Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry¹ offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture⁵. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.     

Cold-Shock and the Mammalian Cell Cycle

no abstract available     

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture

Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G₁ phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G₁ to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs.     

LMO3过表达对胶质细胞增殖的作用

构建重组质粒pcDNA4/HisA-LMO3,转染C8-D1A胶质细胞,对照组为pcDNA4/HisA空载体质粒转染组和未转染组,MTT观察各组细胞的体外生长情况,流式细胞术(FCM)测定各组细胞的细胞周期和凋亡细胞百分比,Western-blot检测LMO3转染后凋亡相关蛋白的表达变化,从而观察LMO3基因对C8-D1A胶质细胞体外生长的影响.RT-PCR、Western印迹显示pcDNA4/HisA-LMO3转染组LMO3mRNA及LMO3蛋白表达水平明显高于对照组;与对照组细胞相比,转染组细胞的增殖能力明显高于空载体对照组及C8细胞(P0.01),S期细胞增加,G0/G1期细胞减少,C8细胞转染LMO3后可以促进C8细胞从G0/G1期进入S期,从而促进细胞的增殖.     

FOX03a过表达对内皮祖细胞周期相关蛋白的调控

目的：观察FOXO3a（forkhead box O3a）的活性改变对内皮祖细胞（endothelial progenitor cells,EPCs）增殖和细胞周期相关蛋白表达的影响。方法：将携带突变激活FOXO3a基因的腺病毒载体Ad-TM（triple mutant）-FOXO3a和阴性对照腺病毒载体Ad-GFP体外感染人脐血来源的EPCs。观察EPCs形态学改变,CCK-8分析转染后EPCs增殖情况,Western blot检测FOXO3a蛋白、细胞周期相关蛋白p27^kip1以及CDK2的表达水平。结果：构建了的2种腺病毒相关载体被成功转染。形态学改变方面,Ad-TM—FOXO3a组EPCs细胞生长缓慢,集落不明显;Westem blot和CCK-8结果显示,Ad-TM—FOXO3a转染组与阴性对照组相比,EPCs增殖被抑制,FOXO3a与p27^kip1蛋白过表达,CDK2表达下调。结论：FOXO3a可能通过上调p27kip1蛋白表达,下调CDK2表达,以抑制EPCs增殖。     

FOXO3a过表达对内皮祖细胞周期相关蛋白的调控

目的:观察FOXO3a(forkhead box O3a)的活性改变对内皮祖细胞(endothelial progenitor cells,EPCs)增殖和细胞周期相关蛋白表达的影响。方法:将携带突变激活FOXO3a基因的腺病毒载体Ad-TM(triple mutant)-FOXO3a和阴性对照腺病毒载体Ad-GFP体外感染人脐血来源的EPCs。观察EPCs形态学改变,CCK-8分析转染后EPCs增殖情况,Western blot检测FOXO3a蛋白、细胞周期相关蛋白p27kip1以及CDK2的表达水平。结果:构建了的2种腺病毒相关载体被成功转染。形态学改变方面,Ad-TM-FOXO3a组EPCs细胞生长缓慢,集落不明显;Western blot和CCK-8结果显示,Ad-TM-FOXO3a转染组与阴性对照组相比,EPCs增殖被抑制,FOXO3a与p27kip1蛋白过表达,CDK2表达下调。结论:FOXO3a可能通过上调p27kip1蛋白表达,下调CDK2表达,以抑制EPCs增殖。     

抗酶1基因转染对HeLa细胞增殖及细胞周期的抑制作用

研究抗酶(antizyme)1对人宫颈癌HeLa细胞增殖与细胞周期的影响,并分析抗酶1对细胞周期蛋白D1(cyclin D1)的表达影响.采用定点突变技术,将抗酶1的frameshift位点缺失,随后将突变基因重组至真核表达载体pEGFP-N1中,鉴定后转染HeLa细胞.通过MTT法检测细胞增殖变化,流式细胞术分析抗酶1对细胞周期的影响.RT-PCR和Western印迹检测抗酶1转染对细胞周期蛋白 D1基因表达的影响.酶切结果显示,抗酶1突变基因成功克隆至pEGFP-N1中.成功转染HeLa细胞后,检测结果显示,抗酶1能够减慢HeLa细胞增殖速度,并使细胞停滞于G₀/G₁期,细胞周期蛋白D1基因的表达同时受到抑制.实验说明,抗酶1基因能够抑制HeLa细胞增殖,通过降低细胞周期蛋白D1的表达阻滞细胞周期.     

TRAIL在细胞中的表达及其诱导凋亡的活性分析

以PCR方法克隆了Trail cDNA全长,构建了其真核表达载体,通过脂质转染HeLa细胞,48小时后利用流式细胞仪分析Trail诱导细胞凋亡的比率,发现发生凋亡的细胞为总细胞数的19％。证实了Trail真核4表达系统的产物的生物学活性高,为从真核表达的途径获得Trail基因工程产品奠定了基础。     

Potassium-Chloride Cotransporter Mediates Cell Cycle Progression and Proliferation of Human Corneal Epithelial Cells

Epidermal growth factor (EGF)-induced proliferation of corneal epithelial cells contributes to its renewal, which maintains the protective and refractive properties of the cornea. This study characterized in human corneal epithelial cells (HCEC) the role of the potassium–chloride cotransporter (KCC) in mediating (i) EGF-induced mitogen-activated protein kinase (MAPK) pathway activation; (ii) increases in cell cycle progression; and (iii) proliferation. The KCC inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA) and KCC activator N-ethylmaleimide (NEM), suppressed and enhanced EGF-induced p44/42MAPK activation, respectively. Such selective modulation was mirrored by corresponding changes in cell proliferation and shifts in cell cycle distribution. DIOA induced a 20% increase in G0/G1-phase cell population, whereas NEM induced a 22% increase in the proportion of cells in the G2/M-phase and accelerated the transition from G0/G1-phase to the S-phase. Associated with these changes, KCC1 content in a plasma membrane enriched fraction increased by 300%. Alterations in regulatory volume capacity were associated with corresponding changes in both KCC1 membrane content and activity. These results indicate that EGF-induced increases in KCC1 activity and content modulate cell volume changes required for (i) activation of the p44/42MAPK signaling pathway, (ii) cell cycle progression, and (iii) increases in cell proliferation.     

Life Cycle Analysis of Mammalian Cells: II. Cells from the Chinese Hamster Ovary Grown in Suspension Culture

A method for life cycle analysis in mammalian cells which utilizes the collection function has been applied to the Chinese hamster ovary grown in suspension. The following durations were found for the various parts of the life cycle: S, 4.13 hours; G1, 4.71 hours; G2, 2.81 hours; mitosis, 0.81 hours. The cell has a total generation time of 12.4 hours as opposed to 20.1 hours for the S3 HeLa cell. However, the relative lengths of each phase of the life cycle are identical within experimental uncertainty in the two cells.     

Effect of Optogenetic Stimulus on the Proliferation and Cell Cycle Progression of Neural Stem Cells

Modulation of stem cell proliferation is a crucial aspect of neural developmental biology and regenerative medicine. To investigate the effect of optical stimulation on neural stem cell proliferation, cells transduced with channelrhodopsin-2 (ChR2) were used to analyze changes in cell proliferation and cell cycle distribution after light stimulation. Blue light significantly inhibited cell proliferation and affected the cell cycle, which increased the percentage of cells in G₁ phase and reduced the percentage in S phase. It is likely that the influence of blue light on cell proliferation and the cell cycle was mediated by membrane depolarization, which induced accumulation of p21 and p27 proteins. Our data provide additional specific evidence that membrane depolarization may inhibit neural stem cell proliferation.     

Effects of Butyrate on Cell Cycle Progression and Polyploidization of Various Types of Mammalian Cells

We studied the effect of butyrate on cell cycle progression and polyploidization in three fibroblast (rat 3Yl, human IMR-90, and human embryo lung HEL) and two epithelial (human embryo kidney HEK and monkey kidney BSC-1) cells. In these cells, except for 3Y1, G1 arrest with butyrate was incomplete, and the production of tetraploid cells was detectable in the presence of butyrate. G2 arrest with butyrate was also incomplete in HEL and BSC-1 cells, and the number of HEL cells increased in the presence of butyrate. On the contrary, most BSC-1 cells that divided in the presence of butyrate were unstable and the number of attached cells decreased. These results indicate that the effect of butyrate on cell cycle progression varies with the cell type and that polyploidization can be induced by a single treatment with butyrate.     

应用微重力旋转生物反应器培养小鼠脂肪干细胞的初步实验研究

目的:观察微重力旋转培养系统(Rotary Cell Culture System,RCCS),对小鼠脂肪干细胞增殖的影响,以寻求一种更有效的促进干细胞扩增的方法.方法:从小鼠的脂肪组织中提取分离、培养脂肪干细胞(ADSCs),并对脂肪干细胞进行流式鉴定后,利用活细胞观察法、Dil免疫荧光标记法、扫描电镜法观察微重力旋转三维培养系统对脂肪干细胞增殖的影响;通过与平面二维培养作对比,血小板计数法记录细胞的增殖情况,并绘制生长曲线.结果:两组的细胞倍增时间具有统计学意义(P＜0.05),模拟微重力旋转三维培养系统较传统平面二维培养系统,脂肪干细胞增殖更明显,生长速度更快.结论:模拟微重力旋转三维培养系统更有利于脂肪干细胞的增殖生长,为后期利用脂肪干细胞修复受损涎腺提供一种更快捷有效的扩增方法.     

动物细胞流加培养的技术现状和发展趋势

流加培养是当前重组蛋白生产的主流培养模式。流加式操作主要是根据细胞对营养物质的不断消耗和需求,设计连续或半连续的流加浓缩营养物,使细胞持续高密度的生长,提高单位反应器体积内目的蛋白产量,从而达到高效生产的目的。流加培养工艺的关键技术主要包培养基的优化设计、流加策略的选择及优化、细胞代谢的调控。     

De-Regulated MicroRNAs in Pediatric Cancer Stem Cells Target Pathways Involved in Cell Proliferation,Cell Cycle and Development

Background

microRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years, the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.

Methodology/Results

Using a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG, Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle, cell proliferation, p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p, hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).

Conclusion

Our findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers.     

Daughter Cell Repair by Mammalian Cells in Culture after Potentially Lethal Radiation Damage

COLONY formation by irradiated surface-attached mammalian cells in culture requires that at least one of the progeny produced at the first post-irradiation division retains the capacity for unlimited division. Abortive colonies result when the irradiated parent cell has experienced sufficient damage to suppress eventual colony formation, but not to prevent the production of a small number of progeny. We now present data which indicate that, if given a suitable environment, progeny from cells “lethally” damaged by X-radiation (not capable of producing a macroscopic colony) can repair damage transferred (sectored) from the parent cell.