Purposes : When Insulin receptor substrae-1(IRS-1), major adaptor protein of Insulin growth factor receptor (IGF receptor), was regulated siRNA and okadaic acid (OA). We investigated the effects of IRS-1 phosphorylation, downstream signaling pathway of IGF-axis. AlsoWe tested effects on cell cycle.

Methods : Using Non-small cell lung cancer 460 and Cos7, we confirmed the effects on cell proliferation with OA treatment by using MTT assay. Anti IRS-1 treatment, siRNA, repressed IRS-1 protein, and the effects on phosphorylation and expressiom of protein stimulating OA and siRNA by using western blotting. Furthermore, the effects on cell cycle progression was studid flow cytometry.

Results :

1. Treatment of NCI-H 460 and Cos7 with OA induced dose-dependent cell proliferation until 5nM. This result exhibited increase about 20% compared control.

2. IRS-1 band intensity did not change but, pser 307IRS-1 was phosphorylated 4h after OA treatment, but not ptyr 632IRS-1. Namely, change in magnitude of phosphorylation of pser 307IRS-1 was not due to chages in IRS-1 protein level.

3. OA induced not AKT and GSK3β phosphorylation, but MAP kinase phosphotylation. Phosphorylation level of MAP kinase have no relevance to MAP kinase protein volume.

4. Anti IRS-1, siRNA, inhibited IRS-1 protein expression.

Because OA treatment and IRS-1 knockdown have influence on cell cycle, treatment of siRNA repressed entry from G0-G1 phase to S phase but OA- stimulated cells promoted cell cycle from S phase to G2-M phase, and treatment of them recovered cell cycle repression from S phase to G2-M phase.

Conclusion : OA-stimulated NCI-H460 and Cos7 induced pser 307IRS-1. By inducing pser 307IRS-1 in OA-stimulated cells, MAP kinase was activated, and induced proteins regulating cell cycle. So we confirmed OA induced entry from S phase to G2-M phase. In addition, regardless of IRS-1, OA prompted cell cycle from S phase to G2-M phase.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA