HUMAN MONOCYTE CHEMOTACTIC PROTEIN-2 - CDNA CLONING AND REGULATED EXPRESSION OF MESSENGER-RNA IN MESENCHYMAL CELLS

Stimulated MG-68 osteosarcoma cells have been used as a source to puri fy and identify the monocyte chemokines MCP-1, MCP-2 and MCP-3. in com parison with MCP-1, the production yields of MCP-2 and MCP-3 in these cells are rather low and variable, Although the protein sequence of hu man MCP-2 is identified, its DNA sequence remains elusive, A degenerat e primer set was used to isolate an MCP-2 gene fragment from the chemo kine YAC contig on human chromosome 17, Based on the gene sequence of MCP-2, a unique primer set was synthesized and used to screen cDNA lib raries for the presence of MCP-2 transcripts by PCR. The complete MCP- 2 cDNA was cloned from a human bone marrow cDNA library and sequenced. The cDNA-derived protein sequence was identical to that of purified n atural MCP-2, except for Gln(46) which replaced Lys(46). There seem th us to exist two MCP-2 allelic variants because at position 46 the codo ns of two residues (Lys(46) and Gln(46)) were detected in individual g enomes. As shown by Northern hybridization, the MCP-2 steady-state mRN A levels in normal diploid fibroblasts were increased by IL-1 beta, IF N-gamma and the double-stranded RNA poly rI:rC, RT-PCR analysis showed induction of MCP-2 mRNA in MG-63 cells by IFN-gamma and IL-1 beta. Th e regulated production of MCP-2 by tumor cells and normal mesenchymal cells is indicative of a role in neoplasia and inflammatory host respo nses. (C) 1997 Academic Press.