Hurta R A, Wright J A
Department of Biochemistry, University of Manitoba, Winnipeg, Canada.
Biochem Biophys Res Commun. 1990 Feb 28;167(1):258-64. doi: 10.1016/0006-291x(90)91759-l.
Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.
核糖核苷酸还原酶催化由核糖核苷二磷酸前体形成脱氧核糖核苷酸，这是DNA合成中的限速步骤。该酶由两个不同的亚基组成，通常称为M1和M2。抗肿瘤药物羟基脲是DNA合成的特异性抑制剂，其作用是破坏核糖核苷酸还原酶M2亚基的酪氨酰自由基。通过将一群小鼠L细胞暴露于浓度不断增加的羟基脲中，分离出了两种高度耐药的细胞系，分别命名为HR-15和HR-30。HR-15和HR-30细胞中核糖核苷酸还原酶活性水平升高，对羟基脲细胞毒性作用的耐药性分别比野生型高68倍和103倍。Northern和Southern印迹分析表明，这两种耐药细胞系中M2 mRNA水平和M2基因拷贝数均升高。用M1特异性cDNA进行的类似研究表明，HR-15和HR-30细胞系中M1信息水平也增加，并显示出M1基因扩增。M1和M2基因表达和拷贝数发生改变的突变细胞系有助于获取与核糖核苷酸还原酶调控及其在DNA合成和细胞增殖中的作用相关的信息。
