Cory A H, Hertel L W, Kroin J S, Cory J G
Department of Biochemistry, East Carolina University School of Medicine, Greenville, NC 27858.
Oncol Res. 1993;5(2):59-63.
2',2'-Difluorodeoxycytidine (Gemcitabine, dFdCyd) is a cytotoxic agent which is active toward a variety of tumor cells. It has been shown that there are multiple intracellular sites of action which include ribonucleotide reductase and DNA polymerase. In these studies, the effects of dFdCyd on wild-type mouse leukemia L1210 cells and variant L1210 cell lines which had alterations at the ribonucleotide reductase site or at the deoxyribonucleoside kinase site were studied. For cell growth, the IC50 value for dFdCyd in wild-type L1210 cells was 3.1 nM. In the variant cell lines, the IC50 values were: hydroxyurea-resistant (HU), 3.3 nM; deoxyadenosine-resistant (Y8), 1.8 nM; pyrazoloimidazole/deoxyadenosine-resistant (ED2), 1.9 nM; and deoxyguanosine-resistant (dGuo-R), 44.7 nM. The dGuo-R cell line had a relatively specific loss of the deoxyribonucleoside kinase responsible for phosphorylating deoxyguanosine and cytosine arabinoside with little loss of the deoxycytidine kinase activity. DFdCyd had no effect on the total uptake of [14C]cytidine into the cells or incorporation into RNA. DFdCyd inhibited the conversion of [14C]cytidine to deoxycytidine nucleotides and incorporation into DNA. However, the incorporation of cytidine into DNA was inhibited to a greater extent than was the inhibition of in situ ribonucleotide reductase activity. Ribonucleotide reductase activity in cell-free extracts prepared from L1210 cells treated with dFdCyd (20 nM) overnight was reduced by 50%. These results show that cell lines which have increased levels of ribonucleotide reductase activity (HU and ED2) or loss of feedback inhibition by dATP (ED2 and Y8) are still sensitive to dFdCyd. The findings indicate that ribonucleotide reductase is not the primary site of inhibition by dFdCyd.(ABSTRACT TRUNCATED AT 250 WORDS)
2',2'-二氟脱氧胞苷（吉西他滨，dFdCyd）是一种对多种肿瘤细胞具有活性的细胞毒性药物。已表明其存在多个细胞内作用位点，包括核糖核苷酸还原酶和DNA聚合酶。在这些研究中，研究了dFdCyd对野生型小鼠白血病L1210细胞以及在核糖核苷酸还原酶位点或脱氧核苷激酶位点发生改变的L1210变异细胞系的影响。对于细胞生长，dFdCyd在野生型L1210细胞中的IC50值为3.1 nM。在变异细胞系中，IC50值分别为：羟基脲耐药（HU），3.3 nM；脱氧腺苷耐药（Y8），1.8 nM；吡唑并咪唑/脱氧腺苷耐药（ED2），1.9 nM；以及脱氧鸟苷耐药（dGuo-R），44.7 nM。dGuo-R细胞系负责磷酸化脱氧鸟苷和阿糖胞苷的脱氧核苷激酶相对特异性缺失，而脱氧胞苷激酶活性几乎没有损失。DFdCyd对[14C]胞苷进入细胞的总摄取或掺入RNA没有影响。DFdCyd抑制[14C]胞苷转化为脱氧胞苷核苷酸并掺入DNA。然而，胞苷掺入DNA的抑制程度大于对原位核糖核苷酸还原酶活性的抑制。用dFdCyd（20 nM）过夜处理的L1210细胞制备的无细胞提取物中的核糖核苷酸还原酶活性降低了50%。这些结果表明，核糖核苷酸还原酶活性水平升高（HU和ED2）或失去dATP反馈抑制（ED2和Y8）的细胞系对dFdCyd仍然敏感。这些发现表明核糖核苷酸还原酶不是dFdCyd的主要抑制位点。（摘要截短至250字）
