Eliminierung des Tumorcounterattack von Kolonkarzinomzellen über gentechnische Suppression der FAS vermittelten Apoptose durch FLIP-Überexpression in humanen T-Zellen

Abstract

Introduction: The FAS/FAS-Ligand-pathway has been recognized as an important mechanism to regulate cell haemostasis of immune competent cells by activation induced cell death (AICD). The expression of Fas-Ligand of tumour cells may enable them to induce apoptosis of activated T-cells wich are especially sensitive to the FAS modulated cell death. This so called tumour counterattack may also explain why tumour cells are capable to survive in the lymphatic and circulation system. Purpose: Our objective was to test the hypothesis that a gene-therapeutically controlled blocking of the AICD mechanism of the T-cells may protect them against tumour counterattack and furthermore re-establish their immune competence towards the tumour cells. Material and Methods: A FLIP transfection was carried out using co-transfection of pMCS LNGFR and pcDNA5FRT-FLIP with X-gene transfection solution. Transfected Jurkat-T-cells were separated from non-transfected cells by paramagnetic beads. FLIP-transfected Jurkat-T-cells and SW620-colon carcinoma cells were co-incubated over 24 h. T-cell survival was shown in a fluorescence reader (Genios, Tecan) by Resazurin-converting vital cells (Celltiter Blue, Promega). The apoptosis rate was quantified by a caspase activity assay (APO-ONE, Promega) and qualified by in situ Annexin V staining, controlled by non-transfected T-cells and T-cells transfected by a contol vector. Results: A FAS-induced tumour counterattack of SW620-colon carcinoma cells against Jurkat-T-cells after coincubation was confirmed in vitro. The positive transfection rate of the FLIP-transfected T-cells was 32 %. The apoptosis rate of the transfected T-cells decreased significantly after coincubation in comparison to the respective control groups. Conclusions: Our results show that a tumour counterattack against activated T-cells can be reduced by c-FLIP-transfection of the Jurkat-T-cells by a targeted deactivation of the AICD cascade. This regain of the immune competence of activated T-cells against the tumour cells may constitute a new and promising strategy of systematic tumour therapy.