Radiation-induced apoptosis of oligodendrocytes and its association with increased ceramide and down-regulated protein kinase B/Akt activity.
OBJECTIVE
Oligodendrocytes are cells responsible for myelination in the central nervous system and have been shown to undergo radiation-induced apoptosis. The roles of ceramide and protein kinase B/Akt (PKB/Akt) were assessed in radiation-induced apoptosis of oligodendrocytes in vitro.


METHODS
Primary cultures of oligodendrocytes were established from neonatal rat brains and cell identity was assessed by immunohistochemistry. Apoptosis was assessed histologically according to its specific morphologic features using 4',6-diaminido-2-phenylindole, and by transferase-mediated deoxynucleotidyl transferase nick end-labelling staining. The ceramide level was measured using a diacyglycerol kinase assay, and PKB/Akt activity was determined using immunoblotting and a protein kinase assay.


RESULTS
Ionizing radiation, C2-ceramide or wortmannin induced apoptosis in oligodendrocytes but not astrocytes. A rapid increase in ceramide was observed in oligodendrocytes after ionizing radiation. Monensin, an inhibitor of acid sphingomyelinase, reduced the apoptotic response in oligodendrocytes after ionizing radiation. Fumonisin B1, an inhibitor of ceramide synthase, showed no such effect in the cells. Radiation-induced apoptosis of oligodendrocytes was associated with a decrease in PKB activity, similar to that observed after treatment with C2-ceramide or wortmannin, but not after dihydro-C2-ceramide. Confocal microscopy revealed a loss of phosphorylated PKB immunostaining in the nucleus of apoptotic oligodendrocytes after ionizing radiation or C2-ceramide treatment. The level of phosphorylated FKHRL1, a transcription factor phosphorylated by PKB, decreased in irradiated oligodendrocytes.


CONCLUSIONS
A ceramide-PKB-mediated signalling pathway might play a role in radiation-induced apoptosis of oligodendrocytes.
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Department of Radiation Oncology, Sunnybrook and Women's College Health Sciences Center, University of Toronto, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada.
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D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000528:radiation effects*; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism*; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D015536:Down-Regulation / Q000187:drug effects / Q000528:radiation effects; D037341:Fumonisins / Q000494:pharmacology; D005720:Gamma Rays; D008985:Monensin / Q000494:pharmacology; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000187:drug effects; D051381:Rats; D011916:Rats, Inbred F344
D002518:Ceramides; D037341:Fumonisins; D011518:Proto-Oncogene Proteins; C056933:fumonisin B1; D008985:Monensin; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt