Polycomb蛋白复合体对细胞衰老的表观遗传学调控

细胞衰老在表观遗传学上的调控越来越受到人们的关注.Polycomb蛋白复合体(polycomb group proteins)通过对组蛋白的修饰,尤其是甲基化修饰发挥对靶基因的沉默作用,并因此广泛参与到发育、增殖、分化以及肿瘤发生等重要生命过程.目前,有一系列的研究报道了polycomb的各组份参与了细胞衰老的调控.本文对polycomb发挥基因沉默作用机制的最新研究进展进行了归纳总结,并以衰老过程中的重要分子p16为重点,详细介绍了polycomb调节p16表达,影响细胞衰老进程的机制.研究表明,多种polycomb成员同时结合在p16INK4a基因座,它们的结合表现出相互依赖的同时又有各自的作用.这为进一步深入理解细胞衰老提供了表观遗传学的证据.     

细胞衰老与肿瘤发生及药物治疗

肿瘤细胞往往具有衰老障碍而表现出无限增殖的能力。细胞衰老障碍与肿瘤化疗和多药耐药关系密切,恢复肿瘤细胞衰老通路是肿瘤治疗和逆转多药耐药的一个很有前途的研究方向。     

Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells

Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3) was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS) generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells.     

A Dominant Role for p53-Dependent Cellular Senescence in Radiosensitization of Human Prostate Cancer Cells

Amplification and overexpression of ErbB2 (HER2/Neu) is one of the most common alterations associated with breast cancer. Activation of ErbB2 via homodimerization in a non-transformed human mammary epithelial cell line, MCF-10A, in basement membrane cultures leads to formation of proliferative structures that share properties with non-invasive early stage lesions. Recently, we have shown that activation of ErbB2 homodimers combined with expression of transforming growth factor (TGF)-beta induces invasive and migratory activity in MCF-10A cells. In this system, migration requires inputs from numerous cellular pathways. We discuss this data and a model for migration induced by ErbB2 and TGF-beta. Concurrent studies by other groups have also shown that ErbB2 and TGF-beta can cooperate to increase metastatic and invasive behavior in murine mammary tumors. Here we discuss these studies and the potential implications of this research on breast cancer therapeutics.     

DUSP1在药物诱导的肝癌细胞衰老中的表达变化及其作用研究

目的:在肝癌的治疗中,化学疗法是重要的治疗手段,尤其在结合外科手术切除的联合治疗中,化疗被广泛的应用于临床。而体内试验证实,化疗药物诱导的细胞衰老在治疗肿瘤的过程中,普遍存在并发挥重要作用,我们着重探讨在药物诱导的人肝癌细胞衰老中,细胞内重要信号途径的变化,发现并利用对细胞衰老有重要意义的蛋白,增强化学治疗肿瘤的效果。在本课题中,我们利用化疗药物阿霉素(doxorubicin)和蛋白酶体抑制剂MG132诱导的人衰老肝癌细胞模型,对MAPK信号途径的负调控因子DUSP家族的表达变化进行检测,筛选表达水平发生显著变化的双特异性磷酸酶(DUSP)家族成员,并初步探讨其在细胞衰老中的作用。方法:利用低剂量的阿霉素和蛋白酶体抑制剂MG132诱导人肝癌细胞衰老;通过Real time RT-PCR和Western blotting检测DUSP家族基因mRNA和蛋白质水平的变化;利用siRNA干扰技术敲降DUSP1,检测药物诱导肝癌细胞衰老水平的变化。结果:经低剂量的阿霉素和蛋白酶体抑制剂短时间处理后,人肝癌细胞系Huh7和SMMC-7721细胞发生明显的细胞衰老;在诱导的衰老细胞中,DUSP1mRNA和蛋白质水平呈显著升高。在敲降DUSP1后,MG132诱导的细胞衰老得到显著的缓解。结论:DUSP1在药物诱导的人肝癌细胞衰老中具有重要的作用,为研究临床治疗中化疗药物引起肿瘤细胞衰老的具体机制带来启发。     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

剪接因子异质核糖核蛋白A2B1在癌细胞和衰老细胞中的相反表达趋势及其对癌细胞衰老的调控

剪接因子异质核糖核蛋白A2/B1(HNRNPA2B1)与人类及小鼠的寿命相关,并在多个癌症的病程进展中发挥重要的作用.然而,HNRNPA2B1能否在细胞衰老这一与个体衰老和抑制癌症密切相关的生物学过程中发挥作用尚不清楚.本研究发现,HNRNPA2B1在多个癌症体系中呈显著上调表达趋势,而在多个细胞衰老体系中则呈显著下调...     

Mesenchymal Stem Cells Suppress Severe Asthma by Directly Regulating Th2 Cells and Type 2 Innate Lymphoid Cells

Patients with severe asthma have unmet clinical needs for effective and safe therapies. One possibility may be mesenchymal stem cell (MSC) therapy, which can improve asthma in murine models. However, it remains unclear how MSCs exert their beneficial effects in asthma. Here, we examined the effect of human umbilical cord blood-derived MSCs (hUC-MSC) on two mouse models of severe asthma, namely, Alternaria alternata-induced and house dust mite (HDM)/diesel exhaust particle (DEP)-induced asthma. hUC-MSC treatment attenuated lung type 2 (Th2 and type 2 innate lymphoid cell) inflammation in both models. However, these effects were only observed with particular treatment routes and timings. In vitro co-culture showed that hUC-MSC directly downregulated the interleukin (IL)-5 and IL-13 production of differentiated mouse Th2 cells and peripheral blood mononuclear cells from asthma patients. Thus, these results showed that hUC-MSC treatment can ameliorate asthma by suppressing the asthmogenic cytokine production of effector cells. However, the successful clinical application of MSCs in the future is likely to require careful optimization of the route, dosage, and timing.     

Shaping Cellular Form and Function by Autophagy

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the g-aminobutyric acid type A receptor (GABA_A receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function.

Addendum to:

Presynaptic Terminals Independently Regulate Synaptic Clustering and Autophagy of GABA_A Receptors in Caenorhabditis elegans

.A.M. Rowland, J.E. Richmond, J.G. Olsen, D.H. Hall and B. A. Bamber

J Neurosci 2006; 26:1711-20     

模拟微重力诱导PC12细胞衰老的初步探讨

目的：构建模拟微重力条件下PC12细胞的培养体系,探讨模拟微重力对PC12细胞衰老的影响。方法：用Cytodex-3型微载体作为PC12细胞的贴附载体,旋转细胞培养系统所提供10-2g的微重力环境进行模拟微重力条件下的细胞培养。在倒置显微镜下观察PC12细胞的生长情况;用扫描电镜观察PC12细胞超微结构的变化;衰老相关β半乳糖苷酶（SA-β-gal）特异性染色对衰老的PC12细胞进行评估。结果：光镜下模拟微重力培养的PC12细胞表现出类衰老细胞的形态,扫描电子显微镜下观察发现其微绒毛增多。SA-β-gal染色的结果显示在模拟微重力的作用下,PC12细胞SA-β-gal的活性升高。结论：模拟微重力可以引起PC12细胞衰老样的形态变化,以及SA-β-gal的活性升高。     

模拟微重力诱导PC12 细胞衰老的初步探讨

目的:构建模拟微重力条件下PC12细胞的培养体系,探讨模拟微重力对PC12细胞衰老的影响。方法:用Cytodex-3型微载体作为PC12细胞的贴附载体,旋转细胞培养系统所提供10-2g的微重力环境进行模拟微重力条件下的细胞培养。在倒置显微镜下观察PC12细胞的生长情况;用扫描电镜观察PC12细胞超微结构的变化;衰老相关β半乳糖苷酶(SA-β-gal)特异性染色对衰老的PC12细胞进行评估。结果:光镜下模拟微重力培养的PC12细胞表现出类衰老细胞的形态,扫描电子显微镜下观察发现其微绒毛增多。SA-β-gal染色的结果显示在模拟微重力的作用下,PC12细胞SA-β-gal的活性升高。结论:模拟微重力可以引起PC12细胞衰老样的形态变化,以及SA-β-gal的活性升高。     

Exosomes Derived from Mesenchymal Stem Cells Suppress Angiogenesis by Down-Regulating VEGF Expression in Breast Cancer Cells

Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules.     

泛素偶联酶2C促进肺癌细胞增殖和迁移与抑制细胞衰老

泛素偶联酶2C与多种肿瘤细胞的增殖密切相关,但其与肺癌发生和发展的关系尚不明确。 本研究以肺癌A549细胞为材料,通过RT-PCR、Western印迹、免疫荧光、SA-β-Gal细胞衰老染色、细胞划痕和Trans-well实验,阐明UBE2C与肺癌细胞的增殖、衰老和迁移能力的关系。结果显示,UBE2C在肺癌细胞中的表达明显高于正常细胞。利用基因修饰技术瞬时过表达或靶向沉默UBE2C后,在肺癌A549细胞中,UBE2C的mRNA和蛋白质水平显著增加3.5倍或减少0.5倍,显著促进或抑制细胞增殖,进而减少或增加细胞的凋亡率。过表达UBE2C后,显著抑制细胞衰老;但沉默UBE2C后,则增加细胞衰老。此外,过表达UBE2C后,下调转移相关基因E-钙黏着蛋白的mRNA和蛋白质表达水平,且上调波形蛋白基因的表达水平,进而促进肺癌细胞的迁移。但靶向敲除UBE2C后,上调E-钙黏着蛋白,同时下调波形蛋白表达水平,进而抑制肺癌细胞的迁移。本研究的开展将明确UBE2C在肺癌中的作用及其机制,为以UBE2C为靶点,提高病人生存期提供了理论基础。     

Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells

Autologous c-kit⁺ cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G₁ phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G₂/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.