lysophosphatidylinositol and Cell-Transformation--Neoplastic

Inositol-containing molecules are involved in important cellular functions, including signalling, membrane transport and secretion. Our interest is in lysophosphatidylinositol and the glycerophosphoinositols, which modulate cell proliferation and G-protein-dependent activities such as adenylyl cyclase and phospholipase A(2). To investigate the role of glycerophosphoinositol (GroPIns) in the modulation of Ras-dependent pathways and its correlation to Ras transformation, we employed a novel liquid chromatography-tandem mass spectrometry technique to directly measure GroPIns in cell extracts. The cellular levels of GroPIns in selected parental and Ras-transformed cells, and in some carcinoma cells, ranged from 44 to 925 microM, with no consistent correlation to Ras transformation across all cell lines. Moreover, the derived cellular inositol concentrations revealed a wide range ( approximately 150 microM to approximately 100 mM) under standard [(3)H]-inositol-loading, suggesting a complex relationship between the inositol pool and the phosphoinositides and their derivatives. We have investigated these pools under specific loading conditions, designing a further HPLC analysis for GroPIns, combined with mass determinations of cellular phosphatidylinositol 4,5-bisphosphate. The data demonstrate that limiting inositol conditions identify a preferred pathway of inositol incorporation and retention into the polyphosphoinositides pool. Thus, under conditions of increased metabolic activity, such as receptor stimulation or cellular transformation, the polyphosphoinositide levels will be maintained at the expense of phosphatidylinositol and the turnover of its aqueous derivatives.

Topics: Cell Transformation, Neoplastic; Chromatography, Liquid; Genes, ras; Humans; Inositol; Inositol Phosphates; Intracellular Fluid; Lysophospholipids; Mass Spectrometry; Tumor Cells, Cultured

Lysophosphatidylinositol (LysoPtdIns) is formed by a constitutively-active phosphoinositide-specific phospholipase A2 in Ras-transformed cells and can stimulate cell proliferation. To evaluate whether LysoPtdIns could function as an autocrine modulator of cell growth, we examined whether LysoPtdIns can be released in the medium of Ras-transformed FRT-Fibro fibroblasts and thyroid cells. Here, we report that LysoPtdIns accumulates in the extracellular space of these lines and reaches levels up to tenfold higher than in the case of normal cells. Moreover, the ionophore A23187 increased the levels of the lysolipid in the extracellular medium. Extracellular LysoPtdIns was rapidly hydrolyzed to inositol 1:2-cyclic phosphate. LysoPtdIns induced thymidine incorporation in FRT-Fibro Ha-Ras fibroblasts, whereas inositol cyclic 1:2-cyclic phosphate did not affect cell growth per se, nor did it interfere with the LysoPtdIns mitogenic activity. We hypothesize that in Ras-transformed fibroblasts the formation and release of LysoPtdIns may function as an autocrine mechanism that participates in the Ras-dependent stimulation of cell growth.

Topics: Animals; Autocrine Communication; Cell Division; Cell Transformation, Neoplastic; Fibroblasts; Genes, ras; Lysophospholipids; Mitogens; Rats; Thyroid Gland

In cell lines stably (KiKi) or reversibly (Ts) transformed by the k-ras oncogene originated from a differentiated rat thyroid line (FRTL5 cells), k-ras-induced transformation has been associated with an increased phospholipase A2 activity. Here we provide evidence that this enzymic activity is phosphoinositide specific and leads to the formation of lysophosphatidylinositol. The levels of this lysolipid increased by 2-3-fold in ras-transformed cells (KiKi cells and Ts cells at the permissive temperature of 33 degrees C) as compared to differentiated cells (FRTL5) or to Ts cells maintained at 39 degrees C, i.e. at the temperature where ras-p21, the product of the ras oncogene, is inactive. Since another lysoderivative, lysophosphatidic acid, has been shown to be a mitogen, we have tested whether lysophosphatidylinositol could have a similar activity on thyroid cells. Lysophosphatidylinositol (10-100 microM) induced a 5-10-fold increase in [3H]thymidine incorporation in both FRTL5 and KiKi cells, whereas lysophosphatidic acid was active only in differentiated cells. Lysophosphatidylinositol (approximately 25 microM) and lysophosphatidic acid (50-100 microM) acted synergistically with insulin in increasing [3H]thymidine incorporation. Moreover, lysophosphatidylinositol at concentrations three-fold higher than those found to be mitogenic, inhibited the activity of the GTPase-activating protein. We conclude that lysophosphatidylinositol is a mitogen that might play a role in the modulation of k-ras transformed cell proliferation.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA; Drug Synergism; Epithelium; Genes, ras; GTPase-Activating Proteins; Insulin; Lysophospholipids; Proteins; ras GTPase-Activating Proteins; Rats; Temperature; Thyroid Gland; Thyrotropin