A Long-Acting Suicide Gene Toxin, 6-Methylpurine, Inhibits Slow Growing Tumors after a Single Administration

Abstract

We have demonstrated antitumor activity against refractory human glioma and pancreatic tumors with 6-methylpurine (MeP) using either a suicide gene therapy strategy to selectively release 6-methylpurine in tumor cells or direct intratumoral injection of 6-methylpurine itself. A single i.p. injection in mice of the prodrug 9-␤-D-[2-deoxyribofuranosyl]-6-methylpurine (MeP-dR; 134 mg/kg) caused sustained regression lasting over 70 days of D54 (human glioma) tumors transduced with the Escherichia coli purine nucleoside phosphorylase (PNP), and a single intratumoral injection of 6-methylpurine (5-10 mg/kg) elicited prolonged delays of the growth of D54 tumors and CFPAC human pancreatic carcinoma. Because the D54 tumor doubling time is Ͼ15 days, the experiments indicate that prodrug activation by E. coli PNP engenders destruction of both dividing and nondividing tumor compartments in vivo and, therefore, address a fundamental barrier that has limited the development of suicide gene strategies in the past. A prolonged retention time of 6-methylpurine metabolites in tumors was noted in vivo (T 1/2 Ͼ24 h compared with a serum half-life of Ͻ1 h). By high-pressure liquid chromatography, metabolites of [3 H]MeP-dR were 5-to 6-fold higher in tumors expressing E. coli PNP. These experiments point to new endpoints for monitoring E. coli PNP suicide gene therapy, including intratumoral enzymatic activity, in situ (intratumoral) prodrug conversion, and tumor regressions after direct injection of a suicide gene toxin. The findings also help explain the strong in vivo bystander killing mechanism ascribed by several laboratories to E. coli PNP in the past.Pancreatic, brain, lung, liver, prostate, and other human cancers often invade locally, become inoperable, and cause death even in the absence of distant metastases. These nonmetastatic, locally invasive cancers account for over 100,000 cases per year in the United States alone, and the majority lead to death (DeVita et al., 1997; SEER Cancer Incidence Public Use Database 1973. Treatment of these types of cancer remains a significant therapeutic challenge. Nonsurgical modalities (chemotherapy and radiotherapy) are ineffective in this setting because these approaches primarily kill proliferating cells. Refractory tumors often have a very low growth fraction (4 -40% of cells actively dividing at any time) (Giangaspero et al., 1987;Sadi and Barrack, 1991;Vescio et al., 1990;Dionne et al., 1998;Springer and Niculescu-Duvaz, 2000). Conventional anticancer agents that are selective for rapidly dividing tumor cells fail to eradicate tumors with a low growth fraction. Compounds designed to kill both proliferating and quiescent tumor cells are limited by toxicity following systemic administration.One proposed solution to this problem is expression of "suicide" genes to generate highly toxic compounds specifically inside growing tumors. An essential question is whether a suicide gene strategy as applied to locally invasive tumors offers an advantage over simply injecting toxins int...

Gene-Directed Enzyme Prodrug Cancer Therapy

“…Both compounds are freely diffusible across cell membranes, allowing their spread from PNP transduced to untransduced cells, and are toxic to both proliferating and nonproliferating cells, thereby achieving a potent bystander effect [81,82]. The bystander activity is facilitated by the nucleotide and nucleobase transporters across membranes in both directions and does not require cellÀcell contact or gap junctions [83].…”

Suicide genes for cancer therapy

“…Furthermore, PNP released into the cell medium is active and stable over long periods, and extracellular conversion of the prodrug following PNP release from dying cells has been described . Additionally, prolonged retention of in situ produced MeP metabolites in tumours has been observed, further improving tumour cell killing efficacy (Gadi et al, 2003).…”

Excellent In vivo Bystander Activity of Fludarabine Phosphate against Human Glioma Xenografts that Express the Escherichia coli Purine Nucleoside Phosphorylase Gene

“…The E. coli PNP gene was excised from pSV-PNP (12) with BamHI and inserted into the pACCMVpLpA adenoviral transfer vector (a gift of Dr. R. D. Gerard). Recombinant adenovirus was constructed by co-transfecting 293 cells in 6-well plates with the plasmid pJM17 (Microbix, Ontario, Canada) and pACCMVpLpA-PNP (13).…”