The sphingolipid ceramide has been recognized as an important second messenger implicated in regulating diverse signaling pathways especially for apoptosis. Very little is known, however, about the molecular mechanisms underlying nonapoptotic cell death induced by ceramide. In the present study, we first demonstrate that ceramide induces nonapoptotic cell death in malignant glioma cells. The cell death was accompanied by several specific features characteristic of autophagy: presence of numerous autophagic vacuoles in the cytoplasm, development of the acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3), and a marked increase in expression levels of two forms of LC3 protein (LC3-I and LC3-II). We additionally demonstrate that ceramide decreases mitochondrial membrane potential and activates the transcription of death-inducing mitochondrial protein, BNIP3, resulting in increased expression levels of its mRNA and protein in malignant glioma cells. Moreover, tumor cells transfected with BNIP3 gene undergo autophagy in the absence of ceramide. These results suggest that ceramide induces autophagic cell death in malignant glioma cells via activation of BNIP3. This study adds a new concept to characterize the pathways by which ceramide acts to induce nonapoptotic autophagic cell death in malignant gliomas.

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DNA-dependent protein kinase (DNA-PK) plays a major role in the repair of DNA double-strand breaks induced by ionizing radiation (IR). Lack of DNA-PK causes defective DNA doublestrand break repair and radiosensitization. In general, the cell death induced by IR is considered to be apoptotic. On the other hand, nonapoptotic cell death, autophagy, has recently attracted attention as a novel response of cancer cells to chemotherapy and IR. Autophagy is a protein degradation system characterized by a prominent formation of doublemembrane vesicles in the cytoplasm. Little is known, however, regarding the relationship between DNA-PK and IR-induced autophagy. In the present study, we used human malignant glioma M059J and M059K cells to investigate the role of DNA-PK in IR-induced apoptotic and autophagic cell death. Lowdose IR induced massive autophagic cell death in M059J cells that lack the catalytic subunit of DNA-PK (DNA-PKcs). Most M059K cells, the counterpart of M059J cells in which DNAPKcs are expressed at normal levels, survived, and proliferated although a small portion of the cells underwent apoptosis. Low-dose IR inhibited the phosphorylation of p70

Programmed cell death (PCD) is involved in a variety of biologic events. Based on the morphologic appearance of the cells, there are two types of PCD as follows: apoptotic (type I) and autophagic (type II). However, the molecular machinery that determines the type of PCD is poorly defined. The purpose of this study was to show whether the presence of the cyclin-dependent kinase (CDK) inhibitor p21, a modulator of apoptosis, determines which type of PCD the cell undergoes. Treatment with C 2 -ceramide was associated with both the cleavage of caspase-3 and poly(ADP-ribose) polymerase and the degradation of autophagy-related Beclin 1 and Atg5 proteins, without a change in the cyclin-CDK activity, which culminated in apoptosis in p21 ؉/؉ mouse embryonic fibroblasts (MEFs). On the other hand, C 2 -ceramide did not cleave caspase-3 or poly(ADP-ribose) polymerase and kept Beclin 1 and Atg5 proteins stable in p21 ؊/؊ MEFs, events that this time culminated in autophagy. When expression of the p21 protein was inhibited by small interfering RNA or when the overexpression of Beclin 1 or Atg5 was induced, autophagy rather than apoptosis was initiated in the p21 ؉/؉ MEFs treated with C 2 -ceramide. In contrast, the exogenous expression of p21 or the silencing of Beclin 1 and Atg5 with small interfering RNA increased the number of apoptotic cells and decreased the number of autophagic cells among C 2 -ceramidetreated p21 ؊/؊ MEFs. ␥-Irradiation, which endogenously generates ceramide, induced a similar tendency in these MEFs. These results suggest that p21 plays an essential role in determining the type of cell death, positively for apoptosis and negatively for autophagy.

We examined the radiological and histological features of, and the influences of the expression of VEGF and its two major receptors, Flt-1 and Flk-1, on the development of peritumoral brain edema (PTBE) in patients with intracranial meningiomas. The expressions of VEGF and VEGF receptors in the immunohistochemical study were analyzed in relation to several factors, including tumor size, location, vascularity, and blood supply, as seen on digital subtraction angiographic studies. The edema volume (P = 0.0003) and edema index (P < 0.0001) had a significantly positive relation to VEGF expression. The positivity of Flt-1 and Flk-1 was mainly observed in tumor vessels; 44 cases (37.2%) were positive for the Flt-1 antibody and 37 cases (31.4%) for the Flk-1 antibody. The mean value of the edema index of the positive-Flt-1 group (5.220 +/- 11.586) was significantly higher than that of the negative-Flt-1 group (1.782 +/- 2.559) (P < 0.0001). The mean value of the edema index of the positive-Flk-1 group (3.925 +/- 5.870) was slightly higher than that of the negative-Flk-1 group (2.671 +/- 8.136) (P < 0.0001). Our data suggest that the expressions of VEGF and VEGF receptors positively relate to each other and to the formation of PTBE in patients with meningiomas.