DNA梯度化分析软件系统在细胞凋亡定量检测中的应用

目的:检验我们设计的DNA梯度化分析软件在细胞凋亡定量检测中的应用价值 方法:用Camptothesin(CAM)诱导U937细胞株凋亡,通过该系统计算DNA琼脂糖凝胶电泳后DNA的梯度值,并与凋亡细胞胞浆寡聚核小体的Elisa检测值进行相关性分析.结果:随着CAM浓度的增加,DNA琼脂糖凝胶电泳后梯度化程度越明显,测出的梯度值也越大,而且与胞浆寡聚核小体的Elisa检测值呈正相关(r=0.989,p<0.01)结论:利用DNA梯度化分析系统可对琼脂糖凝胶电泳后的凋亡细胞DNA片段,进行定量分析,提供简便直接的方法.

转化生长因子β相关激酶1抑制剂对人肝癌细胞系HepG2脂滴积累的影响及其机制

目的观察转化生长因子β相关激酶1(TAK1)抑制剂(NG25)对人肝癌细胞系HepG2脂滴积累的影响,并探讨其作用机制。方法取HepG2细胞并分为两组,处理组细胞加入2μmol/L NG25处理24 h,对照组加入等体积DMSO处理24 h,两组均同时用终浓度为250μmol/L脂肪酸(OA、PA各125μmol/L)共同孵育,采用油红O染色观察细胞脂滴积累情况(油红O染色红光强度),荧光定量PCR法和蛋白免疫印迹法检测细胞过氧化物酶体增殖物激活受体γ(PPARγ)、诱导细胞凋亡DNA分裂因子样效应因子C(CIDEC)mRNA、蛋白相对表达量,双荧光素酶报告基因检测系统检测细胞荧光素酶活性。另取HepG2细胞并分为3组,恢复组细胞预先转染1μg质粒PPARγ后再加入2μmol/L NG25处理24 h,处理组细胞加入2μmol/L NG25处理24 h,对照组细胞加入等体积DMSO处理24 h,3组同时用终浓度250μmol/L脂肪酸(OA、PA各125μmol/L)共同孵育,荧光定量PCR法和蛋白免疫印迹法检测细胞CIDEC mRNA、蛋白。结果与对照组比较,处理组油红O染色红光强度弱(P<0.05)。与对照组比较,处理组PPARγ及CIDEC mRNA、蛋白相对表达量和PPARγ启动子荧光素酶活性降低(P均<0.05);与对照组比较,恢复组和处理组CIDEC mRNA和蛋白相对表达量降低(P均<0.05)。结论NG25对HepG2细胞脂滴积累有抑制作用,机制可能是抑制PPARγ的转录,进而下调CIDEC表达,即NG25通过PPARγ-CIDEC信号途径最终抑制HepG2细胞脂滴积累。

Review:DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis

Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senes- cence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent. Extensive research on tumor suppressor genes, oncogenes, the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are tied to cell proliferation, cell-cycle arrest, cellular senescence and apoptosis. DNA-damage responses are complex, involving “sensor” proteins that sense the damage, and transmit signals to “transducer” proteins, which, in turn, convey the signals to numerous “effector” proteins implicated in specific cellular pathways, including DNA repair mechanisms, cell-cycle checkpoints, cellular senescence and apoptosis. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/recombination and cellular senescence, effects that are generally distinct from their function in apoptosis. In this review, we report progress in understanding the molecular networks that regulate cell-cycle checkpoints, cellular senescence and apoptosis after DNA damage, and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

DNA fragmentation in apoptosis

Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo. Recent work also suggests the existence of additional endonucleases for DNA degradation. Understanding the roles of individual endonucleases in apoptosis, and how they might coordinate to degrade DNA in different tissues during normal development and homeostasis, as well as in various diseased states, will be a major research focus in the near future.

Lethal effect and apoptotic DNA fragmentation in response of D-GalN-treated mice to bacterial LPS can be suppressed by pre-exposure to minute amount of bacterial LPS: Dual role of TNF receptor 1

AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 μg). Both control and experimental groups simultaneously were challenged with LPS plus D-GaIN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GaIN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GaIN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GaIN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.

生长抑制DNA损伤基因45的功能及其作用

许多内源性和外源性的损伤因素均能导致DNA损伤,如活性氧、紫外线辐射、电离辐射、化学致癌药物及烷化剂等。哺乳动物细胞对于致DNA损伤因素的各种刺激会产生一系列应答反应,如细胞周期抑制、DNA修复、DNA去甲基化、细胞生存和细胞凋亡等。生长抑制DNA损伤基因45(Gadd45)在DNA损伤诱导的细胞应答反应中发挥重要作用。细胞内、外环境的多种因素在转录水平、翻译后水平等多个层次对Gadd45进行精确调节。Gadd45家族蛋白与年龄相关疾病、寿命及老化相关。

DDIAS在结直肠癌中的表达及其临床意义

目的探讨DNA损伤诱导的细胞凋亡抑制因子(DDIAS)在结直肠癌组织中的表达及其临床意义。方法回顾性收集2018年1月至2020年12月杭州市富阳区第一人民医院手术切除的176例结直肠癌组织(肿瘤组)和相应的远端切缘组织(正常组),采用免疫组化EnVision染色检测两组标本DDIAS表达情况,分析DDIAS表达与结直肠癌临床病理参数的关系。结果肿瘤组标本DDIAS阳性率为89.8%(158/176),显著高于正常组的44.9%(79/176)(P<0.001)。T1~3浸润深度患者DDIAS阳性率为85.1%(74/87),显著低于T4浸润深度的94.4%(84/89)(P<0.05)。不同年龄、性别、肿瘤分化程度、淋巴结转移、发生部位、肿瘤病理类型、错配修复蛋白表达、Ki-67指数患者DDIAS阳性率比较,差异均无统计学意义(均P>0.05)。结论DDIAS在结直肠癌组织中高表达,并与肿瘤T分期有关。

锌与基因表达的研究进展

锌是机体所必需的一种微量元素,在细胞分化和生长中起着重要作用。细胞内的部分锌存在于细胞核内,在维持遗传稳定性和调控基因表达中起着重要作用。近年来,有关锌对基因表达的影响主要集中于转录水平,即锌通过影响转录因子(含有锌指域)的活性进而调控细胞增殖、分化及细胞死亡。本文就锌调控基因表达的相关方面作一综述。

Actions of human telomerase beyond telomeres

Telomerase has fundamental roles in bypassing cellular aging and in cancer progression by maintaining telomere homeostasis and integrity. However, recent studies have led some investigators to suggest novel biochemical properties of telomerase in several essential cell signaling pathways without apparent involvement of its well established function in telomere maintenance. These observations may further enhance our understanding of the molecular actions of telomerase in aging and cancer. This review will provide an update on the extracurricular activities of telomerase in apoptosis, DNA repair, stern cell function, and in the regulation of gene expression.

Bile acids as endogenous etiologic agents in gastrointestinal cancer

Bile acids are implicated as etiologic agents in cancer of the gastrointestinal (GI) tract, including cancer of the esophagus, stomach, small intestine, liver, biliary tract, pancreas and colon/rectum. Deleterious effects of bile acid exposure, likely related to carcinogenesis, include: induction of reactive oxygen and reactive nitrogen species; induction of DNA damage; stimulation of mutation; induction of apoptosis in the short term, and selection for apoptosis resistance in the long term. These deleterious effects have, so far, been reported most consistently in relation to esophageal and colorectal cancer, but also to some extent in relation to cancer of other organs. In addition, evidence is reviewed for an association of increased bile acid exposure with cancer risk in human populations, in specific human genetic conditions, and in animal experiments. A model for the role of bile acids in GI carcinogenesis is presented from a Darwinian perspective that offers an explanation for how the observed effects of bile acids on cells contribute to cancer development.

New insights into p53 activation

The tumor suppressor p53 is a multifunctional, highly regulated, and promoter-specific transcriptional factor that is uniquely sensitive to DNA damage and cellular stress signaling. The mechanisms by which p53 directs a damaged cell down either a cell growth arrest or an apoptotic pathway remain poorly understood. Evidence suggests that the in vivo functions of p53 seem to balance the cell-fate choice with the type and severity of damage that occurs. The concept of antirepression, or inhibition of factors that normally keep p53 at bay, may help explain the physiological mechanisms for p53 activation. These factors also provide novel chemotherapeutic targets for the reactivation of p53 in tumors harboring a wild-type copy of the gene.

Analysis of cell cycle's correlation of γ-H2AX

Objective： To analyze and discuss cell cycle＇s correlation of y-H2AX, so as to accumulate the data for the further studies of y-H2AX. Methods： MOLT-4 cells, and peripheral blood lymphocytes （PBLs）, with or without 48 h stimulation of phytohemagglutinin （PHA）, were irradiated by ultraviolet rays （UV rays）. Fluorescence-labeled y-H2AX antibody was used to detect γ-H2AX foci at the DNA double-strand breaks （DSBs） in chromatin, DNA damage was analyzed by flow cytometry, cell cycle and cell apoptosis were detected by sub-G1 peak method, the expression of γ-H2AX was detected by Western blot. Results： With the progression of time, sub-G1 peak emerged apparently in the DNA histograms, and the cells of apoptosis increased gradually; with the progression of time, the increase of γ-H2AX emerged and firstly raised, then decreased; PBLs with 48 h stimulation of PHA entered apparently cell cycle, cells of S and G2/M phase emerged, and PBLs without stimulation of PHA did not enter cell cycle; Western blot showed the increase of the expression of γ-H2AX, and the increase also firstly raised, then decreased. Conclusion： γ-H2AX expressed in the cells of stationary phase and proliferative phase, and with the progression of time, the increase of γ-H2AX firstly raised, and then decreased.

The Mechanism pf Weichang'an in Inducing Apoptosis of Gastric Cancer SGC7901 Cells

Objective:To investigate the effect of Chinese medicine Compound Weichang'an(胃肠安)for invig-orating the spleen on apoptosis of gastric cancer SGC7901 cells and its possible mechanism.Methods:The gas-trie cancer SGC-7901 cells were divided into different mass concentration groups(0 mg·L^(-1),500 mg·L^(-1)1000 mg·L^(-1),1500 mg·L^(-1),2000 mg·L^(-1)).CCK8 and monoclonal test were applied to detect prolifera-tion ability;comet assay was used to detect DNA damage.After DCFH-DA fluorescent labeling,the level of ROS activity was detected by flow cytometer;after AnnexinV-FTC/PI double labeling,the proportion of apoptotic ellls was detected by flow cytometer;after JC-1 staining,the mi tochondri almembrane potential was detected by flow cytometer;after FTTC-DEVD-FMK staining,the ratio of Caspase activity was detected by flow cytometer.Results:Weichang an inhibited cell proliferation and reduced cell colony formation in a time-dose-dependent manner;the results of comet electrophoresis showed that Weichang'an could induce DNA damage in gastric cancer cells;com-pared with control group.the ratio of Weichang'an's intervention with the apoptosis of gastric cancer cells in-creased(P<0.05),the mitochondrial membrane potential decreased(P<0.05),the activity of Caspase3 and Caspase9 increased(P<0.05),and the intracellular ROS level increased(P<0.05).Among them,the effect of Weichang'an treatment group(1000 mg·L^(-1))was the most significant.Conclusion:Weichang'an has an inhibi-tory effect on the proliferation of gastric cancer SGC7901 cells and can induce cell apoptosis.Its mechanism may be related with the ROS-mediated pathway of mitochondrial apoptosis and DNA damage.

Cbl negatively regulates JNK activation and cell death

Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apoptosis -- nerve growth factor （NGF） deprivation and DNA damage -- cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation （and most likely, activation） of c-Cbl. Targeting c-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase （JNK） signaling. We demonstrate that Cbl proteins interact with the JNK pathway components mixed lineage kinase （MLK） 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK activation and on cell death.

REV3 and p53 are mutually regulated to affect colon cancer cell growth and apoptosis

REV3 encodes a catalytic subunit of DNA polymerase ; required for translesion DNA synthesis. The inhibition of REV3 expression induces persistent DNA damage and growth arrest in the G1 phase, which is initiated by p53 activation. We speculated thatp53 plays a critical role in regulating apoptosis and cell growth through inhibition of REV3. In this study, we found that experimental suppression of REV3 induced apoptosis and arrested colon cancer at the G1 phase. Surprisingly, suppression of p53 promoted REV3 expression and the accumulation of S-phase cells, suggesting that excessive REV3 activity interferes with replicative DNA synthesis. The above observations collectively reveal genetic interactions between REV3 andp53 in the regulation of apoptosis and cell growth in colon cancer cells.