Regression of Experimental Brain Tumors with 6-Thioxanthine andEscherichia coli gptGene Therapy

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Abstract
The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-thioxanthine, with an ID50 of 4.1 μM, whereas parental C6 cells were not affected at a concentration of 500 μM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This “modified” bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retrovirus producer cells displayed retarded growth upon exposure to 6-thioxanthine (6-TX). In a nude mouse model of intracerebral tumors, grafting of the gpt-retrovirus producer cells leads to an 80% reduction in intracerebral tumor volumes after 6-TX treatment. This reduction results in a 28% increase in the mean time of survival of animals that harbor intracerebral tumors (p < 0.0005). These antitumor effects indicate that the gpt/6-TX enzyme/prodrug pair is a promising alternative to the thymidine kinase gene and ganciclovir combination in the gene therapy of cancer. Gene transfer technologies are being applied in the treatment of neoplasms of the central nervous system. One of the most widely used approaches involves transfer of genes that activate prodrugs into their anticancer metabolites. In this study, producer cells that generate a retrovirus vector that bears the gpt gene from Escherichia coli were grafted into C6 gliomas grown intracerebrally in athymic mice. Partial regression of these tumors was observed after animal treatment with the prodrug 6-thioxanthine (6-TX), leading to a significant prolongation in animal survival. This study shows that a strategy which employs a 6-TX/gpt gene therapy holds promise against malignant tumors of the brain.

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