Fridland A
Cancer Res. 1984 Oct;44(10):4328-32.
We sought to define the cellular activity that mediates resistance in human leukemic cells (CCRF-CEM) to the nucleoside 9-beta-D-arabinofuranosyladenine (ara-A). Stable mutants were obtained by continuous selection at ara-A concentrations of 1 or 2.5 microM in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin. Four clones selected for further investigation were 4- to 11-fold less sensitive to the cytotoxicity of ara-A than the parental CCRF-CEM line. These clones also showed cross-resistance to deoxyadenosine and thymidine, but normal sensitivity to arabinosylcytosine and adenosine, and increased sensitivity to the etoposide VP16-213. No change was found in the activity of kinases that phosphorylate ara-A and the various nucleosides that could account for the resistant phenotype in these mutant lines. Resistance was associated with a 2- to 8-fold increase in the level of all four deoxyribonucleoside triphosphates. The triphosphate pools in the mutants were resistant to the inhibition produced in wild-type cells by addition of deoxy-adenosine or thymidine, although significant activation in the deoxyguanosine triphosphate pool was obtained by higher concentrations of thymidine. An examination of ribonucleotide reductase in extracts of two of the mutants revealed a specific alteration in the normal sensitivity of the enzyme for deoxyadenosine triphosphate and adenosine triphosphate but not 9-beta-D-arabinofuranosyladenine 5'-triphosphate. When the level of ribonucleotide reductase activity was measured, it was found that the ara-A-resistant cells contained approximately twice the wild-type level of cytidine diphosphate reductase activity at physiological adenosine triphosphate level. This combination of increased enzyme activity and alteration in sensitivity to the nucleoside triphosphates could account for both the changes in deoxyribonucleotide pool sizes and the resistant phenotype of the presumed mutants.
我们试图确定介导人白血病细胞（CCRF - CEM）对核苷9 - β - D - 阿拉伯呋喃糖基腺嘌呤（ara - A）产生抗性的细胞活性。在腺苷脱氨酶抑制剂2'-脱氧助间型霉素存在的情况下，通过在1或2.5 microM的ara - A浓度下连续筛选获得稳定突变体。为进一步研究而挑选的四个克隆对ara - A细胞毒性的敏感性比亲本CCRF - CEM细胞系低4至11倍。这些克隆对脱氧腺苷和胸苷也表现出交叉抗性，但对阿糖胞苷和腺苷具有正常敏感性，并且对依托泊苷VP16 - 213的敏感性增加。在这些突变细胞系中，磷酸化ara - A和各种核苷的激酶活性未发现变化，而这些变化本可解释其抗性表型。抗性与所有四种脱氧核糖核苷三磷酸水平增加2至8倍相关。突变体中的三磷酸核苷池对野生型细胞中添加脱氧腺苷或胸苷所产生的抑制具有抗性，尽管较高浓度的胸苷可使三磷酸脱氧鸟苷池有显著激活。对其中两个突变体提取物中的核糖核苷酸还原酶进行检测发现，该酶对三磷酸脱氧腺苷和三磷酸腺苷的正常敏感性发生了特定改变，但对9 - β - D - 阿拉伯呋喃糖基腺嘌呤5'-三磷酸没有影响。当测量核糖核苷酸还原酶活性水平时，发现在生理三磷酸腺苷水平下，ara - A抗性细胞中胞苷二磷酸还原酶活性约为野生型水平的两倍。酶活性增加以及对核苷三磷酸敏感性改变的这种组合可以解释脱氧核糖核苷池大小的变化以及推测突变体的抗性表型。
