The sphingomyelin (SM) pathway is a ubiquitous, evolutionarily conserved signalling system analogous to conventional systems such as the cAMP and phosphoinositide pathways. Ceramide, which serves as second messenger in this pathway, is generated from SM by the action of a neutral or acidic SMase, or by de novo synthesis co-ordinated through the enzyme ceramide synthase. A number of direct targets for ceramide action have now been identified, including ceramide-activated protein kinase, ceramide-activated protein phosphatase and protein kinase Czeta, which couple the SM pathway to well defined intracellular signalling cascades. The SM pathway induces differentiation, proliferation or growth arrest, depending on the cell type. Very often, however, the outcome of signalling through this pathway is apoptosis. Mammalian systems respond to diverse stresses with ceramide generation, and recent studies show that yeast manifest a form of this response. Thus ceramide signalling is an older stress response system than the caspase/apoptotic death pathway, and hence these two pathways must have become linked later in evolution. Signalling of the stress response through ceramide appears to play a role in the development of human diseases, including ischaemia/reperfusion injury, insulin resistance and diabetes, atherogenesis, septic shock and ovarian failure. Further, ceramide signalling mediates the therapeutic effects of chemotherapy and radiation in some cells. An understanding of the mechanisms by which ceramide regulates physiological and pathological events in specific cells may provide new targets for pharmacological intervention.

Stress is believed to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response. Conclusive evidence for this paradigm, however, is lacking. In the present study, we used a genetic approach to address this issue directly. We show that lymphoblasts from Niemann-Pick patients, which have an inherited deficiency of acid sphingomyelinase activity, fail to respond to ionizing radiation with ceramide generation and apoptosis. These abnormalities are reversible up on restoration of acid sphingomyelinase activity by retroviral transfer of human acid sphingomyelinase cDNA. Acid sphingomyelinase knockout mice also expressed defects in radiation-induced ceramide generation and apoptosis in vivo. Comparison with p53 knockout mice revealed that acid sphingomyelinase-mediated apoptosis and p53-mediated apoptosis are likely distinct and independent. These genetic models provide definitive evidence for the involvement of acid sphingomyelinase in one form of stress-induced apoptosis.

Radiotherapeutic doses for malignant gliomas are generally palliative because greater, supposedly curative doses would impart clinically unacceptable damage to nearby vital CNS tissues. To improve radiation treatment for human gliomas, we evaluated microbeam radiation therapy, which utilizes an array of parallel, microscopically thin (<100 microm) planar beams (microbeams) of synchrotron-generated X rays. Rats with i.c. 9L gliosarcoma tumors were exposed laterally to a single microbeam, 27 pm wide and 3.8 mm high, stepwise, to produce irradiation arrays with 50, 75, or 100 microm of on-center beam spacings and 150, 250, 300, or 500 Gy of in-slice, skin-entrance, single-exposure doses. The resulting array size was 9 mm wide and 10.4 mm high (using three 3.8-mm vertical tiers); the beam's median energy was -70 keV. When all data were collated, the median survival was 70 days; no depletion of nerve cells was observed. However, when data from the highest skin-entrance dose and/or the smallest microbeam spacings were excluded, the median survival time of the subset of rats was 170 days, and no white matter necrosis was observed. Others have reported unilateral single-exposure broad-beam irradiation of i.c. 9L gliosarcomas at 22.5 Gy with a median survival of only -34 days and with severe depletion of neurons. These results suggest that the therapeutic index of unidirectional microbeams is larger than that of the broad beams and that an application for microbeam radiation therapy in treating certain malignant brain tumors may be found in the future.

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Radiotherapeutic doses for malignant gliomas are generally palliative because greater, supposedly curative doses would impart clinically unacceptable damage to nearby vital CNS tissues. To improve radiation treatment for human gliomas, we evaluated microbeam radiation therapy, which utilizes an array of parallel, microscopically thin (<100 microm) planar beams (microbeams) of synchrotron-generated X rays. Rats with i.c. 9L gliosarcoma tumors were exposed laterally to a single microbeam, 27 pm wide and 3.8 mm high, stepwise, to produce irradiation arrays with 50, 75, or 100 microm of on-center beam spacings and 150, 250, 300, or 500 Gy of in-slice, skin-entrance, single-exposure doses. The resulting array size was 9 mm wide and 10.4 mm high (using three 3.8-mm vertical tiers); the beam's median energy was -70 keV. When all data were collated, the median survival was 70 days; no depletion of nerve cells was observed. However, when data from the highest skin-entrance dose and/or the smallest microbeam spacings were excluded, the median survival time of the subset of rats was 170 days, and no white matter necrosis was observed. Others have reported unilateral single-exposure broad-beam irradiation of i.c. 9L gliosarcomas at 22.5 Gy with a median survival of only -34 days and with severe depletion of neurons. These results suggest that the therapeutic index of unidirectional microbeams is larger than that of the broad beams and that an application for microbeam radiation therapy in treating certain malignant brain tumors may be found in the future.

The plasma generated from a gas mixture of NH3 plus O2 (NH3 + O2) has been used to impart unique chemical and biological characteristics to polytetrafluoroethylene (PTFE). PTFE treated with NH3 + O2 plasma was physiochemically distinct from surfaces treated with plasma of either NH3 or O2 alone, as determined by electron spectroscopy for chemical analysis (ESCA). The contact angle analysis revealed that the PTFE surfaces became less hydrophobic after plasma treatments. ESCA results indicate the presence of oxygen-containing groups and nitrogen-containing groups at the plasma-treated surfaces. PTFE treated with NH3 + O2 plasma resisted the attachment of platelets and leukocytes in a manner similar to untreated PTFE; however, the attachment of bovine aorta endothelial cells was substantially increased. Once attached, these cells grew to confluency. The increased endothelial cell attachment was higher than that observed following plasma treatment with each gas used separately, which could be attributed to the considerable amount of CF(OR)2-CF2 formed on the NH3 + O2 plasma-treated PTFE surface. At 14 days after subcutaneous implantation in rats, the PTFE wafers treated with NH3 + O2 plasma demonstrated less encapsulation and lower levels of inflammatory cells compared to controls. Collectively, the results suggest that NH3 + O2 plasma treatment imparts a unique character to PTFE and could be useful in certain in vivo applications.

Since radiation therapy remains a primary treatment modality for gliomas, the radioresistance of glioma cells and targets to modify their radiation tolerance are of significant interest. Human apurinic endonuclease 1 (Ape1, Ref-1, APEX, HAP1, AP endo) is a multifunctional protein involved in base excision repair of DNA and a redox-dependent transcriptional co-activator. This study investigated whether there is a direct relationship between Ape1 and radioresistance in glioma cells, employing the human U87 and U251 cell lines. U87 is intrinsically more radioresistant than U251, which is partly attributable to more cycling U251 cells found in G2/M, the most radiosensitive cell stage, while more U87 cells are found in S and G1, the more radioresistant cell stages. But observed radioresistance is also related to Ape1 activity. U87 has higher levels of Ape1 than does U251, as assessed by Western blot and enzyme activity assays (approximately 1.5-2 fold higher in cycling cells, and approximately 10 fold higher at G2/M). A direct relationship was seen in cells transfected with CMV-Ape1 constructs; there was a dose-dependent relationship between increasing Ape1 overexpression and increasing radioresistance. Conversely, knock down by siRNA or by pharmacological down regulation of Ape1 resulted in decreased radioresistance. The inhibitors lucanthone and CRT004876 were employed, the former a thioxanthene previously under clinical evaluation as a radiosensitizer for brain tumors and the latter a more specific Ape1 inhibitor. These data suggest that Ape1 may be a useful target for modifying radiation tolerance.