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GTI-2501 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of R1, the large subunit of ribonucleotide reductase (RNR). In vitro studies, have demonstrated that GTI-2501 decreases mRNA and protein levels of R1 in a sequence-specific and dosedependent manner. Furthermore, GTI-2501 inhibits the growth of human lung, liver, ovary, brain, melanoma, breast and pancreatic tumor cells in colony forming assays. In vivo studies have shown that GTI-2501 significantly inhibits growth of human colon, pancreas, lung, breast, renal, ovarian, melanoma, brain glioblastoma-astrocytoma, and prostatic tumors in CD-1 nude, Balb/c nude and/or SCID mice. GTI-2501 treatment caused total regression of human breast and renal tumor xenografts in mice. These effects are not observed with a scrambled control oligonucleotide containing the same base content but not complementary to R1. GTI-2501 specifically inhibits metastasis of human melanoma cells to the lungs in CD-1 athymic nude mice and prolongs the survival of mice bearing human lymphoma. Taken together these results suggest that an antisense mechanism of action is responsible for growth inhibition in vitro and in vivo and that GTI-2501 can act as a selective and specific anti-tumor agent. Introduction In 2005, it is expected that 1,372,910 new cases of invasive cancer will be diagnosed in the United States and 570,280 people are expected to die from cancer (American Cancer Society, Facts and Figures 2005). Current therapeutic approaches to cancer include surgery, radiation, chemotherapy, hormone and cytokine therapy. Each of these therapies has limited efficacy and can result in toxicity to normal cells. Thus, there is a need for therapies that specifically target tumor cells thereby having more favorable safety profiles. Several therapeutic agents are currently used which inhibit ribonucleotide reductase (RNR) as part of their mechanism of action. These include hydroxyurea (Hydrea®, Bristol-Myers Squibb and Hydroxyurea Capsules, Roxane), gemcitabine (Gemzar®, Eli Lilly) and fludarabine (Fludara®, Berlex). Gemcitabine and fludarabine are not specific inhibitors of ribonucleotide reductase and treatment results in significant side effects that limit their effectiveness. Hydroxyurea is a reversible inhibitor of RNR that requires relatively high concentrations to be effective. Ribonucleotide reductase catalyzes the reaction in which 2'-deoxyribonucleotides (dADP, dGDP, dUDP, and dCDP) are synthesized from the corresponding ribonucleoside 5'diphosphates (ADP, GDP, UDP, and CDP). This step is the rate-limiting reaction in the production of 2'-deoxyribonucleoside 5'-triphosphates required for DNA replication (1). RNR consists of two protein components. R1 is a 160-kDa dimer that contains at least two different effector-binding sites and R2 is a 78-kDa dimer that contains a non-heme iron that participates in catalysis by forming an unusual free radical on the aromatic ring of a tyrosine residue. Expression of both R1 and R2 are required for enzymatic activity. Interestingly, R1 and R2 are encoded by different genes located on separate chromosomes and the mRNAs are differentially expressed throughout the cell cycle (2,3). Consequently, the level of R1 protein remains relatively stable throughout the cell cycle, while R2 is only expressed during late G1/early S phase, when DNA replication occurs. RNR activity is regulated by the amount of enzyme present in the cell and by allosteric control mechanisms involving positive and negative effectors (1,4). Recently, an R2 paralogue, p53R2, was identified that is induced by DNA damage (5-7). Expression of p53R2 is regulated by p53, via a p53 binding sequence in intron 1 of INTERNATIONAL JOURNAL OF ONCOLOGY 28: 469-478, 2006 469 GTI-2501, an antisense agent targeting R1, the large subunit of human ribonucleotide reductase, shows potent anti-tumor activity against a variety of tumors YOON LEE, AIKATERINI VASSILAKOS, NINGPING FENG, HONGNAN JIN, MING WANG, KEYONG XIONG, JIM WRIGHT and AIPING YOUNG Lorus Therapeutics Inc., 2 Meridian Road, Ontario, M9W 4Z7, Canada Received October 6, 2005; Accepted November 29, 2005 _________________________________________ Correspondence to: Dr Yoon Lee, Lorus Therapeutics Inc., 2 Meridian Road, Ontario, M9W 4Z7, Canada E-mail: [email protected] Abbreviations: RNR, ribonucleotide reductase; ODN, oligodeoxynucleotide; AS-ODN, antisense oligodeoxynucleotide; PS-ASODN, phosphorothioate antisense oligodeoxynucleotide; 5' UTR, 5' untranslated region; 3' UTR, 3' untranslated region; SCID, severe combined immune deficient; kDa, kilodalton; NK, natural killer; HU, hydroxyurea; 5-FU, 5-fluorouracil; dNTP, deoxynucleoside triphosphate; FBS, fetal bovine serum; FCS, fetal calf serum; RB, retinoblastoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ·-MEM, minimal essential medium; HUVEC, human umbilical vein endothelial cells; MAPK, mitogen-activated protein kinase-2; PBS, phosphate-buffered saline; HRP, horseradish peroxidase