INTRACELLULAR METABOLISM OF 3'-AZIDO-3'-DEOXYTHYMIDINE (AZT) - A NUCLEAR-MAGNETIC-RESONANCE STUDY ON T-LYMPHOBLASTOID CELL-LINES WITH DIFFERENT RESISTANCE TO AZT

This paper reports the results of P-31 and H-1 nuclear magnetic resona nce (NMR) studies on the uptake and phosphorylation of 3'-azido-3'-deo xythymidine (AZT) in the human CD4(+) T-lymphoblastoid cell line CCRF- CEM (CEM-1.3) and in its AZT-resistant cell variant MT-500, isolated b y prolonged culturing of CEM cells in the presence of increasing AZT c oncentrations. After 3 hr of incubation in the presence of 0.5 mM AZT, both AZT and its monophosphorylated form (AZT-MP) could be detected i n the sensitive cell line in concentrations above the NMR detection le vels. In another cell line, MOLT-4, which is less sensitive to AZT eff ects, the intracellular level of AZT-MP was much lower and was only sl ightly raised by increasing the concentration of AZT in the extracellu lar and intracellular compartments. In the AZT-resistant clone MT-500, characterized by a very low thymidine kinase (TK, EC 2.7.1.21) activi ty with respect to the parental clone, the intracellular AZT-MP concen tration was below detection (<0.02 nmol/10(6) cells). Since, however, not only AZT-MP but also AZT signals failed to be detected in MT-500 e xtracts following cell incubation with AZT, it was concluded that a TK deficiency cannot be the exclusive mechanism of AZT resistance in the se cells. The possible effects of additional mechanisms of drug resist ance, such as specific AZT cell extrusion and limited permeation, are discussed, together with the new prospects offered by NMR spectroscopy to further evaluate the limiting steps for the utilization of antiret roviral nucleoside analogues. (C) 1997 Elsevier Science Inc.