DESTRUCTION OF BYSTANDER CELLS BY TUMOR-CELLS TRANSFECTED WITH INDUCIBLE NITRIC-OXIDE (NO) SYNTHASE GENE

Background: The activation of an enzyme, inducible nitric oxide syntha se (iNOS), catalyzes the production of endogenous nitric oxide (NO). N O, in turn, is associated with cell death, suppression of tumor develo pment, and inhibition of metastasis of murine melanoma cells. Moreover , the in vivo induction of iNOS is associated with regression of estab lished hepatic metastases. Whether this regression required the activa tion of the iNOS gene in every tumor cell or whether NO-producing tumo r cells can also kill bystander (neighboring) cells has been previousl y unknown. Purpose: The goal of this study was to determine whether ce lls producing NO and then undergoing autolysis can also kill bystander cells in vitro and in vivo. Methods: Murine K-1735 C4.parental (C4.P) melanoma cells were transfected with the functional iNOS gene (transf ectant denoted as C4.L8) or with a control truncated-nonfunctional iNO S gene (transfectant denoted as C4.S2). NO-mediated cytolysis and byst ander cell killing were determined in vitro and in vivo. Results: Only the functional iNOS-transfected C4.L8 cells produced NO and underwent autolysis. C4.L8 cells also produced statistically significant lysis of iNOS-negative C4.P cells. This lysis was suppressed by the specific iNOS inhibitor N-G-methyl-L-arginine. NO-producing C4.L8 cells and co ntrol C4.P or C4.S2 cells were injected subcutaneously into syngeneic C3H/HeN mice. Control C4.P and C4.S2 cells produced rapidly growing su bcutaneous tumors, whereas C4.L8 cells did not. The mixture of C4.P an d C4.S2 cells (1:5 ratio) produced rapidly growing subcutaneous tumors , whereas the mixture of C4.P and C4.L8.5 cells (1:5 ratio) produced s low-growing tumors. The subcutaneous growth of C4.P cells was not affe cted by C4.L8.5 cells injected subcutaneously at a distant site. Mixtu res of C4.P cells labeled with [I-125]iododeoxyuridine and C4.L8 cells (NO producing) or C4.S2 cells (control) were injected subcutaneously. The survival rate of the radiolabeled cells indicated that the NO-pro ducing C4.L8.5 cells lysed the bystander C4.P cells. Conclusion: The p roduction of high-level endogenous NO causes autolysis in tumor cells and lysis of bystander cells under in vitro and in vivo conditions. Im plications: NO-mediated cell killing does not require transfection of genes into every cell in a neoplasm.