Desiree Ehleiter

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.

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 α/ actinomycin D and serum withdrawal. Thus, resistance to apoptosis inasmase −/− MEFs was not global but rather stress type specific. Most importantly, the sensitivity to stress could be restored in theasmase −/− 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.

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, ∼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.

Correction: Mitochondrial Ceramide-Rich Macrodomains Functionalize Bax upon Irradiation

The authors would like to correct Fig 5, as errors were introduced in the preparation of this figure for publication. In Fig 5D[b], the image for the Light fraction Bak Western blot and the numbering of the fractions are incorrect. The authors have provided a corrected version of Fig 5 here. Fig 5 Ceramide induces formation of a mitochondrial ceramide-rich macrodomain (MCRM). The authors confirm that these changes do not alter their findings. The authors have provided the original Western blot and additional underlying data as Supporting Information.