摘要:

AbstractActively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin‐containing intermediate filaments that react with a combination of AE1 and AE3 anti‐keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti‐keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non‐proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non‐proliferating RPE cells. In contrast to the presence of keratin‐containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin‐containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reach

ISSN:0021-9541    

DOI:10.1002/jcp.1041450202

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

年代:1990

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐β (TGFβ) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone‐forming cells. TGFβ has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast‐like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGFβ plays a role in regulating mineral deposition in the matrix, the effects of TGFβ on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGFβ (0.1–10 ng/ml) to examine the influence of cell maturation on response to TGFβ. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high‐density cultures, there was further stimulation at 10 ng/ml. There was a dose‐dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose‐dependent increase in phospholipase A2‐specific activity in the plasma membranes and matrix vesicles of both high‐ and low‐density cultures. In agreement with previous studies, TGFβ inhibited cellular proliferation 50%. The results show that addition of TGFβ stimulates the activity of enzymes associated with calcification. The effect of TGFβ is dependent on the stage of maturation of the cell. This study indicates that TGFβ may play an important role in induced bone formation, calcification, and fracture repair in addition to its ro

ISSN:0021-9541    

DOI:10.1002/jcp.1041450203

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe proto‐oncogene c‐myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184A1N4 (A1N4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c‐myc (A1N4‐myc) or SV40T antigen (A1N4‐T). Neither the A1N4, A1N4‐myc, or A1N4‐T cells will grow in soft agar or form tumors in nude mice. However, A1N4‐T or A1N4‐myccells, but not the parental A1N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor α (TGFα), or basic fibroblast growth factor (bFGF). Like EGF and TGFα, bFGF is moderately mitogenic for the anchorage‐dependent growth (ADG) of all three cell lines. Further, co‐cultivation of A1N4‐T or A1N4‐myccells with primary diploid mammary fibroblasts can also induce the anchorage‐independent growth (AIG) and stimulate the ADG of A1N4‐T or A1N4‐myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1N4‐T and A1N4‐myccells and was found to contain immunoreactive TGFα and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFGα or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammar

ISSN:0021-9541    

DOI:10.1002/jcp.1041450204

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe‐L system of amino acid transport is markedly diminished in chronic lymphocytic leukemia (CLL) B‐lymphocytes, with a maximal velocity less than 15% that of normal B‐lymphocytes. Another membrane‐associated function, the activity of the ectoenzyme, gamma‐glutamyl transpeptidase (GGT), is diminished in CLL B‐cells to 30% that of normal B‐cells. In addition to its transpeptidase activity, a role for GGT has been postulated in the transport of amino acids. In the present report, the possible relationship of these two physiologic functions in CLL B‐cells was studied. The L system transport defect in CLL is restored by phorbol ester‐induced cell maturation; following incubation with 0.15 μM tetradecanoyl phorbol acetate (TPA) for 17 hours, the L system initial velocity showed a 20‐fold increase. In contrast, there was no significant effect on GGT activity with cell maturation. Furthermore, an antibody which diminished GGT activity by 50% in lymphoid cells did not inhibit L system transport. Thus, the impaired L system amino acid transport and GGT activity appear to be independent proc

ISSN:0021-9541    

DOI:10.1002/jcp.1041450205

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

年代:1990

数据来源: WILEY

摘要:

AbstractTo analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases [neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)] in primary cultures of renal proximal tubule cells grown in various culture media. The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness. In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)‐supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains. By contrast, in serum‐free hormonally defined medium (DM: insulin, 5 μg/ml; dexamethasone, 5 × 10−8M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule. When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments. This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde‐fixed cells but does not change the total enzymatic activity. This suggests the presence in tubular cells of a dexamethasone‐dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the ap

ISSN:0021-9541    

DOI:10.1002/jcp.1041450206

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

年代:1990

数据来源: WILEY

摘要:

AbstractAn adenosine 3′,5′‐cyclic monophosphate (cAMP)‐dependent growing cell line called CT‐Mat was established by the long‐term cultivation of an interleukin‐2 (IL‐2)‐dependent human T‐cell line, ILT‐Mat, in the presence of cholera toxin instead of IL‐2. CT‐Mat cells can grow in the medium containing either cholera toxin or forskolin or cAMP derivatives. Although the CT‐Mat cell line can still grow dependent on IL‐2, the forskolin‐induced growth of CT‐Mat cells was demonstrated not to be mediated by an autocrine mechanism of IL‐2 or any other growth factor. The intracellular cAMP level was elevated by treatment with the chemical agents but little by treatment with IL‐2. These suggest that cAMP transduces intracellular growth signals different from those through the IL‐2 receptor

ISSN:0021-9541    

DOI:10.1002/jcp.1041450207

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

年代:1990

数据来源: WILEY

摘要:

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 iso

ISSN:0021-9541    

DOI:10.1002/jcp.1041450208

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

年代:1990

数据来源: WILEY

摘要:

AbstractWe have previously reported on the biochemical properties of a Na+,K+,2Cl−‐cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na+,K+,2Cl−‐cotransport in HeLa cells was studied by86Rb+influx and86Rb+/22Na+efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on86Rb+flux, mediated by the bumet‐anide‐sensitive Na+, K+, 2Cl−‐cotransport system and the ouabain‐sensitive Na+/K+‐pump, were investigated. ANP reduced bumetanide‐sensitive86Rb+influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide‐sensitive86Rb+influx was observed in the presence of 8‐bromo‐cGMP, while neither isoproterenol as a β‐receptor agonist nor 8‐bromo‐cAMP‐could alter bumetanide‐sensitive86Rb+influx. Furthermore, efflux of86Rb+and22Na+was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch,Biochim. Biophys. Res. Commun.168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na+, K+, 2Cl−‐cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α‐methyl‐aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na+, K+, 2Cl−‐cotransport‐mediated86Rb+uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na+/K+‐pump was markedly stimulated in the presence of amino acids, while neither ANP and 8‐Br‐cGMP nor isoproterenol and 8‐Br

ISSN:0021-9541    

DOI:10.1002/jcp.1041450209

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

年代:1990

数据来源: WILEY

摘要:

AbstractIn previous studies (Housey et al.:Cell52: 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

ISSN:0021-9541    

DOI:10.1002/jcp.1041450210

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

年代:1990

数据来源: WILEY

摘要:

AbstractTo clarify the modulation of intercellular communication via gap junctions, associated with the growth induction of quiescent 3T3‐L1 cells, we investigated ‐the gap‐junctional intercellular communication in growth‐stimulated cells that were able to bind fibronectin‐coated beads. When quiescent 3T3‐L1 cells were incubated with fibronectin‐coated beads for the first 2 h after the addition of calf serum, 24.0% of the cells bound and phagocytosed beads. Among the cells with bound beads, the percentage of the cells labeled concurrently with bromodeox‐yuridine was 63.7% when examined 13 h after the addition of calf serum. Transient reduction of dye‐coupling, measured with Lucifer Yellow CH, was observed only in the cells with bound beads 2 h after addition of calf serum, but it was not observed in the cells without bound beads. When the quiescent cells were incubated with fibronectin‐coated beads for 2 h from 4–6 h after the addition of calf serum, the percentage of cells with bound beads increased to 53.1 %, but the decrease in dye‐coupling among the cells with bound beads was slight. These results suggest that the induction of cell growth causes a transient reduction in gap‐junctional intercellular communication in 3T3‐L1 cells with boun

ISSN:0021-9541    

DOI:10.1002/jcp.1041450211

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

年代:1990

数据来源: WILEY