Transcriptional basis for the differences in inducible nitric oxide synthase (iNOS) expression between nonmetastatic and metastatic murine melanoma cell lines

An inverse correlation exists between expression of the inducible nitric ox ide synthase (iNOS) gene and the ability of cloned K1735 murine melanoma ce ll lines to metastasize. We have analyzed the basis for the difference in i NOS induction by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) in metastatic and nonmetastatic K1735 cells. Nuclear run-on (NRO) assays re vealed an upregulation of iNOS transcription on treatment with IFN-gamma pl us LPS in nonmetastatic cells but not in a metastatic line. Transcription f actors IFN regulatory factor 1 (IRF-1) and NF-kappa B were induced and func tional in both metastatic and nonmetastatic K1735 lines treated with IFN-ga mma plus LPS, Furthermore, a reporter construct driven by the wild-type iNO S promoter was transcriptionally activated in both nonmetastatic and metast atic cells. The iNOS-inducible phenotype was dominant in somatic cell hybri ds generated by the fusion of nonmetastatic and metastatic cells, suggestin g that no inhibitors of iNOS expression are present in metastatic cells. We conclude that the selective block in iNOS transcription in metastatic K173 5 cells is likely due to an alteration in iNOS gene regulatory sequences. H owever, no such alteration was detected within the 1.7 kb iNOS promoter reg ion in metastatic cells.