Ruiz van Haperen V W, Veerman G, Eriksson S, Boven E, Stegmann A P, Hermsen M, Vermorken J B, Pinedo H M, Peters G J
Department of Oncology, Free University Hospital, Amsterdam, The Netherlands.
Cancer Res. 1994 Aug 1;54(15):4138-43.
2',2'-Difluorodeoxycytidine (gemcitabine, dFdCyd) is a deoxycytidine analogue with promising antitumor activity. In order to be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to dFCyd in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of dFdCyd. The IC50, defined as the concentration of dFdCyd causing 50% growth inhibition, at 72 h exposure increased from 0.6 nM dFdCyd in A2780 to 92 microM in the resistant variant, named AG6000. Although the resistant cell line is routinely cultured in 6 microM dFdCyd, the resistant phenotype can be maintained for at least 10 passages without dFdCyd. AG6000 is cross-resistant to other drug which require activation by dCK, such as 1-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. There was no specific dCK activity in extracts from AG600 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse-transcribed and PCR-amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs. Chromosome spreads from the cell lines showed no major differences between A2780 and AG6000. The latter cell line was also cross-resistant to 2',2'-difluorodeoxyurdine, the deamination product of dFdCyd. Additionally, cross-resistance to the multidrug resistant drugs doxorubicin and vincristine was observed. This was not associated with the induction of P-glycoprotein, as determined by the RNase protection assay. Injection of AG6000 cells s.c. into nude mice demonstrated that the cell line had retained its tumorigenicity; AG6000 xenografts were not sensitive to dFdCyd treatment, in contrast to the parental A2780 tumors. No dFdCyd triphosphate accumulation was found in the resistant tumors, in contrast to the parental A2780 tumors. These results indicate that the dFdCyd resistance phenotype is stable, and mainly due to dCK deficiency.
2',2'-二氟脱氧胞苷（吉西他滨，dFdCyd）是一种具有良好抗肿瘤活性的脱氧胞苷类似物。为了发挥活性，它必须被脱氧胞苷激酶（dCK）磷酸化。我们通过将人卵巢癌细胞系A2780暴露于浓度不断增加的dFdCyd中来诱导其对dFdCyd产生抗性。IC50定义为导致50%生长抑制的dFdCyd浓度，在72小时暴露时，从A2780中的0.6 nM dFdCyd增加到抗性变体（命名为AG6000）中的92 μM。尽管抗性细胞系通常在6 μM dFdCyd中培养，但在没有dFdCyd的情况下，抗性表型至少可维持10代。AG6000对其他需要dCK激活的药物具有交叉抗性，如1-β-D-阿拉伯呋喃糖基胞嘧啶、5-氮杂-2'-脱氧胞苷和2-氯脱氧腺苷。AG600细胞提取物中没有特异性的dCK活性。使用多克隆抗dCK抗体进行的蛋白质印迹分析未在AG6000细胞提取物中检测到任何dCK蛋白。使用特异性dCK引物进行逆转录和PCR扩增的mRNA表明，AG6000除了表达一个500个碱基对的异常扩增子外，还表达一个701个碱基对的正常长度扩增子。细胞系的染色体铺展显示A2780和AG6000之间没有主要差异。后者细胞系对dFdCyd的脱氨产物2',2'-二氟脱氧尿苷也具有交叉抗性。此外，还观察到对多药耐药药物阿霉素和长春新碱的交叉抗性。如通过核糖核酸酶保护试验所确定的，这与P-糖蛋白的诱导无关。将AG6000细胞皮下注射到裸鼠体内表明该细胞系保留了其致瘤性；与亲本A2780肿瘤相反，AG6000异种移植物对dFdCyd治疗不敏感。与亲本A2780肿瘤相反，在抗性肿瘤中未发现dFdCyd三磷酸的积累。这些结果表明，dFdCyd抗性表型是稳定的，主要是由于dCK缺乏。
