【作者】 张旭辉；

【导师】 于晓妉；

【作者基本信息】 中国人民解放军军事医学科学院 ， 细胞生物学， 2007， 博士

【摘要】 研究背景：组蛋白去乙酰化酶抑制剂(HDACi)是一类能够有效抑制抑制肿瘤细胞增殖、诱导细胞分化、促进细胞凋亡的抗肿瘤新药。其杀伤肿瘤的作用机制可能涉及通过表观修饰调节基因表达及抑制分子伴侣蛋白Hsp90的功能。染色体乘客复合体(chromosomal passenger complex，CPC)是近年来发现报道的细胞内一组与染色体结合并负责细胞有丝分裂调控的蛋白分子，主要由Aurora B激酶、着丝粒中心蛋白(Inner centromere protein，INCENP)、Survivin及Borealin／DasarB等蛋白分子组成。研究证实CPC在有丝分裂过程中扮演了重要的角色，涉及纺锤体形成、染色体排列、姊妹染色单体分离、纺锤体检查点信号及胞质分裂等多种重要功能。热休克蛋白90(heat shock proteins，Hsp90)是细胞内重要的分子伴侣蛋白，能够参与细胞内一些重要蛋白分子的构象，稳定性及激酶活性的调节。研究表明CPC的两种重要的蛋白组分Aurora B和Survivin蛋白皆为热休克蛋白90的底物蛋白，我们先前的研究表明HDACi能够使Hsp90分子发生乙酰化而抑制Hsp90和其底物蛋白的结合，从而促进多种底物蛋白包括survivin通过泛素-蛋白酶体通路降解。我们在前期实验中观察到，应用HDACi处理多种肿瘤细胞，都会诱导细胞产生异常的有丝分裂现象。因此，提示我们思考HDACi能否通过影响CPC的正常定位和表达，进而杀伤肿瘤细胞，诱导有丝分裂灾变?围绕这一问题，我们针对HDACi对细胞CPC分子表达和着丝粒定位、相关的着丝粒表观修饰改变、动粒组装和细胞检查点功能等影响进行了系列的深入研究，对HDACi诱导肿瘤细胞发生有丝分裂灾变的分子机制进行探讨。研究内容及结果：1．HDACi诱导肿瘤细胞发生有丝分裂灾变HDAC抑制剂FK228、TSA及SAHA分别处理非小细胞肺癌、前列腺等细胞后，瑞氏-姬姆萨染色显示细胞形态出现了明显改变，表现为细胞胞浆皱缩、胞内颗粒逐渐增多、并出现大的多核细胞。多核细胞计数和有丝分裂各期细胞计数结果显示，FK228处理后多核细胞数明显增多，药物处理后有丝分裂后期和末期细胞的比例明显减少，出现明显前中期阻滞现象。免疫荧光染色结果显示FK228处理后细胞出现以细胞周期前中期阻滞染色体分离排列异常、多核细胞为特征的有丝分裂灾变现象。上述结果表明，HDACi处理能够诱导细胞发生有丝分裂灾变。在后续实验中我们主要以FK228进行相关实验2．FK228影响CPC分子的着丝粒定位和AuroraB激酶活性。免疫荧光染色证实FK228可以在作用12h后明显改变CPC各组成蛋白Survivin、AuroraB、INCENP及Borealin在有丝分裂过程中的着丝粒定位。伴随AuroraB激酶蛋白定位的改变，其底物蛋白组蛋白H3第十位点丝氨酸的磷酸化(H3Ser10-ph)明显降低，着丝粒CENP-A蛋白的磷酸化同样降低，提示AuroraB激酶的活性受到明显抑制。3．FK228对CPC分子定位的影响不是由于抑制Hsp90功能所致蛋白印记实验检测结果显示，FK228可以程度不同地降低A549和H1299细胞中AuroraB和survivin蛋白的表达，其中survivin蛋白的降低出现较早，可以在用药后24小时明显清除，48小时完全清除，而AuroraB蛋白的降低要在用药后48小时较为明显。随后免疫荧光染色证实FK228处理6小时，对survivin蛋白的定位尚无明显影响，12h后，survivin蛋白在着丝粒的定位即开始消失，这一结果明确证实survivin蛋白在着丝粒定位能力的消失早于蛋白水平的下降，即FK228影响CPC蛋白的着丝粒定位并不是由于抑制Hsp90功能，促进AuroraB及survivin蛋白降解所引起的。4．FK228明显改变着丝粒表观遗传修饰特征着丝粒是染色体上的一个特化的结构，由着丝粒DNA (CEN DNA)装配上复杂的染色体蛋白和着丝粒蛋白组成。CEN染色质中其组蛋白修饰谱与常染色质及侧翼的异染色质不同，CEN染色质的表观修饰特征包括组蛋白低乙酰化，同时缺少组蛋白H3第9位赖氨酸的二甲基化修饰(H3K9-Me2)，仅在着丝粒外围呈现组蛋白H3第9位赖氨酸的三甲基化修饰(H3K9-Me3)。研究表明这种不同的修饰方式有助于其独特区域及着丝粒区三维结构的组成，代表着决定着丝粒特性的表观遗传信息，提示表观遗传修饰对着丝粒的功能起着重要的调控作用。我们研究发现FK228可明显改变其表观修饰，其作用包括(1)．FK228作用后6h明显增强组蛋白乙酰化；(2)．H3K9乙酰化的增强明显降低着丝粒H3K9-Me3修饰，但对核质内表达的H3K9-Me2没有影响；(3)．FK228降低H3K9-Me3修饰并影响H3Ser10-ph修饰，直接导致着丝粒旁异染色质蛋白HP1在有丝分裂期解离障碍，表达增强；(4)．CENP-A的磷酸化修饰减弱；5．FK228影响着丝粒的组装和功能着丝粒具有包括动粒形成、纺锤体介导的运动，姊妹染色体结合及有丝分裂检查点等多重的功能。FK228能够改变着丝粒旁的表观遗传修饰特征，而表观遗传修饰在着丝粒区起重要的调控作用，提示我们引一步研究着丝粒区表观遗传修饰特征的改变对着丝粒组装和功能的影响。我们的研究证实FK228对着丝粒具有如下影响：(1)．FK228抑制着丝点(动粒)区外板蛋白CENP-E和CENP-F的定位；(2)．FK228虽不影响动粒区内板蛋白CENP-A定位，但能够降低CENP-A蛋白的磷酸化，而磷酸化修饰对于CENP-A蛋白的功能是重要的；(3)．FK228对内、外板蛋白定位的影响直接影响动粒的组装和功能，表现为着丝粒失去与微管的结合能力；6．FK228灭活细胞检查点功能(1)．FK228降低检查点蛋白BubR1和Bub1蛋白的着丝粒定位；(2)．FK228处理后纺锤体检查点在黏附和张力两方面的功能都受到了抑制；本研究结果首次证实组蛋白去乙酰化酶抑制剂FK228可通过改变着丝粒重要的表观修饰特征，从而影响HP1蛋白在有丝分裂期的释放，并影响着丝粒的正确组装和CPC分子的着丝粒正常定位，导致细胞纺锤体检查点灭活，最终造成细胞有丝分裂灾变。更多还原

【Abstract】 Background:Histone deacetylase inhibitor (HDACi) is a promising class of anticancer agents with high efficiency on inhibiting tumor growth, inducing cell differentiation and/or apoptosis. The possible mechanisms for HDACi to kill cancer cells are related with epigenetic regulation of gene expression and inhibition of heat shock protein 90 (Hsp90 )chaperone function.The chromosomal passenger complex (CPC), is a group of protein consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B. CPC plays important roles in key mitotic events, including kinetochore formation, the spindle assembly checkpoint, assembly of stable bipolar spindle and chromosome bi- directly orientation, and cytokinesis.Hsp90 is the important molecular chaperone of the cells, its function involving the modulation of protein conformation, stability and kinases activities. Previous studies show that CPC member Aurora B and Survivin both are the client protein of Hsp90. Our previous data suggested that HDACi can enhance the acetylation of Hsp90 and interrupt its chaperone function, which leads the depletion of several client proteins including Survivn via ubiquitin-proteasome pathway.Our preliminary data also showed that HDACi treatment induced abnormal mitosis in several tumor cell lines. It is rational to assume that HDACi may exert its effect on inducing mitotic catastrophe via affecting the localization and expression of CPC proteins. To confirm our hypothesis and elucidate the possible mechanisms, the effect of HDACi on the localization and expression of CPC proteins was detected, and the influence of FK228 on centromere epigenetic modification, assembly of centromere and the spindle checkpoint function was detected. We got the following Results and conclusion:1. HDACi induce mitotic catastrophe in tumor cellsAfter treatment with HDACi, including FK228, TSA and SAHA respectively, the morphological changes of non small cell lung cancer cells and prostate cancer cells were observed with Wright-Giemsa stain. The results showed that after drugs exposure, the cells exhibited remarkable cytoplasm crimple and studded with increased mutinuclear cells, the mitosis cycle was arrested and show more prometaphase cells, and less anaphase and telophases cells followed FK228 exposure. Immunofluorescence microscopy confirmed that FK228 induced the mitotic catastrophe. Typical features including mitotic spindle disorganization, failed chromosome segregation such as lagging chromosome and multinucleated cells formation.2. FK228 influents the centromere localization and expression of CPC proteins and reduces the Aurora B kinase activity.The results of immunofluorescence stain showed that FK228 treatment for 12 hours, obviously disrupted the the centromere localization of CPC members including Survivin, AuroraB, INCENP and Borealin. The AuroraB kinase activity was also inhibited as the phosphorylation of its two substrates, H3( Ser10) and CENP-A (ser7) at kinetochores was reduced.3. The FK228-induced disruption of CPC centromere localization is not caused by its effect on inhibiting Hsp90 chaperone function.Western blotting results revealed FK228 depleted both Survivin and Aurora B proteins, followed FK228 treatment for 24hour, Survivin began to decline and disappeared at 48hour. Compared with Surivivn, the depletion of Aurora B protein appeared late until 48 hour. Meanwhile, immunofluorescence experiment showed that FK228-induced decline of Survivin at the centromere appeared as early as 12 hour. It indicated that the effect of FK228 on inhibiting the localization of Survivin at the centromeres happened earlier than its effect on depleting the protein. That is to say the lost of CPC proteins from centromeres is not caused by the downregulation of Survivin and Aurora B proteins through interrupting Hsp90 chaperone function.4. FK228 remarkable changes the epigenetic modification markers of pericentromere.The centromere (CEN) is a specific chromosomal locus that embedded in heterochromatin and contains blocks of histone H3 nucleosomes interspersed with the histone H3 variant CENP-A. CEN chromatin has unique epigenetic characteristics, which are distinct from that of both euchromatin and flanking heterochromatin, including hypoacetylation of histones; absence of H3 Lys4 dimethylation (H3 Lys4-diMe); and H3 Lys9-triMe is enriched in pericentric regions. The specific histone modifications in CEN may contribute to the diverse properties and function of the centromere.Followed FK228 treatment, we found that the epigenetics modification markers of centromere have been changed, including:(1) FK228 increased the acetylation of histone H3.(2) FK228 inhibited the expression of H3K9Me3 at the centromenere, but had no influence on the expression of H3K9Me2.(3) FK228 changed the binary ’methyl/phos switch’ through inhibiting the H3K9Me3 and H3Ser10pho modification, which can inhibit the releasing of HP1 from metaphase chromosome and increase the accumulation of HP1 on pericentromere.(4) Decreased phosphorylation of CENP-A.5. FK228 affects the assembly and function of centromere. The centromere function including kinetochore formation, spindle-mediated movements, sister cohesion and a mitotic checkpoint. FK228 changed the epigenetic modification of pericentromere and centromere, which led us to further analyze whether these changes would influence the assembly and function of centromere. We got the following results:(1) FK228 inhibited the kinetochore proteins CENP-E and CENP-F located in outer plate of the cetromere.(2) FK228 had no effect on the localization of kinetochore inner plate protein CENP-A, but decreased the phosphorylation at ser7, an important modification for its function.(3) FK228 disrupted the attachment between centromere and microtubes during mitosis via inhibiting the kinetochore function.6.FK228 inhibits the function of spindle checkpoint(1) FK228 inhibited the localization of checkpoint proteins Bubl and Bub R1 at thecentromere.(2) FK228 inhibits the activation of mitotic checkpoint on either losing attachment or tension.In this study, we proved for the first time that the mechanisms for FK228-induced cancer cells mitotic catastrophe are related with its effect on changing epigenetic modification of centromere and pericentromere. These epigenetic changes inhibit the releasing of HP1 from metaphase chromosome, disrupt the kinetochore assembly and the localization of CPC proteins at the centromere, and finally inactivate the function of centromere and mitotic checkpoint.更多还原