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Under serum-free culture conditions rapamycin causes G1 phase accumulation and p53- independent apoptosis in rhabdomyosarcoma cells. Rapamycin-induced apoptosis is prevented by exogenous IGF-I through a signaling pathway independent of Ras-Erk-1/2 and Akt (Thimmaiah et al. Cancer Res. 63:364-74, 2003). Studies were extended to explore the involvement of the pro-apoptotic protein Bad in rapamycin-induced cell killing, and IGF-I mediated rescue. IGF-I treatment resulted in rapid and sustained phosphorylation of Bad predominantly on Ser 112 and to a lesser extent on Ser 155, resulting in the association of Bad with 14-3-3 γ. To determine whether Bad phosphorylation is required for IGF-I induced survival signaling, Rh1 cells overexpressing pEGFP-N1 (control), pEGFP-Bad (wild-type) or pEGFP-Bad (S112A) mutant were tested. Exogenous expression of wild-type Bad or Bad S112A mutant decreased the viability considerably without or with treatment with rapamycin. IGF-I protected against rapamycin-induced apoptosis in cells expressing pEGFP-Bad but was less effective in rescuing cells expressing the Bad S112A mutant. These data suggest that IGF-I induced phosphorylation of Bad at Ser112 is important for protecting Rh1 cells from rapamycin-induced apoptosis. We have started to map the IGF-I signaling pathway leading to Ser112 phosphorylation using pharmacological and genetic approaches. Our preliminary results indicate IGF-I signaling to Bad requires activation of PI3K, PKC (PKC-μ /PKC-θ /PKC-ε) but not PKA or p90RSK. Supported by PHS awards CA 77776, CA23099 and by ALSAC.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]