Proceedings of the American Association for Cancer Research (AACR) 96th Annual Meeting, April 16-20, 2005, Anaheim/Orange County, CA, Abstract 3304. Philadelphia, PA: American Association for Cancer Research, 2005 Apr; 46:1
Zidovudine (AZT), a thymidine analogue and a carcinogen, was the first drug found effective against the human immunodeficiency virus (HIV-1). The antiretroviral efficacy of AZT depends on phosphorylation for incorporation into DNA, and requires continuous therapy. The intracellular mono-, di- and tri-phosphorylation steps are catalyzed by thymidine kinase 1 (TK1), thymidylate kinase, and pyrimidine nucleoside diphosphate kinase, respectively. These studies were designed to investigate AZT metabolism in 2 types of human cells, MOLT-3 transformed lymphoblastoid cells and normal mammary epithelial cells under conditions of continuous AZT exposure. MOLT-3 cells were exposed to AZT (800 uM) for 14 passages. During that time levels of TK1 protein expression were determined by western blotting. The data showed that the enzymatically active form of TK1, the 24kDa monomer, was depleted by passage 5 and was not subsequently restored by passage 14. Concomitantly, the enzymatically-inactive TK1 48kDa dimer increased with duration of exposure. Depletion of the TK1 monomer correlated with a transient (5-fold) increase in doubling time (passages 2-4) and a final 2-fold increase in doubling time that was maintained from passage 5 to passage 14. Gene expression analysis of TK1, determined by real time PCR, showed a 5.7-fold down regulation at passage 1 and an 8.3-fold down-regulation at passage 5; these data correlated well with a loss in TK1 enzyme specific activity, by 30%-75%, determined by a radiochemically based enzyme assay. Incorporation of AZT into DNA was measured by radioimmunoassay, and mean values were 59.3, 72.4 and 54.6 molecules of AZT/106 nucleotides at passages 1, 5, and 14, respectively, suggesting that MOLT-3 cells may use an alternate pathway to phosphorylate AZT. In order to examine this phenomenon in normal human cells, we used NHMEC strains having naturally differing levels of TK1 expression. NHMEC strains expressing low levels of TK1 (n=2), determined by western blotting, showed no detectable incorporation of AZT into DNA when exposed to 200 uM AZT for 24 hr, while strains with high levels of TK1 expression(n=2) showed > 200 molecules of AZT/106 nucleotides. In summary, chronic AZT exposure in transformed MOLT-3 cells resulted in loss of TK1 expression and enzyme activity, but not loss of capacity to incorporate AZT into DNA. These data suggest that down-regulation of TK1 may be circumvented through employment of other pathways. In contrast, in the normal breast epithelial cells the capacity to incorporate AZT into DNA appears to be linked to the capacity of individual cell strains to express TK1.
Proceedings of the American Association for Cancer Research (AACR) 96th Annual Meeting, April 16-20, 2005, Anaheim/Orange County, CA