TRANSFECTION WITH THE INDUCIBLE NITRIC-OXIDE SYNTHASE GENE SUPPRESSESTUMORIGENICITY AND ABROGATES METASTASIS BY M-1735 MURINE MELANOMA-CELLS

Previous studies from our laboratory demonstrated an inverse relations hip between the expression level of inducible nitric oxide synthase (i NOS) and the metastatic potential of murine K-1735 melanoma cells. The purpose of this study was to provide direct evidence that the express ion of iNOS suppresses metastatic potential of melanoma cells. Highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iN OS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containi ng 3 mM N-G-methyl-L-arginine (NMA). Positive transfectants were ident ified by Southern and Northern blot analyses and homogeneous staining with a specific anti-iNOS monoclonal antibody. Semiconfluent cultures of C4.P (parental), C4.Neo.3 (control transfection), C4.S2.3 (inactive iNOS), and C4.L8.5 (functional iNOS) were harvested, and viable cells were injected intravenously into syngeneic C3H/HeN mice and allogenei c BALB/c nude mice. C4.P, C4.Neo.3, and C4.S2.3 cells were highly meta static whereas C4.L8.5 cells were not metastatic. Experiments with [I- 125]IdUrd-labeled tumor cells demonstrated that the initial arrest in the lung microvasculature did not differ among the lines, but that C4. L8.5 cells died by 48-72 h after injection. Enhanced survival of all K -1735 C4 cells (including C4.L8.5) was found in mice given twice daily injections of 20 mg NMA. The C4.L8.5 cells produced slow growing subc utaneous tumors in nude mice, whereas the other three lines produced f ast growing tumors. In vitro studies confirmed that in the absence of NMA the expression of iNOS in C4.L8.5 cells induced apoptosis. Collect ively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorig enicity, and abrogation of metastasis.