羟基脲（Hydroxyurea）对细胞周期与珠蛋白基因表达的影响（简报）

Although the hydroxyurea (HU) has been extensively studied, little is known of its molecular mechanism in controlling the expression of human globin gene and in modulating the progression of cell-cycle in K 562 cell. In the present study, the effect of hydroxyurea on proliferative kinetics of K 562 cells was examined by monitoring the number of cells during a period of 8 day's cell culture. Our results showed that there was a dose related decrease in cell growth when K562 cells were incubated with HU. Moreover, cell-cycle analysis demonstrated that HU had profound effect on cell-cycle distribution. In the case of the induced K 562 cells, there was an increased accumulation of cells in S phase and a decreased fraction of cells in G 1 and G 2 + M phase. Furthermore, HU could induce the expression of human beta-globin gene in the induced K 562 cells. Our results indicate that HU has a potential to inhibit the proliferation of K 562 cells and to stimulate the terminal differentiation of this cell.     

MCP-1 in pleural injury: CCR2 mediates haptotaxis of pleural mesothelial cells

Pleural injury results in the death of mesothelial cells and denudation of the mesothelial basement membrane. Repair of the mesothelium without fibrosis requires proliferation and migration of mesothelial cells into the injured area. We hypothesized that monocyte chemoattractant protein-1 (MCP-1) induces proliferative and haptotactic responses in pleural mesothelial cells (PMCs) and that the MCP-1 binding receptor CCR2 mediates the pleural repair process. We demonstrate that PMCs exhibited MCP-1-specific immunostaining on injury. MCP-1 induced proliferative and haptotactic responses in PMCs. PMCs express CCR2 in a time-dependent manner. Fluorescence-activated cell sorting analysis demonstrated that interleukin (IL)-2 upregulated CCR2 protein expression in PMCs, whereas lipopolysaccharide (LPS) downregulated the response at the initial period compared with that in resting PMCs. However, the inhibitory potential of LPS was lost after 12 h and showed a similar response at 24 and 48 h. Haptotactic migration was upregulated in PMCs that were cultured in the presence of IL-2. The increased haptotactic capacity of mesothelial cells in the presence of IL-2 correlated with increased CCR2 mRNA expression. PMCs cultured in the presence of LPS showed decreased haptotactic activity to MCP-1. Blocking the CCR2 with neutralizing antibodies decreased the haptotactic response of PMCs to MCP-1. These results suggest that the haptotactic migration of mesothelial cells in response to MCP-1 are mediated through CCR2, which may play a crucial role in reepithelialization of the denuded basement membrane at the site of pleural injury and may thus contribute to the regeneration of the mesothelium during the process of pleural repair.     

Nuclear heterogeneity and proliferation activity of human adipose derived MSC-like cells

Adipose tissue (AT) is an easily available source of mesenchymal-like stem cells (MSCs) that are appropriate for applications in regenerative medicine. There is conflicting evidence on the morphology of AT-derived stem cells (ADSCs). Here, we described the morphology and proliferation activity of human ADSCs. The cells were plated at a density of 10 cells/sm² and cultivated for 1 month. Twenty-one colonies were grown. In nine out of 17 analyzed colonies, few atypical cells (large nuclei and cytoplasm) were found. ANOVA demonstrated that colonies also differed (p = 0.0025) in the size and diameter of cell nuclei. The size of nuclei and logarithm of cell density were correlated in the reverse proportion (−0.7; p = 0.002). Thus, a culture obtained from the stromal vascular fraction (SVF) is heterogeneous and composed of two types of cells, i.e., highly proliferative and large, low proliferative cells. These cells are typical of the MSC and ADSC cultures described in literature. The cell heterogeneity observed in some colonies probably resulted from variations in the cell-cycle phases.     

99mTc-HMPAO labelling inhibits cell motility and cell proliferation and induces apoptosis of NC-NC cells

(99m)Tc-hexamethyl-propylenamine-oxime ((99m)Tc-HMPAO)-labelled leukocytes have been used in standard diagnostic procedures for the detection of infection and inflammation. Although some investigators have already pointed out that labelling of leukocytes with (99m)Tc-HMPAO has detrimental effects on the cells, still very little is known regarding the effects of ionizing radiation on lymphocyte function. The effects of (99m)Tc-HMPAO-labelling on lymphocyte adhesion, proliferation, mitotic index, migration and apoptosis were evaluated. The lymphoblastoid cell line NC-NC was used as the lymphocyte population. (99m)Tc-HMPAO-labelling decreased cell adhesion, proliferation, mitotic index and motility, whereas it induced apoptosis and cell-cycle arrest. The rate of decrease in cell proliferation was up to 70% (P<0.001) by day 4 after labelling. (99m)Tc-HMPAO-labelling led a 35% decrease (P<0.001) in adhesion ability of the cells on fibronectin at 16h. Using the Boyden chamber motility assay, it was shown that both spontaneous and monocyte chemotactic protein (MCP-1)-induced lymphocyte motility were strongly reduced by (99m)Tc-HMPAO-labelling. The decrease in motility was approximately five-fold (P<0.05). In addition, a 12-fold increase (P<0.05) was observed in apoptosis of the (99m)Tc-HMPAO-treated cells compared with control cells. Besides, it was shown that cell-cycle arrest was induced starting from the 3rd day after treatment with (99m)Tc-HMPAO. Our observations indicate that (99m)Tc-HMPAO-labelling has damaging effects on lymphocyte function including cell adhesion, proliferation, mitotic index, motility and cell cycle under in vitro conditions.     

N-acetylcysteine induces beneficial changes in the acinar cell cycle progression in the course of acute pancreatitis

Oxygen free radicals (OFR) are produced in the course of acute pancreatitis (AP). In addition to injurious oxidative effects, they are also involved in the regulation of cell growth. The aim of the present study was to examine the relationship between the effectiveness of N-acetyl-l-cysteine (NAC) to prevent the generation of OFR and the changes in the cell-cycle pattern of acinar cells in the course of AP induced in rats by pancreatic duct obstruction (PDO). NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Flow-cytometric measurement of OFR generation in acinar cells was carried out using dihydrorhodamine as fluorescent dye. Plasma amylase activity, pancreatic glutathione (GSH) content and TNF-alpha plasma levels were also measured. The distribution of acinar cells throughout the different cell-cycle phases was analysed at different AP stages by flow cytometry using propidium iodide staining. NAC administration reduced the depletion of pancreatic GSH content and prevented OFR generation in acinar cells of rats with PDO-induced acute pancreatitis. As a result, AP became less severe as reflected by the significant improvement of hyper-amylasaemia and maintenance of plasma TNF-alpha levels at values not significantly different from controls were found. NAC administration inhibited progression of cell-cycle phases, maintaining acinar cells in quiescent state at early PDO times. The protection from oxidative damage by NAC treatment during early AP, allows the pancreatic cell to enter S-phase actively at later stages, thereby allowing acinar cells to proliferate and preventing the pancreatic atrophy provoked by PDO-induced AP. The results provide evidence that OFR play a critical role in the progression of acinar cell-cycle phases. Prevention of OFR generation of acinar cells in rats with PDO-induced AP through NAC treatment, not only protects pancreas from oxidative damage but also promotes beneficial changes in the cell cycle progression which reduce the risk of pancreatic atrophy.     

Proliferative status of colonic mucosa in organ culture: 3H-thymidine-labelling studies and computer modelling

3H-thymidine labelling studies and a computer simulation have been employed to assess proliferative status and cellular organisation in colonic explants maintained in culture for 5 to 7 days. The one-hour flash labelling index (Is) for crypts within the middle region of explants (5.2%) was considerably lower than that observed in vivo (8.8%). Crypt length and the distribution of labelled cells appeared similar for both situations. A computer simulation program for crypt-cell proliferation was devised, facilitating the modulation of a number of parameters including the cell-cycle time (Tc) and its component phases, the cut-off position, and cell loss at mitosis. This simulation was employed to model continuous labelling (72 h) data obtained in vitro and provided an estimate of various kinetic parameters. Data for the middle region of explants was fitted with a Tc of 62 h, an S phase of 8 h and a cell loss factor (20%) which was consistent with histological findings. A fit to the experimental data obtained in vitro could be achieved by a model based upon a mode of cellular organisation known to occur within crypts in vivo. Therefore in vitro, the dynamic processes of crypt-cell proliferation and migration appear to be organised in the same manner as seen in vivo.     

3-Hydrogenkwadaphnin induces monocytic differentiation and enhances retinoic acid-mediated granulocytic differentiation in NB4 cell line

Recently, we have reported that 3-hydrogenkwadaphnin (3-HK), a diterpene ester isolated from Dendrostellera lessertii (Thymealeaceae), is very effective against leukemia cell lines without any detectable effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK induces G1 cell-cycle arrest, differentiation and apoptosis in APL NB4 cell line. Indeed, the drug between 24 to 96 h induced 7-65% growth inhibition of NB4 cells. Cell viability was also decreased by 2-55% between 24 to 96 h treatments with the drug, respectively. These effects of the drug were also dose-dependent. According to flow cytomtry results, 3-HK (15 nM) induced a significant G1-arrest up to 24 h which was consequently followed with appearance of sub-G(1) peak at 72 to 96 h. Hoechst 33258 staining and DNA fragmentation assays confirmed the occurrence of apoptosis among the treated cells. On the other hand, NBT reducing assay, Wright-Giemsa staining, phagocytic activity and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation especially toward macrophage-like morphology. Interestingly, 3-HK at 5 and 10 nM enhanced the effects of all-trans retinoic acid (ATRA) in NB4 cells. Based on these results, 3-HK might become an ideal candidate for treatment of APL patients pending full exploration of its biological functions.     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell loss was induced in the descending colon of the rat by temporary ischaemia to investigate whether this would lead to an increase in crypt cell proliferation. Shortly after the temporary ischaemia the number of cells per crypt was markedly reduced, and it was shown that the cell loss occurred mainly from the non-proliferating upper half of the crypt. The number of cells per crypt reached control values again after 24-48 h. There was a marked increase in proliferative activity, as reflected by the labelling index after 3HTdR and by the mitotic index, with peak values at 16 and 24 h after ischaemia. After 48 h the proliferative indices were normal again. The increase in crypt cell proliferation was characterized by an increase in the labelling index as well as in the mitotic index per crypt cell position. No enlargement of the proliferative cell compartment in the crypt was observed. It is most likely then that the increase in crypt cell proliferation was brought about by a shortening of the cell cycle, since the growth fraction in the lower half of the crypt approaches 1.0. The possible implications of the present data for the control of colonic cell proliferation and colonic carcinogenesis are discussed.     

Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes

Expression of cyclin, a non-histone nuclear protein, during recombinant interleukin 2 (rIL2)-driven cell-cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S-phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell-cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25-49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA-encoding murine cyclin was cloned from a cDNA library prepared from IL2-stimulated cloned T cells. The sequence of the 5' end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2-induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.     

Circadian rhythms of the parameters of the mouse esophagus epithelial cell kinetics

Circadian rhythms of the mitotic activity, DNA synthesis and the parameters of the mitotic cells of the mouse esophagus epithelium were studied during the periods of maximum and minimum proliferation. The number of mitoses and DNA-synthetizing cells increases rhythmically at 1--7 a. m. from 22 p. m. to 4 a. m., respectively. When 3H-thymidine was injected to the mice at 2 a. m., tG2min was 1h; tG2+1/2 M was 2h; tS was 7.1; tG1+1/2 M was 2h; tS was 7.1; tG1+1/2 M was 15.9h. When 3H-thymidine was injected at 2 p. m., tS rose up to 8.2 and tG1+1/2 M up to 14.8h. The mitotic cycle in both series of experiments totalled 25 h. Thus, the duration of various phases of the mitotic cycle depends on the time of the day and correlates with circadian rhythms of the mitotic activity and the number of DNA-synthetizing cells. Duration of the mitotic cycle of the cells passing through it at varying time of the day is the same and approximates the period of the circadian rhythm of mitoses and DNA synthesis in esophagus epithelium.     

Morphologic changes in the kidneys in acute kidney failure due to acute pancreatitis

Light optical and electron-microscopic investigations were carried out on 42 dogs with experimental hypertensive-enzymatic acute pancreatitis. It was revealed that microcirculatory changes appeared already within the first hour of experimental pancreatitis. By the 4th hour of the experiment the changes in basal membrane and podocytes, as well as proliferation of mesangial cells and growth of mesangial matrix were noted. Thus, at the initial stages of acute pancreatitis morphological alterations in the kidneys correspond to the pattern of mesangial proliferative diffuse glomerulitis.     

原儿茶酸促进人脂肪干细胞体外增殖的研究

为了寻找能够促进干细胞增殖的药物,观察了中药益智仁（Alpinia oxyphylln）中提取的原儿茶酸对人脂肪干细胞体外增殖的影响,并对其作用机制进行了初步的探讨．人脂肪干细胞能在体外分化为神经元样细胞,并对凋亡的PC-12细胞起到保护作用．原儿茶酸能够促进人脂肪干细胞的增殖,且呈现明显的剂量依赖性和时间依赖性．流式细胞术检测细胞DNA含量的结果显示,原儿茶酸处理组细胞S期所占比例明显增加,其中,1．5mmol／L原儿茶酸处理组细胞S期所占比例与对照组相比增加2倍以上．同时,该组细胞G2／M期所占比例明显增加,G0／G1期所占比例明显下降．蛋白质免疫印迹结果显示,1．5mmol／L原儿茶酸处理组细胞周期素D1（cyclinD1）的表达明显升高．cyclin D1-siRNA转染显著抑制了原儿茶酸对人脂肪干细胞体外增殖的促进作用．流式细胞术检测细胞表面标志物,成骨诱导和脂肪诱导的结果显示,原儿茶酸处理后,人脂肪干细胞仍保持间充质干细胞多分化潜能的特性．上述结果提示,原儿茶酸有可能在人脂肪干细胞介导的干细胞移植治疗中发挥作用．     

Culture procedure of mesothelial cells from the rat parietal pleura

Cultures were made of mesothelial cells obtained by scraping the parietal pleura of the adult rats. The growth was restricted to close polyhedric epithelial-like cells, forming a monolayer. The cellular proliferation continued until the 7th day, followed by a stationary phases. In subcultures the mesothelial cells kept their epithelial type. The cultures were stopped on the 20th day.     

Resveratrol Inhibits Proliferation and Promotes Apoptosis of Neuroblastoma Cells: Role of Sirtuin 1

Resveratrol prolongs lifespan and prevent cancer formation; however, the mechanisms are not understood. Here we evaluated the cell-cycle inhibition and apoptosis of resveratrol in B65 neuroblastoma cells, and we also studied the effects of resveratrol on the mammalian silent information regulator 2 (SIRT1). Results show that resveratrol reduces cell viability and causes apoptosis at 24 h of treatment. Resveratrol partially blocked cell proliferation, and significantly increased the fraction of cells arrested in the S phase. The role of SIRT1 in cell-cycle effects mediated by resveratrol was studied through changes in the expression of SIRT1 using western blot. Exposure to resveratrol decreased SIRT1 content, concomitant with an increase in the acetylated form of sirtuin substrates p53 and NFκ-β. Treatment of B65 neuroblastoma cells with resveratrol also reduced the content of the phosphorylated form of AKT. Exposure to the SIRT1 inhibitors nicotinamide and sirtinol altered neither cell viability nor the fraction of apoptotic cells. Furthermore, when cells were exposed simultaneously to resveratrol and nicotinamide or sirtinol, no changes were observed in the fraction of apoptotic cells. Our results show that a decrease in SIRT1 content, caused by exposure to resveratrol, does not appear to be involved in cell-cycle arrest or activation of apoptosis.     

How the change from FLM to FACS affected our understanding of the G1-phase of the cell cycle

The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DMA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G1 phase variability resulted from constancy of S and G2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G1-phase amount of DMA appeared to be "arrested in G1 phase". The loss of absolute phase length determinations has therefore led to the proposals of G1-phase arrest, G1-phase controls, restriction points, and G0 phase. It is suggested that these G1-phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G1-phase controls.     

How the Change from FLM to FACS Affected Our Understanding of the G1-Phase of the Cell Cycle

The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DNA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G1-phase variability resulted from constancy of S and G2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G1-phase amount of DNA appeared to be “arrested in G1 phase”. The loss of absolute phase length determinations has therefore led to the proposals of G1-phase arrest, G1-phase controls, restriction points, and G0 phase. It is suggested that these G1-phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G1-phase controls.     

Effects of purine cyclic nucleotides on the growth of neonatal rat hepatocytes in primary tissue culture

cGMP and db-cGMP administered for 20–24 h to neonatal rat hepatocytes in primary culture stimulated their DNA synthesis and proliferation only at concentrations higher than the physiological one, whereas at concentrations equal to or lower than the physiological concentration they were ineffective or inhibitory for both activities. Induction of DNA synthesis to be effected by cGMP required 15 h of treatment, preceded, however, by inhibition of the same process between the 6th and the 14th hour of exposure. In contrast, cAMP and db-cAMP stimulated the flow of cultivated hepatocytes into the S and M stages of their mitotic cycle when administered at very wide concentration range, including the physiological for cAMP and the sub-physiological for db-cAMP. cAMP was effective after 12–14 h of treatment. Equimolar mixtures of cGMP with cAMP and of db-cGMP with db-cAMP also stimulated the proliferative activity of primary hepatocytes, but only at very low doses, which induced a first peak of DNA synthesis between the 2nd and the 6th hour of treatment and a second peak at about the 18th hour. These actions of the cyclic compounds, employed singly or in equimolar combination, were shown to be specific, since they could not be reproduced by their main metabolites. The present results strengthen the view that cAMP plays a pre-eminent role in the positive regulation of hepatocyte proliferation. Contrary to the postulate of the dualistic doctrine, cGMP by itself is not proliferogenic in the physiological range; in fact, cGMP acts as an ancillary, possibly dispensable, compound whose physiological role may be to help, in cooperation with cAMP, liver cells to cross the G1/S boundary of their growth-division cycle.     

Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination of fluid streaming. These findings support the hypothesis that a history of PD for a certain period could serve as a trigger of EPS after stoppage of PD.     

Altered expression of genes regulating cell growth,proliferation, and apoptosis during adenosine 3',5'-cyclic monophosphate-induced differentiation of neuroblastoma cells in culture

An elevation of the intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) induces terminal differentiation in neuroblastoma (NB) cells in culture; however, genetic alterations during differentiation have not been fully identified. To investigate this, we used Mouse Genome U74A microarray containing approximately 6000 functionally characterized genes to measure changes in gene expression in murine NB cells 30 min and 4, 24, and 72 h after treatment with cAMP-stimulating agents. Based on the time of increase in differentiated functions and their status (reversible versus irreversible) after treatment with cAMP-stimulating agents, the induction of differentiation in NB cells was divided into three distinct phases: initiation (about 4 h after treatment when no increase in differentiated functions is detectable), promotion (about 24 h after treatment when an increase in differentiated functions occurs, but they are reversible upon the removal of cAMP), and maintenance (about 72 h after treatment when differentiated functions are maximally expressed, but they are irreversible upon the removal of cAMP). Results showed that alterations in expression of genes regulating cell growth, proliferation, apoptosis, and necrosis occurred during cAMP-induced differentiation of NB cells. Genes that were upregulated during the initiation, promotion, or maintenance phase were called initiators, promoters, or maintainers of differentiation. Genes that were downregulated during the initiation, promotion, or maintenance phase were called suppressors of initiation, promotion, or maintenance phase. Genes regulating growth may act as initiators, promoters, maintainers, or suppressors of these phases. Genes regulating cell proliferation may primarily act as suppressors of promotion. Genes regulating cell cycle may behave as suppressors of initiation or promotion, whereas those regulating apoptosis and necrosis may act as initiators or suppressors of initiation or promotion. The fact that genetic signals for differentiation occurred 30 min after treatment with cAMP, whereas cell-cycle genes were downregulated at a later time, suggests that decision for NB cells to differentiate is made earlier and not at the cell-cycle stage, as commonly believed.     

Examining Go-or-Grow Using Fluorescent Cell-Cycle Indicators and Cell-Cycle-Inhibiting Drugs

The go-or-grow hypothesis states that adherent cells undergo reversible phenotype switching between migratory and proliferative states, with cells in the migratory state being more motile than cells in the proliferative state. Here, we examine go-or-grow in two-dimensional in vitro assays using melanoma cells with fluorescent cell-cycle indicators and cell-cycle-inhibiting drugs. We analyze the experimental data using single-cell tracking to calculate mean diffusivities and compare motility between cells in different cell-cycle phases and in cell-cycle arrest. Unequivocally, our analysis does not support the go-or-grow hypothesis. We present clear evidence that cell motility is independent of the cell-cycle phase and that nonproliferative arrested cells have the same motility as cycling cells.