Influence of extracellular calcium on cell permeabilization and growth regulation by the lymphokine leukoregulin

Permeablization of human K562 leukemia cells was measured in the presence and absence of extracellular ionic calcium to examine the relationship of ionic calcium to increased membrane permeability and the inhibition of cell proliferation by this lymphokine. In the absence of extracellular calcium, the ability of leukoregulin to permeabilize the cell membrane is diminished but is fully restored by addition of 1 mM extracellular Ca++ as shown flow cytometrically by loss of intracellular fluorescein. Membrane permeability is also increased by calcium ionophore A23187 but permeablization is completely blocked in calcium-free medium despite the intramembrane presence of the calcium ionophore. Membrane permeablization by the lectin phytohemagglutinin, in contrast, is independent of extracellular calcium. A similar divergence in cell proliferation activity of the three modulators of calcium flux and membrane permeability occurs in the absence of extracellular calcium. Leukoregulin inhibition of cell proliferation is abolished, inhibition by calcium ionophore A23817 is greatly reduced, and inhibition by phytohemagglutinin is unchanged. Leukoregulin permeabilized K562 cells isolated by fluorescence activated cell sorting resume proliferation after 72 h. In contrast cells permeablized by calcium ionophore A23187 or phytohemagglutinin fail to resume proliferation by 7 days. The membrane permeablizing action of leukoregulin is, therefore, partially dependent upon extracellular calcium. It is also effected through a mechanism other than calcium ionophore transport or lectin type transmembrane signaling, and is accompanied by a reversible inhibition of cell proliferation.     

The influence of calcium on the cyclic AMP-mediated stimulation of DNA synthesis and cell proliferation by prostaglandin E 1

Prostaglandin type E₁ (PGE₁) rapidly stimulates cyclic AMP formation and the initiation of deoxyribonucleic acid (DNA) synthesis in rat thymic lymphocytes suspended in vitro by reactions which are not affected by wide variations in the extracellular calcium concentration. On the other hand, the operation of the associated reaction(s) responsible for the subsequent progression of the stimulated cells into mitosis is profoundly affected by the extracellular calcium level. If the maximum intracellular cyclic AMP concentration is in the lower range of stimulatory values (e.g., 150 × 10^?8 picomoles per cell as produced by an exposure to 0.5 μg of PGE₁ per milliliter of medium), an extracellular calcium concentration of 0.5 to 1.0 mM is needed to obtain maximum cell proliferation, but not the maximum stimulation of DNA synthesis. Contrariwise, if the cellular cyclic AMP content is raised to a much higher level (260 × 10^?8 picomoles per cell) by exposure to a greater PGE₁ concentration (5.0 μg per millilter), cell proliferation is maximally stimulated in calcium-free medium and increasing the extracellular calcium concntration above 0.2 mM actually prevents the stimulation of cell proliferation (but does not affect the stimulation of DNA synthesis). Thus, the ultimate translation of PGE₁'s early cyclic AMP-mediated reactions into increased cell proliferation is determined by both the intracellular cyclic AMP level and the extracellular calcium concentration.     

Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes

The role that extracellular calcium plays in activating resting cloned cytotoxic T lymphocytes (CTL) to proliferate and to produce lymphokines was examined. In these cells, stimulation with interleukin 2 (IL-2) induced a proliferative response without a concomitant production of macrophage-activating factor (MAF), whereas stimulation with antigen or lectin (in the absence of IL-2) induced MAF production but not proliferation. In the case of IL-2-induced proliferation, extracellular calcium was required to initiate proliferation as well as to prevent cellular arrest later in the G2 + M phase of the cell cycle. In MAF production extracellular calcium was required both to activate the phosphatidylinositol signal-transducing mechanism and to mobilize intracellular calcium in antigen- or lectin-stimulated cytotoxic T lymphocytes. Further, extracellular calcium was required for only 8 of the 18 hr of stimulation time which was needed to achieve maximal MAF production, indicating that both calcium-dependent and -independent events exist in the signal pathway. Additional experiments with calcium ionophores and activators of protein kinase C indicated that although both intracellular calcium mobilization and de novo protein phosphorylation are involved in MAF production, an optimal increase in the level of intracellular calcium by itself is insufficient to induce the production of this lymphokine.     

神经干细胞体外增殖分化的钙成像研究

神经干细胞具有广阔的应用前景,但对于其增殖和分化的内源机制、外部环境信号还并不十分了解。研究表明,钙信号很可能在其中起到了调控作用。利用钙离子成像技术,观察神经干细胞的单细胞体外增殖和分化过程,记录了在细胞分裂过程中钙信号变化的曲线。发现细胞增殖和分化过程中都会产生钙浓度的变化,但在细胞分裂后期两者钙信号的模式却存在差别。实验结果提示,胞内钙水平的波动只是细胞增殖的伴随产物,但却是细胞分化的必要条件。由此提出钙信号对神经干细胞分化调控机制的假设,并指出其对今后研究的意义。     

Casein phosphopeptides modulate proliferation and apoptosis in HT-29 cell line through their interaction with voltage-operated L-type calcium channels

At the intestinal level, proliferation and apoptosis are modulated by the extracellular calcium concentration; thus, dietary calcium may exert a chemoprotective role on normal differentiated intestinal cells, while it may behave as a carcinogenesis promoter in transformed cells. Calcium in milk is associated with casein and casein phosphopeptides (CPPs), hence is preserved from precipitation. CPPs were demonstrated to induce uptake of extracellular calcium ions by in vitro intestinal tumor HT-29 cells but only upon differentiation. Here, the hypothesis that CPPs could differently affect proliferation and apoptosis in undifferentiated and differentiated HT-29 cells through their binding with calcium ions was investigated. Results showed that CPPs protect differentiated intestinal cells from calcium overload toxicity and prevent their apoptosis favoring proliferation while inducing apoptosis in undifferentiated tumor cells. The CPP effect on undifferentiated HT-29 cells, similar to that exerted by ethyleneglycol-O, O'-bis(2-aminoethyl)-N, N, N', N'-tetraacetic acid (EGTA), is presumably due to the ability in binding the extracellular calcium. The effect on differentiated HT-29 cells is coupled to the interaction of CPPs with the voltage-operated L-type calcium channels, known to activate calcium entry into the cells under depolarization and to exert a mitogenic effect: the use of an agonist potentiates the cell response to CPPs, while the antagonists abolish the response to CPPs (36% of examined cells) or reduce both the percentage of responsive cells and the increase of intracellular calcium concentration. Taken together, these results confirm the potentialities of CPPs as nutraceuticals/functional food and also as modulators of cellular processes connected to the expression of a cancer phenotype.     

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells resuts in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated ⁴⁵Ca⁺², while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression. © 1995 Wiley-Liss, Inc.     

Extracellular Mg concentration and Ca blockers modulate the initial steps of the response of Th2 lymphocytes in co-culture with macrophages and dendritic cells

Magnesium is highly involved in the metabolic network such that even subtle disturbances in its homeostasis affect many cellular functions, including calcium homeostasis, signal transduction, energy metabolism, membrane stability and cell proliferation. Recently, magnesium level has been proposed to modulate the priming and activity of immune cells. We studied the behavior of antigen-presenting cells (APCs) and T lymphocytes after altering the magnesium/calcium balance. We used two different populations of primary APCs, i.e. bone marrowderived dendritic cells and bone marrow-derived macrophages, while D10.G4.1 cells served as a model of responding Th2 cells. Our principal findings are the following: (i) the extracellular magnesium concentration had no significant impact on endocytosis by bone marrow-derived APCs, (ii) high concentrations of extracellular magnesium, with or without calcium antagonists, significantly decreased IL-4 and IL-10 secretion by Th2 cells in a co-culture system of APCsandTh2lymphocytes, (iii) proliferation ofTh2cells in co-culture systems was significantly inhibited by calcium antagonists independently from extracellular magnesium concentrations. Our results suggest that alterations of magnesium and calcium homeostasis impact on some crucial steps of the immune response.     

Influence of calcium and calcium-modulating agents on differentiation of murine erythroleukaemia cells

Since the involvement of calcium ions in the regulation of cell division and differentiation has been proposed, in this study we have examined the effect of extracellular calcium and of calcium-modulating agents on the DMSO-induced differentiation of murine erythroleukaemia cells. Neither proliferation nor differentiation of these cells was affected by calcium deprivation in the culture medium. Moreover, calcium-chelating agents or agents blocking intracellular calcium uptake induced a marked inhibition of cell differentiation. Intracellular calcium antagonists induced inhibition when cells were grown in a calcium-deprived medium. In contrast, murine erythroleukaemia cell differentiation was unaffected by agents that increased intracellular concentration of calcium. Our results indicate that a mobilization of calcium is indispensable for eliciting full cellular response, but the increase in intracellular level of this cation is not sufficient for complete signal transduction. It is likely that a marked alteration of the intracellular calcium system and availability could be responsible for the independence of our cell system from calcium modulation.     

A dominant negative Fas-associated death domain protein mutant inhibits proliferation and leads to impaired calcium mobilization in both T-cells and fibroblasts

Death domain-containing members of the tumor necrosis factor (TNF) receptor family ("death receptors") can induce apoptosis upon stimulation by their natural ligands or by agonistic antibodies. Activated death receptors recruit death domain adapter proteins like Fas-associated death domain protein (FADD), and this ultimately leads to proteolytic activation of the caspase cascade and cell death. Recently, FADD has also been implicated in the regulation of proliferation; functional inhibition of FADD results in p53-dependent impairment of proliferation in activated T-cells. In this study we have further analyzed T-cells derived from transgenic mice expressing a dominant negative FADD mutant (FADD DN) under control of the lck promoter in vitro so as to identify the signaling pathways that become engaged upon T-cell receptor stimulation and that are regulated by death receptors. FADD DN expression inhibits T-cell proliferation, both at the G(0) --> S transition and in the G(1) phase of continuously proliferating cells. We observe a decrease in the release of calcium from intracellular stores after T-cell receptor stimulation, whereas influx of extracellular calcium seems to be unaffected. FADD DN-expressing fibroblasts show a similarly inhibited cell growth and impaired calcium mobilization indicating that the modulation of proliferation and calcium response by death receptors is not cell type-specific.     

Regulation of intracellular pH by electrogenic Na+/HCO3– co-transporters in embryonic neural stem cell-derived radial glia-like cells

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pH_i) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extracellular K⁺ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extracellular calcium was removed, an instant depolarization and recovery of the pH_i, back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by S0859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pH_i recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pH_i regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pH_i regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.     

Growth Control in Colon Epithelial Cells: Gadolinium Enhances Calcium-Mediated Growth Regulation

Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1?C5???M combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet.     

Calcium ion-dependent proliferation of L1210 cells in culture

Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the "pile-up" of the L1210 cells in the GO/Gl phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the G0/G1 phase to S-phase. Cells enriched for S and G2/M phase by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/Gl. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the G0/G1 to S-phase of the cell cycle.     

Investigations into the nature of growth-related proteolysis in human fibroblasts

Previous experiments have shown that cultured human fibroblasts possess a cell-surface proteinase (the growth-related proteinase; GRP) which is essential to cell proliferation. In the present work, proteinase inhibition in defined and complex serum-free media and in pre-conditioned normal medium, still resulted in a corresponding inhibition of cell proliferation. Proteinase inhibition also blocked the action of a range of peptide growth factors and of a phorbol ester. Elevated extracellular calcium concentrations were still mitogenic in the presence of proteinase inhibitors. Proteinase inhibition did not affect the mobilisation of intracellular calcium, nor the metabolism of inositol phosphate derivatives in response to a mitogenic stimulus.     

Cilostazol prevents endothelin-induced smooth muscle constriction and proliferation

Cilostazol is a phosphodiesterase inhibitor that has been shown to inhibit platelet activation. Endothelin is known to be the most potent endogenous growth promoting and vasoactive peptide. In patients and animal models with stroke, the level of circulating endothelin increases and complicates the recovery progress contributed by vascular constriction (an immediate pathology) and vascular proliferation (a long-term pathology). However, the effects of cilostazol on endothelin have not been explored. To demonstrate the dual-antagonizing effects of cilostazol on vasoconstriction and cell proliferation induced by endothelin, we used primary culture of mouse vascular smooth muscle cells in vitro, mouse femoral artery ex vivo, and intracranial basilar artery ex vivo. We show that the dual-inhibition effects of cilostazol are mediated by blocking endothelin-induced extracellular calcium influx. Although cilostazol does not inhibit endothelin-induced intraorganellar calcium release, blockade of extracellular calcium influx is sufficient to blunt endothelin-induced vasoconstriction. We also show that cilostazol inhibits endothelin-induced cellular proliferation by blocking extracellular calcium influx. Inhibition of cAMP-dependent protein kinase (PKA) can block anti-proliferation activity of cilostazol, confirming the downstream role of PKA in cellular proliferation. To further demonstrate the selectivity of the dual-antagonizing effects of cilostazol, we used a different phosphodiesterase inhibitor. Interestingly, sildenafil inhibits endothelin-induced vasoconstriction but not cellular proliferation in smooth muscle cells. For the first time, we show selective dual-antagonizing effects of cilostazol on endothelin. We propose that cilostazol is an excellent candidate to treat endothelin-associated diseases, such as stroke.     

The modulation of growth of normal rat liver epithelial cells in calcium-poor medium by epidermal growth factor,phenobarbital, phorbol ester,and retinoic acid

Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca²⁺, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast, phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal cells, but such an effect does not seem to be a universal property of tumor promoters. This research was supported by National Institutes of Health Grant CA 29323.     

Involvement of cationic channels in proliferation and migration of human mesenchymal stem cells

Human mesenchymal stem cells (HMSCs) have been applied in various clinic settings. Ion channels play an important role in cellular physiology. However, the potential role of cationic channels in regulating the proliferation and migration properties of hMSCs remains to be determined. In the present study, the functional expression of ion channels in hMSCs was investigated by patch clamp. MTT assay and BrdU stainings were used to assess the proliferation of hMSCs. hMSC migration was evaluated by Transwell migration assays. The results show that sodium-, L-type calcium, potassium currents have been identified in hMSCs. TEA (K⁺ channel blocker), nifedipine (Ca²⁺ channel blocker) can inhibit both proliferation and migration of hMSCs. The increase of extracellular Ca²⁺ concentration promoted both proliferation and migration of hMSCs. TTX, a Na⁺ channel blocker, promoted cell proliferation but inhibited cell migration. Our data suggest that cationic channels (sodium, L-type calcium, potassium channels) play important roles in regulating proliferation and migration of hMSCs.     

Extracellular ATP and ADA-buffer enable chick embryo fibroblasts to grow in secondary cultures in protein-free, hormone-free, extracellular growth factor-free medium

The replacement of HEPES with ADA buffer and addition of ATP to the serum-free, protein-free Eagle's minimal essential medium (EMEM) caused stimulation of proliferation in sparse, secondary cultures of resting chick embryo fibroblasts. Better cell growth and highly reproducible results were obtained if the cells had been dispersed from primary cultures with EGTA and thereafter remained without any further contact with serum or other extracellular proteins than when trypsin was used. The frequent changes of culture medium caused no retardation of cell growth. The observed more than 12 fold increase in the cell number after stimulation of the cultures with ATP is interpreted in favour of the conceptions assuming a significant role of intracellular calcium and intracellular pH in regulation of cell proliferation.     

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixtyfold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G₀/G₁ cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling. © 1994 Wiley-Liss, Inc.     

Control of 3T3 cell proliferation by calcium

Summary When a population of 3T3 mouse cells was subcultured regularly at confluency, the original epitheliodid or stellate cells disappeared and, by the ninth passage, they had been replaced by spindle-shaped cells. The original cells proliferated only when the extracellular calcium concentration exceeded 0.1mm, and their proliferative activity became maximum only when the calcium concentration was 0.5mm. The spindle-shaped cells were much more sensitive to proliferative stimulation by calcium. Although these cells also could not proliferate without extracellular ionic calcium, they proliferated maximally in the presence of as little as 0.05mm calcium. Thus, calcium is a major regulator of the proliferation of 3T3 mouse cells. Moreover, it appears that the sensitivity of the proliferative machinery to the calcium ion can vary greatly within an established cell line.