Long-term AZT exposure alters the metabolic capacity of cultured human lymphoblastoid cells

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 (P₁-P₁₄) and cultured for an additional 14 passages (P₁₅-P₂₈) 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 P₁-P₅ of AZT exposure. From P₁₅ to P₂₈, 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/10⁶ nucleotides at the 10μM plasma-equivalent AZT dose at P₁, P₅, and P₁₄, respectively. An exposure-related increase in the frequency of micronuclei (MN) was observed in cells exposed to either 10 or 800μM AZT during P₁-P₁₄. Analysis of the cell cycle profile revealed an accumulation of S-phase cells and a decrease in G₁-phase cells during exposure to 800μM AZT for 14 passages. When MOLT-3 cells were grown in AZT-free media (P₁₅-P₂₉), 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.