Decreased sensitivity to 1-O-octadecyl-2-O-methlyl-glycerophosphocholine in MCF-7 cells adapted for serum-free growth correlates with constitutive association of Raf-1 with cellular membranes

We have previously shown that inhibition of MCF-7 cell proliferation by 1-O -octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OCH3) is linked to a dru g-induced decrease in membrane Raf-1 levels and the subsequent inhibition o f mitogen-activated protein (MAP) kinase activation in response to growth f actor stimulation. We now report that adaptation of MCF-7 cells for growth in a serum-free formulation results in decreased sensitivity to growth inhi bition by ET-18-OCH3. The decrease in ET-18-OCH3 sensitivity occurred progr essively during the adaptation process and correlated with the presence of increasing amounts of inactive Raf-1 that stably associated with MCF-7 cell membranes. ET-18-OCH3, sensitivity could be restored by growing the adapte d cells in serum-containing medium, which resulted in the loss of membrane- associated Raf-1. In human normal mammary epithelial cells, which are insen sitive to ET-18-OCH3 Raf-1 was also associated with membranes in quiescent cells. In both cell: types, incubation with ET-18-OCH3 had no effect on the membrane-Raf-1 levels, suggesting that ET-18-OCH3-induced reduction of Raf -1 levels in growth factor-stimulated MCF-7 cells is due to inhibition of R af translocation, The activation and termination of the MAP kinase pathway in response to growth factors in the adapted MCF-7 cells and HNME cells occ urred without changes to membrane Raf-1 levels, Because membrane translocat ion is not required to activate Raf in these cells, inhibition of Raf trans location by ET-18-OCH3 subsequent to cell stimulation has no effect on the activation of the membrane-bound Raf and, consequently, the activation of t he MAP kinase pathway, The ability of the cells to activate the MAP kinase pathway in the presence of the drugs enables them to resist the growth-inhi bitory effects of the drug, leading to the observed ET-18-OCH3 insensitivit y of the cells.