OBJECTIVE To study whether an adriamycin-resistant cell line(HL-60/ADR) can be sensitized by adriamycin(ADR) to TRAIL-mediated apoptosis.METHODS The mRNA levels of the TRAIL receptor and apoptosis-related signaling molecules involved in the TRAIL-mediated apoptotic pathway were measured by RT-PCR.The protein levels of apoptotic-related signaling molecules involved in the TRAIL-mediated apoptotic pathway and processed caspase-3,caspase-9,and caspase-8 were measured by Western blots.Apoptosis was assessed by flow cytometry.Mitochondrial membrane potential was analyzed by DiOC6(3) staining.Cytotoxicity was determined by the colorimetric MTT viability/ proliferation assay.RESULTS Treatment with a combination of TRAIL and subtoxic concentrations of ADR resulted in synergistic cytotoxicity and apoptosis for both the parental HL-60 and the HL-60/ADR cells.For HL-60,there was a 5-fold potentiation and synergy in cytotoxicity for TRAIL and for HL-60/ADR,cytotoxicity to TRAIL was potentiated 6-fold with ADR.Adriamycin treatment modestly up-regulated TRAIL-R2(DR5),but had no effect on the expression of Fas-associated death domain,c-FLIP,Bcl-2,Bcl-xL,Bax,and IAP family members(cIAP-1,cIAP-2,XIAP,and survivin).The protein levels of pro-caspase-8 and pro-caspase-3 were not affected by ADR,whereas pro-caspase-9 and Apaf-1 were up-regulated.Combined treatment with TRAIL and ADR resulted in activation of caspase-9 and caspase-3,but there was no detectable processing of caspase-8 beyond the background levels.There was signif icant depolarization of the mitochondrial membrane by the combined treatment of both cell lines and it was more pronounced in the parental HL-60 cell line.The combined treatment with TRAIL and ADR resulted in 42.6% of the HL-60/ADR cells undergoing DNA fragmentation,whereas treatment with either ADR or TRAIL alone resulted in 5.46% and 21.3% DNA fragmented cells,respectively.Similar results were obtained with the HL-60 cells.CONCLUSION These fi ndings demonstrate that ADR can still signal ADR-resistant tumor cells,resulting in the modifi cation of the TRAIL-mediated signaling pathway and apoptosis. OBJECTIVE To study whether an adriamycin-resistant cell line (HL-60 / ADR) can be sensitized by adriamycin (ADR) to TRAIL-mediated apoptosis. METHODS The mRNA levels of the TRAIL receptor and apoptosis-related signaling molecules involved in the TRAIL- mediated apoptotic pathway were measured by RT-PCR. The protein levels of apoptotic-related signaling molecules involved in the TRAIL-mediated apoptotic pathway and processed caspase-3, caspase-9, and caspase-8 were measured by Western blots. by flow cytometry. Mitochondrial membrane potential was analyzed by DiOC6 (3) staining. Cytotoxicity was determined by the colorimetric MTT viability / proliferation assay. RESULTS Treatment with a combination of TRAIL and subtoxic concentrations of ADR resulted in synergistic cytotoxicity and apoptosis for both the parental HL-60 and the HL-60 / ADR cells. For HL-60, there was a 5-fold potentiation and synergy in cytotoxicity for TRAIL and for HL-60 / ADR, cytotoxicity to TRAIL was potentiated 6-f old with ADR.Adriamycin treatment modestly up-regulated TRAIL-R2 (DR5), but had no effect on the expression of Fas-associated death domain, c- FLIP, Bcl- 2, Bcl- xL, Bax, and IAP family members ( cPAP-1, cIAP-2, XIAP, and survivin) .The protein levels of pro-caspase-8 and pro-caspase-3 were not affected by ADR, whereas pro- caspase-9 and Apaf-1 were up-regulated. Combined treatment with TRAIL and ADR result in activation of caspase-9 and caspase-3, but there was no detectable processing of caspase-8 beyond the background levels. Where was the sign of icant depolarization of the mitochondrial membrane by the combined treatment of both cell lines and it was more pronounced in the parental HL-60 cell line. The combined treatment with TRAIL and ADR resulted in 42.6% of the HL-60 / ADR cells undergoing DNA fragmentation, treatment with either ADR or TRAIL alone resulted in 5.46% and 21.3% DNA fragmented cells, respectively.Similar results were obtained with the HL-60 cells. CONCLUSION These fi ndings demonstratethat ADR can still signal ADR-resistant tumor cells, resulting in the modification of the TRAIL-mediated signaling pathway and apoptosis.