Activation of c-Jun and suppression of phospho-p44/42 were involved in diphenylhydantoin-induced apoptosis of cultured rat cerebellar granule neurons.
OBJECTIVE
To investigate possible intracellular signal molecules involved in diphenylhydantoin (DPH)-mediated apoptosis of cerebellar granule neurons (CGN) and explore possible molecular mechanisms of neurotoxicity of DPH.


METHODS
Fluorescein diacetate (FDA) stain, hochest 33258 stain, and agar gel electrophoresis were used to test morphological and biological characters of primary CGN and cortical neurons (CN) in the presence or absence of 100 micromol/L DPH; Western blot and RT-PCR were employed to further investigate apoptotic/survival signal moleculars involved in the neuronal apoptotic signal transdution.


RESULTS
DPH 100 micromol/L induced a typical apoptosis of CGN but had no toxicity on CN. Cerebellar granule neural apoptosis induced by 100 micromol/L DPH was significantly inhibited by pre-treatment with SB203580 (10 micromol/L) or CEP-11004 (1 micromol/L) for 1 h. DPH markedly upregulated the levels of phospho-c-Jun (active c-Jun), total c-Jun protein and c-jun mRNA in CGN. The levels of phospho-c-Jun dramatically elevated by DPH at 8 h were significantly inhibited by SB203580 (10 micromol/L) or CEP-11004 (1 micromol/L). Moreover, the activities of p44/42 (ERK1/ERK2), other members of MAP kinases and generally believed to be important survival effetors in CGN, were markedly suppressed. However, the activities of both JNK and p38 were little affected in the process of apoptosis of CGN induced by 100 micromol/L DPH.


CONCLUSIONS
The selective toxicity of DPH on CGN is likely due to its ability to induce apoptosis of CGN, it is a process involved activation of c-Jun and suppression of the activity of p44/42.
Zhao
Ling-Zhi
LZ
Department of Pharmacology, Sun Yat-Sen Medical School, Sun Yat-sen University, Guangzhou, Guangdong 510089, China.
Su
Xing-Wen
XW
Huang
Yi-Jun
YJ
Qiu
Peng-Xin
PX
Yan
Guang-Mei
GM
D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D017209:Apoptosis; D002470:Cell Survival; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000166:cytology; D003594:Cytoplasmic Granules / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010672:Phenytoin / Q000494:pharmacology*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D000927:Anticonvulsants; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D010672:Phenytoin; D019950:Mitogen-Activated Protein Kinase 1