Caspase 3 gene expression and [Ca2+]i homeostasis underlying desipramine-induced C6 glioma cell apoptosis.
OBJECTIVE
To study desipramine (Des)-induced apoptosis and regulation of caspase 3 gene expression and [Ca2+]i homeostasis in rat glioma C6 cells.


METHODS
Apoptotic DNA breaks were quantified by propidium iodide (PI) incorporation using flow cytometry (FCM) and were detected by DNA agarose gel electrophoresis. Expression of apoptotic effector gene caspase 3 was assessed by reverse transcription polymerase chain reaction (RT-PCR). Single cell [Ca2+]i was measured using fluorescence indicator Fura-3/AM with confocal laser scanning microscopy.


RESULTS
Des induced apoptotic DNA breaks in a concentration-dependent manner evidenced by hypodiploid peak on FCM histogram and the apoptotic cell percentage induced by Des 10, 20, and 40 micromol/L for 24 h was 5.2 %, 21.9 %, and 41.9 %, respectively. Apoptotic DNA breaks were further confirmed by a typical "DNA ladder" on agarose gel electrophoresis after exposure to Des 40 micromol/L for 24 h. Meanwhile, expression of caspase 3 gene was observed following Des 20 micromol/L treatment. Des 40 micromol/L resulted in an early sustained increase in [Ca2+]i over 28 min and the elevation magnitude was greatly decreased by removal of extracellular free [Ca2+]i with calcium-chelator egtazic acid, suggesting that Des elicited [Ca2+]i influx rather than intracellular calcium mobilization.


CONCLUSIONS
Up-regulation of caspase 3 gene expression and disturbance of homeostasis in calcium signaling system might play pivotal roles in Des-induced apoptotic DNA breaks of C6 cells.
Qi
Hong
H
Department of Pharmacology, Shanghai Second Medical University, Shanghai 200025, China.
Chen
Hong-Zhuan
HZ
Jin
Zheng-Jun
ZJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001693:Biological Transport, Active; D001921:Brain / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis* / Q000235:genetics; D004249:DNA Damage; D003891:Desipramine / Q000494:pharmacology*; D015870:Gene Expression; D005910:Glioma / Q000473:pathology*; D006706:Homeostasis / Q000502:physiology*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D002118:Calcium; D003891:Desipramine