Cell cycle dependence of calmodulin levels during HL-60 proliferation and myeloid differentiation: No changes during pre-commitment

The putative role of Ca²⁺ and calmodulin in regulating cell proliferation and differentiation was tested in HL-60 human promyelocytic leukemia cells. The dependence of retinoic acid (RA)-induced terminal myeloid differentiation of HL-60 promyelocytic leukemia cells on calmodulin levels and calcium ion flux was ascertained. RA-treated and untreated control cells were stained for cellular DNA with a Hoechst dye. Populations of G1/0, S and G2+M phase cells were isolated by fluorescence activated cell sorting (FACS). Cytosolic calmodulin levels were then measured as a function of cell cycle phase for RA-treated and untreated cells using a radioimmunoassay. RA-treated cells were measured at early times, corresponding to the precommitment state, and late times, when significant cell differentiation had occurred. Cellular calmodulin levels increased with progression through the cell cycle. In contrast, no difference in calmodulin levels was observed between RA-untreated or -treated cells in the same cell cycle phases at early or late times. RA-induced HL-60 terminal myeloid differentiation was thus apparently not regulated by cellular cytosolic calmodulin levels. These conclusions were supported by the effects of calmodulin antagonists and calcium flux inhibitors. The calmodulin antagonists trifluoperazine and compound 48/80 both retarded cell growth in a concentration-dependent manner. But at concentrations where cellular effect was evidenced by slight growth inhibition, neither antagonist inhibited RA-induced cell differentiation or G1/0 growth arrest. The same was true of the gated calcium channel inhibitors, verapamil and nitrendipene, and the passive calcium flux inhibitor, CoC1₂. RA-induced HL-60 cell differentiation and arrest in G0 was thus apparently not strongly dependent on cellular calmodulin levels or calcium flux. This is in strong contrast to murine erythroleukemia cells. The results argue against a central regulatory role for calmodulin or calcium flux in control of HL-60 growth arrest or differentiation.     

Calmodulin and the cell cycle: Involvement in regulation of cell-cycle progression

Calmodulin levels are elevated twofold at late G1 and/or early S phases during the growth cycle of CHO-K1 cells. These levels are maintained throughout the remainder of the cell cycle until cytokinesis. The G1 daughter cells then contain half the intracellular calmodulin level found prior to cell division. Elevation of calmodulin at the G1-S boundary is independent of the length of G1, and the increase in calmodulin appears to be related to progression into S phase. The importance of calmodulin for G1-S progression is suggested by the ability of the anticalmodulin drug W13 to elicit specific and reversible progression delays into and through S phase.     

Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.) : Evidence for Posttranslational Control of Calmodulin Expression

The expression of calmodulin mRNA and protein were measured during a growth cycle of carrot (Daucus carota L.) cells grown in suspension culture. A full-length carrot calmodulin cDNA clone isolated from a λgt10 library was used to measure steady-state calmodulin mRNA levels. During the exponential phase of culture growth when mitotic activity and oxidative respiration rates were maximal, calmodulin mRNA levels were 4- to 5-fold higher than they were during the later stages of culture growth, when respiration rates were lower and growth was primarily by cell expansion. Net calmodulin polypeptide synthesis, as measured by pulse-labeling in vivo with [³⁵S]methionine, paralleled the changes in calmodulin steady-state mRNA level during culture growth. As a consequence, net calmodulin polypeptide synthesis declined 5- to 10-fold during the later stages of culture growth. The qualitative spectrum of polypeptides synthesized and accumulated by the carrot cells during the course of a culture cycle, however, remained largely unchanged. Calmodulin polypeptide levels, in contrast to its net synthesis, remained relatively constant during the exponential phases of the culture growth cycle and increased during the later stages of culture growth. Our data are consistent with increased calmodulin polypeptide turnover associated with periods of rapid cell proliferation and high levels of respiration.     

Staurosporine引起正常和肿瘤细胞内Ca~(2 )和CaM水平发生变化

低剂量Staurosporine可以使正常细胞可逆地阻断于G1期,但对肿瘤细胞的周期运行不发生任何影响。本文利用显微光度术,测定了单个细胞内Ca~(2 )、活化钙调素和总钙调素的含量,结果表明:5ng/mL staurosporine作用于细胞18h,使正常细胞2BS G1和S期总CaM含量降低;而BGC-823细胞各周期时相总钙调素含量不发生改变;钙活化钙调素增加。Staurosporine阻断2BS细胞于G1期而不影响BGC-823的周期运行可能与Stauro-Sporine使2BS G1期细胞的钙调素降低以及抑制了2BS细胞的P~(107)磷酸化有关。     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.     

The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans.

The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin-regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth-arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle-dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A. nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.     

Changes in calmodulin and its mRNA accompany reentry of quiescent (G0) cells into the cell cycle

Release of CHO-K1 cells from plateau or stationary phase and reentry into the cell cycle is specifically and reversibly blocked at two distinct sites by the anticalmodulin drug W13. The first block occurs early during release while the cells are still at G0/G1, whereas the second occurs later in reentry during early S phase. As determined by radioimmunoassay, calmodulin levels undergo changes at three distinct steps in plateau-phase entry and release. First, the entry of exponentially growing cells into plateau phase is accompanied by an increase in the calmodulin level. The second change is a reduction in the calmodulin content of cells within the first hour following release from plateau phase. The third change is the subsequent increase in calmodulin levels, which precedes entry of the cells into S phase. Analysis of calmodulin mRNA levels by dot-blot hybridization demonstrates that the changes in calmodulin protein are preceded by changes in calmodulin mRNA. Furthermore, whereas a decrease in CaM mRNA is observed within the first hour following plateau release, no such decrease is observed for beta-actin mRNA, suggesting that this decrease may be selective for calmodulin. This selectivity is further substantiated by the fact that identical changes in calmodulin and calmodulin mRNA are observed in cells released from plateau by two different techniques. Taken together, these data suggest that calmodulin may play an important role in the reentry of cells into the cell cycle.     

The role of calmodulin in the proliferation of transformed and phenotypically normal tsASV-infected rat cells

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, behaved as if nontransformed at a nonpermissive 40 degrees C and were rendered quiescent by serum deprivation. These serum-deprived cells were stimulated to start entering S phase about 7 hours after serum addition at 40 degrees C or about 9 hours after shifting the cultures to 36 degrees C, a temperature allowing the production of active viral pp60src and expression of the transformed phenotype. The transit of both serum- and temperature-stimulated tsLA23-NRK cells through later G1 was inhibited by the unrelated calmodulin antagonists W7 and R24571. The former drug was found to block the cells at a point in the cell cycle no more than 2 hours from the G1/S transition. The weaker calmodulin antagonist, W5, was less effective in impairing progression. Thus, calmodulin is likely required for the transit of both transformed and phenotypically normal tsLA23-NRK cells through the later stages of their G1 phases. Cells neoplastically transformed by ASV contain more calmodulin than uninfected, non-neoplastic cells. At the nonpermissive 40 degrees C, the calmodulin content of the tsLA23-NRK cells dropped to the non-neoplastic level. When these phenotypically nontransformed cells were enabled to reenter the cell cycle while still in low-serum medium by a 40 to 36 degrees C shift, they passed through the G1 and S phases and divided without a concomitant rise in the total calmodulin content. Thus, a calmodulin rise does not appear to be required for the expression of one characteristic of transformed cells, i.e., reduced requirement for exogenous growth factors.     

The distribution of calmodulin and Ca^2＋—activated calmodulin in cell cycle of mouse erythroleukemia cells

Cell proliferation is accompanied with changing levels of intracellular calmodulin (CaM) and its activation.Prior data from synchronized cell population could not actually stand for various CaM levels in different phases of cell cycle.Here,based upon quantitative measurement of fluorescence in individual cells,a method was developed to investigate intracellular total CaM and Ca^2 -activated CaM contents. Intensity of CaM immunoflurescence gave total CaM level,and Ca^2 -activated CaM was measured by fluorescence intensity of CaM antagonist trifluoperazine (TFP).In mouse erythroleukemia (MEL) cells,total CaM level increased from G1 through S to G2M,reaching a maximum of 2-fold increase,then reduced to half amount after cell division.Meanwhile,Ca^2 -activated CaM also in creased through the cell cycle(G1,S,G2M).Increasing observed in G1 meant that the entry of cells from G1 into S phase may require CaM accumulation,and,equally or even more important,Ca^2 -dependent activation of CaM.Ca^2 -activated CaM decreased after cell division.The results suggested that CaM gene expression and C^2 -modulated CaM activation act synergistically to accomplish the cell cycle progression.     

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry

Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth. While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d. A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27%. Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior.     

Calmodulin levels in the yeast and mycelial phases of Ceratocystis ulmi.

The calmodulin content of the yeast and mycelial phases of Ceratocystis ulmi was determined by radioimmunoassay. Calmodulin levels increased at the G1-S boundary of the cell cycle, coinciding with the first visible appearance of buds or germ tubes. However, in both phases the cellular calmodulin levels were equivalent. No differential synthesis was observed.     

Functional expression of chicken calmodulin in yeast

The coding region of a chicken calmodulin cDNA was fused to a galactose-inducible GAL1 promoter, and an expression system was constructed in the yeast Saccharomyces cerevisiae. Expression of calmodulin was demonstrated by purifying the heterologously expressed protein and analyzing its biochemical properties. When the expression plasmid was introduced into a calmodulin gene (cmd1)-disrupted strain of yeast, the cells grew in galactose medium, showing that chicken calmodulin could complement the lesion of yeast calmodulin functionally. Repression of chicken calmodulin in the (cmd1)-disrupted strain caused cell cycle arrest with a G2/M nucleus, as observed previously with a conditional-lethal mutant of yeast calmodulin. These results suggest that the essential function of calmodulin for cell proliferation is conserved in cells ranging from yeast to vertebrate cells.     

Flow cytometric cell cycle analysis of cultured brown bear fibroblast cells

The aim of this study was to assess by flow cytometry the cell cycle of brown bear fibroblast cells cultured under different growth conditions. Skin biopsies were taken in Cantabria (Spain) from a live, anaesthetized brown bear. DNA analysis was performed by flow cytometry following cell DNA staining with propidium iodide. Serum starvation increased (P<0.01) the percentage of G0/G1 phase cells (92.7+/-0.86) as compared to cycling cells (39.7+/-0.86) or cells cultured to confluency (87.3+/-0.86). DMSO included for 48h in the culture significantly increased (P<0.01) the percentage of G0/G1 phase of the cell cycle at all concentrations used and decreased percentages of S phase in a dose-dependent fashion. Roscovitine increased the G0/G1 phase of the cell cycle (P<0.01) at 15microM concentration. Interestingly, the G2/M stage significantly increased at 30 and 50microM compared to the control and 15microM (P<0.02). The cell cycle of brown bear adult fibroblast cells can be successfully synchronized under a variety of culture conditions.     

Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans.

Calcium and calmodulin have been widely implicated in the control of cell proliferation. We have created a strain of the genetically tractable filamentous fungus, Aspergillus nidulans, that is conditional for calmodulin expression. This was accomplished by replacing the unique endogenous calmodulin gene with one regulated by the inducible alcohol dehydrogenase (alcA) gene promoter by homologous recombination. This strain cannot grow when the cells are incubated in medium containing a carbon source that represses the alcA promoter. Characterization of the arrested cells shows that 83% are blocked in the G2 phase of the cell cycle. The block is due to very low levels of calmodulin and is fully reversible upon changing to medium that contains an inducer of the alcA promoter. The rate of cell proliferation in this strain is dependent upon both the intracellular calmodulin and extracellular Ca2+ concentrations. Raising the calmodulin concentration by inducing the alcA promoter not only causes the cells to enter the proliferative cycle more quickly and to grow faster, but also decreases the concentration of extracellular Ca2+ required to support growth by 10-fold, as compared with cells grown in noninducing medium. Thus both the intracellular calmodulin and extracellular Ca2+ concentrations are important and interactive factors in regulating the nuclear division cycle of Aspergillus nidulans.     

Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342,rabbit anti calmodulin antibody,FITC-labeled goat anti rabbit IgG and SR101(sulfo rhodamine 101)simultaneously to stain individual normal and transformed cells,the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus,calmodulin and total protein respectively,could be recognized in individualj cells without interference,The phase of the cell cycle was determined by DNA content(Hoechst 33342),We found that in transformed cells(NIH3T3) tsRSV-LA90,cultured at 33℃ and transformed C3H10T1/2 Cells),the ration of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV-LA90 cells,cultured at 39℃ and C3H10T1/2 cells)in every cell cycle phase,This ration increased obviously only from G1 to S phase in either normal or transformed cells.The results showed that calmodulinreally increased during the transformation,and its increase was specific.In the meantime when cells proceeded from G1 to S.the intraceollular calmodulin content also increased specifically.     

Calmodulin regulates the post-anaphase reposition of centrioles during cytokinesis

INTRODUCTIONCytokinesis is a very complicated and carefully orches-trated process. During the last step of this process, anintercellular bridge is formed between the two daughtercells. A number of studies suggest that this intercellularbridge is not merel…     

The presence of parvalbumin in a nonmuscle cell line attenuates progression through mitosis

Based on studies that have examined the effect of calcium chelators on cells, it has been proposed that this cation plays a role in regulating cell proliferation. In this study a novel approach was used to indirectly examine the role of calcium in cell cycle progression. A cDNA for the Ca2+-binding protein parvalbumin has been expressed in mouse C127 cells, using a bovine papilloma virus-based expression vector. The normal role of parvalbumin is that of a calcium buffer in vertebrate fast twitch muscle, and the C127 cells do not normally express this protein. The presence of parvalbumin had several effects on the growth of C127 cells. The most striking phenotype was an increase in cell cycle duration which analysis showed was the result of an increase the length of G1 and mitosis (predominantly at prophase). Since changes in cell cycle duration typically occur as a result of changes in G1 duration, the observed increase in the length of mitosis is most unusual. The present results indicate that the previously observed increase in the rate of cell proliferation in cells with elevated calmodulin levels is not the result of a general increase in the level of cytoplasmic calcium-binding protein, but is specific to calmodulin. In addition, the results suggest that calcium regulates progression through mitosis by both calmodulin-dependent (metaphase transition) and -independent (prophase) mechanisms.     

人羊水来源干细胞的体外培养及其生物学性状分析

目的:体外分离培养人羊水来源干细胞(hAFSC),观察分析其基本的生物学性状。方法:取孕中期产前诊断所抽取的羊水,离心收集细胞,然后贴壁培养获得hAFSC。在细胞培养的基础上,观察hAFSC的形态;研究其增殖能力;用流式细胞术测定细胞周期及不同代次细胞表面阶段特异性胚胎抗原4(SSEA-4)表达率的变化;利用RT-PCR方法检测细胞干性基因的表达;利用诱导培养基诱导,检测其向肝样细胞与成骨细胞分化的潜能。结果:在体外培养条件下,hAFSC表现为成纤维细胞形态;具有良好的增殖能力,群体倍增时间约为36 h;细胞周期检测G0/G1期细胞约占86.7%,S+G2/M期细胞约占13.3%;流式细胞术检测结果表明,hAFSC表达SSEA-4,且SSEA-4阳性率随传代次数呈现先上升后下降的趋势;hAFSC在mRNA水平上表达Nanog、Oct4和Rex1等胚胎干细胞标志性基因;经诱导培养基诱导后,hAFSC可以向肝样细胞与成骨细胞分化。结论:hAFSC是一群呈成纤维细胞样,有良好增殖能力,且具有多向分化潜能的细胞,加之其来源方便,伦理学限制较少,因此在细胞治疗及组织工程等方面有着广泛的应用前景。     

Evaluation of human MSCs cell cycle, viability and differentiation in micromass culture

Mesenchymal stem cells (MSCs) have the potential to differentiate into distinct mesenchymal tissue cells. They are easy to expand while maintaining their undifferentiated state, which suggests that these cells could be an attractive cell source for tissue engineering of cartilage. In vitro high density micromass culture has been widely used for chondrogenesis induction. Our objective was to investigate human MSCs cell cycle, viability and differentiation in these conditions. Therefore, to induce human MSCs chondrogenesis, micromasses were cultured in the presence of transforming growth factor-beta1 in serum free medium for 21 days. Cell cycle, cell viability and cell phenotype were analyzed by flow cytometry. From day 0 to 7, the G0/G1 phase increased, whereas the S phase decreased gradually, but cell cycle phases (S, G0/G1 and G2/M) did not significantly change after day 7. Less than 10% of cells were apoptotic, but no necrosis was observed, even at day 21. We observed a decrease in CD90 and CD105 expression, from day 0 to 21. In conclusion, our results demonstrate a good viability of human MSCs in micromass culture during the whole period of culture. Moreover, micromass culture allowed human MSCs to be synchronized at the G0/G1 phase, while their phenotype suggested some degree of differentiation.