Differential responses of FLIPLong and FLIPShort-overexpressing human myeloid leukemia cells to TNF-alpha and TRAIL-initiated apoptotic signals.
OBJECTIVE
Clonal marrow cells from patients with early myelodysplastic syndrome (MDS) undergo apoptosis in response to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Cells from advanced MDS are resistant to TRAIL. Two isoforms of the Flice inhibitory protein (FLIP) short (FLIPS) and FLIP long (FLIPL), which modulate TRAIL signals, showed disease-stage-dependent differential regulation. Therefore, we aimed at characterizing potential differential effects of FLIPL and FLIPS, on TRAIL and TNF-alpha-induced apoptosis in model leukemic cell lines.


METHODS
Using lentiviral constructs, FLIPL and FLIPS, as well as a green fluorescent protein control were overexpressed in ML-1 cells, which constitutively express very low levels of FLIP and are highly sensitive to apoptosis induction. Cells were then exposed to TRAIL or TNF-alpha, and effects on the extrinsic and intrinsic pathways of apoptosis induction were assessed.


RESULTS
Overexpression of FLIP reduced TRAIL and TNF-alpha-induced apoptosis in ML-1 cells. However, while FLIPL completely abrogated apoptosis, FLIPS allowed for BID cleavage and caspase-3 activation. Concurrently, there was a decline of Bcl-xL and X-linked inhibitor of apoptosis protein (XIAP) in FLIPS cells followed by apoptosis. Further, inhibition of nuclear factor-kappaB (NF-kappaB) activation in TNF-alpha-treated cells resulted in profound apoptosis in FLIPS, but not in FLIPL-overexpressing cells, consistent with the observations in patients with early stage MDS. Inhibition of NF-kappaB had only minimal effects on TRAIL signaling.


CONCLUSIONS
Thus, FLIPL and FLIPS exerted differential effects in myeloid leukemic cell lines in response to TRAIL and TNF-alpha. It might be possible to therapeutically exploit those differences with effector molecules specific for the FLIP isoforms.
Seal
Sudeshna
S
Fred Hutchinson Cancer Research Center, Department of Chemistry, University of Washington School of Medicine, Seattle, WA 98109-1024, USA.
Hockenbery
David M
DM
Spaulding
Emily Y
EY
Kiem
Hans-Peter
HP
Abbassi
Nissa
N
Deeg
H Joachim
HJ
D017209:Apoptosis / Q000235:genetics; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000096:biosynthesis* / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000235:genetics; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics; D006801:Humans; D016086:Lentivirus; D015470:Leukemia, Myeloid, Acute / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
C494811:BCL2L1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D016328:NF-kappa B; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D051020:bcl-X Protein; C505407:CASP3 protein, human; D053148:Caspase 3