The role of the cell cycle on the efficiency of photochemical gene transfection

The efficiency of gene transfection mediated by nonviral vectors is limited because of nonoptimal intracellular trafficking of transfecting DNA. Most nonviral vectors deliver transfecting DNA into a cell through endocytosis. However, poor escape from endocytic vesicles and inefficient transport of DNA into the nucleus often limits a success of gene transfection. Photochemical transfection is a new method, based on light-induced permeabilisation of endocytic vesicles, liberating transfecting DNA into the cytosol, concurrently increasing the chances for DNA to enter the nucleus.The aim of this study was to investigate the role of the cell cycle for the efficiency of photochemical transfection. It was demonstrated that in asynchronous human colon carcinoma HCT 116 cells photochemical treatment increased the transfection mediated by the nonviral vectors, the cationic polypeptide polylysine and the cationic lipid N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide/dioleoylphosphatidylethanolamine (beta AE-DMRIE/DOPE), by 30- and 2.5-fold, respectively. In aphidicolin-synchronised cells, photochemical transfection mediated by polylysine was dependent on the cell cycle: transfection level was 4-fold higher when illumination, inducing photochemical reactions, was performed during the G2/M phase as compared to the G1/early-S phase. The cell cycle influence on photochemical transfection mediated by beta AE-DMRIE/DOPE was very low: only 20% difference between G2/M and the G1/S phase was observed. We suggest that transgenes, photochemically liberated close/during mitosis, perhaps have the highest opportunity to enter the nucleus and be expressed. However, the dependence of photochemical transfection on the cell cycle might be partially disguised by various factors induced by photochemical treatment.     

pemt2-cDNA的转染抑制大鼠CBRH-7919肝癌细胞c-Met及Bcl-2的表达并诱发凋亡

为探讨磷脂酰乙醇胺 N 甲基转移酶 2 (phosphatidylethanolamine N methyltransferase 2 ,PEMT2 )的转染抑制大鼠肝癌CBRH 7919细胞增殖的分子机理 ,构建了带有完整的pemt2 cDNA基因质粒载体 ,经转染和鉴定 ,确知其在大鼠肝癌细胞系CBRH 7919细胞中稳定高表达 .用细胞培养、免疫细胞化学、Western印迹、流式细胞仪和琼脂糖凝胶电泳等技术研究c Met(HGF的受体 )及抗凋亡蛋白Bcl 2在此过程中的表达和是否诱发凋亡 .免疫组织化学及Western印迹分析显示在转染高表达细胞中 ,c Met及Bcl 2的表达下调 .流式细胞仪分析表明 ,在转染pemt2 cDNA的高表达细胞中 ,G1期细胞增加 ,S期细胞减少 ,并在G0 期出现一个凋亡峰 .琼脂糖凝胶电泳谱显示梯状条带 .结果表明 :pemt2的转染可使大鼠肝癌细胞的c Met及Bcl 2的表达下调 ,并发生凋亡 .     

Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field

Electropermeabilization is a promising nonviral method for gene therapy. However, despite the fact that it is widely used to transfer genes into living cells, the steps that limit DNA transfer remain to be determined. Here, we report the effect of cell synchronization on membrane permeabilization and gene delivery by electric fields.Chinese hamster ovary (CHO) cells were synchronized by aphidicolin or butyrate treatment. Electro-mediated transfection of these cells was evaluated under electric field conditions leading to the same level of membrane permeabilization.Aphidicolin cell synchronization in G2/M phase leads to a slight increase in plasma membrane permeabilization but to a three-fold increase in percentage of transfected cells and to an eight-fold increase in gene expression. This increase in cell transfection is specifically due to the G2/M synchronization process. Indeed, cell synchronization in G1 phase by sodium butyrate has no effect on cell permeabilization and transfection.Our results suggest that the enhanced transfection level in G2/M phase is not simply due to enhanced permeabilization, but reinforce the statement that the melting of the nuclear membrane facilitates direct access of plasmid DNA to the nucleus.     

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.     

Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.     

Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint

The regulation of the Dictyostelium cell cycle has remained ambiguous owing to difficulties in long-term imaging of motile cells and a lack of markers for defining cell cycle phases. There is controversy over whether cells replicate their DNA during development, and whether spores are in G1 or G2 of the cell cycle. We have introduced a live-cell S-phase marker into Dictyostelium cells that allows us to precisely define cycle phase. We show that during multicellular development, a large proportion of cells undergo nuclear DNA synthesis. Germinating spores enter S phase only after their first mitosis, indicating that spores are in G2. In addition, we demonstrate that Dictyostelium heterochromatin is copied late in S phase and replicates via accumulation of replication factors, rather than recruitment of DNA to pre-existing factories. Analysis of variability in cycle times indicates that regulation of the cycle manifests at a single random transition in G2, and we present the first identified checkpoint in Dictyostelium, which operates at the G2-M transition in response to DNA damage.     

G2 phase cell cycle disturbance as a manifestation of genetic cell damage

The predominant cell cycle change induced by X-rays and clastogens in peripheral blood mononuclear cells is the accumulation of cells in the G2 phase of the cell cycle. We show that this accumulation consists of cells that are either delayed or arrested within the G2 phase. Since both X-rays and DNA crosslinking chemicals are known to damage DNA, the G2 phase inhibition caused by these agents is thought to be one of the primary manifestations of (unrepaired) DNA damage. This interpretation is supported by two additional findings. (1) Older individuals have elevated baseline levels of mononuclear blood cells that are delayed and/or arrested in the G2 phase of the cell cycle. This coincides with the increased chromosomal breakage rates reported for older individuals. (2) Irrespective of their age, individuals with inherited genetic instability syndromes (such as Fanconi anemia and Bloom syndrome) exhibit elevated G2 phase cell fractions. We show that the method used to detect such induced or spontaneous cell cycle changes, viz. BrdU-Hoechst flow cytometry, is a rapid and highly sensitive technique for the assessment of genetic cell damage.Dedicated to Professor Ulrich Wolf on the occasion of his 60th birthday     

Transfection-mediated cell-cycle signaling: considerations for transient transfection-based cell-cycle studies.

Transient transfection of recombinant genes into cells is a commonly used approach for analyzing cell-cycle- and/or apoptotic-related activities of cell-cycle control proteins. In this approach, information regarding the functional consequence of expressing a recombinant protein transiently is garnered by comparing against results obtained from cells which are transfected with either a control expression plasmid and/or with mutant expression plasmids. In general however, little attention is paid to whether the transfection procedure itself influences these experiments. Using the calcium phosphate transfection method, we show that the introduction of DNA into cells induces signaling of the cell-cycle control machinery. In Hela cells, a transient increase in G0/G1 cells is observed 8 h after transfection. Furthermore, the introduction of DNA into several cell lines induces apoptosis. Transfection-mediated apoptosis can be elicited through a p53-independent mechanism, suggesting the possible extrapolation to many tumor cell lines. Last, we show that due to a likely cell-cycle-specific entry of marker genes into the nucleus, a highly biased cell-cycle distribution is observed in successfully transfected cells at early times following transfection. The importance of these issues in the interpretation as well as the design of transient transfection-based cell-cycle experiments is discussed.     

A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

Immunoreactivity to antinucleoside antibodies in camptothecin treated HeLa cells

HeLa cells, incubated with camptothecin during the G 1 phase of the cell cycle, show nuclear fluorescence with fluorescein-labeled antinucleoside antibodies. If the G 1 cells are washed free of the drug, the cells no longer demonstrate nuclear fluorescence. Since these antibodies react only with single-stranded DNA, the positive staining in camptothecin-treated G 1 cells suggests that the drug induces denatured regions in DNA. Fluorescent antinucleoside antibodies may be a useful technique for the observation of drug-induced changes in DNA during the G1 phase of the cell cycle.     

Determination of growth inhibitory action point of interferon gamma on WISH cells in cell cycle progression and the window of responsiveness of the cells to the interferon

We had earlier shown that human foetal epithelial cells (WISH), growth-inhibited by interferon gamma (IFNgamma), were reversibly detained at a point prior to DNA synthesis. In the present study, we determined the window of action of IFNgamma in the G1 phase duration and the exact point of detention of WISH cells in cell cycle progression with respect to the known points of detention by the inhibitors of DNA replication initiation (aphidicolin and carbonyl diphosphonate) and of activation of replication protein A (6-dimethylaminopurine), of which RPA activation being the earlier event compared to DNA replication initiation in cell cycle progression. WISH cells, which were released from IFNgamma-induced arrest, permeabilised and exposed independently to these inhibitors show that IFNgamma detains WISH cells prior to initiation of DNA synthesis. Further, exposure of IFNalpha-synchronized (at G0/G1) or mimosine-synchronized (at G1/S) WISH cells to IFNgamma, which was added at different time points post-release from the synchronizing agent, showed that the cells were promptly responsive to the growth inhibitory action of IFNgamma only during the first 11h in G1 phase. Taken together, these results suggest that IFNgamma inhibits growth of WISH cells by detaining them at a point prior to initiation of DNA synthesis and that the IFN acts within the first 11h in G1 phase of the cell cycle.     

Effect of transfection manipulations on mouse cell cycle progression.

BalB/C-3T3 mouse fibroblasts and a temperature-sensitive derivative, ts 2e, were transfected by the calcium phosphatedimethyl sulphoxide procedure to examine the effect of this manipulation on cell cycle progression. Cells were synchronized by growth to confluence in the presence of [2-14C]thymidine to generally label cellular DNA, and then subcultured from the G0 state. Plasmid pSV3-neo or pSV2-neo DNA was added to cells at 24 h post-plating, at peak S phase. At designated intervals prior to, during, and after the transfection procedure, cells were labelled with [methyl-3H]thymidine for 1 h to monitor nascent DNA synthesis and thereby assess cell cycle position. In all experiments performed, irrespective of the time of DNA addition, the transfection manipulations resulted in a reproducible, transient interruption of cell cycle progression, of about 5 h, and manifested as a delay in movement across the subsequent G1-S interface. Thereafter, the cycle resumed normally. The results indicated that the temporal sequence of the cell duplication cycle is altered when cells are exposed to exogenous DNA:Ca3 (PO4)2.     

Phosphoinositide 3-kinase signaling overrides a G2 phase arrest checkpoint and promotes aberrant cell cycling and death of hematopoietic cells after DNA damage

DNA damage activates arrest checkpoints to halt cell cycle progression in G1 and G2 phases. These checkpoints can be overridden in hematopoietic cells by cytokines, such as erythropoietin, through the activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. Here, we show that PI3K activity specifically overrides delayed mechanisms effecting permanent G1 and G2 phase arrests, but does not affect transient checkpoints arresting cells up to 10 hours after gamma-irradiation. Assessing the status of cell cycle regulators in hematopoietic cells arrested after gamma-irradiation, we show that Cdk2 activity is completely inhibited in both G1 and G2 arrested cells. Despite the absence of Cdk2 activity, cells arrested in G2 phase did retain detectable levels of Cdk1 activity in the absence of PI3K signaling. However, reactivation of PI3K promoted robust increases in both Cdk1 and Cdk2 activity in G2-arrested cells. Reactivation of Cdks was accompanied by a resumption of cell cycling, but with strikingly different effectiveness in G1 and G2 phase arrested cells. Specifically, G1-arrested cells resumed normal cell cycle progression with little loss in viability when PI3K was activated after gamma-irradiation. Conversely, PI3K activation in G2-arrested cells promoted endoreduplication and death of the entire population. These observations show that cytokine-induced PI3K signaling pathways promote Cdk activation and override permanent cell cycle arrest checkpoints in hematopoietic cells. While this activity can rescue irradiated cells from permanent G1 phase arrest, it results in aberrant cell cycling and death when activated in hematopoietic cells arrested at the G2 phase DNA damage checkpoint.     

Cyclin D1 expression and cell cycle response in DNA mismatch repair-deficient cells upon methylation and UV-C damage

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.     

The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage

We investigated the role of initial DNA and chromosome damage in determining the radiosensitivity difference between the variant murine leukemic lymphoblast cell lines L5178Y-S (sensitive) and L5178Y-R (resistant) and the difference in cell cycle-dependent variations in radiosensitivity of L5178Y-S cells. We measured initial DNA damage (by the neutral filter elution method) and chromosome damage (by the premature chromosome condensation method) and compared them with survival (measured by cloning) for both cell lines synchronized in G1 or G2 phase of the cell cycle (by centrifugal elutriation) and irradiated with low doses of X rays (up to 10 Gy). The initial yield of DNA and chromosome damage in G2 L5178Y-S cells was almost twice that in G1 L5178Y-S cells and G1 or G2 L5178Y-R cells. In all cases DNA damage expressed as relative elution corresponded with chromosome damage (breaks in G1 chromosomes, breaks and gaps in G2 chromosomes). Also we found that the initial DNA and chromosome damage did not determine cell age-dependent radiosensitivity variations in L5178Y-S cells, as there was less initial damage in the more sensitive G1 phase than in the G2 phase. L5178Y-R cells showed only small changes in survival or initial yield of DNA and chromosome damage throughout the cell cycle. Because survival and initial damage in sensitive and resistant cells irradiated in G2 phase correlated, the difference in radiosensitivity between L5178Y-S and L5178Y-R cells might be determined by initial damage in G2 phase only.     

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.     

Concentration-dependent effects of potassium dichromate on the cell cycle

Hexavalent chromium is found to be a strong mutagen, and it also is a potential carcinogen in man. DNA flow cytometry, growth measurements, and determinations of mitotic index show that 1-2 microM K2Cr2O7 produces a prolongation of the G2 phase of the cell cycle in NHIK 3025 cells. By increasing the chromate concentrations (greater than 2 microM K2Cr2O7) the cells are also arrested in G2 phase. We have found, using synchronized cells and measuring cell cycle time, that the most chromate-sensitive part of the cell cycle is S phase. This phase is also somewhat prolonged, and the cells became arrested in early S phase at high toxic K2Cr2O7 concentrations (8 microM). Our results thus indicate that K2Cr2O7 has an effect within S phase--maybe on DNA/RNA synthesis--and also interferes with processes necessary for progression through the G2 phase.     

Cell cycle-dependent phosphorylation of human DNA polymerase alpha

The expression of DNA polymerase alpha, a principal chromosome replication enzyme, is constitutive during the cell cycle. We show in this report that DNA polymerase alpha catalytic polypeptide p180 is phosphorylated throughout the cell cycle and is hyperphosphorylated in G2/M phase. The p70 subunit is phosphorylated only in G2/M phase. This cell cycle-dependent phosphorylation is due to cell cycle-dependent kinase(s) and not to phosphatase(s). In vitro evidence indicates the involvement of p34cdc2 kinase in the mitotic phosphorylation of DNA polymerase alpha. Tryptic phosphopeptide maps demonstrate that peptides phosphorylated in vitro are identical to those phosphorylated in vivo. DNA polymerase alpha from mitotic cells is found to have lower affinity for single-stranded DNA than does polymerase alpha from G1/S phase cells. These results imply that the mitotic phosphorylation of polymerase alpha may affect its physical interaction with other replicative proteins and/or with DNA at the replication fork.