Cell proliferation in chorionic villi at different gestational ages, as analyzed by premature chromosome condensation

The technique of premature chromosome condensation (PCC) was adapted to human first-trimester chorionic villi cells to analyze the cell-cycle kinetics of interphase chromatin. Uncultured cells of the cytotrophoblast (CT) and the mesenchymal core (MC) were obtained by a two-step digestion. PCC was induced by fusion of the chorionic interphase cells with mitotic Chinese hamster ovary or HeLa cells. Cells showing PCC in G1 (classes 1-6), S, and G2 were found. To analyze further the proliferation stages of chorionic G1 interphases, the proliferation potential index (PPI) of 34 placentae recovered between the 8th and 12th week of gestation was determined. The mean PPI found in the CT and MC cells ranged from 18% to 73%, values similar to those described for intensely proliferating tissues. The highest mean PPI value (73%) was observed in CT cells from placentae recovered at the 9th week of gestation, indicating a high specific proliferative activity of CT cells at this developmental stage.     

Ⅰ型磷酸酶抑制亚基-1对人宫颈癌HeLa细胞增殖的抑制作用

PPI1(Inhibitor-1 ofprotein phosphatase 1)是I型磷酸酶的抑制亚基之一,其活化依赖于35位苏氨酸蛋白激酶(PKA)的磷酸化而发挥抑制作用.本研究目的在于探讨PPIl持续活化型突变体的表达对人宫颈癌细胞株增殖的影响及其可能的作用机制.利用PPI1野生型和活化型突变体表达质粒分别转染HeLa细胞,首先通过H~3TdR掺入实验、迁移实验观察PPI1基因对HeLa细胞增殖能力的影响,研究结果表明:在活化型突变体表达的HeLa细胞株中,细胞~3H掺入量和迁移能力明显受抑.其次通过流式细胞术、Giemsa染色法分析PPIl对HeLa细胞的细胞周期的影响;FACS分析表明HeLa细胞G2/M期比例明显升高;经脱氧胸苷同步化后,该组细胞进入有丝分裂期明显滞后.最后利用Western blot分析PPI1对MAPK信号转导通路的影响,Western blot分析显示该组细胞的ERK磷酸化水平明显下降.研究表明PPI1活化型突变体的表达可抑制人宫颈癌细胞株的增殖,这与其诱导HeLa细胞G2/M期停滞、有丝分裂的进入延缓有关,其中涉及到MAPK信号转导通路的活化受抑制.     

Evaluation of Efficiency of Modified Polypropylenimine (PPI) with Alkyl Chains as Non-viral Vectors Used in Co-delivery of Doxorubicin and TRAIL Plasmid

In this study, co-delivery system was achieved via plasmid encoding TNF related apoptosis inducing ligand (pTRAIL) and doxorubicin (DOX) using carrier based on polypropylenimine (PPI) modified with 10-bromodecanoic acid. Incorporation of alkylcarboxylate chain to PPIs (G4 and G5) could improve transfection efficiency via overcoming the plasma membrane barrier of the cells and decrease cytotoxicity of PPI. Characterization of fabricated NPs revealed that PPI G5 in which 30% of primary amines were substituted by alkyl carboxylate chain (PPI G5-Alkyl 30%) has higher drug loading as compared to the other formulations. PPI G5-Alkyl 30% indicated a decreased drug release may be due to alkyl chains on the surface of PPI, which serve as an additional hindrance for drug diffusion. In vitro cytotoxicity experiments demonstrated that co-delivery system induced apoptosis of tumor cells more efficiently than each of delivery system alone. Furthermore, these results revealed that our combined delivery platform of pTRAIL and DOX using Alkyl-modified PPI G5 can significantly improve the anti-tumor activity and this strategy might develop a new therapeutic window for cancer treatment.     

Heat stress stimulates inositol trisphosphate release and phosphorylation of phosphoinositides in CHO and Balb C 3T3 cells

Exposure of eukaryotic cells to elevated temperature leads to profound switches in cell metabolism and gene expression which may be involved in cellular homeostatic mechanisms. We have investigated the effect of heat shock (45 degrees C) on the metabolism of the phosphoinositides, a class of phospholipids involved in the function of Ca2+ -linked membrane receptors. Heat shock led to stimulation of phosphoinositide turnover in HA1-CHO and Balb C 3T3 cells, resulting in the rapid accumulation of inositol trisphosphate (IP3). Mitogenic and alpha 1 adrenergic stimulation, with serum or phenylephrine, led to similar increases in IP3. Heat shock also caused rapid increase in phosphorylation of polyphosphoinositides (PPI). Prolonged exposure to heat greater than 15 min at 45 degrees C led to progressive cellular toxicity which was associated with depletion of PPI. This decline in PPI concentration appeared to result from inhibition of PPI resynthesis. In this respect, heat may resemble some other types of cellular stresses in stimulating membrane phospholipases to deplete classes of membrane phospholipids. The induction of PPI turnover may, therefore, be involved in both pleiotropic responses to brief heat shock and toxicity resulting from prolonged thermal stress.     

Direct stimulation by thyrotropin-releasing hormone (TRH) of polyphosphoinositide hydrolysis in GH3 cell membranes by a guanine nucleotide-modulated mechanism

Polyphosphoinositide hydrolysis was examined in membranes from thyrotropin-releasing hormone (TRH)-responsive GH3 pituitary cells. [3H]Inositol phosphates (IP2 and IP3) were generated upon incubation of membranes from [3H]inositol-labeled cells indicating the presence of a membrane-associated polyphosphoinositide phosphodiesterase (PPI PDE). Membrane PPI PDE activity was found to be stimulated by TRH and by GTP-gamma-S in Ca2+-modulated manner. In addition, TRH-stimulated PPI hydrolysis was potentiated by GTP. These results demonstrate direct in vitro effects of a hormone on PPI turnover and suggest the involvement of a GTP-binding component in transmembrane signalling by TRH.     

Time-course network analysis reveals TNF-α can promote G1/S transition of cell cycle in vascular endothelial cells

MOTIVATION: Tumor necrosis factor-alpha (TNF-α), a major inflammatory cytokine, is closely related to several cardiovascular pathological processes. However, its effects on the cell cycle of vascular endothelial cells (VECs) have been the subject of some controversy. To investigate the molecular mechanism underlying this process, we constructed time-course protein-protein interaction (PPI) networks of TNF-α induced regulation of cell cycle in VECs using microarray datasets and genome-wide PPI datasets. Then, we analyzed the topological properties of the responsive PPI networks and calculated the node degree and node betweenness centralization of each gene in the networks. We found that p21, p27 and cyclinD1, key genes of the G1/S checkpoint, are in the center of responsive PPI networks and their roles in PPI networks are significantly altered with induction of TNF-α. According to the following biological experiments, we proved that TNF-α can promote G(1)/S transition of cell cycle in VECs and facilitate the cell cycle activation induced by vascular endothelial growth factor. CONTACT: shaoli@mail.tsinghua.edu.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Prolongation of duration of G2 arrest delays and finally blocks entry into M phase in contrast to stable and reversible G1 arrest: study of a G1/G2 temperature-sensitive mutant of rat 3Y1 fibroblasts

Proliferation of 3Y1tsF121 cells was arrested in G1 and G2 phases after a shift up to 39.8 degrees C (restrictive temperature). Both arrests were reversible: after a shift down to 33.8 degrees C (permissive temperature), these cells effectively entered the next phases. However, the entry into M phase of the G2-arrested cells was delayed depending on the time in arrest. The G2-arrested cells finally became incapable of entering M phase with a prolonged incubation at 39.8 degrees C. Under the same condition, G1-arrested cells did not lose their ability to proliferate, and their delay of entry into S phase was slight. Therefore, cells in G2 phase are, in a sense, more unstable than the cells in G1 phase. These results also suggest that the time required for entry into M phase may depend on the preparedness for the initiation of M phase and, that it may be prolonged under the condition where the preparedness for entry into M phase is diminished.     

Identification of Multiple Phosphoinositide-Linked Receptors on Human SK-N-MC Neuroepithelioma Cells

The biochemical and pharmacological characteristics of receptor-stimulated phosphoinositide (PPI) hydrolysis in human SK-N-MC neuroepithelioma cells have been examined. Of 11 ligands tested, the addition of four, i.e., norepinephrine, oxotremorine-M, endothelin-1, and ATP, each resulted in an increased release (three- to eightfold) of inositol phosphates from [3H]inositol-prelabeled cells. Agonist-stimulated PPI turnover was sustained for at least 30 min and required the addition of Ca2+ for full effect. An increased release of inositol phosphates could also be elicited by the addition of the Ca2+ ionophore, ionomycin. All four agonists enhanced the release of radiolabeled inositol mono- and bisphosphates, inositol 1,3,4-trisphosphate, and inositol tetrakisphosphate. Increases in inositol 1,4,5-trisphosphate were smaller and only consistently observed in the presence of norepinephrine or oxotremorine-M. Norepinephrine-stimulated PPI turnover was potently inhibited by prazosin, WB-4101, and 5-methylurapidil (Ki less than 2.5 nM), but was relatively insensitive to chlorethylclonidine pretreatment. This pharmacological profile is consistent with the involvement of an alpha 1A-receptor subtype. The presence of an M1 muscarinic cholinergic receptor is also indicated, because pirenzepine blocked oxotremorine-M-stimulated inositol phosphate release (Ki = 35 nM) with a 30-fold greater potency than the M2-selective antagonist, AF-DX 116. Of the three endothelins tested, only the addition of endothelin-1 and endothelin-2 promoted PPI hydrolysis, whereas endothelin-3 was essentially inactive. A P2 nucleotide receptor of broad agonist specificity is also present on these cells and activates PPI turnover in the absence of a generalized increase in plasma membrane permeability. These results indicate that SK-N-MC cells express at least four PPI-linked receptors. Because the functional coupling of three of these receptors, i.e., alpha 1A-adrenergic, endothelin, and P2 nucleotide, has not been extensively characterized previously in neural tissues, the SK-N-MC cell line may provide a useful model system for studies of these receptors and their regulation.     

Transport of newly synthesized ribosome precedes initiation of S phase in root meristem cells in Helianthus annuus L

According to earlier results, cold treatment blocks most of cells in G1 phase; after 3 h of postincubation at 20 degrees C these cells initiate S phase. Simultaneous cytophotometric (DNA stained with Methyl Green) and autoradiographic (3H-uridine, 3H-arginine) analyses of cold pretreated cells have shown that transport of 3H-RNA into cytoplasm is faster in G2 than in G1 cells: radioactivity of cytoplasm is faster in G2 cells becomes higher than that of nucleoli as early as between 2 h and 2.5 h postincubation, while in G1 cells--not until between 2.5. and 3.0 h. Simultaneous cytophotometric measurements of DNA (Feulgen) and protein (Naphthol Yellow S) contents demonstrate the considerable increase in cytoplasmic protein contents during the 2nd h of postincubation at 20 degrees C; therefore it precedes the export of more than 50% of 3H-RNA synthesized after cold treatment. The results of these experiments indicate two separate events in G1 cells: increase in cytoplasmic protein amounts (up to 2nd h of postincubation) and enhanced transport of newly-synthesized ribosome (2.5 h of postincubation), the later event immediately precede the entry of cells into S phase.     

Preparation of Effective and Safe Gene Carriers by Grafting Alkyl Chains to Generation 5 Polypropyleneimine

Gene therapy is a novel method to treat a variety of diseases including genetic disorders and cancer. Nonviral gene carriers have now gained considerable attention as gene carrier systems. Polyamidoamine (PAMAM) and polypropyleneimine (PPI) are the two most widely used denderimers in gene delivery studies. The aim of the current study was to investigate the effects of modification of generation 5 polypropyleneimine (G5 PPI) dendrimers with alkanoate groups as hydrophobic moieties on DNA transfection and cytotoxicity. Six, 10, and 16 carbon derivatives of bromoalkanoic acids were conjugated onto PPI with 10%, 30%, and 50% of surface amine grafting. Ethidium bromide exclusion assay results proved the ability of modified carriers to condense DNA. Transfection assay showed higher DNA delivery potential for 30% and 50% grafting with decanoate moieties compared to native G5 PPI and Superfect^TM. 3-(4,5-Dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) and apoptosis experiments showed lower toxicity for modified carriers compared to unmodified PPI. The hemolytic effect of grafted carriers was not significantly different from G5 PPI. Size and zeta potential measurements revealed that polyplex size was less than 200 nm and electrical charges were in the range 14–25 mV. The hydrophobic modifications improved transfection activity and toxicity of G5 PPI without negatively affecting hemocompatibility. These modified carriers are therefore promising candidates for further in vivo investigations.KEY WORDS: gene delivery, hydrophobic modification, nonviral vector, polypropyleneimine, transfection     

Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

Cell cycle analysis of p53-induced cell death in murine erythroleukemia cells.

A temperature-sensitive mutant of murine p53 (p53Val-135) was transfected by electroporation into murine erythroleukemia cells (DP16-1) lacking endogenous expression of p53. While the transfected cells grew normally in the presence of mutant p53 (37.5 degrees C), wild-type p53 (32.5 degrees C) was associated with a rapid loss of cell viability. Genomic DNA extracted at 32.5 degrees C was seen to be fragmented into a characteristic ladder consistent with cell death due to apoptosis. Following synchronization by density arrest, transfected cells released into G1 at 32.5 degrees C were found to lose viability more rapidly than did randomly growing cultures. Following release into G1, cells became irreversibly committed to cell death after 4 h at 32.5 degrees C. Commitment to cell death correlated with the first appearance of fragmented DNA. Synchronized cells allowed to pass out of G1 prior to being placed at 32.5 degrees C continued to cycle until subsequently arrested in G1; loss of viability occurred following G1 arrest. In contrast to cells in G1, cells cultured at 32.5 degrees C for prolonged periods during S phase and G2/M, and then returned to 37.5 degrees C, did not become committed to cell death. G1 arrest at 37.5 degrees C, utilizing either mimosine or isoleucine deprivation, does not lead to rapid cell death. Upon transfer to 32.5 degrees C, these G1 synchronized cell populations quickly lost viability. Cells that were kept density arrested at 32.5 degrees C (G0) lost viability at a much slower rate than did cells released into G1. Taken together, these results indicate that wild-type p53 induces cell death in murine erythroleukemia cells and that this effect occurs predominantly in the G1 phase of actively cycling cells.     

Effects of polyphyllin I on growth inhibition of human non-small lung cancer cells and in xenograft

Polyphyllin I (PPI), a small molecular monomer extracted from Rhizoma of Paris polyphyllin, shows strong anticancer effects in previous study. Human lung adenocarcinoma A549 cells, human lung squamous cell carcinoma SK-MES-1 cells, and human lung large cell carcinoma H460 cells were cultured and then treated with PPI. Cell proliferation and apoptosis were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, flow cytometry, western blot analysis, and DNA ladder. Athymic nude mice bearing tumors were injected with PPI, and tumor growth was recorded. Our results showed that PPI significantly inhibited the proliferation of three non-small cell lung cancer (NSCLC) cell lines, with the inhibitory concentrations (IC50) of 1.24, 2.40, and 2.33 μg/ml for A549, H460, and SK-MES-1 cells, respectively. After being treated with 2.5 μg/ml of PPI for 24 h, the apoptotic rate of A549 cells was 39.68%, which was remarkably higher than that of the control. Tumor growth was significantly inhibited in the PPI-treated group compared with the group treated with cisplatin (DDP) or PBS in the nude mice. PPI exhibits antitumor ability in NSCLC cells in vitro and in vivo, which might be related to the apoptosis induced by PPI.     

Individualized therapy for gastroesophageal reflux disease: potential impact of pharmacogenetic testing based on CYP2C19

The main therapeutic agent for gastroesophageal reflux disease (GERD) is a proton pump inhibitor (PPI). Plasma levels and the acid inhibitory effect of PPIs depend on the activity of cytochrome P450 (CYP) 2C19, which is polymorphic. Genotypes of CYP2C19 are classified into three groups: rapid metabolizers (RMs: *1/*1), intermediate metabolizers (IMs: *1/*X), and poor metabolizers (PMs: *X/*X), where *1 and X represent the wild type and the mutant allele, respectively. RMs include ultra-rapid metabolizers, who possess the CYP2C19*17 allele. The pharmacokinetics and pharmacodynamics of PPIs differ among different CYP2C19 genotype groups. Plasma PPI levels and intragastric pH values during PPI treatment are lowest in the RM group, intermediate in the IM group, and highest in the PM group. These CYP2C19-genotype-dependent differences in the pharmacokinetics and pharmacodynamics of PPIs influence the healing and recurrence of GERD during PPI treatment, suggesting the need for CYP2C19 genotype-based tailored therapy for GERD. CYP2C19 pharmacogenetics should be taken into consideration for the personalization of PPI-based therapy. However, the clinical usefulness of CYP2C19 genotype testing in GERD therapy should be verified in clinical studies.     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.     

Effect of serum on heat response of synchronized mouse neuroblastoma cells: protection of cell cycle progression, protein synthesis and survival

The effect of serum and temperature elevation on proliferation has been studied in synchronized mouse neuroblastoma (Neuro-2A) cells. The effects of serum were studied on the induction of (a) mitotic delay due to a non-lethal heat treatment (30 min at 42.7 degrees C) and (b) the loss of colony-forming capacity after a more extensive heat treatment (45 min at 44 degrees C or a continuous 42.7 degrees C heat treatment). The following results were obtained. Under conditions of serum depletion, cell cycle extension of heated G1 phase cells was more than that of heated G2 phase cells. Serum protected against heat-induced alterations of cell cycle progression in G1- but not in G2 phase cells. This effect of serum could be mimicked by a supplement to the medium of human transferrin, bovine pancreas insulin and selenium, and was correlated with protection of protein synthesis. Serum also affected heat-induced cell killing. Under conditions of serum depletion, G1 phase cells were more resistant to heat compared to G2 cells. The presence of serum during heat treatment further increased the thermoresistance of G1 phase cells, but did not affect sensitivity of G2 phase cells. This effect of serum could not be mimicked by a supplement of transferrin, insulin and selenium. These results indicate that serum protects G1 phase cells for heat-induced changes of cell cycle progression as well as on cell survival, but the mechanisms involved in both phenomena seem to be different.     

GABA/progesterone-induced polyphosphoinositide (PPI) breakdown and its role in the acrosome reaction of guinea pig spermatozoa in vitro

To investigate whether GABA/progesterone (P4) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa2+ for 5.5 h and then labeled with [32P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5×107 cells/mL and exposed to 2 mmol/L Ca2+, 5 mmol/L GABA, 10 mmol/L P4 and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermato-zoa were treated with GABA, 32P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists tested；their potency followed the order A23187＞progesterone≥GABA; (iii) GABA-induced PIP2 hydrolysis and rise in PA and the AR were prevented by inclusion of 10 mmol/L neomycin; (iv) the loss of PIP2 labeling and the increase in PA labeling abolished when spermatozoa were exposed to EGTA or Ca2+ channel blocker. These re-sults indicate that GABA or P4-induced PPI breakdown is an important and essential event in the series of changes to membrane fusion during the AR of guinea pig spermatozoa and this effect is mediated via calcium by activation of phosphatidylinositol-specific phospholipase C.     

Formation of proliferative tetraploid cells after treatment of diploid cells with sodium butyrate in rat 3Y1 fibroblasts

When randomly proliferating rat 3Y1 fibroblasts were treated with sodium butyrate, more than 90% of their cells were arrested reversibly with a 2C DNA content at least 12 h before the G1/S boundary. When cells synchronized in the early S phase were treated with butyrate, approximately 70% of all cells were arrested with a 4C DNA content. The arrests in both G1 and G2 phases by the single inhibitor suggest that the two phases share a common mechanism. The ability of cells to undergo mitosis on time was quickly lost with time of arrest in the G2 phase. Upon removal of the inhibitor, the cells arrested with a 4C DNA content entered a new S phase without intervening mitosis. The tetraploid cells thus produced kept proliferating as fast as diploid cells. These results suggest that the inhibition of the normal G2 traverse is somehow responsible for the formation of the proliferative polyploid cells.     

The cell cycle dependence of thermotolerance. III. HeLa cells heated at 45.0 degrees C

We have extended our studies on the cell cycle dependence of thermotolerance to include HeLa cells heated at 45.0 degrees C to compare the results to Chinese hamster ovary (CHO) cells. We found that asynchronous HeLa cells were more resistant to heat than CHO cells but showed a similar development and decay of thermotolerance. Flow cytometry (FCM) was used to study redistributions in the cell cycle after an initial heat dose. Cells heated for 35 min at 45.0 degrees C were delayed in G1 by about 7 h compared to controls, with delays in late S and G2/M phase also. The heat sensitivity varied through the cell cycle; G1 cells were the most resistant to heat, while S-phase cells were uniformly sensitive throughout S phase, and G2 cells were resistant. Thermotolerance could be induced and expressed in early or late S-phase cells, but to a lesser extent than for G1 cells. The results were similar in many respects to CHO cells, but there were significant differences.