Heat Shock Protein-70 Inhibits The Apoptosis Mediated By Apoptosis Inducing Factor In BCR/ABL Expressing Cells

The formation of Bcr/Abl fusion protein is a hallmark of human chronic myeloid leukemia (CML).This fusion protein has a elevated activity of tyrosine activity and activates various signaling pathways as PI3K-AKT、 Ras and STAT5 to render cells malignant transformation with abnormal proliferation and inhibition of apoptosis. Heat shock protein-70(Hsp-70) has been detected in Bcr/Abl expression cells with an increased level. Hsp-70 is a protein involved in cellular stress response and has received extensive attentions for its role in tumorigenesis. Hsp-70 can inhibit Bax localization in mitochondria and promote the apoptosis. Hsp-70 can inhibit Bax localization in mitochondria and promote the apoptosis.It can also inhibit caspase-9 recruitment by Apaf-1 and block the activation of caspase-dependent apoptosis.Apoptosis-inducing factor (AIF) is a major factor in non-caspase apoptosis pathway. AIF localizes in mitochondria by a localization signal at its N terminal and helps the process in respiration chain. When apoptosis occurs, AIF re-localizes into nucleus, induces genome DNA breaks and promotes cell apoptosis. In MEFs cells, overexpression of Hsp70 can block AIF localization in nucleus thus neutralize its apoptosis promotion. Residue 150-226 in AIF is the binding site for AIF which mediates the direct mediation between AIF and Hsp70. We speculated that AIF and the caspase-independent apoptosis pathway may be deactivated in CML cells. This mechanism could be related in CML molecular pathology.To address this hypothesis, we first investigated the status of AIF and caspase-indepented pathway in the presence of apoptosis signals. K562、 32Dp210、Ba/F3 p21 and their corresponding Bcr/Abl negative controls HL-60、32D、Ba/F3 were pre-treated with caspase inhibitor Z-VAD-FMK to block the pathway. Vp-16 is used to induce apoptosis. The apoptotic cells were quantifized by FCM whereas AIF translo cation was detected by WB and immunoflourescence (IFT).AIF mutant tagged HA were expressed in K562 which lack 1-120 mitochondria localization sequence (dMLS-AIF) and can target nucleus and thus initiate apoptosis. WB and IFT were carried out to detect the localization of AIF; flow cytometry and Wright’s stain were to detect cell apoptosis. Secondly, we asked if over expressed Hsp-70 in Bcr/Abl expressing cells inhibits the redistribution of released AIF. Western blotting was conducted to examine the Hsp-70 expression level in K562 and 32D p210, Ba/F3p210,two Bcr/Abl transformed cells lines with their Bcr/Abl nagetive counterparts HL-60,32D and Ba/F3 cells, respectively. After the treatment of IM to inhibit the kinase activity, Hsp-70 and Hsf-1 level were tested.Next, SiRNA against Hsp-70 was employed to reduce the level of Hsp-70 in K562 cells for test the cell vablitity,cell cycle and apoptosis were then analyzed by MTT and FCM. Nucleus accumulation of AIF triggered by Vp-16 was then detected by immunoflourescence and WB. FCM and TEM were carried out to detect apoptosis after the inhibition of caspase pathway.Finally, we asked that how does up-regulated Hsp-70 inhibited the translocation of AIF into nucleus.By Co-IP assay, we detected that,in Bcr/Abl cells, the binding of Hsp-70 and AIF which released from mitochondria, as well as the binding of Hsp-70 in K562 cells and HA-tagged dMLS-AIF. By pull-down, we examined the binding of Hsp-70 in cytoplasm from different cells and HIS-AIF for proving the binding efficiency is related to the Hsp-70 expression level. To demonstrate that AIF binding to over expressed Hsp-70 is the cause to hinder AIF translocation, we disrupted the interaction between Hsp-70 and AIF by expressing AIF mutant which lacks the Hsp-70 binding domain (dHBD-AIF)and we expressed it by adenovirus in K562 cells.Subcellular location of this mutant was assayed by western blotting and immunoflourescence. Potential of this mutant to initiate apoptosis was measured by FCM and TEM.Our data demonstrated that, firstly, Bcr/Abl nagetive cells underwent apoptosis even the caspase pathway was inhibited by Z-VAD-FMK, suggesting that caspase-independent pathway was intact in these cells. However, apoptosis in Bcr/Abl cells was fully blocked by caspase inhibitor. We also found that AIF failed to redistribute in nucleus in Bcr/Abl expressing cells treated with Vp-16, in addition, the nucleus-targeted dMLS-AIF mutant, which was loaded in adenovirus vector and then was expressed in K562 cells, was mainly detected in cytoplasm.Secondly, our results showed that, compared with Bcr/Abl nagetive cells, Hsp-70 was significantly overexpressed to 4-6 fold in Bcr/Abl positive cells. Compared with healthy people, Hsp70 in CML patients can reach to over 100 fold. Overexpression of Hsp-70 seemed to be mediated by Bcr/Abl, because inhibition of Bcr/Abl activity by IM reduced the level of Hsp-70 in a time and dose dependent manner. Besides, Hsp-70 silencing can affect cell functions in a silencing efficiency dependent manner. When Hsp-70 level was effectively reduced by siRNA, cell cycle arrest and induction of apoptosis were detected as a result of Hsp-70 suppression. Moreover, siRNA assisted AIF translocation in to nucleus in the presence of Vp-16, cell death occurred in a caspase-independent manner. These results indicated that high level of Hsp-70 in Bcr/Abl cell was responsible for the failure of AIF nucleus accumulation, leading to the inactivation of caspase-independent pathway.Fanally, we found that released AIF in Bcr/Abl cells was captured by Hsp-70.Hsp-70 interacted with AIF in a dose-dependentent manner in 32D and HL-60 cells.Hsp-70 seemed insufficient to block the import of AIF into nucleus.Besides, gain-of-function of AIF can be achieved by disruption of Hsp-70 by expressing AIF mutant lack Hsp-70 binding domain.The dHBD-AIF was exclusively expressed in nucleus and triggered cell death in a time dependent manner.In conclusion, we found that Bcr/Abl neutralized AIF through up-regulation of HSP-70. Down regulation of Hsp70 can inhibit cell proliferation and promote apoptosis, at the same time, helps AIF translocation in nucleus. By deciphering the mechanism of caspase independent pathway and the players involved, we can further understand how tumor cells escapes from apoptosis which could be a new potential for drug development.