p53-Independent ceramide formation in human glioma cells during γ-radiation-induced apoptosis

Abstract

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to c-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to c-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-Nmethyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiationinduced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by c-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to c-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to cradiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to c-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from c-radiation of cells with endogenous functional p53.

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo

“…Although our data are in agreement with previous findings using B13 (Selzner et al, 2001;Ogretmen and Hannun, 2004) or NOE (Hara et al, 2004) to block CDases, it has been reported that primary human SV-40 transformed skin fibroblasts or lymphocytes from Farber disease patients, a genetic disorder characterized by a decrease in acid CDase, were as sensitive to DNRinduced cell death as cells from healthy controls (Burek et al, 2001). This reported discrepancy with our findings might well be owing to the tumor specificity or type of cells used.…”

Neutral Sphingomyelinase-3 Is a DNA Damage and Nongenotoxic Stress-Regulated Gene That Is Deregulated in Human Malignancies

“…Although speculative, our results also tend to suggest that nSMase3 may not be responsible for lengthened periods of ceramide production that occur in hours to days following treatment with g-radiation or Adriamycin (11,13,25). The possibility therefore exists that one of several other ceramidegenerating pathways may be responsible for the extended production of ceramide.…”

“…To the best of our knowledge, this is the first report of p53 modulating the expression of an individual nSMase; however, the notion that p53 facilitates decreased ceramide levels is not without precedence. For instance, Hara et al (25) have reported that g-radiation causes a p53-dependent increase in acid ceramidase, an enzyme that hydrolyzes proapoptotic ceramide to form growth-promoting sphingosine. Reciprocally, previous studies have shown the p53-dependent increases in ceramide levels via down-regulation of sphingosine-kinase-1, thus forming the concept that p53 promotes ceramide generation rather than promoting its breakdown or abrogating its synthesis (52).…”

“…For example, activation of death receptors TNF receptor 1 and Fas (CD95) is known to stimulate ceramide production through activation of aSMase (23,24). Similarly, the activation of aSMase by ionizing radiation is well established (16,25) and increased aSMase activity following the DNA-damaging drug Adriamycin (doxorubicin) has also been reported (26). The importance of aSMase-mediated ceramide production is exemplified in aSMase À/À mice and in lymphoblasts from Niemann-Pick patients that lack aSMase activity, where generation of ceramide and induction of apoptosis following exposure to ionizing radiation is abrogated (16).…”

The potential role of DFNA5, a hearing impairment gene, in p53-mediated cellular response to DNA damage

“…We established the p53 knock-down (HepG2-p53-KD) and the control (HepG2-p53-Cont) cell lines as described previously (Hara et al 2004), which were derived from a hepatoblastoma cell line (HepG2 that contains wild-type p53). In brief, HepG2 cells were infected with SI-MSCV-puro-H1R-p53Ri retrovirus for down-regulation of p53 expression and with SI-MSCV-puro-H1R retrovirus for negative control.…”