摘要:

AbstractSwiss 3T3 cells express receptors for both the polypeptide epidermal growth factor (EGF) and the tetradecapeptide bombesin and respond mitogenically to these substances. These cells thus provide a system to analyze potential signal transduction pathways involved in mitogenic stimulation. Here we have determined and compared the early ionic responses elicited by EGF and bombesin and their relation to diacylglycerol (DG) and inositolphosphate (InsPn) production. Whereas EGF fails to cause any significant change in intracellular Ca2+bombesin effectively induces prompt and transient Ca2+mobilization from intracellular stores. Further support of the idea that these receptors utilize distinct signalling pathways comes from the measurements of cytoplasmic pH (pHi). As in most target cells, EGF induces a delayed (1 min) but sustained intracellular alkalinization that reaches a new steady state after ∼︁10 min. Bombesin, in contrast, elicits a biphasic response; within seconds, a rapid but transient rise in pHiis observed, followed by a further slower sustained alkalinization. Inhibition of the Na+/H+exchanger prevents both EGF as well as bombesin‐induced alkalinization. However, under these conditions, bombesin evokes a rapid and sustained acidification related to the Ca2+response. Apparently, bombesin initiates a Ca2+‐dependent acidifying process immediately after binding of the hormone to its receptor. Furthermore, we could demonstrate that the bombesin‐induced alkalinization depends on protein kinase C activation whereas the EGF response does not. Determination of the total DG and InsPnaccumulation revealed that EGF is ineffective in stimulating phospholipase C‐mediated production of these second messengers. In contrast, bombesin causes a rapid DG and InsPnproduction coinciding with the Ca2+response and the first phase of the rise in pHifollowed by a slower DG accumulation coinciding with the second alkalinization phase. Our results show that in Swiss 3T3 cells the bombesin receptor activates the hydrolysis of inositol lipids as a mechanism of signal transduction, which consequently causes changes in Ca2+iand pHi. Clearly, the EGF receptor utilizes different pathways to evoke mitogenisis and stimulates Na+/H+exchange independently of DG production and protein kinase C

ISSN:0021-9541    

DOI:10.1002/jcp.1041420302

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

年代:1990

数据来源: WILEY

摘要:

AbstractATP and ADP, in concentrations ranging from 1—100 μM, increased the release of [3H]choline and [3H]phosphorylcholine (P‐choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP‐γS and ADPβS) and adenosine 5′‐(β,γ imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5‐(α,β methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Yreceptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 μM), the tumor promoter phorbol 12‐myristate 13‐acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 μM). Down‐regulation of protein kinase C, following a 24‐hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P‐choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Yrece

ISSN:0021-9541    

DOI:10.1002/jcp.1041420303

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

年代:1990

数据来源: WILEY

摘要:

AbstractStimulation of A‐431 carcinoma cells with epidermal growth factor (EGF) causes dramatic morphologic responses including ruffling, rounding, and bulk‐phase pinocytosis. In attempts to explore the mechanisms responsible for changes in plasmalemma topography. we have investigated the effects of exogenous sterols thought to alter membrane fluidity.Light and scanning electron microscopy revealed a time‐ and concentration‐dependent inhibition of ruffling (>90%) by cholesterol. This effect could be duplicated by preincubation of the cells with comparable levels of low‐density lipoproteins (LDL). EGF‐stimulated bulk‐phase endocytosis also is inhibited by treatment with cholesterol. No alteration of EGF binding, kinase stimulation, or internalization was detected in cells incubated in cholesterol‐enriched medium (175 μg/ml in 0.5% ethanol), nor did cholesterol or LDL have any effect on EGF‐stimulated rounding.Morphometry of electron micrographs from cholesterol‐treated cells revealed a selective depletion of interdigitating lateral surface membrane that normally appears to be recruited to generate apical ruffles. Thus, the sterol inhibition of ruffling may be due to redistribution of plasmalemma rather than to changes in membrane viscosity. Together with previous observations, these data suggest that EGF‐stimulated ruffling and bulk‐phase pinocytosis are related phenomena, whereas EGF‐stimulated cell rounding

ISSN:0021-9541    

DOI:10.1002/jcp.1041420304

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe role of phospholipase (PLase) activation and lipid metabolism in lymphotoxin (LT)‐ and tumor necrosis factor (TNF)‐mediated destruction of murine L929 cells was examined. At the levels of LT and TNF employed, cell destruction began at 4–6 h and was 99% complete by 30 h. Cell membrane phospholipids (PL), labelled in situ at the C2 position with14C arachidonic acid, were analyzed by two‐dimensional thin‐layer chromatography and quantiated over a 30 h time course after LT or TNF treatment. The ratio of radiolabel incorporation relative to the actual amount of each PL present was determined by inorganic phosphate analysis. Radiolabelled arachidonic acid, eicosanoids, and neutral lipids were released into the medium prior to the onset of cell death (4–6 h) and continued to accumulate linearly throughout the destructive reaction. There was a quantitative relationship between the appearance of radiolabelled metabolites in the media and the loss of radiolabelled cellular PL. Cellular phosphatidylethanola‐mine was the primary PL deacylated by PLase action, showing a 75% reduction in radiolabel. The PLase inhibitors—quinacrine, hydrocortisone, dexamethasone, and indomethacin—were potent inhibitors of LT‐ and TNF‐mediated cell destruction, suggesting that selective deacylation of specific membrane PL by PLase activation is an important step in the events that lead to LT‐ and TNF‐mediated cell

ISSN:0021-9541    

DOI:10.1002/jcp.1041420305

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

年代:1990

数据来源: WILEY

摘要:

AbstractTreatment of M5076 tumor cells with the phorbol esters 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA) and phorbol 12, 13 dibutyrate (PdBu) inhibited cellular proliferation, whereas 1,2‐dioctanoyl‐glycerol (DiC8) and 1‐oleoyl2‐acetyl‐glycerol (OAG) did not affect cell growth. Inhibition of cellular proliferation in this cell line appears to be a consequence of protein kinase C (PKC) down‐regulation since phorbol esters, but not a single application of diacylglycerols (DGs) down‐regulated cellular PKC levels. By repeated application of DGs, PKC down‐regulation was achieved and correlated with inhibition of proliferation. Phorbol ester‐induced PKC down‐regulation was reversible, upon removal of the phorbol ester, and the reappearance of PKC was associated with resumption of proliferation. The mitogenic responsiveness of these cells to added serum depended upon cellular PKC levels. Phorbol esters also caused the phosphorylation of two proteins which were not phosphorylated in response to DG treatment. Inhibition of growth of M5076 cells appears to be associated with phosphorylation of two novel proteins

ISSN:0021-9541    

DOI:10.1002/jcp.1041420306

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

年代:1990

数据来源: WILEY

摘要:

AbstractNeovascularization, a common occurrence in chronic inflammatory lesions, requires endothelial cell (EC) proliferation. Because this form of inflammation is often mediated by immunologically generated cytokines, the effects of such cytokines on human umbilical vein EC proliferation in vitro were investigated. Low concentrations of recombinant interferon gamma (rIFN‐γ) (10—100 U/ml), but not a higher concentration (1,000 U/ml), enhanced both basal and endothelial cell growth factor (ECGF)‐stimulated EC proliferation. Recombinant interleukin 1 (rIL‐1) and recombinant tumor necrosis factor‐α (rTNF) had minor effects on basal EC proliferation, but significant inhibition was observed in the presence of ECGF. A combination of rIFN‐γ and rTNF induced marked suppression of EC proliferation, which appeared to be due to a cytotoxic effect on the EC, as demonstrated by51Cr release. In contrast, the combination of rIFN‐γ and rIL‐1 had only an additive effect on EC proliferation, with no evidence of cytotoxicity. These results suggest that cytokines have important regulatory roles in local vascular proliferation. These effects varied not only with the individual cytokine, but also with the combination of cytokines used. The most striking effects were (1) the stimulation of proliferation by IFN‐γ at a low concentration and (2) the inhibition by both rIL‐1 and rTNF of ECGF

ISSN:0021-9541    

DOI:10.1002/jcp.1041420307

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

年代:1990

数据来源: WILEY

摘要:

AbstractEpidermal G1‐chalone and transforming growth factor‐β (TGFβ), two endogenous inhibitors of epidermal cell proliferation, were compared with regard to several effects on epidermis in vivo and in vitro. Both factors inhibited DNA labeling in a rat tongue epithelial cell line, with similar kinetics and half‐maximal effects at approximately 1 pg/ml (enriched chalone) and 1 ng/ml (TGFβ). For primary neonatal mouse keratinocytes, (TGFβ) was found to be a rather strong inhibitor of cell proliferation, whereas chalone showed only a weak effect on cells grown in medium containing 1.2 mM Ca2+and no effect at all in the presence of 0.06 mM Ca2+. Vice versa, upon i.p. injection, only chalone was able to inhibit mouse epidermal DNA synthesis in vivo, whereas TGFβ had no effect at all. A moderate increase of transglutaminase activity in neonatal primary mouse keratinocytes was induced by both factors at concentrations of about 300 pg TGFβ/ml and 10 pg chalone fraction/ml. Chalone did not compete with [125]TGFβ for specific binding sites on primary murine keratinocytes. A polyclonal "chalone antiserum" did not interact with TGFβ, and a neutralizing TGFβ antibody that inhibited the effect of TGFβ on cell proliferation could not block the inhibitory effect of chalone on RTE2 cells. In contrast to TGFβ, epidermal G1‐chalone did not induce proliferation of NIH‐3T3 cells. These results indicate that epidermal G1‐chalone and TGFβ are two different inhibitors of epide

ISSN:0021-9541    

DOI:10.1002/jcp.1041420308

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

年代:1990

数据来源: WILEY

摘要:

AbstractEarly passage normal diploid Syrian hamster (SH) fetal cell cultures contain a transient subpopulation of contact‐insensitive (CS−) cells which lack density‐dependent inhibition of cell division. The size of this CS−subpopulation decreases during in vitro passage by conversion of the CS−cells to contact‐sensitive (CS+) cells. Approximately 10—15 population doublings after the frequency of the CS−cells has declined to below 0.001%, mass cultures cease proliferating and exhibit cellular senescence. Cultures with higher initial numbers of CS−cells exhibit longer in vitro proliferative life spans than cultures with smaller initial numbers of CS−cells. Active tumor promoting phorbol esters (12‐O‐tetra‐decanoyl‐phorbol‐13‐acetate [TPA] and phorbol‐12, 13‐didecanoate [PDD]) retard the decline in the proportion of CS−cells during in vitro passage, while the inactive tumor promoting phorbol ester, 4α‐phorbol‐12, 13‐didecanoate (4αPDD) has no effect on the rate of loss of the CS−cells. In addition, continuous treatment from secondary culture with TPA or PDD extends by approximately twofold the in vitro proliferative life span of SH fetal cell cultures. Treatment must, however, begin at passage 1 or 2 when the CS−cells are still present. After the proportion of the CS−cells has decreased to<0.001%, as in passage 6 cultures, promoters have no effect on the life span of the culture. This finding that exposure to promoters results in both a prolonged maintenance of the CS−cellular subpopulation, as well as an extention of in vitro proliferative life span, suggests that the conversion of CS−cells to CS+ce

ISSN:0021-9541    

DOI:10.1002/jcp.1041420309

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

年代:1990

数据来源: WILEY

摘要:

AbstractProstaglandin production by cultured human endothelial cells varies with growth conditions. We observed a marked diminution in both spontaneous and inducible production of prostacyclin (PGI2) by human umbilical vein and saphenous vein endothelial cells when they were cultured in the presence of the heparin‐binding growth factor, acidic fibroblast growth factor (aFGF) and heparin, compared with PGI2production during culture in medium lacking these factors. Decreased PGI2production was related to duration of exposure of the cells to aFGF and heparin and depended on the concentration of both substances. Heparin (1—100 μg/ml) strongly potentiated the effects of aFGF but had a limited and variable effect alone. The decrease in PGI2production correlated with a reduction in the cellular content of immunoreactive prostaglandin H synthase and prostacyclin synthase. Arachidonate deacylation was not decreased. In addition, the eicosanoid profile of endothelial cells was changed by exposure to aFGF and heparin. These studies indicate that heparin acts as a modulator of prostaglandin synthesis in endothelial cells through its interaction with aFGF, mediated by alterations in two key enzymes in the arachidonate metabolic pat

ISSN:0021-9541    

DOI:10.1002/jcp.1041420310

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

年代:1990

数据来源: WILEY

摘要:

AbstractTerminal deoxynucleotidyl transferase (TdT) is a template‐independent DNA polymerase that is transiently expressed during the normal development of T and B lymphocytes. Phorbol 12‐myristate 13‐acetate (PMA) has been reported to induce maturation‐like changes, including the loss of TdT, in many leukemic cell lines. We investigated the mechanism of TdT repression by PMA in an early thymocyte‐like cell line, RPMI 8402. At a concentration of 8 nM, PMA caused both repression of TdT synthesis and arrest of proliferation. At greater concentrations of PMA, these same changes initially occurred, but then cell proliferation resumed, and TdT was reexpressed. At both 8 and 160 nM PMA, TdT biosynthesis and TdT mRNA became undetectable within 8 hours, while cell proliferation and DNA synthesis were not significantly reduced until 16 hours. Growth arrest induced by serum starvation did not result in a similar reduction of TdT RNA even after 48 hours. With 160 nM PMA, TdT mRNA could be detected again by 24 hours, and proliferation resumed. Transcription run‐off assays indicated that TdT RNA synthesis ceased within 1 hour after exposure to both 8 and 160 nM PMA. T cell receptor α (TcR α) RNA was induced when TdT RNA was repressed. TcR β RNA levels were unchanged, and TcR γ RNA was up‐regulated. TdT gene repression and modulation of cell proliferation as well as induction of TcR gene expression are normal events during intrathymic T cell maturation. This cell model provides a system for analyzing the molecular regulation of these significant dev

ISSN:0021-9541    

DOI:10.1002/jcp.1041420311

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

年代:1990

数据来源: WILEY