摘要:

AbstractOur previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c‐AMP in prostaglandin mediated cell cycle arrest, we use the‐49 lymphoma variant (cyc−) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A1(dmPGA1) inhibits DNA synthesis and cell growth in cyc−cells. DNA synthesis is inhibited 42% by dmPGA1(50 μM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the α,β unsaturated ketone ring. Dimethyl PGA1is most effective in inhibiting DNA synthesis in cyc−cells, with prostaglandins PGE1and PGB1being less potent inhibitors of DNA synthesis. DmPGE2caused a significant stimulation of DNA synthesis. S‐49 cyc‐variant cells exposed to (30–50 μm) dmPGA1, arrested in the G1phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S‐49 cyc−cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G1, S, G2, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S‐phase DNA synthesis from the G1cell cycle block. The S‐49 cyc−cells are known to have a G1/S boundary through M phase transition time of 14.8 h, making the location of the prostaglandin cell cycle arrest at or very near the G1/S interface. The oncogenes,c‐fosandc‐mycwhich are normally expressed during G1in proliferating cells have a 2–3 fold enhanced expression in prostaglandin G1arrested cells. These data using the S‐49 variants demonstrate that dmPGA1inhibits DNA synthesis and arrests the cell cycle independent of cAMP‐mediated effects. The prostaglandin arrested cells maintain the gene expression of a G1synchronous cell which suggests a unique molecular mechanism for prostaglandin action in arresting cell growth. These properties indicate that this compound may be an effective tool to study molecular mec

ISSN:0730-2312    

DOI:10.1002/jcb.240540302

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

年代:1994

数据来源: WILEY

摘要:

AbstractWe have used differential hybridization to isolate and characterize two novel cDNAs expressed in chondrocytes and some osteoblastic cells. A rat osteosarcoma ROS17/2.8 cDNA library was screened and cDNA clones hybridizing strongly to radiolabeled porcine calvaria cDNA but weakly to a control radiolabeled cDNA were isolated. Two clones were obtained—p.6.1 and p.10.15. A radiolabeled probe of p10.15 was shown to hybridize specifically to a 2.3 Kb message RNA from a chondrogenic clonal cell population from rat calvaria‐RCJ 3.1C5.18, and the mRNA was downregulated by 1,25 (OH)2D3, which inhibits chondrogenesis in these cells. The other clone, p6.1, was found to hybridize to a 0.95 Kb message that is expressed in rat liver, kidney, lung, muscle, and brain, but not expressed in spleen and expressed only in low levels in thy

ISSN:0730-2312    

DOI:10.1002/jcb.240540303

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe level of c‐myctranscripts is increased in cells exposed to extremely low frequency (elf) electromagnetic (EM) fields at 60 Hz. The aim of the present experiments was to determine if regulatory regions upstream of the c‐mycgene modulate the response to EM fields. DNA upstream of P1 of both mouse and human c‐mycgenes was transfected into cells as CAT constructs. The presence of DNA 5′ to the human or mouse myc genes results in increased expression of CAT following 20 min exposures of cells to 60 Hz elf EM fields. Specific portions of the human upstream DNA were deleted and introduced into cells. The region responsive to EM fields is located between −353 and −1,257 relative to the

ISSN:0730-2312    

DOI:10.1002/jcb.240540304

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

年代:1994

数据来源: WILEY

摘要:

AbstractA number of studies suggest that an inverse correlation exists between the epidermal growth factor–receptor and the estrogen receptor expression in primary human breast carcinoma as well as in established human breast carcinoma cell lines. Recent studies suggest that the epidermal growth factor–receptor does not regulate the estrogen receptor gene expression. Whether the estrogen receptor regulates the epidermal growth factor–receptor gene expression is not known. We addressed this question by stably transfecting the estrogen receptor cDNA into the estrogen receptor–negative human breast carcinoma cell line MDA‐MB‐231. Constitutive expression of functional estrogen receptors in the transfactants resulted in increased mRNA levels of both epidermal growth factor–receptor and transforming growth factor α. Estradiol treatment of transfected cells, although enhancing transforming growth factor α mRNA levels, did not modulate epidermal growth factor–receptor mRNA levels. The estrogen receptor–transfected cells grown in estrogenic regular medium, however, exhibited lower constitutive levels of epidermal growth factor–receptor mRNA than in steroid‐stripped medium, suggesting that estrogens coupled with some factors normally present in the regular medium may indeed downmodulate epidermal growth factor–receptor mRNA. Sodium butyrate treatment enhanced epidermal growth factor–receptor mRNA levels in nontransfected cells grown in regular estrogenic as well as in steroid stripped medium. Sodium butyrate enhancement of epidermal growth factor–receptor mRNA levels was completely abolished in estrogen receptor–transfected cells grown in regular estrogenic medium and blunted in steroid stripped medium. Using various epidermal growth factor–receptor gene promoter‐CAT constructs in transient transfection assays, we further demonstrate that sodium butyrate enhanced transcription of the epidermal growth factor–receptor gene. The putative sodium butyrate responsive element(s) appears to localize within the proximal 384 bp of the epidermal growth factor–receptor gene promoter region. Although the interactions between estrogen receptor and epidermal growth factor–receptor are rather complex, taken together, our data suggest that estrogen receptor can indeed modulate the epiderm

ISSN:0730-2312    

DOI:10.1002/jcb.240540305

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

年代:1994

数据来源: WILEY

摘要:

AbstractRecombinant human apolipoprotein E3 (apoE), purified fromE. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose‐ and time‐dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin‐binding growth factors. Thus, at low serum concentrations (0–2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF‐dependent proliferation was dramatically inhibited by apoE with an IC50≈ 50 nM. Under conditions where cell proliferation was mainly serum‐dependent, apoE also suppressed growth but required higher concentrations to be effective (IC50≈ 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC50values obtained with these cells were generally 3–5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growt

ISSN:0730-2312    

DOI:10.1002/jcb.240540306

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

年代:1994

数据来源: WILEY

摘要:

AbstractA novel pathway for physiological “cross‐talk” between the insulin receptor and the regulatory Gi‐protein has been demonstrated. We tested the hypothesis that a coupling defect between Giand the insulin receptor is present in the liver of obese patients with and without type li diabetes. Insulin 1 × 10−9M (∼ ED50) and 1 × 10−7M (Max) inhibited pertussis toxin‐catalyzed ADP ribosylation of Giin human liver plasma membranes from lean and obese nondiabetic patients. However, 1 × 10−7M insulin was without effect in membranes from patients with type II diabetes. This coupling defect was not intrinsic to Gi, since Mg2+and GTPγS inhibited pertussis toxin‐catalyzed ADP ribosylation in both diabetic and nondiabetic patients. Binding of insulin of the α‐subunit and activation of the tyrosine kinase intrinsic to the β‐subunit of the insulin receptor are not responsible for the coupling defect.125I insulin binding is the same in obese patients with or without diabetes. Tyrosine kinase of the insulin receptor is decreased in diabetes. However, a monoclonal antibody to the insulin receptor (MA‐20) at equimolar concentrations with insulin equally inhibits pertussis toxin‐catalyzed ADP ribosylation of Giwithout activating tyrosine kinase or insulin receptor autophosphorylation. Immunodetection of G‐proteins suggested that Gi3αwas normal in diabetes and Gi1‐2αwas decreased by 40% in the diabetic group as compared to the obese nondiabetic group but was normal when compared to the lean non diabetic group. We conclude that the novel pathway of insulin signaling involving the regulatory Giproteins via biochemical mechanisms not directly involving the tyrosine kinase of the insulin receptor is altered in obese type II diabetes and offers a new target for the search of

ISSN:0730-2312    

DOI:10.1002/jcb.240540307

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

年代:1994

数据来源: WILEY

摘要:

AbstractWe have previously characterized the stimulation of HeLa cell surface peptidase activity directed toward a nonapeptide substrate in response to low fluences of ultraviolet irradiation [Brown et al. (1993): J Cell Biochem 51:102–115]. To explore the hypothesis that this comprised a global response to cell stress featuring the interruption of DNA synthesis, a variety of agents affecting macromolecular synthesis were applied to HeLa cell cultures. Actinomycin D, 5,6‐dichloro‐1 β‐ribofuranosyl benzimadazole, mitomycin C, ultraviolet light, and cycloheximide at doses which inhibited cell growth, but fell short of increasing the proportion of cells which had lost cell membrane impermeability to trypan blue, resulted in the concentration dependent increase in both amino‐ and endo‐peptidase activities of intact HeLa cell cultures. γ‐Irradiation, despite inhibiting an increase in cell number over a 20‐h observation period, had no effect on the expressed level of cell surface peptidase activity nor did the accumulation of cells in S or G2phase by thymidine parasynchronization. Some of these agents were found to increase the proportion of cells in the culture undergoing apoptosis (programmed cell death), and a strong correlation was found between the extent of apoptosis and the degree of elevation in cell surface peptidase activity. Higher concentrations of perturbants in some instances increased the percentage of cells that were nonviable and an associated release of intracellular proteases overwhelmed the linear correlation with apoptotic cells. The present data do not distinguish between a homogeneous elevation of surface peptidase activity in all cells of treated cultures or the heterogeneous increase in only preapoptotic or apoptotic cells. Since sunburn of the skin increases both the occurrence of apoptotic keratinocytes (sunburn cells) in the affected epidermis and the release of membrane bound cell activators such as transforming growth factor α, it is suggested by way of extrapolation of these in vitro results, that the increase in cell surface proteolytic activity plays an integral part in the reparative responses of the epide

ISSN:0730-2312    

DOI:10.1002/jcb.240540308

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

年代:1994

数据来源: WILEY

摘要:

AbstractTamoxifen has been an effective antiestrogen in suppressing breast cancer growth which is estrogen‐responsive or dependent. Early studies have provided circumstantial evidence that transforming growth factor‐β (TGF‐β) may be an autocrine mediator of tamoxifen action. Therefore, it is both fundamentally important and clinically relevant to investigate the relationship between tamoxifen and TGF‐β. In this study, we demonstrated that CAMA‐1 cells, which are sensitive to tamoxifen inhibition, did not respond to TGF‐β growth inhibition. The type I and II TGF‐β receptors were undetectable by the radio‐ligand affinity labeling technique. Despite the presence of a normal TGF‐β type II receptor gene, the mRNA transcript of the gene was undetectable by the extremely sensitive Intron‐differential RNA/PCR method. The possibility that the lack of TGF‐β receptors might be intimately linked to the absence of normal retinoblastoma (Rb) gene products, as suggested by previous studies of retinoblastoma cells, was further investigated. The lack of TGF‐β receptor expression was found due to reasons other than the absence, deletion or abnormality of the Rb gene because a normal Rb gene and its hyper‐ and hypo‐phosphorylated protein products were detected in CAMA‐1 cells. In conclusion, our results suggest that the TGF‐β system is not obligatory for anties

ISSN:0730-2312    

DOI:10.1002/jcb.240540309

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

年代:1994

数据来源: WILEY

摘要:

AbstractHuman C‐reactive protein (CRP) is an acute phase blood component that accumulates at sites of tissue damage and necrosis and is degraded by neutrophils to biologically active peptides. A dodecapeptide composed of amino acids 27–38 of CRP mediates cell attachment in vitro. This peptide was designated the cell‐binding peptide (CB‐Pep) of CRP. Characterization of the interaction between fibroblasts and modified synthetic peptides with sequential deletions from either the N‐terminus or C‐terminus revealed that the minimal sequence for cell attachment or inhibition of cell attachment to the CB‐Pep wasPhe‐Thr‐Val‐Cys‐Leu, which corresponds to residues 33–37 within each of the five 206 amino acid subunits of CRP. The pentapeptide by itself mediated cell attachment. Substitutions for each residue within the CB‐Pep indicated that the critical residues for activity were Phe‐33 and Thr‐34. This cell‐binding pentapeptide represents a recognition motif for cell adhes

ISSN:0730-2312    

DOI:10.1002/jcb.240540310

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

年代:1994

数据来源: WILEY

摘要:

AbstractMitochondrial response to the effect of a hydroxylase (PH) inhibitor was tested in marrow stromal cells during stimulation of osteoprogenitor cell (OPC) differentiation. The rationale for this was to explore pathways of regulatory interactions between procollagen synthesis and mitochondrial respiration that could be linked to the commitment of OPCs to mineralization. Stimulated OPCs exposed to the PH inhibitor cis‐hydroxyproline (cis‐HP), compared to the noninhibiting isomer trans‐HP, for 11 days showed a dose‐dependent decrease in cell proliferation, the surviving cells showed increased alkaline phosphatase activity. Trans‐HP did not influence the cis‐HP effect on ALP and on proliferation arrest. Short time exposures, 2–3 days, to cis‐HP at different periods suggested that Days 0–3 and 3–5 were critical for the commitment to Day 21 mineralization of OPCs. On Days 0–3 cells were most sensitive to cis‐HP, since on Day 11, 8 days after removal of cis‐HP, they were too scarce to be counted by the staining method. However, the presence of 5.0 mM cis‐HP in the cultures during Days 3–5 has induced on Day 21 close to 24‐fold more mineralization/cell than controls, compared to the trans‐HP effect, which was only close to 3‐fold. The presence of cis‐HP in the cultures on Days 0–3 has augmented the mitochondrial Day 3 retention of rhodamine 123 (Rho) in the stromal cells, relative to controls, compared to the presence of trans‐HP. However, the presence of cis‐HP during Days 3–5 or 3–6 resulted in lower Day 5 Rho retention, relative to controls, which was not significantly different from the retention that resulted from trans‐HP. Since Rho retention is related to the final result of aerobic respiration level, these results are interpreted as a cis‐HP inhibitory effect on procollagen peptidyl‐proline hydroxylation, which may in turn release oxygen surpluses, to be available for mitochondrial consumption. The fall in Rho retention responses to cis‐HP between Days 0–3 and 3–5 is suggesting either abrupt decrease in proline hydroxylation or p

ISSN:0730-2312    

DOI:10.1002/jcb.240540311

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

年代:1994

数据来源: WILEY