bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamic acid potentiates hepatocyte response to mitogens in primary culture
Authors: Hasegawa Kaoru , Miyata Yasuji , Carr Brian I.
Published: February 2005

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L‐15 medium, their response to EGF was low compared with that in Williams' medium E or Koga's medium L. Furthermore, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes in L‐15 medium. Addition of glutamic acid (1–20 μM) to EGF‐containing L‐15 medium not only enhanced DNA synthesis > tenfold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Aspartic acid, glutamine, or ornithine at 20 mM did not replace the glutamic acid effect on DNA synthesis. Proline also enhanced EGF‐induced DNA synthesis, although it was less effective than glutamic acid. Therefore, this effect may be specific to a high concentrations of glutamic acid. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested. In the presence of glutamic acid, EGF showed a dose‐dependent (0.5–20 ng/ml) stimulation of DNA synthesis with a maximal effect at 10 ng/ml. Almost the same effect was obtained with transforming growth factor alpha (0.5–20 ng/ml). Glutamic acid also induced an expansion of the mitogenic action of angiotensin II. Since glutamic acid did not affect [125I]EGF binding to hepatocytes or its processing, the effect may occur internal to the receptor. These results suggest that glutamic acid modulates the sensitivity of the hepatocyte response to mitogens © 1994 Wiley‐Liss, Inc.
Cross‐talk of parathyroid hormone‐responsive dual signal transduction systems in osteoblastic osteosarcoma cells: Its role in PTH‐induced homologous desensitization of intracellular calcium response
Authors: Sugimoto Toshitsugu , Ikeda Kazuto , Kano Junichi , Yamaguchi Toru , Fukase Masaaki , Chihara Kazuo
Published: February 2005

The present study was designed to characterize the cross‐talk of parathyroid hormone (PTH)‐responsive dual signal transduction systems (cAMP‐dependent protein kinase (PKA) and calcium/protein kinase C [PKC]) and its participation in PTH‐induced homologous desensitization of intracellular calcium ([Ca2+]i) in osteoblastic UMR‐106 cells. Although our recent study revealed that prolonged (more than 2 h) pretreatment with PKC‐activating phorbol ester, phorbol 12‐myristate 13‐acetate (PMA) significantly decreased the PTH‐stimulated cAMP production, pretreatment with PMA (10−7 and 10−6 M) but not 10−6 M 4alphaphorbol 12,13‐didecanoate (PDD), incapable of activating PKC for 30 min significantly augmented 10−7 M hPTH‐(1‐34)‐stimulated cAMP production. H‐7 (50 uM), a PKC inhibitor, significantly antagonized this PMA‐induced effect. Pretreatment with 10−6 M PMA for 30 min did not affect PTH receptor binding but significantly augmented a cAMP responsiveness to 10−5 M forskolin and 1 ug/ml cholera toxin. Pertussis toxin (0.5 ug/ml) did not affect the PMA‐induced augmentation of the PTH‐stimulated cAMP production. PTH caused a complete homologous desensitization of [Ca2+]i response within 30 min. Pretreatment with 10−4 M dibutyryl cAMP for 30 min and 6 h significantly reduced and completely blocked the PTH‐induced increase in [Ca2+]i, respectively. Pretreatment with 10−4 M Sp‐cAMPS, a direct PKA activator, for 30 min completely blocked the PTH‐induced increase in [Ca2+]i. Rp‐cAMPS (10−4 M), an antagonist of PKA, slightly but significantly antagonized the PTH‐induced homologous desensitization of [Ca2+]i response. The present study indicates that the time of exposure to PKC activation is a critical determinant in modulating the cAMP system, while PKA activation counterregulatorily acts on the [Ca2+]i system, and that PKA activation is linked to the PTH‐induced homologous desensitization of [Ca2+]i response. © 1994 Wiley‐Liss, Inc.
Vanadate reduces sodium‐dependent glucose transport and increases glycolytic activity in LLC‐PK1 epithelia
Authors: Madsen Karen L. , Porter Valerie M. , Fedorak Richard N.
Published: February 2005

The effect of vanadate pentoxide on apical sodium‐dependent glucose transport in LLC‐PK1 epithelia was examined. Epithelia grown in the presence or absence of 1 μM vanadate formed confluent monolayers and exhibited no differences in DNA, protein, or ultrastructure. Vanadate‐supplemented epithelia demonstrated a lower steady‐state α‐methyl‐D‐glucopyranoside (AMG) concentrating capacity and a twofold reduction in apical AMG uptake Jmax. This decreased AMG transport occurred as a consequence of a reduction in the number of transport carriers and was not associated with a change in the sodium electrochemical gradient. The vanadate‐induced reduction in apical glucose carrier functional activity and expression was accompanied by a stimulation of intracellular glycolytic flux activity, as evidenced by increased glucose consumption, lactate production, PFK‐1 activity, and intracellular ATP. There was no difference in intracellular cAMP levels between vanadate‐supplemented and non‐supplemented epithelia. These results demonstrate an association between stimulation of glycolytic pathway activity and an adaptive response in the form of a reduction in the function and expression of the sodium‐dependent apical glucose transporter in LLC‐PK1 epithelia. © 1994 Wiley‐Liss, Inc.
Effect of staurosporine on MOLT‐4 cell progression through G2 and on cytokinesis
Authors: Traganos Frank , Gong Jianping , Ardelt Barbara , Darzynkiewicz Zbigniew
Published: February 2005

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G1, most virus transformed or tumor cells are unaffected in G1 but arrest in G2 phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nucleolar p120 protein, or protein reactive with Ki‐67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT‐4 cells in the presence of 0.1 μM SSP.MOLT‐4 cells did not arrest in G1 or G2 phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G1 phase at higher DNA ploidy (tetraploidy; G1T), and then progressed through ST (rereplication) into G2T and MT. The rates of entrance to G2 and G2T were essentially identical, indicating that the rates of cell progression through S and ST as well as through G2 and G2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 μM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT‐4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis. © 1994 Wiley‐Liss, Inc.
Recombinant human bone morphogenetic protein‐2 enhances expression of interleukin‐6 and transforming growth factor‐β1 genes in normal human osteoblast‐like cells
Authors: Zheng Ming H. , Wood David J. , Wysocki Stan , Papadimitriou John M. , Wang Elizabet H. A.
Published: February 2005

The process of recombinant human bone morphogenetic protein‐2 (rhBMP‐2)‐induced endochondral ossification involves (1) the proliferation and differentiation of mesenchymal cells into chondroblasts and osteoblasts; (2) the production and maturation of cartilage and bone matrix; and (3) the differentiation of circulating osteoclast precursor cells into osteoclasts. Currently the molecular mechanisms of these complex sequential events are unknown. It seemed reasonable to us to assume that communication between cells through soluble mediators during bone induction by rhBMP‐2 may play an important role in the sequential differentiation of chondroblasts, osteoblasts, and osteoclasts. We have therefore used a human osteoblast‐like initial transfectant cell line (HOBIT) to study the effect of rhBMP‐2 on gene expression of interleukin‐6 (IL‐6) and transforming growth factor‐β1 (TGF‐β1), both of which affect osteogenesis and ostoeclastogenesis. Our results have demonstrated that rhBMP‐2 acts on HOBIT cells to stimulate expression of IL‐6 and TGF‐β1 genes and the production of IL‐6. Enhancement of gene expression of IL‐6 and TGF‐β1 by rhBMP‐2 was both sensitive (half maximal effect at approximately 10 ng/ml) and potent (maximum induction was approximately four and threefold greater than controls, respectively). Time course studies showed that the induction of TGF‐β1 and IL‐6 mRNA occurs within short periods—4 and 8 hours after exposure to rhBMP‐2, respectively. Interestingly, these effects, however, were not accompanied by the mitogenic action of rhBMP‐2. It suggests that rhBMP‐2 enhances IL‐6 and TGF‐β1 production during osteogenesis and at least in part mediates the complex sequential differentiation of chondroblasts, osteoblasts, and osteoclasts during rhBMP‐2‐induced endochondral ossification. © 1994 wiley‐Liss, Inc.
Failure of infinite life span human cells from different immortality complementation groups to yield finite life span hybrids
Authors: Ryan P. Ann , Maher Veronica M. , Justin McCormick J.
Published: February 2005

The observation that fusion of infinite life span cells with finite life span cells produces hybrid cells with finite life spans led to the conclusion that an infinite life span in culture is a recessive trait resulting from loss of the function of a gene or genes that contribute to an active program for cellular senescence. Furthermore, finding that certain pairs of infinite life span cells, when fused to one another, can complement each other to yield finite life span hybrids allowed 30 infinite life span cell lines to be assigned to four immortality complementation groups (Pereira‐Smith and Smith, 1988, Proc. Natl. Acad. Sci. U.S.A., 85:6042). In the present study, we fused a chromosomally stable, near diploid, morphologically normal, infinite life span cell strain, designated MSU‐1.1, with its normal, finite life span, precursor cell strain and obtained finite life span hybrids, as expected if infinite life span in culture is a recessive trait. However, 14 of the 14 hybrids from our fusions of MSU‐1.1 cells with representative cell lines from each of the four immortality complementation groups, and 38 of the 39 hybrids from our fusions of infinite life span cells that have been reported to complement each other, failed to exhibit finite life spans. This result suggests that infinite life span cells cannot complement each other to yield finite life span hybrids. In examining this unexpected result, we obtained evidence that long‐term dual drug selection can be deleterious to hybrid cells even though they carry resistance markers for both drugs, indicating that the cell death of such hybrids observed in other studies may have resulted from the cytotoxic effect of long‐term drug selection, rather than from senescence. © 1994 wiley‐Liss, Inc.
IL‐1α and TNFα act synergistically to stimulate production of myeloid colony‐stimulating factors by cultured human bone marrow stromal cells and cloned stromal cell strains
Authors: Caldwell Jerry , Emerson Stephen G.
Published: February 2005

Human bone marrow stromal cells repond to stimulation by the monokines IL‐1 and TNF by producing colony‐stimulating factors such as GM‐CSF and G‐CSF. In this study we show that IL‐1α and TNFα act synergistically to stimulate GM‐CSF and G‐CSF production by cultured marrow stromal cells. We further show that IL‐1α and TNFα synergistically stimulate production of GM‐CSF and G‐CSF by a clonal stroma‐derived cell strain. Although IL‐1 and TNF share many of the same biological activities, we show that IL‐1α and TNFα have an unequal ability to induce myeloid‐CSF production by both cultures, with IL‐1α being the more potent inducer. We found that induction by IL‐1α and TNFα was independent of cell proliferation. The effect of IL‐1α and TNFα on production of the two myeloid‐CSFs by the clonal cells was significantly greater than the unfractionated passaged stromal cultures, having the greater effect on G‐CSF production. The clonally derived stromal cells constitutively produced colony‐stimulating activity, in particular GM‐CSF, at levels easily detected by ELISA. These findings show that, in addition to the overlapping and additive activities of IL‐1α and TNFα, they can interact synergistically. Our findings further suggest that a small subpopulation of stroma cells may be the major producer of G‐CSF in the marrow microenvironment during immune response. © 1994 wiley‐Liss, Inc.
Retinoids upregulate phagocytosis by human dermal microvascular endothelial cells
Authors: Vora Meena , Karasek Marvin A.
Published: February 2005

Animals fed a diet deficient in vitamin A show severe physiological changes that often result in death. At the cellular level, retinoids have been shown to induce differentiation of cells dervied from a wide spectrum of tissues, including the vasculature. To understand further the mechanisms for these events, we studied the effects of 13‐cis‐retinoic acid, all trans‐retinoic acid, all‐trans‐retinol, and all‐trans‐retinol acetate on human dermal microvascular endothelial cells (HDMEC). Concentrations of retinoids in the physiological range from 0 to 1 μM were used in our experiments. These concentrations were nontoxic to HDMEC. Here we report that in addition to the known effect of retinoids on keratinocytes and sebacytes, retinoids induced morphological and functional changes in HDMEC that gave these cells macrophage like characteristics. 13‐Cis‐retinoic acid and all‐trans‐RA induced HDMEC to phagocytize and to increase the production of hydrogen peroxide and superoxide anion. These two retinoids also changed the morphology of endothelial cells from typical small compact cuboidal epithelioid cells to cells with larger cytoplasm and indistinct cell membranes. The retinoidstimulated HDMEC deposited increased amounts of extracellular matrix. All‐trans‐retinol and all‐trans‐retinol acetate did not significantly affect HDMEC in all parameters tested. The induction of these properties provides a new model with which to study how retinoids regulate gene expression using a normal, nontransformed cell line. © 1994 wiley‐Liss, Inc.
Heat sensitivity of Hela S3 Cell DNA topoisomerase II
Authors: Warters Raymond L. , Barrow Louis R.
Published: February 2005

The sensitivity of HeLa DNA topoisomerase II to 45°C heat shock was measured both in the intact cell and in vitro. In the intact cell, DNA topoisomerase II activity was estimated by measuring the formation and reversal of enzyme‐DNA cleavable complexes by alkaline filter elution of cells exposed to the enzyme poison 4′‐(9‐acridinylamino)(methanesulfon‐m‐anisidide). In vitro enzymatic activity was estimated by measuring changes in the topological state of plasmid and kinetoplast DNA produced by sonicates of nuclei from previously heated cells. The capacity of the enzyme to form, or reverse, enzyme‐DNA cleavable complexes was inactivated during 45°C heating with a reciprocal slope of 120 or 15 min, respectively. In vitro estimates of the activity of the enzyme from previously heated cells indicated that the enzyme was inactivated with a reciprocal slope of 99, 45, and 21 min after 45, 46 and 47°C heating, respectively. DNA topoisomerase I activity was inactivated with a reciprocal slope of 130 min at 45°C. The cumulative results indicate that during 45°C heat shock, thermal inactivation of neither DNA topoisomerase I nor II is rate limiting for either cell survival or for DNA replication. While DNA topoisomerase II is resistant in situ to heat inactivation, in vivo assays indicate that the enzyme's capacity to function in the intact cell may be compromised by hyperthermic changes in the enzyme's environment. © 1994 wiley‐Liss, Inc.
Understanding the dynamics of cellular responsiveness to modifications of metabolic substrates in perifusion
Authors: Brand Rhonda M. , Lyons R. H. , Midgley A. Rees
Published: February 2005

A novel microperifusion system with capabilities for continuous, real‐time, potentiometric monitoring of extracellular hydrogen ion concentration has been used to define the response of HeLa cells to abrupt changes in extracellular energy sources or introduction of an inhibitor of glycolysis. Glycolytic inhibition, induced by removal of glucose or introduction of iodoacetate, each led to a rapid, continuous decrease in acid release. The response to iodoacetate took longer than removal of glucose, perhaps due to the time required for binding and activation. Once inhibition began, however, the rate of change was greater than following glucose removal. Conversely, recovery time following iodoacetate inhibition was much slower than with glucose removal. Unlike the response to short‐term glucose depletion, a second pulse of iodoacetate resulted in a faster response followed by an even longer recovery time. The response to switching between glucose and glutamine began almost without evident delay. The response patterns revealed that HeLa cells prefer glutamine to glucose, but, in the presence of both energy sources, some glucose continues to be used. In summary, these results indicate that continuous, real‐time monitoring of the kinetics of hydrogen‐ion release can be used to gain new insights into the dynamics of cellular response to perturbations of extracellular energy sources. © 1994 Wiley‐Liss, Inc.
Effects of epoxyeicosatrienoic acids on 86Rb uptake in renal epithelial cells
Authors: Staudinger R. , Abraham N. G. , Schwartzman M. L. , Escalante B.
Published: February 2005

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites formed endogenously via the cytochrome P450 pathway in rat, rabbit, and human kidney. We characterized the effects of the four regioisomeric EETs on ion transport in the renal epithelial cell line, LLC‐PK1. Among the EETs, 14, 15‐EET was the most potent inhibitor of 86Rb uptake. Its effect was concentration‐dependent (IC50 = 75 nM) and stereoselective to the 14S, 15R‐EET. Experiments measuring 14, 15‐EET–induced 86Rb uptake inhibition in the presence of inhibitors of Na+‐K+‐ATPase activity (ouabain), Na+‐K+‐Cl− cotransporter (furosemide), and Na+‐H+ exchanger (amiloride) suggested that 14, 15‐EET inhibits ion transport via an amiloride‐sensitive mechanism. These results, together with previous reports demonstrating their endogenous production in the kidney, suggest an important role for EETs, specifically 14, 15‐EET, in the regulation of ion and water reabsorption in the kidney and implicate their function in renal pathophysiology. © 1994 Wiley‐Liss, Inc.
Involvement of Na+, K+‐ATPase inhibition in K562 cell differentiation induced by bufalin
Authors: Numazawa Satoshi , Shinoki Masa‐Aki , Ito Hisatomi , Yoshida Takemi , Kuroiwa Yukio
Published: February 2005

Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na+, K+ ‐ATPase prepared from K562 cells in vitro. N+, K+ ‐ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1‐T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of 3H‐bufalin and 3H‐ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of 3H‐bufalin was considerably higher than 3H‐ouabain. The saturated level of 3H‐bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of 3H‐bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N+, K+ ‐ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin. © 1994 Wiley‐Liss, Inc.
Reactive oxygen metabolite‐induced toxicity to cultured bovine endothelial cells: Status of cellular iron in mediating injury
Authors: Hiraishi Hideyuki , Terano Akira , Razandi Mahnaz , Pedram Ali , Sugimoto Tsuneaki , Harada Takashi , Ivey Kevin J.
Published: February 2005

We aimed to determine the status of iron in mediating oxidant‐induced damage to cultured bovine aortic endothelial cells. Chromium‐51‐labeled cells were exposed to reaction mixtures of xanthine oxidase/hypoxanthine and glucose oxidase/glucose; these produce superoxide and hydrogen peroxide, or hydrogen peroxide, respectively. Xanthine oxidase caused a dose dependent increase of 51Cr release. Damage was prevented by allopurinol, oxypurinol, and extracellular catalase, but not by superoxide dismutase. Prevention of xanthine oxidase‐in‐duced damage by catalase was blocked by an inhibitor of catalase, aminotriazole. Glucose oxidase also caused a dose‐dependent increase of 51Ci release. Glucose oxidase‐induced injury, which was catalase‐inhibitable, was not prevented by extracellular superoxide dismutase. Both addition of and pretreatment with deferoxamine (a chelator of Fe3+) prevented glucose oxidase‐induced injury. The presence of phenanthroline (a chelator of divalent Fe2+) prevented glucose oxidase‐induced 51Cr release, whereas pretreatment with the agent did not. Apotransferrin (a membrane impermeable iron binding protein) failed to influence damage. Neither deferoxamine nor phenanthroline influenced cellular antioxidant defenses, or inhibited lysis by non‐oxidant toxic agents. Treatment with allopurinol and oxypurinol, which inhibited cellular xanthine oxidase, failed to prevent glucose oxidase injury. We conclude that (1) among the oxygen species extracellularly generated by xanthine oxidase/hypoxanthine, hydrogen peroxide induces damage via a reaction on cellular iron; (2) deferoxamine and phenanthroline protect cells by chelating Fe3+ and Fe2+, respectively; and (3) reduction of cellular stored iron (Fe3+) to Fe2+ may be a prerequisite for mediation of oxidantinduced injury, but this occurs independently of extracellular superoxide or cellular xanthine oxidase‐derived superoxide. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Laminin SIKVAV peptide‐induced angiogenesis in vivo is potentiated by neutrophils
Authors: Kibbey Maura C. , Kleinman Hynda K. , Corcoran Marta L. , Wahl Larry M.
Published: February 2005

Angiogenesis has been investigated in vivo using subcutaneously injected reconstituted basement membrane (Matrigel) supplemented with angiogenic factors. Previously we found that the laminin‐derived synthetic peptide containing SIKVAV (ser‐ile‐lys‐val‐ala‐val) promoted angiogenesis in vivo. In parallel studies, it was observed that new vessel formation in response to this peptide occurred several days after basic fibroblast growth factor‐induced angiogenesis. Since this delay suggested that SIKVAV‐induced angiogenesis may be secondary to other events, we investigated here earlier time points to determine if both indirect and direct mechanisms of angiogenesis are involved. We found that neutrophils are continuously recruited to the SIKVAV‐containing plugs between 4 hours to 3 days following the initial injection. By day 7, columns of endothelial cells begin to migrate into the plug and form small blood vessels. In contrast, neutropenic mice had a 62% reduction in SIKVAV‐induced angiogenesis when compared to control mice. Freshly isolated neutrophils also degraded laminin, the major component of the basement membrane Matrigel. These cells also produced factors in response to SIKVAV peptide which induced proliferation of human umbilical vein endothelial cells relative to a control peptide. In vitro experiments utilizing human neutrophils demonstrated that these cells migrate to the SIKVAV peptide and possess a specific cell surface SIKVAV binding protein of ∼56 kD. These data suggest that neutrophils are induced to migrate to the Matrigel plugs, at least in part, by SIKVAV peptide, where they may release their own angiogenic factors and degrade the matrix, thus physically facilitating cell migration and liberating additional angiogenic matrix fragments and/or cytokines. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America
Inhibition of angiogenesis by tissue inhibitor of metalloproteinase
Authors: Johnson Mark D. , Kim Hyeong‐Reh Choi , Chesler Louis , Tsao‐Wu Gladys , Polverini Peter J. , Bouck Noel
Published: February 2005

Matrix proteases play a critical role in cell invasion and migration, including the process of angiogenesis. The ability of specific factors to induce angiogenic responses correlates with their stimulation of matrix protease synthesis and release. Using an in vivo angiogenesis assay, the endothelial cell response to known angiogenic factors, basic fibroblast growth factor (bfGF) and adipocyte conditioned medium, was blocked by an inhibitor of matrix metalloproteinase activity, TIMP‐1. The TIMP effect was mediated, at least in part, through the inhibition of endothelial cell migration, as determined by the ability of TIMP to block chemotaxis in a Boyden chamber assay. These results indicate that the inhibition of migration is a direct effect on the endothelial cells and does not require accessory cells. An additional observation was that the RNA levels for TIMP were significantly reduced in differentiated adipocytes, compared to undifferentiated F442A controls. Therefore, the acquisition of an angiogenic phenotype may involve not only the induction of positive factors, but also the suppression of angiogenesis inhibitors. © 1994 Wiley‐Liss, Inc.
Senescence and cell density of human diploid fibroblasts influence metabolism of insulin‐like growth factor binding proteins
Authors: Grigoriev Vitalii G. , Moerman Elena J. , Goldstein Samuel
Published: February 2005

In order to analyze changes in metabolism of insulin‐like growth factor binding proteins (IGFBPs) related to cell senescence and cell density, we compared human diploid fibroblasts (HDF) in the proliferatively vigorous first half (young cells) and senescent HDF in the last 10% (old cells) of the replicative lifespan after seeding cells over an eighfold range and proliferation to high density. Increasing the seeding cell density of both young and old HDF led to elevated rates of IGFBP‐3 secretion, an increasing ratio of the 42/38 kDa species of IGFBP‐3, and degradation of all species of IGFBPs derived from both the fetal bovine serum component of the culture medium and from HDF. At a given seeding density old HDF produced more IGFBP‐3 and degraded more IGFBPs than young HDF. IGFBP‐4 was degraded by a protease that appeared to be different from the protease(s) involved in degradation of the other IGFBPs. Young HDF at all seeding densities contained a cell‐associated 29 kDa IGFBP, whereas this protein could not be detected in old cells. Thus, although certain changes in IGFBP metabolism are similar in young HDF seeded at high densities and in old HDF, young and old phenotypes can be distinguished by characteristic qualitative and quantitative changes in IGFBPs derived from fetal bovine serum and from HDF. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America
Stable differentiation of a human colon adenocarcinoma cell line by sodium butyrate is associated with multidrug resistance
Authors: Ho Samuel B. , Yan Pei‐Sha , Dahiya Rajvir , Neuschwander‐Tetri Brent A. , Basbaum Carol , Kim Young S.
Published: February 2005

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat‐appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T‐D. The LS174T‐D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T‐D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)‐IV and aminopeptidase. The activities of DAP‐IV and aminopeptidase were increased 5.6‐fold and 3.4‐fold, respectively, in LS174T‐D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T‐D cells were more resistant to doxorubicin (93‐fold), cisplatin (23‐fold), 5‐fluorouracil (12‐fold), 5‐fluorodeoxyuridine (31‐fold), and methotrexate (12.5‐fold). Intracellular uptake of (3H)‐5‐fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr‐1 p‐glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione‐S‐transferase activities were increased in LS174T‐D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL‐buthionine sulfoximine reversed LS174T‐D resistance to cisplatin. Long‐term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione‐mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America
Regulation of γ‐actin gene expression by insulin
Authors: Messina Joseph L.
Published: February 2005

Insulin exerts rapid effects on cellular metabolism and can cause morphological changes by inducing rearrangements of cytoskeletal components. The regulation of specific cytoskeletal genes by insulin, however, has not been studied extensively. In the present work insulin was found to rapidly, but transiently, increase transcription of the cytoskeletal γ‐actin gene in rat H4IIE (H4) hepatoma cells. Insulin‐induced transcription of the γ‐actin gene was evident within 5 min and was maximal by 15 min at 10‐fold above control levels. The stimulation of transcription was transient, with a return towards basal levels by 120 min. Transcription of γ‐actin was increased at insulin concentrations as low as 1 × 10−11 M and was maximal at 1 × 10−9 to 1 × 10−8 M. Transcription of several control genes (skeletal and cardiac α‐actin and β‐tubulin) were unaltered by insulin administration. Messenger RNA (mRNA) levels for the γ‐actin gene increased, but to a lesser degree than transcription. Since the γ‐actin message is an abundant and stable mRNA, its levels would not be expected to change dramatically from a transient induction of transcription. Like insulin, phorbol esters transiently increased transcription of the γ‐actin gene. In addition, pretreatment of cells with phorbol esters for 24 h reduced the ability of insulin to induce γ‐actin transcription. These data support our hypothesis that insulin and phorbol esters share intracellular signalling pathways in the control of transcription of specific genes. © 1994 Wiley‐Liss, Inc.
Cultured rat hepatocytes adapt their cellular glycolytic activity and adenylate energy status to tissue oxygen tension: Influences of extracellular matrix components, insulin and glucagon
Authors: Ohno Kouji , Maier Peter
Published: February 2005

The influence of extracellular matrix components, insulin, and glucagon on the cellular response to periportal‐ or pericentral‐equivalent tissue oxygen tension was investigated in freshly isolated rat hepatocytes cultured at 13% O2 or 4% O2 in Teflon membrane dishes. With extended culture time, significant increases in lactate release and cellular lactate content were observed in cultures at 4% O2 compared with 13% O2. This shift toward glycolysis was detectable when hepatocytes were cultured on dishes coated with rat liver crude membrane fraction (CMF/COL) but not in collagen type I‐coated dishes. This indicates that extracellular matrix components are involved in the process of adaptation. ATP and total adenylate content in cells cultured at 4% O2 were up to 40% lower than in cells cultured at 13% O2. However, the adenylate energy charge was not affected, suggesting that an adequate energy supply was maintained also in hepatocytes cultured at pericentral‐equivalent oxygen tension. This adaptation was reversible. When hepatocytes were transferred either from 4% to 13% O2 or from 13% to 4% O2, they adapted the corresponding metabolic profile to the new oxygen tension within 2 days. This demonstrates that hepatocytes are not fully unidirectionally programmed. The modulation of the glycolytic activity by insulin and glucagon was effective in cultures at pericentral‐equivalent oxygen tension (4% O2) only. Insulin (0.1‐100 nM) counteracted the effect of insulin in a dose‐dependent manner. Clearly, oxygen tension is the principal regulator in the hepatic glycolytic activity, whereas the hormones (insulin and glucagon) act as secondary modulators. © 1994 Wiley‐Liss, Inc.
Enhancement of proliferation in cultures of Chinook salmon embryo cells by interactions between inosine and bovine sera
Authors: Ganssin R. C. , Tran Q. H. , Rabgey T. F. , Bols N. C.
Published: February 2005

The influence of inosine on DNA synthesis by Chinook salmon embryo cells (CHSE‐214) was investigated because previously cell number was shown to increase from six‐ to thirtyfold if inosine was added to the basal medium (L‐15) supplemented with either dialyzed fetal bovine serum (dFBS), calf serum (CS), or dCS. Relative to L‐15, 3H‐thymidine incorporation was inhibited by these sera alone but elevated in nondialyzed (intact) FBS. Inosine at 10 μM stimulated 3H‐thymidine incorporation from ten‐ to seventyfold in dFBS, CS, and dCS but was only slightly stimulatory in FBS and in L‐15 alone. As well as inosine, hypoxanthine, cIMP, IMP, IDP, and ITP were just as stimulatory, but the nonsalvageable purines (xanthine, xanthosine, and XMP) were not. The stimulatory action of inosine was highest in low density cultures. Dipyridamole and S‐(p‐nitrobenzyl)‐6‐thioinosine (NBTI), inhibitors of facilitated nonconcentrative nucleoside transport, did not completely block the enhancement of cell number by inosine and by themselves increased proliferation in CS and dCS. Overall, these results suggest that exogenous inosine promoted CHSE‐214 proliferation by overcoming factors in the nondialyzable fraction of sera that led to purine loss and by raising intracelular purine nucleotides to levels necessary for cells to respond to growth factors in the nondialyzable fraction of sera. © 1994 Wiley‐Liss, Inc.
Global change of gene expression at late G1/S boundary may occur in human IMR‐90 diploid fibroblasts during senescence
Authors: Pang Jong Hwei , Chen Kuang Yu
Published: February 2005

The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR‐90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263: 11431–11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR‐90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR‐90 cells but not in old IMR‐90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age‐dependent attenuation in activities. In contrast, expression of growth‐related genes such as elF‐5A, c‐Ha‐ras, and β‐actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp‐1 binding site as the most common cis‐element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled. © 1994 Wiley‐Liss, Inc.
Autocrine downregulation of fibroblast growth factor receptors in F9 teratocarcinoma cells
Authors: Moscatelli David
Published: February 2005

The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind 125I‐basic FGF (FGF‐2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of 125I‐FGF‐2 was due to an increase in the number of receptors with no change in their affinity. When 125I‐FGF‐2 was cross‐linked to cell surface receptors, an increase in FGF‐2‐receptor complexes with molecular weights of 140,000–160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF‐4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF‐receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased 125I‐FGF‐2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF‐4 increased the 125I‐FGF‐2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells. © 1994 Wiley‐Liss, Inc.
Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands
Authors: Métioui Mourad , Grosfils Katrina , Dehaye Jean‐Paul
Published: February 2005

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura‐2.

In the absence of extracellular magnesium and calcium ([Ca2+]o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca2+]o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura‐2; 10 m̈M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca2+]in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca2+‐free/Ca2+ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca2+]in and the calcium uptake but potentiated the response to ATP.

These results suggest that, after binding to purinergic receptors, extracellular ATP4‐ increases [Ca2+]in. ATP4‐ does not mobilize thapsigargin‐sensitive intracellular calcium pools (among which is the IP3‐sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley‐Liss, Inc.
Signalling the molecular stress response to nephrotoxic and mutagenic cysteine conjugates: Differential roles for protein synthesis and calcium in the induction of c‐fos and c‐myc mRNA in LLC‐PK1 cells
Authors: Yu Kefu , Chen Qin , Liu Hong , Zhan Yi , Stevens James L.
Published: February 2005

Nephrotoxic and mutagenic cysteine conjugates (NCC) are activated by the enzyme cysteine conjugate, β‐lyase, to reactive acylating species which bind covalently to cellular macromolecules. We now show that an early event after treatment of LLC‐PK1 cells with NCC is the induction of mRNA for both c‐fos and c‐myc. Treatment with S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC) induced c‐fos (53‐fold) and c‐myc mRNA (20‐fold) and increased transcription about 3‐fold for both genes. Covalent binding was required for induction of both mRNAs. Dithiothreitol partially prevented induction of both c‐fos and c‐myc RNA. Buffering the DCVC‐induced increase in cytosolic free calcium had no effect on c‐fos mRNA but partially blocked c‐myc mRNA induction. Cycloheximide blocked the induction of c‐myc mRNA in the absence of an effect on c‐fos induction. The data suggest that the increase in c‐fos mRNA is a primary response to DCVC toxicity and occurs without a requirement for protein synthesis or an increase in intracellular free calcium. In contrast, c‐myc induction requires protein synthesis, suggesting that the presence of another primary response factor may regulate induction either transcriptionally or posttranscriptionally. The data suggest that different signalling pathways regulate induction of c‐fos and c‐myc mRNA in response to stress caused by reactive acylating species. © 1994 Wiley‐Liss, Inc.
Native TIMP‐free 70 kDa progelatinase (MMP‐2) secreted at elevated levels by RSV transformed fibroblasts
Authors: Stefansson Steingrimur , Aimes Ronald T. , Seward Nancy B. , Alexander Daniela S. , Quigley James P.
Published: February 2005

Rous sarcoma virus‐transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase‐2 (MMP‐2). Affinity‐purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP‐2 and a native 70 kDa progelatinase free of any detectable TIMP‐2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS‐PAGE and nondenaturing, native gel electrophoresis. The purified TIMP‐free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP‐free progelatinase with the organomercurial, p‐aminophenylmercuric acetate (APMA), results in rapid (5‐60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP‐free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP‐complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6‐24 hours and the specific enzyme activity of the preparation is 8 to 18‐fold lower. Purified avian TIMP‐2 added to the TIMP‐free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP‐free progelatinase than the normal CEF cultures which produce predominantly TIMP‐complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP‐free progelatinase, the suppression of its rapid activation by TIMP‐2, and the potential effect of the altered balance between TIMP‐free and TIMP‐complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed. © 1994 Wiley‐Liss, Inc.
Insulin‐like growth factor (IGF) binding to a murine mammary epithelial cell line
Authors: Hadsell Darryl L. , Gibson C. Anne , Baumrucker Craig R.
Published: February 2005

The goal of this study was to relate insulin‐like growth factor (IGF) binding with IGF‐stimulated growth in a murine mammary epithelial (COMMA‐D/MME) cell line. Affinity crosslinking with [125I]IGF‐I showed major bands at 224,000 and 148,000 Mr that were ablated by the inclusion of IGF‐I or ‐II at 130 nM. Scatchard‐transformed [125I]IGF‐I binding data was best fit by a two‐site model, with 24,000 sites possessing a Kd of 0.33 nM and 1,000 sites possessing a Kd of 8.09 nM per cell. Competition analysis showed ED50 values for IGF‐I, ‐II, and insulin to be 0.90 ± 0.03, 7.15 ± 4.27, and 335 ± 104 nM, respectively. Affinity crosslinking of [125I]IGF‐II showed three major bands of 230,000, 148,000, and 61,000 to 58,000 Mr. Unlabeled IGF‐II ablated all bands, while IGF‐I and insulin ablated only the 148,000 Mr band. Competition analysis showed ED50 values for unlabeled IGF‐I and ‐II to be 0.10 ± 0.01 and 5.31 ± 2.04 nM, respectively. In spite of the receptor affinity differences, no significant difference was noted in IGF‐I and ‐II in capacity to stimulate cell growth. These data indicate that COMMA‐D/MME cells express IGF receptors and that both IGFs are mitogenic. © 1994 Wiley‐Liss, Inc.