Genome-Wide and Functional Analysis of Acquired Resistance to TRAIL/APO2l Mediated Apoptosis of Leukemia Cell Lines.

BACKGROUND: Acute leukemia comprises malignant diseases of clonal character, to which specific treatment remains limited. Apoptosis induced by death receptor activation (i.e. by tumor necrosis factor-related apoptosis inducing ligand, TRAIL/APO2L) represents a potential method for cancer therapy. A loss of sensitivity to apoptosis represents one of the key molecular mechanisms of cancer cell resistance to chemo/immuno/radiotherapy. TRAIL, a member of the TNF family of death ligands, appears to specifically and efficiently kill tumor cells of diverse origin while sparing normal tissues. The TRAIL receptor family consists of five receptors: two death receptors (DR4, DR5), two decoy receptors (DcR1, DcR2), and osteoprotegerin (OPG). AIMS: Analysis of the molecular basis of TRAIL resistance and functional analysis of individual TRAIL receptors in HL60 myeloid leukemia cells. MATERIALS AND METHODS: TRAIL-resistant cells were selected from the original HL60 population using pressure of recombinant TRAIL. The expression of TRAIL receptors and other apoptosis regulating molecules and CD14 was analyzed by flow cytometry and by real time RT-PCR. Percentage of apoptotic cells was measured by flow cytometry using Annexin-V-FITC/PI apoptosis detection kit. The contribution of individual TRAIL receptors on the transmission of apoptotic signal was measured using blocking antibodies to TRAIL receptors. The TRAIL resistance related genome aberrations were analyzed by comparative genomic hybridization (CGH). RESULTS: Blocking antibodies to DR4 receptors significantly reduced the number of apoptotic HL60 cells compared to untreated controls. The blockage of DR5 receptors did not significantly inhibited TRAIL-induced cell death. Combination of anti-DR4 and anti-DR5 antibodies almost completely abrogated TRAIL-induced HL60 cell death and significantly reduced apoptosis compared to control or anti-DR4 antibody alone (p<0.01). Blocking of decoy receptors (DcR1, DcR2, and OPG) did not significantly affect the apoptotic signaling in HL60 TRAIL-sensitive and TRAIL-resistant cell lines. The TRAIL-resistant HL60 phenotypes were characterized by the decreased expression of TRAIL receptors DR4, DR5, DcR1, and DcR2, decreased or increased expression of CD14, and unchanged or undetectable expression of OPG as compared to control TRAIL-sensitive HL60 cells. CGH showed the loss of genomic material of long arm of chromosome 8, at 8q2, in all tested HL60 TRAIL-resistant lines (n=6). Further, a loss of genomic material in at least two TRAIL-resistant lines was detected at 1p3, 8p11-22, and chromosome 19. The gain of genomic material in at least two TRAIL-resistant lines was detected at 1q21-23, 6q2, and 15q11.1-21.1. Other identified chromosomal aberrations were unique for individual TRAIL-resistant leukemia lines derived from HL60 cell line. SUMMARY/CONCLUSIONS: HL60 cells transduced TRAIL-specific apoptotic signal predominantly through TRAIL receptor DR4. Decoy receptors, including OPG, did not play a significant role in TRAIL resistance. The identified TRAIL-resistant phenotypes are associated with distinct genomic changes. Support: IGA MZ NR8317-4, NR8930-4, GAUK 50/2004/c, MSM 0021620813.