骨髓增殖性疾病的研究进展:后JAK2突变时代的几个临床问题

 细胞死亡与细胞增殖是对立面,是机体的基本细胞生物学机制之一。近年来报道的越来越多的细胞死亡方式令人困惑。最近细胞死亡命名委员会（NCCD）的细胞死亡分类建议书梳理了相关的研究进展,使概念更清晰。文章概述其主要精神,补充一些文献资料,并探讨细胞死亡研究的意义。     

DCs中NLRP3炎性复合体活化诱导IL-1依赖的获得性抗肿瘤免疫应答

细胞死亡有多种形式,可以分为免疫性的和非免疫性的。正常细胞更新中的细胞死亡可认为是非免疫性的;而放射治疗或者化学治疗药物引起的细胞死亡是免疫性的,可以引起免疫应答,并且对于治疗效应是必须的。     

自噬性细胞死亡研究进展

自噬性细胞死亡（autophagic cell death）为Ⅱ型程序性细胞死亡,同细胞凋亡（Ⅰ型程序性细胞死亡）一样被发现多年,但近些年才引起人们的关注。现已证实自噬性细胞死亡是除细胞凋亡外另一重要的死亡调控机制。自噬性细胞死亡在参与细胞存活、分化、增殖和衰老的同时还可能参与多种疾病的发生发展,尤其与肿瘤及其电离辐射的关系越来越密切,因此研究自噬发生及调控的分子机制对于进一步了解自噬与疾病的关系,探索临床治疗方案具有重要的意义。本文就近些年自噬性细胞死亡的研究进展作一详细综述。     

自噬与肿瘤治疗

自噬是细胞内蛋白、细胞器降解的渐变过程.目前,自噬性细胞死亡被认为是第Ⅱ类程序化细胞死亡.对肿瘤细胞自噬的抑制可导致不同的结果--生存或死亡,因此自噬是细胞死亡机制还是细胞保护机制存在争议.总的来说,对自噬的利用可推动新的肿瘤治疗方法的进展.     

肿瘤放射治疗中的编程性细胞死亡

肿瘤放射治疗中的编程性细胞死亡傅晓颖综述曹世龙审校（上海医科大学附属肿瘤医院２０００３２）一、编程性细胞死亡概念编程性细胞死亡（ａｐｏｐｔｏｓｉｓ）是不同于坏死（ｎｅｃｒｏｓｉｓ）的有其形态学和生物化学特征的受基因调控的细胞主动死亡形式。编程性细胞死...     

p53 基因相关的细胞非经典死亡

[摘要] 程序性细胞死亡是多细胞生物死亡的重要生物学过程,其调控方式复杂,对维持细胞内环境稳定十分重要。在过去的几年中,除了凋亡之外,对程序性细胞死亡的非凋亡形式的研究也取得了进展。p53 作为经典的肿瘤抑制因子,除了控制细胞增殖和凋亡外,也参与非典型细胞死亡调控。p53 通过对其下游靶点的转录调控以及与关键蛋白的直接作用,直接或间接调节细胞的非典型死亡。本文对p53 在凋亡、铁中毒、细胞程序性坏死、自噬性细胞死亡、有丝分裂灾难、副凋亡等几种非典型细胞死亡模式中的作用作一综述,为肿瘤抑制机制的阐明及癌症药物研制提供相应参考。     

雷帕霉素上调Beclin 1诱导肝癌细胞死亡的体外研究

 目的研究雷帕霉素诱导肝癌细胞死亡的方式以及时效与量效关系。方法锥虫蓝排斥实验分析雷帕霉素诱导肝 癌细胞死亡的时效、量效关系；透射电镜技术检测雷帕霉素诱导肝癌细胞死亡方式；Western blot检测Beclin 1表 达变化。结果雷帕霉素诱导肝癌细胞死亡具有时效和量效关系；可诱导肝癌细胞的凋亡和自噬性细胞死亡；可上调 Beclin 1蛋白的表达。结论雷帕霉素可能是一种新的有效的肝癌化疗药物。     

凋谢

一、概念自Virchow时代以来,人们一直把细胞死亡与坏死混为一谈。实际上,细胞死亡有两种截然不同的方式,一种是病理状态下的坏死,另一种则是广泛存在于生理及病理情况下的凋谢。凋谢的形态为人们所熟知,     

肾癌中调节性细胞死亡的新动向和未来展望

肾癌是泌尿系统常见的恶性肿瘤之一。近年来,我国肾癌的发病率呈逐年上升的趋势,严重威胁着人们的健康。调节性细胞死亡是由一种细胞主动有序的死亡方式,普遍存在于生命活动过程中,在维系生命活动的平衡中发挥着至关重要的作用。近期Science杂志上报道了一种新的调节性细胞死亡方式即铜死亡,进一步强化了生命体中细胞死亡的重要性。随着对调节性细胞死亡认识的不断深入,越来越多的研究显示不同的调节性细胞死亡（如铁死亡、焦亡、自噬等）均与肾癌的发生、发展密切相关。如诱导细胞铁死亡将显著抑制肾癌的侵袭和转移、并与肾癌患者的更好预后密切相关;细胞焦亡不仅可以诱导肾癌细胞死亡还可以激活抗肾癌的免疫应答;自噬在肾癌中具有“双向”作用,增强自噬可抑制肾癌细胞生长,但也可能减弱联合用药治疗的效果;抑制细胞凋亡和坏死性凋亡可以显著促进肾癌细胞的增殖、侵袭等。本文将综述铁死亡、细胞焦亡、自噬、细胞凋亡和坏死性凋亡的分子机制和在肾癌发生、发展中作用的研究进展并进行展望,为探索肾癌的发病机制和潜在的治疗靶点提供新的视角。     

细胞凋亡与肝癌研究进展

细胞增殖、分化和死亡是多细胞个体发育过程中三项最基本的生命活动,三者都以保证个体的整体生存和生命活动的正常进行为目标,其中任一过程发生异常都会引起机体病变.近年来,细胞死亡与疾病的关系逐渐得到人们的关注和重视.业已发现,恶性肿瘤的发生、发展不仅是细胞增殖失控、分化失调的结果,细胞死亡的异常在其中也起着重要作用.细胞死亡有坏死(necrosis)与凋亡(apoptosis)二种不同的基本形式.目前对细胞凋亡的研究已有较大进展,为探索恶性肿瘤的防治方法提供了一个崭新的思路.1 细胞凋亡的概念细胞凋亡于1972年由Kerr等首次提出.机体正常生长发育过程中的细胞死亡称为程序性细胞死亡(programmedcell death,PCD),指细胞在一定条件下,受自身基因调控,自行结束生命活动的过程.术语PCD强调的是这一过程机制     

Combined low dose ionizing radiation and green tea-derived epigallocatechin-3-gallate treatment induces human brain endothelial cells death

The microvasculature of brain tumors has been proposed as the primary target for ionizing radiation (IR)-induced apoptosis. However, the contribution of low dose IR-induced non-apoptotic cell death pathways has not been investigated. This study aimed to characterize the effect of IR on human brain microvascular endothelial cells (HBMEC) and to assess the combined effect of epigallocatechin-3-gallate (EGCg), a green tea-derived anti-angiogenic molecule. HBMEC were treated with EGCg, irradiated with a sublethal (≤10 Gy) single dose. Cell survival was assessed 48 h later by nuclear cell counting and Trypan blue exclusion methods. Cell cycle distribution and DNA fragmentation were evaluated by flow cytometry (FC), cell death was assessed by fluorimetric caspase-3 activity, FC and immunoblotting for pro-apoptotic proteins. While low IR doses alone reduced cell survival by 30%, IR treatment was found more effective in EGCg pretreated-cells reaching 70% cell death. Analysis of cell cycle revealed that IR-induced cell accumulation in G2-phase. Expression of cyclin-dependent kinase inhibitors p21(CIP/Waf1) and p27(Kip) were increased by EGCg and IR. Although random DNA fragmentation increased by approximately 40% following combined EGCg/IR treatments, the synergistic reduction of cell survival was not related to increased pro-apoptotic caspase-3, caspase-9 and cytochrome C proteins. Cell necrosis increased 5-fold following combined EGCg/IR treatments while no changes in early or late apoptosis were observed. Our results suggest that the synergistic effects of combined EGCg/IR treatments may be related to necrosis, a non-apoptotic cell death pathway. Strategies sensitizing brain tumor-derived EC to IR may enhance the efficacy of radiotherapy and EGCg may represent such a potential agent.     

Retention of Cell Adhesion and Growth Capability in Human Cervical Cancer Cells Deprived of Cell Anchorage

Cell adhesion is linked to various regulatory processes of growth as well as apoptotic cell death in normal and transformed epithelial cells. We investigated changes of cellular responses to the deprivation of cell anchorage associated with immortalization or malignant transformation. Normal human ectocervical keratinocytes (NCE cells) deprived of cell anchorage become susceptible to apoptosis, and in parallel they lose their adhesion to the culture substratum. The loss of cell adhesion is not directly due to apoptosis. NCE16 cells, an immortalized but not malignantly transformed subline of NCE, underwent apoptosis and lost cell adhesion in suspension, as the NCE cells did. By contrast, apoptosis was not inducible in human cervical cancer-derived C33A cells in suspension. Of other cell lines derived from human cervical cancer, SiHa cells showed a weak apoptotic response and Caski cells were highly sensitive to apoptosis in the absence of cell anchorage. Unlike NCE or NCE16 cells, all these cancer cells retained cell adhesion as well as growth capability in suspension cultures. These results indicate that retention of cell adhesion and growth capability in the absence of cell anchorage is more closely associated with cancer cell lines than resistance to apoptosis upon the deprivation of cell anchorage.     

Micromolar taxol, with or without hyperthermia, induces mitotic catastrophe and cell necrosis in HeLa cells

Purpose: Although the mode of action of taxol, when used in nanomolar or micromolar concentrations during long periods, is extensively studied, there are few data available on taxol-mediated cytotoxicity when the drug is applied for a short time alone or in combination with hyperthermia. We studied the effect of taxol and hyperthermia on cell cycle kinetics, proliferation, and mode of cell death in human cervical carcinoma HeLa cells, following a scheme which resembles the one currently used in regional chemotherapy. methods: Cells were incubated with micromolar doses of taxol for two h under normothermic or hyperthermic conditions and then cultured in drug-free medium for several days. Cell viability was assessed via an MTT assay. Necrotic and apoptotic cell death was determined using Trypan blue staining and TUNNEL assay, respectively. Flow cytometry was used for the analysis of cell cycle kinetics and the counting of apoptotic cells. Mitotic index, nuclear morphology and nuclear envelope organization were analyzed by fluorescence microscopy. results: Cells exposed to micromolar doses of taxol for 2 h and then transferred to a drug-free medium for 24 h were arrested at G2/M or M phase. When treated cells were cultured in normal media for longer periods, most of them remained in a tetraploid state, became multinucleated without properly completing cytokinesis and died mostly by necrosis. Hyperthermia alone exerted a cytotoxic effect, inhibited proliferation and caused minor changes in cell cycle kinetics. When combined with taxol treatment, hyperthermia modified the cell cycle-arresting effects of the drug, but did not alter significantly taxol-mediated cytotoxicity. conclusions: From these data we conclude that short time incubation of HeLa cells under normothermic or hyperthermic conditions with micromolar concentrations of taxol is sufficient enough to induce extended cell growth arrest and cell death by necrosis.     

Toll样受体4在淋巴瘤细胞株中的表达及其对细胞增殖和耐药的影响

目的 探讨Toll样受体4（Toll-like receptor 4,TLR4）在人淋巴瘤细胞株中的表达及其对细胞增殖和耐药的影响。方法 采用RT-PCR、qPCR和Western blot检测8株淋巴瘤细胞株中TLR4 mRNA及蛋白的表达情况并筛选出TLR4高表达株,对高表达株进行基因测序以排除MyD88 L265P基因突变。将TLR4高标达细胞株分为空白对照组、TAK-242组、细菌脂多糖（lipopolysaccharides, LPS）组和LPS+TAK-242组,分别进行细胞增殖和阿霉素耐药实验。采用CCK-8试剂盒检测其增殖活性和细胞杀伤率,用Western blot法检测增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）和P-糖蛋白（P-glycoprotein, P-gp）的变化。结果 8株淋巴瘤细胞株中TLR4 mRNA和蛋白广泛表达,其中Burkitt淋巴瘤细胞株Raji的表达水平最高。Raji细胞MyD88基因为野生型。与空白对照组相比,TAK-242和LPS+TAK-242组Raji细胞的增殖活性无明显改变（P=2.19, P=1.85）,LPS组Raji细胞的增殖活性明显升高（P=0.016）, LPS组PCNA蛋白表达明显升高（P=0.009）。阿霉素在半数抑制浓度下各组杀伤率分别为：空白对照组（49.23±2.03）%、TAK-242组（51.41±1.12）%、LPS组（24.65±3.17）%、LPS+TAK-242组（41.17±2.69）%,可见LPS组细胞杀伤率明显降低（P=0.002）。P-gp蛋白表达明显升高（P=0.001）。结论 TLR4分子在淋巴瘤细胞株中广泛表达,尤以Burkitt淋巴瘤细胞株Raji最高,激活TLR4可以促使肿瘤细胞增殖和耐药,其机制可能与PCNA和P-gp的表达上调有关。     

Paclitaxel induced apoptosis in breast cancer cells requires cell cycle transit but not Cdc2 activity

Purpose Paclitaxel (PTX) is a widely used chemotherapy agent and may cause cell death by apoptosis subsequent to microtubule (MT) disruption. In this paper, we have investigated whether cell cycle transit and or Cdc2 (Cdk1) activity is required for the apoptosis induced by PTX.Methods Cell cycle was analyzed by flow cytometry, Cdc2 was assayed bio chemically. Cdc2 activity was decreased by siRNA and dominant negative (dn) Cdc2 expression. Cells were arrested by chemical or biological inhibitors in a G₁or S phase. Apoptosis was measured by DNA fragmentation and examination of nuclei by microscopy. JNK and AKT activations were assessed as well.Results Cell cycle inhibition was highly effective in decreasing PTX induced apoptosis. MT morphology was not altered by these inhibitors. PTX induced JNK activity or AKT mediated BAD phosphorylation was unaffected by cell cycle inhibitors. Abrogation of Cdc 2 activity was without effect on PTX induced apoptosis.Conclusions While cell cycle transit is required for PTX induced apoptosis; Cdc2 activity is not required.     

Ganoderma Lucidum Polysaccharides Target a Fas/Caspase Dependent Pathway to Induce Apoptosis in Human Colon Cancer Cells

Ganoderma lucidum polysaccharides (GLP) extracted from Ganoderma lucidum have been shown to inducecell death in some kinds of cancer cells. This study investigated the cytotoxic and apoptotic effect of GLP onHCT-116 human colon cancer cells and the molecular mechanisms involved. Cell proliferation, cell migration,lactate dehydrogenase (LDH) levels and intracellular free calcium levels ([Ca2+]i) were determined by MTT,wound-healing, LDH release and fluorescence assays, respectively. Cell apoptosis was observed by scanningand transmission electron microscopy. For the mechanism studies, caspase-8 activation, and Fas and caspase-3expression were evaluated. Treatment of HCT-116 cells with various concentrations of GLP (0.625-5 mg/mL)resulted in a significant decrease in cell viability (P< 0.01). This study showed that the antitumor activity ofGLP was related to cell migration inhibition, cell morphology changes, intracellular Ca2+ elevation and LDHrelease. Also, increase in the levels of caspase-8 activity was involved in GLP-induced apoptosis. Western blottingindicated that Fas and caspase-3 protein expression was up-regulated after exposure to GLP. This investigationdemonstrated for the first time that GLP shows prominent anticancer activities against the HCT-116 humancolon cancer cell line through triggering intracellular calcium release and the death receptor pathway.     

Histone deacetylase inhibitor‐mediated cell death is distinct from its global effect on chromatin

Romidepsin and vorinostat are histone deacetylase inhibitors (HDACis) that have activity in T‐cell lymphomas, but have not gained traction in solid tumors. To gain deeper insight into mechanisms of HDACi efficacy, we systematically surveyed 19 cell lines with different molecular phenotypes, comparing romidepsin and vorinostat at equipotent doses. Acetylation at H3K9 and H4K8 along with 22 other histone lysine acetylation and methylation modifications were measured by reverse phase proteomics array (RPPA), and compared with growth inhibition (IC50), and cell cycle arrest. These assays typically used to assess HDACi effect showed that acetylation and methylation of specific lysine residues in response to HDACis were consistent across cell lines, and not related to drug sensitivity. Using a treatment duration more reflective of the clinical exposure, cell death detected by annexin staining following a 6 h drug exposure identified a subset of cell lines, including the T‐cell lymphoma line, that was markedly more sensitive to HDAC inhibition. Kinetic parameters (Km values) were determined for lysine acetylation and for cell cycle data and were themselves correlated following HDACi exposure, but neither parameter correlated with cell death. The impact on cell survival signaling varied with the molecular phenotype. This study suggests that cellular response to HDACis can be viewed as two distinct effects: a chromatin effect and a cell death effect. All cells undergo acetylation, which is necessary but not sufficient for cell death. Cells not primed for apoptosis will not respond with cell death to the impact of altered histone acetylation. The divergent apoptotic responses observed reflect the variable clinical outcome of HDACi treatment. These observations should change our approach to the development of therapeutic strategies that exploit the dual activities of HDACis.     

Molecular mechanisms of celery seed extract induced apoptosis via s phase cell cycle arrest in the BGC-823 human stomach cancer cell line

Background: Mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Loss of cell cycle control, leading to uncontrolled proliferation, is common in cancer. Therefore, the identification of potent and selective cyclin dependent kinase inhibitors is a priority for anti-cancer drug discovery. There are at least two major apoptotic pathways, initiated by caspase-8 and caspase-9, respectively, which can activate caspase cascades. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. This is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane. Anti-tumor effects of celery seed extract (CSE) and related mechanisms regarding apoptosis were here investigated in human gastric cancer BGC-823 cells. Methods: CSE was produced by supercritical fluid extraction. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and apoptosis by flow cytometry using Annexin/PI staining and DAPI staining and a laser scanning confocal microscope (LSCM). Cell cycling was evaluated using PI staining with flow cytometry and expression of cell cycle and apoptosis-related proteins cyclin A, CDK2, bcl-2 and bax was assessed by immunohistochemical staining. Results: CSE had an anti-proliferation effect on human gastric cancer BGC-823 cells in a dose- and time-dependent manner. After treatment, the apoptotic rate significantly increased, with morphological changes typical of apoptosis observed with LSCM by DAPI staining. Cell cycle and apoptosis related proteins, such as cyclin A, CDK2 and bcl-2 were all down-regulated, whereas bax was up-regulated. Conclusions: The molecular determinants of inhibition of cell proliferation as well as apoptosis of CSE may be associated with cycle arrest in the S phase.     

Chinese Medicinal Herb, Acanthopanax gracilistylus, Extract Induces Cell Cycle Arrest of Human Tumor Cells in vitro

We investigated the effect of a Chinese medicinal herb, Acanthopanax gracilistylus (AG), extract (E) on the growth of human tumor cell lines in vitro. AGE markedly inhibited the proliferation of several tumor cell lines such as MT-2, Raji, HL-60, TMK-1 and HSC-2. The activity was associated with a protein of 60 kDa, which was purified by gel-filtration chromatography. Cell viability analyses indicated that the treatment with AGE inhibits cell proliferation, but does not induce cell death. The mechanism of AGE-induced inhibition of tumor cell growth involves arrest of the cell cycle at the G₀/G₁ Stage without a direct cytotoxic effect. The cell cycle arrest induced by AGE was accompanied by a decrease of phosphorylated retinoblastoma (Rb) protein. Furthermore, cyclin-dependent kinases 2 and 4 (Cdk2 and Cdk4), which are involved in the phosphorylation of Rb, were also decreased. These results suggest that AGE inhibits tumor cell growth by affecting phosphorylated Rb proteins and Cdks.     

Cell killing and sensitization to heat shock by hypothermic incubation of asynchronous and synchronized mouse neuroblastoma cells

The effect of hypothermia on cell survival and on subsequent response to hyperthermia was studied in asynchronous and synchronized Neuro-2A cells. Cell cycle progression was blocked at temperatures below 27 degrees C. Immediately after shift to hypothermic temperatures, cells became more sensitive to hyperthermia. Development of thermosensitization was time and temperature dependent. Thermosensitization of cells by hypothermia was high at 0 degrees C and 15 degrees-30 degrees C and less at 5 degrees-10 degrees C. Sensitization started to occur before hypothermic cell death became manifest and developed gradually. Hypothermic cell death was observed when the cells were incubated for more than 1 day at temperatures of 0 degrees-24 degrees C with a minimal cell death during incubation at 6 degrees C. Thermosensitization of cells by hypothermia depended on the position of the cell in the cell cycle at the time of shift to hypothermic temperatures. Cells in late G1 and early S phase became more thermosensitive than did cells in G1 or late S-G2 phase. Furthermore G1-S cells were more sensitive to prolonged hypothermia alone than were G1 or late S-G2 cells. In contrast, late S-G2 cells were most sensitive to hyperthermia alone. It is concluded that the temperature- and cell cycle-dependent way of hypothermic induced cell death was similar to the thermosensitization of cells by hypothermia. But thermosensitization became manifest prior to the actual cell death, following hypothermic treatment.