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The Ubiquitin-proteasome Pathway Involved in ATRA-induced Leukemia Cells G1/G0 Phase Arrest and Transition into Differentiation

Author: LinMeiHua
Tutor: HeQiaoJun;YangBo
School: Zhejiang University
Course: Pharmacology
Keywords: ubiquitin-proteasome pathway ATRA cyclins CDKs G1/G0 arrest
CLC: R733.7
Type: Master's thesis
Year: 2009
Downloads: 26
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Abstract

Objective: All-trans retinoic acid （ATRA） is mandatory in the treatment of acute promyelocytic leukemia （APL） in clinic. It is a powerful inducer of terminal differentiation of human myeloid leukemia cells in vitro, and elicits complete remission of APL with striking clinical benefits. ATRA-induced cell differentiation is often accompanied with the G1/G0 phase arrest, while how ATRA couples cell cycle arrest to differentiation remains largely unknown. Many proteins related to cell differentiation and cell cycle arrest are degraded by the ubiquitin-proteasome pathway （UPP）. However, systematic studies focusing on the exact role of the UPP in the differentiation of myeloid cells are still rare. So here we investigate the roles of the UPP in controlling the proliferation/differentiation transition in myeloid leukemia cells.Methods: 1) For proliferation assay, human myeloid leukemia cells NB4 and HL-60 were treated with ATRA, cell growth curve was performed by counting cells each day. For cell cycle analysis, cells were stained with PI and analyzed by flow cytometry. For cell differentiation analysis, CD11b, a potent differentiation marker of leukemia cell was detected by flow cytometry. The expressions of cell cycle related proteins （cyclin D1, cyclin E, CDK2, CDK4, CDK6, pRb） were shown by Western blot, as were the level of poly-ubiquitinated conjugates and theα,β-subunits of 20S cylindrical catalytic core. The transcription level of ubiquitin was determined by real time PCR. 20S proteasome activity was determined by using fluorogenic peptide substrate Suc-LLVY-AMC. 2) The proteasome-specific inhibitor MG132, was used to further explore the involvement of the UPP in controlling the proliferation/ differentiation transition. The cell cycle profile and cell differentiation were determined by flow cytometric analysis under the treatment of ATRA alone or in combination with MG132. The expression of G1/S related protein （cyclin D1, cyclin E, CDK2, CDK4, CDK6, pRb and p-pRb） were detected by Western blot. For identification of the UPP dependent degradation of cyclin E and CDK2, the mRNA level of cyclin E and CDK2 were shown by RT-PCR. The Co-IP and Western blot were performed to detect the interaction between Ub and cyclin E or CDK2.Results:1) Here we report that ATRA （0.1 or 1μM） and ATRA （1 or 5μM） suppresses proliferation in NB4 and HL-60 leukemia cells by inducing G1/G0 phase arrest through cells counting and FACS. ATRA-induced cell differentiation is accompanied with the G1/G0 phase arrest shown by the elevation of CD11b expression, which reachs a peak on third day.2) By using Western blot, we found that ATRA treatment caused a time-dependent reduction in protein level of cyclin E, CDK2, CDK4, CDK6 and p-pRb, most of which took on an evident reduction after 3 days of ATRA stimuli and decreased more significantly, approaching 90-100% reduction after 5 days of ATRA treatment in both cell lines. Unexpectedly, the expression of cyclin D1 was on an increase. All of these suggest that the down-regulation of cell cycle related proteins is in correlation with ATRA-induced G1/G0 phase arrest.3) Here we report that the activity of the UPP was increased in ATRA-induced NB4 and HL-60 cell differentiation. By using RT-PCR, we observed that the ubiquitin mRNA was up-regulated in a time-dependent manner. By using Western blot, we found that the poly-ubiquitin protein conjugates were up-regulated after ATRA treatment and a peak level was observed on the third day. Theα,β-subunits of 20S cylindrical catalytic core were increased with time going on. Proteasome activity was determined by hydrolysis of the fluorogenic peptide Suc-LLVY-AMC. We observed that 20S proteasome activity increased in both cells with time going on after exposure to ATRA.4 ) Besides exploring the variation of the UPP activity in ATRA-treated HL-60R （RA-resistant cell line） and human neuroblastoma cell CHP126, we also treated NB4 cells with other known differentiation-inducing agents, DMSO, TPA and 1,25（OH）₂D₃ to ascertain the activity of UPP. By using Western blot, we found that DMSO-induced granulocytic differentiation was associated with marked up-regulation of poly-ubiquitinated proteins. Meanwhile, the proteasome activity was also enhanced determined by hydrolysis of the fluorogenic peptide Suc-LLVY-AMC, with a peak level on the second day. By contrast, there’s no change in the activity of UPP in TPA induced-monocytic/macrophagic differentiation since the poly-ubiquitinated proteins was not up-regulated by TPA as well as the proteasome activity, which was even decreased on the third day of TPA treatment. Neither is the activity of UPP in 1,25（OH）₂D₃-induced NB4 cell differentiation. Thus it is couluded that the activity of UPP may be cell type specific and in association with the the type of differentiation as well.5) To further determine whether the up-regulation of UPP was indispensable in ATRA-induced cell cycle arrest and differentiation, proteasome inhibitor MG132 was employed to test this hypothesis. By using flow cytometric analysis, we found that MG132 partially blocked the cell cycle arrest and differentiation induced by ATRA. The percentage of cells arrested in the G1/G0 phase was reduced to 59% in the NB4 cells treated with both ATRA and MG132 compared with 74% in the cells treated with ATRA alone. And the expression of CD11b decreased from 70% in ATRA treatment to 40% in ATRA and MG132 co-treatment. In HL-60 cells, compared with ATRA stimuli alone, co-treatment with MG132 also reduced the number of cells arrested at G1/G0 phase presented by the data that 67% （treated with ATRA alone） and 54% （treated with ATRA and MG132）. And the percentage of CD11b positive cells decreased from 50% in ATRA treatment to 30% in ATRA and MG132 co-treatment. Data in both cell lines demonstrated the indispensable role of UPP in ATRA-induced P/D transition.6) By using Western blot, we observed that in the NB4 cells, ATRA-induced CDK2 degradation was partial abrogated in the presence of MG132 and no changes were observed in cyclin E protein level. However, the protein level of cyclin E/CDK2 complex in HL-60 cells was in the opposite. The declined levels of cyclin E could be overcame by the addition of MG132 in HL-60 cells, while the decrease of CDK2 level after ATRA treatment could not be reversed by MG132. Furthermore, in contrast to the results that the reversion of ATRA-induced down-regulation of pRb （ser-780） by MG132 was seen in HL-60 cells, there was virtually no change of pRb （ser-780） levels in NB4 cells. Additionally, compared with ATRA stimuli alone, the cells with co-treatment of MG132 had no alterations on the expression of cyclin D1/ CDK4/CDK6 complex in both cell lines.7) Here we demonstrated that the exact protein degraded by UPP in G1/S transition was different in those two cell lines, namely, CDK2 in NB4 cells and cyclin E in HL-60 cells through analysis of the results of RT-PCR, Co-IP and Western blot. All of these results leading us to focus on the UPP-dependent degradation of cyclin E/ CDK2 complex in ATRA-induced myeloid leukemia cell proliferation/differentiation transition, and the role of each individual protein mediated by the UPP may be cell type specific. Conclusion: Data presented here demonstrated the importance role of UPP in ATRA-induced leukemia cells differentiation. And the exact proteins degraded through this pathway may actually have an effect on controlling the proliferation/differentiation transition. Understanding these pathways may provide some hints toward making the UPP a very interesting topic and developing new treatments for human myeloid leukemia.

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