bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular expression of bone‐related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures
Authors: Malaval Luc , Modrowski Dominique , Gupta Ashwani K. , Aubin Jane E.
Published: February 2005

Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, β‐glycerophosphate and 10−8M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317–330; Aubin et al., 1990, J. Bone Miner. Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of osteoblastic markers. Immunocytochemistry was combined with Northern blotting, enzymatic assay, and radioimmunoassay to analyze the expression of bonerelated proteins during the growth and differentiation sequence. By mRNA levels, protein production and/or enzymatic activity, expression of osteocalcin, bone sialoprotein, and alkaline phosphatase increased concomitantly with the development of bone nodules, while osteopontin mRNA levels decreased and those of SPARC/osteonectin did not change significantly. In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteocalcin, declined. Immunolabeling revealed that cells in early cultures stained poorly for SPARC/osteonectin and strongly for thrombospondin. Later, SPARC/osteonectin staining increased in most cells, while thrombospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity. At all time points studied, osteoblasts within bone nodules stained homogeneously for thrombospondin and alkaline phosphatase, and with marked heterogeneity of intensity amongst cells for SPARC/osteonectin and osteocalcin. Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cells which Intensely labels osteoblasts and osteocytes (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339–1352), co‐localized with osteocalcin. Alkaline phosphatase activity and the amount of osteocalcin determined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules. These studies indicate that the bone marrow stromal cell system is a useful model to study the temporal and spatial expression of bone‐related proteins during osteogenesis and formation, mineralization, and maturation of bone nodules. Further, immunolabelling at the individual cell and single bone nodule level allowed discrimination of marked variability of expression of osteoblast markers during the differentiation sequence. © 1994 Wiley‐Liss, Inc.
Activation of serpins and their cognate proteases in muscle after crush injury
Authors: Festoff Barry W. , Reddy Rajendra B. , Vanbecelaere Marnie , Smirnova Irina , Chao Julie
Published: February 2005

Direct muscle injury was induced in rats in order to evaluate alterations in the balance of serine proteases and inhibitors (serpins) as a response to tissue damage. It was previously found that certain proteases, specifically urokinase‐like plasminogen activator (uPA) and others, required activation in order to effect regeneration. We hypothesized that the magnitude and temporal sequence of serpin activation would follow, pari passu, activation of their cognate proteases. In addition to uPA, tissue PA (tPA) and tissue kallikrein were the proteases studied. The serpins we analyzed were protease nexin I (PNI), PA inhibitor 1 (PAI‐1), and the kallikrein‐binding protein (KBP). uPA nearly doubled 48 h after injury, while there was no change in amidolytic activity after addition of fibrin monomer as an estimation of tPA activity. Tissue kallikrein activity, barely detectable in normal muscle, slowly increased, nearly tripling at 7 days after injury. Greater magnitude and more rapid changes in muscle serpins occurred over the same post‐injury time course. By 24 h PNI increased threefold, while PAI‐1 increased more slowly, reaching double the control values by 5 days after injury. Surprisingly, KBP, the serpin‐class inhibitor of tissue kallikrein, had the most robust response, increasing tenfold over control 48 h after crush injury of muscle. These results further implicate the serpin:protease balance in tissue injury. Participation of complex receptors, such as the α2‐macroglobulin receptor/low density lipoprotein receptor‐related protein (LRP), various growth factors, cytokines, and other molecules, in regulating this balance is implicated by these data. © 1994 wiley‐Liss, Inc.
Stimulation of hepatocyte DNA synthesis by prostaglandin E2 and prostaglandin F2α additivity with the effect of norepinephrine, and synergism with epidermal growth factor
Authors: Refsnes Magne , Thoresen G. Hege , Dajani Olav F. , Christoffersen Thoralf
Published: February 2005

Previous data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE2 and PGFF2α have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGFα]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum‐free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE2, PGF2α, PGD2, and the synthetic analog dimethyl‐PGE2 markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE2 and PGF2α on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE2 and PGF2α showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest (a) that PGE2 and PGF2α facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and (b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine. © 1994 wiley‐Liss, Inc.
Coordinate and independent regulation of αB‐crystallin and HSP27 expression in response to physiological stress
Authors: Head Mark W. , Corbin Elaine , Goldman James E.
Published: February 2005

α‐Crystallins share structural and functional properties with the stress protein hsp27. These polypeptides are expressed at low constitutive levels in many tissues including brain, and αB‐crystallin and hsp27 can accumulate in central nervous system glia in a variety of neurological conditions. We report here that heat shock and exposure to transition metals result in an increase in the steady state mRNA level of αB‐crystallin and hsp27 in primary cultures of rat forebrain astrocytes. Both exposure to tumour necrosis factor‐α and hypertonic conditions result in αB‐crystallin mRNA accumulation but no change in the hsp27 mRNA level. Under some of these conditions increased synthesis and accumulation of αB‐crystallin and hsp27 protein are also evident. We are unable to detect αA‐crystallin mRNA in resting or stressed astrocytes. A novel phenomenon involving a transitory change in stress protein mRNA mobility in Northern blots during induction is reported, which is stress type and cell type independent. The results demonstrate multiple stress regulation of αB‐crystallin and hsp27 in cultured astrocytes, suggesting that they can legitimately be regarded as stress proteins in the central nervous system. © 1994 wiley‐Liss, Inc.
Dependence of collagen remodelling on α‐smooth muscle actin expression by fibroblasts
Authors: Arora Pamela D. , McCulloch Christopher A. G.
Published: February 2005

To study the relation between expression of the putative myofibroblast marker α‐smooth muscle actin and the remodelling of extracellular matrix, immunocytochemical, gel electrophoresis, and collagen gel contraction studies were performed on two human fibroblast subtypes. Double immunolabelling for total actins and α‐smooth muscle (sm) actin as well as affinity labelling of filamentous and monomeric actins in gingival fibroblasts demonstrated that α‐sm was colocalized in stress fibres and in regions with high levels of monomeric actin throughout the cytoplasm. α‐sm comprised up to 14% of total cellular actin as assessed by 2D gel electrophoresis. Thirteen different gingival and seven different periodontal ligament fibroblast lines constitutively expressed on α‐sm actin. These cells exhibited up to 60% inter‐line variations of fluorescence due to α‐sm actin and up to 70% and 45% inter‐line variation in the rate of collagen gel contraction. Quantitative, single cell fluorimetry of α‐sm actin immunoreactivity demonstrated a linear relation between gel contraction and α‐sm actin (correlation coefficients of 0.71 for gingival and 0.61 for periodontal ligament cells), but there was no detectable relationship between total actin content and gel contraction. In contrast, flow cytometry demonstrated that 99% of the total gated cells from cell lines exhibiting rapid gel contraction showed α‐sm actin staining above background fluorescence as compared to only 35% of cells with slow rates of gel contraction. Contracting collagen gels stained with FITC‐phalloidin showed cells with well‐developed stress fibres that were progressively more compact and elongated during the time of maximal gel contraction. To examine the dependence of gel contraction on assembly of monomeric actin into actin filaments, cells were electroporated in the presence of phalloidin or cytochalasin D. Collagen gels exhibited up to 100% inhibition of gel contraction that was dose‐dependent. Gel contraction was inhibited 93% by electroinjection of cells with α‐sm actin antibody prior to incubation, but the antibody did not inhibit actin assembly after attachment and spreading on substrates. These data indicate that gel contraction is dependent on α‐sm actin expression and that α‐sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix. © 1994 wiley‐Liss, Inc.
Electrogenic NaK pump current in rat skeletal myoballs
Authors: Li Kai‐Xun , Sperelakis Nicholas
Published: February 2005

Catecholamines and insulin have been reported to hyperpolarize skeletal muscle fibers via stimulation of the electrogenic Na‐K pump (Flatman and Clausen, 1979, Nature, 281:580–581). Therefore, the electrogenic Na‐K pump current was investigated in cultured colcemid‐treated rat skeletal myoballs using whole‐cell voltage clamp. Skeletal muscles were taken from newborn rat hindlegs, trypsin digested, and cultured. By day 7, all myoblast cells fused into myotubes. After treatment with the microtubule disrupter colcemid (10−7 M) for 2 days, some of the myotubes became transformed into spherical myoballs, having an average diameter of 41.2 ± 1.5 μm (n = 21). The resting membrane potential averaged ‐56.8 ± 1.7 mV (n = 40). Ouabain (1 mM) quickly depolarized the myoballs to ‐51.1 ± 1.1 mV (n = 27), showing the existence of an electrogenic Na‐K pump in the skeletal myoball preparation. The values for the specific membrane resistance and capacitance were 5.5 ± 1.0 KΩ‐cm2 (n = 21) and 3.7 ± 0.3 μF/cm2 (n = 21), respectively. The pump current averaged 0.28 ± 0.03 pA/pF (n = 10), with the membrane potential at ‐60 mV and 10 mM intrapipette Na+. The Na‐K pump contribution to resting membrane potential was calculated to be 5.7 mV, matching the ouabain‐induced rapid depolarization. When the Na‐K pump was stimulated with 50 mM intrapipette Na+, the pump current was about doubled (0.52 ± 0.08 pA/pF; n = 10). Isoproterenol (1 μM) and 8‐Br‐cAMP (1 mM) also significantly increased pump current by 50% (0.42 ± 0.04 pA/pF; n = 9) and 64% (0.46 ± 0.09 pA/pF; n = 7), respectively. In contrast, although insulin and phorbol ester also increased pump current, this increase was not statistically significant. The ineffectiveness of insulin and phorbol ester may be due to colcemid interfering with Na‐K pump translocation from internal vesicles to the sarcolemma. © 1994 wiley‐Liss, Inc.
Role of membrane surface potential and other factors in the uptake of non‐transferrin‐bound iron by reticulocytes
Authors: Quail Elizabeth A. , Morgan Evan H.
Published: February 2005

Reticulocytes suspended in low ionic strength media such as isotonic sucrose solution efficiently take up non‐transferrin‐bound iron and utilize it for heme synthesis. The present study was undertaken to determine how such media facilitate iron utilization by the cells. The effects of changes in membrane surface potential, membrane permeability, cell size, transmembrane potential difference, oxidation state of the iron, and lipid peroxidation were investigated. Iron uptake to heme, cytosol, and stromal fractions of cells was measured using rabbit reticulo‐cytes incubated with 59Fe‐labelled Fe(II) in 0.27 M sucrose, pH 6.5. Suspension of the cells in sucrose led to increased membrane permeability, loss of intracellular K+, decreased cell size, and increased transmembrane potential difference. However, none of these changes could account for the high efficiency of iron uptake which was observed. The large negative membrane surface potential which occurs in sucrose was modified by the addition of mono‐, di‐, tri‐, and polyvalent cations to the solution. This inhibited iron uptake to a degree which for many cations varied with their valency. Other cations (Mn2+, Co2+, Ni2+, Zn2+) were also very potent inhibitors, probably due to direct action on the transport process. Ferricyanide inhibited iron uptake, while ferrocyanide and ascorbate increased the uptake of Fe(III) but not Fe(II). It is concluded that the high negative surface potential of reticulocytes suspended in sucrose solution facilitates iron uptake by aiding the approach of iron to the transport site on the cell membrane. The iron is probably transported into the cell in the ferrous form. © 1994 wiley‐Liss, Inc.
Nitric oxide and energy production in articular chondrocytes
Authors: Stefanovic‐Racic M. , Stadler J. , Georgescu H. I. , Evans C. H.
Published: February 2005

Addition of human, recombinant interleukin‐1β (hrIL‐1β) to cultures of lapine articular chondrocytes provoked a delayed increase in the production of both nitric oxide (NO) and lactate. These two phenomena followed a similar time course and shared a parallel dose‐response sensitivity to hrIL‐1β. A causal relationship is suggested by the ability of N‐monomethyl‐L‐arginine (NMA), an inhibitor of NO synthase, to blunt the glycolytic response to hrIL‐1β. Furthermore, addition of S‐nitroso‐N‐acetylpenicillamine (SNAP), which spontaneously generates NO in culture, increased lactate production to the same degree as IL‐1. However, 8‐Br‐cGMP and isobutylmethylxanthine (IBMX) had no effect either in the presence or absence of IL‐1. Even under standard, aerobic, cell culture conditions, chondrocytes consumed little oxygen, either in the presence or absence of IL‐1 or NMA. Furthermore, cyanide at concentrations up to 100 μM had no effect upon NO synthesis or lactate production. Thus, the increases in glycolysis under study were not secondary to reduced mitochondrial activity. Although cells treated with IL‐1 had increased rates of glycolysis, their concentrations of ATP fell below those of untreated chondrocytes in a time‐dependent, but NMA‐independent, manner. Transforming growth factor‐β (TGF‐β) and synovial cytokines (CAF) also increased lactate production. However, TGF‐β failed to induce NO, and its effect on glycolysis was independent of NMA. Furthermore, cells treated with TGF‐β were not depleted in ATP. These data are consistent with hypotheses that rates of proteoglycan synthesis are, in part, regulated by the intracellular concentration of ATP or by changes in pericellular pH. These two possibilities are not mutually exclusive. © 1994 wiley‐Liss, Inc.
Changes in mitochondrial shape and distribution induced by ethacrynic acid and the transient formation of a mitochondrial reticulum
Authors: Soltys Bohdan J. , Gupta Radhey S.
Published: February 2005

We have examined the effect of ethacrynic acid on mitochondrial morphology and distribution as well as on cellular toxicity in cultured human fibroblasts, African Green Monkey B‐SC‐1 kidney cells, and Chinese hamster ovary cells. Treatment of the above cells with 66 μM ethacrynic acid causes no reduction in cell viability after 2 h but is cytotoxic upon prolonged (6–7 days) exposure. Ethacrynic acid treatment for up to 2 h is found to cause novel shape changes and redistribution of mitochondria, as assessed by immunofluorescence and electron microscopy. Early effects include the transient formation of a mitochondrial reticulum involving the majority of mitochondria, and these reticula are aligned along microtubules. At later times within 2 h, mitochondrial distributions become disoriented (show no association with microtubules), and an aggregation and final positioning of mitochondria around the nucleus is observed. Whole mount electron microscopy shows that mitochondria in treated cells increase in length and form junctions, indicating reticula result from mitochondrial fusion. Electron microscopy of sections through ethacrynic acid induced reticula demonstrates structural continuity in mitochondria at branch points and the presence of regular cristae. Staining of endoplasmic reticulum and mitochondria in intact cells with the cyanine dye 3,3′‐dihexyloxacarbocyanine iodide provides evidence of concurrent aggregation of endoplasmic reticulum. Rhodamine 123 staining of living cells followed by immunofluorescent labeling of mitochondria in the same cells indicates that all mitochondria retain a transmembrane potential during the druginduced shape changes and redistributions. The described effects of ethacrynic acid on mitochondrial morphology as well as on cellular toxicity are completely prevented by 0.5 mM dithiothreitol, indicating that ethacrynic acid is acting as a sulfhydryl reagent to produce the observed effects. The above observations also indicate that ethacrynic acid effects on mitochondrial morphology are an early event in the drug‐induced cytotoxicity. The generation of varied mitochondrial morphologies by fusion and fission of mitochondria and its modulation by agents such as ethacrynic acid are discussed. © 1994 wiley‐Liss, Inc.
Staurosporine, a non‐specific PKC inhibitor, induces keratinocyte differentiation and raises intracellular calcium, but Ro31–8220, a specific inhibitor, does not
Authors: Jones K. T. , Sharpe G. R.
Published: February 2005

The responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non‐specific inhibitor), and Ro31–8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31–8220 had little effect on morphology or growth and blocked both the TPA‐induced growth inhibition and calcium rise. Ro31–8220 had no effect on staurosporine‐induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31–8220 inhibits TPA‐induced differentiation. © 1994 wiley‐Liss, Inc.
Role of cell density/cell‐cell contact, and growth state in expression of differentiated properties by the LLC‐PK1 Cell
Authors: Amsler Kurt
Published: February 2005

Populations of the renal epithelial cell line, LLC‐PK,1, acquire many properties characteristic of the proximal tubular cell at confluence. At confluence cells both enter a nonproliferative state and develop extensive cell‐cell contacts. To determine if one or both factors is responsible for acquisition of the differentiated phenotype, growth arrest was initiated in populations of varying densities by two procedures (serum deprivation and thymidine block) and expression of several differentiated properties (Na‐hexose symport activity, gamma‐glutamyl transpeptidase activity, alkaline phosphatase activity, and villin protein) was examined. Induction of growth arrest resulted in expression of all differentiated properties even in subconfluent populations. The level of expression in a population was proportional to cell density at the initiation of growth arrest; higher density was associated with increased expression. Evidence indicated the existence of some minimal density below which cells could not express detectable levels of differentiated properties in response to induction of growth arrest. The procedure used to initiate growth arrest did not affect this behavior, indicating that initiation of cell growth arrest rather than hormone deprivation was the inducing factor. These results indicate that both cell growth state and cell density independently modulate expression of differentiated properties by the LLC‐PK1 cell. These results are incorporated into a model in which cells in the absence of “appropriate” cell‐cell contact arrest at a differentiation‐incompetent cell cycle point. In the presence of appropriate cell‐cell contact (as yet undefined) cells arrest at a distinct differentiation‐competent cell cycle point and initiate expression of the differentiated phenotype. © 1994 wiley‐Liss, Inc.
Engagement of the T‐cell antigen receptor by anti‐CD3 monoclonal antibody causes a rapid increase in lymphocyte F‐actin
Authors: Phatak Pradyumna D. , Packman Charles H.
Published: February 2005

Activation of protein kinase C (PKC) causes a rapid and sustained increase in the F‐actin of T lymphocytes. Because the phosphatidylinositol pathway and the cytoskeleton play a role in lymphocyte activation, we examined the relationship between signal transduction and the F‐actin increase in human blood T cells. Anti‐CD3 monoclonal antibodies (mAbs) initiate signals which result in activation of T lymphocytes through the T‐cell receptor (TCR), involving the phosphatidylinositol pathway, activation of PKC, and increasing intracellular calcium (Cai2+). The fluorescent probe NBD‐phallacidin was used to examine the conformational state of actin following stimulation of T lymphocytes with anti‐CD3 mAb. Each of three different murine anti‐CD3 mAbs caused rapid increases in lymphocytic F‐actin content, which was enhanced by cross‐linking with a goat anti‐mouse IgG. A maximally effective dose of the mAb Leu 4 caused a rise in cellular F‐actin of 1.8‐fold at 2 minutes and a three‐fold increase in Cai2+. Ionomycin, 100 nM, caused a Cai2+ rise similar in magnitude to that caused by anti‐CD3 mAb but had no effect on F‐actin content. Inhibitors of PKC, 1(5‐isoquinolinylsulfonyl)‐2methylpiperazine (H7), sphingosine, and sphinganine lowered the resting cellular F‐actin and partially blocked the increase in F‐actin caused by either anti‐CD3 mAb or ionomycin; however, they had no effect on the rise in Cai2+. Cells leached of Ca2+ with EGTA and ionomycin exhibited no Cai2+ increase in response to anti‐CD3 mAb or ionomycin; such cells retained the F‐actin increase caused by anti‐CD3 mAb. We conclude that stimulation of human T lymphocytes via the TCR causes an early rapid increase in F‐actin content. Activation of PKC may play a role but the concomitant Cai2+ increase is neither sufficient nor necessary for the F‐actin increase. © 1994 wiley‐Liss, Inc.
Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid‐induced decreases in prostaglandin production and prostaglandin synthase activity
Authors: Chromiak Joseph A. , Vandenburgh Herman H.
Published: February 2005

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avlan skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10−8 M Dex reduced PGF2α production 55–65% and PGE2 production 84–90% after 24–72 h of incubation. Repetitive 10% stretch‐relaxations of non‐Dex‐treated cultures increased PGF2α efflux 41% at 24 h and 276% at 72 h, and increased PGE2 production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex‐treated cultures increased PGF2α production 162% after 24 h, returning PGF2α efflux to the level of non‐Dex‐treated cultures. At 72 h, stretch increased PGF2α efflux 65% in Dex‐treated cultures. Mechanical stimulation of Dex‐treated cultures also increased PGE2 production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8–24 h of incubation, and mechanical stimulation of the Dex‐treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex‐induced declines in PGHS activity and prostaglandin production. © 1994 wiley‐Liss, Inc.
Uneven distribution of protein kinase C‐α and ‐β isozymes in human sarcomas and carcinomas
Authors: Cuadrado Antonio , Issing Wolfgang , Fleming Timothy P. , Molloy Christopher J.
Published: February 2005

Protein kinase C (PKC) represents a family of structurally related Ser/Tre kinases which are involved in mitogenic signalling and may contribute to human neoplasia. To address this issue, the messenger RNA and protein levels of PKC isoenzymes α and β were analyzed in several human sarcoma‐ and carcinoma‐derived cell lines. Carcinomas contained low or undetectable levels of either PKC‐α or PKC‐β. Sarcomas exhibited similar or increased PKC expression compared to human diploid fibroblasts. Moreover, sarcoma cell lines expressing one PKC isoform did not contain detectable levels of the other. When PKC was depleted from the tumor cells, we observed that the PKC overexpressing sarcomas had reduced their malignant properties as determined by their ability to grow in semisolid medium. In addition, epidermal growth factor‐stimulated and erbB2‐transformed fibroblasts exhibited enhanced cell growth in the absence of PKC. We propose a model for the effect of PKC as a negative regulator of proliferation in epithelial cells and a growth promoter in fibroblasts. © 1994 wiley‐Liss, Inc.
Phenotypic modulation of keratins, vimentin, and α‐fetoprotein in cultured rat liver epithelial cells after chemical, oncogene, and spontaneous transformation
Authors: Bisgaard Hanne Cathrine , Ton Phuongnga T. , Nagy Peter , Thorgeirsson Snorri S.
Published: February 2005

Several lines of evidence have indicated that rat liver epithelial (RLE) cell lines may be related to a dormant stem cell compartment in the liver in vivo. We have demonstrated that keratin 14 (K14) is expressed together with vimentin in undifferentiated RLE cells. However, upon spontaneous transformation and differentiation to hepatoblast‐like progeny the expression of these intermediate filaments (IF) is abrogated, while expression of another set of genes, among others keratin 18 (K18) and α‐fetoprotein (AFP), is induced (Bisgaard et al., 1994, J. Cell. Physiol., in press). To better understand the mechanisms underlying IF expression during transformation and differentiation of RLE cells we examined the expression and regulation of IFs in clonal cell lines of chemically, oncogene, and spontaneously transformed RLE cells and their resulting tumors. These clonal lines provided a wide variety of tumor phenotypes including trabecular, solid and tubular adeno‐carcinomas, undifferentiated carcinomas, and spindle cell carcinomas. Northern blot analysis of the cell lines confirmed the differential expression of IF mRNAs. While keratin 8 (K8) was expressed at similar steady‐state levels in all cell lines, K14 and vimentin but not K18 were expressed in the majority of cell lines chemically transformed with aflatoxin B1 or by transduction of oncogenes. In contrast, cell lines transformed spontaneously by prolonged passage in vitro expressed K18, while K14 and vimentin were absent. The keratin expression pattern in vitro was retained in the majority of the resulting tumors. However, the keratins expressed in vitro did not accurately predict the tumor phenotype in vivo. In particular, in tumors typed morphologically as adenocarcinomas, the keratin pair typically expressed in chemically transformed tumor cells was K8/K14, whereas K8/K18 was expressed in the tumors derived from spontaneously transformed cell lines. Finally we showed by nuclear run‐on and in vitro translation analyses that the expression of K14, K18, and vimentin in transformed RLE cell lines was regulated at the transcriptional level, whereas that of K8 appeared to be posttranslational. These findings suggest that events controlling the differential expression of IF genes are involved in the processes leading to transformation and differentiation of the RLE cell lines. We conclude that the transformed RLE cell lines provide a valuable model to further examine the regulatory mechanisms involved in hepatic differentiation of undifferentiated “progenitor‐like” RLE cells. © 1994 wiley‐Liss, Inc.
NIH 3T3 cells transfected with a yeast H+‐ATPase have altered sensitivity to insulin, insulin growth factor‐I, and platelet‐derived growth factor‐AA
Authors: Peterson Eric P. , Martinez Gloria M. , Martinez‐Zaguilan Raul , Perona Rosario , Gillies Robert J.
Published: February 2005

The role of intracellular pH (pHin) in the regulation of cell growth in both normal and transformed cells is a topic of considerable controversy. In an effort to study this relationship NIH 3T3 cells were stably transfected with the gene for the yeast H+‐ATPase, constitutively elevating their pHin. The resulting cell line, RN1a, has a transformed phenotype: The cells are serum independent for growth, clone in soft agar, and form tumors in nude mice. In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum‐independent growth, using defined media to investigate the effects of specific growth factors on the transfected and parental NIH 3T3 cells. While both cell lines show similar growth increases in response to platelet‐derived growth factor (PDGF)‐BB and epidermal growth factor (EGF), they respond differently to insulin, insulin‐like growth factor‐I (IGF‐I) and PDGF‐AA. RN1a cells exhibit increased growth at nanomolar concentrations of insulin but the parental cells had only a relatively minor response to insulin at 10 μM. Both cell lines showed some response to IGF‐I in the nanomolar range but the response of RN1a cells was much larger. Differences in insulin and IGF‐I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF‐AA. RN1a cells are relatively insensitive to stimulation by PDGF‐AA and express fewer PDGF α receptors as shown by Northern blots and receptor‐binding studies. We propose a unifying hypothesis in which the H+‐ATPase activates a downstream element in the PDGF‐AA signal transduction pathway that complements insulin and IGF‐I signals, while leading to downregulation of the PDGF α receptor. © 1994 wiley‐Liss, Inc.
Tenascin induction in tenascin nonproducing carcinoma cell lines in vivo and by TGF‐β1 in vitro
Authors: Sakai Takao , Kawakatsu Hisaaki , Ohta Masatsugu , Saito Masaki
Published: February 2005

Tenascin, a novel six‐armed extracellular‐matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis in association with stromal‐epithelial interactions. In this study, we have tested the hypothesis that tenascin expression depends upon the change of the cellular environment from in vitro to in vivo. The distribution and alterations in the expression of tenascin were compared between in vitro and in vivo studies in a variety of human epithelial‐ and nonepithelial‐derived cell lines. When cell lines were transplanted into nude mice, all xenografts induced host‐mouse‐stroma‐derived tenascin. Four carcinoma‐derived cell lines and all sarcoma‐derived lines, which secreted tenascin in vitro, were found to produce human tenascin after transplantation. Furthermore, three carcinoma‐derived cell lines, A431, HEp‐2, and MCF7, which did not synthesize tenascin in vitro, did synthesize human tenascin after transplantation. These tenascin nonproducing carcinoma cell lines did not express tenascin mRNA in vitro. The addition of TGF‐β1 to the culture medium induced the synthesis and secretion of tenascin, but TGF‐β2 and bFGF were less effective. TGF‐β1 also induced other extracellular‐matrix components, fibronectin and laminin. TGF‐β1 did not induce tenascin in tenascin nonproducing carcinoma cell lines, such as WiDr and A549, in which human tenascin was not induced after transplantation. We have established an in vitro system in which tenascin is induced by the diffusible factor TGF‐β1. This system could shed light on the mechanism of induction of human tenascin observed in vivo in tenascin nonproducing carcinoma cell lines. © 1994 wiley‐Liss, Inc.
IL‐3 and ribavirin induce high level expression of megakaryocytic markers and messages during long‐term treatment of a megakaryocytic leukemia cell line
Authors: Majumdar Alokes , Kerby Stephen , Mullikin Brian , Seidman Michael M. , Stenberg Paula E. , Beckstead Jay H. , Cooney David A.
Published: February 2005

Megakaryocyte differentiation is a lengthy process with cells moving through a continuum delineated by the sequential expression of specific gene products. The limited number of primary cells available from marrow for analysis has brought attention to some leukemic cell lines which show enhanced megakaryocyte marker expression following incubation with inducing agents, the most common of which is phorbol myristate acetate (PMA). We developed an alternative induction protocol for the megakaryocytic leukemic cell line CMK, which involved incubation of the cells with IL‐3 and the nucleoside analog, ribavirin, for 1–2 weeks. This treatment was neither toxic nor cytostatic and yielded increased levels of the surface glycoproteins GPIIb/IIIA and GPIb‐IX. Levels of some megakaryocytic messages (GPIIIa, GPIX) showed a marked rise by 12 days of incubation in the inducer combination. This was due to a synergistic interaction between IL‐3 and ribavirin which influenced both transcriptional and posttranscriptional events. Light and electron microscopy demonstrated the presence of large polyploid cells, with morphological features similar to those of megakaryocytes, in the induced cultures. Analysis of the heterogeneity of response in the cell population to the induction regimen after several days of treatment suggested that cells which failed to display surface markers had been stimulated by the inducers but did not have sufficient time to complete expression of that marker. The results were consistent with the view that the cells in the starting population were distributed along a temporal expression pathway, and those which were first to express the earliest marker would also lead in the expression of a later marker. The order of expression was the same as that during normal megakaryocyte development. © 1994 Wiley‐Liss, Inc.
Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid‐stimulating hormone and tumor‐promoting phorbol ester in cultured porcine thyroid cells
Authors: Bellon Georges , Chaqour Brahim , Wegrowski Janusz , Borel Jacques‐Paul , Antonicelli Frank , Claisse Didier , Haye Bernard
Published: February 2005

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid‐stimulating hormone (TSH) and tetradecanoyl phorbol‐13‐acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin‐EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L‐lysine)‐coated dishes. Cells differentiated into follicular or vesicular‐like structures were distinguished by their ability to organify Na[125l] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450–480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131l]‐labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450–480 resolved to Mr 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti‐human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin l. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin‐resistant polypeptides ranging from Mrs 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6‐fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factor 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium. © 1994 Wiley‐Liss, Inc.
Ca2+‐conductances in cultured rat retinal pigment epithelial cells
Authors: Strauss Olaf , Wienrich Marion
Published: February 2005

Membrane conductances for Ca2+ in cultured rat pigment epithelial cells were studied in the whole‐cell configuration of the patch‐clamp technique using barium (10 mM) as a charge carrier. Two types of voltage‐dependent and verapamiland diltiazem‐sensitive Ba2+ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba2+ currents indicating the presence of L‐type Ca2+ channels. In order primary cultures a low‐voltage activated channel was observed with properties different from T‐type calcium channels or TTX‐sensitive calcium conducting sodium channels. © 1994 Wiley‐Liss, Inc.
Transforming growth factor β1 inhibits interleukin‐1‐induced but enhances ionomycin‐induced interferon‐γ production in a t cell lymphoma: Comparison with the effects of rapamycin
Authors: Dumont Francis J. , Kastner Carolyn A.
Published: February 2005

Transforming growth factor β1 (TGF‐β1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF‐β1 on the production of IFN‐γ measured at the mRNA and protein levels in the YAC‐1 cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL‐1 or IL‐1 + PMA, the production of IFN‐γ is suppressed by both TGF‐β1 (ED50 = 1.9 pM) and RAP (ED50 = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10‐fold by TGF‐β (ED50 = 1.8 pM) and 1.5—3‐fold by RAP. Therefore, in YAC‐1 cells, TGF‐β1 exerts opposite effects on IFN‐γ production depending on the mode of activiation, and these effects parallel those of RAP. To further analyze the mode of action of TGF‐β1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN‐γ mRNA induced by IL‐1 insensitive to TGF‐β1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin‐mediated induction of IFN‐β mRNA by either TGF‐β1 or RAP. Hence, the up‐regulation of IFN‐β production by TGF‐β1 and RAP may involve a different biochemical mechanism that that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF‐β1 and RAP modulate IFN‐γ production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC‐1 cells selected for resistance to the inhibition of IL‐1‐mediated IFN‐γ induction by RAP. Moreover, the IFN‐γ modulatory action of RAP in YAC‐1 cells was accompanied by an antiproliferative effect, whereas TGF‐β1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF‐β1 may result from the dis ruption of biochemical processes related to, although distinct from, those affected by RAP. © 1994 Wiley‐Liss, Inc.
Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells
Authors: Kalra Vijay K. , Ying Yong , Deemer Kathleen , Coates Thomas D. , Natarajan Rama , Nadler Jerry L.
Published: February 2005

Cigarette smoking is ranked among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanisms, however, that link cigarette smoking to increased incidence of atherosclerosis are not understood. The adherence of circulating monocytes to the endothelium, migration into the subendothelium, and subsequent formation of foam cells are principal initial events in the development of atherosclerosis. We therefore determined whether cigarette smoke caused increased adherence of monocytes to endothelial cells and the cellular mechanism of this increased adherence. Cigrette smoke condensate (CSC), the particulate fraction of cigarette smoke derived from 2R1 standard research cigarettes, at a concentration of 25–30 μg/ml (average yield of CSC is 26.1 mg/cigarette), augmented (70–90%) basal adherence of human peripheral blood monocytes to a cultured monolayer of endothelial cells derived from bovine aorta (BAEC) and human umbilical vein (HUVEC). There was a concomitant increase in the expression of CD11b ligand on the surface of monocytes as determined by flow cytometry, utilizing FITC conjugated Mab MO‐1 (CD11b). However, nicotine (1–15 μg/ml) and cadmium sulfate (10 μg/ml), constituents of CSC, individually or in combination had no effect either on CD11b expression or adherence of monocytes to endothelial cells. Treatment of HUVEC with CSC for 60 min also resulted in an increased expression of ICAM‐1 and ELAM‐1 as determined by mean fluorescence intensity of ICAM‐1 and ELAM‐1 labeled cells in flow cytometric analysis. The CSC induced expression of CD11b in monocytes was optimal at 25–30 min and was inhibited by protein kinase C inhibitors, staurosporine and H‐7, and also by baicalein, a lipoxygenase inhibitor. Similarly, CSC induced ICAM‐1 and ELAM‐1 expression in HUVEC was inhibited by protein kinase C inhibitors. CSC stimulated the adherence of human monocytes but not the monocytic cell lines HL‐60, U937, and THP‐1 to endothelial cells. The CSC stimulated adherence of human monocytes was inhibited (80%) by MAb to CD11b and 50% by Mab to ICAM‐1 and ELAM‐1. These results suggest that cigarettee smoke particulate constituents activate protein kinase C, leading to increased surface expression of adhesive ligand CD11b on peripheral blood monocytes and counter receptor(s) ICAM‐1 and ELAM‐1 in endothelial cells. The expression of ligand and counter receptor leads to potentiated adherence of monocytes to endothelial cells, an initial event in the pathogenesis of cigarette smoke induced inflammatory response in the vessel wall. © 1994 Wiley‐Liss, Inc.
Complex pattern of insulin‐like growth factor binding protein expression in primary rat osteoblast enriched cultures: Regulation by prostaglandin E2, growth hormone, and the insulin‐like growth factors
Authors: McCarthy Thomas L. , Casinghino Sandra , Centrella Michael , Canalis Ernesto
Published: February 2005

Primary osteoblast‐enriched (Ob) cultures from fetal rat bone synthesize insulinlike growth factor (IGF) I and IGF‐II, which each enhance Ob function. While a number of agents modulate IGF‐I production, IGF‐II is constitutively expressed in this culture model. Independent of their expression, however, the activity of the IGFs can be modified by a small group of proteins termed IGF binding proteins (IGFBPs), but little is known about the regulation of individual IGFBPs that are synthesized by Ob cells. Northern blot analysis revealed that serum‐deprived primary rat Ob cells. Northern blot analysis revealed that serum‐deprived primary rat Ob cells express transcripts encoding IGFBP‐2, IGFBP‐3, IGFBP‐4, IGFBP‐5, and IGFBP‐6, but undetectable levels of IGFBP‐1 transcripts. Western ligand blots of Ob culture medium probed with 125I‐IGF‐I or 125I‐IGF‐II showed predominant IGFBPs migrating at 30/32 kDa, with minor bands at 24 and 38–47 kDa. Western antibody analysis identified IGFBP‐2 and IGFBP‐5 within the 30/32 kDa complex, while gel mobility shift on SDS‐PAGE following deglycosylation determined that IGFBP‐3 comprised the 38–47 kDa complex. By Northern analysis, 6 h treatment with prostaglandin E2 (PGE2), growth hormone (hGH), IGF‐I, or IGF‐II revealed a complex pattern of regulatory effects on steady‐state IGFBP transcript expression. PGE2 increased the transcript levels of IGFBP‐3, IGFBP‐4, and IGFBP‐5, (∼22‐, ∼2‐, and ∼4‐fold respectively), but had no effect on IGFBP‐2 or IGFBP‐6 transcripts. hGH enhanced IGFBP‐3 and IGFBP‐5 transcripts (each approximately twofold). IGF‐I and IGF‐II had no effect on IGFBP‐2 steady‐state transcript levels but enhanced the level of IGFBP‐5 transcripts (approximately fourfold). By Western ligand blot analysis, 24 h treatment with PGE2 elevated the 24 and 38–47 kDa IGFBPs and to a lesser extent the 30/32 kDa complex, hGH elevated the 38–47 kDa IGFBPs, and IGF‐I and IGF‐II each increased the 30/32 kDa IGFBP complex. Therefore, a comparison of results obtained from Northern, Western ligand, and Western antibody studies indicates that multiple IGFBPs are expressed by primary rat Ob cultures. While IGFBP‐2 and IGFBP‐6 synthesis in Ob cultures is relatively unaffected by short‐term treatment with PGE2, hGH, or the IGFs, these agents modify IGFBP‐3, IGFBP‐4, and IGFBP‐5 expression with individual patterns of effects. In addition, some changes in IGFBP polypeptide levels that are independent of alterations in transcript expression may result from the formation of complexes between IGFs and certain IGFBPs, which could serve to store IGFs for future utilization in the formation phase of bone remodeling. © 1994 Wiley‐Liss, Inc.