【作者】 林美花；

【导师】 何俏军； 杨波；

【作者基本信息】 浙江大学 ， 药理学， 2009， 硕士

【摘要】 研究目的:全反式维甲酸（all-trans retinoic acid,ATRA）治疗急性早幼粒白血病（acute promyelocytic leukemia,APL）能特异性诱导APL细胞分化成熟,具有疗效显著、完全缓解率高、毒副作用小等特点,因此成为临床上治疗APL的首选药物。但至今为止,ATRA诱导细胞分化的作用机制并未得到系统阐述。据文献报道,维甲酸诱导细胞分化过程伴随着明显的周期阻滞现象,同时与细胞分化、周期阻滞密切相关的蛋白均通过泛素-蛋白酶体通路降解。但是,关于泛素-蛋白酶体通路在维甲酸诱导白血病细胞分化中的作用并未见文献报道。本实验主要研究泛素-蛋白酶体通路在全反式维甲酸（ATRA）诱导白血病细胞增殖/分化转变过程中的作用,并探讨其可能的作用机制。研究方法:1)选用人白血病细胞株NB4和HL-60为研究对象,以全反式维甲酸（ATRA）,二甲基亚砜（DMSO）等分化诱导剂处理来评价泛素蛋白酶体通路活性的变化与细胞分化间的相关性;采用血细胞计数板计数来观察细胞增殖情况,描绘细胞生长曲线;细胞经碘化丙锭（PI）染色后采用流式细胞术分析细胞周期的变化情况;细胞经CD11b-PE抗体结合后采用流式细胞术检测细胞表面分化抗原CD11b表达;应用Western blot检测细胞周期相关蛋白（cyclin D1、cyclin E、CDK2、CDK4、CDK6、pRb）和泛素化蛋白,及20S核心颗粒α亚基、β亚基的表达;应用Real-time PCR检测细胞内泛素单体mRNA表达水平;采用荧光底物Suc-LLVY-AMC的水解检测细胞内20S蛋白酶体活性。2)以蛋白酶体抑制剂MG132合用ATRA与单用ATRA处理细胞比较进一步观察UPP在细胞增殖/分化转变过程中作用。采用流式细胞术检测细胞周期变化及细胞表面分化抗原CD11b表达;应用Western blot检测细胞内与G1/S时相转换的相关蛋白（cyclin D1、cyclinE、CDK2、CDK4、CDK6、pRb及p-pRb）的表达;应用Real-time PCR检测细胞内关键周期蛋白cyclin E和CDK2 mRNA表达水平;应用Co-IP和Western blot检测周期蛋白cyclinE和CDK2同泛素蛋白的结合情况。研究结果:1)通过血细胞计数板计数,显示ATRA（0.1或1μM）和ATRA（1或5μM）可分别显著抑制NB4和HL-60细胞增殖。流式细胞术分析细胞周期情况,显示ATRA（0.1μM）和ATRA（5μM）能使白血病细胞的细胞周期阻滞在G1/G0期,同时诱导了细胞的分化,CD11b的表达率明显增加,都在第3天基本上达到峰值。2)Western blot检测结果表明,相关周期蛋白cyclin E、CDK2、CDK4、CDK6和pRb均随作用时间增加而下调,并在第3天的时候变化最明显,到第5天时基本上不表达。而cyclin D1却呈相反的变化趋势。说明ATRA诱导细胞周期阻滞同关键周期蛋白的下调是相关的。3)泛素蛋白酶体活性在ATRA诱导的NB4和HL-60细胞分化过程中明显上调。RT-PCR检测结果表明泛素单体Ub mRNA的表达是呈时间依赖性增加的,Westernblot检测结果显示多聚泛素蛋白水平是上调的,在第3天达到最大值。20S核心颗粒α亚基、β亚基蛋白量是随作用时间的增加而增加。20S蛋白酶体活性检测结果表明其活性也是呈时间依赖性增加的。4)ATRA处理维甲酸耐受细胞株HL-60R和神经母细胞瘤细胞CHP126后,及其它的分化诱导剂如DMSO、TPA和1,25（OH）₂D₃诱导NB4细胞不同类型分化过程中UPP活性变化的检测。Western blot和20S蛋白酶体活性检测结果显示,ATRA处理HL-60R和CHP126后,多聚泛素蛋白水平和蛋白酶体活性并未被上调。而在DMSO诱导的粒性细胞分化过程中多聚泛素蛋白水平和蛋白酶体活性都是上调的,并在第2天达到最大值,而TPA和1,25（OH）₂D₃诱导的单核/巨噬细胞系分化过程中两者都没有明显变化,其活性在第3天反而有所下降。上述结果表明UPP活性的变化同细胞的类型及细胞的分化类型是相关的。5)采用蛋白酶体抑制剂MG132进一步验证UPP在ATRA诱导的细胞周期阻滞和分化过程中的必要性,及其可能的作用机制。流式细胞术结果显示,在药物诱导的NB4细胞中,单用ATRA（0.1μM）作用72小时后G1/G0期细胞比例为74%,CD11b表达率为70%,而当MG132和ATRA共同处理后,G1/G0期细胞比例下降到59%,CD11b表达也下调至40%。同样地,在HL-60细胞中也有此现象,采用ATRA（5μM）先作用细胞48小时,再用MG132（1μM）作用最后的24小时。结果发现MG132和ATRA共同处理后,G1/G0期细胞比例为54%,CD11b表达率为40%,较之单用ATRA作用72小时后G1/G0期细胞比例的67%和CD11b表达率的50%都明显下调。由此证明了MG132确能有效地逆转ATRA引起的细胞周期阻滞和分化效应。6)Western blot检测结果表明MG132和ATRA共同处理细胞后,同单用ATRA相比,与G1/S时相转换相关的关键蛋白在NB4和HL-60细胞上的变化情况具有细胞特异性。其中在NB4细胞株中,MG132能逆转ATRA作用后CDK2蛋白的下调,而在HL-60细胞上,则是cyclin E和p-pRb（ser-780）蛋白被累积。其它的周期蛋白cyclin D1、CDK4和CDK6基本上没有变化。提示通过蛋白酶体途径降解的关键蛋白可能同ATRA诱导的细胞增殖/分化转变相关。7)RT-PCR,Co-IP和Western blot检测结果显示,ATRA诱导的NB4细胞中的CDK2蛋白和HL-60细胞内的cyclin E蛋白确是通过UPP通路降解的,是泛素化介导的蛋白酶体依赖性的。纵观上述结果,在ATRA诱导的髓性细胞分化过程中UPP活性上调是必需的,参与UPP途径的蛋白是细胞特异性的,但最终都通过调控细胞G1/S“节点”,进而调节细胞增殖/分化转变这一过程。结论:泛素蛋白酶体通路（UPP）可能参与调节全反式维甲酸（ATRA）诱导白血病细胞分化的过程,其作用机制可能同经泛素蛋白酶体通路降解的与周期阻滞、细胞分化相关的蛋白有关。本研究为维甲酸诱导细胞分化的分子机制研究提供了全新的内容,并将UPP作为一个新的靶点,有利于全面了解其作用机制,进而为临床髓性白血病的治疗提供一定的理论指导。更多还原

【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.更多还原