Destruction of Bystander Cells by Tumor Cells Transfected With Inducible Nitric Oxide (NO) Synthase Gene

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Abstract
The activation of an enzyme, inducible nitric oxide synthase (iNOS), catalyzes the production of endogenous nitric oxide (NO). NO, in turn, is associated with cell death, suppression of tumor development, and inhibition of metastasis of murine melanoma cells. Moreover, the in vivo induction of iNOS is associated with regression of established hepatic metastases. Whether this regression required the activation of the iNOS gene in every tumor cell or whether NO-producing tumor cells can also kill bystander (neighboring) cells has been previously unknown. The goal of this study was to determine whether cells producing NO and then undergoing autolysis can also kill bystander cells in vitro and in vivo. Murine K-1735 C4.parental (C4.P) melanoma cells were transfected with the functional iNOS gene (transfectant denoted as C4.L8) or with a control truncated-nonfunctional iNOS gene (transfectant denoted as C4.S2). NO-mediated cytolysis and bystander cell killing were determined in vitro and in vivo. 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 control C4.P or C4.S2 cells were injected subcutaneously into syngeneic C3H/HeN mice. Control C4.P and C4.S2 cells produced rapidly growing subcutaneous tumors, whereas C4.L8 cells did not. The mixture of C4.P and 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 slow-growing tumors. The subcutaneous growth of C4.P cells was not affected by C4.L8.5 cells injected subcutaneously at a distant site. Mixtures of C4.P cells labeled with [(125)I]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-producing C4.L8.5 cells lysed the bystander C4.P cells. The production of high-level endogenous NO causes autolysis in tumor cells and lysis of bystander cells under in vitro and in vivo conditions. NO-mediated cell killing does not require transfection of genes into every cell in a neoplasm.