Disruption of cell cycle kinetics and cyclin-dependent kinase system by ethanol in cultured cerebellar granule progenitors.
An in vitro model of neuronal precursors, primary culture of cerebellar granule progenitors (CGPs), was used to investigate the mechanisms underlying ethanol-induced cell cycle damage. The CGP cultures were generated from 3-day-old rats. Ethanol significantly inhibited the proliferation of the CGPs in culture. Analysis of cell cycle kinetics by a cumulative 5-bromo-2'-deoxyuridine (BrdU) labeling technique demonstrated that ethanol exposure increased the duration of the cell cycle and decreased the growth fraction (the cycling population). The duration of the S-phase and total cell cycle was significantly prolonged by ethanol exposure by 220% and 135%, respectively, while the growth fraction was decreased from 44% in the control groups to 22% in the ethanol-exposed cultures. Cyclin-dependent kinase 2 (Cdk2) is a key protein that regulates both the passage from G1 into S, and the S phase progression. The results from in vitro phosphorylation assay and Western blot demonstrated that ethanol dramatically down-regulated both the activity and the expression of Cdk2. In addition, ethanol significantly decreased the expression of Cyclin A and Cyclin D(2). Further studies using in situ TUNEL assay and DNA fragmentation ELISA showed that ethanol caused a delayed apoptosis, i.e. the ethanol-induced apoptosis was evident only after chronic exposure. On the other hand, ethanol did not affect the necrotic index. In conclusion, ethanol decreases the cycling pool of CGPs by inducing cell cycle delay and promoting apoptosis. Ethanol-mediated disturbance of the cyclin-dependent kinase system may be an important mechanism to account for cell cycle arrest in neuronal precursor cells.
Li
Z
Z
Department of Microbiology, Immunology and Cell Biology, Robert C. Byrd Health Science Center, West Virginia University School of Medicine, P.O. Box 9177, Morgantown, WV 26506-9177, USA.
Lin
H
H
Zhu
Y
Y
Wang
M
M
Luo
J
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D042846:CDC2-CDC28 Kinases; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D002531:Cerebellum / Q000166:cytology*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D063647:Fetal Alcohol Spectrum Disorders / Q000378:metabolism / Q000473:pathology; D020287:In Situ Nick-End Labeling; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology
D002492:Central Nervous System Depressants; D000431:Ethanol; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases