Establishment and Characterization of a Fas-Resistant T Cell Line

Abstract

Fas is a cell surface receptor that controls a signal transduction pathway leading to apoptosis. We established an antihuman Fas monoclonal antibody (mAb)-resistant variant, kit-225-FR, from the human T cell line, kit-225. Flow cytometric analysis revealed that the expression of Fas molecules on kit-225-FR was preserved. The defect in Fas molecule was not detected either by reverse transcription polymerase chain reaction (PCR) analysis of the Fas transcript or by PCR-single strand conformation polymorphism analysis of the Fas gene in kit-225-FR. Although kit-225-FR was resistant to a high concentration of anti-Fas mAb, apoptosis could be induced, as with the wild type, by exogenous C2-ceramide exposure. MORT1/FADD was expressed at wild-type level in kit-225-FR, as determined by Western blot analysis. It therefore appears that the apoptotic signal transduction in kit-225-FR is defective between FADD and the sphingomyelin-ceramide pathway. By comparing the differences from the wild-type kit-225, kit-225-FR would serve as a useful cell line for analyzing Fas-specific signal transduction pathways in detail.