INSULIN-RECEPTOR SUBSTRATE-1 ANTISENSE EXPRESSION IN AN HEPATOMA-CELLLINE REDUCES CELL-PROLIFERATION AND INDUCES OVEREXPRESSION OF THE SRCHOMOLOGY-2 DOMAIN AND COLLAGEN PROTEIN (SHC)

In mammalian cells, the insulin receptor substrate 1 protein (IRS-1) i s a specific substrate for insulin and IGF-1 receptor tyrosine kinases which is involved in mediating metabolic and mitogenic actions of ins ulin and IGFs. In order to determine if IRS-1 is also essential in a c hicken derived hepatoma cell line (LMH cells), IRS-1 gene has been inv alidated in these cells. For this, we subcloned chicken IRS-1 gene in an antisense orientation into a mammalian expression vector driven by the cytomegalovirus early promoter. LMH cells were stably transfected with this construct or with the empty vector carrying only the neomyci n resistance gene and selected for cIRS-1 expression. One subclone, C2 , showed a complete repression of cIRS-1 expression at both protein an d mRNA levels. Proliferation of C2 cells was dramatically reduced (54% ) compared with Neo(I) cells. Furthermore this reduction was accompani ed by a decrease in insulin-dependent [H-3]thymidine incorporation, in dicating a reduction in DNA synthesis. Insulin-dependent [U-C-14]gluco se incorporation into cellular lipids was also significantly reduced i n C2 cell line suggesting an alteration in lipogenesis. In wild type L MH cells, SHC which is involved in Pas pathway, also served as a subst rate for insulin receptor tyrosine kinase. In C2 cells, SHC expression , its association with the insulin receptor and its tyrosine phosphory lation were largely increased. Two forms of the regulatory subunit of PI 3-kinase were present: p85 and p55 forms. Furthermore, C2 cells dis played increased basal phosphatidylinositol (PI) 3'-kinase activity. T his report demonstrates a role for cIRS-1 in the metabolic and mitogen ic actions of insulin in LMH cells. However, the overexpression of cIR S-1 antisense did not completely abolish cell proliferation. This may be explained by the exacerbation of an alternative pathway that only p artly compensate for the knocking out of cIRS-1 gene: the overexpressi on of SHC. (C) 1998 Published by Elsevier Science Ireland Ltd. All rig hts reserved.