异硫氰酸苯己酯对Molt-4细胞组蛋白甲基化、乙酰化调控的实验研究

目的 研究异硫氰酸苯己酯（PHI）在体外对淋巴细胞白血病Molt-4细胞系的作用，观察PHI对Molt-4细胞组蛋白甲基化、乙酰化调控的影响。方法 采用MTT法、克隆抑制实验观察PHI对Molt-4细胞增殖的影响；采用流式细胞术检测PHI诱导细胞凋亡和对细胞周期的影响；用Western blot法观察PHI作用后细胞的组蛋白乙酰化酶、组蛋白甲基化及乙酰化状态的变化。结果 PHI可上调Molt-4细胞组蛋白乙酰化酶P300／CBP水平，显著提高组蛋白H3、H4乙酰化及H3K4甲基化水平，抑制组蛋白甲基化H3K9表达，阻滞细胞于G0／G1期，并诱导细胞凋亡。结论 PHI可能是一种组蛋白去乙酰化酶抑制剂，同时能调控组蛋白甲基化，影响其表观遗传学，可能作为新的抗白血病治疗药物。     

丙戊酸钠抑制人多发性骨髓瘤U266细胞增殖及组蛋白乙酰化调控的研究

本研究探讨丙戊酸钠（valproic acid sodium,VPA）对U266细胞增殖的抑制作用及对组蛋白乙酰化修饰调控的影响。采用CCK-8法检测VPA对U266细胞增殖的抑制作用,流式细胞术检测VPA作用前后U266细胞周期的变化,半定量RT—PCR检测VPA作用后U266细胞中组蛋白去乙酰化酶1（HDAC1）mRNA的表达变化,Western blot方法检测VPA作用后HDAC1及组蛋白H3、H4乙酰化蛋白水平的变化。结果表明,VPA对U266细胞增殖的抑制作用呈时间一浓度依赖性;2．5mmol／L的VPA处理细胞48小时后,细胞周期检测显示G0／G1期细胞比例增高,S期细胞比例下降,细胞被阻滞在G0／G1期（P〈0．05）;RT—PCR证实VPA能够抑制HDAC1mRNA的表达水平;Western blot方法证实不同浓度VPA作用细胞48小时后HDAC1蛋白水平降低,组蛋白H3、H4乙酰化表达水平提高。结论：VPA能够抑制U266细胞增殖,使细胞阻滞于G0／G1期,这可能与VPA抑制HDAC1表达,上调组蛋白H3、H4乙酰化水平有关。     

Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty.

Vascular smooth muscle cell (VSMC) proliferation after arterial injury is important in the pathogenesis of a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. Thus, a better understanding of the molecular mechanisms underlying VSMC proliferation in response to arterial injury would have important therapeutic implications for patients with atherosclerotic vascular disease. The p21 protein is a negative regulator of mammalian cell cycle progression that functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase, and by binding to and inhibiting the DNA polymerase delta co-factor, proliferating cell nuclear antigen (PCNA). In this report, we show that adenovirus-mediated over-expression of human p21 inhibits growth factor-stimulated VSMC proliferation in vitro by efficiently arresting VSMCs in the G1 phase of the cell cycle. This p21-associated cell cycle arrest is associated both with significant inhibition of the phosphorylation of the retinoblastoma gene product (Rb) and with the formation of complexes between p21 and PCNA in VSMCs. In addition, we demonstrate that localized arterial infection with a p21-encoding adenovirus at the time of balloon angioplasty significantly reduced neointimal hyperplasia in the rat carotid artery model of restenosis. Taken together, these studies demonstrate the important role of p21 in regulating Rb phosphorylation and cell cycle progression in VSMC, and suggest a novel cytostatic gene therapy approach for restenosis and related vascular proliferative disorders.     

急性白血病组蛋白甲基化、乙酰化修饰异常的研究

目的 研究急性白血病患者组蛋白H3K4和H3K9甲基化及H3、H4乙酰化的修饰异常.方法 用Western blot方法检测19例急性白血病患者组蛋白H3K4、H3K9甲基化水平及15例急性白血病患者H3、H4乙酰化水平,同时选取9例非肿瘤患者及4名健康志愿者作为对照.结果 19例急性白血病患者组蛋白H3K4甲基化水平(0.220±0.096)低于对照组(0.447±0.186)(P＜0.01);H3K9甲基化水平(0.409±0.106)高于对照组(0.168±0.015)(P＜0.01).15例急性白血病患者组蛋白H3乙酰化水平(0.128±0.013)低于对照组(0.386±0.104)(P＜0.01);H4乙酰化水平(0.096±0.008)低于对照组(0.341±0.096)(P＜0.01).结论 急性白血病细胞的组蛋白甲基化、乙酰化修饰存在异常,表现为组蛋白H3K4甲基化水平减低,H3K9甲基化水平增高,组蛋白乙酰化水平呈明显的减低,这些表达的异常均与染色质结构致密、基因转录抑制有关.组蛋白甲基化、乙酰化调控修饰异常与急性白血病的发病可能有关,有可能作为急性白血病治疗的靶点.     

The cHS4 chromatin insulator reduces gammaretroviral vector silencing by epigenetic modifications of integrated provirus

The cHS4 chromatin insulator has been shown to improve the expression of integrating gene transfer vectors by reducing the impact of silencing chromosomal position effects. To better understand the underlying mechanisms of this protection, we investigated the influence of this element on the epigenetic modifications of a gammaretroviral reporter vector. In HT1080 cells, we found that a fourfold increase in the level of green fluorescent protein (GFP) reporter expression from the cHS4-insulated vector was correlated with a twofold increase in acetylation at lysines 9 and 14 of histone H3, but not with CpG methylation. In a mouse bone marrow transduction and transplantation model, we found that a 10-fold increase in the likelihood of GFP expression from the cHS4-insulated vector was correlated with an eightfold increase in histone H3 acetylation, as well as a fourfold decrease in CpG methylation. Histone hyperacetylation peaked at the cHS4 core, and in vivo diminished nearly threefold through the central portion of the vector. Taken together, these studies demonstrate that the cHS4 chromatin insulator reduces gammaretroviral vector silencing by modulating epigenetic modifications of integrated provirus, and identify a specific topological distribution of these modifications that may prove informative for future vector designs.     

Transforming growth factor-beta-dependent growth inhibition in primary vascular smooth muscle cells is p38-dependent

Vascular smooth muscle cells (VSMCs) constitute the major cellular component of the vessel tunica media. VSMC proliferation is a key feature in developing vessels and pathological states such as atherosclerosis and restenosis. Transforming growth factor (TGF)-beta is a key regulator of VSMCs, but its effect on VSMC proliferation and apoptosis are controversial. Here, we characterized TGF-beta effects on basal-, serum-, and platelet-derived growth factor-BB-induced primary mouse VSMC proliferation. TGF-beta led to potent growth inhibition of VSMCs isolated from normal mouse aortae without inducing apoptosis. Growth inhibition by TGF-beta was due to G0/G1 arrest. Next, we explored distinct signaling pathways activated by TGF-beta and the effects of pharmacological inhibition of these. TGF-beta led to activation of Smad2/3, p38, p42/44, and c-Jun NH2-terminal kinase (JNK) pathways, assessed by phosphorylation, immunofluorescence, and reporter gene analysis. TGF-beta-dependent growth inhibition was specifically attenuated by pharmacological blockade of the TGF-beta type I receptor (TbetaRI) kinase or p38 mitogen-activated protein kinase pathways, whereas blockade of p42/44 or JNK kinases did not influence the effect of TGF-beta. TbetaRI kinase inhibition blocked all downstream pathways including Smad and p38 phosphorylation. In contrast, p38 inhibition did not alter Smad function, as assessed by translocation or reporter gene expression, but selectively inhibited p38 activity. These results demonstrate that TGF-beta acts as a potent antiproliferative mediator in VSMCs, irrespective of the proliferative stimulus, without inducing apoptotic effects. The anti-proliferative effect of TGF-beta is due to G0/G1 arrest and mediated primarily by the p38 pathway, suggesting that p38 kinase is central to TGF-beta-mediated growth inhibition in primary mouse VSMCs.     

Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy

Xenograft models are suitable for in vivo study of leukemia’s pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.     

组蛋白去乙酰化酶抑制剂LBH589对多发性骨髓瘤细胞MM1R的抑制作用

本研究探讨新一代组蛋白去乙酰化酶抑制剂LBH589单药或者联合蛋白酶体抑制剂硼替佐米,对多发性骨髓瘤(MM)细胞的抗瘤效应。采用MTT法检测LBH589(10、20、50 nmol/L)及50 nmol/L分别联合硼替佐米(10、20 nmol/L)作用于人多发性骨髓瘤MM1R细胞24,48 h后的细胞增殖抑制作用;采用流式细胞术检测LBH589对MM1R细胞周期和细胞凋亡的影响;采用Western blot分析LBH589(10、20、50 nmol/L)作用MM1R细胞24 h后组蛋白H4乙酰化的程度。结果表明,LBH589单药及与硼替佐米联合均能够抑制MM1R细胞增殖,并与药物浓度和作用时间呈正相关。MM1R细胞经药物作用48 h后,G0/G1期细胞逐渐增多,G2/M期及S期细胞逐渐减少,细胞阻滞在G0/G1期,同时可见MM1R细胞的凋亡率增加,作用呈浓度依赖性,且LBH589与硼替佐米联合作用均较单药作用更加明显(均P<0.001);Western blot分析显示,不同浓度LBH589作用MM1R细胞24 h后组蛋白H4乙酰化的程度上调,呈浓度依赖性。结论:LBH589能够抑制MM1R细胞增殖,阻滞细胞周期,诱导细胞凋亡,且与硼替佐米联合对骨髓瘤细胞有协同作用。     

姜黄素调节NB4细胞P53和组蛋白H3乙酰化抑制细胞增殖的研究

目的 研究姜黄素对NB4细胞组蛋白H3和非组蛋白P53的乙酰化和细胞增殖的作用，探讨姜黄素抗白血病机制。方法 应用小同浓度（50，25，12．5，6．25，3．125μmol／L）的姜黄素作用NB4细胞不同时间（0，4，8，12，24h），MTT法测定姜黄素对NB4细胞增殖的影响，Western blot法检测乙酰化组蛋白H3和乙酰化P53的水平。结果 姜黄素以时间和剂量依赖方式抑制NB4细胞增殖，在24h和36h的IC值分别为40μmol／L和25μmol／L；能明显上调组蛋白H3的乙酰化水平，促进P53的表达和P53的乙酰化。结论 姜黄素具有去乙酰化酶抑制剂作用，能上调组蛋白H3乙酰化水平，促进肿瘤抑制凶子P53表达和活化，抑制白血病细胞增殖。     

Genetic Variation Stimulated by Epigenetic Modification

Homologous recombination is essential for maintaining genomic integrity. A common repair mechanism, it uses a homologous or homeologous donor as a template for repair of a damaged target gene. Such repair must be regulated, both to identify appropriate donors for repair, and to avoid excess or inappropriate recombination. We show that modifications of donor chromatin structure can promote homology-directed repair. These experiments demonstrate that either the activator VP16 or the histone chaperone, HIRA, accelerated gene conversion approximately 10-fold when tethered within the donor array for Ig gene conversion in the chicken B cell line DT40. VP16 greatly increased levels of acetylated histones H3 and H4, while tethered HIRA did not affect histone acetylation, but caused an increase in local nucleosome density and levels of histone H3.3. Thus, epigenetic modification can stimulate genetic variation. The evidence that distinct activating modifications can promote similar functional outcomes suggests that a variety of chromatin changes may regulate homologous recombination, and that disregulation of epigenetic marks may have deleterious genetic consequences.     

A new type of vasodilator, HA1077, an isoquinoline derivative, inhibits proliferation of bovine vascular smooth muscle cells in culture

The effects of a newly developed vasodilator agent, HA1077 [1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride], were investigated on the proliferation of cultured bovine aortic vascular smooth muscle cells (VSMC). HA1077 (10-100 microM) inhibited both fetal calf serum-induced proliferation and [3H]thymidine incorporation into DNA of the growth-arrested VSMC in a dose-dependent manner. When quiescent cells were stimulated with platelet-derived growth factor followed by insulin, HA1077 (1-30 microM), administered together with either stimulation, showed dose-dependent inhibition of [3H]thymidine incorporation. Further reduction of [3H]thymidine incorporation was observed when HA1077 was present at both stimulations, suggesting that HA1077 suppresses DNA synthesis acting in both competence and progression stages. After stimulation with fetal calf serum, quiescent VSMC started and ceased DNA synthesis in 15 to 18 hr and 24 hr, respectively. HA1077 inhibited [3H]thymidine incorporation when it was added either from 12 hr to 15 hr or from 21 hr to 24 hr after serum stimulation. In addition, when percent inhibition of [3H]thymidine incorporation by continuous exposure to HA1077 was examined as a function of the time it was added, reductions of the value were observed at 0 to 3 hr, 12 to 18 hr and 21 to 24 hr. Thus, we concluded that HA1077 suppresses DNA synthesis of bovine VSMC acting at the G0/G1 and the G1/S phase transitions and also in the S phase of the cell cycle. It is suggested that this agent may act as a potent inhibitor of VSMC proliferation as well as a vasodilator.     

细胞周期蛋白A对培养大鼠血管平滑肌细胞起始识别复合物1表达水平的影响

目的 探讨细胞周期蛋白A （Cyclin A）对培养大鼠血管平滑肌细胞（VSMCs）周期起始识别复合物1（ORC1）表达水平的影响.方法 采用组织块贴片法原代培养的大鼠胸主动脉血管平滑肌细胞,利用双胸苷使VSMCs达到细胞周期同步化,应用逆转录聚合酶链反应技术和流式细胞仪检测不同细胞周期VSMCs的ORC1和Cyclin A mRNA和蛋白表达.结果 经鉴定培养的细胞为VSMCs,采用血清饥饿法、双胸苷阻断法和秋水仙素阻抑法分别获得了处于不同细胞周期的同步化的VSMCs：G0/G1期（89.22 ±3.54）％,G1/S交界期（66.74±7.16）％,S期（63.24±4.06）％,G2/M期（51.64±11.18）％,各期比较差异有统计学意义（P均＜0.01）.Cyclin A在静止状态对VSMCs的ORC1 mRNA和蛋白无影响,在G2/M期达到高峰[（52.133 3 ±2.122 1）％],与G1/S期[（10.9167±0.531 1）％]、S期[（7.656 7±0.412 4）％]比较,差异有统计学意义（P均＜0.01）.ORC1在G2/M期达到最低值[（1.276 7 ±0.161 7）％],与G1/S期[（13.371 0±1.057 3）％]、S期[（3.043 3 ±0.538 0）％]比较,差异有统计学意义（P均＜0.01）,可能原因是Cyclin A阻止ORC1结合在VSMCs染色质上.结论 CyclinA可能调控ORC1在VSMCs细胞周期的启动.