The mechanism of chemotherapeutic drug-induced apoptosis in leukaemic cells was studied to further investigate whether Fas/FasL system was involved in apoptosis induced by chemotherapeutic drugs and assess their effects when used in combination with soluble FasL (sFasL). The expression of Fas on human leukaemic cell lines K562, HL-60 and U937 treated with daunorubicin (DNR) or cytosine arabinoside (Ara-C) was detected by using flow cytometry. The activities of sFasL, DNR and Ara-C inducing apoptosis of leukaemic cells, in the absence or presence of neutralizing anti-Fas IgG antibody, were detected by using flow cytometry and TUNEL. The results showed that flow cytometric profiles of K562, HL-60 and U937 cells treated with DNR or Ara-C failed to show any significant increase in Fas expression over 18 h (P>0.05). Anti-Fas monoclonal antibody (IgG) could not block the apoptosis in leukaemic cells induced by DNR or Ara-C, but could block the apoptosis induced by sFasL. A role of sFasL in a cytotoxic synergistic effect when used in combination with chemotherapeutic drugs was revealed. It was concluded that chemotherapeutic drug-induced apoptosis in human leukaemic cells (UG37, HL-60) is independent of the Fas/FasL system, but combination of sFasL and drug treatment produces a synergistic cytotoxic effect on human luekaemic cells.
The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated. FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology, PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine 2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels of mFasL protein on NIH3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3 cells membrane was investigated by the inducing apoptosis of Fas + Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately co-cultured with Yac-1 cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants from twelve resistant clones was 8.5×10 5 colony-forming-unit (CFU)/ml. The NIH3T3 cells transfected by above supernatants had a higher level of mFasL (53.81±6.9 %), and significantly induced the apoptosis of Fas + Yac-1 cells (56.78±4.5 %), as both were cocultured for 5 h at 1∶1 ratio, whereas it is 7.08±3.4 % in control group (P<0.01). Supernatant from PLFIN-PA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.
