bluuebunny/crossref_metadata_2025_split huggingface dataset:
Serum factor revealing two distant phases of negative proliferation control in mitogen‐stimulated normal fibroblasts
Authors: Böhmer Ralph M. 
Published: February 2005

AbstractWhen quiescent normal skin fibroblasts are stimulated by mitogens such as epidermal growth factor (EGF), the proportion of cells entering a division cycle decreases with increasing cell density. The presence of a synthetic double‐stranded RNA (poly I:C) enhances this density‐related restriction. Fetal calf serum (FCS) as well as human serum (HS) and human platelet‐poor plasma (HPPP) completely abrogate the inhibiting effect of cell density on EGF mitogenicity, both in the presence and absence of poly I:C. HS and HPPP are up to ten times more potent than FCS in overcoming density‐related restriction of EGF mitogenicity in human skin fibroblasts, whereas the mitogenic potencies of FCS, HS and HPPP in the absence of EGF are identical. Thus the mitogenic activity of FCS, HS and HPPP and their ability to overcome the density‐related restriction of EGF‐induced proliferation may be due to different molecules. Addition of FCS or HS at various times after EGF exposure reveals two distinct control points within the prerepli‐cative phase: one within the first 2 hours and the other between 10 and 20 hours after the beginning of EGF exposure. Thus, the interactions of EGF, serum, and poly I:C reveal the kinetics of a cell density‐related mechanism of negative proliferation control.
Expression of multiple proteins structurally related to gamma‐glutamyl transpeptidase in non‐neoplastic adult rat hepatocytes in vivo and in culture
Authors: Mathis Georg A. , Wyss Peter A. , Schuetz Erin G. , Hughey Rebecca P. , Sirica Alphonse E. 
Published: February 2005

AbstractFreshly isolated hepatocytes from normal adult rat liver do not express measurable gamma‐glutamyl transpeptipdase (GGT) mRNA in contrast to the significant GGT mRNA levels expressed by normal adult rat kidney and hyperplastic bile ductular tissue from bile duct‐ligated rats. However, the induction of GG activity in rat hepatocytes by two‐thirds hepatectomy was accompanied by the appearance of a high level of GGT mRNA. We are now able to demonstrate that normal adult rat hepatocytes express 5 protein bands which cross‐react with 2 different anti‐rat kidney GGT antisera. The apparent molecular weights were 26.9, 58.0, 63.9, 73.5, and 83.4 kDa, respectively. Expression of the 26.9‐ and 58.0‐kDa proteins strikingly parallels the pattern of induction of GGT enzymatic activity. This suggests that these 2 proteins correspond to the active dimeric enzyme previously described in kidney and neoplastic hepatocellular tissue. In normal hepatocytes, the 73.5‐kDa protein represents 50% of the total GGT‐immunoreactive protein, contrast to kidney, where this band contains less than 4% of the GGT protein. The kinetics of expression of the 73.5‐kDa protein upon induction of GGT activity in hepatocytes, as well as in culture turnover studies, suggests that this protein is a precursor form of the active enzyme, such as the described 78/79‐kDa single‐chain glycoprotein propeptide of GGT. It appears that in normal hepato‐cytes, this precursor is not processed to the same extent as in kidney or in hyperplastic bile ductular tissue.
Interactions of dimethyl sulfoxide and granulocyte‐macrophage colony‐stimulating factor on the cell cycle kinetics and phosphoproteins of G1‐enriched HL‐60 cells: Evidence of early effects on lamin B phosphorylation
Authors: Brennan J. K. , Lee K. S. , Frazel M. A. , Keng P. C. , Young D. A. 
Published: February 2005

AbstractWe have found that GM‐CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL‐60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL‐60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombiniant human GM‐CSF and/or 0–1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]‐orthophosphate‐labeled cell proteins separated by giant 2‐D gel electrophoresis. Results were correlated with (1) intracellular pH, determined by measurement of BCECF fluorescence; (2) [32P]‐orthophosphate uptake; (3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide‐stained cells; and (4) growth, determined by cell volume and concentration. GM‐CSF stimulated and DMSO inhibited the GM‐CSF‐stimulated phosphorylation of 1 protein (∼65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1 not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15–30 minutes), cell volume change (16–24 hr), and cell concentration increase (28–32 hr). GM‐CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synch onization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM‐CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM‐CSF‐DMSO antagonism is exerted through the same mitogenic pathway, that a major locus of cytokinetic effect is on G1 to S transit, and that nuclear envelope protein phosphorylation is an important early event.
Retinoic acid priming potentiates the induction of urokinase‐type plasminogen activator by cyclic adenosine monophosphate in mouse mammary carcinoma cells
Authors: Mira‐Y‐Lopez Rafael 
Published: February 2005

AbstractInteractive regulation of gene expression by retinoic acid (RA) and cyclic adenosine monophosphate (cAMP) in mammary tumor cells was explored using Shionogi mouse mammary carcinoma cells (SC115) as a model and urokinase‐type plasminogen activator (uPA) as a target gene product. Twenty‐four hour treatment of SC115 cells with 100 nM RA, 1 mM 8‐bromo‐cAMP (BrcAMP), and 100 nM RA + 1 mM BrcAMP resulted in extracellular uPA activity increases of 1.4‐fold, sevenfold, and 20‐fold, respectively. These effects were dose‐dependent with regard to both interacting members. Similar responses were obtained if 1 nM cholera toxin or 10 μM forskolin was used instead of the cAMP analog. Retinoids lacking the carboxylic acid function were inactive. The changes in uPA activity were accompanied by similar changes in uPA antigen concentration, as seen via Western blot analysis, and uPA mRNA abundance, as seen via Northern blot analysis. Actinomycin D, an inhibitor of RNA synthesis, blocked uPA stimulation by BrcAMP, suggesting that mRNA levels were transcriptionally regulated. The effect of BrcAMP on extracellular uPA activity was first evident at 2 h and peaked at 6 h; the effect of RA alone and the synergistic response to joint treatment, however, followed a slower time course, requiring at least 12 h for initial expression and increasing gradually with time up to at least 48 h. Priming with RA for 48 h followed by extensive washing of the cells resulted in a threefold enhancement of the stimulatory effect of BrcAMP on uPA. Experiments utilizing the casein/plasminogen overlay method for the detection of uPA secretion by single cells showed that the enhanced response to BrcAMP was due to an increased rate of uPA secretion per cell rather than to an increased fraction of uPA‐secreting cells. Initial investigation of the mechanism of RA potentiation of cAMP responsiveness showed that RA did not alter cellular cAMP levels or total cAMP‐dependent protein kinase A activity. Finally, the tumor promoter phorbol myristate acetate, an activator of protein kinase C, also increased SC115 cell uPA activity and synergized with RA. This raised the possibility that the enhancement of cAMP responsiveness by RA was indirectly mediated via an effect on protein kinase C. Experiments with protein kinase C‐depleted cells, however, showed that this was not the case. In conclusion, RA treatment of SC115 cells potentiates the effect of cAMP on uPA expression at the single cell level via a partially irreversible mechanism independent of protein kinase C. The molecular target of RA and whether SC115 cell differentiation underlies the effect of RA remain to be established.
cAMP promotes branching of laminin‐induced neuronal processes
Authors: Weeks Benjamin S. , Papadopoulos Vassilios , Dym Martin , Kleinman Hynda K. 
Published: February 2005

AbstractLaminin is a potent stimulator ofneurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium‐proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108‐15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin‐induced neurites. A four‐fold increase in the number of branches per neurite and a two‐to‐three‐fold increase in the number of neurites per cell were observed in both NG108‐15 and PC12 cells cultured on laminin when either 8‐BrcAMP or forskolin was added. The cAMP‐induced branching was also observed when PC12 cells were cultured on a laminin‐derived synthetic peptide (PA22‐2), which contains the neurite‐promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22‐2 were axonal, not dendritic, and the cAMP‐induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin‐mediated neurite outgrowth, but cAMP can modulate the effects of laminin.
Role of TGFα and its receptor in proliferation of immortalized rat tracheal epithelial cells: Studies with tyrphostin and TGFα antisera
Authors: Ferriola Patrice C. , Earp H. Shelton , Di Augustine Richard , Nettesheim Paul 
Published: February 2005

AbstractWe have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGFα and do so regardless of the mechanism by which they were transformed. In order to determine whether TGFα is an autocrine growth regulator of immortalized RTE ‐cells, we have examined the function of TGFα/EGF receptors and the growth requirements for TGFα in these cells. The level of immunoprecipitated TGFα/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGFα/EGF receptors. Scatchard analysis of TGFα/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high‐affinity (Kd = 0.4nM) and low‐affinity (Kd = 9.8nM) binding sites. A tyrphostin TGFα/EGF receptor tyrosine kinase inhibitor decreased in a dose‐dependent manner the proliferation as well as EGF‐induced autophosphorylation of the TGFα/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF‐dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGFα antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGFα and its receptor.
Role of putrescine in interleukin 1β production in human histiocytic lymphoma cell line U937
Authors: Tahara Hideki , Otani Shuzo , Matsui‐Yuasa Isao , Koyama Hidenori , Nishizawa Yoshiki , Morisawa Seiji , Morii Hirotoshi 
Published: February 2005

AbstractTreatment with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and incubation with lipopolysaccharide (LPS) induces Interleukin 1β (IL‐1β) production in the histiocytic lymphoma cell line U937. Here we investigated the effect of treatment with both TPA and 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3) on LPS‐induced IL‐1β production in U937 cells. To clarify the mechanism of IL‐1β production, the possible role of polyamines in this process was examined. Combined treatment with TPA and 1,25(OH)2D3 for 72 h followed by incubation with LPS for 24 h caused synergistic induction of both IL‐1β release and mRNA expression. On the other hand, TPA increased the numbers of vitamin D3 receptors, which may be one mechanism of this synergistic induction. Ornithine decarboxylase (ODC), a rate‐limiting enzyme for polyamine biosynthesis, was also induced by these compounds biphasically: the first peak of ODC activity was observed at 4 h of the incubation with the two compounds and the second peak was at 4 h after the addition of LPS. To find whether these peaks were related to IL‐1β production, DL‐α‐difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, was added together with TPA and 1,25(OH)2D3. DFMO decreased the cellular levels of putrescine and spermidine and suppressed IL‐1β release and IL‐1β mRNA expression by 65%. Exogenous putrescine, but not spermidine, abrogated these kinds of inhibition. Similar results were obtained with DFMO and the polyamines during the differentiation of the cells up to the monocyte or macrophage stage. These results thus suggest that changes in either of these intracellular polyamines, especially putrescine, help to regulate the differentiation of U937 cells, resulting in partial control of the regulation of IL‐1β production.
A toxin‐resistant mouse L‐cell mutant defective in protein transport along the secretory pathway
Authors: Laurie S. M. , Robbins A. R. 
Published: February 2005

AbstractUsing methods designed for isolation of mutants defective in receptor‐mediated endocytosis, a novel L‐cell mutant was obtained that exhibits resistance to three different protein toxins as well as alterations in secretion. This rrutant, LEFIC, is resistant to modeccin, Pseudomonas exotoxin, and ricin. Thesi toxins, which enter the cytoplasm via receptor‐mediated endocytosis, are thought to penetrate into cells at the level of late endosomes or the trans Golgi network. Early endosomal acidification appears to be normal in the mutant based on its accumulation of iron from transferrin and its sensitivity to diphtheria toxin A chain‐transferrin conjugate. Within the secretory pathway twodelays in transport ofvesicular stomatitis virus (VSV) G protein wereobserved in LEFIC: a 20–30 min delay in acquisition of Endo H resistance and a 1–2 hr delay in appearance of newly synthesized G protein on the cell surface. Movement of endogenous proteins along the secretory pathway was also affected in LEFIC. Fibronectin secretion was delayed by 15 min, and membrane proteins were delayed in arrival at the cell surface. The phenotype of LEFIC is consistent with a defect in a component or compartment shared by both the late endocytic and constitutive secretorypathways.
Growth‐associated gene expression is not constant in cells traversing G‐1 after exiting mitosis
Authors: Cosenza Stephen C. , Carter Ruth , Pena Angel , Donigan Anne , Borrelli Michael , Soprano Dianne Robert , Soprano Kenneth J. 
Published: February 2005

AbstractAnalysis of gene expression following stimulation of growth‐arrested cells has beei the main approach for identification of growth‐associated genes. Since the activation of these gene sequences is dependent on both the stimulatory agent and theitate of quiescence of the cell, the activation and role of the same genes may be entirely different in non‐growth arrested, actively proliferating cells. We have addressed the question of growth‐associated gene expression during active growth by analyzing gene expression during G‐1 of cells which have jusl exited mitosis without first leaving the cell cycle. We were able to isolate, by a non‐inductive, drug free system, a population of highly synchronized Swiss 3T3 cells within mitos is (>90%) in numbers sufficient to determine the pattern of expression pf a large number of representative growth‐associated genes. Our results show that after replating the mitotic ceils into conditioned medium: (1) growth‐associated gene expression is not constant during G‐1 of actively proliferating cells, and (2) while a number of genes (e.g., JE, c‐myc, ODC, p53, and histone) exhibited patterns of expression similar to that reported in the quiescent systems, others (e.g., nur‐77, vimentin, calcyclin) exhibited patterns which were completely different. From these results, we can begin to construct a temporal map of G‐1 progression during active growth.
Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells
Authors: Ridsdale J. A. , Clegg J. S. 
Published: February 2005

AbstractMouse L929 cells were exposed to the nonionic detergent Brij 58. As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase. In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58. The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not scape immediately. This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij. Data are presented that are most readily interpreted as protein leakage having occured fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent. The perrneabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium. Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent. Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.
Correlation of the inability to sustain growth in defined serum‐free medium with the suppression of tumorigenicity in Wilms' nephroblastoma
Authors: Dowdy Steven F. , Weissman Bernard E. , Stanbridge Eric J. 
Published: February 2005

AbstractWe report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum‐free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175–180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell‐mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) Chromosome from the nontumorigenic microcell hybrid cells resulted In the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum‐free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (ECF) or 5 μg/ml of insulin did not alter this effect. However, the addition of 5 μg/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum‐free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum‐free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.
Effect of recombinant human interleukin‐6 on nitrite production of mouse myeloid leukemia cells
Authors: Takagi Kuniaki , Hosaka Tsutomu , Suketa Yasunobu 
Published: February 2005

AbstractThe effect of recombinant human interleukin‐6 (rhlL‐6) on induction of nitrite (NO) production was investigated in a mouse myeloid leukemia cell line (M1) and a subclone (Mm1). NO was induced by rhlL‐6 (>50 U/ml) in these cell lines. Pretreatment with rhlL‐6 (100 U/ml) for 1 day synergistically enhanced the production of NO in Mm1 by LPS. Furthermore, pretreatment withl lL‐6 (100 U/ml) shortened the lag time of induction. These results indicate that lL‐6 is involved in the regulation of the NO production in macrophage‐like cells.
Association of c‐myc protein with enzymes of DNA replication in high molecular weight fractions from mammalian cells
Authors: Studzinski George P. , Shankavaram Uma T. , Moore Dorothy C. , Reddy Prem Veer 
Published: February 2005

AbstractOncoprotein c‐myc is expressed in proliferating but not quiescent mammalian cells, and its overexpression or inappropriate expression is associated with malignant transformation. However, in spite of an intense interest, the normal function of this protein has remained elusive. As a step towards the elucidation of the function of c‐myc protein, we studied its distribution within several types of cells, including HL 60, K 562, COLO 320, and CHEF/18 cells. In all of the cells studied, c‐myc protein was detected in high molecular weight protein fractions, in 350–600 Kd range, in gel‐exclusion chromatography and sucrose gradient centrifugation. This distribution of c‐myc protein coincided with the distribution of DNA polymerase α and several other enzymes necessary for DNA replication. The data suggest that c‐myc product may be a component of the replitase complex of enzymes involved in nuclear DNA replication.