DEVELOPMENT OF ZIDOVUDINE (AZT) RESISTANCE IN JURKAT T-CELLS IS ASSOCIATED WITH DECREASED EXPRESSION OF THE THYMIDINE KINASE (TK) GENE AND HYPERMETHYLATION OF THE 5'-END OF HUMAN TK GENE

The T-cell line Jurkat E6-1 was rendered resistant to zidovudine (AZT) in vitro by exposure to low but gradually increased concentrations of the drug. Biochemical pharmacology studies of [H-3]AZT in the AZT-res istant T-cell lines showed a significant reduction of AZT phosphorylat ion to the mono-, di-, and triphosphate anabolites. Peripheral blood m ononuclear cells (PBMCs) from pediatric patients with human immunodefi ciency virus type 1 (HIV-1) infection showed a similar pattern of decr eased AZT anabolism. Enzymatic studies with purified thymidine kinase (TK) preparations from these cell lines showed a gradual decline in V- max related to their level of resistance to AZT. The Jurkat/AZT-20 and Jurkat/AZT-100 cells were studied in greater detail with reverse tran scriptase/polymerase chain reaction (RT/PCR) cloned probes to determin e possible molecular mechanisms of resistance to AZT. TK mRNA was sign ificantly decreased (similar to 5- to 10-fold) in the AZT-resistant T- cell lines. Southern blot analyses indicated that there were no major rearrangements or deletions of the TK gene, but the 5' end of the gene in the AZT-resistant cells is highly methylated when compared to wild -type cells. No apparent differences were seen in thymidylate kinase ( dTMPk) mRNA levels in the same T-cell lines. Thus the decreased expres sion of TK mRNA and resultant TK enzymatic activity is responsible for the observed reduction in the AZT anabolism in the resistant T-cell l ines. Decreased T-cell TK activity could allow wild-type, AZT-sensitiv e HIV-1 to replicate in the presence of subinhibitory AZT triphosphate (AZT-TP) cellular concentrations enabling a genetic variant with drug resistance to emerge and outgrow the AZT-sensitive, wild-type virus.