bluuebunny/crossref_metadata_2025 huggingface dataset:
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

A 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

It 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

Basic 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

In 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

We 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

The 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

To 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

Fluctuation 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

Rat 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

Factors 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

Epithelial 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.
A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity
Authors: Mascardo Renato N. , Thompson Wayne , Gallo Mary Ann , Chambers Timothy C. , Eilon Gabriel
Published: February 2005

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E‐64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl‐leucyl‐agmatine (SLA), an analogue of E‐64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on en‐dothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetato (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P‐labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.
Effect of inflammatory cytokines on human endothelial cell proliferation
Authors: Saegusa Yasuhiro , Ziff Morris , Welkovich Linda , Cavender Druie
Published: February 2005

Neovascularization, 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‐stimulated proliferation.
Dexamethasone‐induced killing of neoplastic cells of lymphoid derivation: Lack of early calcium involvement
Authors: Bansal Neelam , Houle Antoinette G. , Melnykovych George
Published: February 2005

The role of calcium influx in dexamethasone‐induced fragmentation of DNA was studied in the glucocorticoid‐sensitive human lymphoid line of T cell derivation (CEM‐C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmida‐zolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM‐C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM‐C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).
Cytoskeleton as a target in menadione‐induced oxidative stress in cultured mammalian cells. I. Biochemical and immunocytochemical features
Authors: Bellomo G. , Mirabelli F. , Vairetti M. , Iosi F. , Malorni W.
Published: February 2005

Cytoskeletal abnormalities occurring during oxidative stress generated by the metabolism of the redox cycling compound 2‐methyl‐1,4‐naphtoquinone (menadione) have been investigated in different mammalian cells in culture. Extraction of the whole cytoskeleton as well as the intermediate filament‐ and the microtubule‐enriched fractions from menadione‐treated cells revealed a marked depletion of protein sulfhydryl groups. The analysis of the whole cytoskeletal fraction by PAGE showed a menadione‐dependent and thiol‐sensitive oxidation of actin, leading to the formation of high‐molecular‐weight aggregates. In addition, the extraction of this fraction with high concentrations of KCl entailed only a partial solubilization of actin. The comparative cytochemical analysis performed on treated cells showed a menadione‐dependent clustering of actin microfilaments. The metabolism of menadione induced microtubule depolymerization and inhibition of GTP‐induced microtubule assembly from soluble cytosolic components. The latter phenomenon was prevented by previously treating the cytosolic fraction with thiol reductants such as dithiothreitol. Menadione increased the protein content of the intermediate‐size filament fraction, partially purified by one or more cycles of disassembly/assembly, and particularly enriched in polypeptides reacting with antikeratin antibodies. Furthermore, a reversible and oxidation‐dependent change of the electrophoretic mobility of some polypeptides in this fraction was detected. The immunocytochemical investigation of intermediate‐size filament distribution in menadione‐treated cells, however, revealed only minor modifications mainly consisting of perinuclear condensation of cytokeratin structures. These findings suggest that cytoskeletal structures (actin microfilaments, microtubules, and intermediate‐size filaments) are actually significant targets in quinone‐induced oxidative stress.
Growth‐associated modifications of low‐molecular‐weight thiols and protein sulfhydryls in human bronchial fibroblasts
Authors: Atzori Luigi , Dypbukt Jeannette M. , Sundqvist Kristina , Cotgreave Ian , Edman Charlotte C. , Moldéus Peter , Grafström Roland C.
Published: February 2005

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol‐reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin pertubs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein‐bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low‐molecular‐weight thiols and protein‐bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol‐depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein‐bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cystine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low‐molecular‐weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.
Regulation of Na+/H+ and Cl−/HCO3− antiports in vero cells
Authors: Tønnessen Tor Inge , Aas Alf Tore , Ludt Jannikke , Blomhoff Heidi Kiil , Olsnes Sjur
Published: February 2005

The effect of serum, phorbol‐12‐myristate‐13‐acetate (TPA), and forskolin on the activity Na+/H+ antiport and the Na+‐coupled and Na+‐independent Cl−/ HCO3− antiport was studied in Vero cells by measuring 22Na+ and 36Cl− fluxes and changes in cytosolic pH (pHi). The Na+‐independent Cl−/HCO3− antiport, which acts as an acidifying mechanism, is strongly pH‐sensitive. In serum‐starved cells it is activated at alkaline cytosolic pH, with a half‐maximal activity at pHi ∼7.20. Incubation with serum increased the activity of the Na+‐independent Cl−/HCO3− antiport at pHi values from 6.8 to 7.2. Thus serum appeared to alter the pHi sensitivity of this antiporter such that the threshold value for activation of the antiport was shifted to a more acidic value. Na+ /H+ antiport was somewhat stimulated initially by addition of serum, but further incubation with serum (> 45 min) decreased its activity. The activity of the Na+ ‐coupled Cl−/HCO3− antiport, which is the major alkalinizing antiport in Vero cells, was not altered by short‐term incubation with serum (< 10 min) but decreased after prolonged incubation (> 45 min). Our findings with TPA and forskolin indicate that the effect of serum is partly mediated by the protein kinase C pathway, whereas the cyclic adenosine monophosphate pathway does not appear to play an important role. The net effect of serum on the pHi‐regulating antiports was a slight decrease in intracellular pH.
Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation
Authors: Nånberg Eewa , Morris Clive , Higgins Theresa , Vara Francisco , Rozengurt Enrique
Published: February 2005

To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two‐dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13‐dibutyrate were indistinguishable. Down‐regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C‐dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.
Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA Class 1, HLA Class 2, and neoplastic epithelial antigens) in the apical cell membrane
Authors: Magnusson Karl‐Eric , Gustafsson Mikael , Holmgren Kajsa , Johansson Birgitta
Published: February 2005

We have studied the effect of maturation to small intestinal‐like epithelial cells of the human colonic calcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21‐day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM‐Gal. In comparison to control cells grown in DMEM‐Glu medium, the more small intestinal‐like cells grown in DMEM‐Gal displayed no alteration of the lateral mobility of either cholera toxin (B subuni)‐labelled ganglioside GM1 (diffusion coefficient, D [x 108] = 0.8–0.9 cm2s−1; mobile fraction, R = 50‐60%) or antibody‐stained Class 2 histocompatibility (HLA‐DR) antigen (D [x 109] = 2 cm2s−1; R = 60–70%). However, antibody‐labelled β2‐microglobulin of HLA Class 1 antigen displayed increased mobility in HT29‐Gal cells; D was × 1.4 and R × 1.8 larger in the HT29‐Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were × 0.60 and × 0.69 of the values seen in HT29‐Glu cells. It is thus concluded that DMEM‐Gal‐induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobil‐ity of different membrane components.
Regulation of polyamine transport in Chinese hamster ovary cells
Authors: Byers Timothy L. , Pegg Anthony E.
Published: February 2005

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC−) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with α‐difluoromethylornithine in CHO cells or via polyamine starvation of CHODC− cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylgiyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.
Intracellular distribution of the c‐fos antigen during the cell cycle
Authors: Rahm Magnus , Hultgardh‐Nilsson Anna , Jiang Wei‐Qin , Sejersen Thomas , Ringertz Nils R.
Published: February 2005

The subcellular localization of the c‐fos proto‐oncogene product was studied in the G1, S, G2, and mitotic phases of the cell cycle by indirect immunofluores‐cence. For these analyse c‐fos transfected L6J1 rat skeletal myoblasts and adult rat aortic smooth muscle cells in secondary culture, and c‐fos‐ and c‐myc co‐transfected mouse Swiss 3T3 fibroblasts were used. During G1, S, and G2, the c‐fos protein was evenly distributed in the nucleus, with exclusion of the nucleoli. In mitotic prophase the c‐fos antigen was dissociated from the condensed chromosomes and became diffusely distributed in the cell cytoplasm, where it remained until telophase, when, again, it appeared to be associated with chromatin in the re‐assembling nucleus. When comparing the subnuclear distribution of the c‐fos product with that of densely packed DNA, stained with the fluorochrome Hoechst, an inverse relationship was found. Dispersed chromatin regions with weak Hoechst DNA fluorescence showed a stronger fos immunofluorescence than regions that contained a higher concentration of DNA. The localization of c‐fos antigen partially overlapped with that of antigens typical of small nuclear ribonucleoprotein complexes participating in transcription and splicing.

To examine if the c‐fos protein would bind preferentially to specific interphase chromosomes the nucleus was fragmented into micronuclei containing single, or groups of, chromosomes. Immunofluorescence analysis showed that the majority of micronuclei were fos‐positive. Possible roles of the c‐fos proto‐oncogene product are discussed in relation to other nuclear antigens.
Sodium‐dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats
Authors: Chen Ming L. , King Rebecca S. , Armbrecht H. James
Published: February 2005

The transport of phosphate by primary cultures of renal cells from young (5‐6 weeks) and adult (10–12 months) rats was studied. Renal tubule cells isolated from young and adult groups exhibited typical epithelial morphology and similar growth rates. The Na‐dependent phosphate uptake was saturable with a Km of 5–7 μM over a substrate range of 1–500 μM. A decrease in Na‐dependent phosphate uptake in adult cells (30%) was found compared to that of young cells. The Na‐independent component of phosphate uptake did not vary with age. In addition, the inhibition of phosphate uptake by a variety of compounds (ouabain, gramicidin, 2,4‐dinitrolphenol, KCN, and arsenate) were similar in both age groups. Kinetic analysis showed that a significant reduction in Vmax (4.4 ± 0.4 vs. 3.1 ± 0.2 nmol Pi/mg protein/10 min in young and adult cells, respectively), but not Km, resulted in this decreased uptake of phosphate in adult groups. There was no difference in the efflux of phosphate from both age groups. When cells were preincubated in a phosphate‐free medium for 24 hours, the uptake of phosphate was increased to 46% and 24% of their corresponding controls in young and adult cells, respectively. The decreased phosphate uptake and limited adaptation to a phosphate‐free medium by the adult renal cells may account for the hypophosphatemia and phosphaturia seen in adult and old animals in vivo.
Membrane potential of rat calvaria bone cells: Dependence on temperature
Authors: Massas Ruth , Korenstein Rafi , Bincmann Dieter , Tetsch Peter
Published: February 2005

The membrane potentials of bone cells derived from calvaria of new born rats was shown to be strongly dependent on temperature. When we lowered the temperature from 36°C to 26°C, cells with spontaneous resting membrane potentials (MP) of ‐80 to‐50 mV depolarized (mean amplitude 8 mV; n = 33), and the membrane resistance increased by ∼ 80% (n = 20). The temperature response depended on the actual MP, the reversal potential being in the range of −80 to −90 mV. With the application of ouabain (0.1−1 mmol/liter; n = 12), cells depolarized. Simultaneously, the reversal potential of the temperature response was shifted towards more positive values and approached the actual MP level of the cells. Consequently, the depolarization amplitudes induced by lowering temperature were reduced at spontaneous MP levels. The rise of the membrane resistance during cooling was unaffected. When the extracellular chloride concentration was reduced from 133 to 9 mmol/liter, temperature‐de pendent depolarizations persisted at spontaneous MP values (n = 5). The findings indicate that the marked effects of temperature changes on the MP of bone‐derived cells are mainly determined by changes of the potassium conductance.
Effect of butylated hydroxytoluene on biliineage differentiation of the human HL‐60 myeioblastic leukemia cell
Authors: Burns C. Patrick , Petersen Eric S.
Published: February 2005

Butylated hydroxytoluene (BHT), which has both antioxidant and membrane active properties, has been reported to affect cellular differentiation. We studied its effect on the bipotent lineage differentiation of the important HL‐60 human my‐eloblastic leukemia cell line using reduction of nitroblue tetrazolium, cell cycle analysis, population growth rote, monoclonal antibodies, and morphology. BHT markedly accelerated retinoic acid‐induced myelocytic differentiation and dihydroxyvitamin D3‐induced monocvtic differentiation in a concentration and time‐dependent manner. Butylated hydroxyanisole (BHA) had a comparable effect. Preincubation with the compounds was not necessary to evoke the acceleration. Other antioxidants and inhibitors of eicosanoid synthesis were inactive. We conclude that the important food preservatives BHT and BHA accelerate the kinetics of terminal differentiation of human leukemia and that this effect is likely due at least in part to their membrane active properties.
Lipopolysaccharide induces competence genes JE and KC in Balb/C 3T3 cells
Authors: Tannenbaum Charles S. , Major Jennifer A. , Poptic Earl J. , DiCorleto Paul E. , Hamilton Thomas A.
Published: February 2005

The expression of the early genes JE and KC has been examined in Balb/c 3T3 cells treated with bacterial lipopolysaccharide (LPS). Previous studies showed that JE and KC mRNAs are induced in murine peritoneal macrophages treated with LPS, suggesting a role for these genes in inflammatory responses. Consistent with this possibility are recently published cDNA sequences which document that both genes are members of a superfamily of inflammation‐ and/or growth‐related cytokines. In the present study, we provide evidence that the mRNAs for JE and KC are specifically induced by LPS treatment of Balb/c 3T3 cells. The LPS‐stimulated expression of JE and KC was dose dependent, and exhibited a transient time course; message levels were maximal between 2 and 4 hr and declined by 8 hr. The LPS‐augmented accumulation of JE and KC occurred even in the presence of cyclohexamide, which additionally had a superinducing effect on the expression of both genes. Cyclohexamide alone, in the absence of LPS, also induced JE and KC mRNA accumulation. LPS‐stimulated JE and KC mRNA expression was dependent upon the stimulation of transcription as determined by nuclear “run‐on” studies. Comparative analyses indicated that, under the conditions employed, LPS was a somewhat less effective stimulant of JE expression than PDGF or EGF, and was more effective than PDGF and equivalent to EGF in its ability to augment KC accumulation. Unlike PDGF and EGF, LPS did not stimulate DNA synthesis by Balb/c 3T3 cells at any time over the 72 hr period examined. The ability of the inflammatory, non‐mitogenic stimulus LPS to selectively induce JE and KC mRNA expression by fibroblasts may reflect their participation in inflammation and wound healing as secretory cells.
Studies on the role of basic fibroblast growth factor in vivo: Inability of neutralizing antibodies to block tumor growth
Authors: Dennis Phillip A. , Rifkin Daniel B.
Published: February 2005

Affinity‐purified polyclonal rabbit antibodies prepared against recombinant basic fibroblast growth factor (bFGF) neutralized the ability of bFGF to stimulate plasminogen activator (PA) production and endothelial cell migration in vitro. After iodination and intraperitoneal injection of the antibodies in mice, approximately 76% of the maximum circulating level of 125l‐anti‐bFGF antibodies (AF) was found as intact IgG after 24 hr. Furthermore, the circulating 125I‐AF retained the ability to bind bFGF. Studies were performed to determine whether the growth of three different murine tumors (CT26, EHS, or B16/BL6) could be inhibited with affinity‐purified neutralizing antibodies against bFGF. Tumors were injected subcutaneously in syngeneic mice, and neutralizing antibodies against bFGF were injected daily into the peritoneum. All studies, which varied in tumor burden, antibody dose, and study length, indicated that neutralizing antibod es against bFGF had no effect on tumor size, tumor growth, or tumor histology.
Acquisition of a lysosomal enzyme by myoblasts in tissue culture
Authors: Beauchamp Jonathan R. , Olsen Irwin , Partridge Terence A.
Published: February 2005

Skeletal muscle myoblasts from different sources acquired high levels of the lysosomal enzyme β‐glucuronidase, when they were cultured together with mitogen‐activated lymphocytes. Immunofluorescent staining, thermal stability, and electrophoretic mobility showed that the increase in enzyme activity in the myoblasts was due to the presence of the lymphocyte form of the enzyme. Although myoblasts were able to take up exogenous β‐glucuronidase from the culture medium by mannose 6‐phophate receptor‐mediated endocytosis, enzyme acquisition during co‐culture with lymphocytes was independent of this pathway. Enzyme transfer from the lymphocytes was found to require direct cell‐cell contact with the muscle cells, and was accompanied by an increase in β‐glucuronidase activity in the lymphocytes themselves. Since this additional activity was also due to the presence of the lymphocyte form of the enzyme, these results indicate that interaction with the muscle cells induced the de novo synthesis of β‐glucuronidase in the lymphocytes.
Concanavalin A‐ and fetal‐calf‐serum‐induced rounding and myosin light chain phosphorylation in cultured smooth muscle cells
Authors: Sasaki Yasuo , Iwata Kunie , Sasaki Yasuharu
Published: February 2005

Rabbit aorta smooth muscle cells (SMC) in long‐term culture retracted in less than 10 min in response to a sequential order of stimulations by concanavalin A (Con A) and fetal calf serum (FCS). With additional continuous stimulation by FCS, the SMC took on a circular shape and were anchored to the substrate by retraction fibrils. This rounding was observed only when the cells were sequentially stimulated by Con A and FCS. Depletion of intracellular Ca2+ stores by the addition of EGTA and Ca2+ ionophores inhibited the rounding. Transient phosphorylation of MLC20 was observed in the initial stage during the SMC rounding. The extent of monophosphorylated MLC20 increased for up to 5 min to a maximal value of 49%. The diphosphorylated form reached a maximal value of 29% within 2 min; then both forms of MLC20 decreased. The process of the SMC rounding was inhibited by antimycin A or cytochalasins, in a dose‐dependent manner, findings which suggested a dependency on both metabolic energy and actin‐containing microfilaments. The smooth‐muscle‐relaxing agent, HA1077, also inhibited the process of SMC rounding. These observations suggest that a cellular contractile process might be involved in rounding of SMC.