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 (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.
Olivero
Ofelia A
OA
Laboratory of Cancer Biology and Genetics, Carcinogen-DNA Interactions Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA. oliveroo@exchange.nih.gov
Vazquez
Irma L
IL
Cooch
Catherine C
CC
Ming
Jessica
J
Keller
Emily
E
Yu
Mia
M
Borojerdi
Jennifer P
JP
Braun
Hannan M
HM
McKee
Edward
E
Poirier
Miriam C
MC
D019380:Anti-HIV Agents / Q000633:toxicity*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000187:drug effects; D018736:DNA Adducts / Q000187:drug effects; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D006801:Humans; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced; D015162:Micronucleus Tests; D010766:Phosphorylation; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015215:Zidovudine / Q000633:toxicity*
D019380:Anti-HIV Agents; D018736:DNA Adducts; D015215:Zidovudine; D004247:DNA; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D013936:Thymidine