Release of Carcinoembryonic Antigen from Human Tumor Cells by Phosphatidylinositol-Specific Phospholipase C: Highly Effective Extraction and Upregulation from LS-174T Colonic Adenocarcinoma Cells

Abstract

Carcinoembryonic antigen (CEA), produced by gastrointestinal tumor cells, is anchored to cell membrane by a glycosyl-phosphatidylinositol moiety which can be cleaved with phosphatidylinositol-specific phospholipase C (PI-PLC). We studied the extraction of CEA from living human colon carcinoma (LS-174T, HT-29, COLO-205, and HRT-18) and pancreatic carcinoma (CAPAN) cells by PI-PLC from Bacillus cereus. The total CEA content of LS-174T cells, quantitated by Triton X-1 14 extraction followed by radioimmunoassay or by immunohistochemistry, was 3.5-fold higher than that of other cells ( P < 0.001). The spontaneous release of CEA from LS-174T cells into culture medium was also higher than that from other cells ( P < 0.00 1), reaching 620 ng/107 cells (≈28% of cellular content) after 24 h. Overall, living LS-174T cells were highly susceptible to CEA extraction by PI-PLC, which was dependent on PI-PLC dose and on treatment time, leading in optimal conditions to the solubilization of 4100 ng/10 7 cells after 24 h (≈75% of total CEA). After 24 h treatment at the highest PI-PLC dose, cell lines remained viable and growing, and membrane CEA expression was not exhausted but only reduced as compared to untreated cells. At the same time, the amount of CEA solubilized by P1-PLC exceeded the CEA reduction in membranes, suggesting that enzyme treatment increased CEA turnover. This was particularly true for LS- 174T cells which maintained 54% of the expression of untreated cells, whereas the amount of CEA extracted by PI-PLC reached 190% of this expression. Growing LS-174T cells thus constitute an effective material for producing high quantities of CEA by PI-PLC cleavage, especially since these cells probably "regenerate" be cause of enhanced turnover during PI-PLC action, thus allowing continuous CEA production. These experimental conditions also provide an interesting model for studying the modulation of CEA expression and release.