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
Sj. Wu et al., 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, Journal of acquired immune deficiency syndromes and human retrovirology, 8(1), 1995, pp. 1-9
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.