Caspase 3 gene expression and [Ca2+]i homeostasis underlying desipramine-induced C6 glioma cell apoptosis.
Abstract
AIM: 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.
CONCLUSION: 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.
Keywords:
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.
CONCLUSION: 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.