bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of differentiation and keratin protein expression by vitamin A in primary cultures of hamster tracheal epithelial cells
Authors: Edmondson Susan W. , Wu Reen , Mossman Brooke T. 
Published: February 2005

AbstractHamster tracheal epithelial (HTE) cells maintained in primary culture show the induction of specific keratin species under vitamin A‐deficient conditions. A comparison was made between the morphology and the expression of keratins in HTE cells in vivo and in primary culture with and without vitamin A. HTE cells cultured in serum‐free, vitamin A‐supplemented medium formed a simple cuboidal, ciliated monolayer and produced four simple epithelial keratins (7, 8,18, and 19). In contrast, vitamin A‐deficient HTE cells, which were squamous‐like and stratified in culture, produced a more complex keratin pattern, with the induction of four additional keratin species (5,6, 14, and 17). A keratin pair whose expression serves as a marker of stratified epithelia was induced, as well as a single keratin species unique to lesions of squamous metaplasia in vitamin A‐deficient hamster tracheal organ cultures. Thus it appears that HTE cells retain the ability to respond to a deficiency in vitamin A through squamous differentiation and increased keratin production when removed from the intact organ and maintained in primary culture in a chemically defined medium. This system may be useful for the study of mechanisms underlying the squamous differentiation of respiratory epithelial cells in the development of bronchogenic tumors.
Distinct pathways regulate transforming growth factor β1‐stimulated proto‐oncogene and extracellular matrix gene expression
Authors: Howe Philip H. , Cunningham Muriel R. , Leof Edward B. 
Published: February 2005

AbstractThe effect of pertussis toxin (PT) on transforming growth factor β1 (TGFβl)‐induced proto‐oncogene expression was investigated in AKR‐2B fibroblasts. PT substantially abolished c‐sis and c‐myc mRNA expression following TGFβl stimulation. This inhibitory effect was specific for TGFβ1‐stimulated proto‐oncogene expression and associated with the ADP‐ribosylation of a 41‐kDa substrate. Actinomycin D decay and nuclear run‐on experiments demonstrated that the inhibitory effects of PT are a result of decreased transcriptional activation and not to an increased decay of proto‐oncogene message. PT did not, however, affect TGFβl‐stimulated fibronectin and collagen mRNA accumulation nor did it have any inhibitory effect on TGFβl‐induced morphological transformation. These data indicate that TGFβl‐stimulated gene expression is coupled to multiple pathways distinguished by their sensitivity to PT.
Mitogenic response of near‐diploid mouse cell line m5S/1M induced by epidermal growth factor
Authors: Watanabe Sachihiko , Umeda Masato , Nomura Tomoko , Kobayashi Tetsuyuki , Nakano Tohoru , Arita Hitoshi , Utsumi Hiroshi , Sasaki Masao S. , Inoue Keizo 
Published: February 2005

AbstractA nonmalignant near‐diploid cell line m5s/1M, established by Sasaki and Kodama (J. Cell. Res., 131:114–122, 1987), was shown to respond to the epidermal growth factor (EGF). The m5s/1M cells showed high sensitivity to post‐confluence inhibition of cell division and formed a uniform monolayer after the cells had become confluent. The addition of EGF resulted in loss of contact‐dependent inhibition of growth and caused a massive piling up of a multilayered array of cells after they had become confluent. When EGF was removed from the medium, the cell number decreased rapidly, and the cells formed a uniform monolayer at the density observed in the absence of EGF. m5S/1M cells have high‐ and low‐affinity receptors for EGF (approximately 40,000 receptors per cell), and the apparent dissociation constants of the EGF‐binding reactions were 3.3 nM and 0.15 nM, respectively. The effect of EGF on the intracellular mobilization of Ca2+ and the formation of inositol phosphates was studied by using the calcium‐sensitive fluorescent indicator fura 2 and [3H]inositol. EGF had no effect either on the mobilization of cytosolic free calcium ([Ca2+]i) or on the formation of inositol phosphates in m5/1M cells, whereas bradykinin induced a rapid increase in both [Ca2+]i and inositol phosphates. Analysis of the gly‐cosphingolipid (GSL) composition of m5S/1M cells showed that globotriaosyl‐ceramide (Gb3Cer), which is known to be a Burkitt lymphoma‐associated antigen, is specifically expressed in the EGF‐treated cells. The expression of Gb3Cer is dependent on the presence of EGF, with a reversible shift in GSL composition being observed in the presence or absence of EGF.
Evidence that density‐dependent growth arrest is a two‐stage process in WI‐38 cells
Authors: Owen Thomas A. , Carter Ruth , Whitman Mary Maureen , Soprano Dianne Robert , Soprano Kenneth J. 
Published: February 2005

AbstractIt was the goal of this study to determine whether during long‐term quiescence WI‐38 cells gradually lose labile components which then need to be resynthesized before a stimulated cell can progress through G‐1 and enter S. The metabolic and molecular status of WI‐38 cells was systematically analyzed as they entered and were maintained for an extended period of time in a state of density‐dependent growth arrest. Our results indicate that growth arrest in WI‐38 cells can be divided into two stages. The first, which we call “early” growth arrest, occurs during the first 7–10 days following cessation of DNA synthesis and mitosis. It is characterized by few biochemical changes compared to actively proliferating cells. During this period of early growth arrest cells do not exhibit a prolongation of the prereplicative stage following serum stimulation. In contrast, WI‐38 cells growth arrested for 10–20 days exhibit a number of changes at the molecular and biochemical level(i.e., a twofold decrease in total protein and total RNA content, and decreased levels of most proteins, but an increased amount of fibronectin and collagen). Also, quiescent WI‐38 cells stimulated at any time during “later” or “deep” growth arrest do exhibit a prolonged prereplicative phase. Although changes were also observed in the patterns of expression of ten representative growth‐associated genes (i.e., histone H‐3, p53, c‐Ha‐ras, 2A9/calcyclin, 4F1/vimentin, LDL‐receptor, insulin receptor, collagen, and fibronectin), these occurred mostly at the time when the cells ceased synthesis of DNA and mitosis and became quiescent. No changes in the steady‐state levels of the growth‐associated transcripts analyzed occurred while the cells were maintained in the growth‐arrested state. Thus, these experiments show that although WI‐38 cells do cease to incorporate thymidine and divide under crowded culture conditions, the “quiescent” cells continue to undergo changes, are metabolically active, and certainly do not grossly deteriorate.
Basic fibroblast growth factor and transforming growth factor‐α are hepatotrophic mitogens in vitro
Authors: Hoffmann Birgit , Paul Dieter 
Published: February 2005

AbstractBasic fibroblast growth factor (bFGF) and transforming growth factor‐α (TGFα) have been identified as potent hepatotrophic mitogens. bFGF and TGFα induce DNA synthesis in fetal and adult rat hepatocytes in primary culture and support fetal rat hepatocyte multiplication in chemically defined medium. No additional exogenous growth or progression factors are required by the cells for traversing the cell cycle or for cell division. These mitogenic polypeptides, previously identified in various cell types including liver and endothelial cells, platelets, and macrophages may act locally in a paracrine mode in controlling hepatocyte multiplication in the liver during development and regeneration.
Inositol tetrakisphosphate‐induced sequestration of Ca2+ replenishes an intracellular pool sensitive to inositol trisphosphate
Authors: Hill Timothy D. , Boynton Alton L. 
Published: February 2005

AbstractIn a permeable neoplastic rat liver epithelial (261 B) cell system, inositol 1,3,4,5‐tetrakisphosphate—Ins (1,3,4,5)P4—induces sequestration of Ca2+ released by inositol 2,4,5‐trisphosphate—Ins(2,4,5)P3; a non‐metabolized inositol trisphosphate (InsP3) isomer—and Ca2+ added exogenously in the form of CaCl2. Studies were performed to identify the Ca2+ pool filled after Ins(1,3,4,5)P4 treatment. Both Ins(2,4,5)P3 and inositol 1,4,5‐trisphosphate—Ins(1,4,5)P3—dose‐dependently release Ca2+ from permeable 261B cells‐Ins(1,4,5)P3 having a threefold greater potency—but differ in that Ca2+ released by Ins(1,4,5)P3 is readily sequestered, while the Ca2+ released by Ins(2,4,5)P3 is not. Maximal release of Ca2+ by 6 μM Ins(2,4,5)P3 blocked the action of Insd (2,4,5)P3, demonstrating that these two isomers influence the same intracellular Ca2+ pool through a shared membrane receptor. Addition of 2 μM Ins(2,4,5)P3 to discharge partially the Ca2+ pool reduced the amount of Ca2+ released by a maximal dose of Ins(1,4,5)P3 (2 μM). Ins(1,3,4,5)P4 combined with Ins(2,4,5)P3 produced a Ca2+ release and sequestration response, which replenished the InsP3‐sensitive pool as indicated by a recovery of full Ca2+ release by 2 μM Ins(1,4,5)P3. Induction of Ca2+ sequestration by Ins(1,3,4,5)P4 occurred dose‐dependently, with a halfmaximal response elicited at a dose of 0.9 μM. Further studies of the effect of Ins(1,3,4,5)P4 apart from the influence of Ins(2,4,5)P3 using a model in which the Ca2+ levels are raised by an exogenous addition of CaCl2 showed that Ins(1,4,5)P4 released twice the amount of Ca2+ from the storage pool following Ins(1,3,4,5)P4‐induced Ca2+ sequestration. These results demonstrate that the Ca2+ uptake induced by Ins(1,3,4,5)P4 preferentially replenishes the intracellular Ca2+ storage sites regulated by Ins(1,4,5)P3 and Ins(2,4,5)P3.
Regulation of α‐smooth muscle actin and other polypeptides in proliferating and density‐arrested vascular smooth muscle cells
Authors: Liau Gene , Janat Mohamed F. , Wirth Peter J. 
Published: February 2005

AbstractWe have examined α‐smooth muscle actin (α‐SM actin) protein and mRNA levels in proliferating and density‐arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two‐dimensional (2‐D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2‐D gel analysis, we consistently detected increased expression of 12 polypeptides in growth‐arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth‐arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high‐density growth‐arrested cells. Under these conditions we observed no change in relative α‐SM actin protein content as determined by 2‐D gel analysis and Western blots. This was corroborated by high levels of α‐SM actin mRNA levels in both proliferating and high‐density growth‐arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (α‐SM actin) in a proliferation‐ and density‐independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically‐ and explant‐derived SMC contained high levels of α‐SM actin as determined by immunofluorescence and by Northern analysis.
Effects of dexamethasone on induction of monocytic differentiation in human U‐937 cells by dimethylsulfoxide
Authors: Nakamura Takashi , Kharbanda Surender , Spriggs David , Kufe Donald 
Published: February 2005

AbstractThe present studies demonstrate that dimethylsulfoxide (DMSO) treatment of human U‐937 myelomonocytic: leukemia cells is associated with induction of monocytic differentiation. The DMSO‐induced U‐937 monocytic phenolype was associated with (1) growth inhibition, (2) loss of clonogenic survival, (3) increases in á‐naphthyl acetate esterase (NSE) staining, and (4) increases in cell surface expression of the monocyte marker Mac‐1. DMSO treatment of U‐937 cells was also associated with down‐regulation of c‐myc and c‐myb gene expression as well as with increases in tumor necrosis factor (TNF) mRNA levels. The results further demonstrate that induction of U‐937 monocytic differentiation by DMSO is accompanied by increases in phospholipase A2 activity. Moreover, this stimulation of phospholipase A2 was sensitive to dexamethasone. We therefore studied the effects of dexamethasone on DMSO‐induced differentiation of U‐937 cells. Although dexamethasone had no effect on growth inhibition or loss of clonogenic survival by DMSO, this glucocorticoid blocked increases in NSE staining and cell surface Mac‐1 expression. Dexamethasone also had no effect on the down‐regulation of c‐myc and c‐m/b expression but blocked the reappearance of c‐myb transcripts after 6 hr of DMSO treatment. Finally, dexamethasone inhibited DMSO‐induced increases in TNF gene expression. Taken together, the results demonstrate that dexamethasone inhibits multiple characteristics, including the stimulation of phospholipase A2 activity, associated with DMSO‐induced monocytic differentiation of U‐937 cells.
Modulation of tyrosine, serine, and threonine phosphorylation and intracellular processing of the epidermal growth factor receptor by antireceptor monoclonal antibody
Authors: Sunada Hironobu , Yu Peter , Peacock Jeffrey S. , Mendelsohn John 
Published: February 2005

AbstractTo investigate the functional significance of epidermal growth factor (EGF) receptor phosphorylation, experimental systems were explored in which receptor phosphorylation on tyrosine and serine/threonine could be differentially stimulated. Exposure of A431 cells to 20 nM EGF at 37°C results in phosphorylation of serine, threonine, and tyrosine sites on the receptor. Monoclonal antibody (mAb) 225 binds to the EGF receptor with affinity comparable to EGF and competes with the binding of EGF. Exposure of A431 cells to 20 nM EGF in the presence of 300 nM anti‐EGF receptor mAb 225 (15‐fold excess) selectively activated serine and threonine phosphorylation of the receptor, but not tyrosine phosphorylation. This observation indicates that EGF‐mediated receptor phosphorylation on tyrosine and on serine/threonine residues is dissociable. The intracellular fate of the EGF receptor was examined under conditions that produce different phosphorylation states of receptor amino acids. Exposure of A431 cells to EGF decreased the half‐life (T1/2) of the receptor from 17.8 h to 5.6 h, with activation of tyrosine, serine, and threonine phosphorylation. Incubation with mAb 225 augmented the degradation rate (T1/2 = 8.5 h) without activation of receptor phosphorylation. Concurrent exposure to EGF (20 nM) and mAb 225 (300 nM) resulted in comparable enhanced degradation (T1/2 = 9.5 h), with increased phosphorylation only on serine and threonine residues. These results suggest that serine/threonine phosphorylation is irrelevant to the augmentation of receptor degradation. Methylamine, an inhibitor of lysosomal function that did not affect phosphory‐lation of the EGF receptor, completely protected EGF receptors from rapid degradation induced by EGF, but it only slightly altered the rate of EGF receptor degradation elicited by mAb 225 or by EGF plus 15‐fold excess mAb 225. In contrast, mAb 455, Which binds to the receptor but does not inhibit EGF binding and EGF‐induced activation of phosphorylation on tyrosine, serine, and threonine residues, did not influence EGF‐induced rapid, methylamine sensitive degradation of EGF receptor. The results suggest that when EGF receptors are internalized under conditions that do not activate the receptor tyrosine kinase, they are sorted into a nonlysosomal pathway that differs from the methylamine‐sensitive lysosomal pathway traversed following activation by EGF. The data indicate the possibility of a function for tyrosine kinase activation and tyrosine autophosphorylation in determining the lysosomal intracellular pathway of EGF receptor processing and degradation.
New mouse somatic cell mutants resistant to cadmium affected in the expression of their metallothionein genes
Authors: Chopra Arvind , Thibodeau Jacques , Tam You Cheuk , Marengo Claude , Mbikay Majambu , Thirion Jean‐Paul 
Published: February 2005

AbstractFluctuation tests à la Luria and Delbruck were performed with mouse LMTK cells, and the results indicate that the appearance of variants resistant to cadmium is due to random spontaneous mutations and not to epigenetic events. The rate of spontaneous mutations leading to cadmium resistance was calculated to be 0.92 × 10−6 per cell per generation. This rate increased 14‐fold on treatment with ethyl methane sulfonate. Several stable mutant cell lines resistant to cadmium were selected and characterized with respect to metallothionein (MT) induction. Based on the copy number of mt+ genes and the levels of MT proteins and mRNA, the mutants could be divided into two classes, A and B. Although group A mutants have the same number of mt1+ and mt2+genes as wild‐type cells, upon induction with cadmium, the amount of MT proteins and mRNA in the mutants are greatly increased over wild‐type levels. This observation strongly suggests a mutation that regulates MT gene transcription in these cells. In group B mutants, the mt+genes are amplified about three‐ to fourfold, and their MT protein and mRNA basal levels are, as expected, much higher than in the wild‐type cells, under uninduced and induced conditions.
Rat vascular smooth muscle cells immortalized with SV40 large T antigen possess defined smooth muscle cell characteristics including growth inhibition by heparin
Authors: Reilly Christopher F. 
Published: February 2005

AbstractRat aortic smooth muscle cells (SMC) have been established by retroviral delivery of the complementary DNA (cDNA) for the simian virus 40 large T antigen (SV40LT) and examined for SMC phenotypic markers and growth characteristics, including responsiveness to the antiproliferative effects of heparin. The transfected cells (SV40LT–SMC) maintain defined SMC characteristics for more than 215 population doublings (PD) as judged by muscle‐specific actin expression and growth inhibition by heparin. SV40LT–SMC > 129 PD become transformed while SV40LT–SMC < 77 PD resemble nontransfected SMC morphologically and are nontumorigenic. SV40LT–SMC apparently release a growth factor which acts in an autocrine fashion, since (1) suramin inhibits SV40LT–SMC proliferation, (2) SV40LT–SMC‐conditioned medium (CM) contains mitogenic activity, and (3) SV40LT–SMC CM suppresses the binding of platelet‐derived growth factor to SMC. Heparin (10‐100 μg/ml) is a potent inhibitor of both early (80 PD) and late‐passage (> 80 PD) SV40LT–SMC proliferation. The antiproliferative effects of heparin are similar to those previously observed for SMC by several criteria; the dose‐response inhibition curves are indistinguishable from those obtained with nontransfected cells, other glycosaminoglycans have little effect on SV40LT–SMC growth, the antiproliferative effects of heparin are reversed in the presence of epidermal growth factor, and heparin displays high‐affinity saturable binding to SV40LT–SMC. In conclusion, SV40LT–SMC are a continuous line of SMC‐like cells that are sensitive to the growth inhibitor, heparin. SV40LT–SMC should facilitate studies of heparin inhibition and may be applicable for the study of other SMC characteristics as well.
Differences in inhibition by IDF45 (an inhibitory diffusible factor) of early RNA synthesis stimulation induced by pp60 v‐src and various mitogens
Authors: Delbe J. , Villaudy J. , Blat C. , Desauty G. , Golde A. , Harel L. 
Published: February 2005

AbstractFactors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v‐src expression in CEF was poorly inhibited by IDF45.In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v‐src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v‐src expression. By contrast, stimulation of RNA synthesis induced by IGF‐I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis.These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v‐src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF‐binding protein and as an inhibitory molecule in DNA stimulation induced by serum.
Eicosanoid release from human bronchial epithelial cells upon exposure to toluene diisocyanate in vitro
Authors: Mattoli Sabrina , Masiero Mauro , Calabrò Francesco , Mezzetti Maurizio , Plebani Mario , Allegra Luigi 
Published: February 2005

AbstractEpithelial injury and inflammation are involved in airway hyperresponsiveness and asthma induced by toluene diisocyanate. In that isocyanates are insoluble and highly reactive compounds, bronchial epithelial cells may represent the most important target cells of their toxic effect. We hypothesized that damage to airway epithelium by toluene diisocyanate may result in the release of metabolites of arachidonic acid, which are known to promote inflammation and to alter epithelial cell function and airway smooth muscle responsiveness. To test this hypothesis we examined eicosanoid products in the culture media of bronchial epithelial cells exposed in vitro to 8 and 18 ppb toluene diisocyanate. Epithelial cells derived from human bronchi obtained at surgery were cultured to confluency on collagen‐coated microporous membranes. Those cells, which expressed differentiated characteristics of epithelial cells (they showed keratin‐containing filaments and had a cobblestone appearance), were alternatively exposed to toluene diisocyanate or air for 30 min in a specially designed in vitro chamber. The production of me‐tabolites of arachidonic acid was assessed by measuring the release of immunor‐eactive products into the cell medium at the end of the exposure and during a 2 hr period after exposure. This method revealed a predominant isocyanate‐induced release of immunoreactive 15‐ hydroxyeicosatetraenoic acid. Release rate of this compound tended to be dose related and was associated with cell damage as assessed by the release of lactate dehydrogenase in the medium.