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Establishment and characterization of a Fas-resistant T cell line
M Higuchi1, T Horiuchi, T Sawabe
1First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
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.
Insights
A new human T cell variant, kit-225-FR, is resistant to anti-Fas monoclonal antibody (mAb) but retains Fas expression. This resistance indicates a defect in the Fas apoptotic signaling pathway downstream of FADD.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Fas is a crucial cell surface receptor regulating apoptosis.
- Understanding Fas-mediated apoptosis is vital for immunology and disease research.
Purpose of the Study:
- To investigate the molecular mechanisms underlying resistance to anti-Fas monoclonal antibody (mAb).
- To characterize a novel anti-Fas mAb-resistant human T cell variant, kit-225-FR.
Main Methods:
- Flow cytometry to assess Fas expression.
- Reverse transcription polymerase chain reaction (PCR) and PCR-single strand conformation polymorphism analysis to examine Fas gene and transcript.
- Western blot analysis to evaluate MORT1/FADD expression.
- Apoptosis induction assays using anti-Fas mAb and C2-ceramide.
Main Results:
- The kit-225-FR variant exhibited preserved Fas molecule expression.
- No defects were found in the Fas transcript or gene in kit-225-FR.
- Apoptosis could still be induced by C2-ceramide, despite resistance to anti-Fas mAb.
- MORT1/FADD expression was at wild-type levels.
Conclusions:
- The apoptotic signaling pathway in kit-225-FR is defective between FADD and the sphingomyelin-ceramide pathway.
- The kit-225-FR cell line is a valuable tool for detailed analysis of Fas-specific signal transduction.