Ethanol metabolism results in a G2/M cell-cycle arrest in recombinant Hep G2 cells

Dahn L. Clemens, Lilian E. Calisto, Michael F. Sorrell, Dean J. Tuma

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Previous studies using the Hep G2-based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation-mediated impairment of DNA synthesis we analyzed the cell-cycle progression of VA cells. These studies showed approximately a 6-fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin-dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co-immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol-treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3-fold. Thus, the impairment in cell-cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell-cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2.

Original languageEnglish (US)
Pages (from-to)385-393
Number of pages9
JournalHepatology
Volume38
Issue number2
DOIs
StatePublished - Aug 1 2003

Fingerprint

G2 Phase Cell Cycle Checkpoints
Cyclin B1
Hep G2 Cells
Ethanol
Cell Cycle
Phosphorylation
Cyclin-Dependent Kinases
G2 Phase
DNA
Immunoprecipitation
Cell Division
Cell Culture Techniques
Amino Acids

ASJC Scopus subject areas

  • Hepatology

Cite this

Ethanol metabolism results in a G2/M cell-cycle arrest in recombinant Hep G2 cells. / Clemens, Dahn L.; Calisto, Lilian E.; Sorrell, Michael F.; Tuma, Dean J.

In: Hepatology, Vol. 38, No. 2, 01.08.2003, p. 385-393.

Research output: Contribution to journalArticle

@article{cf00b4ab6fd94343a63be13551468cd6,
title = "Ethanol metabolism results in a G2/M cell-cycle arrest in recombinant Hep G2 cells",
abstract = "Previous studies using the Hep G2-based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation-mediated impairment of DNA synthesis we analyzed the cell-cycle progression of VA cells. These studies showed approximately a 6-fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin-dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co-immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol-treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3-fold. Thus, the impairment in cell-cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell-cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2.",
author = "Clemens, {Dahn L.} and Calisto, {Lilian E.} and Sorrell, {Michael F.} and Tuma, {Dean J.}",
year = "2003",
month = "8",
day = "1",
doi = "10.1053/jhep.2003.50332",
language = "English (US)",
volume = "38",
pages = "385--393",
journal = "Hepatology",
issn = "0270-9139",
publisher = "John Wiley and Sons Ltd",
number = "2",

}

TY - JOUR

T1 - Ethanol metabolism results in a G2/M cell-cycle arrest in recombinant Hep G2 cells

AU - Clemens, Dahn L.

AU - Calisto, Lilian E.

AU - Sorrell, Michael F.

AU - Tuma, Dean J.

PY - 2003/8/1

Y1 - 2003/8/1

N2 - Previous studies using the Hep G2-based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation-mediated impairment of DNA synthesis we analyzed the cell-cycle progression of VA cells. These studies showed approximately a 6-fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin-dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co-immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol-treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3-fold. Thus, the impairment in cell-cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell-cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2.

AB - Previous studies using the Hep G2-based VA cells showed that ethanol metabolism resulted in both cytotoxicity and impaired DNA synthesis, causing reduced accumulation of cells in culture. To further characterize the ethanol oxidation-mediated impairment of DNA synthesis we analyzed the cell-cycle progression of VA cells. These studies showed approximately a 6-fold increase in the percentage of cells in the G2/M phase of the cell cycle after 4 days of ethanol exposure. The G2/M transition requires activity of the cyclin-dependent kinase, Cdc2. Cdc2 is positively regulated by association with cyclin B1, and negatively regulated by phosphorylation of amino acids Thr14 and Tyr15. Immunoblot analysis revealed that ethanol metabolism had little affect on total Cdc2 content in these cells, but resulted in the accumulation of up to 20 times the amount of cyclin B1, indicating that cyclin B1 was available for formation of Cdc2/cyclin B1 complexes. Co-immunoprecipitation revealed that 6 times more Cdc2/cyclin B1 complexes were present in the ethanol-treated cells compared with the controls. Investigation of the phosphorylation state of Cdc2 revealed that ethanol oxidation increased the amount of the phosphorylated inactive form of Cdc2 by approximately 3-fold. Thus, the impairment in cell-cycle progression could not be explained by a lack of cyclin B1, or the ability of Cdc2 and cyclin B1 to associate, but instead resulted, at least in part, from impaired Cdc2 activity. In conclusion, ethanol oxidation by VA cells results in a G2/M cell-cycle arrest, mediated by accumulation of the phosphorylated inactive form of Cdc2.

UR - http://www.scopus.com/inward/record.url?scp=0042265643&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0042265643&partnerID=8YFLogxK

U2 - 10.1053/jhep.2003.50332

DO - 10.1053/jhep.2003.50332

M3 - Article

C2 - 12883482

AN - SCOPUS:0042265643

VL - 38

SP - 385

EP - 393

JO - Hepatology

JF - Hepatology

SN - 0270-9139

IS - 2

ER -