Identifying the factors and signal pathways necessary for anchorage-independent growth of Ha-ras oncogene-transformed NIH/3T3 cells

Abstract

Ha-ras Val 12 overexpression was positively correlated with colony formation by NIH/3T3 derivative “2–12” cells harboring an inducible Ha-ras Val 12 transgene. The ras-farnesylation inhibitor, Lovastatin, completely suppressed colony formation at higher dosages. However, Ha-ras oncogene overexpression alone could not stimulate colony formation under serum-deprived conditions, suggesting that ras is required but not sufficient for supporting colony formation. Substituting cow colostrum (AC-2®) for serum did not result in colony formation from 2–12 cells in soft agar, suggesting the colostrum lacked or contained insufficient amounts of factors that stimulate colony formation. Supplementation of AC-2®-containing medium with growth factors, such as insulin-like growth factor-1 (IGF-1), partially restored the capability of anchorage-independent cell growth induced by Ha-ras overexpression. Consistently, antibodies specific for IGF-1 receptors only partially blocked colony formation from 2–12 cells. The data indicate that multiple factors, including IGF-1, are required for Ha-ras-dependent colony formation. Signal transduction studies revealed that, under Ha-ras overexpression conditions, IGF-1 utilizes phosphatidyl inositol 3-kinase and NF-κB to transduce colony formation-related signaling.