'Loop' domain deletional mutant of bcl-x(L) is as effective as p29bcl- x(L) in inhibiting radiation-induced cytosolic accumulation of cytochrome C (cyt c), caspase-3 activity, and apoptosis

Purpose/Objective: To investigate the effect of the enforced expression of p29Bcl-x(L) or its loop deletional mutant, p1SBcl-x(L)Δ, on irradiation- induced apoptosis and cell-cycle distribution of HL-60 cells. Materials and Methods: We compared the irradiation-induced molecular cascade of apoptosis in control human AML HL-60/neo versus Bcl-x(L) overexpressing (~8-fold) (HL- 60/Bcl-x(L)) and HL-60/Bcl-x(L)Δ cells that express the loop domain deletional mutant construct (Δ26-83 AA) of Bcl-x(L). The three cell lines were irradiated with 6MV photons to varying doses up to 20 Gy. Following this, cytosolic cyt c levels, caspase-3 activity, and the Bcl-2 family of proteins were evaluated utilizing Western blot analysis (whole cell lysate or cytosolic S-100 fraction). Apoptosis was assessed by internucleosomal DNA fragmentation, Annexin-V staining and FACS analysis, as well as by morphologic criteria. The cell-cycle effects of radiation were analyzed by flow cytometry. Results: Eight hours following irradiation (12 Gy) of HL- 60/neo cells, a marked increase (~8-fold) in the cytosolic accumulation of cyt c in the S-100 fraction was observed. This was associated with the cleavage of caspase-3, as well as the generation of its poly (ADP-ribose) polymerase (PARP) and DFF (DNA fragmentation factor)-45 cleavage activity. Twenty-four to forty-eight hours after irradiation, internucleosomal DNA fragmentation and positive Annexin-V staining (32.3 ± 3.3%) was detected in HL-60/neo cells. In contrast, in both HL-60/Bcl-x(L) and HL-60/Bcl-x(L)Δ cells, a significantly lower percentage of apoptotic cells (p< 0.05) were detected and internucleosomal DNA fragmentation was not induced. Following irradiation, Western analysis neither demonstrated any significant alteration in Bcl-2, p29Bcl-x(L), p18Bcl-x(L)Δ, or Bax; nor induced CD95 (Fas receptor) or Fas ligand expression in any cell type. However, in all cell types, irradiation produced approximately a 2-fold increase in the percentage Of cells in the G₂/M phase of the cell cycle. Conclusion: These results demonstrate that an intact loop domain is not necessary for the full antiapoptotic function of Bcl-x(L) against irradiation-induced cytosolic accumulation of cyt c, caspase activation, and apoptosis of HL-60 cells. Additionally, the cell-cycle effects of ionizing radiation in HL-60 cells are not affected by enforced expression of Bcl-x(L) or Bcl-x(L)Δ.