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.
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