Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.
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PMID:G-proteins mediate intestinal chloride channel activation. 170 25

Three Carcinoembryonic Antigen (CEA) gene family members: CEA, Non-specific cross-reactive antigen 50/90 (NCA) and biliary glycoprotein (BGP) were expressed in the colon carcinoma cell lines LS174T and HT29. The CEA, NCA50/90 and four alternatively spliced BGP transcripts (BGP a-d) were identified. The molecular weights of the mature glycoproteins were: CEA, 180kD; NCA50/90, 70-100 kD; BGP, 85, 120 and 140 kD. Pulse chase experiments demonstrated that CEA first appears as a 165 kD high mannose precursor which is trimmed to a 160 kD intermediate and finally transformed into the mature 180 kD glycoprotein. The precursor form of NCA had a molecular weight of 50 kD. CEA and NCA50/90, but not BGP, were linked to the cell membrane via glycosyl phosphatidyl inositol and could be released from the intact tumor cells by glycosyl phosphatidyl inositol-specific phospholipase C. CEA on isolated membranes and in cell lysates, but not on intact cells, was also cleaved by fresh human serum or purified glycosyl phosphatidyl inositol-specific phospholipase D.
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PMID:Biosynthesis of carcinoembryonic antigen (CEA) gene family members expressed in human tumor cell lines: evidence for cleavage of the glycosyl phosphatidyl inositol (GPI) anchor by GPI-PLC and GPI-PLD. 181 6

Monoclonal antibody (MAb) D612 recognizes an antigen expressed on the cell surface of normal and malignant gastrointestinal epithelium. It is a murine IgG2a/kappa which has been previously shown to mediate killing of human colon carcinoma cells using human effector cells (which could be enhanced in the presence of interleukin-2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of MAb D612 immunoprecipitates of extracts of L-[35S]methionine-, L-[3H]leucine-, and D-[3H]glucosamine-labeled human colon carcinoma cells showed that the D612 antigen is a Mr 48,000 glycoprotein. Similar estimates of molecular mass were obtained from SDS-PAGE analyses of MAb D612 immuno-precipitates of radioiodinated extracts of surgically resected colon carcinoma and adjacent normal colonic mucosa. D612 antigen was not detectable in immunoprecipitates of supernatant media from radiolabeled cell cultures, suggesting that the antigen is not readily shed from the surface of cultured cells. The D612 antigen was shown to be clearly distinct from previously described gastrointestinal carcinoma-associated glycoproteins: the D612 antigen shows a migration pattern of SDS-PAGE distinct from those of the antigens recognized by MAbs KS1/4 and GA733, and reciprocal immunodepletion analyses of D-[3H]glucosamine-labeled colon carcinoma cells utilizing MAbs D612 and GA733 revealed no cross-reactivity between these antibodies. Similarly, competitive binding studies between MAbs 17-1A and KS1/4 and MAb D612 revealed no similarity between the epitopes recognized by MAb D612 and MAbs 17-1A and KS1/4. MAbs D612 and 17-1A were also titered in immunoperoxidase staining assays on serial frozen sections of normal and malignant colon. MAb D612 showed a higher titer of immunostaining reactivity with both normal and malignant colon than did MAb 17-1A. MAb D612 showed roughly equivalent immunostaining titers against normal and malignant colon; whereas MAb 17-1A showed higher titer of immunostaining reactivity against the normal colon tissue than against the malignant colon. Flow cytometric analysis of phosphatidylinositol-specific phospholipase C-treated colon carcinoma cells revealed no loss of D612 antigen from the cell surface, suggesting that the mechanisms of attachment of the D612 antigen to the cell surface does not involve linkage to a phosphatidylinositol glycan.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the colorectal carcinoma-associated antigen defined by monoclonal antibody D612. 198 33

The levels of expression of phosphoinositide-specific phospholipase Cs (PLCs) were examined in a series of primary human colon carcinomas and in eight colon carcinoma cell lines by using monoclonal antibodies and cDNA probes for PLC gamma 1, PLC beta 1, and PLC delta 1. Western and northern blot analyses of PLC gamma 1 revealed elevated expression of this isozyme at both the protein and mRNA levels in most tumors when compared with paired adjacent normal mucosa samples (in 11 of 13 pairs in the western blots and 8 of 9 pairs in the northern blots). On the other hand, decreased levels of the PLC delta 1 protein were seen in most colon carcinomas (12 of 13 paired samples). The levels of PLC beta 1 protein were too low to detect possible differences between the carcinoma and normal mucosa samples. Relatively high expression of PLC gamma 1 was found in almost all of the eight human colon carcinoma cell lines at both the protein and mRNA levels. Only weak expression of PLC beta 1 was detected in these cell lines, by both western and northern blot analyses, and PLC delta 1 protein was not detected in any of the carcinoma cell lines. These findings provide evidence that colon carcinomas display altered expression of individual isoforms of PLCs and suggest that increased expression of PLC gamma 1 may play an important role in colon carcinogenesis.
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PMID:Expression of phospholipases gamma 1, beta 1, and delta 1 in primary human colon carcinomas and colon carcinoma cell lines. 789 68

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-114 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 from other cells (P < 0.001), reaching 620 ng/10(7) cells (approximately 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 (approximately 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 PI-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" because 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.
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PMID:Release of carcinoembryonic antigen from human tumor cells by phosphatidylinositol-specific phospholipase C: highly effective extraction and upregulation from LS-174T colonic adenocarcinoma cells. 821 92

The large clostridial cytotoxins toxin A and toxin B from Clostridium difficile are major virulence factors known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Both toxins mono-glucosylate and thereby inactivate small GTPases of the Rho family. Recently, it was reported that toxin B, but not toxin A, induces pore formation in membranes of target cells under acidic conditions. Here, we reassessed data on pore formation of toxin A in cells derived from human colon carcinoma. Treatment of 86Rb+-loaded cells with native or recombinant toxin A resulted in an increased efflux of radioactive cations induced by an acidic pulse. The efficacy of pore formation was dependent on membrane cholesterol, since cholesterol depletion of membranes with methyl-beta-cyclodextrin inhibited 86Rb+ efflux, and cholesterol repletion reconstituted pore-forming activity of toxin A. Similar results were obtained with toxin B. Consistently, methyl-beta-cyclodextrin treatment delayed intoxication of cells in a concentration-dependent manner. In black lipid membranes, toxin A induced ion-permeable pores only in cholesterol containing bilayers and at low pH. In contrast, release of glycosylphosphatidylinositol-anchored structures by phosphatidylinositol specific phospholipase C treatment did not reduce cell sensitivity toward toxins A and B. These data indicate that in colonic cells toxin A induces pore formation in an acidic environment (e.g. endosomes) similar to that reported for toxin B and suggest that pore formation by clostridial glucosylating toxins depends on the presence of cholesterol.
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PMID:Cholesterol-dependent pore formation of Clostridium difficile toxin A. 1651 41

Initiation of cell growth and neoplastic transformation frequently involves activation of growth factor receptor-coupled tyrosine kinases and stimulation of the phosphoinositide second messenger system. Altered expression of CD44 variants was reported in several malignant tumor types with possible implications for tumor progression and prognosis. CD44 variant expression was reported to be associated with second messenger activation and differentiation. We therefore investigated the effects of butyrate-induced short-term differentiation on phosphoinositide signaling, phospholipase C and protein kinase C activity and alteration of CD44 variant expression in human HT-29 colon carcinoma cells. HT-29 cells were cultured with sodium butyrate for 6 days. Phosphoinositide turnover was measured by [32P]orthophosphate incorporation and phospholipase C activity by determination of the release of [3H]inositolphosphates from [3H]myoinositol prelabeled cells. Protein kinase C activity was determined by histone III-S phosphorylation, PKC subtype expression by RNase protection analysis, and CD44 variant expression was determined by RT-PCR using variant-specific primers. Treatment of HT-29 human colon carcinoma cells with sodium butyrate caused a dose-dependent inhibition of cell proliferation (IC50, 2.5 mM) with morphologic signs of an enterocytic differentiation following 6 days of treatment. The phosphoinositide turnover as determined by 32P-incorporation under non-equilibrium conditions showed a 30-40% inhibition of labeled phosphoinositides and phosphatidic acid and a dose-dependent inhibition of cholinergically stimulated phospholipase C activity as a secondary event following butyrate-induced enterocytic differentiation. However, long-term incubation of HT-29 cells with phorbol ester or an inhibitor of classical and novel PKC subtypes did not affect cell proliferation. In butyrate-treated HT-29 cells activation of calcium-dependent protein kinase C by cholinergic stimulation or phorbolester treatment induced an increase in membrane-bound cPKC activity, while expression of distinct high- molecular CD44 variant transcripts v3 (670 bp), v5 (940 bp) and v8 (535 bp) were drastically reduced after butyrate pretreatment. Enterocytic differentiation of HT-29 colon carcinoma cells seems to be associated with alterations in phosphoinositide resynthesis, phospholipase C activity and ligand/receptor-induced PKC translocation. The observed reduction of distinct high-molecular CD44v3, v5 and v8 variants following butyrate-induced differentiation indicates an association of specific CD44 variant expression with the malignant phenotype of HT-29 colon cancer cells, thus being possible targets for new diagnostic and therapeutic strategies.
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PMID:Butyrate-induced alterations of phosphoinositide metabolism, protein kinase C activity and reduced CD44 variant expression in HT-29 colon cancer cells. 1936 Mar 23

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