bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of differentiation‐inducing agents on maturation of human MCF‐7 breast cancer cells
Authors: Guilbaud Nicolas F. , Gas Nicole , Dupont Marie Ange , Valette Annie 
Published: February 2005

AbstractThe effects of the differentiation inducing agents (DIAs), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF‐7) were compared. All these agents reversibly caused a concentration‐dependent growth inhibition in monolayers and markedly reduced colony‐forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 μM), but cell replication ceased with NaBu (1 mM), forskolin (50 μM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in GO/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 μM)‐treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 μM)‐treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.
Reversal of lovastatin‐mediated inhibition of natural killer cell cytotoxicity by interleukin 2
Authors: Cutts J. Lynn , Bankhurst Arthur D. 
Published: February 2005

AbstractThe activation of human natural killer (NK) cell cytotoxicity by interleukin 2 (IL‐2) is well established, although the biochemical mechanisms ofthis stimulation have not yet been fully delineated. Earlier, we reported that treatment of NK cells with an inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase such as compactin or lovastatin significantly abrogates the in vitro killing of a susceptible human erythroleukemic cell line and that this inhibition can be completely reversed by 2 hr of exposure to mevalonate (J. Cell. Physiology 139:550–557, 1989). We report here that 24 hr of treatment with IL‐2 also reverses lovastatin inhibition of NK cell function. In addition to natural cytotoxicity, IL‐2 also restores chemotactic and antibody dependent cellular cytotoxicity functions to lovastatin‐treated cells. IL‐2 does not stimulate proliferation of these cells during this time period, nor does it affect the phenotypic composition of the NK cell preparations. Although IL‐2 was able to reverse the lovastatin‐mediated inhibition of every cell function we examined, it had no effect on the inhibition of cholesterol biosynthesis as measured by [3H]acetate incorporation into non‐saponifiable lipids, nor did it stimulate HMG CoA reductase activity. These findings support the hypothesis that there is a non‐sterol isoprenoid product which is required for NK cell cytotoxicity and chemotaxis. In addition, the data suggest that IL‐2 stimulation of NK cells proceeds by an isoprenoid‐independent pathway.
Growth factor‐induced DNA synthesis in cells that overproduce protein kinase C
Authors: Hoshina Sadayori , Ueffing Marius , Weinstein I. Bernard 
Published: February 2005

AbstractIn previous studies (Housey et al.: Cell 52: 343–354, 1988), our laboratory demonstrated that a cell line R6‐PKC3 that stably overproduces high levels of the βisoform of PKC displayed several abnormalities in growth control, and these phenotypic changes were also markedly enhanced when the cells were exposed to TPA. The present studies indicate that these cells also display marked changes in their response to certain growth factors. A striking finding was that several agents when tested alone in serum‐free medium, including EGF, PDGF, TPA, teleocidin, and OAG, stimulated DNA synthesis in quiescent R6‐PKC3 cells but had a negligible effect in quiescent R6‐C1 cells, a vector control cell line with jiormal levels of PKC. R6‐PKC3 cells also show an exaggerated response to very low concentrations of serum, when compared to R6‐C1 control cells. These studies provide direct genetic evidence that alterations in ce lular levels of PKC can markedly influence the responses of cells to specific growth factors.
Establishment of an osseous cell line from fetal rat calvaria using an immunocytolytic method of cell selection: Characterization of the cell line and of derived clones
Authors: Bernier Suzanne M. , Desjardins Johanne , Sullivan Arthur K. , Goltzman David 
Published: February 2005

AbstractUsing selective media and complement‐mediated lysis of primary cultures of a fetal rat calvarial cell population, we have developed a cell line (OBCK6) that exhibits osteoblastic characteristics. OBCK6 cells demonstrated enhanced para‐thyroid hormone (PTH)‐stimulated adenylate cyclase activity relative to the primary, calvarial population, production of alkaline phosphatase activity and type I collagen, and the capacity to form mineralized nodules in unsupplemented medium after prolonged (22–26 day) culture. Two sublines, CFK1 and CFK2, which were isolated by dilution cloning, differed morphologically and with respect to growth rate. CFK1 cells demonstrated high PTH and prostaglandin E2‐stimulated adenylate cyclase activity, whereas only low PTH‐stimulated activity was observed in CFK2 cells. Retinoic acid and 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] each reduced PTH‐stimulated adenylate cyclase activity in both the cell types. Retinoic acid and dexamethasone reduced and 1,25(OH)2D3 enhanced alkaline phosphatase activity in these cells. PTH significantly augmented alkaline phosphatase activity to a much greater extent in CFK1 than in CFK2 cells. Both CFK1 and CFK2 cells expressed type I but not type III collagen, and neither expressed osteocalcin. Strong Alcian blue staining of CFK2 cells was suggestive of a cartilaginous phenotype. These three cell lines, therefore, demonstrated discrete characteristics of skeletal cell function and should provide important models for evaluation of mechanisms of mineralization and for control of skeletal cell growth and mesenchymal differentiation in vitro.
Signal transduction in N‐formyl‐methionyl‐leucyl‐phenylalanine and concanavalin A stimulated human neutrophils: Superoxide production without a rise in intracellular free calcium
Authors: Liang Songlin , Woodlock Timothy J. , Whitin John C. , Lichtman Marshall A. , Segel George B. 
Published: February 2005

AbstractChanges in intracellular ionized free calcium ([Ca]i), inositol triphosphate (IP3), and‐sn‐1,2‐diacylglycerol (DAG) were determined in relation to agonist‐induced human neutrophil superoxide (O2−) production. With 0.1 μM N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) stimulation, generation of IP3 and a peak rise in [Cai] occurred at 30 sec, preceding maximal O2− production (1.5 min) and the maximal rise in DAG mass (4 min). FMLP‐induced O2 production was inhibited by pertussis toxin. In cytochalasin B‐primed, concanavalin A (Con A) stimulated neutrophils, a peak rise in [Ca], but not IP3 proceeded O2 production, and pertussis toxin did not inhibit O2− production. EGTA inhibited the cytochalasin B/fMLP‐induced increment in [Ca]i and O2− production by 75% and 50% respectively, and completely ablated the response to cytochalasin B/Con A, suggesting a role for extracellular as well as intracellular calcium in the respiratory burst. However, three types of experiments indicate that an increase in [Ca]i is neither sufficient nor always required for O2 − production. First, treatment with ionomycin resulted in a marked increase in [Ca]i but did not cause O2− production. Second, pertussis toxin inhibited both fMLP‐induced IP3 generation and O2− production but did not inhibit the rise in [Ca]i Third, following neutrophil priming with dioctanoylglycerol (diC8), maximal O2− production occurred in response to 0.015 μM fMLP or Con A without a rise in [Ca]i, and diC8/fMLP‐induced O2− production was not inhibited by EGTA. Taken together, these data suggest that (1) an increment in [Ca]i is not strictly essential for neutrophil O2− production, (2) unlike fMLP, Con A‐induced O2− production does not proceed through a pathway involving the pertussis toxin‐sensitive G protein, and (3) regulation of neutrophil [Ca]i involves mechanisms independent of IP3 concentration.
Involvement of Hexose transport in myogenic differentiation
Authors: Kudo P. A. , Lo T. C. Y. 
Published: February 2005

AbstractA high (HAHT) and a low (LAHT) affinity hexose transport system are present in undifferentiated rat L6 myoblasts; however, only the latter can be detected in multinucleated myotubes. This suggests that HAHT is either down‐regulated or modified as a result of myogenesis. The present investigation examined the relationship between HAHT and myogenic differentiation. While myogenesis could be inhibited by the potent hexose transport inhibitor phloretin, it was not affected by phlorizin which had no effect on hexose transport. This relationship was further explored using six different HAHT‐defective mutants. All six mutants, altered in either the HAHT transport affinity (Type I mutants) or capacity (Type II mutants), were impaired in myogenesis. Since these mutants were selected from both mutagenized and non‐mutagenized cells with different reagents, or with different concentrations of the same reagent, the deficiency in myogenesis was likely due to changes in HAHT properties. This notion was confirmed by the observation that growth of Type I mutants in high D‐glucose concentrations could rectify the defect in myogenesis. D‐glucose was unlikely to rectify the defect in myogenesis, if this defect was due to a second unrelated mutation that may have arisen during isolation of the mutants. Since both types of mutants were not altered in LAHT, D‐glucose should still be taken up into the cells. The fact that the glucose‐mediated increase in fusion could not be observed in Type II mutants (deficient in the HAHT transporter) suggested that myogenesis was dependent on the presence of D‐glucose or its metabolites in specific HAHT‐accessible compartments. It is tempting to speculate that trans‐acting regulators involved in myogenesis may be synthesized from the glucose metabolites in these specialized HAHT‐accassible compartments.
Platelet‐derived growth factor‐induced destabilization of smooth muscle alpha‐actin mRNA
Authors: Corjay Martha H. , Blank Randal S. , Owens Gary K. 
Published: February 2005

AbstractWe have previously shown that treatment of postconfluent, quiescent rat vascular smooth muscle cells (SMC) with platelet‐derived growth factor (PDGF) dramatically reduced smooth muscle (SM) alpha‐actin synthesis and SM alpha‐actin mRNA abundance, suggesting a role for this mitogen in the control of SMC differentiation. In the present studies, we explored the molecular mechanisms whereby PDGF decreases SM alpha‐actin mRNA levels. Treatment of postcon‐fluent SMC with both platelet PDGF and recombinant PDGF‐BB resulted in a dramatic and concentration‐dependent decrease in SM alpha‐actin mRNA levels. We observed no differences in efficacy between platelet PDGF and PDGF‐BB, indicating that the PDGF‐A chain is not required for the effect. The rate of decrease in SM alpha‐actin mRNA abundance in PDGF‐treated SMC was greater than that observed in cells treated with the transcriptional inhibitor, actinomycin D, with or without PDGF, indicating that PDGF induced a transcriptionally dependent destabilization of the cytosolic SM alpha‐actin mRNA pool. This effect appeared selective for SM alpha‐actin, in that there was no evidence of a similar change in non‐muscle (NM) beta‐actin mRNA stability following PDGF treatment. Results of nuclear run‐on analyses showed no differences in SM alpha‐actin transcription between PDGF‐ and vehicle‐treated SMC at either 4 or 24 hours following treatment, demonstrating that decreases in transcription of the SM alpha‐acti gene did not contribute to PDGF‐induced changes in SM alpha‐actin mRNA abundance, Results of these studies support a possible role for PDGF in regulation of SMC differentiation via a post‐transcriptional control mechanism.
Significance of extracellular zinc‐binding ligands in the uptake of zinc by human fibroblasts
Authors: Ackland M. Leigh , McArdle Harry J. 
Published: February 2005

AbstractExtracellular zinc (Zn)‐binding ligands were investigated as vehicles for uptake of Zn by human fibroblasts. The uptake of α‐macroglobulin, a major serum Zn‐binding protein proposed to have a function in Zn transport, was less than 1/200 that of the Zn uptake rate. The fibroblast growth medium, BME with 10% FBS, contains several Zn‐binding ligands. These were separated into components of MW greater than 30,000 and components of MW less than 30,000 using an Amicon microconcentrator. Cells accumulated Zn from both fractions; however, there was more uptake from the filtrate (MW 30,000), containing ligands with low affinity for Zn, hence with greater free Zn concentration. Zn uptake from a number of ligands with a range of affinities for Zn was examined and found to be inversely proportional to the Ka value for the ligands and therefore proportional to the free Zn concentration. When histidine and desferrioxamine, two structurally different Zn‐binding ligands were compared, analysis of the concentration curves of calculated free Zn against Zn uptake gave similar Vmax and Km values (S.E.M.) (±S.E.M.) of 373 ± 6 pmol/μg DNA'h and 0.08 ± 0.004 μM for histidine, and 349 ± 10 pmol/μg DNA/h and 0.06 ± 0.008 μM for DFO, suggesting that the same transport mechanism was operating in both systems. We conclude that no specific ligands are essential for transport of Zn into fibroblasts, but that ‘free’ Zn is acquired by the cell.
Cell‐cell interactions in the process of differentiation of thyroid epithelial cells into follicles: A study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles
Authors: Munari‐Silem Yvonne , Mesnil Marc , Seimi Samia , Bernier‐Valentin Françoise , Rabilloud Rachida , Rousset Bernard 
Published: February 2005

AbstractThyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36–48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell‐cell contacts involved in (a) the formation of the thyroid follicular lumen and (b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC‐Dextran and Cascade Blue conjugated to bovine serum albumin: CB‐BSA). LY microinjected into IL of 2–9‐day‐old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 μm. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC‐Dextran or CB‐BSA microinjected into IL behaved as LY; the probes were restrained into the iumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15–30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2+‐dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FiTC‐Dextran or CB‐BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell‐to‐cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell‐to‐cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.
Evidence that the adenylate cyclase secreted from Bordetella pertussis does not enter animal cells by receptor‐mediated endocytosis
Authors: Donovan Maura G. , Storm Daniel R. 
Published: February 2005

AbstractBordetella pertussis, the pathogen responsible for whooping cough, produces a calmodulin‐sensitive adenylate cyclase. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels (Confer and Eaton:Science 217:948–950, 1982; Shattuck and Storm: Biochemistry 24:6323–6328, 1985). However, the mechanism for entry of the catalytic subunit of this adenylate cyclase into animal cells is unknown. It has been reported that the B. pertussis adenylate cyclase extracted from bacterial cells with urea does not enter animal cells by receptor‐mediated endocytosis. There is, in addition to the cell associated form of the B. pertussis adenylate cyclase, a cell‐invasive form of the enzyme secreted into the bacterial culture media. The properties of the cell‐associated and secreted enzymes are significantly different (Masure and Storm: Biochemistry 28:438–442, 1989). In this study, we report evidence that the secreted form of the B. pertussis adenylate cyclase enters animal cells by a mechanism distinct from receptor‐mediated endocytosis.
M‐07e human leukemic factor‐dependent cell line provides a rapid and sensitive bioassay for the human cytokines GM‐CSF and IL‐3
Authors: Avanzi Gian Carlo , Brizzi Maria Felice , Giannotti Joann , Ciarletta Agnes , Yang Yu‐Chung , Pegoraro Luigi , Clark Steven C. 
Published: February 2005

AbstractWe have isolated a subline of the M‐07 human megakaryoblastic leukemia cell line, designated M‐07e, that requires either interleukin‐3 (IL‐3) or granulocyte macrophage colony‐stimulating factor (GM‐CSF) for growth, even in the presence of fetal calf serum. This cell line will not grow long term in any other cytokine although it responds slightly to IL‐2, IL‐4, IL‐6, IL‐9, and interferon‐γ. We have used the M‐07e subline to develop a quantitative bioassay for the measurement of levels of either GM‐CSF or IL‐3. This assay is as sensitive to either factor as the human bone marrow colony assay (CFU‐GM) or the chronic myelogeneous leukemic (CML) blast cell proliferation assay for these factors and is much more convenient and reliable than either. With this assay, as little as 25–50 pg/ml of either IL‐3 or GM‐CSF can be detected, a level that should render the assay useful for analysis of these molecules in samples from patients undergoing colony‐stimulating factor therapy and from conditioned media from natural sources of the factors. In these cases, neutralizing antisera to each cytokine are required to demonstrate the specificity of the assay. This assay, in combination with quantitative immunoassays should greatly facilitate the analysis of the roles of IL‐3 and GM‐CSF in regulating hematopoiesis both in patients and in natural sources of the cytokines.
Epithelioid macrophages secrete a deactivating factor for superoxide release
Authors: Camarero Valeria C. P. C. , Junqueira Virginia B. , Colepicolo Pio , Karnovsky Manfred L. , Mariano Mario 
Published: February 2005

AbstractThe release of superoxide anion (O) by inflammatory macrophages, multinucleated giant cells, and epithelioid cells, obtained by the insertion of round glass coverslips into the subcutaneous tissue of mice, was investigated. O was shown to be spontaneously released by cells on the surface of glass coverslips implanted up to 7 days but not by cells obtained 14 or 21 days after coverslip implantation. The formel showed increased O release when stimulated by phorbol myristate acetate, whereas cells harvested after 14 or 21 days implantation did not. The induction of delayed type hypersensitivity around coverslips implanted for 5 days increased spontaneous O release by these cells by 40%. On the other hand, when the same protocol was used with coverslips implanted for 14 days, O release was not detected. These results were viewed in regard to the composition of the cell population at each time point. When coverslips were removed after 14 days of implantation and the cells incubated for 30 minutes in vitro, the medium so conditioned inhibited O release by cells of 5 day old preparations. This indicates the release by cells on the longer term coverslips of a substance that inhibits O production by cells of coverslips implanted for 5 days only. This inhibitory activity could be suppressed by treating the conditioned medium with proteases. The factor was, however, heat stable and exerted its effects even when the test cells were exposed to phorbol myristate acetate.
Evidence for c‐myc in the signaling pathway for TGF‐β in well‐differentiated human colon carcinoma cells
Authors: Mulder Kathleen M. , Humphrey Lisa E. , Choi Ho Gene , Childress‐Fields Karla E. , Brattain Michael G. 
Published: February 2005

AbstractPreviously, we described a model culture system for comparing responsiveness of poorly differentiated and well‐differentiated human colon carcinoma cells to exogenous growth factors. While polypeptide growth stimulators elicited an up‐regulation of c‐myc, as well as a mitogenic response in the well‐differentiated cells, the poorly differentiated cells were insensitive to exogenous growth stimulators. We now show, by thymidine incorporation experiments and autoradiographic analysis, that transforming growth factors, β1 (TGF‐β) abrogated the mitogenic responses to the growth factors epidermal growth factor + insulin + transferrin (lC,50 = 0.8 ng/ml), as well as to nutrients (basal medium; lC50 = 0.2 ng/ml) in the well‐differentiated cells. The poorly differentiated cells did not respond to TGF‐β. Moreover, TGF‐β (10 ng/ml) completely abrogated the growth factor‐stimulated up‐regulation of c‐myc in the TGF‐β responsive, well‐differentiated colon carcinoma cells. Addition of TGF‐β to the TGF‐β‐responsive, well‐differentiated cells, at a time after c‐myc had been transiently up‐regulated in response to growth stimulatory factors, resulted in a loss of responsiveness to TGF‐β. Addition of TGF‐β to these cells at increasing time periods after EIT stimulation also resulted in a loss of the TGF‐β‐induced repression of c‐myc. The results suggest an important role for c‐myc in the mechanism of action of TGF‐β in well‐differentiated human colon carcinoma cells.