calcimycin and Urinary-Bladder-Neoplasms

Our laboratory demonstrated that hyperhomocysteinemia accelerates atherosclerosis in mouse models through ER stress and activation of the unfolded protein response (UPR). In this study, we tested the hypothesis that homocysteine-induced ER stress may arise from ER-Ca(2+) disequilibria. We found that homocysteine-induced cytosolic Ca(2+) transients in T24/83 cells and human aortic smooth muscle cells (HASMCs). These calcium effects occurred at concentrations of homocysteine in the external medium (1-5 mM) that increase intracellular homocysteine in these cell types. Prolonged homocysteine treatment (5 h) at these exogenous concentrations reduced ER-Ca(2+) emptying evoked by thapsigargin. However, these homocysteine-induced effects on ER-Ca(2+) emptying were of a much smaller magnitude than those evoked by A23187 or thapsigargin (ER stressors known to induce ER stress through ER-Ca(2+) depletion). T24/83 cells stably overexpressing the Ca(2+)-binding ER chaperone GRP78 showed diminished cytosolic Ca(2+) transients induced by homocysteine and reduced ER-Ca(2+) emptying evoked by thapsigargin. Prevention of the homocysteine-induced UPR by cycloheximide pretreatment normalized GRP78 expression and ER-Ca(2+) emptying evoked by thapsigargin. These results are inconsistent with a mechanism of ER stress induction by homocysteine through ER-Ca(2+) depletion.

Topics: Aorta; Calcimycin; Calcium; Cell Line, Tumor; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Fura-2; Heat-Shock Proteins; Homocysteine; Humans; Ionophores; Molecular Chaperones; Muscle, Smooth, Vascular; Oxidative Stress; Thapsigargin; Time Factors; Urinary Bladder Neoplasms

Interleukin 1 alpha (IL-1 alpha) is synthesized as a 33 kDa form and proteolytically processed into a 17 kDa form. Although IL-1 alpha has no signal peptide, it is released from cells. To investigate the relationship between the processing and release of IL-1 alpha, human bladder carcinoma cells (HTB9 5637) which express IL-1 alpha constitutively, were treated with calcium ionophore (A23187). A23187 induced the processing of 33 kDa IL-1 alpha and selectively released processed 17 kDa IL-1 alpha, without any change in the release of 33 kDa IL-1 alpha. When extracellular calcium was chelated by EGTA, or when intracellular calpain was inhibited by the cell-permeable cysteine-protease inhibitor, E64d, the processing of 33 kDa IL-1 alpha was significantly blocked, the release of 33 kDa IL-1 alpha being unchanged. These results indicate that the release of IL-1 alpha was accompanied by the processing of 33 kDa IL-1 alpha.

Topics: Calcimycin; Calcium; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Egtazic Acid; Gene Expression; Humans; Interleukin-1; L-Lactate Dehydrogenase; Leucine; Methionine; Molecular Weight; Protein Processing, Post-Translational; Sulfur Radioisotopes; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Prostaglandin (PG) H synthase and eicosanoid products of arachidonic acid metabolism have been implicated in several steps in the carcinogenic process. This study assessed these parameters using primary cultures of human urothelial cells. To determine the possible presence of permeability barriers to agonist stimulation, incubations were performed with adherent cells in the presence or absence of thioglycolate pretreatment or with cell suspensions. No evidence for permeability barriers was observed. With adherent cells in the absence of thioglycolate, radioimmunoassayable PGE2 was stimulated by epinephrine less than 12-O-tetradecanoylphorbol-13-acetate = thrombin less than bradykinin = A23187 much less than arachidonic acid. Tumor promoters but not non-tumor promoters stimulated PGE2 synthesis. 1-Oleoyl-2-acetylglycerol which like 12-O-tetradecanoylphorbol-13-acetate activates protein kinase C also increased PGE2 synthesis. Cells prelabeled with [14C]arachidonic acid were exposed to agonists and the profile of eicosanoids synthesized was assessed by high performance liquid chromatography. With bradykinin, the pattern of eicosanoids synthesized was 6-keto-PGE1 alpha (12% of total 14C label), thromboxane B2 (0.4%), PGF2 alpha (1.7%), PGE2 (18%), PGD2 (1%), leukotrienes (0.4 to 1%), 12-hydroxy-5,8,10-heptadecatrienoic acid (3%), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (4%), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (0%) and 5-hydroxy-5,8,12,14-eicosatetraenoic acid (2%). Thus, human urothelial cells contain both prostaglandin H synthase and lipoxygenase pathways with the former being more prominent. These pathways may participate in urinary bladder carcinogenesis.

Topics: Arachidonic Acids; Bradykinin; Calcimycin; Carcinogens; Dinoprostone; Epinephrine; Epithelium; Humans; Prostaglandins E; Tetradecanoylphorbol Acetate; Thrombin; Ureter; Urinary Bladder Neoplasms