Gröschel Bettina, Kaufmann Andreas, Höver Gerold, Cinatl Jaroslav, Doerr Hans Wilhelm, Noordhuis Paul, Loves Willem J P, Peters Godefridus J, Cinatl Jindrich
Department of Hematology & Oncology, Institute of Medical Virology, JWG University Frankfurt/M., Paul-Ehrlich Strasse 40, 60596 Frankfurt/M., Germany.
Biochem Pharmacol. 2002 Jul 15;64(2):239-46. doi: 10.1016/s0006-2952(02)01109-7.
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.
在3'-叠氮基-2',3'-双脱氧胸苷(AZT)存在的情况下连续培养T淋巴细胞系H9细胞,产生了一种对胸苷和脱氧胞苷类似物均具有交叉抗性的细胞变体。与亲代细胞相比,连续暴露于AZT的H9细胞中,AZT、2',3'-双脱氢-3'-脱氧胸苷以及不同的脱氧胞苷类似物(如2',3'-双脱氧胞苷、2',2'-二氟-2'-脱氧胞苷(dFdC)和1-β-D-阿拉伯呋喃糖基胞嘧啶(Ara-C))的细胞毒性作用显著降低(分别降低>8.3倍、>6.6倍、>9.1倍、5×10⁴倍、5×10³倍)。此外,在对AZT耐药的H9细胞中,AZT、d4T、ddC和2',3'-双脱氧-3'-硫代胞苷(3TC)的抗HIV-1作用也显著减弱(分别减弱>222倍、>25倍、>400倍、>200倍)。对AZT耐药的H9细胞中对嘧啶类似物交叉抗性的细胞机制研究表明,胸苷激酶-1(TK1)的mRNA水平降低,且缺乏脱氧胞苷激酶(dCK)的mRNA表达。通过对dCK蛋白的蛋白质印迹分析以及dCK酶活性测定,证实了dCK基因表达的缺失。此外,在对AZT耐药的细胞中,TK1和TK2的酶活性也降低。为了确定dCK的缺乏是否影响脱氧胞苷类似物dFdC的活性三磷酸酯的形成,在H9亲代细胞和对AZT耐药的细胞中,在暴露于1和10μM dFdC后测量了dFdCTP的积累和保留情况。亲代H9细胞在4小时后积累了约30和100 pmol dFdCTP/10⁶个细胞,而在对AZT耐药的细胞中未检测到dFdCTP积累。这些结果表明,在AZT存在的情况下对H9细胞进行连续处理,选择出了一种对胸苷类似物耐药的细胞变体,该变体由于TK1、TK2和dCK的缺乏而对脱氧胞苷类似物具有交叉抗性。