XAF1 induced mitotic catastrophe through modulation of G2/M checkpoint and interaction with checkpoint kinase 1

Abstract

Background: XAF1 was first recognized as an antagonist in XIAP-suppressed caspase 3 activity. It has lower expression in cancer cells than normal tissue and sensitizes TRAILand etoposide-triggered apoptosis. However, its role in cell growth and the underlying mechanism have not been studied. Methods: Transfectants of gastrointestinal cancer cell lines AGS, Lovo and SW1116 stably expressing XAF1 and vector control were established. Cell growth, apoptosis, morphology, mitotic status and cell cycle distribution were assessed. Mitotic catastrophe was identified by occurrence of aberrant nuclei and centrosomal amplification. The expression and activity of G2/M proteins were detected by immunoblotting and kinase assay. Interaction between XAF1 and checkpoint kinase 1 (Chk1), was demonstrated by co-immunoprecipitation and the subcellular co-localization while the role of Chk1 in XAF1-induced G2/M arrest was evaluated by RNA interference. Results: Over-expression of XAF1 suppressed serum-dependent cancer cell growth, induced cell accumulation in mitosis, mitotic catastrophe and G2/M cell cycle arrest. Interestingly, XAF1 was only expressed in G2/M phase after cell cycle synchronization. In addition, XAF1 interacted with and activated Chk1, inactivated Cdc25C, and Cdc2/cyclin B complex. Suppression of Chk1 abrogated XAF1-induced G2/M arrest. Furthermore, over-expression of XAF1 increased the susceptibility of cancer cell to serum-deprivation-induced apoptosis. Conclusions: These findings suggest that XAF1 promotes apoptosis secondary to mitotic catastrophe through modulating G2/M checkpoint, implicating a novel XIAP-independent function pathway of XAF1. Low expression of XAF1 in cancer cell might correlate to the surveillance defect of transformed cell in gastrointestinal tract, thus inferring XAF1 as a potential target for the therapy of gastrointestinal cancers.