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Selective Inhibition of Phosphatidylinositol Phospholipase C by Cytotoxic Ether Lipid Analogues¹

Department of Pharmacology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 [G. P., M. J. S., C. G., D. M., J. R.], and Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 [E. J. M.]

The ether lipid analogue 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) has been shown to be a direct inhibitor of Swiss 3T3 fibroblast and BG1 ovarian adenocarcinoma cell cytosolic phosphoinositide selective phospholipase C (PIPLC) using [³H]-phosphatidylinositol-(4, 5)-bisphosphate ([³H]PIP₂) as the substrate. The inhibition occurred when ET-18-OCH₃ was incorporated into the [³H]PIP₂ substrate micelles, with 50% inhibition (IC₅₀) occurring at a ET-18-OCH₃: [³H]PIP₂ ratio of 0.04, or an assay concentration of 0.4 µM, and when ET-18-OCH₃ was added directly to the incubation, with an IC₅₀ of 9.6 µM. Lipid prepared from cells exposed to cytotoxic concentrations of ET-18-OCH₃ for 18 h also inhibited PIPLC with an IC₅₀ < 1 µM. The noncytotoxic analogue 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine inhibited PIPLC when incorporated into the [³H]PIP₂ substrate micelles, but lipid from cells grown with 5 µM 1-O-alkyl-2-hydroxy-sn-glycero-3-phosphocholine did not inhibit PIPLC. BG1 cells, which were more sensitive than Swiss 3T3 fibroblasts to growth inhibition by ET-18-OCH₃, had a cytosolic PIPLC activity one-third that of Swiss 3T3 cells. NIH 3T3 cells exhibited the same sensitivity to growth inhibition by ET-18-OCH₃ as Swiss 3T3 cells and had a similar level of PIPLC. v-sis NIH 3T3 cells were relatively resistant (>3-fold) to growth inhibition by ET-18-OCH₃ and had a cytosolic PIPLC activity more than twice that of the wild type cells. ET-18-OCH₃ was a weak inhibitor, IC₅₀ >100 µM, of phospholipase D activity in NIH 3T3 cell membranes. In intact NIH 3T3 cells ET-18-OCH₃ at cytotoxic concentrations did not inhibit phospholipase D or phosphatidylcholine-selective phospholipase C activity. The results show that the ether lipid analogues at cytotoxic concentrations are selective inhibitors of PIPLC and that the inhibition of PIPLC may be related to the growth inhibitory activity of the ether lipid analogues.

¹ Supported by NIH Grants 42286 and 48725 (G. P.) and CA41314 (E. J. M.).

² To whom requests for reprints should be addressed, at Arizona Cancer Center, University of Arizona, 1501 North Campbell Ave., Tucson, AZ 85724.

Received 11/12/91. Accepted 3/ 9/92.

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