TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL. We found that parental (PT) HL60 cells were very sensitive to a 6 h incubation in the presence of TRAIL and died by apoptosis. In contrast, AR cells were resistant to TRAIL concentrations as high as 2 microg/ml for 24 h. Two pharmacological inhibitors of PI3K, Ly294002 and wortmannin, restored TRAIL sensitivity of AR cells. AR cells stably overexpressing PTEN had lower Akt1 activity and were sensitive to TRAIL. Conversely, PT cells stably overexpressing a constitutive active form of Akt1 became TRAIL resistant. TRAIL activated caspase-8 but not caspase-9 or -10 in HL60 cells. We did not observe a protective effect of Bcl-X(L) or Bcl-2 against the cytotoxic activity of TRAIL, even though TRAIL induced cleavage of BID. There was a close correlation between TRAIL sensitivity and intranuclear presence of the p50 subunit of NF-kappaB. Higher levels of the FLICE inhibitory protein, cFLIP(L), were observed in TRAIL-resistant cells. Both the cell permeable NF-kappaB inhibitor SN50 and cycloheximide lowered cFLIP(L)expression and restored sentivity of AR cells to TRAIL. Our results suggest that Akt1 may be an important regulator of TRAIL sensitivity in HL60 cells through the activation of NF-kappaB and up-regulation of cFLIP(L) synthesis.