摘要:

AbstractCells of gut and skin frequently suffer mechanically‐induced plasma membrane disruptions in vivo, and bioactive molecules, including basic fibroblast growth factor (bFGF), could enter and leave cytoplasm through these disruptions. We here provide three lines of evidence that bFGF is released with surprising efficiency through plasma membrane disruptions, resembling those known to occur in vivo, produced by scraping endothelial cells from their culturing substratum. First, 41% of the total of bFGF extractable in 1 M NaCl by freeze‐thaw and sonication was released simply by scraping the endothelial cells. Second, relative to release of lactate dehydrogenase, cells wounded by scraping under conditions promoting>60% cell survival released a significantly larger amount (up to twofold more) of growth promoting activity than did cells uniformly killed and irreversibly permeabilized by scraping in the cold or by freezing and thawing. Last, cells that survived membrane disruptions released, and contained, less bFGF on each subsequent wounding, consistent with release of bFGF through transient (i.e., survivable) membrane disruptions. A polyclonal antibody against bFGF completely neutralized the growth promoting activity released by scraping, confirming that bFGF is released through endothelial cell plasma membrane disruptions. Cell fractionation and immunolocalization, including a novel permeabilization technique for electron microscope immunolocalization, demonstrated a cytosolic location of bFGF. We conclude that many characteristics of bFGF–its broad spectrum of producing and target cell types, cytosolic location, efficient release through biologically and pathologically relevant plasma membrane wounds, and its release from cells that survive membrane wounds–make it a strong candidate as a “wound hormone” for rapidly initiating the cell growth required for routine maintenance of tissue integrity and/or repair a

ISSN:0021-9541    

DOI:10.1002/jcp.1041480102

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full‐term human placenta. These purified membranes were enriched 25‐fold in Na+/ K+‐adenosine triphosphatase (ATPase), 37‐fold in [3H]dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+uptake was observed, which followed Michaelis‐Menten kinetics, with a KmCa2+of 0.18 ± 0.05 μM and Vmaxof 0.93 ± 0.11 nmol/mg/min. The addition of Mg2+to the incubation medium significantly decreased this uptake in a concentration‐dependent manner, with a maximal inhibition at 3 mM Mg2+and above. The Lineweaver‐Burk plots of Ca2+uptake in the absence and in the presence of 1 mM Mg2+suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+had no significant effect on Ca2+uptake, eliminating the hypothesis of a Ca2+/Mg2+exchange mechanism. This ATP‐independent Ca2+uptake was not sensitive to 10−6M nitrendipine nor to 10−4M verapamil. An ATP‐dependent Ca2+transport was also detected in these BPM, whose Km Ca2+was 0.09 ± 0.02 μM and Vmax3.4 ± 0.2 nmoles/mg/3 min. This Ca2+transport requires Mg2+, the optimal concentration of Mg2+being approximately 1 mM. Preincubation of the membrane with 10−6M calmodulin strongly enhanced the initial ATP‐dependent Ca2+uptake. Finally, no Na+/Ca2+exchang

ISSN:0021-9541    

DOI:10.1002/jcp.1041480103

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

年代:1991

数据来源: WILEY

摘要:

AbstractHuman granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) (0.1 nM) down‐modulates its receptor in IL‐3/GM‐CSF dependent M‐07e cells, in KG‐1 cells and normal granulocytes, whereas phorbol esters 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (2 nM) down‐modulates the GM‐CSF receptor in M‐07e cells and granulocytes but not in KG‐1 cells. As data analysis shows by nonlinear regression, the decreased binding ability depends on a reduction of the binding sites with no significant change of their dissociation constant. To gain insight into the mechanisms involved in the GM‐CSF receptor regulation, we investigated the role of protein kinase C (PKC). GM‐CSF, unlike TPA, was unable to activate PKC in all the cells studied. Moreover, unlike TPA, GM‐CSF was still able to down‐modulate its receptor in cells where PKC was inhibited by 1‐(5‐isoquinolonesulphonyl)‐2‐methylpiperazine (H7) and staurosporine or in cells where PKC was exhausted by prolonged incubation with 1 μM TPA. Finally, the receptor re‐expression rate was accelerated by protein kinases inhibitors. These results, taken together, indicate the presence of a PKC‐dependent and ‐independent down‐modulation mechanism and a negative role of the endo

ISSN:0021-9541    

DOI:10.1002/jcp.1041480104

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe HT‐29 human colon carcinoma cell line differentiates in glucose‐free medium to an enterocytic phenotype. We previously isolated a series of HT‐29 subclones selected for high levels of expression of secretory component (SC), the epithelial receptor for polvmeric immunoglobulins. To develop a model system for studying effects of cell polarity on SC expression and release from the cell surface, the HT‐29.74 subclone was induced to differentiate in glucose‐free medium. Expression of SC was induced by glucose deprivation in both the parental HT‐29 cell line and, to an even greater extent, in the HT‐29.74 subclone. Prolonged glucose deprivation of HT‐29.74 cells resulted in morphological changes consistent with enterocytic differentiation. Metabolic radiolabeling of SC in differentiated HT‐29.74 cells indicated that proteolytic cleavage of membrane‐bound to free SC occurred both on the cell surface and intracellularly, possibly in a vacuolar apical compartment or intrapeithelial lumen. To study effects of cell polarity on SC release, differentiated HT‐29.74 cells were depolarized by culturing in low calcium medium. Within 2 hours after transfer of the cells into low calcium medium, a burst of SC release was observed concomitant with cell depolarization. Subsequently, release of SC declined significantly and remained low as long as cells were maintained in a depolarized state. The extent of cell depolarization could be controlled by varying the extracellular calcium concentration or by substituting the divalent cation Sr++, which partially prevents depolarization, for Ca++. In either case, the magnitude of the initial burst and subsequent decline in release of SC was proportional to the extent of cell depolarization. We conclude that cell polarity plays an important role in controlling the release of SC in intestinal epithelial cells, most likely by regulating the distribution of membranebound SC and SC protease, which are on the basolateral and apical cell surfaces, respectively, in

ISSN:0021-9541    

DOI:10.1002/jcp.1041480105

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe have examined effects of horse serum (HS) and various fractions (1 million‐1M, 300K, 100K, and 30K nominal molecular weight limit) obtained by ultrafiltration on expression of TTX‐sensitive Na‐channels and on activities of the Na‐K pump and glucose transport systems in cultured myotubes obtained from 1‐2‐day‐old neonatal rat pups. Five‐day‐old cells were transferred to serum‐free medium with no hormone or growth factor supplements (DMEM) for 24 hr and then treated with the various serum fractions for 48 hr. Measurements were made of specific [3H]‐saxitoxin (STX) binding, action potential properties,86Rb‐uptake and 2‐deoxyglucose (2‐DG) uptake. HS significantly increased all parameters compared to DMEM (increases in STX‐binding, 69%; Rb‐uptake, 65%; 2‐DG uptake, 93%). Results of treatment with the separate fractions showed that the 300K fraction caused a significantly greater increase in STX‐binding than either HS or the other fractions. In contrast, the increases in Rb and 2‐DG uptakes induced by the different fractions were not different from that obtained with HS. We conclude that serum contains a factor that selectively increases expression of TTX‐s

ISSN:0021-9541    

DOI:10.1002/jcp.1041480106

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe human kidney cell line 293 was generated by transfection of adenovirus DNA into normal human embryonic kidney (HEK) cells (Graham et al., 1977), whereas the human kidney cell lines ST‐1i and STt‐4i were generated by transfection of HEK cells with plasmids encoding SV40 viral oncogenes (Abcouwer et al., 1989). In this study, we examined kidney‐specific enzyme activity levels in 293, ST‐1i, and STt‐4i cells to determine their ability to exhibit kidney‐specific gene expression. Enzymes examined were leucine aminopeptidase (LAP), γ‐glutamyl transpeptidase (γ‐GTP), and the disaccharidases trehalase and maltase. Enzymatic activity levels were compared to three other kidney cell lines (MDCK, OK, and LLC‐PK1) as well as to normal human embryonic kidney (HEK) cells and the human hepatoma cell line, Hep G2. Modulation of kidney‐specific enzyme activities was assessed in response to several differentiation‐inducing agents (adenosine, n‐butyric acid, hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), N, N′‐dimethylformamide (DMF), isobutyl methyl xanthine (IBMX), di butyryl cAMP, and retinoic acid). ST‐1i and STt‐4i exhibit elevated levels of LAP, γ‐GTP, trehalase, and maltase, consistent with their kidney cell origin, whereas 293 cells exhibit elevated levels of just γ‐GTP and maltase. Maltase and γ‐GTP enzyme activities in ST‐1i and STt‐4i cells were very responsive to the various inducing agents; 293 cells were less responsive at the inducer concentrations examined. None of the three human cell lines formed domes under any of the experimental conditions. In summary, ST‐1i and STt‐4i are comparable to normal HEK cells in expression of kidney‐specific enzymes and in responsiveness to differentiation‐inducing agen

ISSN:0021-9541    

DOI:10.1002/jcp.1041480107

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara‐C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara‐C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritlated thymidine (3HTdR) “suicide” technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara‐C concentrations gave very similar dose‐response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara‐C did not lose colony‐forming ability even though the same population was sensitive to3HTdR. The hydrocortisone effect was dose and time related; protection from ara‐C increased from 10−8to 10−5M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara‐C even while the cells are

ISSN:0021-9541    

DOI:10.1002/jcp.1041480108

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe previously cloned a set of primary response genes, which we call TIS (TPA‐Inducible Sequence) genes, from a cDNA library prepared from Swiss 3T3 cells treated with tetradecanoyl phorbol acetate (TPA) and cyclohexirnide. TPA, polypeptide growth factors, and serum induce TIS gene expression in 3T3 cells. We now report that cadmium and zinc elevate mRNA levels for the TIS genes, including TIS28(c‐fos), in Swiss 3T3 cells. The time‐Course of TIS gene mRNA accumulation after metal exposure is delayed in comparison to the accumulation of TIS gene mRNA after treatment with TPA and growth factors. Cadmium induction of the TIS gene message accumulation is blocked by actinomycin D. Moreover, cadmium treatment does not significantly stabilize TIS gene messages. TIS gene induction by metal is a primary response; TIS8, which encodes a zinc‐finger transcription factor, and TIS28(c‐fos), can be induced in the presence of cadmium and cycloheximide, an inhibitor of protein synthesis. Downregulation of protein kinase C does not attenuate TIS gene induction by hea

ISSN:0021-9541    

DOI:10.1002/jcp.1041480109

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe cis‐acting elements governing transcription from the murinec‐mycP2 promoter have not been well defined. To gain a better understanding of the nature of the protein‐DNA interactions that take place on the P2 promoter, protein binding assays were performed. The ME1a2 and E2F factors appear to be the predominant proteins bound to a region spanning positions −140 to −24 relative to the P2 transcription start site. By a number of criteria, these factors appear to be distinct. Whenc‐mycpromoter sequences were coupled to the chloramphenicol acetyltransferase gene (CAT) and transiently transfected into tissue culture cells it was found that optimal transcription from P2 was heavily dependent on the ME

ISSN:0021-9541    

DOI:10.1002/jcp.1041480110

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

年代:1991

数据来源: WILEY

摘要:

AbstractPlatelet‐derived growth factor (PDGF), epidermal growth factor (EGF), insulin‐like growth factor‐1 (IGF‐1), and insulin protect density‐inhibited murine Balb/c‐3T3 fibroblasts against death by distinctive mechanisms. Determination of the cell survival‐enhancing activity of growth factors by cell enumeration and neutral red uptake measurement gives equivalent results. PDGF displays a steep doseresponse relationship in the 1−5 ng/ml range. The other factors display shallow log‐linear relationships in the following ranges: EGF: 0.2−5 ng/ml; IGF‐1: 2−80 ng/ml; and insulin: 57−4,500 ng/ml. Agonists that lead to the activation of protein kinase A, including forskolin, 8‐bromoadenosine 3′:5′‐cyclic monophosphate (Br‐cAMP) and N6,2′‐O‐dibutyryladenosine 3′:5′‐cyclic monophosphate (db‐cAMP), markedly increase both short‐term (5‐h) and long‐term (20‐h) survival of cells. 2‐lsobutyl‐1‐methylxanthine (IBMX) markedly enhances short‐term survival, but its effect decays with time. The protein kinase C agonist 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) has a moderate protective effect at concentrations of 16‐32 nM, and 64 nM TPA is highly effective. The synthetic diacylglycerols 1,2‐dioctanoylglycerol (DiC8) and 1‐oleoyl‐2‐acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplemation of EGF with a protein kinase A or C agonist results in a varying additive increase in short‐term (5‐h) cell survival and supplementation of EGF+insulin or PDGF+EGF+insulin increases further the already high level of protection given by the growth factor combinations. Combining a protein kinase A and a protein kinase C agonist in the absence of growth factors gives an approximately additive increase in cell survival. Results obtained with kinase, RNA, and protein synthesis inhibitors suggest that: (1) activated protein kinase C catalyzes one or more phosphorylation events in quiescent Balb/c‐3T3 cells that lead to gene expression with the protein product(s) mediating protection of quiescent cells against death, and (2) phosphorylation events Catalyzed by protein kinase A largely

ISSN:0021-9541    

DOI:10.1002/jcp.1041480111

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

年代:1991

数据来源: WILEY