2-3-5-(triglutathion-s-yl)hydroquinone and Cell-Transformation--Neoplastic

Topics: Animals; Carcinogens; Carcinoma, Renal Cell; Cell Division; Cell Transformation, Neoplastic; Glutathione; Humans; Hydroquinones; Kidney; Kidney Neoplasms; Loss of Heterozygosity; Mutagens; Repressor Proteins; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2(+/+) and Tsc-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250) © 2015 Wiley Periodicals, Inc.

Topics: Animals; Carcinoma, Renal Cell; Cell Line; Cell Transformation, Neoplastic; Glutathione; Humans; Hydroquinones; Kidney Neoplasms; Male; Neoplasms, Experimental; Phosphorylation; Protein Domains; Protein Processing, Post-Translational; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; Rats; RNA Splicing; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase signaling cascades have been implicated in a number of human cancers. The tumor suppressor gene tuberous sclerosis-2 (Tsc-2) functions as a negative regulator of mTOR. Critical proteins in both pathways are activated following treatment of Eker rats (Tsc-2(EK/+)) with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ), which also results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. Western blot analysis of kidney tumors formed following treatment of Tsc-2(EK/+) rats with TGHQ for 8 months revealed increases in B-Raf, Raf-1, pERK, cyclin D1, 4EBP1, and p-4EBP1-Ser65, -Thr70, and -Thr37/46 expression. Similar changes are observed following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (quinol-thioether rat renal epithelial [QTRRE] cells) that are also null for tuberin. These cells exhibit high ERK, B-Raf, and Raf-1 kinase activity and increased expression of all p-4EBP1s and cyclin D1. Treatment of the QTRRE cells with the Raf kinase inhibitor, sorafenib, or the MEK1/2 kinase inhibitor, PD 98059, produced a significant decrease in the protein expression of all p-4EBP1s and cyclin D1. Following siRNA knockdown of Raf-1, Western blot analysis revealed a significant decrease in Raf-1, cyclin D1, and all p-4EBP1 forms noted above. In contrast, siRNA knockdown of B-Raf resulted in a nominal change in these proteins. The data indicate that Raf-1/MEK/ERK participates in crosstalk with 4EBP1, which represents a novel pathway interaction leading to increased protein synthesis, cell growth, and kidney tumor formation.

Topics: Animals; Carcinoma, Renal Cell; Carrier Proteins; Cell Culture Techniques; Cell Line; Cell Transformation, Neoplastic; Cyclin D1; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Hydroquinones; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kidney Neoplasms; Loss of Heterozygosity; Male; MAP Kinase Kinase Kinases; Phosphoproteins; Protein Biosynthesis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; Rats; Rats, Mutant Strains; Receptor Cross-Talk; RNA, Small Interfering; Signal Transduction; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Although hydroquinone (HQ) is a rodent carcinogen, because of its lack of mutagenicity in standard bacterial mutagenicity assays it is generally considered a nongenotoxic carcinogen. 2,3,5-Tris-(glutathion-S-yl)HQ (TGHQ) is a potent nephrotoxic metabolite of HQ that may play an important role in HQ-mediated nephrocarcinogenicity. TGHQ mediates cell injury by generating reactive oxygen species and covalently binding to tissue macromolecules. We determined the ability of HQ and TGHQ to induce cell transformation in primary renal epithelial cells derived from the Eker rat. Eker rats possess a germline inactivation of one allele of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene that predisposes the animals to renal cell carcinoma. Treatment of primary Eker rat renal epithelial cells with HQ (25 and 50 microM) or TGHQ (100 and 300 microM) induced 2- to 4-fold and 6- to 20-fold increases in cell transformation, respectively. Subsequently, three cell lines (The QT-RRE 1, 2, and 3) were established from TGHQ-induced transformed colonies. The QT-RRE cell lines exhibited a broad range of numerical cytogenetic alterations, loss of heterozygosity at the Tsc-2 gene locus, and loss of expression of tuberin, the protein encoded by the Tsc-2 gene. Only heterozygous (Tsc-2(EK/+)) kidney epithelial cells were susceptible to transformation by HQ and TGHQ, as wild-type cells (Tsc-2(+/+)) showed no increase in transformation frequency over background levels following chemical exposure. These data indicate that TGHQ and HQ are capable of directly transforming rat renal epithelial cells and that the Tsc-2 tumor suppressor gene is an important target of TGHQ-mediated renal epithelial cell transformation.

Topics: Animals; Blotting, Western; Carcinoma, Renal Cell; Cell Division; Cell Transformation, Neoplastic; Cytogenetic Analysis; DNA Primers; Epithelial Cells; Gene Silencing; Genes, Tumor Suppressor; Glutathione; Hydroquinones; Kidney; Kidney Neoplasms; Polymerase Chain Reaction; Rats; Rats, Mutant Strains; Repressor Proteins; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins