Study On The Effects And Mechanisms Of CSN Complex In ATRA-induced APL Cell Differentiation

Objective:As the first example of clinically cyto-differentiating agent, all-trans retinoic acid(ATRA) triggers growth arrest and terminal differentiation of acute promyelocytic leukemia(APL) by prompting PML/RARa protein degradation. The striking clinical benefits of ATRA in treating APL gave rise to enthusiasm in clarifying the mechanisms of its action and a variety of ATRA targets have been reported, the mechanisms responsible for its beneficial effects remain unclear.Recent data demonstrate that retinoic acid induced gene G(RIG-G),which was originally cloned in ATRA-induced differentiation of acute promyelocytic leukemia cell line NB4, could alter the cellular distribution of CSN5 and disrupt the integrity of CSN holocomplex, which strongly implied that CSN may play an important role in ATRA-induced differentiation. So the aim of this study was to investigate the involvement of CSN in ATRA-induced APL cells differentiation. The current study was divided into three sections: 1) to demonstrate the changes of levels of CSN in ATRA-induced APL cells differentiation. 2) to investigate the effect of ATRA on CSN-CRLs-E3 signaling pathway in ATRA-induced differentiation of APL cells. 3)to explore the effect and mechanism of CSN-CRLs-E3 signaling pathway on the regulating degradation of PML/RARα protein by autophagy pathway.Method:1) Human acute myeloid leukemia cells NB4 was treated with ATRA.Morphologic changes in the cells were assessed by Giemsa staining. Cell surface marker CD11 b was detected by flow cytometry. The transcriptional level of CSN was measured through Real-time PCR. Cell lysates were subjected to Western blot analysis for the detection of CSN protein level. Using RT-PCR to detect the expression of CSN3 and CSN5 in 11 cases of newly diagnosed APL patients.2) Human acute myeloid leukemia cells NB4 was treated with ATRA. Cell lysates were subjected to Western blot analysis for the detection of cullin1,cullin3(scaffold proteins of CRLs), the substrate recognition proteins(Skp2, βTr CP,KLHL20) and the downstream target proteins of CSN-CRLs pathway.3) Human acute myeloid leukemia cells NB4 was treated with ATRA. Western blot and immunoprecipitation were employed to detect the ATRA-induced autophagy in NB4 cells. 293 T cells were transfected to express DAPK1 and PML-RARα. Using Western blot to observe the effect of DAPK1 on PML/ RARαprotein and cell autophagy, comparing the effect of proteasome inhibitor(MG132)and autophagy inhibitor(3-MA).Results:1) Exponentially growing NB4 cells were treated with ATRA,separately for0,24,48,72 hrs. Morphologic changes assessed by Giemsa staining revealed that the ATRA-treated NB4 cells acquired a granulocytic morphology. The number of NB4 cells expressing CD11 b, a marker of myeloid differentiation, increased significantly in a time-dependent manner. In parallel, ATRA caused a substantial, time-dependent reduction in CSN protein and m RNA levels in NB4 cells. Meanwhile, the expression levels of CSN3 and CSN5 in newly diagnosed APL patients were much higher than those in healthy group. The levels were obviously down-regulated when APL patients achieved complete remission. These data indicated that ATRA can down-regulate the expression of CSN.2) Results obtained from Western blot demonstrated that the expression levels of cullin1, cullin3(scaffold proteins of CRLs), and their substrate recognition proteins Skp2, βTr CP, KLHL20 were significantly down-regulated in ATRA-induced differentiation. Meanwhile levels of the downstream target proteins of CSN-CRLs pathway, such as DEPTOR, p27, DAPK1 were up-regulated by ATRA. These results illustrated that CSN-CRLs-E3 signaling pathway may play an important role in the pathogenesis of APL and ATRA-induced differentiation.3) ATRA can enhance autophagy of APL cells and promote the degradation of PML/RARα protein. Since DEPTOR, DAPK1 play an important role in the regulation of autophagy, do they also have impacts on the regulating degradation of PML/RARα protein by autophagy pathway? Next, lysates of 293 T cells co-transfected with p IRES2-EGFP-PML/RARα and p CDNA3.1-DAPK1 plasmidswere subjected to Western blot analysis. The data revealed that co-expression of DAPK1 decreased the levels of PML/ RARα protein and enhanced cell autophagy.And the turnover of DAPK1 was blocked by the autophagy inhibitors(3-MA). These data indicated that DAPK1 could facilitate the degradation of PML/RARα protein,which might be related to cell autophagy mediated by DAPK1.Conclusion:The data presented herein demonstrated that CSN was high expressed in APL patients and NB4 cells, and ATRA can down-regulate the expression of CSN.Secondly, CSN-CRLs-E3 signaling pathway could be down-regulated in ATRA-induced differentiation, causing the increase of DAPK1, which could facilitate the degradation of PML/RARα protein. In summary, the study explored the effects of CSN on ATRA induced differentiation of APL cells and the concrete mechanism, providing fresh insights into the design of new differentiation therapies against leukaemia.