Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis

Abstract

Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, required for DNA synthesis. Ribonucleotide reductase consists of two dissimilar protein components often called M1 and M2, which are independently regulated during cell proliferation. The M1 component contains multiple effector binding sites and is responsible for the complex allosteric regulation of the enzyme, whereas the M2 protein contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction. Since the reaction is rate limiting for DNA synthesis, ribonucleotide reductase plays an important role in regulating cell division, and hence, cell proliferation. There are many inhibitors of ribonucleotide reductase and perhaps the most valuable one from a cell biology, biochemistry, and clinical point of view is the hydroxamic acid, hydroxyurea. This drug has also been very useful as a selective agent for isolating a variety of mammalian mutant cell lines altered in ribonucleotide reductase gene expression. Regulatory, structural, and biological characteristics of ribonucleotide reductase are reviewed, including evidence that ribonucleotide reductase, particularly the M2 protein, has an important early role to play in tumor promotion. In addition, modifications in the expressions of genes altered in hydroxyurea-resistant mutants and cultured in the absence or presence of hydroxyurea are discussed, with emphasis on changes in M2 protein, M1 protein, and the iron-storage protein ferritin.(ABSTRACT TRUNCATED AT 250 WORDS)

A link between ferritin gene expression and ribonucleotide reductase R2 protein, as demonstrated by retroviral vector mediated stable expression

“…Fax: (1).(204).787-2190. target for the drug) and ferritin, as we have demonstrated in previous studies [3,4,6,7]. The R2 gone is frequently amplified in mammalian cells resistant to hydroxyurea, and the elevated expression of genes like ornithine decarboxylase and a gone coding for a disulphide isomerase activity in these cells appears to be due to the co-amplification of these genes, which are located on the same chromosome close to the R2 gone [6][7][8][9].…”

“…As shown in Fig. 1, the anti-myc monoclonal antibody detected a protein band of 45 kDa corresponding to the R2 protein in myc-R2 vector infected cells [4][5][6], providing an example of stably expressed R2 eDNA in mammalian cells. To determine if the myc-R2 protein is biologically active, the sensitivity of cells to the cytotoxic effects of hydroxyurea was evaluated in colony forming assays.…”

“…We were interested in determining whether the elevated levels of R2 would be accompanied by increased expression of the ferritin genes, as has been observed with cell lines selected for resistance to hydroxyurea [4][5][6]. Fig.…”

“…This is likely due to the iron centre of the protein which is required for enzyme activity, since a loss of iron destroys ribonucleotide reductase activity preventing DNA synthesis and cell proliferation [4]. Free iron is extremely toxic to cells [1,2].…”

“…Treatment of cells with the antitumour agent, hydroxyurea, destabilizes the iron centre, and destroys a tyrosyl free radical in the R2 protein, resulting in a loss of enyzme activity and an inhibition of DNA synthesis and cell proliferation [5]. We have shown that cells selected for resistance to the cytotoxic effects of hyd,'oxyurea contain elevated levels of the R2 protein of ribonucleotide reductase, which is rate-limiting for enzyme activity [3,4]. Interestingly, these drug resistant cells also showed increased ferritin gene expression, suggesting that ferritin plays an important role as a storage protein for detoxifying excess iron released from inactive R2, and/or as an intracellular reservoir for iron that is required for activation of the R2 protein [5,6].…”