摘要:

AbstractThe islated osteoclast bone resorption assay has proved to be a useful means of examining the response of mammalian and avian osteoclasts to a variety of stimuli. The assay has traditionally been performed over a period of 24 hours. By extending the duration of the osteoclast bone resorption assay, we have been able to assess the long‐term effects of carboxyl‐terminal parathyroid hormone‐related protein (hPTHrP[107‐139]), salmon calcitonin (sCT) and hPTH[1‐34]on bone resorption and TRACP‐positive osteoclast‐like cell number. We found that, in control cultures over a period of up to 144 hours, the osteoclast‐like cells not only remained viable but their numbers also increased. The number of mononucleated and multinucleated osteoclast‐like cells doubled in the first 48 hours before stabilizing over the remainder of the incubation period. Osteoblasts also proliferated, resulting in a resorption response to hPTH [1‐34] being evident from 48 hours onward. hPTHrP[107‐139]persistently inhibited basal and PTH‐stimulated bone resorption for at least 96–144 hours. Where as “escape” from the inhibijtory effect of sCT was seen after 48–72 hours. Decreased numbers of bothe mononucleated and multinucleated TRACP‐positive osteoclast‐like cells were seen by 48 hours in cultures treated with sCT. In contrast, hPTHrP[107‐139] reduced the number of mononuclear TRACP‐positive cells with only a late effect on multinucleated cells. Furthermore, the incsreased number of osteoclast‐like cells seen in response to hPTH[1‐34]was inhibited by carboxyl‐terminal PTHrP. In summary, this study indicates that the extended bone resorption assay system is a complex one where bothe osteoclastic resorption and osteoclast maturation are evident. Using this syste, we have shown that hPTHrP[107‐139] acts as a potent long‐term inhibitor of osteoclastic bone resorption, without evidence of escape from its effect. Its action to reduce the number of mononucleated osteoclst‐like cells suggests that it affects several asp

ISSN:0021-9541    

DOI:10.1002/jcp.1041550102

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

年代:1993

数据来源: WILEY

摘要:

AbstractThe contraction of collagen lattices made with forskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracelluar cyclic AMP raising drugs including cholera toxin (CT), forskolin, and dibutyryl‐cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor‐β (TGF‐β). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin‐induced intracellular cyclic AMP increase was attenuated by TGF‐β, but not by aFGF and insulin, when added simultaneously. Thus, TGF‐β may attenuate CT's inhibition on collagen lattice contraction by attenuating CTinduced intracellualr cyclic AMP increse, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMPindependent mechannism. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041550103

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

年代:1993

数据来源: WILEY

摘要:

AbstractIn the present study, we have examined the regulation of attachment, onset of proliferation and the subsequent growth, in vitro, of chick retinal pigmented epithelial (RPE) cells as a function of the nature of the substratum and of either the activation or inhibition of protein kinase C (PKC). The RPE cells have an adhesive preference for protein carpets which contain laminin. This preference disappears gradually with time in culture. The adhesion of RPE cells to fibronectin is shown to be a receptor‐mediated process which involves the RGD recognition signal. This study also demonstrates that a PKC activator, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), affects RPE cell adhesion in a substratum‐dependent manner. Exposure of RPE cells to TPA lowers the cell attachment efficacy to ECM protein substrata but does not affect cell attachment to plastic. The onset of cell proliferation is accelerated by TPA on all of the substrata tested. The minimal duration of an effective TPA pulse exerting a long‐lasting influence on RPE cell proliferation is between 1.5 and 3.5 hr. Stimulation of cell proliferation by TPA in long‐term cultures is independent of the nature of the growth substratum. The acceleration of the onset of cell proliferation by TPA is sensitive to 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7), an inhibitor of conventional PKC, and thus appears to be dependent on the activation of conventional PKC. H7 also affects cell‐cell contacts, causing an alteration in the shape (“squaring”) of RPE cells packed into large colonies. Conversely, the effects of TPA on both the attachment and the long‐term proliferation of RPE cells are not dependent a conventional PKC isotype, since H7 cannot abolish the influence of TPA on either process. We conclude that the effect of TPA on long‐term proliferation of RPE cells is either dependent on a novel PKC isotype or indepen

ISSN:0021-9541    

DOI:10.1002/jcp.1041550104

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

年代:1993

数据来源: WILEY

摘要:

AbstractIn this study, we investigated the mechanism of synergistic effects of cytokine and hyperthermia on cytotoxicity in HT‐29. When cells were heated at 42°C in the presence of recombinant human tumor necrosis factor (rhTNF‐α), recombinant interferon‐gamma (rhIFN‐γ), or in a combination of both, a synergistic increase in the cytotoxic effects of the respective drugs was observed. We hypothesized that alteration of cytokine or heat‐induced polypeptides synthesis was responsible for a synergistic interaction between heat and cytokine.Five heat shock proteins (HSPs, Mr110,000, 100,000, 90,000, 70,000, and 28,000) were preferentially synthesized during chronic heating at 42°C. In contrast, the synthesis of two proteins (Mr60,000 and 29,000) was induced by treatment with rhIFN‐γ (1,000 U/ml). Although the combination of chronic hyperthermia (42°C) with TNF‐α, IFN‐γ, or TNF‐α + IFN‐γ increased cytotoxicity, alteration/induction of polypeptides was not correlated with the synergistical cytotoxic effects of cytokine and heat. Thus, the synergistic effects of cytokine and hyperthermia are not mediated through an induction of polypep

ISSN:0021-9541    

DOI:10.1002/jcp.1041550105

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

年代:1993

数据来源: WILEY

摘要:

AbstractWe studied the effects of parathyroid hormone (PTH) on two markers of the osteoblast phenotype: alkaline phosphatase (AP) (activity and mRNA) and cyclic adenosine monophosphate (cAMP) accumulation. Osteoblast‐like cells derived from fetal rat (ROB) and mouse (MOB) calvariae were isolated by collagenase treatment. Cells were cultured in α‐Minimal Essential Medium (MEM) with 2% fetal calf serum (FCS) for 4 days. In ROB and MOB bPTH(1‐34) induced a fast increase (up to 5 minutes) in cAMP accumulation. When equal amounts of cells were seeded, the cAMP accumulation was higher in MOB than in ROB. No difference in basal AP activity was observed between ROB and MOB. When bPTH(1‐34) was added to ROB for the last 24 or 48 hr, AP activity decreased dose dependently. However, MOB treated with bPTH(1‐34) for the last 24 or 48 hours showed an increase of AP activity. Basal AP activity was positively correlated with the seeding density of ROB and MOB cultures. Basal AP activity influenced the degree of inhibition (ROB) or stimulation (MOB) after incubation with bPTH(1‐34). © 1993 Wi

ISSN:0021-9541    

DOI:10.1002/jcp.1041550106

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

年代:1993

数据来源: WILEY

摘要:

AbstractThis study examines the effects of extracellular Ca2+concentrations, [Ca2+]o, and of treatments known to modulate intracellular Ca2+levels on the extent and timing of DNA synthesis in primary cultures of adult rat hepatocytes. In cultures exposed to insulin and EGF, the extent of DNA synthesis between 40 h and 70 h in culture was independent of [Ca2+]oin the range 25–1,800 μM, although the peak of DNA synthesis occurred 5–10 h earlier with 1.2 mM Ca2+than with 25 μM Ca2+. Complete removal of extracellular Ca2+using EGTA blocked DNA synthesis if Ca2+was removed on the second day after EGF addition but not if Ca2+was absent only on day 1. Treatment of cultures in 1.2 mM Ca2+‐containing media with Ca2+‐ionophore A23187 or with thapsigargin, agents expected to raise cytosolic [Ca2+], failed to augment the stimulation of DNA synthesis by EGF. These observations suggest that hepatocytes may have a permissive requirement for [Ca2+]o>0 at least late in the sequence of events leading from growth factor stimulation to DNA synthesis. However, sustained elevation of cytosolic [Ca2+] does not appear to be important as an early signalling event either in mediating or augmenting EGF action in hepatocytes. The ability of liver tumor promoters α‐hexachlorocyclohexane or DDT to stimulate DNA synthesis in combination with EGF was independent of [Ca2+]o. By contrast, the skin tumor‐promoting phorbol ester, TPA, or liver tumor promoter, phenobarbital, were without effect or inhibitory at low [Ca2+]obut in combination with EGF, stimulated DNA synthesis at [Ca2+]o>0.4 mM, suggesting that Ca2+may have some role in mediating or modulating the stimulatory effects of these agents. © 1993

ISSN:0021-9541    

DOI:10.1002/jcp.1041550107

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

年代:1993

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐β1 (TGFβ) is a regulator of protein synthesis in cultured chicken embryo cells (CEC). Preceding a gradual increase in overall protein synthesis, members of the Hsp70 family (Hsp70, Hsc70, and Grp78) and the Hsp90 family (90‐2 and 90‐3) of molecular chaperones are induced rapidly and represent a new class of TGFβ‐inducible proteins (I.M. Takenaka and L.E. Hightower, J. Cell. Physiol.,152:568–577, 1992). Herein,32P‐labeled cDNA probes encoding Hsc70 and Hsp90 were used to show that levels of the corresponding mRNAs increased as a fraction of total RNA and in polysomes within five hours of treatment of CEC with TGFβ. This cytokine did not increase rates ofhsc70andhsp90gene transcription as measured by run‐on transcription assays of isolated nuclei. However, the Hsp RNA inductions were inhibited by dactinomycin, indicating a requirement for newly synthesized RNA. Both Hsc70 and Hsp90 mRNAs had relatively short half‐lives, measured by Northern blot analyses of dactinomycin chases, which were not altered substantially in TGFβ‐treated cells. In contrast, Hsp mRNA half‐lives increased in heat shocked CEC exposed to dactinomycin during recovery, revealing a difference in regulation of these genes in stressed cells compared with TGFβ‐treated cells. Our results support the conclusion thathsc70andhsp90gene expression is regulated posttranscriptionally in TGFβ‐treated CEC, and the mechanism likely involves a nuclear event such as increasing the half‐lives of nuclear RNA transcripts, processing, or transport into the c

ISSN:0021-9541    

DOI:10.1002/jcp.1041550108

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

年代:1993

数据来源: WILEY

摘要:

AbstractRepetitive mechanical stimulation of cultured avian skeletal muscle increases the synthesis of prostaglandins (PG) E2and F2αwhich regulate protein turnover rates and muscle cell growth. These stretch‐induced PG increases are reduced in low extracellular calcium medium and by specific phospholipase inhibitors. Mechanical stimulation increases the breakdown rate of3H‐arachidonic acid labelled phospholipids, releasing free3H‐arachidonic acid, the rate‐limiting precursor of PG synthesis. Mechanical stimulation also increases3H‐arachidonic acid labelled diacylglycerol formation and intracellular levels of inositol phosphates from myo‐[2‐3H]inositol labelled phospholipids. Phospholipase A2(PLA2), phosphatidylinositol‐specific phospholipase C (PLC), and phospholipase D (PLD) are all activated by stretch. The stretch‐induced increases in PG production,3H‐arachidonic acid labelled phospholipid breakdown, and3H‐arachidonic acid labelled diacylglycerol formation occur independently of cellular electrical activity (tetrodotoxin insensitve) whereas the formation of inositol phosphates from myo‐[2‐3H]inositol labelled phospholipids is dependent on cellular electrical activity. These results indicate that mechanical stimulation increases the lipid‐related second messengers arachidonic acid, diacylglycerol, and PG through activation of specific phospholipases such as PLA2and PLD, but not by activation of phosphatidylinositol‐specif

ISSN:0021-9541    

DOI:10.1002/jcp.1041550109

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

年代:1993

数据来源: WILEY

摘要:

AbstractEstrogen (E), progesterone (P), and epidermal growth factor (EGF) are involved in the growth and development of the normal mammary gland. While studies have been carried out to investigate the in vivo effects of EGF in the immature mammary gland, nothing is known about the growth effects of EGF or its potential interactions with E and/or P in the adult mammary gland. The present studies were undertaken to investigate the effects of EGF, E, and P on mammary cell proliferation in immature, peripubertal vs. adult, sexually mature mice. We have found that EGF promotes epithelial and stromal cell proliferation in both the immature and adult mammary glands. In the immature gland, the end bud epithelium is most responsive to the proliferative effects of EGF and there is no apparent interaction between EGF, E, and/or P. In contrast, in the mature gland EGF adds to the proliferative effects of E+P in the ductal epithelium resulting in more extensive ductal sidebranching. Thus these results demonstrate that the developmental state of the mammary gland determines the nature and extent of the interactions between EGF, E, and P in growth and development. © 1993 Wiley‐Liss, I

ISSN:0021-9541    

DOI:10.1002/jcp.1041550110

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

年代:1993

数据来源: WILEY

摘要:

AbstractCell density negative control (CDNC) of normal human fibroblast proliferation occurs after stimulation by mitogens with different signal transduction mechanism. Delayed exposure to agents that interfere with CDNC, such as doublestranded RNA and vanadate, reveals the existence of a biochemical event, involved in CDNC, that occurs 5–8 hr after the beginning of mitogenic stimulation. This is earlier than the point of “mitogenic commitment,” defined by the duration of mitogen exposure required for cell cycle entry (8–18 hr). Phosphorylation of the retinoblastoma gene product (pRB) begins 8–10 hr after mitogen stimulation and is nearly complete at 18 hr, just as the first cells enter S‐phase. CDNC prevents pRB phosphorylation. Interferon β delays pRB phosphorylation by up to 20 hr but has little effect on the timing of mitogenic commitment. Thus mitogenic commitment is located in time between CDNC and pRB phosphorylation. When agents that cause a release from CDNC are applied to dense, negatively controlled cultures after 18 hr of EGF stimulation, pRB phosporylation occurs 6–8 hr after release. This suggests that the negatively controlled cells process the mitogenic signal but accumulate at a restriction point. The relatively early timing of CDNC‐related events in the prereplicative phase raises the possibility that pRB phosphorylation is a consequence rather than a prerequisite for release from cell density negative control. © 199

ISSN:0021-9541    

DOI:10.1002/jcp.1041550111

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

年代:1993

数据来源: WILEY