Tagger A Y, Wright J A
Department of Biochemistry, University of Manitoba, Winnipeg, Canada.
Int J Cancer. 1988 Nov 15;42(5):760-6. doi: 10.1002/ijc.2910420522.
Ribonucleotide reductase consists of 2 protein components frequently called M1 and M2. Hydroxyurea specifically inhibits DNA synthesis by interacting with the M2 protein and destroying a unique tyrosyl-free radical. We have carried out a molecular and cellular characterization of 2 Chinese hamster ovary cell lines exhibiting either low (HN(R)-AT) or relatively high (H(R)-R2T) resistance to the cytotoxic effects of hydroxyurea. Both drug-resistant lines have an increased level of ribonucleotide reductase activity. EPR measurements for tyrosyl-free radical content and studies with M1-specific antibodies indicated that the elevation in enzyme activity was entirely due to an increase in the M2 component. Studies with M1 cDNA showed that both drug-resistant cell lines contained a wild-type level of M1 mRNA and a wild-type M1 gene copy number. Studies with M2 cDNA indicated that the 2 drug-resistant lines possessed elevated levels of M2 message that could explain the observed increase in M2 component. The elevation of M2 mRNA in the most resistant line, H(R)-R2T, was due to an increase in M2 gene copy number. The low resistant cell line, HN(R)-AT, exhibited a wild-type M2 gene copy number, indicating that the increase in M2 gene message occurred through a process other than gene amplification. Enzyme kinetic studies with partially purified preparations from both drug resistant lines showed reduced sensitivity to hydroxyurea and to the negative allosteric effector, dATP. In addition to hydroxyurea, H(R)-R2T cells were also resistant to several other drugs whose site of action is the M2 component. Furthermore, H(R)-R2T cells were not cross-resistant to colchicine or puromycin, suggesting that hydroxyurea-resistant cells do not share the multi-drug resistance phenotype, which is frequently associated with cross-resistance to these drugs.
核糖核苷酸还原酶由两种蛋白质成分组成，通常称为M1和M2。羟基脲通过与M2蛋白相互作用并破坏一种独特的无酪氨酸自由基来特异性抑制DNA合成。我们对两种中国仓鼠卵巢细胞系进行了分子和细胞特性分析，这两种细胞系对羟基脲的细胞毒性作用表现出低抗性（HN(R)-AT）或相对高抗性（H(R)-R2T）。两种耐药细胞系的核糖核苷酸还原酶活性水平均有所提高。对无酪氨酸自由基含量的电子顺磁共振测量以及使用M1特异性抗体进行的研究表明，酶活性的升高完全归因于M2成分的增加。对M1 cDNA的研究表明，两种耐药细胞系都含有野生型水平的M1 mRNA和野生型M1基因拷贝数。对M2 cDNA的研究表明，两种耐药细胞系的M2信使水平升高，这可以解释观察到的M2成分增加。抗性最强的细胞系H(R)-R2T中M2 mRNA的升高是由于M2基因拷贝数的增加。低抗性细胞系HN(R)-AT表现出野生型M2基因拷贝数，这表明M2基因信使的增加是通过基因扩增以外的过程发生的。对两种耐药细胞系部分纯化制剂的酶动力学研究表明，它们对羟基脲和负变构效应物dATP的敏感性降低。除了羟基脲，H(R)-R2T细胞对其他几种作用位点为M2成分的药物也具有抗性。此外，H(R)-R2T细胞对秋水仙碱或嘌呤霉素没有交叉抗性，这表明羟基脲抗性细胞不具有多药耐药表型，而多药耐药表型通常与对这些药物的交叉抗性相关。
