formazans and Urinary-Bladder-Neoplasms

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.

Topics: Antimetabolites, Antineoplastic; Blotting, Western; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Survival; Chromones; Cyclin-Dependent Kinase Inhibitor p27; Deoxycytidine; Drug Screening Assays, Antitumor; Elafin; Formazans; Gemcitabine; Gene Expression Regulation; Gene Silencing; Humans; Morpholines; RNA, Small Interfering; Spectrometry, Mass, Electrospray Ionization; Tetrazolium Salts; Ubiquitins; Urinary Bladder Neoplasms

Based on the granulocyte-macrophage colony-stimulating factor (GM-CSF) dependency of a newly established human myeloid cell line GM/SO, we developed a highly specific and sensitive bioassay for human GM-CSF. The presence of bioactive GM-CSF could be determined by measuring the formazan concentration produced from MTT by the cells that survived and proliferated in the presence of either natural or recombinant human GM-CSF. With this assay we were able to quantify the level of GM-CSF in two human sera as well as in conditioned media from human bladder cell carcinoma cell line 5637, a human fibroblast line, and phytohemagglutinin-stimulated peripheral blood mononuclear cells. The sensitivity of the assay allows measurement of concentrations of GM-CSF as low as 0.1 U/ml.

Topics: Biological Assay; Cell Division; Cell Survival; Culture Media; Cytokines; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Formazans; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytes, Mononuclear; Phytohemagglutinins; Recombinant Proteins; Tumor Cells, Cultured; Urinary Bladder Neoplasms