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The effects of 2-chloro-2′-deoxyadenosine, β-D-arabinofuranosyl-2-fluoroadenine, and 5-aza-2′-deoxycytidine on promoter methylation of the selected tumor suppressor genes (i.e., ERα, BRCA1, E-cadherin, PTEN, and APC) were estimated using methylation-sensitive restriction analysis (MSRA) in K562 cells (human erythroleukemic cell line) and MCF-7 cells (human breast cancer cell line). In both cell lines all tested drugs completely reduced methylation of PTEN and APC promoters. The results indicate that the tested nucleoside analogues, which are known inhibitors of DNA synthesis, also are implicated in indirect (or direct in the case of 5-aza-dCyd) regulation of post-replicative DNA modifications (i.e., DNA methylation).  相似文献   

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《生物磁学》2011,(20):3801-3804,3808
目的:探讨抑制甲基转移酶(DNMT)对K562细胞中癌-睾丸抗原表达的影响及其机制。方法:分别采用针对DNMT家族不同成员的siRNA转染K562细胞,采用RT—PCR检测细胞中DNMT及癌-睾丸抗原的水平表达,并采用甲基化特异PCR(MSP)检测部分癌.睾丸抗原基因启动子的甲基化状态。结果:经siRNA干扰后,K562细胞中DNMT1、DNMT3a和DNMT3b的表达量均明显降低,癌-睾丸抗原CTl0的启动子区序列发生了去甲基化,但处于非甲基化状态的MAGE.A1启动子区没有发生任何改变。干扰DNMT组的K562细胞,再表达癌-睾丸抗原CT10、PRAME和CT9,而MAGE-A1、ssx-1的表达上调,但是NY-ESO—1、HCA587和HCA661的表达状况均没有任何影响。结论:在K562细胞中,干扰DNMT可使部分癌-睾丸抗原基因的启动子区发生去甲基化,从而导致相应的癌-睾丸抗原分子的再表达或表达增加。  相似文献   

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Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA topology and topoisomerase activity.  相似文献   

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In this paper the mean number N of nucleoli of cell nuclei and the relative frequency PN of cell nuclei with N nucleoli are investigated. To solve the first problem, the stereological model of convex shaped nuclei and nucleoli and the assumption of randomly-isotropically distributed objects in space are used. The model is developed for non-vanishing thickness T of tissue sections. The relationship between the unknown number N of nucleoli in cell nuclei and the mean number n of nucleoli in nuclear sections determined by counting is N = n (D+T)/(d + T). Here D and d are the mean values of caliper diameter for nuclei and for nucleoli, resp. The second problem is illustrated by means of some biomedical examples: The relative frequency PN of cell nuclei with N nucleoli can be approximated by a generalized Poisson distribution in all investigated cases. Therefore the mean nucleolar number N is the essential parameter to describe the frequencies of cell nuclei with different numbers of nucleoli.  相似文献   

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According to the method used in our laboratory, our group synthesized (DIPP-Trp)2-Lys-OCH3. It inhibited the proliferation of K562 and HeLa cells in a dose-and time-dependent manner with an IC50 of 15.12 and 42.23 µM, respectively. (DIPP-Trp)2-Lys-OCH3 induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)2-Lys-OCH3-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)2-Lys-OCH3 not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G2/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)2-Lys-OCH3 is a potential anticancer drug that intervenes in the signalling pathway.  相似文献   

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Different populations of DNA polymerase alpha in HeLa cells   总被引:3,自引:0,他引:3  
Three different populations of HeLa DNA polymerase alpha have been distinguished using a novel preparation of chromatin isolated using an isotonic salt concentration, which contains intact DNA. One synthesizes DNA in vitro at 85% of the rate in vivo, is found only in S-phase nuclei tightly associated with the nucleoskeleton and requires unbroken DNA in the form of chromatin as a template: we assume this is the authentic S-phase activity. On incubation at 37 degrees C, this activity dissociates from the nucleoskeleton to give a soluble activity that prefers broken templates. This soluble activity is in turn heterogeneous, containing active complexes of about 0 X 75 X 10(6) and 3 X 10(6) Mr. The third activity is also soluble and released by lysing cells at any stage of the cell cycle. It, too, prefers broken templates. The authentic activity is obscured by the soluble ones if broken templates are provided.  相似文献   

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Taurine Levels in Discrete Brain Nuclei of Rats   总被引:6,自引:1,他引:6  
Concentrations of taurine have been measured in 44 microdissected rat brain nuclei or areas. Taurine is ubiquitously present and distributed unevenly in the rat brain: the ratio of the highest (pyriform cortex) to lowest (midbrain reticular formation) concentrations is 4.7:1. High taurine levels were found in cerebral cortical areas, caudate-putamen, cerebellum, median eminence, and supraoptic nucleus. Acute pain stress reduced taurine levels in the hypothalamus and the lower brainstem nuclei but not in cortical areas. Increased locomotor and behavioral activities following a high dose of amphetamine elevated taurine concentrations significantly in the substantia nigra and locus ceruleus.  相似文献   

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目的:通过二烯丙基二硫诱导白血病K562细胞发生自噬性死亡,探讨其作用机制。方法:40 mg/LDADS作用K562细胞12小时后,透射电镜观察K562细胞超微结构,MDC染色荧光显微镜观察自噬泡及流式细胞仪定量检测自噬率,RT-PCR检测Beclin1mRNA的表达水平。结果:DADS作用后的K562细胞后,透射电镜可观察到胞质内出现大量自噬体;MDC染色荧光显微镜观察显示,K562细胞胞浆中的自噬泡明显增多,而空白组与溶媒组胞浆中的自噬泡很少;流式细胞术定量测定空白对照组、溶媒对照组、DADS药物组自噬率分别为(7.27±5.60)%、(7.10±5.13)%、(27.39±6.51)%(P〈0.05);空白对照组为0.658±0.007,溶媒对照组为0.671±0.012,两者的Beclin1mRNA的表达强度无明显差异(P〉0.05),DADS药物组为0.911±0.008,高于对照组(P〈0.05)。结论:二烯丙基二硫可诱导白血病k562细胞发生自噬性死亡,其机制可能与Beclin1的上调有关。  相似文献   

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双氢青蒿素对人白血病细胞K562增殖及凋亡的影响   总被引:1,自引:0,他引:1  
目的:研究双氢青蒿素对人白血病细胞增殖及凋亡的作用,探讨其对白血病的作用机制,为进一步研究提供依据。方法:体外培养K562细胞,用细胞计数法绘制生长曲线;流式细胞仪及荧光显微镜检测药物作用前后细胞凋亡作用;Western-blot测定药物作用前后线粒体、细胞浆细胞色素c的表达。结果:双氢青蒿素的浓度为1×10~(-4),1×10~(-5),1×10~(-6)l/L时,细胞生长受到显著抑制,并呈剂量依赖性;流式细胞仪检测出凋亡峰;Hoechst33342/PI双荧光染色可观察到明显的核浓缩、凝集等细胞凋亡表现;Western-blot检测1×10~(-5)mol/L药物处理细胞后线粒体细胞色素c表达水平下调1,细胞浆出现明显细胞色素c蛋白奈带。结论:双氢青蒿素能显著抑制人白血病细胞K562的生长,并诱导其凋亡,可能与线粒体途径有关。  相似文献   

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Dinucleosome formation is the first step in the organization of the higher order chromatin structure. With the ultimate aim of elucidating the dinucleosome structure, we constructed a library of human dinucleosome DNA. The library consists of PCR-amplifiable DNA fragments obtained by treatment of nuclei of erythroid K562 cells with micrococcal nuclease followed by extraction of DNA and adaptor ligation to the blunt-ended DNA fragments. The library was then cloned using a plasmid vector and the sequences of the clones were determined. The dominating clones containing the Alu elements were removed. A total of 1002 clones, which comprised a dinucleosome database, contained 84 and 918 clones from the clones before and after removing Alu elements, respectively. Approximately 70% of the clones were between 300 and 400 bp in size and they were distributed to various locations of all chromosomes except the Y chromosome. The clones containing A(2)N(8)A(2)N(8)A(2) or T(2)N(8)T(2)N(8)T(2) sequences were classified into three types, Type I (N shape), Type II (V shape) and Type III (M shape) according to DNA curvature plots. The locations of experimentally determined curved DNA segments matched well with the calculated ones though the clones of Types I and III showed additional curved DNA segments as revealed by the curvature plots. The distributions of complementary dinucleotides in the nucleosome DNA, at the ends of the dinucleosome DNA clones, allowed us to predict the positions of the nucleosome dyad axis, and estimate the size of the nucleosome core DNA, 125nt. The distributions of AA and TT dinucleotides, as well as other RR and YY dinucleotides, showed a periodicity with an average period of 10.4 bases, close to the values observed before. Mapping of nucleosome positions in the dinucleosome database based on the observed periodicity revealed that the nucleosomes were separated by a linker of 7.5+ approximately 10 x n nt. This indicates that the nucleosome-nucleosome orientations are, typically, halfway between parallel and antiparallel. Also an important finding is that the distributions of AA/TT and other RR/YY dinucleotides, apparently, reflect both DNA curvature and DNA bendability, cooperatively contributing to the nucleosome formation.  相似文献   

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The growth fraction, estimated by the monoclonal antibody Ki-67 labeling, and DNA content, assessed by ethidium bromide staining, were determined simultaneously in K562 leukemic cells by flow cytometry. A multiparametric analysis enabled the fraction of the cell population with G1, S, and G2 + M contents in Ki-67-positive and Ki-67-negative cells to be evaluated. Butyric acid (BUT) was used as positive control. The fraction of Ki-positive cells decreased with the BUT concentration, while the proportion of cells with G1 DNA content increased only in the Ki-negative cells. Adriamycin, aclacinomycin A, and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreased the fraction of Ki-positive cells by more than 50% for both anthracyclines and by 25% for fagaronine. Following treatment, Ki-negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.  相似文献   

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目的构建稳定表达红色荧光蛋白(red fluorescent protein,RFP)和嘌呤霉素(puromycin)抗性的K562.PM.RFP细胞株,便于慢性粒细胞性白血病研究中K562细胞的观察和筛选。方法采用PCR法获得RFP片段,将其插入到慢病毒pGC-FU-3FLAG-IRES—Puromycin载体中获得pGC—PM—RFP重组质粒,经脂质体转染到293T细胞中获得慢病毒LV—PM—RFP,有限稀释法检测慢病毒在293T细胞中的转染效率,用包装获得的慢病毒感染K562细胞,经嘌呤霉素筛选获得RFP阳性的K562-PM—RFP细胞株。结果PCR及测序结果证实目的基因RFP正确克隆至慢病毒质粒中,经慢病毒LV—PM-RFP感染的K562细胞能在嘌呤霉素抗性培养基中存活,并稳定表达RFP。结论成功构建了慢病毒重组质粒pGC—PM-RFP,并获得了携带RFP及嘌呤霉素抗性基因的K562-PM—RFP细胞株。  相似文献   

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Template-activating factors I (TAF-I) α and β have been identified as chromatin remodeling factors from human HeLa cells. TAF-Iβ corresponds to the protein encoded by thesetgene, which was found in an acute undifferentiated leukemia as a fusion version with thecangene via chromosomal translocation. To determine the localization of TAF-I, we raised both polyclonal and monoclonal antibodies against TAF-I. The proteins that react to the antibodies are present not only in human cells but also in mouse, frog, insect, and yeast cells. The mouse TAF-I homologue is ubiquitous in a variety of tissue cells, including liver, kidney, spleen, lung, heart, and brain. It is of interest that the amounts of TAF-Iα and β vary among hemopoietic cells and some specific cell types do not contain TAF-Iα. The level of the TAF-I proteins does not change significantly during the cell cycle progression in either HeLa cells synchronized with an excess concentration of thymidine or NIH 3T3 cells released from the serum-depleted state. TAF-I is predominantly located in nuclei, while TAF-I that is devoid of its acidic region, the region which is essential for the TAF-I activity, shows both nuclear and cytoplasmic localization. The localization of TAF-I in conjunction with the regulation of its activity is discussed.  相似文献   

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《Epigenetics》2013,8(1):54-65
DNA methylation has been viewed as a stable component of the epigenome, which is established during development and fixed thereafter. We show here using nearest neighbor analysis, immunocytochemistry, and high performance capillary electrophoresis that the DNA methylation pattern varies in HeLa cells during a single cell cycle. Immunocytochemical analysis in primary human fibroblasts shows similar variations. The global levels of DNA methylation decreased in G1 and increase during the S phase of the cell cycle. Since there was little change in the DNA methylation levels in repetitive sequences throughout the cell cycle, we examined the DNA methylation pattern of unique sequences using a human CpG island microarray. Hybridization with methylated DNA from G1 and S phase of the cell cycle revealed that 174 CG-containing sequences were differentially methylated between G1 and S. 75% of all the variations in DNA methylation detected in unique sequences represented hypomethylation at G0, with changes occurring in both CpG islands and non-CpG islands. Bisulfite mapping confirmed these changes in methylation in the regions identified by the microarray. This is the first demonstration of a dynamic DNA methylation pattern within a single cell cycle of a mature somatic cell. These data are important for our understanding of the stability of DNA methylation patterns in somatic cells.  相似文献   

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The time-course of ELF-EMF application to biological systems is thought to be an important parameter determining the physiological outcome. This study investigated the effect of ELF-EMF on the differentiation of K562 cells at different time courses. ELF-EMF (50?Hz, 5?mT, 1?h) was applied at two different time-courses; first at the onset of hemin induction for 1?h, and second, daily 1?h for four days. While single exposure to ELF-EMF resulted in a decrease in differentiation, ELF-EMF applied everyday for 1?h caused an increase in differentiation. The effect of co-stressors, magnesium, and heat-shock was also determined and similar results were obtained. ELF-EMF increased ROS levels in K562 cells not treated with hemin, however did not change ROS levels of hemin treated cells indicating that ROS was not the cause. Overall, these results imply that the time-course of application is an important parameter determining the physiological response of cells to ELF-EMF.  相似文献   

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莪术醇诱导慢性粒细胞白血病K562细胞分化的研究   总被引:2,自引:0,他引:2  
林海  李晓辉 《现代生物医学进展》2007,7(11):1674-1676,F0003
目的:以人慢性粒细胞白血病细胞株K562细胞为对象,研究莪术醇(curcumenol)诱导K562细胞分化作用,并同莪术油做了比较研究。方法:通过测定细胞内酶变化判定莪术醇等诱导K562细胞的分化;用RT-PCR测定莪术醇等作用后K562细胞bcr/abl mRNA表达量的变化,同时测定莪术醇等对K562细胞微核影响,探讨诱导K562细胞的分化机理。结果:莪术醇能够诱导K562细胞向成熟分化,30μg/mL莪术醇作用72h后,Gimsa染色后可见K562细胞向终末细胞分化,出现杆状及分叶核细胞,细胞内酶学指标也呈分化表现;同时,莪术醇作用后bcr/abl融合基因的表达降低,K562细胞的微核率减少。结论:莪术醇对畸变的K562细胞染色体有作用,影响bcr/abl融合基因的表达,使K562细胞向成熟分化。  相似文献   

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