Cyclins D1, E, A expression and synergistic cytotoxicity to a cholangiocarcinoma cell line from recombinant TNF-alpha and PHA supernate.
We studied the cytotoxic effects of recombinant TNF-alpha and supernate of phytohemagglutinin stimulated peripheral blood mononuclear cells individually and in combination against a cholangiocarcinoma cell line. Levels of cyclins D1, E and A in the cell line were detected by immunoblotting, and the cell cycle stage was assayed by propidium iodide staining followed by flow cytometry analysis. Viable and apoptotic cells were assessed by trypan blue dye exclusion, DAPI staining, agarose DNA laddering and propidium iodide staining. At the beginning of each experiment, the majority of cholangiocarcinoma cells expressed cyclin A and were in S phase as determined by propidium iodide staining. Treatment of such cells with recombinant TNF-alpha resulted in cytotoxic effects clearly evident at 36 hours post exposure. There was a synergistic killing effect when recombinant TNF-alpha was combined with PHA supernate and this effect could be partly neutralized by monoclonal anti TNF-alpha, interleukin (IL)-2, IL-12 and IFN-gamma.
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Molvibha
M
Vongsakul
Ajchara
A
Aksomboon
D000328:Adult; D001650:Bile Duct Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism*; D001653:Bile Ducts, Intrahepatic / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism; D018281:Cholangiocarcinoma / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism*; D019925:Cyclin A / Q000096:biosynthesis* / Q000187:drug effects* / Q000276:immunology; D019938:Cyclin D1 / Q000096:biosynthesis* / Q000187:drug effects* / Q000276:immunology; D019927:Cyclin E / Q000096:biosynthesis* / Q000187:drug effects* / Q000276:immunology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects* / Q000276:immunology; D004198:Disease Susceptibility; D004359:Drug Therapy, Combination; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008297:Male; D010835:Phytohemagglutinins / Q000276:immunology / Q000627:therapeutic use*; D011994:Recombinant Proteins / Q000276:immunology / Q000627:therapeutic use*; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000627:therapeutic use*
D014408:Biomarkers, Tumor; D019925:Cyclin A; D019927:Cyclin E; D010835:Phytohemagglutinins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019938:Cyclin D1