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 [3H]AZT in the AZT-resistant T-cell lines showed a significant reduction of AZT phosphorylation to the mono-, di-, and triphosphate anabolites. Peripheral blood mononuclear cells (PBMCs) from pediatric patients with human immunodeficiency virus type 1 (HIV-1) infection showed a similar pattern of decreased AZT anabolism. Enzymatic studies with purified thymidine kinase (TK) preparations from these cell lines showed a gradual decline in Vmax related to their level of resistance to AZT. The Jurkat/AZT-20 and Jurkat/AZT-100 cells were studied in greater detail with reverse transcriptase/polymerase chain reaction (RT/PCR) cloned probes to determine possible molecular mechanisms of resistance to AZT. TK mRNA was significantly decreased (approximately 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 expression of TK mRNA and resultant TK enzymatic activity is responsible for the observed reduction in the AZT anabolism in the resistant T-cell lines. Decreased T-cell TK activity could allow wild-type, AZT-sensitive 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.
Wu
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Department of Pediatrics, USC School of Medicine, Childrens Hospital Los Angeles 90027, USA.
Liu
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Solorzano
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Kwock
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Avramis
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D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002478:Cells, Cultured; D002648:Child; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D004351:Drug Resistance; D015971:Gene Expression Regulation, Enzymologic; D015658:HIV Infections / Q000150:complications / Q000188:drug therapy; D015497:HIV-1; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000201:enzymology / Q000821:virology; D008745:Methylation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology*; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D015215:Zidovudine / Q000494:pharmacology*
D000998:Antiviral Agents; D017931:DNA Primers; D012333:RNA, Messenger; D015215:Zidovudine; D004247:DNA; D013937:Thymidine Kinase