bromochloroacetic-acid and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Kidney stone disease is associated with renal fibrosis by the unclear mechanisms. We hypothesized that calcium oxalate (CaOx), a major crystalline component of kidney stones, could induce secretion of fibrotic factors from macrophages leading to "epithelial mesenchymal transition/transdifferentiation" (EMT) of renal tubular cells. Western blot analysis revealed an increased level of vimentin (mesenchymal marker) but decreased levels of E-cadherin and cytokeratin (epithelial markers) in MDCK cells treated with "secreted products from CaOx-exposed macrophages" (CaOx-M-Sup). Immunofluorescence study confirmed the increased level of vimentin and decreased level of cytokeratin, and also revealed the increased level of fibronectin (another mesenchymal marker). The data also showed decreased levels and disorganization of F-actin (cytoskeletal marker) and zonula occludens-1 (ZO-1) (tight junction marker) induced by CaOx-M-Sup. ELISA demonstrated the increased level of transforming growth factor-β1 (TGF-β1), the well-defined EMT inducer, in CaOx-M-Sup. Downstream signaling of TGF-β1 was involved as demonstrated by the decreased level of RhoA. Interestingly, pretreatment with a proteasome inhibitor (MG132) could restore RhoA to its basal level, most likely through ubiquitin-proteasome pathway (UPP). Moreover, MG132 successfully sustained cytoskeletal assembly and tight junction, and could prevent the cells from EMT. Altogether, these data demonstrate for the first time that CaOx-M-Sup could induce EMT in renal tubular cells by TGF-β1 signaling cascade via RhoA and UPP. This may be, at least in part, the underlying mechanism for renal fibrosis in kidney stone disease.

Topics: Animals; Cadherins; Calcium Oxalate; Cell Line; Cysteine Proteinase Inhibitors; Dogs; Epithelial-Mesenchymal Transition; Humans; Keratins; Kidney Tubules; Leupeptins; Macrophages; Madin Darby Canine Kidney Cells; Proteasome Endopeptidase Complex; rhoA GTP-Binding Protein; Signal Transduction; Transforming Growth Factor beta1; Ubiquitin; Vimentin; Zonula Occludens-1 Protein

To investigate the role of Epstein-Barr virus (EBV) in epithelial tumors, we compared the expression pattern of cellular genes in the EBV-infected gastric carcinoma cell line, NU-GC-3, and its uninfected control. Subtractive suppression hybridization (SSH) was combined with high-density DNA array screening to identify differentially expressed genes. We have discovered that EBV infection upregulated a truncated variant of human basic hair keratin 1 (hHb1-DeltaN), a gene that had previously been identified in metastatic breast carcinoma. We verified the differential expression of hHb1-DeltaN in 3 independent EBV-positive and -negative NU-GC-3 clones by Northern blotting. We further verified the EBV-dependent upregulation of hHb1-DeltaN in 3 other carcinoma cell lines (AGS, TWO3 and DLD1) by RT-PCR. Inhibition of CpG methylation by 5-Aza-CdR induced hHb1-DeltaN mRNA expression in the EBV-negative clones but did not alter the expression in the EBV-positive clones. The expression of hHb1-DeltaN protein was detectable by immunofluorescence and Western blotting in EBV-positive but not in EBV-negative NU-GC-3 clones after proteasome inhibitor (MG132) treatment. hHb1-DeltaN protein formed fibrous structures in the cytoplasm and accumulated in distinct nuclear bodies in the euchromatic areas of the cell nucleus. We suggest that the unstable hHb1-DeltaN protein may inhibit some of the functions of the keratin cytoskeleton and/or interfere with transcription regulation. It also may establish a link between EBV and the low differentiated or anaplastic status of the carcinomas that carry the virus.

Topics: Azacitidine; Blotting, Northern; Blotting, Western; CpG Islands; Cysteine Proteinase Inhibitors; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Humans; Keratins; Leupeptins; Oligonucleotide Array Sequence Analysis; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms; Tumor Cells, Cultured; Up-Regulation

Quinidine inhibits proliferation and promotes cellular differentiation in human breast tumor epithelial cells. Previously we showed quinidine arrested MCF-7 cells in G(1) phase of the cell cycle and led to a G(1) to G(0) transition followed by apoptotic cell death. The present experiments demonstrated that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine. The cells accumulated lipid droplets, and the cytokeratin 18 cytoskeleton was reorganized. Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h. Quinidine-treated MCF-7 cells showed elevated p21(WAF1), hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1, similar to the cell cycle response observed with cells induced to differentiate by histone deacetylase inhibitors, trichostatin A, and trapoxin. Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro. HDAC1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium. The proteasome inhibitors MG-132 and lactacystin completely protected HDAC1 from the action of quinidine. We conclude that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC1 via a proteasomal sensitive mechanism.

Topics: Acetylation; Acetylcysteine; Animals; Anti-Bacterial Agents; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Division; Chickens; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeleton; Down-Regulation; Enzyme Inhibitors; Female; G1 Phase; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immunoblotting; Keratins; Leupeptins; Multienzyme Complexes; Peptides; Phosphorylation; Proteasome Endopeptidase Complex; Quinidine; Retinoblastoma Protein; Time Factors; Tumor Cells, Cultured