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Preferential Cytotoxicity of Cells Transduced with Cytosine Deaminase Compared to Bystander Cells after Treatment with 5-Flucytosine¹

Departments of Radiation Oncology [T. S. L., A. R., E. Y. N.] and Pharmacology [P. L. S.], University of Michigan Medical Center, Ann Arbor, Michigan 48109-0010; and Department of Pediatrics and Human Genetics, Michigan State University, East Lansing, Michigan 48224 [M. W., J. E. T.]

In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect. Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells. We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU). To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells. We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5-FC does not depend on GJIC. However, our most striking finding was that both communication-competent and -incompetent CD-transduced cells were preferentially killed, by a factor of up to 500, compared to bystander cells. The lesser dependence of the CD/5-FC system on GJIC, combined with the finding that most cancer cells lack the capacity for GJIC, suggest that the CD/5-FC system may be superior to the HSV-TK/GCV approach for gene therapy. However, the premature death of the CD-transduced 5-FU "factory" suggests that other strategies may be necessary to produce a sufficient quantity of 5-FU for a duration long enough to produce permanent tumor regression.

¹ Supported by NIH Grants PO1 CA42761 (to T. S. L.) and RO1 CA21104 (to J. E. T.).

² To whom requests for reprints should be addressed, at Department of Radiation Oncology, University of Michigan, 1500 East Medical Center Drive, UH-B2C490, Ann Arbor, MI 48109-0010. Phone: (313) 647-9955; Fax: (313) 763-7371; E-mail: tsl@umich.edu.

Received 12/22/97. Accepted 4/16/98.

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