LONG-TERM EXPOSURE TO ZIDOVUDINE AFFECTS IN-VITRO AND IN-VIVO THE EFFICIENCY OF PHOSPHORYLATION OF THYMIDINE KINASE

The purpose of this study was to investigate the mechanism of acquired cellular resistance to AZT, a mechanism that has been described as a potential source of drug resistance in addition to viral mutations. To study this phenomenon the kinetics parameters of thymidine kinase (TK ) activity have been defined in CEMazt, a cell line previously selecte d for resistance to AZT, in comparison with the parental AZT-sensitive CEM cells. The results revealed that the value of the maximum velocit y (V-max) of TK activity for deoxythymidine (dThd) phosphorylation is decreased in CEMazt as compared to the wild-type cell line (V-max: CEM = 105.3+/-17.6 nmol/hr/mg of protein; CEMazt = 0.3+/-0.02 nmol/hr/mg of protein; p < 0.001). Furthermore, the enzyme affinity versus dThd i s lower in CEMazt as compared to CEM (K-m: CEM = 0.9+/-0.2 mu M; CEMaz t = 1.6+/-0.2 mu M; P < 0.01). Consequently phosphorylation efficiency , expressed as the ratio between V-max and K-m, is also reduced in CEM azt (p < 0.001). To evaluate whether such a phenomenon may also occur in patients, ex vivo experiments were carried out by using PBMCs from HIV-infected patients, treated or not treated with AZT. The results (m ean values from 10 patients for each group) indicate that a prolonged treatment (>6 months) with AZT may modify the enzymatic kinetics of TK , leading to a significant reduction in the phosphorylation efficiency of the enzyme (4.07+/-1.7 in treated patients versus 13.5+/-1.7 in un treated patients; p < 0.001). These results indicate that AZT treatmen t can also induce a defect in TK activity in patients.