Effects of the dilution rate on cell cycle distribution and PEI-mediated transient gene expression by CHO cells in continuous culture

In this study, a continuous culture system was applied to mammalian cells on large scale, and polyethyleneimine (PEI) mediated transient gene expression (TGE). PEI MAX 40,000 was chosen as a superior reagent from three types of PEI. The cell cycle distribution of cells in batch and continuous cultures was determined, in which the effects of cell cycle distribution on transfection efficiency, post-transfection proliferation and recombinant prothrombin expression were evaluated. Compared with cells from end-log and plateau phase in batch culture, cells from mid-log phase possessed a larger fraction of S and G2/M phase cells and a smaller fraction of G1 phase cells. In the continuous culture, the fraction of cells in the S and G2/M phases increased and the fraction of cells in the G1/G0 phase decreased with increasing dilution rates. Cells from the continuous culture run at highest dilution rate had excellent proliferation, transfection efficiency and protein expression. These results were confirmed by transfecting cells synchronized to different phases. The G2/M arrested cells exhibited a nearly 10-fold increase in recombinant human prothrombin production relative to that of non-dividing cells. The use of continuous culture for large scale transfection demonstrated a better cell physiological state for TGE process.     

Genes adopt non‐optimal codon usage to generate cell cycle‐dependent oscillations in protein levels

The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle‐regulated genes with that of other genes, we discovered that there is a significant preference for non‐optimal codons. Moreover, genes encoding proteins that cycle at the protein level exhibit non‐optimal codon preferences. Remarkably, cell cycle‐regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non‐optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl‐, threonyl‐, and glutamyl‐prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose that non‐optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell‐cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle‐dependent dynamics of proteins.     

病毒感染对细胞周期调控影响的研究进展

大量研究表明,病毒感染细胞时,病毒编码的蛋白或DNA可以扰乱细胞周期通路:促进细胞向S期转化或者使细胞静息于G2/M期。在细胞内,细胞周期的调控机制十分复杂,其包含了由DNA损伤导致的细胞通路活化及其他方式。关于病毒对细胞周期的调控方式及细胞周期的改变对于病毒感染的研究已取得一定进展。对于病毒的此类研究可以揭示细胞活动中的关键调控因子及细胞周期检查点的具体分子机理。对病毒调控宿主细胞周期以达到自身最大化复制的机理进行综述。     

RNAi-mediated knock-down of gene mN6A1 reduces cell proliferation and decreases protein translation

Methyltransferases play essential roles in modulating important cellular and metabolic processes. A mouse putative N6-DNA methyltransferase gene (GenBank No AY456393) is a novel gene named mN6amt1(mN6A1). To investigate its function in cell fate and protein translation, RNA interference (RNAi)-mediated knock-down method was established. Cell cycle analysis suggests that the cell proliferation decreases after RNAi with mN6A1. The expression plasmid of luciferase was used to detect protein translation, and the results showed that luciferase expression decreased after RNAi with mN6A1, whereas increased after over-expression of mN6A1 or/and eRF1. The binding between mN6A1 and eRF1 was identified by co-immunoprecipitation and pull-down experiments. It might be suggested that mN6A1 participates in protein translation through interaction with eRF1.     

Uncoupling of bait‐protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP‐1 and the PKA Cβ1 subunit

An expression‐uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP‐bait protein from the target cells. Here, the TAP‐tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue‐specific binding partners of the protein kinase A catalytic subunit Cβ1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor‐dependent interaction of Cβ1 with the cell cycle and apoptosis regulatory protein‐1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell‐ and tissue‐specific protein complexes.     

Cyclin D1与细胞周期调控

细胞周期是细胞生命活动中一个最重要的过程,其关键是G1 期的启动.细胞周期蛋白(Cyclin)、细胞周期蛋白依赖性激酶(CDKs)和CDK抑制因子(CKIs)是参与钿胞周期调控的主要因子.Cyclin D1是调控细胞周期G1期的关键蛋白,是一个比其他Cyclins更加敏感的指标,对细胞周期调控至关重要.综述Cyclin D1的结构和功能及其在肿瘤组织中的表达特征,初步分析Cyclin D在昆虫细胞周期调控的研究.     

miR-155 通过p53/p21 调控前列腺癌细胞周期

目的:探讨miR-155对前列腺癌细胞周期的影响及其分子机制。方法:通过转染anti-miR-155抑制前列腺癌DU145和PC-3细胞中miR-155水平后,采用流式细胞术观察细胞周期的变化,western blot和RT-PCR观察p53和p21蛋白及CDK2和cyclin蛋白和m RNA表达的变化。结果:与对照组相比,DU145和PC-3细胞转染anti-miR-155后,G2/M期细胞阻滞,S期细胞数比例显著增加(P0.05),p53和p21蛋白和m RNA表达水平显著增加(P0.01),CDK2和cyclin E蛋白和m RNA表达均显著降低(P0.01)。结论:miR-155可影响人前列腺癌细胞的周期,可能与其调节p53、p21及其下游的CDK2和cyclin E的表达相关。     

HTm4基因在造血细胞周期调控中的作用

为了研究HTm4基因在造血细胞细胞周期调控中的作用,以佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导K562细胞分化为模型,利用流式细胞术(FACS)及半定量RT-PCR对分化过程中细胞周期的变化及HTm4基因的表达进行了分析,并利用Tet-Off调控表达系统,将HTm4基因以及C端功能域缺失的HTm4-ct转染K562细胞,观察对细胞周期的影响。结果表明,PMA同时引起了K562细胞的增殖和分化,G0／G1期细胞的比例以及HTm4基因的表达均呈现出波浪形的变化趋势,说明HTm4基因可能参与了细胞退出细胞周期的过程。HTm4基因转染后引起K562细胞滞留于G0／G1期,但C端功能域缺失的HTm4-ct没有此作用,说明C端功能域在HTm4基因调控细胞周期的功能中发挥重要作用。     

Ubiquitin Protein Ligase Ring2 Is Involved in S‐phase Checkpoint and DNA Damage in Cells Exposed to Benzo[a]pyrene

Previous studies in our laboratory demonstrated that Ring2 may affect DNA damage and repair through pathways other than through regulating the expression of the nucleotide excision repair protein. In a series of experiments using wild‐type cell (16HBE and WI38) and small interfering RNA (siRNA) Ring2 cells exposed to benzo[a]pyrene (BaP), we evaluated the cell cycle and DNA damage. The benzo(a)pyrene‐7,8‐dihydrodiol‐9,10‐epoxide (BPDE–DNA) adduct assay demonstrated that in vitro exposure to BaP increased DNA damage in a time‐ and dose‐dependent manner in wild‐type and siRNA Ring2 cells. Analysis of covariance showed that a decrease of Ring2 caused DNA hypersensitivity to BaP. Flow cytometry results and proliferating cell nuclear antigen levels indicated that inhibition of Ring2 attenuated the effect of BaP on S‐phase arrest. Taken together, these data implied that the lower proportion of cells in the S phase induced by inhibition of Ring2 may play an important role in DNA hypersensitivity to BaP.     

Robustness and topology of the yeast cell cycle Boolean network

Yeast cell cycle Boolean network was used as a case study of robustness to protein noise. Robustness was interpreted as involving stability of G1 steady state and sequence of gene expression from cell cycle START to stationary G1. A robustness measure to evaluate robustness strength of a network was proposed. Robust putative networks corresponding to the same steady state and sequence of gene expression of wild-type network were sampled. Architecture of wild-type yeast cell cycle network can be revealed by average topology profile of sampled robust putative networks.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

Microarrays and the relationship of mRNA variation to protein variation during the cell cycle

Microarray analyses have led to the postulated existence and identification of numerous genes that are believed to be expressed and presumably to act in a cell-cycle-specific manner because their expression varies during the cell cycle. It is important to see how protein variation can be produced from mRNA variation. We have calculated the protein content throughout the cell cycle resulting from cell-cycle-specific mRNA expression, and compared the result to protein content resulting from constant, cell-cycle independent, mRNA expression. For stable proteins, cell-cycle-specific mRNA expression leads to a maximum 2-fold change in protein content compared to proteins synthesized from constantly expressed mRNA. More realistic sinusoidal patterns of mRNA expression exhibit much smaller ratios of 1.25 or lower, even for extremely large amplitudes in mRNA expression. For unstable proteins that have a cycle-independent half-life, only at extremely short protein half-lives does mRNA variation have a significant impact on variation of protein content during the division cycle. We also apply these findings to proteins with a cycle-specific decay pattern. mRNA variations during the eukaryotic division cycle variation of mRNA during the cell cycle can have only a minimal affect on the variation of protein content during the cell cycle. We conclude that mRNA variations during the division cycle, as measured by microarrays, cannot by themselves, identify cycle-specific functions related to protein variations.     

Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后,其增殖能力明显减慢。CyclinD1基因表达降低,细胞主要停滞于G0/G1期。在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖,通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用     

Overexpression of WDR79 in non‐small cell lung cancer is linked to tumour progression

WD‐repeat protein 79 (WDR79), a member of the WD‐repeat protein family, acts as a scaffold protein, participating in telomerase assembly, Cajal body formation and DNA double‐strand break repair. Here, we first report that WDR79 is frequently overexpressed in cell lines and tissues derived from non‐small cell lung cancer (NSCLC). Knockdown of WDR79 significantly inhibited the proliferation of NSCLC cells in vitro and in vivo by inducing cell cycle arrest and apoptosis. WD‐repeat protein 79 ‐induced cell cycle arrest at the G0/G1 phase was associated with the expression of G0/G1‐related cyclins and cyclin‐dependent kinase complexes. We also provide evidence that WDR79 knockdown induces apoptosis via a mitochondrial pathway. Collectively, these results suggest that WDR79 is involved in the tumorigenesis of NSCLC and is a potential novel diagnostic marker and therapeutic target for NSCLC.