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11-Deoxy-16,16-dimethyl PGE2 (DDM-PG[E.sub.2]) protects renal proximal tubule epithelial cells (LLC-P[K.sub.1) against the toxicity induced by 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. We have now determined the ability of DDM-PGE2 to protect against other renal toxicants and report that DDM-PG[E.sub.2] only protects against oncotic cell death, induced by [H.sub.2][O.sub.2], iodoacetamide, and TGHQ, but not against apoptotic cell death induced by cisplatin, mercuric chloride, or tumor necrosis factor-[alpha]. DDM-PG[E.sub.2]-mediated cytoprotection is associated with the upregulation of at least five proteins, including the major endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 (Grp78). To elucidate the role of Grp78 in oncotic cell death, we used LLC-P[K.sub.1] cells in which induction of grp78 expression was disrupted by stable expression of an antisense grp78 RNA (pkASgrp78). As anticipated, DDM-PG[E.sub.2] failed to induce Grp78 in pkASgrp78 cells, with a concomitant inability to provide cytoprotection. In contrast, DDM-PG[E.sub.2] induced Grp78 and afforded cytoprotection against [H.sub.2][O.sub.2], iodoacetamide, and TGHQ in empty vector transfected cells (pkNEO). These data suggest that Grp78 plays an essential role in DDM-PG[E.sub.2]-mediated cytoprotection. Moreover, TGHQ-induced p38 MAPK activation is disrupted under conditions of a compromised ER stress response in pkASgrp78 cells, which likely contributes to the loss of cytoprotection. Finally, using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we found that DDM-PG[E.sub.2] induced several proteins in pkNEO cells, but not in pkASgrp78 ceils, including retinol-binding protein, myosin light chain, and heat shock protein 27. The findings suggest that additional proteins may act in concert with Grp78 during DDM-PG[E.sub.2]-mediated cytoprotection against oncotic cell death. quinone-thioether; endoplasmic reticulum stress; heat shock protein 27; thromboxane receptor; proteomics

11-Deoxy-16,16-dimethyl PGE2 (DDM-PGE2) protects renal proximal tubule epithelial cells (LLC-PK1) against the toxicity induced by 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone. We have now determined the ability of DDM-PGE2 to protect against other renal toxicants and report that DDM-PGE2 only protects against oncotic cell death, induced by H2O2, iodoacetamide, and TGHQ, but not against apoptotic cell death induced by cisplatin, mercuric chloride, or tumor necrosis factor-α DDM-PGE2-mediated cytoprotection is associated with the upregulation of at least five proteins, including the major endoplasmic reticulum (ER) chaperone glucose-regulated protein 78 (Grp78). To elucidate the role of Grp78 in oncotic cell death, we used LLC-PK1 cells in which induction of grp78 expression was disrupted by stable expression of an antisense grp78 RNA (pkASgrp78). As anticipated, DDM-PGE2 failed to induce Grp78 in pkASgrp78 cells, with a concomitant inability to provide cytoprotection. In contrast, DDM-PGE2 induced Grp78 and afforded cytoprotection against H2O2, iodoacetamide, and TGHQ in empty vector transfected cells (pkNEO). These data suggest that Grp78 plays an essential role in DDM-PGE2-mediated cytoprotection. Moreover, TGHQ-induced p38 MAPK activation is disrupted under conditions of a compromised ER stress response in pkASgrp78 cells, which likely contributes to the loss of cytoprotection. Finally, using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we found that DDM-PGE2 induced several proteins in pkNEO cells, but not in pkASgrp78 cells, including retinol-binding protein, myosin light chain, and heat shock protein 27. The findings suggest that additional proteins may act in concert with Grp78 during DDM-PGE2-mediated cytoprotection against oncotic cell death. [ABSTRACT FROM AUTHOR]

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.

We previously reported a protein expression profiling experiment conducted on human pancreatic tissues using 2D gel electrophoresis and mass spectrometry. Here, 18 spots that were identified in the gel at molecular weights more than 10 kDa lower than database values are characterized. The matrix-assisted laser desorption/ionization mass spectrometry coverage is sufficient to identify the protein region present in each spot. Most of the fragments correspond to processed chains and known structural or functional domains, which may result from limited proteolysis.