Ribonucleotide Reductase Subunit One as Gene Therapy Target

RNR catalyzes the reaction in which 2 -deoxyribonucleotides are generated from the corresponding ribonucleotide 5 diphosphates. This is the rate-limiting step in the production of 2 -deoxyribonucleoside 5 -triphosphates required for DNA replication. RNR consists of two protein subunits, R1 and R2. The R1 subunit is a Mr 160 KD homodimer that contains the catalytic site, two allosteric effector-binding sites, and redox active disulfides that participate in the reduction of substrates (1, 2). The R2 subunit is a Mr 78 KD homodimer that contains a nonheme iron that participates in catalysis by forming an unusual free radical on the aromatic ring of a tyrosine residue. Both the R1 and the R2 subunits are required to form the active site of the enzyme, with its genes located on separate chromosomes and the corresponding mRNAs differentially expressed throughout the cell cycle. The level of R1 protein remains relatively stable throughout the cell cycle, whereas R2 expression is cell cycle dependent, with highest expression concurrent with DNA replication. The RNR enzymatic activity is also regulated by allosteric control mechanisms involving positive and negative effectors (2, 3). Although best characterized as the large subunit of the RNR complex, R1 may also be the large subunit component of the complex responsible for the generation of 2 -deoxyribonucleoside 5 -triphosphate for DNA repair. p53R2, a recently identified p53-regulated R2 paralogue, is the putative small subunit of the ribonucleotide reductase complex involved in DNA repair (4–6). R1 has been shown to be up-regulated in response to DNA damage, consistent with a role in DNA repair (7). Further support for a role for R1 in DNA repair is found in a recent study that demonstrated that R1 can form a functional complex with p53R2 (8). The requirement of R1 for more than one reductase complex would explain the uncoupled nature of R1 and R2 expression. Several recent studies provide renewed interest in targeting RNR in the development of anticancer therapeutics (Fig. 1). An intriguing observation is that the RB tumor suppressor suppresses R1 and R2 as one of the mechanisms by which it controls progression through the cell cycle (9). RB inactivation, often observed in tumors, leads to increased dNTP levels and a concomitant resistance of tumor cells to drugs such as 5-fluorouracil (5-FU) and hydroxyurea (HU; Ref. 10). The R2 protein and the R1 subunit appear to play an additional role in determining the malignant potential of tumor cells. The R2 protein determines this via cooperation with a number of activated oncogenes (11, 12). The decrease in anchorage-independent growth for the R1 subunit was accompanied by marked suppression of malignant potential in vivo (12). Studies also show elevated expression of mouse Rl leads to suppression of transformation, tumorigenicity, and metastatic properties of tumor cells. Increased expression of R2 has been found to increase the drug-resistant properties of cancer cells and to increase invasive potential, whereas R2 expression in antisense orientation led to the reversal of drug resistance and resulted in decrease proliferation of tumor cells (13–19). Taken together, these studies indicate that, apart from the antiproliferative effect of RNR inhibition, the specific inhibition of R2 expression would likely provide additional antineoplastic benefits. The R1 gene has been mapped to chromosome 11p15.5 (20). Interestingly, the centromeric part of 11p15.5 contains a region of frequent LOH in many solid malignancies including lung, breast, ovarian, and stomach cancers (21–27). LOH resulting from an allelic loss of a polymorphic locus is often useful in the identification of tumor suppressor genes. The frequency of LOH within this region is also correlated with metastatic tumor spread (28, 29). Genetic complementation studies using chromosome 11 and fragments containing segment 11p15.5 resulted in reduced tumorigenesis in a syngeneic mouse model and growth inhibition of a number of tumor cell lines in vitro (30–35). Results suggest that R1, encoded within the region of frequent allele loss, may account for at least part of the observed tumor-suppressing activity in the 11p15.5 region. The article by Cao et al. (36) investigates the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1). A recombinant adenoviral vector, rAd5-R1, was produced by site-specific recombination and in vitro expression studies showed adenovirusmediated overexpression of R1 compared with vehicle and rAd5-LacZ controls. Transduction of rAd5-R1 into human colon adenocarcinoma cells (Colo320 HRS) induced antiproliferative effects in a doseand time-dependent manner. It was observed that the proliferation of normal cells is not affected by R1 overexpression, which is consistent with R1 acting as a tumorspecific growth suppressor. These inhibitory effects were demonstrated in animal models. The rAd5-R1 not only suppress tumorigenesis after ex vivo treatment of Colo320 HRS cells, but it also suppressed the growth of Colo320 HRS xenografts when injected intratumorally. Both experiments revealed a significant effect on tumor growth compared with the effect after treatment with a lacZ-expressing recombinant virus, which demonstrated Received 8/8/03; accepted 8/26/03. 1 To whom requests for reprints should be addressed, at City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 910103000. Phone: (626) 359-8111, extension 64442; Fax: (626) 301-8233; E-mail: [email protected]. 2 The abbreviations used are: RNR, ribonucleotide reductase; RB, retinoblastoma (tumor suppressor); LOH, loss of heterozygosity. 3 R1 and R2 are also called M1 and M2 in the human system to distinguish the RNR subunits from the rodent system. 4304 Vol. 9, 4304–4308, October 1, 2003 Clinical Cancer Research