摘要:

Abstractα2‐Macroglobulin (α2M), epidermal growth factor (EGF), and vesicular stomatitis virus (VSV) each enter cultured fibroblasts by receptor‐mediated endocytosis. The present study defines some basic ionic requirements in the cell culture medium which are necessary for the maximal rate of endocytosis of these three ligands. Na+and HCO 3−were both necessary for maximal endocytosis of125I‐α2M,125I‐EGF, and35S‐VSV at 37°C. The ion specificities for both the anion and cation requirements were established. The binding of125I‐α2M to its cellular receptors at 4°C was unaffected by the absence of Na+and HCO 3−in the culture medium. In addition, the absence of Na+and HCO 3−in the culture medium did not reduce cellular uptake of horseradish peroxidase by fluid phase endocytosis. Na+and HCO 3−may be general requirements

ISSN:0021-9541    

DOI:10.1002/jcp.1041130302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractCells of the established preadipose line, 3T3‐L1, appear to be undifferentiated fibroblasts during exponential growth. When cells become quiescent, a small percentage of them accumulate triglyceride and become morphologically indistinguishable from mature adipocytes. When insulin is added to quiescent cultures, up to 50% of the cells differentiate into adipocytes. The distribution of lipid‐containing cells which appear in clusters of varying sizes was analyzed to determine whether commitment to differentiation occurred after quiescence or during exponential growth and whether insulin was required as an inducer of commitment. The spatial arrangement of 3T3‐L1 cells at quiescence on some culture dishes was destroyed by replating. This resulted in random distribution of these cells. The distribution of adipocytes among replated and nonreplated cells in these experiments was compared to a computer generated random distribution of differentiated among undifferentiated cells. Dispersal of cells at confluence resulted in a distribution of fat among nonfat cells not significantly different from the computer generated random distribution. In undisturbed cultures, the distribution of fat cells is not random and is consistent with a commitment event in single cells at any cell division during exponential growth followed by divisions of both committed and uncommitted cells. Since insulin affected the number of mature adipocytes only when added after cessation of exponential growth, insulin is not the inducer of commitment but merely enhances lipid production in previously committed

ISSN:0021-9541    

DOI:10.1002/jcp.1041130303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractChicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage‐independent colony formation in gelified medium, at the restrictive temperature (41°5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of thesrcgene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viralsrcgene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by E

ISSN:0021-9541    

DOI:10.1002/jcp.1041130304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractBovine adrenal cortex cells maintained on extracellular matrix (ECM)‐coated dishes will proliferate actively when serum is replaced by HDL (25 μg protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures. A requirement for transferrin (1 μg/ml) becomes apparent only when cells have been grown for at least four generations in the absence of serum.Early passage (P1–P3) bovine adrenal cortex cells cultured in serum‐free medium responded to ACTH (10−8M) with increased 11‐deoxycortisol production; this effect was not observed in later passage cells (P7–P15). The cells' ability to utilize LDL‐derived cholesterol and to respond to db cAMP (1mM) by increased steroid release was preserved in cells cultured for over 60 generations in the serum‐free medium. HDL, although also able to increase steroid production in early‐passage cultures exposed to ACTH or to ACTH and dibutyryl cyclic AMP (db cAMP), was 10 fold less potent than LDL. It did not support steroidogenesis in cultures not exposed to these trophic agents.The life span of bovine adrenal cortex cells grown in the serum‐free medium on fibronectin (FN)‐ versus ECM‐coated dishes was compared. Cells seeded in serum‐containing medium and grown in serum‐free medium had a life span of 34 versus 60 generations when maintained on fibronectin‐ or ECM‐coated dishes, respectively. Cells seeded in the complete absence of serum in the serum‐free medium on ECM‐ or fibronectin‐coated dishes could be passaged for 26 or 13 generations, respectively. While FGF was an absolute requirement for cells cultured on fibronectin‐coated dishes, it was not required when cells were maintained on ECM. These observations demonstrate the influence of the ECM not only in promoting cell growth and differentiation bu

ISSN:0021-9541    

DOI:10.1002/jcp.1041130305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractProliferation and death were measured in synchronously growing cultures of HeLa S3 cells during treatment with up to 30 mM caffeine. Changes in the number of colony‐forming cells were determined by single‐cell plating, while changes in the total number of cells were measured both by electronic counting and by monitoring cell division and physiological death cinemicrographically. At concentrations between 2 and 5 mM, cell killing occurs over several days during which the cells traverse the generation cycle once or a few times before losing colony‐forming ability, with consequent proliferation of non‐colony‐forming cells. This indicates that lethal damage is accumulated with time. Death occurs more rapidly at higher concentrations, without proliferation, the kinetics of inactivation being strongly dependent on the phase of the cycle (cell age) at which treatment is initiated. G1cells are killed more slowly in 10 mM caffeine than are S cells, but G1cells respond rapidly to 20 mM caffeine, suggesting the inception of an additional mode of killing. The incidence of sister‐cell fusion increases with increasing caffeine concentration above 1 mM. On addition of 10 mM caffeine to a culture prepared from collected mitotic cells, the cells undergo a transient rounding and then respread after several hours; with 20 mM, they never respread. The generation cycle is prolonged in a concentration‐dependent fashion, as is the duration of G1; the generation time is doubled in 5–6 mM caffeine. G2and M are also prolonged at concentrations above 3 mM, but S is not prolonged even by

ISSN:0021-9541    

DOI:10.1002/jcp.1041130306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe intracellular Ca2+content of nontransformed Balb/c3T3 cells is two to three times higher than that of a spontaneously transformed derivative. Depriving either cell type of extracellular Mg2+causes a 2‐ to 3‐fold increase in their Ca2+content over a 24‐hr period. Restoring Mg2+to the medium decreases the Ca2+content of the cells to their original values in about the same time. The increase in Ca2+content is not blocked by cycloheximide suggesting that normal rates of protein synthesis are not required to produce this effect. Mg2+deprivation also decreases the initial rate of Ca2+efflux from the transformed cells and increases the size of the slowly exchanging fraction of Ca2+to the levels found in the nontransformed cells. Since Mg2+deprivation normalizes the appearance and growth behavior of the transformed cells, the possible intermediary role of Ca2+in this normalization was studied. Large changes in extracellular Ca2+produced large changes in the Ca2+content of the transformed cells with little change in appearance or thymidine incorporation rate. Ca2+deprivation did inhibit thymidine incorporation in early passage nontransformed cells; however with repeated passage, this effect decreased, as did the Ca2+content of these cells. The possible role of Mg2+in regulating cellular Ca2+content and distribution is discussed, as is the relation of Ca2+content and distribution to the development of the transformed

ISSN:0021-9541    

DOI:10.1002/jcp.1041130307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractThe Na+uptake into neuroblastoma x glioma hybrid cells was measured in Hepes‐buffered EMEM containing 10% calf serum and 5 mM ouabain in the presence and absence of amiloride (1.0 mM). Amiloride was found to markedly inhibit net Na+influx (by approximately 50%). Examination of the effect of amiloride on net Na+influx in the absence of calf serum revealed that a significant amiloride‐sensitive Na+influx remains even under serum‐deprived conditions, although the degree of amiloride inhibition (35%) is substantially lower than that found in the presence of serum. The amiloride‐insensitive portion of Na+influx was found to be independent of serum effects. Estimation of resting membrane potential was made by measurement of the steady state distribution of the lipophilic cation, TPP+, in the presence and absence of amiloride. A large, immediate increase in TPP+uptake, indicative of a membrane hyperpolarization, was seen upon addition of amiloride. Determination of the effect of amiloride on resting membrane potential of serum‐deprived cells showed that cells are hyperpolarized to a greater extent in the presence than in the absense of amiloride, and that serum exerts a depolarizing effect on the cells. Thus, serum‐stimulation of Na+influx results in a depolarization of resting membrane potential, while amiloride inhibition of Na+influx causes a hyperpolarization. These data strongly suggest that NG108‐15 cells possess an electrogenic Na+influx pathway that is sensitive to amiloride inhibition and enha

ISSN:0021-9541    

DOI:10.1002/jcp.1041130308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractWe examined the role of the monovalent cations Na+and K+in the events encompassing the release of O 2−by alveolar macrophages after stimulation with formyl methionyl phenylalanine (FMP). This was accomplished by determining the effect of changing the extracellular [Na+] and/or [K+] on FMP‐stimulated O 2−production; and measuring22Na+42K+and86Rb+influx and efflux and intracellular [K+] for control and FMP‐stimulated alveolar macrophages. Stimulated O 2−production was relatively insensitive to changes in extracellular K+or Na+concentrations until the [Na+] was decreased below 35 mM. At 4 mM [Na+], the rate of O 2−production remained at 75% of the maximal rate observed at physiological concentrations of [Na+]. Both influx and efflux of22Na+were stimulated above control rates by FMP. The increased rates of fluxes lasted for a few minutes suggesting a transient increase in membrane permeability to Na+. Ouabain partially inhibited22Na+efflux but had no effect on O 2−release. The influx of86Rb+and42K+was not altered by the addition of FMP but was virtually abolished in the presence of 10 μM ouabain or 1 mM quinine. In the presence of extracellular calcium, FMP‐stimulated a prolonged (>20 minutes) increase in86Rb+or42K+efflux which was inhibitable by 1 mM quinine. In the absence of extracellular calcium, FMP stimulation of K+efflux was greatly diminished and was not affected by quinine, although quinine still inhibited O 2−production under these conditions. It was also observed that there was a loss of intracellular K+when cells were stimulated by FMP in the presence of Ca+2, but not in the absence of Ca+2. Taken together, these results suggest a minimal direct role, if any, for K+in the events that lead to FMP‐stimulated O

ISSN:0021-9541    

DOI:10.1002/jcp.1041130309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractHuman diploid fibroblasts in culture were examined for production of glycopeptide hormones. Forty‐one percent of the strains produced human chorionic gonadotropin (hCG) under normal growth conditions. Constitutive hCG synthesis was apparently unrelated to donor age, length of time in culture, or number of passages. Follicle stimulating hormone (FSH) was not found in any strain investigated. Only one cell strain produced free α‐chains of glycopeptide hormones. Hydroxyurea (HU) at a concentration of 1 mM mediated a small, statistically significant increase in hCG production (p<0.01) in all constitutive strains, but had no effect on non‐hCG‐producing fibroblast strains. Sodium butyrate (Bu) was effective in increasing hCG synthesis in only one constitutive strain, derived from a newborn foreskin. HU treatment had no apparent effect on cell structure. All Bu‐treated strains, both those producing hCG and the nonproducers, showed morphological alterations; cells were flattened and they contained ordered arrays of refractile granules. It is suggested that hCG synthesis in cultured human diploid fibroblasts may result from a localized chromosomal event in which the loci responsible for this hormone are activated. Human diploid fibroblasts in culture are shown to be amenable to the study of gene expression and its

ISSN:0021-9541    

DOI:10.1002/jcp.1041130310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY

摘要:

AbstractChanges in transepithelial electrical resistance and cyclic nucleotide levels were monitored accompanying chemical induction of domes in a clonal subline of MDCK kidney epithelial cells. Confluent cell monolayers grown on nitrocellulose filters exhibited a relatively high mean transepithelial resistance (387 ohms · cm2). Hexamethylene bisacetamide, a potent inducer of dome formation (Lever, 1979b), stimulated significantly increased transmonolayer resistance as well as elevated levels of intracellular cyclic AMP. By contrast, dimethylformamide, an equally potent inducer of dome formation in MDCK cells, did not appreciably alter either resistance values or cyclic nucleotide levels. These results suggest that induction of dome formation in epithelial cell cultures by compounds generally known as inducers of differentiation may involve multiple and separate mechanisms

ISSN:0021-9541    

DOI:10.1002/jcp.1041130311

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1982

数据来源: WILEY