A voltage-dependent calcium current in mouse Swiss 3T3 fibroblasts

Patch-clamp experimens in the whole-cell mode have been performed in Swiss 3T3 mouse fibroblasts. Depolarizations from negative holding potential (V _h<–60mV) gave rise to a rapidly activating, fully inactivating, inward current of few tenths of nA in physiological saline at 35°C. The current persisted when external Na⁺ was replaced by impermeant TMA⁺ and disappeared in O Ca²⁺, 1 mM EGTA. The current was reversible blocked by Co²⁺ and it was slightly reduced when external Ca²⁺ was substituted by Ba²⁺. Finally its reversal potential changed with Nernstian slope with increasing external Ca²⁺ concentrations. It is concluded that these cells possess a voltagedependent Ca²⁺ channel.     

Early Signals in the Mitogenic Response of Swiss 3T3 Cells: A Comparative Study of Purified PDGF Homodimers

Abstract

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB. Two distinct receptor subunits, α and β, have been identified which bind either all three isoforms of PDGF (α) or PDGF-BB only (β). Here, we have compared the effect of purified PDGF homodimers on the early intracellular signaling events and mitogenesis in Swiss 3T3 cells, which possess equivalent numbers of the α and β subunits. Both PDGF-AA and PDGF-BB stimulated receptor phosphorylation, inositol phosphate formation, activation of protein kinase C, calcium mobilization, EGF receptor transmodulation, sodium uptake, arachidonic acid release, cyclic AMP accumulation, and c-fos induction in a comparable, dose-dependent manner (half-maximal values for all these responses were in the 2–10 ng/ml range for both homodimers). At high concentrations of PDGF (> 10 ng/ml), the BB homo-dimer effect on early membrane and cytosolic signals was 20–30% greater than PDGF-AA, reflecting the greater number of available binding sites for PDGF-BB. DNA synthesis studies indicated that PDGF-AA and PDGF-BB were potent mitogens for Swiss 3T3 cells, displaying identical dose-response effects. Moreover, the mitogenic activities of both homodimers were equally potentiated in the presence of insulin. These results indicate that both PDGF-AA and PDGF-BB stimulate the full complement of molecular responses required for the synergistic interactions mediating long-term mitogenesis. We conclude that α and β receptor subunits do not differ in their ability to transduce PDGF-mediated signals leading to DNA synthesis in Swiss 3T3 cells.     

Mitogenic effect of HIV-infected human T cell lines on mouse B cells mediated by surface immunoglobulin

Following HIV-1 infection, a number of disorders are induced in both normal T and B cells by virus products derived from infected CD4⁺ T cells. In the present study, we found that HIV-infected, but not uninfected, human T cell lines generated vigorous blastogenesis and proliferation of freshly isolated mouse B cells in a short-term culture. Neither human B cells nor rat B cells showed significant responses to the HIV-infected T cell lines in the present condition. The mitogenic effect of HIV-infected human T cell line requires direct cell–cell interaction between mouse B cells and HIV-infected T cell lines. Since either mitomycin c treatment or paraformaldehyde fixation of HIV-infected T cell lines resulted in complete loss of the mitogenic effect, it seems that de novo synthesized viral products are responsible for this effect. Furthermore, anti-mouse immunoglobulin antibody inhibited completely the B cell stimulation by the HIV-infected human T cell lines. Thus, surface immunoglobulin (sIg) on mouse B cells appears to be an essential molecule which transduces activation signals from HIV-infected human T cells into cytoplasm of the B cells.     

Mitogenic effects of partially purified interleukin 2 on thymocyte subpopulations and spleen T cells of the mouse

Partially purified interleukin 2 (IL-2) promotes proliferation of mouse spleen T, but not B cells, and of peanut-agglutinin-negative (PNA^?), and cortisone-resistant “mature” thymocytes, but not of PNA⁺ “immature” thymocytes. Within the cortisone-resistant thymocyte population, IL-2-responsive cells were found in the blast cell fraction. Proliferation was measured by [³H] thymidine incorporation and subsequent increase in viable cells. The mitogenic effect of IL-2 could not be inhibited by 50 mM methyl-α-D -mannoside which excludes contaminating concanavalin A (Con A) as a cause of mitogenicity. The relative increase in viable cells in IL-2 vs. control cultures was abrogated by 1.5 mM hydroxyurea. A possible effect of IL-2 on cell survival is thus ruled out. IL-2, when acting as comitogen with Con A, affected only PNA^? and cortisone-resistant thymocytes. These cells also showed high intrinsic IL-2 release when stimulated with Con A such that a comitogenic effect of externally added IL-2 was only seen at low cell concentrations. PNA⁺ thymocytes could neither be induced to release IL-2 nor did these cells become Con A-responsive under the influence of IL-2, thereby excluding an IL-2-mediated maturation.     

Mitogenic stimulation of mouse lymphoid cells by Aleuria aurantia agglutinin

An investigation was undertaken to determine if an agglutinin isolated from Aleuria aurantia possesses a mitogenic activity. Proliferation response of mouse lymphoid cell cultures was measured by 3H-thymidine uptake. The results revealed that Aleuria aurantia agglutinin at the concentration of 1 microgram/ml is mitogenic for Thy-1+ splenocytes and cortisone-resistant thymocytes.     

Mitogenic effect of anti-Friend leukemia virus antiserum on T cells

It has been previously reported that goat- and rat antisera directed against Friend leukemia virus (aFLV) are mitogenic for some B cells, but not for T cells. Here we report that activation of T cells by concanavalin A (Con A) rendered T cells responsive to the mitogenic activity of aFLV. This activity was contained in the immunoglobulin fraction and could be absorbed by purified FLV preparations. Optimal conditions for measuring the mitogenc activity of aFLV include 48 h preincubation of thymocytes with 3 μg/ml Con A followed by reculturing the activated thymocytes for 28 h with aFLV. The acquisition of an aFLV-responsive state was dependent on early protein synthesis during the Con A-induced activation period. aFLV did not substitute for interleukin 2 (IL2) in a costimulator assay. Evidence is presented that aFLV acted in a cocultivation assay via growth factor(s). In contrast to control cultures, aFLV-treated lymphoblasts contained, in their supernatants, IL2 activity as demonstrated by their effect on an IL2-dependent T cell line. The data suggest that aFLV acted upon activated T cells by enhancing the endogenous production and/or release of IL2.     

Transduction of Mitogenic Signals in T Lymphocytes

Treatment of human T lymphocytes with mitogenic ligands, such as concanavalin A (Con A), induces a rapid activation of the enzyme ornithine decarboxylase (ODC). This activation occurs within minutes and is completely inhibited when the cells are treated with 1 mM Li+ (in an inositol-free medium) prior to stimulation with Con A. In the presence of 1 mM myo-inositol Li+ has no effect on the Con A-induced activation of ODC. To elucidate why inositol is needed for the mitogen-induced activation of ODC in T lymphocytes, we tested the ability of different inositol metabolites to reverse the inhibitory effect of Li+. Here we report that inositol phospholipids, in addition to inositol, reverse the Li+-induced inhibition of ODC activation, while all other inositol derivatives tested were ineffective. This indicates that Li+ does not block the activation of ODC by inhibiting the generation of inositol phosphates, but rather by a mechanism which is circumvented if inositol phospholipids are added. The molecular mechanisms involved in the rapid activation of ODC by mitogens in human T lymphocytes apparently require inositol phospholipids, but are not directly mediated by inositol-1,4,5-trisphosphate (IP3) alone, diacylglycerol alone, or other inositol phosphates.     

Mitogenic effect of beryllium sulfate on mouse B lymphocytes but not T lymphocytes in vitro

Beryllium metal and its salts can produce disease in man and in animal models. Beryllium disease is thought to involve cell-mediated immunity and an antigen-dependent response by beryllium-specific T cells. Beryllium salts have been shown to stimulate lymphocyte proliferation and release of lymphokines, and to induce granuloma formation and delayed-type hypersensitivity reactions in mice, guinea pigs and man. The studies described here were designed to test the hypothesis that a second lymphocyte population, B cells, may be responding nonspecifically to beryllium. Different populations of BDF1 mouse lymphocytes were cultured in the presence of varying concentrations of beryllium sulfate (BeSO4), and the increase in 125-iodouracildeoxyriboside uptake after 72 h in culture was determined. The data show that BeSO4 is weakly mitogenic for normal mouse spleen cells. Furthermore, BeSO4 is mitogenic for normal and nude mouse spleen B cells and not for spleen T cells or thymocytes in vitro. These findings suggest that BeSO4 can stimulate B cells nonspecifically, and support the hypothesis that polyclonal activation of B cells by beryllium may occur.     

Organization of cyclic AMP-dependent connexin 43 in Swiss 3T3 cells attached to a cellulose substratum.

We have previously shown that the adenylyl cyclase, which produces cyclic AMP (cAMP) in Swiss 3T3 cells, is activated by their attachment to a cellulose substratum (Cuprophan, CU). This substratum adsorbs vitronectin poorly, prevents cell spreading and causes them to aggregate. By contrast, cells spread out on polystyrene and contain low concentrations of cAMP. We have found that Connexin 43 (Cx 43) gap junction plaques are involved in this cell aggregation. MDL 12330 A, a specific inhibitor of adenylyl cyclase, prevented cell aggregation on CU and abolished Cx 43 channel clustering. But forskolin, a direct activator of adenylyl cyclase, and SBr cAMP, a cell-permeable analogue of cAMP, caused Cx 43 channel clustering in cells attached to polystyrene. Hence, Cx 43 channel clustering is regulated by cAMP in Swiss 3T3 cells. In addition, neither brefeldin A nor monensin (inhibitors of transit through the endoplasmic reticulum and Golgi apparatus), abolished Cx 43 channel clustering in cells aggregated on CU. Thus, the Cx 43 that form clusters in cells attached to CU are not dependent upon the trafficking of Cx 43 from intracellular storage sites, but are probably reorganised from the plasma membrane.     

Homodimeric forms of bombesin act as potent antagonists of bombesin on Swiss 3T3 cells.

Two Lys3-bombesin dimers were prepared by crosslinking epsilon-amino groups Lys3-bombesin with noncleavable (glutaraldehyde) and cleavable [dimethyl-3,3'-dithiobispropionimidate (DTBP)] crosslinkers. The dimers were purified by HPLC ion-exchange chromatography and were shown to have retained immunoreactivity with an anti-bombesin monoclonal antibody directed against the C-terminal binding region of bombesin. The glutaraldehyde cross-linked bombesin dimer specifically inhibited binding of 125I-GRP to its receptor on Swiss 3T3 cells. Bombesin, at 0.6-60 nM induced mitogenesis in quiescent Swiss 3T3 cells, whereas, incubation of cells with the glutaraldehyde bombesin dimer at concentrations up to 124 nM did not. In competition assays, the bombesin dimer exhibited a dose dependent inhibition of bombesin-induced mitogenic activity and intracellular Ca++ mobilization. The bombesin dimer was 100 to 1000-fold more potent than D-Phe12Leu14-bombesin and D-Phe12bombesin, respectively, in inhibiting bombesin-induced mitogenesis on quiescent Swiss 3T3 cells. Similarly, the DTBP-bombesin dimer was not mitogenic to Swiss 3T3 cells, however, cleavage of the disulfide crosslinker with DTT of cell bound DTBP dimer restored mitogenic activity. Finally, the glutaraldehyde bombesin dimer also inhibited growth of bombesin receptor positive H345 SCLC cells in vitro. These findings suggest that the dimeric forms of bombesin are potent antagonists of bombesin.     

Restricted replication of adenovirus type 2 in mouse balb/3T3 cells

Summary The nature of the infection of mouse B3T3 cells by adenovirus type 2 (Ad2) has been studiedin vitro. Following infection with an adsorbed MOI of 225, more than 90 percent of the cells synthesized both early and late virus-specific antigens. In contrast, the yield of progeny virus varied from only 2 × 10⁴ to 2 × 10⁶ FFU/2 × 10⁵ cells. The range in yields was related, in part, to the number of cell generations from the time of the initial subcloning, the yield increasing with passage level. Infectious center analysis suggested that fewer than 0.5 percent of infected cells synthesized progeny virus.Analysis of DNA synthesis in infected multiplying B3T3 cells demonstrated that cellular DNA synthesis began to be shut off at 12 hours p.i., a time when viral DNA synthesis was beginning. The maximum rate of viral DNA synthesis was approximately 12 percent of that in infected human cells. In contrast to infected multiplying cells, infection of quiescent B3T3 cell cultures resulted in the induction of cellular, along with viral, DNA synthesis. Analysis of late gene expression detected synthesis of most viral polypeptides, but revealed greater than 90 percent reductions in the rate of synthesis of polypeptides II, III, IV, and IX, as compared with infected human cells.With 8 FiguresThese studies were supported by NIH Research Grant No. CA-08851 and NIH Training Grant No. CA-09069.     

A nonselective analysis of SV40 transformation of mouse 3T3 cells

Mouse cells transformed by simian virus 40 show many alterations in their growth properties in vitro. In order to investigate the coordinate nature of these changes, we have analyzed the growth properties of 40 randomly selected colonies arising after SV40 infection of 3T3 cells. Clones of cells, established from these colonies, were characterized as to saturation density and doubling time in 10% and 1% calf serum, growth in methyl cellulose suspension, colony formation on monolayers of normal cells, and presence of viral antigens. This analysis revealed that only 5 of the clones were indistinguishable from 3T3 cells; the remaining 35 clones differed from 3T3 cells in that they grew as rapidly in 1% calf serum as standard SV40 transformed cells. Of these 35 clones, ten corresponded to standard transformants previously described. Another ten showed other growth properties intermediate between 3T3 cells and standard transformants. These intermediate clones had lower levels of viral T-antigen than standard transformants and showed considerable heterogeneity in staining from cell to cell. The remaining 15 clones were T-antigen negative and had saturation densities slightly higher than that of 3T3 cells. These changes in cellular behavior are stable on recloning.     

A voltage-dependent calcium current in mouse MC3T3-E1 osteogenic cells

MC3T3-E1 osteogenic cells in a growing state were voltage-clamped by the whole-cell patch-clamp method. The MC3T3-E1 cells exhibited a transient, fast-inactivating Ca inward current upon depolarizing pulses from a holding potential of -80 mV. This current had a threshold of activation of about -50 mV and was insensitive to the dihydropyridine, nifedipine. These results show that MC3T3-E1 cells have a voltage-dependent Ca channel corresponding to the "T-type."     

Early signals in the mitogenic response of Swiss 3T3 cells: a comparative study of purified PDGF homodimers

Platelet-derived growth factor (PDGF) occurs as three dimeric isoforms, AA, BB, and AB. Two distinct receptor subunits, alpha and beta, have been identified which bind either all three isoforms of PDGF (alpha) or PDGF-BB only (beta). Here, we have compared the effect of purified PDGF homodimers on the early intracellular signaling events and mitogenesis in Swiss 3T3 cells, which possess equivalent numbers of the alpha and beta subunits. Both PDGF-AA and PDGF-BB stimulated receptor phosphorylation, inositol phosphate formation, activation of protein kinase C, calcium mobilization, EGF receptor transmodulation, sodium uptake, arachidonic acid release, cyclic AMP accumulation, and c-fos induction in a comparable, dose-dependent manner (half-maximal values for all these response were in the 2-10 ng/ml range for both homodimers). At high concentrations of PDGF (greater than 10 ng/ml), the BB homodimer effect on early membrane and cytosolic signals was 20-30% greater than PDGF-AA, reflecting the greater number of available binding sites for PDGF-BB. DNA synthesis studies indicated that PDGF-AA and PDGF-BB were potent mitogens for Swiss 3T3 cells, displaying identical dose-response effects. Moreover, the mitogenic activities of both homodimers were equally potentiated in the presence of insulin. These results indicate that both PDGF-AA and PDGF-BB stimulate the full complement of molecular responses required for the synergistic interactions mediating long-term mitogenesis. We conclude that alpha and beta receptor subunits do not differ in their ability to transduce PDGF-mediated signals leading to DNA synthesis in Swiss 3T3 cells.     

槲皮素对小鼠体内和体外NIH-3T3细胞的抗氧化作用及其机理

目的 比较槲皮素对小鼠体内和体外H2O2处理前/后的NIH-3T3细胞抗氧化效应的差异并探讨其作用机理。方法 将NIH-3T3细胞分为4组,槲皮素前保护组（Qb）：槲皮素后保护组(Qa)：H2O2组 (H2O2)和对照组(C)。 用试剂盒检测细胞内T-AOC、SOD、GSH-Px、GSH、MDA、NOS和NO2-/NO3-的水平;MTT和TUNEL法检测细胞凋亡率和生存率;免疫印迹和免疫组化技术检测细胞的cyclin D1、PTEN、NF-kB、HSP-70、Bcl-2、Bax、 及caspase-3的表达。另将20只Wistar大鼠等分为对照组和实验组,检测槲皮素灌胃前、后1、2及24h血浆内T-AOC、SOD、GSH-Px、GSH、MDA、NOS和NO2-/NO3-的水平。结果H2O2组细胞凋亡率增高,cyclin D1、PTEN及Bcl-2的表达下调;Bax、HSP-70、NF-kB及caspase-3的表达上调;T-AOC,SOD,GSH-Px及GSH水平降低,NOS、NO2-/NO3-及MDA增高。动物灌胃后1h/2h血浆中T-AOC、SOD、GSH-Px、GSH,NOS及NO2-/NO3-的水平增高,MDA降低;24h后基本恢复。结论 槲皮素在体内外皆可通过调节抗氧化酶体系发挥抗氧化作用,其效应依据H2O2处理与否和处理前/后等细胞的异质性而呈现一定差异。     

Accumulation of cyclic AMP in Swiss 3T3 cells adhering to a cellulose biomaterial substratum through interaction with adenylyl cyclase

Controlling cell shape induced by cell-substrata interaction appears of prime importance to influence subsequent biological processes such as cell migration, proliferation, differentiation or apoptosis. Studies on Swiss 3T3 fibroblasts have recently provided evidence that cell spreading is mediated by integrins and RhoA. Our previous studies showed that on Cuprophan, a cellulose membrane (CU) to which vitronectin adhesive protein is poorly adsorbed, Swiss 3T3 cells are rounded and undergo cAMP-dependent aggregation. In contrast, on a polyacrylonitrile membrane (AN69) that favours the adsorption of vitronectin and fibronectin, cells spread out and contain low concentrations of cAM P. We have now examined the parts played by the three components in the cAMP pathway (receptors, G-proteins and adenylyl cyclase itself) in cAM P-dependent cell aggregation on CU. Experiments with intact cells showed no interaction between the CU and receptors, or between the CU and G-proteins. Assays on membrane preparations plus the Mn-ATP substrate, which uncouples G-proteins and adenylyl cyclase, demonstrated that activation of the cAMP pathway by CU depends primarily on the catalytic activity of the adenylyl cyclase. These investigations provide essential data for the development of biomaterials that favour cell functionality.     

MC3T3-HA复合培养后的细胞凋亡检测

目的:探讨羟基磷灰石(Hydroxyapatite,HA)压片材料对小鼠MC3T3成骨细胞凋亡的影响,提供一种方便可行的组压片材料的生物相容性检测方法。方法:小鼠MC3T3成骨细胞株接种在HA压片材料上,应用光学显微镜和PI-Hoechst33342双染色荧光显微镜观察细胞,采用RT-PCR方法分析HA对Id2(Inhibitor of differentiation 2,分化抑制因子2)和OPN(osteopontin,骨桥蛋白)等基因表达的影响。结果:HA压片材料组MC3T3细胞凋亡率(78.7%)显著高于正常对照组(24.6%)(t’=28.1,P=0.000)。HA压片材料双荧光染色可以直接检测种子细胞的凋亡。结论:HA压片可促进小鼠MC3T3成骨细胞凋亡。     

Mechanisms of the ATP potentiation of hyposmotic taurine release in Swiss 3T3 fibroblasts

Reducing osmolarity by 35% increased ³H-taurine efflux from Swiss 3T3 fibroblasts from 0.5% to a peak of 5.7%. The presence of ATP (10–100 µM; EC₅₀ 1.5 µM) increased taurine efflux up to 10%, and decreased the set point for hyposmotically stimulated taurine release (HTR). ATP potentiation was mimicked by UTP, reduced by addition of suramin and pyridoxal phosphate-6-azophenyl-2,4-disulphonic acid (PPADS) and unaffected by ADP, ,-methylene-ATP (,-ATP) or 2-methylthio-ATP (Me-ATP), suggesting its mediation by purinergic P2Y₂ and P2Y₄ metabotropic receptors. Under isosmotic conditions ATP increased the cytosolic [Ca²⁺] ([Ca²⁺]_i) markedly, but did not increase taurine release. HTR was independent of external Ca²⁺ but was reduced (by 56–59%) by BAPTA-AM, thapsigargin-induced depletion of intracellular Ca²⁺ stores, or phospholipase C (PLC) inhibition. Blockade of calmodulin (CaM) or calmodulin kinase II (CaMKII) reduced HTR by 54% and 76%, respectively. The ATP-mediated potentiation was prevented fully by all these treatments. HTR was reduced by 30–50% by blockers of protein tyrosine kinases (AG18), phosphoinositide 3-kinase (PI3K) (wortmannin), p21rho (toxin B), p21rho-kinase (Y27632) and the stress-activated kinase p38 (PD169316). ATP-mediated potentiation was reduced similarly by these blockers. Simultaneous inhibition of PI3K and CaMKII abolished HTR. Altogether, these results suggest a modulatory effect of ATP, probably exerted by a potentiation of the Ca²⁺-dependent fraction of HTR. This fraction has as signalling elements a PLC-dependent [Ca²⁺]_i increase, resulting from Ca²⁺ released from thapsigargin-sensitive internal stores, followed by activation of CaM/CaMKII reactions. The Ca²⁺/ATP effect operates only when the Ca²⁺-independent, tyrosine kinase-mediated pathway is already activated. Suggested elements of cross-talk between the two pathways are PLC, PI3K and CaMKII.