The different actions of normal and supranormal calcium concentrations on the proliferation of BALB/c 3T3 mouse cells

Summary In the presence of a normal (1.25 to 1.80 mM) calcium concentration, addition of fresh bovine calf serum or completely changing the medium induces proliferatively quiescent BALB/c 3T3 mouse cells in dense cultures to start a growth division cycle and initiate DNA synthesis about 12 hr later. Fresh, low-calcium (0.02 mM physiologically available) medium also causes cells to start a growth-division cycle. However, the development of such stimulated, calcium-deprived cells stops just before the expected time of initiation of DNA synthesis, which can then be rapidly induced by restoration of the normal calcium concentration. Simply raising the calcium concentration to nonphysiologically high levels (without otherwise altering the medium) can mimic the action of fresh serum or fresh whole medium by inducing some of the cells in proliferatively quiescent confluent cultures to start a growth-division cycle and initiate DNA synthesis 22 hr later. Issued as NRCC No. 15371.     

Plate-Induced tumors of BALB/3T3 cells exhibiting foci of differentiation into pericytes,chondrocytes, and fibroblasts

The BALB/3T3 clone A31 mouse embryo cell line has been used by many investigators as a model “normal” “fibroblast” line for a variety of in vitro studies. It has been shown, however, that these cells are not “normal” because they will produce tumors within 2–4 months if 3 × 10⁴ cells are implanted subcutaneously in BALB/c mice attached to 0.2 × 5 × 10-mm plastic plates. Previous studies also suggested that these cells were not fibroblasts because they gave rise to tumors with the characteristics of vascular endothelium not fibroblasts. We now report that BALB/3T3 (clone A31), BALB/3T3-T, a proadipocyte subclone of clone A31 cells, and six recent subclones of BALB/3T3-T cells show additional differentiation patterns when tumors derived by implantation of these cells attached to plastic plates are examined. Differentiation into pericytes, chondrocytes, and fibroblasts was observed. We conclude that the BALB/3T3 clone A31 cell line and related lines are multipotent mesenchymal cells which are capable of differentiation into a variety of cell types.     

Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation.     

Apoptosis observed in BALB/3T3 cells having ingested Staphylococcus aureus.

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 microg/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I-infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 +/- 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron-dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2- to 6-hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.     

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.     

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells

The transforming activity of sodium fluoride was studied in the SHE and the BALBl3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75–125 g/ ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 g/ml. In the BALBl3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L₈ (2⁷) of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 g/ ml to a 50 g/ ml concentration which is highly toxic for BALBl3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.Abbreviations CE cloning efficiency - NaF sodium fluoride - SHE Syrian hamster embryo - TF transformation frequency     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Epidermal growth factor (EGF) is required only during the traverse of early G1 in PDGF stimulated density-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells that had received a transient exposure to PDGF and were then transferred to medium containing only EGF and somatomedin C (Sm-C) began DNA synthesis after the G0/G1 lag. Supraphysiological concentrations of insulin could be employed to replace the Sm-C requirement. This G0/G1 lag phase was bisected by the requirement for the exogenous presence of EGF. Our data indicated that EGF was required during the traverse of only the first half of G0/G1 phase (6 h) and not during the traverse of late G1. Subphysiological serum concentrations of Sm-C were also necessary to be present with EGF for progression through early G0/G1; however, traverse of the final half of G0/G1 and commitment to DNA synthesis required the presence of Sm-C. It was found that physiological concentrations of Sm-C were required for the traverse late G1. The requirement for Sm-C for G0/G1 traverse of BALB/c-3T3 cells as opposed to human fibroblasts or glial cells may be due to a difference in endogenous synthesis of an insulin-like growth factor. Our data are in close agreement with previous reports that EGF is only required for approximately the first 8 h during traverse of the G0/G1 phase. The requirement for EGF to be present for the first 6 h of G0/G1 could result from a continued or repetitious event or by more than one distinct EGF-requiring event.     

Macromolecular synthesis and stability in growth-arrested BALB/c-3T3 cells

Concentrations of methylglyoxal bis-(guanylhydrazone) (mGBG) that inhibited serum-stimulated BALB/c-3T3 cells in late G1 caused a marked inhibition of 3H-leucine incorporation during a 20-min incubation. No decrease was observed in the incorporation of 3H-uridine during a 20-min incubation; however, the amount of acid-insoluble 3H-uridine in mGBG-treated cultures was decreased when the incubation period was longer than 20 min. The amount of the decrease in the accumulation of incorporated 3H-uridine was directly proportional to the length of the incorporation time. Between 10 and 12 h after quiescent BALB/c-3T3 cells were serum-stimulated in mGBG no additional 3H-uridine was accumulated. The stability of the incorporated 3H-uridine, as determined by acid-insoluble radioactivity remaining after the addition of actinomycin D, was less in cells cultured in mGBG. Exogenous spermine or spermidine reversed the inhibition of 3H-uridine accumulation in acid-insoluble material produced by mGBG as well as the decrease in stability of the incorporated 3H-uridine in acid-insoluble material. The effects of mGBG on both the incorporation of 3H-uridine and the stability of the incorporated 3H-uridine can apparently be accounted for by an effect on ribosomal RNA.     

Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using BALB/c 3T3 mouse embryo fibroblasts

Summary The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (¹²⁵I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15°C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with K _d =59.6 pM and an estimated 1.57×10⁵ receptors/cell. Half-maximal displacement of bound¹²⁵I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing halfmaximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 μg/ml, respectively. Epidermal growth factor, transforming growth factor type α, and acidic and basic fibroblast growth factors did not compete for¹²⁵I-IGF-I binding at 1 μg/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that¹²⁵I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Inhibition of the Na+/K+ cotransport system by cyclic AMP and intracellular Ca2+ in human red cells

Human erythrocytes are able to incorporate cyclic AMP (cAMP) in amounts larger than those required to saturate cAMP-dependent protein kinase. In contrast to previous observations in avian red blood cells in which cAMP stimulates the Na⁺/K⁺ cotransport system, we demonstrate that cAMP inhibits this system in human erythrocytes. The cotransport inhibition is enhanced by addition of phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine to the incubation medium. The cAMP concentration giving half-maximal cotransport inhibition showed a wide variation among different individuals (from 0.1 to 5 mM external cAMP concentration). In contrast to cAMP, cyclic GMP showed little effect on the cotransport system. Ca²⁺ introduced into the cell interior was an inhibitor of the Na⁺/K⁺ cotransport system. These results suggest that in human cells in which endogeneous levels of cAMP and Ca²⁺ are modulated by hormones, the Na⁺/K⁺ cotransport system may be under hormonal regulation.     

Cell cycle parameters of 3T3 cells cultured as aggregates

Summary BALB/c 3T3 cells cultured as aggregates were examined by two independent techniques to determine whether or not cells accumulated at a specific point in the cell cycle, and if so to determine the point at which they accumulate. Replating cells onto dishes followed by pulse labeling with [³H]thymidine and autoradiography indicated that aggregate-cultured cells were in the same phase of the cell cycle as cells cultured as confluent monolayers. Flow microfluorometry confirmed that 75% of the aggregate-maintained cells were arrested in G₀ or G₁, with 25% distributed throughout the rest of the cell cycle. Labeling and mitotic indices of cells in aggregates were also consistent with about 20 to 25% of the cells being in S+G₂=M phases of the cell cycle at any time. This work was supported by PHS Grant CA20323 and NSF Grant PCM 74-15092 to H. G., who is also a Harry H. Pinney Cancer Scholar.     

Adenosine 3′:5′-cyclic monophosphate,adenylate cyclase and a cyclic AMP binding-protein in Phaseolus vulgaris

The validity of using the binding-protein method for determining cyclic AMP in purified and partially purified extracts of Phaseolus tissues has been examined and confirmed. Measurement of cyclic AMP concentration by binding-protein gave similar results to those obtained by direct spectrophotometry of purified extracts. A cyclic AMP binding-protein and adenylate cyclase were demonstrated in Phaseolus extracts. Isolated intact chloroplasts were shown to possess adenylate cyclase activity but persistent cyclic AMP phosphodiesterase activity obviated quantitative assessment.     

Online monitoring of BALB/3T3 metabolism and adhesion with multiparametric chip-based system

A multiparametric chip-based system was employed to measure cell adhesion, metabolism, and response to metal compounds previously classified as cytotoxic in immortalized mouse fibroblasts (BALB/3T3 cell line). The system measures in parallel, online, and in label-free conditions the extracellular acidification rates (with pH-sensitive field effect transistors [ISFETs]), the cellular oxygen consumption (with amperometric electrode structures [Clark-type sensors]), and cell adhesion (with impedimetric interdigitated electrode structures [IDESs]). The experimental protocol was optimized to monitor metabolism and adhesion of the BALB/3T3 cell line. A total of 70,000 cells and a bicarbonate buffer-free running low-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal clone serum III and 1mM Hepes were selected to maintain cells in good conditions on the chip during the measurements performed under perfusion conditions. Cells were exposed to sodium arsenite, cadmium chloride, and cis-platinum at concentrations ranging from 1 to 100 microM. The kinetics of cell response to these compounds was analyzed and suggests that the Clark-type sensors can be more sensitive than IDESs and ISFETs in detecting the presence of high chemical concentration when short exposure times (i.e., 2h) are considered. The cytotoxicity data obtained from the online measurements of acidification, respiration, and adhesion at 24h compare well, in terms of half-inhibition concentration values (IC(50)), with the ones obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and colony-forming efficiency (CFE) assay. The results show a good sensitivity of the system combined with the advantages of the online and label-free detection methods that allow following cell status before, during, and after the treatment in the same experiment.     

Dibutyrylic cyclic AMP and theophylline inhibit proliferation of accessory cells in primary cultures of adrenomedullary cells

Summary Studies on isolated adrenal chromaffin cells in primary cultures may be seriously hampered by the presence of non-chromaffin, mainly fibroblast-like cells, which always occur in dissociates of adrenal medullary tissue and often outnumber the chromaffin cells by the end of the first week of culture, when no measures are taken to control their proliferation. The present study offers a new means to inhibit effectively the proliferation of these accessory cells by treating the cultures with dibutyrylic cyclic AMP (dbcAMP, 0.1 or 0.01 mM) and equimolar amounts of the phosphodiesterase inhibitor theophylline. With this treatment cultures of young rat adrenal chromaffin cells remain virtually free of accessory cells for two weeks of culture. Cultures of bovine adrenomedullary cells retain their initial amounts of non-chromaffin cells, which largely depends upon whether the primary cell suspensions have undergone differential plating prior to seeding. Suppression of accessory cell proliferation with dbcAMP and theophylline is partly due to maintaining differentiation of cortical cells, which otherwise dedifferentiate into rapidly dividing fibroblast-like elements. However, a more direct action of dbcAMP on accessory cells in terms of growth control is also conceivable. DbcAMP and theophylline in the doses applied do not impair the viability, ultrastructure and catecholamine-storing capacity of cultured chromaffin cells.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

Effects of bovine platelet-poor plasma on the proliferation of normal and transformed BALB/c 3T3 cells

Summary Platelet-poor plasma, as well as autologous platelet-rich serum, was prepared from freshly-drawn bovine whole blood. Bovine platelet-poor plasma had properties similar to those previously decribed for human platelet-poor plasma; e. g., it would (a) support the growth of virally transformed but not normal BALB/ c 3T3 cells, (b) act synergistically with either partially purified platelet-derived growth factor or fibroblast growth factor to initiate cell replication in quiescent 3T3 cells, and (c) act sequentially with platelet-derived growth factor to initiate 3T3 replication. It appears that bovine serum contains both competence and progression factors and that stimulation of fibroblasts with bovine serum involves at least two sequential stages analogous to those described for stimulation with human serum.     

Response of cytosolic calcium,cyclic AMP,and cyclic GMP in Dimethylsulfoxide-differentiated HL-60 cells to modulated low frequency electric currents

The action of interferential current (IFC), an amplitude-modulated 4000 kHz current used in therapeutic applications, upon intracellular calcium, adenosine 3′:5′-cyclic monophosphate (cAMP), and guanosine 3′:5′-cyclic monophosphate (cGMP) was investigated. Human promyelocytes (HL-60) were differentiated to granulocytes by dimethylsulfoxide (DMSO) treatment and exposed for 5 min at 25, 250, and 2500 μA/cm² current density. No significant changes in cytosolic free calcium were detected as a function of modulation frequency of the IFC. However, intracellular cAMP reacted in a complex way to modulation frequency, resulting in stimulations and depressions within the range of frequencies studied (0–125 Hz). The “windows” of modulation frequency, where statistically significant increases or decreases in cAMP were noted, coincided with those published earlier for mouse fibroblasts. Cellular cGMP content was always lowered by IFC treatment. Furthermore, no significant influence of IFC current density upon the three second messengers was noted. These results, which also include data relating to treatment with sinusoidal 50 Hz current, contribute to a more detailed understanding of the primary biophysical mechanisms of signal transduction by time-varying electric fields. Bioelectromagnetics 19:452–458, 1998. © 1998 Wiley-Liss, Inc.