β 2 ‐microglobulin induces caspase‐dependent apoptosis in the CCRF‐HSB‐2 human leukemia cell line independently of the caspase‐3, ‐8 and ‐9 pathways but through increased reactive oxygen species

Exogenous β 2 ‐microglobulin (β 2 m) induces significant apoptosis in the CCRF‐HSB‐2 human lymphoblastic leukemia cell line as detected by DNA fragmentation, DAPI staining and annexin V binding assay. β 2 m treatment induced the release of cytochrome c and apoptosis‐inducing factor (AIF) from the mitochondria, but no change in mitochondrial membrane potential (ΔΨm) was observed during apoptosis, suggesting that cytochrome c may be released through a mechanism independent of mitochondrial permeability transition (MPT) pore formation. Moreover, the β 2 m‐induced release of cytochrome c and AIF from the mitochondria in CCRF‐HSB‐2 cells was caspase‐independent, since Z‐VAD‐fmk, a general inhibitor of caspases, did not block the release of these factors. However, Z‐VAD‐fmk treatment significantly blocked β 2 m‐induced apoptosis, while Western blot analysis revealed that caspases‐1, ‐2, ‐3, ‐6, ‐7, ‐8 and ‐9 are not activated during β 2 m‐induced apoptosis in these cells. These results collectively indicate that a post‐mitochondrial caspase‐dependent mechanism is involved in β 2 m‐induced apoptosis. Moreover, β 2 m significantly enhanced the production of reactive oxygen species (ROS) during 12–48 hr treatment, and β 2 m‐induced apoptosis was almost totally inhibited in cells pre‐treated with the antioxidant N‐acetylcysteine (NAC), providing evidence that β 2 m‐induced apoptosis in CCRF‐HSB‐2 cells is ROS‐dependent. Therefore, these results reveal that β 2 m‐induced apoptosis in CCRF‐HSB‐2 cells may occur through an unknown caspase‐dependent and ROS‐dependent mechanism(s) that is associated with cytochrome c and AIF release from mitochondria, but is independent of the caspase ‐3, ‐8 and ‐9 pathways. © 2002 Wiley‐Liss, Inc.