bluuebunny/crossref_metadata_2025 huggingface dataset:
Heparin‐like molecules bind differentially to prion‐proteins and change their intracellular metabolic fate
Authors: Gabizon Ruth , Meiner Zeev , Halimi Michele , Ben‐Sasson Shmuel A.
Published: February 2005

PrPSc is the only known component of the scrapie prion. The difference between PrPSc and its normal isoform PrPc is probably conformational, since no difference has been found in the amino acid sequence or postranslational modifications between both proteins. Heparan sulfate (HS) has been shown to be a component of amyloid plaques in a number of diseases including the prion diseases. We now present evidence that PrP can specifically bind to heparin‐like compounds and that this interaction might have a physiological significance. HS can increase the concentration of PrP in normal neuroblastoma cells, whereas low molecular weight heparin (LMWH) does not. In contrast, LMWH and other heparin‐like molecules, excluding HS, can inhibit the synthesis of PrPSc in scrapie infected cells and reverse their phenotype back to normal as judged by measurement of PrPSc by immunoblotting and by infectivity experiments. Whether an interaction between PrP and glycosaminoglycans plays a direct role in the conversion of PrPc into PrPSc remains to be established. © 1993 Wiley‐Liss, Inc.
Specific simple sugars promote chemotaxis and chemokinesis of corneal endothelial cells
Authors: Vogel Tikva , Blake Diane A. , Whikehart David R. , Guo Neng‐Hua , Zabrenetzky Vivian S. , Roberts David D.
Published: February 2005

Bovine corneal endothelial cells showed a strong migratory response to specific simple sugars (D‐glucose and sucrose, but not L‐glucose, sorbitol, lactose, or D‐galactose) at concentrations above 10 mM. Checkerboard analysis of the migratory responses in modified Boyden chambers indicated both chemotactic and chemokinetic effects. Serum starvation of the cultures increased the chemotaxis towards D‐glucose and 2‐deoxy‐D‐glucose, but not towards sucrose. Migration to sucrose and glucose was inhibited by chelation of extracellular calcium or by inhibition of Na+,K+ ATPase with ouabain. To date, this migratory response has been found only in corneal endothelial cells. Neither human melanoma cells, human breast carcinoma cells, bovine aortic endothelial cells, nor bovine microvascular endothelial cells migrated towards simple sugars, although all cell types migrated toward fibronectin in chemotaxis assays. After 16–19 passages in culture, bovine corneal endothelial cells retained their ability to migrate towards fibronectin, but lost their ability to migrate towards sugars. This loss of migratory response was accompanied by a sevenfold decrease in Na+,K+ ATPase activity. Although loss of Na+,K+ ATPase activity accompanied the loss of migratory response, pretreatment of cell cultures with 25 mM glucose did not stimulate, but rather lowered Na+,K+ ATPase activity in low or high passage cultures. © 1993 Wiley‐Liss, Inc.
Coupling of glucose transport and phosphorylation in Xenopus oocytes and cultured cells: Determination of the rate‐limiting step
Authors: Whitesell Richard R. , Aboumrad Mireille K. , Powers Alvin C. , Regen David M. , Le Chinh , Beechem Joseph M. , May James M. , Abumrad Nada A.
Published: February 2005

The initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose. To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes. The method is based on the use of intracellular [2‐3H]glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of [2‐3H]glucose efflux and of 3H2O released by phosphorylation. The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphory lation activities can be regulated by expression of mRNA or injection of foreign protein. Oocytes microinjected with [2‐3H]glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Km of 40 7μM and a Vmax of 0.1 nmol/min/oocyte. Co‐injection of yeast hexokinase increased glucose phosphorylation by five‐fold. Expression of human glucose transporter (GLUT1) mRNA resulted in a 25‐30‐fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes. The kinetics of transport and phosphorylation of [2‐3H]glucose were analyzed by a multiple curve‐fitting program that provided estimates of kinetic coefficients for both processes from a single time course. The analysis showed that expression of GLUT1 shifted the rate‐limiting step in glucose utilization from transport to phosphorylation. A similar shift occurred at a three‐fold lower extracellular concentration of 2‐deoxyglucose. In a pancreatic beta cell line both transport and phosphorylation showed high Km values, with phosphorylation as the limiting step. The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models. © 1993 Wiley‐Liss, Inc.
Response of stratified cultures of human keratinocytes to disruption of proteoglycan synthesis by p‐nitrophenyl‐β‐D‐xylopyranoside
Authors: Haggerty John G. , Bretton Randolph H. , Milstone Leonard M.
Published: February 2005

Proteoglycans play a role in regulating proliferation and adhesion of cells to each other and to the basal lamina. Synthesis of proteoglycans is disrupted by β‐xylosides, which serve as alternate substrate sites for glycosaminoglycan chain attachment and therefore prevent glycosylation of the core protein. We have investigated the effects of p‐nitrophenyl‐β‐D‐xylopyranoside (PNP‐xyloside) on cultured human keratinocytes. Stratified cultures were incubated for 7 days with PNP‐xyloside (0.05–2.0 mM). Concentrations as low as 0.05 mM increased the secretion of free chondroitin sulfate by 10–15‐fold over untreated cultures. Cellassociated proteoglycan decreased as PNP‐xyloside concentration increased. At 2 mM PNP‐xyloside, heparan sulfate as well as chondroitin sulfate addition to core proteins was disrupted: the core protein of epican, a heparan sulfate form of CD44 found on keratinocytes, was detected immunologically but lacked heparan sulfate. 2.0 mM PNP‐xyloside reduced the number of attached cells by 20–25% after 7 days, but had little effect on morphology or protein synthesis. These results indicate that intact proteoglycans are not critical for maintaining epidermal keratinocyte stratification, cell‐cell adhesion, or growth. © 1994 Wiley‐Liss, Inc.
Injuries to cultivated BJA‐B cells by ajoene, a garlic‐derived natural compound: Cell viability, glutathione metabolism, and pools of acidic amino acids
Authors: Scharfenberg Klaus , Ryll Thomas , Wagner Roland , Wagner Karl G.
Published: February 2005

Ajoene (4,5,9‐trithiadodeca‐1,6,11‐triene‐9‐oxide), a garlic‐derived natural compound, which had been shown to have cytostatic/cytotoxic properties, was tested with a B cell lymphoma‐derived cell line (BJA‐B cells) in order to elucidate its mechanism of cytotoxic action. Viability of the cells was determined by the Trypan blue exclusion test and the colorimetric tetrazolium (MTT) assay, whereas metabolic disturbance was evaluated by measuring the pools of reduced (GSH), oxidized glutathione (GSSG) and the acidic amino acids, Glu and Asp. Fast uptake of ajoene was accompanied by an immediate reduction of the CSH and increase in the GSSG levels. The extent of these changes, as well as the further development of the metabolite pools, depended on the ajoene dose per cell. At a sublethal ajoene dose the GSH and GSSG pools rose at the later stages to levels much higher than in the control experiment. Bleb formation at the cytoplasmic membrane was a further rapid phenomenon, although injuries detected by Trypan blue exclusion developed only at a later stage. The MTT assay, performed in a parallel experiment (48 h after ajoene addition), showed, however, that reduction of cell viability was established at the very beginning of ajoene exposure. Altogether, the action of ajoene strongly resembled oxidative stress (i.e., interference with SH homeostasis and its pleiotropic consequences to cell physiology and metabolism). © 1994 Wiley‐Liss, Inc.
Transrepression of type II collagen by TGF‐β and FGF is protein kinase C dependent and is mediated through regulatory sequences in the promoter and first intron
Authors: Bradham Douglass M. , Wiesche Beatrix In Der , Precht Patricia , Balakir Richard , Horton Walter
Published: February 2005

Transforming growth factor beta and basic fibroblast growth factor are multipotential factors found in bone and cartilage that may be involved in both the proliferation and differentiation of chondrocytes. It was previously reported that TGF‐β plus FGF caused a modulation of chondrocyte phenotype that included the downregulation of steady‐state level of the collagen II transcript. In this report, the results of nuclear run‐off data indicate that repression of transcript initiation from the collagen II gene is the primary mechanism involved in the growth factor induced inhibition. Transient transfection assays with CAT expression vectors containing portions of the collagen II gene show that the TGF‐β/FGF induced transrepression requires a region in the first intron previously reported to have transcriptional enhancer activity and to bind chondrocyte nuclear proteins. In addition, silencer elements in the promoter also appear to play a role. Protein data as well as transient transfection experiments indicate that the activation of protein kinase C is necessary for the growth factor–induced down‐regulation of collagen II expression. These studies suggest that a cascade initiating with PKC activation is responsible for modifying transcription factors that interact with regulatory sequences in the collagen II gene. A detailed understanding of the factors involved in cartilage‐specific gene regulation in chondrocytes would facilitate development of therapeutic protocols for the repair of degenerated cartilage in diseases such as osteoarthritis. © 1994 Wiley‐Liss, Inc.
Regulation of insulin‐like growth factor binding protein synthesis and secretion in human retinal pigment epithelial cells
Authors: Feldman Eva L. , Randolph Ann E.
Published: February 2005

Cultured human retinal pigment epithelial cells (RPE) secrete insulin‐like growth factor binding proteins (IGFBPs), a family of polypeptides which modulate the actions of the insulin‐like growth factors. RPE cells secrete two IGFBPs with Mr estimates of 34,000 and 46,000, respectively. Treatment of RPE cells with IGF‐I markedly stimulated the secretion of the 46,000 Mr form. This stimulation occurred via an IGF‐I receptor independent mechanism because both [QAYL]IGF‐I (an IGF‐I analogue with decreased affinity for the IGFBPs but normal affinity for the IGF‐I receptor) and α‐IR3 (a blocking monoclonal antibody against the IGF‐I receptor) had no effect on IGF‐I stimulated increases in IGFBPs. Additionally, [QAYL]IGF‐I enhanced RPE cell proliferation to the same magnitude as IGF‐I. Treatment with IGF‐I, [QAYL]IGF‐I, or α‐IR3 had no effect on steady‐state levels of the 2.5 kb IGFBP‐3 or the 1.3 kb IGFBP‐6 mRNA transcripts as measured by Northern blotting and quantitative autoradiography. Forskolin and a group of candidate growth factors, including platelet‐derived growth factor, epidermal growth factor, and acidic and basic fibroblast growth factor, modestly increased IGFBP secretion when compared to untreated cells, but these effects were small when compared to IGF‐I treatment. Fetal calf serum enhanced the presence of the 2.5 kb IGFBP‐3 mRNA transcript in a dose‐dependent fashion but had no effect on the 1.3 kb IGFBP‐6 mRNA transcript. IGF‐I, forskolin, and the candidate growth factors had no effect on either IGFBP‐3 or IGFBP‐6 mRNA. These data suggest that the production of IGFBPs in human RPE cells is regulated by distinct mechanisms which include (1) an IGF‐I receptor independent interaction of IGF‐I with secreted IGFBPs and (2) de novo synthesis of IGFBPs by serum‐containing factors. © 1994 Wiley‐Liss, Inc.
Invadopodia promote proteolysis of a wide variety of extracellular matrix proteins
Authors: Kelly Thomas , Mueller Susette C. , Yeh Yunyun , Chen Wen‐Tien
Published: February 2005

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus invade the extracellular matrix (ECM) using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Normal

Throughout this manuscript normal cells refers to chicken embryo fibroblasts and transformed cells refers to these cells infected with Rous sarcoma virus.

cells neither form invadopodia nor degrade the ECM. Here we show that cells expressing invadopodia degrade and enter into a fibronectin‐rich matrix produced by normal fibroblasts. Within 6 h after seeding onto the matrix, the invasive cells create an area devoid of matrix fibrils surrounding the cell body. Proteolysis mediates this matrix clearing because sevenfold more radiolabeled matrix is released into the growth media by the transformed cells relative to the normal cells. In addition to this assembled matrix, transformed cells were grown on thin layers of purified ECM proteins, revealing that invadopodia can degrade fibronectin, collagen type I, collagen type IV, and laminin. A 160 kDa protease that is extracted from transformed cells by Triton X‐114 partitions into the detergent phase and is prominent in ventral plasma membranes that contact the ECM suggesting that it is a membrane associated protease. © 1994 Wiley‐Liss, Inc.
DNA strand breakage during physiological apoptosis of the embryonic chick lens: Free 3' OH end single strand breaks do not accumulate even in the presence of a cation‐independent deoxyribonuclease
Authors: Chaudun E. , Arruti C. , Courtois Y. , Ferrag F. , Jeanny J. C. , Patel B. N. , Skidmore C. , Torriclia A. , Counis M. F.
Published: February 2005

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosmes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single‐strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP‐ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP‐ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double‐strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation. © 1994 Wiley‐Liss, Inc.
Activated platelet‐derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line
Authors: Vassbotn Flemming S. , Östman Arne , Langeland Nina , Holmsen Holm , Westermark Bengt , Heldin Carl‐Henrik , Nistér Monica
Published: February 2005

Human glioblastoma cells (A172) were found to concomitantly express PDGF‐BB and PDGF β‐receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up‐regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U343MGa 31L) or on Ki‐ras‐transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transtormed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists. © 1994 Wiley‐Liss, Inc.
Involvement of alkaline phosphatase in the modulation of receptor signaling in osteoblasts: Evidence for a difference between human parathyroid hormone‐related protein and human parathyroid hormone
Authors: Fukayama Shoichi , Tashjian Armen H.
Published: February 2005

We have previously reported that alkaline phosphatase (ALPase) is functionally involved in calcium uptake by several osteoblast‐like cell lines. We have extended these studies to investigate the actions of ALPase on the cAMP response to and the receptor binding of human parathyroid hormone (hPTH) and human parathyroid hormone‐related protein (hPTHrP). Pretreatment of human osteoblast‐like SaOS‐2 cells with human placental ALPase (hpALPase) inhibited the cAMP response to hPTH(1‐34) but had no effect on the actions of hPTHrP(1‐34) or vasoactive intestinal peptide. The inhibitory effect was reversed by L‐Phe‐Gly‐Gly, an inhibitor of hpALPase. Treatment of SaOS‐2 cells with hpALPase modestly reduced the binding of hPTH to 70% of control values, with little or no effect on the binding of hPTHrP. Bovine kidney and calf intestine ALPases were without effect on either the cAMP response or binding of hPTH or hPTHrP in SaOS‐2 cells. In rat osteoblast‐like ROS 17/2.8 cells, hpALPase had no effect on cAMP production stimulated by hPTH(1‐34) or hPTHrP(1‐34), arguing against a nonspecific effect of hpALPase. We suggest that, in SaOS‐2 cells, the common PTH/PTHrP receptor can differentiate between the agonist activities of hPTH and hPTHrP by a mechanism that is sensitive to hpALPase. © 1994 Wiley‐Liss, Inc.
Distinct protein tyrosine phosphorylation during mitogenesis induced in quiescent sv40‐transformed 3T3 T cells by insulin or vanadate
Authors: Wang Hanlin , Scott Robert E.
Published: February 2005

Insulin and vanadate selectively induce mitogenesis in quiescent SV40 large T antigen‐transformed 3T3 T cells (CSV3–1) but not in quiescent nontransformed 3T3 T cells. Insulin and vanadate mediate this effect in CSV3–1 cells by distinct signal transduction mechanisms that involve protein tyrosine kinase activity. To further study these processes, changes in protein tyrosine phosphorylation induced by insulin and vanadate were investigated. Using immunoprecipitation and Western blotting techniques with antiphosphotyrosine antibodies, we report distinct protein phosphorylation characteristics in insulin‐ and vanadate‐stimulated CSV3–1 cells. The insulin receptor β‐subunit is phosphorylated within 2 min after insulin stimulation of transformed CSV3–1 cells. Insulin also stimulates a rapid increase in tyrosine phosphorylation of the 170 kDa insulin receptor substrate‐1 and complex formation between the phosphorylated insulin receptor substrate‐1 and the 85 kDa subunit of phosphatidylinositol 3'‐kinase. In contrast, vanadate does not initially increase detectable phosphorylation of any proteins, including neither the insulin receptor nor the insulin receptor substrate‐1. After 60 min, however, a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins is observed in vanadate‐treated CSV3–1 cells. Furthermore, treatment of CSV3–1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation but only minimally inhibits the effects of insulin. Finally, insulin stimulates the phosphorytion of a 33 kDa protein, whereas vanadate does not. By comparison, in nontransformed 3T3 T cells, insulin induces a delayed and weaker tyrosine phosphorylation of the insulin receptor β‐subunit and vanadate does not enhance the tyrosine phosphorylation of the 55 and 64 kDa proteins. These data together indicate that the mitogenic effects of insulin and vanadate are associated with distinct protein phosphorylation patterns that appear to be differentially regulated in SV40‐transformed and nontransformed 3T3 T cells. © 1994 Wiley‐Liss, Inc.
Calpain activation in apoptosis
Authors: Squier Margaret K. T. , Miller Anita C. K. , Malkinson Alvin M. , Cohen J. John
Published: February 2005

Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane‐bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium‐dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well‐known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium‐dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low‐level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both “induction” and “release” models of apoptotic cell death. © 1994 wiley‐Liss, Inc.
Progressive loss of sensitivity to endothelium‐derived growth inhibitors expressed by human melanoma cells during disease progression
Authors: Rak Janusz W. , Hegmann Erik J. , Lu Chao , Kerbel Robert S.
Published: February 2005

Tumor progression is frequently associated with changes in responsiveness of tumor cells to paracrine growth factors. A potential major source of such paracrine factors in solid tumors are endothelial cells since this type of cell can constitute a sizeable fraction of the cellular composition of solid tumors. As an initial step to examining the possible effects of endothelial cell‐associated growth factors on tumor cell growth, a panel of human melanoma cell lines representative of different stages of tumor progression was employed for studies utilizing endothelial cell‐derived growth modulators. Macrovascular or microvascular human endothelial cells from umbilical vein or from skin, respectively, inhibited melanoma cell growth in direct coculture experiments. The potency of this inhibitory effect diminished as a function of melanoma progression. Conditioned media from endothelial cell cultures mimicked the effect of the cell coculture experiments, suggesting the involvement of soluble growth factor(s). Approximately 50–75% of the conditioned media inhibitory effect was abrogated by addition of the neutralizing antibody to interleukin‐6 (IL‐6). Gel filtration chromatography revealed the presence of additional inhibitors in endothelial cell conditioned medium. Two peaks of activity were detected with apparent molecular weights of approximately 100–150 Kd and 20–30 Kd, the latter containing IL‐6 activity. Whereas early‐stage radial growth phase (RGP) primary tumor‐derived melanoma cells were sensitive to at least three different endothelial products of high or low molecular weight (including IL‐6), melanoma cells from more advanced metastatic lesions were resistant to the latter activities, and retained only partial sensitivity to the high molecular weight inhibitor. More advanced vertical growth phase (VGP) primary melanoma cell lines expressed intermediate inhibition‐sensitive phenotypes. Thus human melanoma development appears to be associated with progressive loss of sensitivity to the growth inhibitory effects of IL‐6 and other factors produced by endothelial cells. This is likely to be a result of a selection process when tumor cells are confronted with adjacent vasculature during the progress of tumor angiogenesis. © 1994 wiley‐Liss, Inc.
Temporal expression of myogenic regulatory genes during activation, proliferation, and differentiation of rat skeletal muscle satellite cells
Authors: Smith C. K. , Janney M. J. , Allen R. E.
Published: February 2005

The satellite cell is responsible for growth and repair of postnatal skeletal muscle. We investigated the expression of the myogenic regulatory gene (MRG) family in these cells in the stages from quiescence to fusion. Using polymerase chain reaction amplification of reverse‐transcribed RNA (RT‐PCR) isolated from adult rat satellite cells, we demonstrated a temporal sequence of gene activation, which is distinct from that previously observed in embryonic somitic cells. No MRG expression was detected in predominantly quiescent cells. MyoD is activated by 12 h in cell culture, prior to the first evidence of proliferation. MRF4 and myf‐5 appear by 48 h and may be associated with the first division cycle. Myogenin is not detectable until 72 h after satellite cell recovery from the muscle fiber, coincidental with the first evidence of differentiation. © 1994 wiley‐Liss, Inc.
Transforming growth factor‐β regulation of retinoblastoma gene product and E2F transcription factor during cell cycle progression in mouse fibroblasts
Authors: Kim Tae‐Aug , Ravitz Michael J. , Wenner Charles E.
Published: February 2005

The mechanism by which transforming growth factor beta (TGFβ) exerts growth stimulatory effects was examined in C3H/10T1/2 mouse fibroblasts by study of cell cycle regulation of the retinoblastoma gene product (p110Rb) and transcriptional regulation of the p110Rb‐associated transcription factor, E2F. Northern blotting analysis shows that TGFβ and/or epidermal growth factor (EGF) stimulate by three to sixfold the level of Rb mRNA which is also reflected by the increased levels of p110Rb. p110Rb becomes phosphorylated in mid‐G1 and further phosphorylated at the G1/S transition. Hyperphosphorylation of p110Rb by TGFβ can be observed when cells are in S phase. TGFβ stimulates by three to fourfold the activity of cdk2 kinase consistent with the observed phosphorylation of p110Rb and also with the possibilit that the kinase is involved in phosphorylating p110Rb close to the G1/S transition. Thus, TGFβ as a growth stimulator induces, as does EGF, the phosphorylation of p110Rb during cell cycle progression. Transient transfection of E2F promoter constructs was used to analyze the effect of TGFβ on the modulation of E2F‐mediated transcription. The data revealed that TGFβ can stimulate wild‐type adenoviral E2 promoter activity by 12‐fold. Taken together, TGFβ‐induced phosphorylation of p110Rb in mouse fibroblasts appears to exert a positive regulatory function upon genes that have a pivotal role in the G1/S transition of the cell cycle. © 1994 Wiley‐Liss, Inc.
Age and development‐related changes in osteopontin and nitric oxide synthase mRNA levels in human kidney proximal tubule epithelial cells: Contrasting responses to hypoxia and reoxygenation
Authors: Hwang Shiaw‐Min , Denhardt David T. , Wilson Patricia D. , Laskin Jeffrey D.
Published: February 2005

Osteopontin (OPN) encodes a secreted glycosylated phosphoprotein containing a GRGDS motif that can mediate cell attachment through the αvβ3 integrin, and has recently been shown to down‐regulate nitric oxide synthase (NOS) expression. We report here that primary cultures of renal proximal tubule epithelial (PTE) cells prepared from human kidneys of different developmental stages and ages show a positive correlation between developmental age and the expression, at the mRNA level, of both OPN and constitutive NOS. However, OPN and NOS responded in different manners, as assessed by mRNA measurements, to hypoxia‐reoxygenation injury. The OPN mRNA level, assessed by Northern blotting, increased slightly during 60 min of hypoxia and more substantially during subsequent reoxygenation of primary PTE cells derived from the kidneys of young but not of aged donors. The abundance of NOS mRNA, measured using a cDNA probe to the constitutive form of the enzyme, was enhanced during hypoxia in kidneys derived from humans of all ages, and then decreased during reoxygenation—possibly as the result of increased OPN expression. PTE cells from aged kidneys are more susceptible to cell death under hypoxic conditions than PTE cells from young kidneys. An investigation of the effect of an oxidant on OPN and NOS mRNA levels revealed that within 30 min of exposure to H2O2, NOS mRNA levels decreased simultaneously with an increase in OPN mRNA levels. Nitric oxide (NO), the product of NOS, is at low levels an important signal transduction molecule participating in the regulation of vascular tone and renal reabsorption; at high levels it is cytotoxic. We suggest that the diminished ability of cells from old kidneys to down‐regulate NO production and to increase OPN expression after hypoxia‐reoxygenation may contribute to their increased susceptibility to oxidant injury. © 1994 Wiley‐Liss, Inc.
Correlation of transformation from epithelial to mesenchymal‐like morphology and endogenous bFGF levels in human nasopharyngeal carcinoma cells
Authors: Chen Jan‐Kan , Shen Ling‐Shang , Chao Hsiou‐Hsian
Published: February 2005

CG‐1 human nasopharyngeal carcinoma cells in monolayer culture formed both cohesive, epithelial‐like colonies and scattered, fibroblastic‐like colonies in mixed proportions. In the presence of exogenously added bFGF (4 ng/ml), about 85% of the colonies formed were fibroblastic‐like. CG‐1 cells were capable of synthesizing and releasing bFGF, and, when compared by the immunological method, cells in fibroblastic‐like colonies were found to contain higher levels of endogenous bFGF than cells in the epithelial‐like colonies. Furthermore, cells in the peripheral region of the epithelial‐like colonies, which were fibroblastic‐like in morphology, also appeared to contain higher levels of endogenous bFGF. In addition, in the presence of suramin, neutralizing antibody to bFGF, or neutralizing antibodies to bFGF and EGF, the number of cohesive colonies formed was greatly increased. Moreover, addition of the 2 M NaCl‐eluted heparin‐Sepharose fraction of the CG‐1 cell‐coditioned medium promoted the formation of dispersed colony in a dose‐dependent manner. The results suggest that bFGF can regulate CG‐1 cell phenotype in an autocrine manner. © 1994 Wiley‐Liss, Inc.
Cleavage of the epidermal growth factor receptor by a membrane‐bound leupeptin‐sensitive protease active in nonionic detergent lysates of senescent but not young human diploid fibroblasts
Authors: Carlin Cathleen , Phillips Paul D. , Brooks‐Frederich Katherine , Knowles Barbara B. , Cristofalo Vincent J.
Published: February 2005

Numerous studies suggest that epidermal growth factor (EGF) signaling is impaired in nonproliferating senescent human diploid fibroblasts downstream of receptor binding. One possible explanation for these results is that senescent cells possess unique enzymatic activities capable of regulating functional levels of the EGF receptor. To test that hypothesis, nonionic detergent lysates of young and senescent cells were compared for proteolytic activity directed towards the EGF receptor, and a protease that cleaves the 170 kDa EGF receptor was identified in lysates from senescent but not young cells. Although studies presented here were carried out with WI‐38 cells, our data indicate that other senescent fibroblasts possess a similar activity. The degradation product immunoprecipitated by a monoclonal antibody specific for an EGF receptor exocytosolic epitope had an approximate molecular weight of 100,000. This product was also detected following cell surface labeling with 125I, and by cross‐linking 125I‐EGF to intact cells with disuccinimidyl suberate. The proteolytic activity in senescent cell lysates was specifically inhibited by leupeptin and did not require divalent cations; it was also inactivated by aprotic solvents such as dimethylsulfoxide (DMSO) or ethylene carbonate. Interestingly, this protease was not active during ligand‐induced intracellular processing of the EGF receptor, suggesting that it does not normally function in endocytic or lysosomal compartments. The susceptibility of the protease to inactivation by cell surface trypsinization is consistent with a plasma membrane localization. Since EGF receptor cleavage is not observed unless senescent cells are solubilized with nonionic detergents, it seems likely that the protease is confined to specialized regions of the plasma membrane. Whether or not the EGF receptor is a physiologic target for this protease is unclear. Its expression at the cell surface is nevertheless significant, since it suggests there are mechanisms for regulating membrane‐bound proteins, or biologically active peptides in the extracellular space, in senescent cells that are either absent or inactive in young cells. © 1994 Wiley‐Liss, Inc.
Calmodulin‐like effect of oncomodulin on cell proliferation
Authors: Blum Janaki K. , Berchtold Martin W.
Published: February 2005

Oncomodulin (OM) is a Ca2+ binding protein (CABP) structurally closely related to parvalbumin. Expression of OM is restricted to early embryonic stages, the placental cytotrophoblasts, and neoplastic tissues. The function of OM as a calmodulin (CaM)‐like enzyme modulator is controversial. Two types of experiments demonstrate that OM may act in an analogous fashion to CaM in T14 and T10 cancerous cell lines, which both express OM. First, both OM transcript and protein levels increased at the G1/S boundary in a similar manner to CaM, though not to the same extent, in the chemically transformed rat fibroblast cell line T14 synchronized at mitosis by nocodazole. Second, antisense oligonucleotides specific to the OM ATG region inhibited growth of T14 in a similar dose‐dependent manner as observed with CaM‐specific antisense probes. © 1994 Wiley‐Liss, Inc.
cDNA from human ocular ciliary epithelium homologous to βig‐h3 is preferentially expressed as an extracellular protein in the corneal epithelium
Authors: Escribano Julio , Hernando Nati , Ghosh Sikha , Coca‐Prados Miguel , Crabb John
Published: February 2005

The non‐pigmented ciliary epithelium is largely responsible for the formation of aqueous humor in the mammalian eye. To provide a basis for studies at the molecular level, a directional expression cDNA library was constructed in Uni‐ZAP XR vector from poly A+ RNA of the human non‐pigmented ciliary epithelial derived ODM‐2 cell line. Fifty‐three cDNA clones were isolated from the library and characterized by partial sequence analysis. Approximately 49% of the clones exhibited homology with known genes in the GenBank/EMBL databases. The putative identification of these clones may reflect the transcriptional activity of the ODM‐2 cells in culture. One of the identified clones, ODM‐42‐I, was found to be specific and highly expressed in the corneal epithelium. This clone had an exact match with a recently discovered human gene, βig‐h3 (Skonier et al., 1992, DNA Cell Biol., 11: 511–522), which codes a surface recognition protein, inducible by transforming growth factor beta (TGF‐β), and containing a putative binding site (RDG) for integrins. The ODM‐42‐I cDNA clone displays a distinctive pattern of expression found in the human eye, expressed almost exclusively in the cornea. Further studies, using sera from a synthetic peptide to the carboxy‐terminal region of ODM‐42‐I, reveal that the protein is heterogeneous in charge and is preferenctially expressed on the extracellular surface of corneal epithelial cells, and might share immunologic properties with integrins β1. © 1994 Wiley‐Liss, Inc.
Prostaglandin‐stimulated second messenger signaling in bone‐derived endothelial cells is dependent on confluency in culture
Authors: Ida Rodney , Lee Austin , Huang Jason , Yamaguchi Dean T. , Brandi Maria Luisa
Published: February 2005

New bone formation is associated with an increase in blood flow by the invasion of capillaries. Endothelial cells that line the capillaries can produce paracrine factors that affect bone growth and development, and in turn, could be affected by products produced by bone cells, in particular the osteoblasts. Since osteoblasts produce prostaglandins E2 and F2α (PGE2, PGF2α), it was investigated if these PGs were agonists to bone‐derived endothelial cells (BBE) by assessing changes in cAMP and free cytosolic calcium concentration ([Ca2+]i) second messenger generation. We found that confluent cultures of BBE cells, a clonal endothelial cell line derived from bovine sternal bone, responded to 1 μM PGE2 by an increase in cAMP. PGF2α at the same concentration was less potent in stimulating an increase in cAMP production in confluent BBE cells. Subconfluent cells with a morphology similar to that of fibroblastic cells were not as sensitive to PGE2‐stimulated cAMP generation. PGF2α failed to elicit any cAMP production in subconfluent cultures. PGE2 and PGF2α both stimulated an increase in [Ca2+]i concentration in a dose‐dependent manner. The potency of PGE2 was similar to that of PGF2α in stimulating an increase in [Ca2+]i. The Ca2+ response was mostly independent of extracellular Ca+, was unchanged even with prior indomethacin treatment, was unaffected by caffeine pretreatment, but was abolished subsequent to thapsigargin pretreatment. The PG‐induced increase in [Ca2+]i was also dependent on the confluency of the cells. In a subconfluent state, the responses to PGE2 or PGF2α were either negligible, or only small increases in [Ca2+]i were noted with high concentrations of these two PGs. Consistent, dose‐dependent increases in [Ca2+]i were stimulated by these PGs only when the cells were confluent and had a cobblestoned appearance. Since it was previously demonstrated that BBE cells respond to parathyroid hormone (PTH) by the production of cAMP, we tested if bovine PTH(1‐34) amide bPTH(1—34) also increased [Ca2+]i in these cells. No change in [Ca2+]i was found in response to bPTH (1—34), although bPTH (1—34) stimulated a nine to tenfold increase in cAMP. We conclude that BBE cells respond to PGE2 and PGF2α but not to bPTH(1—34) by an increase in [Ca2+]i probably secondary to stimulation of phospholipase C and that the cAMP and [Ca2+]i second messenger responses in BBE cells are dependent on the state of confluency of the cells. © 1994 Wiley‐Liss, Inc.
Thyroid‐induced changes in the growth of the Liver, Kidney, and Diaphragm of Neonatal Rats
Authors: Canavan James P. , Holt Janet , Easton Juliet , Smith Kaye , Goldspink David. F.
Published: February 2005

Eu‐, hypo‐ and hyper‐thyroid rats were studied 12 days postpartum. Hypothyroidism was induced by administering propylthiouracil (PTU) via the mother's drinking water beteen late gestation and throughout lactation. This procedure effectively blocked the normal early postnatal surge of T3 and T4. In contrast, hyperthyroidism was induced in the young pups by daily injections of T4 from day 3 postpartum. The effects of these experimental manipulations of thyroid status on the rates of protein turnover and growth of the liver, kidney, and diaphragm were studied and compared with measurements made on appropriate euthyroid control tissues. Tissue rates of protein synthesis were decreased in response to hypothyroidism with consequent growth retardation of all three tissues and the whole animal. In contrast, the three body tissues responded very differently to the induction of hyperthyroidism. Hepatic rates of protein synthesis and growth were completely unaffected by thyroid excess. The response of the diaphragm was essentially the reverse of that seen with hypothyroidism, i.e., the enhanced rates of protein synthesis and protein degradation leading to muscle hypertrophy. The rates of protein turnover in the kidney were also increased, but unlike the diaphragm the net result was renal atrophy. Clearly, thyroid hormones influence the normal rapid growth of the neonate and its individual tissues. However, beyond a certain concentration the threshold of responsiveness to these hormones seems to vary between individual tissues. © 1994 Wiley‐Liss, Inc.
Growth and intestinal differentiation are independently regulated in HT29 colon cancer cells
Authors: Schroy Paul C. , Rustgi Anil K. , Ikonomu Ekaterini , Liu Xiu P. , Polito Joseph , Andry Chris , O'Keane J. Connor
Published: February 2005

The polar‐planar compound hexamethylene bisacetamide (HMBA) can inhibit HT29 colon carcinoma cell growth and induce a more benign phenotype, as defined by decreased anchorage‐independent clonogenicity, loss of a cell surface malignancy marker, and decreased in vivo tumorigenicity. The principle aim of this study was to determine whether HMBA's effects on HT29 cell growth and biologic behavior correlate with effects on intestinal differentiation. Parallel studies were performed with sodium butyrate (NaBT), a potent inducer of intestinal differentiation HT29 cell growth, proliferation, and markers of intestinal differentiation were assayed after short‐ and long‐term treatment with HMBA, NaBT, or the combination. Both 5 mM HMBA and 5 mM NaBT were potent inhibitors of monolayer growth; in combination their effects were nearly additive. Inhibition of DNA synthesis was detectable within 6 h of treatment and was preceded by down‐regulation of c‐myc expression. Soft agar clonogenicity was also decreased by 90%, > 99%, and > 99% by HMBA, NaBT, and the combination, respectively. Despite these parallel effects on growth and in vitro markers of a benign phenotype, effects on intestinal differentiation were discordant. NaBT induced significant increases in membrane‐associated alkaline phosphatase activity, cytosolic mucin content, PAS+/diastase‐resistant cells, and ultrastructural evidence of intestinal cell differentiation. HMBA not only failed to induce markers of intestinal differentiation, but attenuated NaBT's effects when used in combination. These data suggest that growth and intestinal differentiation may be independently regulated in HT29 cells. They also suggest that expression of intestinal markers of differentiation is not a prerequisite for the acquisition of a more benign phenotype. © 1994 Wiley‐Liss, Inc.
Modulation of potassium channels by protein tyrosine kinase inhibitors
Authors: Saad A. H. , Kuo S. S. , Koong A. C. , Hahn G. M. , Giaccia A. J.
Published: February 2005

Exposure of non‐excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA‐insensitive slow‐inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA‐sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation‐induced currents are inhibited by pretreatment with the free radical scavenger, N‐Acetyl L‐Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non‐excitatory cells in a closed state. Following exposure to ionizing radiation, free radical‐induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels. © 1994 Wiley‐Liss, Inc.
Effect of tumor necrosis factor‐α and interferon‐γ on the growth of a human salivary gland cell line
Authors: Wu Ava J. , Kurrasch Regina H. , Katz Joseph , Fox Philip C. , Baum Bruce J. , Atkinson Jane C.
Published: February 2005

Interferon‐γ (IFN‐γ) is a product of activated T‐lymphocytes, and tumor necrosis factor‐α (TNF‐α) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF‐α and IFN‐γ on a human submandibular gland epithelial cell line (HSG). IFN‐γ caused a concentration‐dependent decrease in HSG cell growth (∼70% in 6 days). Conversely, TNF‐α alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF‐α and 1,000 units/ml of IFN‐γ), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine‐induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3–6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN‐γ alone and that of IFN‐γ and TNF‐α in combination were blocked completely using an antibody to the IFN‐γ receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up‐regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM‐1) and histocompatibility antigen HLA‐DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN‐γ, with or without TNF‐α, resulted in increased levels of ICAM‐1 and the acquisition of HLA‐DR expression. These aggregate data suggest that IFN‐γ alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF‐α. © 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.