophoresis of DNA extracted from irradiated bovine aortic endothelial cells showed that both TPA (30 ng/ml; 30 min) and bFGF (1 ng/ml) inhibited the apoptotic degradation of DNA induced in these cells by radiation exposure (500 cGy). Both the bFGF- and the TPA-mediated inhibition of apoptosis could be reversed by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (20 microM). These data demonstrate the involvement of PKC in the inhibition of radiation-induced apoptosis by bFGF and the rescue of endothelial cells from this mode of radiation-induced cell death.

Effect of age on the expressed B cell repertoire: role of B cell subsets.

Aged humans and experimental animals are impaired in their responses to most foreign antigens although they produce greater amounts of autoantibodies. We have examined the effect of age on the production of antibodies to a prototypic foreign antigen, sheep erythrocytes (SRBC), and to a prototypic autoantigen, bromelain-treated mouse erythrocytes (BrMRBC), in young and old mice before and after immunization with SRBC. Old mice express more anti-BrMRBC plaque-forming cell (PFC) antibodies before and an even greater number after immunization with SRBC than young mice. Conversely, old mice produce far fewer anti-SRBC PFC than young mice following immunization with SRBC. We hypothesized that the differences in the responses of old mice to BrMRBC and SRBC reflects differences in the activity of CD5+ and CD5- B cells. To test this hypothesis we immunized young and old mice with foreign antigens reported (and confirmed in our studies) to stimulate CD5+ B cells [TNP-ficoll and phosphorylcholine-keyhole limpet hemocyanin (KLH)] or CD5- B cells (SRBC and TNP-KLH). We found that the PFC response of old mice to antigens mediated by CD5+ B cells was equal to or greater than that of young mice. In contrast the PFC response of old mice induced by antigens mediated by CD5- B cells was only 10% that of young mice. Thus it appears that the immune response of old mice is well maintained for antigens which elicit a CD5+ B cell response but not for those which elicit a CD5- B cell response.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant vaccinia virus expressing the PR/8 influenza hemagglutinin gene overcomes the impaired immune response and increased susceptibility of old mice to influenza infection.

Elderly humans have increased morbidity and mortality after viral influenza despite immunization. A mouse model of influenza infection was used to search for a more effective way to induce immunity to influenza. Old (18 months) BALB/c mice were more susceptible to influenza pneumonia than young (2 months) BALB/c mice after intranasal challenge with PR/8 influenza virus despite prior immunization with influenza virus. The decreased resistance to live influenza virus challenge was associated with an impaired generation of anti-hemagglutinin antibody and cytotoxic T lymphocytes in old mice. In contrast, immunization of old mice with a recombinant vaccinia virus expressing the PR/8 influenza hemagglutinin gene protected them from intranasal challenge with live influenza virus and generated high levels of anti-PR/8 influenza virus hemagglutinin antibody and PR/8-specific cytotoxic T cells. Recombinant vaccine overcame the age-associated immune defect that follows the administration of conventional viral vaccine.

Prothymosin alpha expression occurs during G1 in proliferating B or T lymphocytes.

To gain insight into possible functions for prothymosin alpha in the proliferative cycle of lymphocytes, we examined the kinetics of prothymosin alpha mRNA expression in mitogen stimulated murine lymphocytes. This mRNA increases after mitogen stimulation, peaking in mid G1. This kinetics is compatible with induction of the prothymosin alpha gene by the c-myc protein (Eilers, M., Schirm, S. and Bishop, J.M. (1991) EMBO J., 10, 133-141). Thus, although prothymosin alpha mRNA is found throughout the cell cycle, the elevated expression in G1 may be associated with an increased requirement for prothymosin alpha during the G1/S transition or the S phase of the cell cycle.