Effect of Interferon-r on Fas-mediated Apoptosis of Colon Cancer Cell Line HT-29

Kang, Jin Kyung; Park, In Suh; Kim, Won Ho; Ha, Sung Ho

Abstract

BACKGROUND/AIMS: Recent evidence suggests that alterations in regulation of apoptosis contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, degenerative diseases and inflammatory diseases. Fas antigen(APO-1, CD95) is a cell surface receptor protein that is broadly expressed in normal and neoplastic cells and can mediate apoptosis in susceptible cells. Fas is involved in immune-related apoptosis including T-cell selection in thymus, down regulation of immune response and cytotoxic T-cell mediated cytotoxicity. In contrast to immune system, little is known about the function of Fas antigen expressed on epithelial cells. We, therefore, studied the functional role of Fas in apoptosis of colon cancer cell line HT-29.
METHODS
Cell surface Fas expression was measured by flow cytometry using IgM anti-Fas monoclonal antibody(CH-11). Fas mRNA expression was measured by RT-PCR. Cytotoxicity and cell survival were assessed by LDH assay and MTT assay, respectively. Apoptosis was detected by confocal microscopic observation of chromatin condensation after DAPI stain and confirrned by dernonstration of DNA fragmentation in agarose gel electrophoresis as well as .TUNEL assay. DNA content was determined by flow cytometry after staining with propidium iodide and sub-Gl peak was considered as apoptotic cells.
RESULTS
Twenty to thirty percent of control HT-29 expressed Fas antigen on their surface. Nevertheless, Fas ligation by IgM anti-Fas monoclonal antibody(CH-11) failed to induce apoptosis in control HT-29. Fas protein as well as Fas mRNA expression was enhanced by IFN-y. In addition, Fas ligation in IFN-y pretreated HT-29 induced apoptosis dose-dependently. Cycloheximide and actinomycin D induced apoptosis in IFN-y pretreated HT-29, whereas they failed to induce apoptosis independently.
CONCLUSIONS
Fas antigen expressed on the surface of colon cancer cell line HT-29 is not sufficient to induce apoptosis. Cellular activation by IFN-y not only enhances Fas expression but also sensitizes HT-29 to Fas-mediated apoptosis. Apoptosis inducing effect of IFN-p pretreatment is complexly mediated by enhancing Fas expression as well as other mechanism yet undetermined.