摘要:

AbstractAnti‐p185HER2monoclonal antibodies often show intense reactivity with the basement membrane of tumor cells that overexpress the HER2/neu gene product (p185HER2). To evaluate a possible interaction between p185HER2and adhesion molecules or their receptors, the polarity of p185HER2was tested in lung carcinoma cell line Calu‐3, which overexpresses this protein, in cultures grown as confluent monolayers or as aggregates. MAb immunostaining patterns indicated that p185HER2is concentrated on the baso‐lateral membrane of cells and that it colocalizes with the integrin α6β4 at the cell‐cell junctions where laminin is also found. The same membrane region showed intense reactivity with antiphosphotyrosine antibodies. Furthermore, integrin clustering induced by the specific antibody was accompanied by the clustering of p185HER2, as indicated by immunoelectron microscopy, and by a subsequent increase in p185HER2tyrosine phosphorylation. Treatment with exogenous laminin also resulted in increased basal levels of p185HER2phosphorylation. These data suggest a physical interaction between the integrin and the oncoprotein that might be functionally relevant in directly controlling the tyrosine phosphorylation of the catalytic domain of p185HER2. © 1994 Wiley

ISSN:0730-2312    

DOI:10.1002/jcb.240550402

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

年代:1994

数据来源: WILEY

摘要:

AbstractA tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti‐proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)‐plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U‐24067 and U‐42129), heparin, suramin, interferon alpha‐2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen‐dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha‐2a reduce urokinase‐type PA (u‐PA) and PA inhibitor‐1 activity, while heparin and retinoic acid increase u‐PA activity. Suramin reduces cell‐associated u‐PA activity and greatly increases PAI‐1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti‐angiogenesis, and that anti‐angiogenesis and anti‐proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti‐angiogenesis is likely to be varied

ISSN:0730-2312    

DOI:10.1002/jcb.240550403

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe calcium‐regulating hormone 1,25‐dihydroxyvitamin D3[1,25(OH)2D3] is recognized as an immuno‐modulator affecting the activities of macrophages and lymphocytes. We have shown that macrophages harvested from vitamin D–deficient mice (–D MPs) exhibit impaired phagocytic and tumoricidal activities as compared with control cells (+D MPs), and that bone marrow–derived macrophage (BMDM) differentiation is modulated by 1,25(OH)2D3. The release of tumor necrosis factor–α (TNF‐α) by macrophages is considered a major mechanism by which these cells exert their tumoricidal function. This cytokine was also implicated in modulation of bone resorption. In the present study we examine the role of 1,25(OH)2D3in TNF‐α synthesis and release. BMDMs were harvested from +D and −D mice, cultured in vitro, and their conditioned media were analyzed for the presence of TNF‐α. BMDMs did not release measurable amounts of TNF‐α without stimulation. Addition of endotoxin (LPS) to the cultures resulted in a marked stimulation of TNF‐α release. 1,25(OH)2D3increased the stimulatory action of LPS, but failed to elicit a stimulatory effect in the absence of LPS. The use of another macrophage activator, interferon‐γ (IFN‐γ), yielded essentially similar results. +D and −D mice were injected with LPS and TNF‐α levels in the serum were measured. A marked reduction (∼ fourfold) in the TNF‐α levels was observed in the serum of −D mice as compared with +D mice. Western blot and immunoprecipitation analyses suggested that the main effect of 1,25(OH)2D3is on TNF‐α synthesis. Our findings suggest that 1,25(OH)2D3plays a role in the regulation of TNF‐α secr

ISSN:0730-2312    

DOI:10.1002/jcb.240550404

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

年代:1994

数据来源: WILEY

摘要:

AbstractHepatocyte growth factor (HGF) and epidermal growth factor (EGF) are major hepatacyte mitogens, but HGF, also known as scatter factor (SF), has also been shown as a potent motogen for epithelial and endothelial cells. The mechanisms by which HGF is a stronger motogen compared to other mitogens are not understood. Here we report a comparative study of the effect of the two growth factors on cultured primary rat hepatocytes regarding their differential effects on morphology, mitogenicity, and motility as well as the phosphorylation of cytoskeletal‐associated proteins. Using three different motility assays, both HGF and EGF increased the motility of hepatocytes, but HGF consistently elicited a significantly greater motility response than EGF. Additionally, HGF induced a more flattened, highly spread morphology compared to EGF. To examine if HGF and EGF phosphorylated different cytoskeletal elements as signal transduction targets in view of the observed variation in morphology and motility, primary cultures of32P‐loaded rat hepatocytes were stimulated by either HGF or EGF for up to 60 min. Both mitogens rapidly stimulated four isoforms of MAP kinase with similar kinetics and also rapidly facilitated the phosphorylation of cytoskeletal‐associated F‐actin. Two cytoskeletal‐associated proteins, however, were observed to undergo rapid phosphorylation by HGF and not EGF during the time points described. One protein of 28 kDa was observed to become phosphorylated fivefold over controls, while the EGF‐stimulated cells showed only a slight increase in the phosphorylation of this protein. Another protein with an apparent mwt of 42 kDa was phosphorylated 20‐fold at 1 min and remained phosphorylated over 50‐fold over control up to the 60 min time point. This protein was observed to become phosphorylated by EGF only after 10 min, and to a lesser extent (20‐fold). Taken together, the data suggest that HGF and EGF stimulate divergent as well as redundant signal transduction pathways in the hepatocyte cytoskeleton, and this may result in unique HGF‐ or EGF‐specific motility, morphology, and mitogenicity in hepatocytes.

ISSN:0730-2312    

DOI:10.1002/jcb.240550405

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

年代:1994

数据来源: WILEY

摘要:

AbstractInduction of mitosis inXenopus laevisoocytes by hormones and the oncogenicras‐p21 protein has been shown to correlate with a cascade of phosphorylations of the Ser/Thr family of kinases. However, the exact hierarchy of enzymes and their mutual interdependency has not been fully elucidated yet. We have used theXenopus laevissystem to investigate the mechanism of activation of the Ser/Thr kinases cascade and their relationship. Comparison between progesterone‐induced germinal vesicle breakdown (GVBD), a hallmark of mitosis in oocytes, to that triggered byras‐p21, revealed the existence of at least two independent mechanisms to activate the MAP kinase enzyme in vivo. While progesterone function is dependent of cdc2 protein kinase activity,ras‐p21 is independent of this enzyme. However, both progesterone andras‐p21 converge at the MAP kinase level, and depletion of MAP kinase activity inhibits the GVBD and S6 kinase II activation induced by both progesterone andras‐p21. These results provides further evidence that MAP kinase is a critical step for regulation of the cell cycle in oocytes and a critical point whererasand progesterone signaling converge. © 1994 Wil

ISSN:0730-2312    

DOI:10.1002/jcb.240550406

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe production of the second messenger molecules diacylglycerol and inositol 1,4,5‐trisphosphate is mediated by activated phosphatidylinositol‐specific phospholipase C (PLC) enzymes. We report the enhancement of the phosphoinositide metabolism pathway in KMS‐4 and KMS‐8 cells, both of which are human colorectal carcinoma cell lines derived from familial adenomatous polyposis patients. In these cells, the cellular contents of diacylglycerol and inositol 1,4,5‐trisphosphate were constitutively increased and the PLC activity in vitro was significantly high, as compared with those in normal colon cells or in other sporadic colorectal carcinoma cells. Northern and Western analyses showed the high expression levels of both PLC‐γ1 and PLC‐δ1 in KMS‐4 and KMS‐8 cells. Moreover, we detected the enhancement of protein–tyrosine kinase activity and tyrosine phosphorylation of PLC‐γ1 in these KMS cells. These results suggest the involvement of activated phosphoinositide signaling pathways in the colorectal tumorigenesis of familial adenomatous polyposis

ISSN:0730-2312    

DOI:10.1002/jcb.240550407

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe presence and inducibility of the major cadmium (Cd) chelating protein metallothionein (MT) in testicular cells has been controversial. In this study, the induction and production of MT in testicular cells were studied using mouse Leydig and Sertoli cell lines. Metal accumulation was studied by subjecting the cells to increasing levels of Cd. The presence of transcription factors for MT synthesis was analyzed by transfecting the cells with a reporter gene under the control of the MT promoter. The dose‐ and time‐dependent induction of MT were conducted by Northern analyses. Expression of MT genes occurred in both Leydig and Sertoli cells. To avoid cross hybridization of the MT probe with mRNAs encoding testicular metal binding proteins and to investigate the integrity of MT mRNA, isoMT mRNA identification and primer extension experiments were performed. Those studies show that the induced mRNA indeed encodes MT. The biosynthesis of MT was confirmed by following35S‐cysteine incorporation into the protein. Finally, cadmium tolerance of testicular cells is compared with that of fibroblast cells. By these studies, we conclude that the MT genes are functional and inducible in testicular cells. © 1994 Wiley‐L

ISSN:0730-2312    

DOI:10.1002/jcb.240550408

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe indol alkaloid staurosporine is a potent inhibitor of protein kinase C, but has also been shown to have certain effects paradoxically similar to those of protein kinase C–activating phorbol esters. We show here that collagenase mRNA expression is stimulated by 10 nM staurosporine in normal andras‐oncogene–transformed rat fibroblasts. The kinetics of collagenase mRNA induction by staurosporine were slow compared to induction by phorbol ester. Staurosporine induction of the collagenase promoter appeared to be mediated via the TPA response element (TRE). Induction did not involve any increase injunmRNA expression and did not require expression of c‐Jun. Prolonged treatment with phorbol ester to deplete protein kinase C did not inhibit stimulation of the collagenase promoter by staurosporine. Instead, involvement of cAMP‐dependent protein kinase (PKA) was indicated by inhibition of staurosporine induction by the PKA inhibitor H‐89. In addition, raised levels of cAMP were observed during the first hour of staurosporine treatment. Altogether, our data indicate that staurosporine induces a PKA‐dependent pathway leading to c‐Jun–independent activation of the collagenase TRE element. © 19

ISSN:0730-2312    

DOI:10.1002/jcb.240550409

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe proto‐oncogenes c‐fos and c‐jun have been shown in numerous model systems to be induced within minutes of growth factor stimulation, during the G0/G1transition. In this report we use the mitotic shake‐off procedure to generate a population of highly synchronized Swiss 3T3 cells. We show that both of these immediate‐early, competence genes are also induced during the M/G1transition, immediately after completion of mitosis. While c‐fos mRNA levels drop to undetectable levels within 2 hr after division, c‐jun mRNA levels are maintained at a basal level which is ∼ 30% maximum throughout the remainder of G1. In order to access the functional significance of these patterns of c‐fos and c‐jun expression, antisense oligodeoxynucleotides specific to c‐fos or c‐jun were added to either actively growing Swiss 3T3 cells or mitotically synchronized cells, and their ability to inhibit DNA synthesis and cell division determined. Our results show that treatment of Swiss 3T3 cells with either c‐fos or c‐jun antisense oligodeoxynucleotides, while actively growing, during mitosis, or in early G1, results in a reduction in ability to enter S and subsequently divide. This was also true if Swiss 3T3 cells were treated during mid‐G1with c‐jun antisense oligodeoxynucleotides. These results demonstrate that the regulation of G1progression following mitosis is dependent upon the expression and function of the immediate‐early, competence proto‐oncogenes c‐

ISSN:0730-2312    

DOI:10.1002/jcb.240550410

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

年代:1994

数据来源: WILEY

摘要:

AbstractNuclear matrix is a nuclear protein–DNA superstructure believed to be the exclusive site of DNA replication, transcription, repair, and recombination. The attachment regions of chromatin loops to the nuclear matrix, called MARs, nest origins of replication, have transcriptional enhancer activity, and via their interaction with protein transcription factors may govern gene switch during development and tissue‐specific gene expression. In this study the 967 bp MAR of the chicken α‐globin gene is analyzed for the presence of hexanucleotides from a number (83 in total) of vertebrate protein transcription factors and core origins of replication. A total number of 760 hexanucleotides from factor sites or origins of replication were used for this search. We found that: (1) The occurrence of protein transcription factor binding sites overall on the MAR fragment as well as on the enhancer and promoter regions of other genes is only about 1.2–1.5 times higher than in random DNA, something consistent for all MAR and enhancer sequences examined. However, a high concentration (up to 2.7 times over random sequences) of hexanucleotide factor sites is observed on small stretches of the α‐globin gene MAR. (2) Some regulatory protein binding sites are underrepressented whereas others are overrepresented, giving to an MAR a particular transcription factor flavor. (3) The DNA curvature map of the MAR sequence and the potential sites of positioned nucleosomes suggest the sites where a competition between core histone octamers and protein transcription factors for DNA might be found. This approach might provide a novel technique to diagnose for the regulatory or nonregulatory function of a stretch of DNA. Furthermore, MARs are proposed to constitute important regulatory elements of genes in addition to enhancers, promoters, silencers, locus control regions, and origins of replication. Additional parameters such as interaction of a transcription factor with other transcription factors fixed at vicinal sites, DNA methylation, intrinsic DNA curvature torsional strain, and nucleosome positioning might also determine the high‐affinity binding of a transcription factor to its functional sites and its exclusion from or low affinity binding to other nonregulatory regions. © 1994 W

ISSN:0730-2312    

DOI:10.1002/jcb.240550411

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

年代:1994

数据来源: WILEY