3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells.
Continuous cultivation of T-lymphoid H9 cells in the presence of 3'-azido-2',3'-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2',3'-didehydro-3'-deoxythymidine as well as different deoxycytidine analogs such as 2',3'-dideoxycytidine, 2',2'-difluoro-2'-deoxycytidine (dFdC) and 1-ss-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5 x 10(4)-, 5 x 10(3)-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2',3'-dideoxy-3'-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 microM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10(6) cells after 4hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.
Gröschel
Bettina
B
Department of Hematology & Oncology, Institute of Medical Virology, JWG University Frankfurt/M., Paul-Ehrlich Strasse 40, 60596 Frankfurt/M., Germany.
Kaufmann
Andreas
A
Höver
Gerold
G
Cinatl
Jaroslav
J
Doerr
Hans Wilhelm
HW
Noordhuis
Paul
P
Loves
Willem J P
WJ
Peters
Godefridus J
GJ
Cinatl
Jindrich
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D019380:Anti-HIV Agents / Q000494:pharmacology*; D015153:Blotting, Western; D003570:Cytidine Triphosphate / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000172:deficiency / Q000378:metabolism*; D004352:Drug Resistance, Microbial / Q000502:physiology; D015497:HIV-1 / Q000187:drug effects; D006801:Humans; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000201:enzymology; D013937:Thymidine Kinase / Q000172:deficiency / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism; D015215:Zidovudine / Q000494:pharmacology*; D000093542:Gemcitabine
D019380:Anti-HIV Agents; D012333:RNA, Messenger; D003841:Deoxycytidine; D015215:Zidovudine; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; C105339:thymidine kinase 2; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D003842:Deoxycytidine Kinase; D014544:Uridine Triphosphate; D000093542:Gemcitabine