ABSTRACT
The antiretroviral efficacy of 3'-azido-3'-deoxythymidine (AZT) is dependent upon intracellular mono-, di-, and triphosphorylation and incorporation into DNA in place of thymidine. Thymidine kinase 1 (TK-1) catalyzes the first step of this pathway. MOLT-3, human lymphoblastoid cells, were exposed to AZT continuously for 14 passages (P(1)-P(14)) and cultured for an additional 14 passages (P(15)-P(28)) without AZT. Progressive and irreversible depletion of the enzymatically active form of the TK-1 24-kDa monomer with loss of active protein was demonstrated during P(1)-P(5) of AZT exposure. From P(15) to P(28), both the 24- and the 48-kDa forms of TK-1 were undetectable and a tetrameric 96-kDa form was present. AZT-DNA incorporation was observed with values of 150, 133, and 108 molecules of AZT/10(6) nucleotides at the 10 microM plasma-equivalent AZT dose at P(1), P(5), and P(14), respectively. An exposure-related increase in the frequency of micronuclei (MN) was observed in cells exposed to either 10 or 800 microM AZT during P(1)-P(14). Analysis of the cell cycle profile revealed an accumulation of S-phase cells and a decrease in G(1)-phase cells during exposure to 800 microM AZT for 14 passages. When MOLT-3 cells were grown in AZT-free media (P(15)-P(29)), there was a reduction in AZT-DNA incorporation and MN formation; however, TK-1 depletion and the persistence of S-phase delay were unchanged. These data suggest that in addition to known mutagenic mechanisms, cells may become resistant to AZT partially through inactivation of TK-1 and through modulation of cell cycle components.
Subject(s)
Anti-HIV Agents/toxicity , T-Lymphocytes/drug effects , Zidovudine/toxicity , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , DNA/drug effects , DNA Adducts/drug effects , Down-Regulation , Drug Resistance, Neoplasm/drug effects , Humans , Micronuclei, Chromosome-Defective/chemically induced , Micronucleus Tests , Phosphorylation , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Thymidine/metabolism , Thymidine Kinase/antagonists & inhibitors , Thymidine Kinase/metabolismABSTRACT
A mainstay of the antiretroviral drugs used for therapy of HIV-1, zidovudine (AZT) is genotoxic and becomes incorporated into DNA. Here we explored host inter-individual variability in AZT-DNA incorporation, by AZT radioimmunoassay (RIA), using 19 different strains of normal human mammary epithelial cells (NHMECs) exposed for 24 h to 200 microM AZT. Twelve of the 19 NHMEC strains showed detectable AZT-DNA incorporation levels (16 to 259 molecules of AZT/10(6) nucleotides), while 7 NHMEC strains did not show detectable AZT-DNA incorporation. In order to explore the basis for this variability, we compared the 2 NHMEC strains that showed the highest levels of AZT-DNA incorporation (H1 and H2) with 2 strains showing no detectable AZT-DNA incorporation (L1 and L2). All 4 strains had similar (> or =80%) cell survival, low levels of accumulation of cells in S-phase, and no relevant differences in response to the direct-acting mutagen bleomycin (BLM). Finally, when levels of thymidine kinase 1 (TK1), the first enzyme in the pathway for incorporation of AZT into DNA, were determined by Western blot analysis in all 19 NHMEC strains at 24 h of AZT exposure, higher TK1 protein levels were found in the 12 strains showing AZT-DNA incorporation, compared to the 7 showing no incorporation (p=0.0005, Mann-Whitney test). Furthermore, strains L1 and L2, which did not show AZT-DNA incorporation at 24 h, did have measurable incorporation by 48 and 72 h. These data suggest that variability in AZT-DNA incorporation may be modulated by inter-individual differences in the rate of induction of TK1 in response to AZT exposure.
