CLONING AND FUNCTIONAL EXPRESSION OF HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE (NOS) CDNA FROM A GLIOBLASTOMA CELL-LINE A-172

Nitric oxide (NO) is a messenger molecule with diverse functions throughout the body. The inducible type of nitric oxide synthase (NOS) is considered to be a key molecule in the immune responses to bacteria, parasites, and tumors, and its gene expression is regulated by cytokines. We isolated 3 overlapping partial inducible NOS cDNA clones from a human glioblastoma cell line A-172 induced by IL-1, TNF-alpha, and IFN-gamma. The 3,963-bp human glioblastoma inducible NOS cDNA contained the longest open reading frame of 3,459 bp, which encoded a polypeptide of 1,153 amino acids with a calculated molecular mass of 131 kDa. This human inducible NOS possessed consensus recognition sites for the cofactors FMN, FAD, and NADPH and calmodulin recognition sites, and displayed 48.1% sequence identity with the endothelial type, 43.1% with the neuronal type, and 99.3% with the inducible type from hepatocytes, and 99.9% with the inducible type from chondrocytes and adenocarcinoma. An expression plasmid consisting of pSG5 expression vector and cDNA containing the entire putative coding sequence was constructed and transfected into COS-1 cells. COS-1 cells showed nitric oxide synthase activity together with a 130 kDa immunoreactive band on Western blot analysis.