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A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10c12 CLA dose dependently decreased viable cell number (55 +/- 3% reduction 96 h after adding 5 microM t10c12 CLA). t10c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight (M(r)) IGF-II and higher M(r) forms of IGF-II. The levels of the higher M(r) and the mature form of IGF-II were decreased 50 +/- 3% and 22 +/- 2%, respectively, by 5 microM t10c12 CLA. c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using 125I-labeled IGF-II revealed that t10c12 CLA slightly decreased IGFBP-2 production; c9t11 CLA had no effect. Exogenous IGF-II reversed t10c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.

The human insulin-like growth factor II (IGF-II) gene constitutes a complex transcriptional unit that contains nine exons and four promoters. Expression of the IGF-II gene yields a family of mRNAs that all encode prepro-IGF-II. In addition, a stable 1.8 kb RNA is formed that is derived from the 3' untranslated region of exon 9. Recently, we have shown that this RNA species arises by site-specific endonucleolytic cleavage of IGF-II mRNAs and not by transcription from a separate promoter. In the present study we establish that two widely separated sequence elements of approximately 300 nucleotides, both located within exon 9, are required for this cleavage reaction. The first element encompasses about 200 nucleotides upstream and 100 nucleotides downstream of the cleavage site, while the second element is located within a region of 330 nucleotides about 2 kb upstream of the cleavage site. Interestingly, site-specific cleavage also occurred when a fragment from exon 9 of the IGF-II gene containing these two elements was inserted into the 3' untranslated part of the beta-globin gene. Apparently, the expressed hybrid beta-globin-IGF-II mRNA contains all the regulatory elements to confer site-specific endonucleolytic cleavage.

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