Both G0/G1 Cell Cycle Arrest and Continuous Activation of MAPK Induce Autophagic Cell Death in bcr-abl-Positive Leukemic Cells.

Back ground;

Accumulating evidence suggests that programmed cell death is not defined as apoptosis but cells use different pathways for active self-destruction as reflected by different morphology. Autophagic cell death (APCD), which also be designated type II programmed cell death and appears to be a phylogenetically old phenomenon, is observed in physiological and disease state. Indeed, APCD is observed in several neurodegenerative diseases and cancer cells, however, the APCD in leukemic cells has not been reported yet. Here, we found APCD was occurred in bcr-abl-expressing CML cell lines.

Methods;

Bcr-abl-positive cell line, K562, were cultured with 2nM of TPA up to 48hours. Subsequently, we examined the morphological change by light microscopy and electronmicroscopy. To evaluate the APCD, TPA-treated K562 cells were labeled with autofluorescent agent Monodansylcadaverin(MDC), which specifically accumulates in autophagosomes and we measured accumulation of autophagosmes in cytoplasm by fluorescent microplate reader. TPA continuous activated MAPK of K562 cells and induced cell cycle arrest in G0/G1 phase. In order to elucidate the role of activation of MAPK in APCD, MAPK in K562 cells were inactivated by MAPK inhibitor, U0126 or dominant negative MAPK-transfection, and then, the cells were treated with TPA. Furthermore, to evaluate the involvement of cell cycle arrest in APCD, bcr-abl-transfected murine leukemic cell line, BAF3, was treated with cell cycle arrest inducer, mimosine. In addition, we investigated the expression of an autophagy-related molecule, Beclin 1 by immunohistochemical analysis.

Result;

The 48 hours’ treatment with TPA induced cell death in K562 cells, which accumulated plenty of autophagosomes in cytoplasm. MDC labeling assay revealed that accumulation of autophagosomes were increased in time- and dose-dependent manner. On the other hand, MAPK-inactivated K562 cells exhibited resistance to TPA-induced APCD. Furthermore, the bcr-abl-transfected BAF3 cells exhibited continuous activation of MAPK and underwent APCD by cell cycle arrest by mimosine. Immunohistochemical analysis revealed that Beclin 1 shuttled between nuclear and cytoplasms and co-localized with MAPK during APCD progression.

Conclusion:

APCD in bcr-abl-positive leukemia cells were closely involved in G0/G1 cell cycle arrest and continuous activation of MAPK. These findings are useful in developing the novel strategy to treatment of Ph¹ positive leukemia via alternative pathways and another type of cell death.