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Bystander Killing of Melanoma Cells Using the Human Tyrosinase Promoter to Express the Escherichia coli Purine Nucleoside Phosphorylase Gene¹

Departments of Physiology and Biophysics [B. W. H., Z. B., V. K. G., E. J. S.], Pathology [A. H. W.], and Medicine [R. I. G., E. J. S.], University of Alabama and Birmingham Veterans Administration Medical Center, Birmingham, Alabama 35294, and Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama 35255-5305 [W. B. P.]

We used a gene transfer-based system to generate highly toxic purine bases in tumor cells transfected with the Escherichia coli purine nucleo-side phosphorylase (PNP) gene. Because these toxic purines are membrane permeant, they mediate effective killing of neighboring cells that do not express E. coli PNP ("bystander" toxicity). In mixed cultures containing increasing percentages of cells with gene expression, 100% cancer cell growth arrest and total population killing was demonstrated when as few as 1–2% of cells expressed E. coli PNP. We used E. coli PNP to test bystander killing of human melanoma cells. A 529-bp region upstream of the human tyrosinase gene start site was shown to direct melanoma-specific expression in human cell lines. When this human tyrosinase regulatory region was used to control E. coli PNP expression, profound toxicity was observed in melanoma cells after treatment with the relatively nontoxic substrate 6-methylpurine-deoxyriboside, which is converted by E. coli PNP into the highly toxic purine base 6-methylpurine. Bystander toxicity was estimated as at least 100 cells killed for each cell expressing E. coli PNP, a level substantially higher than that of other tumor sensitization genes currently being used in clinical trails. These results suggest that the high bystander activity of the system could lead to significant antimelanoma responses in vivo.

¹ This work is supported by National Cancer Institute Grant P0134200 and the Lucille P. Markey Trust. E. J. S. is a Lucille P. Markey Scholar in the Biomedical Sciences.

² To whom requests for reprints should be addressed, at Department of Medicine, University of Alabama, 760 Basic Health Sciences Building, 1918 University Boulevard, Birmingham, AL 35294-0005.

Received 12/28/94. Accepted 5/31/95.

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