Ethanol decreases the efficiency of phosphorylation of thymidine kinase in a human T-lymphocytic cell line.
BACKGROUND
Thymidine kinase (TK) and thymidylate kinase (TMPK) are the two rate-limiting enzymes in the cascade of activation of the anti-human immunodeficiency virus (HIV) drug 3'-azido-3'-deoxythymidine (AZT) to its active triphosphate form. We examined the effect of ethanol and a combination of ethanol and AZT on TK and TMPK activities in human Jurkat T cells.


METHODS
Jurkat T cells were exposed to 0.2 and 0.5% (v/v) ethanol concentrations alone or in combination with 5 or 10 microM AZT for 48 hr in growth medium. TK and TMPK activities were determined by measuring the conversion of [3H] substrates (thymidine or AZT for TK and thymidine monophosphate for TMPK) to their respective monophosphate or diphosphate forms. The effect of ethanol on the transcriptional activity of TK was determined by reverse transcription-polymerase chain reaction and on the growth of Jurkat cells by [3H]-thymidine incorporation and cell-cycle analysis.


RESULTS
Treatment of Jurkat cells with 0.2 and 0.5% ethanol concentrations resulted in 25 and 50% decreases (p < or = 0.05) in TK activity, respectively. No significant changes were observed in the TMPK activities. However, ethanol decreased the formation of thymidine diphosphate from thymidine in coupled TK/TMPK reactions, suggesting that decreases in TK activity could result in an overall decrease in the phosphorylation of AZT. The effect of ethanol on TK was independent of its transcript level. AZT in combination with ethanol decreased the inhibitory effect of ethanol on TK activity. However, it did not block the ethanol effect even at higher concentrations. Ethanol significantly decreased the proliferative capacity and cell cycle progression of the Jurkat cells.


CONCLUSIONS
Our in vitro study in human Jurkat T cells indicates that at physiologically achievable concentrations in humans, ethanol can decrease TK activity through decreases in cell proliferation, and it suggests that ethanol ingestion in HIV-1-infected individuals could compromise activation of AZT and related drugs through decreased TK activity.
Prakash
Om
O
Laboratory of Molecular Oncology, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, New Orleans, Louisiana 70121, USA. oprakash@ochsner.org
Tang
Zhen-Ya
ZY
Zhou
Peng
P
Peng
Xiaochan
X
Kolls
Jay
J
Shellito
Judd E
JE
Nelson
Steve
S
D019380:Anti-HIV Agents / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007399:Interphase / Q000187:drug effects; D019169:Jurkat Cells / Q000187:drug effects / Q000201:enzymology; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D013601:T-Lymphocytes / Q000187:drug effects* / Q000201:enzymology*; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D015215:Zidovudine / Q000494:pharmacology
D019380:Anti-HIV Agents; D002492:Central Nervous System Depressants; D000431:Ethanol; D015215:Zidovudine; D013937:Thymidine Kinase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase