bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell cycle dependent gene expression in quiescent stimulated and asynchronously cycling arterial smooth muscle cells in culture
Authors: Campan Michel , Desgranges Claude , Gadeau Alain‐Pierre , Millet Dominique , Belloc Francis
Published: February 2005

The expression of a set of cell cycle dependent (CCD) genes (c‐fos, c‐myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c‐fos mRNA were rapidly and transiently expressed very early in the G1 phase; c‐myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c‐fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c‐fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c‐myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.
Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line
Authors: Gamet Laurence , Murat Jean‐Claude , Remaury Anne , Remesy Christian , Valet Philippe , Paris Hervé , Denis‐Pouxviel Colette
Published: February 2005

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time‐dependent efficacy of the treatment. The cells were arrested in GO/G1 phase by incubation for 24 h in serum‐free medium and proliferation was re‐initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10−5 M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10−7 M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long‐lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell‐growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.
Shear‐induced platelet‐derived growth factor gene expression in human endothelial cells is mediated by protein kinase C
Authors: Hsieh Hsyue‐Jen , Li Nan‐Qian , Frangos John A.
Published: February 2005

Our previous studies have shown that steady shear stress causes a transient increase of platelet‐derived growth factor (PDGF) A and B chain mRNA levels in human umbilical vein endothelial cells (HUVEC). In the present study, we elucidated the signaling pathway of shear stress in HUVEC by examining the roles of protein kineses, intracellular calcium, cyclooxygenase, and guanine nucleotide‐binding proteins (G proteins) in the PDGF gene induction by shear. The protein kinase C inhibitors, H7 and staurosporine, strongly inhibited the shear‐induced PDGF gene expression in HUVEC. In contrast, HA1004, a cAMP‐ and cGMP‐dependent protein kineses inhibitor, was only slightly inhibitory. BAPTA/AM, an intracellular calcium chelator, partially (50%) inhibited the shear‐induced PDGF gene expression. The cyclooxygenase inhibitors, ibuprofen and indomethacin, were slightly inhibitory. A 35–50% inhibition of shear‐induced PDGF gene expression was found with GDP‐β‐S, an inhibitor of G proteins. These results suggest that shear‐induced PDGF gene expression in HUVEC is mainly mediated by protein kinase C activation and requires intracellular calcium. Furthermore, G proteins seem to be involved in this process, whereas prostaglandin synthesis via cyclooxygenase pathway is not. We propose a mechanism of shear‐induced gene expression in HUVEC: Shear stress, either directly or indirectly (G protein‐mediated), enhances the membrane phosphoinositide turnover via phos‐pholipase C, producing diacylglycerol, an activator of protein kinase C. The activated protein kinase C then triggers the subsequent PDGF gene expression.
The differential effects of N,N‐dimethylformamide and transforming growth factor‐β1 on a human ovarian cancer cell line (HOC‐7)
Authors: Grunt Thomas W. , Somay Canatay , Ellinger Adolf , Pavelka Margit , Dittrich Evelyn , Dittrich Christian
Published: February 2005

We have compared the effects of N, N‐dimethylformamide (DMF) and transforming growth factor (TGF)‐β1 on the growth and phenotype of HOC‐7 ovarian cancer cells. Previous density gradient fractionation of untreated HOC‐7 cells suggested that rapidly growing small polygonal medium density cells revert spontaneously into less malignant flattened low density cells. Here we demonstrate that DMF and TGF‐β1 induce similar flattened cell phenotypes. Both agents induce qualitatively similar alterations in the cells. DMF, however, exerted stronger effects than TGF‐β1. The cells become flattened, develop cytoplasmic extensions, and reduce DNA‐synthesis as well as anchorage‐dependent and ‐independent growth. These effects are reversible after removal of the inducers, indicating that the cells have not become terminally differentiated. Electron microscopy demonstrates prominent filament bundles in treated cells. Immunofluorescence further shows that these cells contain large amounts of cytokeratin. Immunocytochemistry and ELISA demonstrate 1‐ to 5‐fold higher amounts of desmoplakin and fibronectin after DMF‐ or TGF‐β1‐exposure. The described differentiation‐like responses of HOC‐7 cells can be used for recognition of pharmacologically induced maturation of ovarian cancer cells. © 1992 Wiley‐Liss, Inc.
Cyclical strain effects on production of vasoactive materials in cultured endothelial cells
Authors: Carosi Joseph A. , Eskin Suzanne G. , McIntire Larry V.
Published: February 2005

Mechanical forces due to fluid flow and cyclical strain can alter endothelial cell morphology and function, including the release of vasoactive materials endothelin, prostacyclin (PGI2), and tissue plasminogen activator (t‐PA). In this study, effects of cyclical strain were modeled by culturing bovine aortic endothelial cells on fibronectin‐coated elastic membranes of silicone rubber (Silastic®) or poly‐etherurethane urea (Mitrathane™). After growing to confluence under static conditions of 37°C in humidified air with 5% CO2, cells were strained cyclically at membrane elongations of 5% or 10% for 24 hours at 1 Hz. Controls were maintained under static conditions or were exposed to fluid motions similar to the strained cells but without stretching. Secretion rates were constant throughout experiments in the strain chamber with no initial burst in metabolism associated with the initiation of strain. Secretion rates were not altered by choice of elastic membrane. At a physiological level of 10% cyclical strain, prostacyclin and endothelian secretion rates were increased by 2.5‐fold and 1.7‐fold, respectively, above stationary controls. Endothelin production demonstrated a dose‐dependent response with cyclical strain, while PGI2 appeared to require a threshold strain before an increase in secretion occurred. No significant differences in t‐PA levels were seen in cyclically strained cells compared with controls. These results indicate that endothelial cells respond metabolically to cyclical strain and suggest that mechanical strain may modulate secretion of selective vasoactive materials by vascular endothelial cells. © 1992 Wiley‐Liss, Inc.
Changes in luminal flow rate modulate basal and bradykinin‐stimulated cell [Ca2+] in aortic endothelium
Authors: Oliver Juan A. , Chase Herbert S.
Published: February 2005

Hemodynamic forces influence many endothelial cell functions. The coupling between hemodynamic forces and cell function could be mediated by mechano‐sensitive ion channels present in the plasma membrane of endothelial cells. Because one of these channels is permeable to Ca2+, we tested whether hemodynamic forces influence endothelial cell Ca2+ ([Ca2+]i). Bovine aortic endothelial cells were grown inside cylindrical glass tubes, loaded with fura‐2, and perfused at different pressures and flow rates on the stage of a fluorescence microscope. Decreasing flow from 110 to 2 ml.min−1 raised [Ca2+]i from 57 ± 11 to 186 ± 29 nM (mean ± SEM, p < 0.01) by increasing the entry of extracellular Ca2+ into the cytoplasm. Increasing flow from 2 to 110 ml.min−1 transiently decreased [Ca2+]i from 62 ± 3 to 33 ± 5 nM (p < 0.01) apparently due to reduced Ca2+ entry and concomitant extrusion by the plasma membrane Ca2+‐ATPase. The rise in [Ca2+]i induced by bradykinin was magnified during a decrease in flow; in control cells, 10−7 M bradykinin increased [Ca2+]i by 162 ± 26 nM, whereas [Ca2+]i increased 350 ± 67 nM (p < 0.05) in cells previously exposed to 110 ml.min−1. These observations suggest that flow‐induced changes in [Ca2+]i might be a signal‐transduction mechanism for endothelial functions responsive to hemodynamic forces and may also modulate the magnitude of hormonally mediated increases in [Ca2+]i. © 1992 Wiley‐Liss, Inc.
Secretion of stromelysin by cultured dermal papilla cells: Differential regulation by growth factors and functional role in mitogen‐induced cell proliferation
Authors: Goodman Linda V. , Ledbetter Steven R.
Published: February 2005

To understand better the molecular nature of the epithelial‐mesenchymal interactions that govern folliculogenesis and hair growth, we have studied the behavior of cultured rat dermal papilla cells (rDP), the mesenchymal component of the hair follicle. Basic fibroblast growth factor (bFGF) and platelet‐derived growth factor (PDGF) both potentiated the growth of rDP in culture, and transforming growth factor‐β (TGF‐β) inhibited rDP proliferation. Biosynthetic labeling studies demonstrated that both PDGF and bFGF induced synthesis of a major secreted protein(s) with Mr = 55–60 kD. It was noted that PDGF and bFGF differentially regulated synthesis of this major secreted protein; PDGF‐mediated induction was found to be transient, while bFGF allowed prolonged synthesis of the protein. Sodium dodecyl sulfate (SDS)‐substrate gel analysis of rDP‐conditioned media revealed that this protein is a metalloproteinase with casienolytic activity and Mr ≈︁ 51 kD (unreduced). We have identified the growth factor‐regulated rDP protein as the matrix metalloproteinase stromelysin by immunoprecipitation. Northern analysis established that increased secretion of stromelysin was accompanied by an increased expression of stromelysin‐specific mRNA. Remarkably, stromelysin antisera interfere with stimulation of dermal papilla cell growth, demonstrating that stromelysin production serves a functional role in mitogen‐induced proliferation in these cells. These findings provide insight into the mechanism by which the connective tissue remodeling required for formation of hair embryonically and the postembryonic hair cycle may be regulated. © 1992 Wiley‐Liss, Inc.
Repression of SV40 T oncoprotein expression by DMSO
Authors: Witte Michael M. , Parker Robert F. , Wang Hanlin , Scott Robert E.
Published: February 2005

SV40 large T oncoprotein‐transformed murine mesenchymal 3T3 T stem cells (CSV3 cells) can be induced to growth arrest and then differentiate into adipocytes. When differentiation occurs, SV40 T oncoprotein expression is repressed (Estervig et al., J Virol 63:2713, 1989). To determine if repression of T oncoprotein expression can also be induced pharmacologically, the effect of a variety of agents that have been reported to effect differentiation in various cell types but not in 3T3 T or CSV3 cells was tested. This rationale suggests that if any of these agents repress T oncoprotein expression in CSV3 cells, then the results would establish that repression of T oncoprotein expression can be mediated by mechanisms independent of overt differentiation. The results show that dimethylsulfoxide (DMSO) is the only agent tested that represses T oncoprotein expression in CSV3 cells. Repression occurs in a dosage‐dependent manner within 24–96 hours after exposure to DMSO. The effect of DMSO on T oncoprotein expression is mediated by posttranslational mechanisms that decrease the stability of the T oncoprotein. DMSO‐induced repression of T oncoprotein expression is also associated with reversion of the transformed phenotype in CSV3 cells as demonstrated by the loss of responsiveness to a specific transformation‐associated mitogen. These data support the conclusion that the pharmacological repression of T oncoprotein expression represents a form of cancer suppressor activity that can be mediated by a distinct molecular mechanism. © 1992 Wiley‐Liss, Inc.
Peroxisome assembly mutations in humans: Structural heterogeneity in Zellweger syndrome
Authors: Santos Manuel J. , Hoefler Sigrid , Moser Ann B. , Moser Hugo W. , Lazarow Paul B.
Published: February 2005

Empty membrane ghosts of peroxisomes were found in fibroblasts from a patient with Zellweger's syndrome, a genetic disease of humans (Santos et al: Science 239:1536–1538, 1988). Import of soluble matrix proteins into the organelle was defective. We have now studied fibroblasts from seven patients representing five complementation groups of the syndrome (defined by complementation for peroxisome enzyme function). We find that empty peroxisome ghosts are present in all seven cell samples. Three patients, representing three complementation groups, give the same membrane pattern by immunofluorescence: few large ghosts. Three other patients, representing two complementation groups, give a second pattern: many large ghosts. The seventh patient's pattern is distinct. Thus, all seven of these patients exhibit Peroxisome IMport (PIM) mutations. Since membrane assembly occurs in these cells, the results indicate that biogenesis of organelle content and membrane proteins proceed by different mechanisms. Growth and division of the empty peroxisomal membrane must occur, but are modified by the mutations (ghost size and abundance vary). Cell fusion and immunofluorescence analyses of peroxisome size and catalase packaging formally demonstrate genetic complementation groups for peroxisome assembly in Zellweger syndrome. © 1992 Wiley‐Liss, Inc.
Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo
Authors: Umezawa Akihiro , Maruyama Tatsuya , Segawa Kaoru , Shadduck Richard K. , Waheed Abdul , Hata Jun‐Ichi
Published: February 2005

Several murine marrow stromal cells were established from murine bone marrow cultures. Stromal cell lines transfected with a tumor‐inducing polyoma virus middle T antigen (MTAg) were inoculated into nude mice subcutaneously. KUSA‐MTAg cells, one of these cell lines, led to the rapid local development of bone marrow consisting of trilineage hemantopoietic cells and bone; other cell lines produced spindle cell sarcoma or hemangiosarcoma. These results suggested that a single stromal cell line, KUSA‐MTAg cells, may induce hematopoietic stem cells or early progenitors of three lineages of hematopoietic cells in vivo. Interestingly, untransfected KUSA cells expressed three new mesenchymal phenotypes, osteocytes, adipocytes, and myotubes, after treatment with 5‐azacytidine. © 1992 Wiley‐Liss, Inc.
Mechanism of zinc uptake by microvilli isolated from human term placenta
Authors: Aslam Nadim , McArdle Harry J.
Published: February 2005

The mechanism of zinc (Zn) uptake by microvillous membrane vesicles prepared from human term placenta has been studied. The uptake was complex, with two processes being identified. In the first process, uptake was rapid, reaching equilibrium within 2 min, and was temperature dependent, with a Q10 of 1.5. Equilibrium Zn levels were sensitive to osmotic pressure, with Zn binding at infinite osmolarity being 69% iso‐osmotic value. The uptake was saturable, with a Vmax of 58 ± 2 nmol/mg protein/min and an apparent Kt of 128 ± 13 μM. Uptake was inhibited by increasing extravesicular K+ concentration, dropping from 0.91 ± 0.03 nmol/mg/min at 0 extravesicular K+ to 0.47 ± 0.03 at an extravesicular K+ concentration of 150 mM ([Zn] = 1.0 μM). In the presence of both valinomycin, an electrogenic ionophore, and nigericin, an electroneutral exchanger, an outwardly directed K+ gradient stimulated Zn uptake. Similarly, preloading vesicles with Zn and imposing an inward gradient resulted in a temperature dependent efflux of Zn. The data suggest that there is a K+ dependent Zn transporter in vesicle membranes, and we suggest that the evidence is biased in favour of a Zn/K+ exchanger rather than Zn being dependent on the membrane potential. © 1992 Wiley‐Liss, Inc.
Identification of multiple PKC isoforms in Swiss 3T3 cells: Differential down‐regulation by phorbol ester
Authors: Olivier Andrée R. , Parker Peter J.
Published: February 2005

The expression of members of the Ca2+ and phospholipid‐dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC‐α, the presence of immunoreactive PKC‐δ, ‐ε, and ζ was detected. Treatment with 500 nM 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) led to the down‐regulation of α, δ, and ε isoforms, but not that of ζ. Higher concentrations of TPA similarly had no effect on the level of PKC‐ζ. In contrast to PKC‐α, the membrane localization of PKC‐δ, ‐ε, and ‐ζ was not enhanced by extraction in Ca2+‐containing buffers, whereas acute TPA treatment increased membrane association of PKC‐α, ‐δ, and ‐ε but not that of PKC‐ζ. © 1992 Wiley‐Liss, Inc.
Epidermal growth factor dependence and TGFα autocrine growth regulation in primary rat tracheal epithelial cells
Authors: Ferriola Patrice C. , Robertson Alice T. , Rusnak David W. , Diaugustine Richard , Nettesheim Paul
Published: February 2005

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGFα autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGFα peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGFα mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGFα antiserum and by a tyrphostin compound that blocks TGFα/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGFα as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation. © 1992 Wiley‐Liss, Inc.
Transforming growth factor‐β1 is a heparin‐binding protein: Identification of putative heparin‐binding regions and isolation of heparins with varying affinity for TGF‐β1
Authors: McCaffrey Timothy A. , Falcone Domenick J. , Du Baoheng
Published: February 2005

Previous studies indicated that a major factor in heparin's ability to suppress the proliferation of vascular smooth muscle cells is an interaction with transforming growth factor‐β1 (TGF‐β1). Heparin appeared to bind directly to TGF‐β1 and to prevent the association of TGF‐β1 with α2‐macroglobulin (α2‐M). The present studies indicate that 20–70% of iodinated TGF‐β1 binds to heparin‐Sepharose and the retained fraction is eluted with ∼0.37 M NaCI. Native, unlabelled platelet TGF‐β1, however, is completely retained by heparin‐Sepharose and eluted with 0.9–1.2 M NaCI. Using synthetic peptides, the regions of TGF‐β1 that might be involved in the binding of heparin and other polyanions were examined. Sequence analysis of TGF‐β1 indicated three regions with a high concentration of basic residues. Two of these regions had the basic residues arranged in a pattern homologous to reported consensus heparin‐binding regions of other proteins. The third constituted a structurally novel pattern of basic residues. Synthetic peptides homologous to these three regions, but not to other regions of TGF‐β1, were found to bind to heparin‐Sepharose and were eluted with 0.15 M‐0.30 M NaCI. Only two of these regions were capable of blocking the binding of heparin to 125I‐TGF‐β. Immobilization of these peptides, followed by affinity purification of heparin, indicated that one peptide was capable of isolating subspecies of heparin with high and low affinity for authentic TGF‐β1. The ability of TGF‐β1 to bind to heparin or related proteoglycans under physiological conditions may be useful in understanding the biology of this pluripotent growth and metabolic signal. Conversely, a subspecies of heparin molecules with high affinity for TGF‐β1 may be a factor in some of the diverse biological actions of heparin. © 1992 Wiley‐Liss, Inc.
Dissociation between parathyroid hormone‐stimulated cAMP and calcium increase in UMR‐106‐01 cells
Authors: Merritt Bradley S. , Yamaguchi Dean T. , Green Jacob , Kleeman Charles R. , Muallem Shmuel
Published: February 2005

We used the osteogenic sarcoma cell line, UMR‐106‐01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two × 10−7 M of the PTH antagonist 8,18Nle 34Tyr‐bPTH(3–34) amide ([Nle, Tyr]bPTH(3–34)A) was required to inhibit 10−9 M bPTH(1–34)‐stimulated cAMP generation by 50%. 10−7 M bPTH(1–34) completely overcame the inhibition induced by 10−6 M [Nle, Tyr]bPTH(3–34)A. Only 7 × 10−8 M and 2.7 × 10−7 M [Nle, Tyr]bPTH(3–34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 × 10−8 and 10−7 M bPTH(1–34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH‐evoked [Ca2+]i increase. Short incubation with PGF2α augmented the PTH‐evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH‐stimulated cAMP increase. Finally, preincubation with 1.5 × 10−9 M bPTH(1–34) for 20 minutes almost completely blocked the effect of 10−7 M bPTH(1–34) on [Ca2+]i, while preincubation with 5 × 10−9 M bPTH(1–34) for 4 hours was required to inhibit the effect of 10−8 M bPTH(1–34) on cAMP production by 50%. The differences in the regulation of the two PTH‐stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain. © 1992 Wiley‐Liss, Inc.
Restoration of proliferating cell nuclear antigen (PCNA) complex formation in xeroderma pigmentosum group a cells following cis‐diamminedichloroplatinum (II)‐treatment by cell fusion with normal cells
Authors: Miura Masahiko , Domon Masaharu , Sasaki Takehito , Kondo Seiji , Takasaki Yoshinari
Published: February 2005

We examined the role of the factor deficient in xeroderma pigmentosum group A (XP‐A) cells in the formation of proliferating cell nuclear antigen (PCNA) complex with DNA in the DNA repair process in human fibroblasts following cisdiamminedichloroplatinum (CDDP)‐treatment. Immunofluorescence staining after methanol fixation was used to detect the PCNA complex formation. When quiescent normal cells were PCNA‐stained at 3 h after 100 μM CDDP treatment for 1 h, almost all nuclei of the cells showed a punctuated staining pattern. On the other hand, nuclei of XP‐A cells were not stained. These results were the same with the findings following 10J/m2 of ultraviolet light (UV)‐irradiation. The quantitative analysis of the PCNA immunofluorescence intensity of normal cells revealed that the mean intensity was increased by 4.8 times by the CDDP‐treatment and 6.1 times by the UV‐irradiation, compared with that of untreated cells. The intensities among nuclei ranged widely in both treatments. In contrast, the mean intensity was not increased in XP‐A cells by the same treatments. However, when XP‐A cells were fused with normal cells with polyethylene glycol (PEG) treatment, the nuclei of the XP‐A cells showed positive PCNA‐staining following CDDP‐treatment or UV‐irradiation in almost all cases. These results suggest that the PCNA complex formation may play a role in the DNA repair process after the step where the factor deficient in XP‐A cells is involved following CDDP‐treatment as well as following UV‐irradiation. © 1992 Wiley‐Liss, Inc.
Repetitive 5‐azacytidine treatments of Fao cells induce a stable and strong expression of gamma‐glutamyl transpeptidase
Authors: Baik Ja‐Hyun , Chikhi Naima , Bulle Frédérique , Giuili Galicia , Guellaën Georges , Siegrist Sylvie
Published: February 2005

The role of DNA methylation in the expression of the rat gamma‐glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5‐azacytidine. Ten repetitive treatments of the cells, with 8 μM 5‐azacytidine for 24 h, led to 13‐ and 80‐fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5‐azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5‐Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase BmRNAs (12‐ to 16‐fold) as wells as for tubulin, gluthathione transferese, and tyrosine aminotransferase mRNAs (2‐to 5‐fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired α‐fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype. © 1992 Wiley‐Liss, Inc.
Platelet endothelial cell adhesion molecule, PECAM‐1, modulates cell migration
Authors: Schimmenti Lisa A. , Yan Horng‐Chin , Madri Joseph A. , Albelda Steven M.
Published: February 2005

Cell migration is an important process in such phenomena as growth, development, and wound healing. The control of cell migration is orchestrated in part by cell surface adhesion molecules. These molecules fall into two major categories: those that bind to extracellular matrix and those that bind to adjacent cells. Here, we report on the role of a cell‐cell adhesion molecule, platelet‐endothelial cell adhesion molecule‐1, (PECAM‐1), a member of the lg superfamily, in the modulation of cell migration and cell‐cell adhesion. PECAM‐1 is a 120–130 kDa integral membrane protein that resides on endothelial cells and localizes at sites of cell‐cell contact. Since endothelial cells express PECAM‐1 constitutively, we studied the effects of PECAM‐1 on cell‐cell adhesion and migration in a null‐cell population. Specifically, we transfected NIH/3T3 cells with the full length PECAM‐1 molecule (two independent clones). Transfected cells containing only the neomycin resistance gene, cells expressing a construct coding for the extracellular domain of the molecule, and cells expressing the neu oncogene were used as controls. The PECAM‐1 transfectants appeared smaller and more polygonal and tended to grow in clusters. Indirect immunofluorescence of PECAM‐1 transfectants showed peripheral staining at sites of cell‐cell contact, while the extracellular domain transfectants and the control cells did not. In two quantitative migration assays, the full‐length PECAM‐1 transfectants migrated more slowly than control cells. Thus, PECAM‐1 transfected into a null cell appears to localize to sites of cell‐cell contact, promote cell‐cell adhesion, and diminish the rate of migration. These findings suggest a role for this cell‐cell adhesion molecule in the process of endothelial cell migration. © 1992 Wiley‐Liss, Inc.
In vitro model of angiogenesis using a human endothelium‐derived permanent cell line: Contributions of induced gene expression, G‐proteins, and integrins
Authors: Bauer Jeffrey , Margolis Michael , Schreiner Clara , Edgell Cora‐Jean , Azizkhan Jane , Lazarowski Eduardo , Juliano R. L.
Published: February 2005

The EA hy926 cell line is a continuous, clonable, human cell line that displays a number of features characteristic of vascular endothelial cells (Edgell et al., 1983). Here we report that when EA hy926 cells (EA cells) are plated on an extracellular matrix material [Matrigel®], they undergo a process of morphological re‐organization leading to the formation of a complex network of cord or tubelike structures. These events seem to resemble, in some respects, an in vitro process of angiogenesis. The morphological re‐arrangement occurs within a 12–16 hr period and seems to require expression of new messenger RNA and protein, since it is completely blocked when actinomycin D or cycloheximide are present at the time the cells are plated on Matrigel. This is not due to overt toxicity of the drugs, since exposure of cells to actinomycin D at 2 hr or more after plating on Matrigel has little effect on the formation of the tubelike structures. The process of Matrigelinduced tube formation also apparently involves a G‐protein mediated signal. Treatment of the EA cells with pertussis toxin completely blocks the process and causes the ADP‐ribosylation of a 42 kD protein that is recognized by an antibody to Gi‐alpha subunits. In contrast, concentrations of pertussis toxin sufficient to block tube formation have only modest effects on the adhesion or motility of EA cells on purified matrix components such as laminin or collagen IV. The process of Matrigel‐induced tube formation also involves integrins since monoclonal antibodies to integrin alpha6 or beta 1 subunits can completely block the process. The concentrations of anti‐integrin antibodies needed to block tube formation are much lower than those required to block cell adhesion on purified matrix components and are sufficient to occupy less than 10% of the alpha6 or beta 1 subunits available at the cell surface. These results suggest that integrins may be involved in this potential model of angiogenesis in processes beyond their usual role in cell adhesion. Based on these results, it seems likely that the EA hy 926 cell line will prove to be a useful model for in vitro study of angiogenic processes. © 1992 Wiley‐Liss, Inc.
Electrochemical potentials of potassium in skeletal muscle under different metabolic states
Authors: Khuri Raja N. , Agulian Samuel K. , Abdulnour‐Nakhoul Solange , Nakhoul Nazih L.
Published: February 2005

Intracellular potassium and membrane potential were measured simultaneously by means of double‐barrelled liquid ion‐exchange microelectrodes in single fibers of rat thigh muscle in vivo in rats maintained in seven different metabolic states. The K+ equilibrium potential (Ek) was more negative than the simulataneously measured membrane potential (Em) in the normal state by 18.4 mV. K+ loading, acute and chronic, resulted in depolarization of Em due to increased serum K+ (hyperkalemia) with no increase in intracellular K+. K+ depletion resulted in hyperpolarization of Em as plasma K+ decreased proportionately more than intracellular K+. Low Na+ diet had no effect. Intracellular K+ was decreased in acute acidosis but not in the chronic state. Thus K+ depletion and acute acidosis are associated with intracellular K+ decrease. The fact that hyperpolarization exists in the former and not the latter is a reflection that hypokalemia accompanies the former condition. The hyperpolarizing states of K+ depletion and chronic acidosis are accompanied by decreased excitability and muscle weakness. © 1992 Wiley‐Liss, Inc.
Establishment of a chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses A23187 induction of both GRP78 and GRP94
Authors: Li Li‐Jing , Li Xuan , Ferrario Angela , Rucker Natalie , Liu Eva S. , Wong Sam , Gomer Charles J. , Lee Amy S.
Published: February 2005

GRP78, a 78,000 dalton protein residing in the endoplasmic reticulum, is postulated to play important roles in protein folding and cell survival during calcium and other physiologcial stress. Here we describe the construction of an eukaryotic expression vector for the constitutive expression of grp78 antisense RNA and the creation of a CHO cell line, 78WO, which expresses high levels of the grp78 antisense RNA through amplification of the stably transfected antisense vector. We observed that whereas 78WO maintains a basal levelof GRP78 similar to that of control cells, GRP78 is no longer inducible by A23187. The 78WO cells have undergone a compensatory increase in grp78 transcription such that the effects of antisense are cancelled out at the protein level under nonstressed conditions. In these same cells, GRP94, a 94,000 dalton ER protein, is also rendered noninducible by A23187. This provides the first evidence that the regulation of two ER proteins might be coupled such that the failure to induce GRP78 results in the down‐regulation of GRP94. The 78WO cell line grows with a doubling time of about 26 hr and exhibits decreased tolerance to A23187, suggesting the GRPs contribute to cell viability under calcium stress. The establishment of this cell line, which can be stably maintained, will provide a useful tool for testing whether the induction of the GRPs is important for protein folding or transport and whether their enhanced synthesis is the cause or consequence of a variety of physiological adaptations. © 1992 Wiley‐Liss, Inc.
Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo
Authors: Grant Derrick S. , Kinsella James L. , Fridman Rafael , Auerbach Robert , Piasecki Barbara A. , Yamada Yoshihiko , Zain Mona , Kleinman Hynda K.
Published: February 2005

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1(CDPGYIGSR) chains block the capillary‐like tube formation on a laminin‐rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic‐biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22‐2) on endothelial cells. The SIKVAV‐containing peptide was as active as the YIGSR‐containing peptide for endothelial cell attachment but was less active than either the RGD‐containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the ‐SIKVAV‐site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury. © 1992 Wiley‐Liss, Inc.
Cell cycle regulated expression of nucleolar antigen P120 in normal and transformed human fibroblasts
Authors: Fonagy Anna , Swiderski Carol , Wilson Amy , Bolton Wade , Kenyon Norma , Freeman James W.
Published: February 2005

Normal and SV40 virus‐transformed WI‐38 human lung fibroblasts were serum starved and refed, or synchronized by double thymidine block and released from the block. At different time points in the cell cycle, steady state levels of P120 mRNA and P120 protein content of the cells were determined by densitometric scans of Northern and Western blots. At the same time points, [3H]thymidine uptake was measured and flow cytometric analysis performed for DNA content and P120 antigen staining. Levels of P120 protein and P120 mRNA were approximately 4 times greater in non‐synchronous, exponentially growing transformed cells than in similarly growing normal cells. Early G1‐cells, synchronized either with serum deprivation or with metabolic block, contained only a trace amount of P120 protein and mRNA. The P120 gene was transcribed early in G1 and P120 protein synthesis initiated in middle G1. A dramatic increase of P120 protein level occurred in S‐phase with a corresponding mRNA peak preceding the P120 protein peak. These results indicate that P120 is overexpressed in transformed WI‐38 cells and that P120 is temporally regulated during the cell cycle of both transformed and normal fibroblasts. The dramatic increase in P120 protein expression at the G1 to S boundary suggests that P120 may play a role in the regulation of cell cycle and increased nucleolar activity that is associated with cell proliferation. © 1993 Wiley‐Liss, Inc.
Pulsatile and steady flow induces c‐fos expression in human endothelial cells
Authors: Hsieh Hsyue‐Jen , Li Nan‐Qian , Frangos John A.
Published: February 2005

The effects of pulsatile and steady fluid flow on the mRNA levels of proto‐oncogenes c‐fos, c‐jun, and c‐myc in cultured human umbilical vein endothelial cells (HUVEC) were investigated. c‐fos mRNA levels in stationary cultures were very low. A 1 Hz pulsatile flow with an average shear stress of 16 dynes/cm2 induced a dramatic increase of c‐fos mRNA levels in HUVEC 0.5 h after the onset of flow, which declined rapidly to basal levels within 1 h. Steady flow with a similar shear stress also induced a transient increase of c‐fos mRNA levels, but to a lesser extent. In addition, increased, c‐fos mRNA levels were observed when low shear (2–6 dynes/cm2) was replaced by high shear (16–33 dynes/cm2). Pulsatile and steady flow caused a slight increase of c‐jun and c‐myc mRNA levels. The role of pulsatility was also investigated in platelet‐derived growth factor (PDGF) expression. Pulsatile flow induced a transient increase of PDGF A‐ and B‐chain mRNA levels with peaks at 1.5–2 h. Pulsatile flow, which was more stimulatory in mediating c‐fos expression, however, was less stimulatory than steady flow in mediating PDGF expression. By using various inhibitors, protein kinase C was found to be an important mediator in flow‐induced c‐fos expression, with the involvement of G proteins, phospholipase C, and intracellular calcium. Protein kinase C was previously shown as a possible major mediator in flow‐induced PDGF expression which, at least partly, appeared to follow the induction mechanism of c‐fos, suggesting a possible connection between c‐fos and PDGF induction. However, the c‐fos antisense treatment, which significantly inhibited c‐fos transcription, failed to block the flow‐induced PDGF expression, suggesting that flow‐induced c‐fos expression may not play an important role in the mechanism of flow‐induced PDGF expression. The difference in the induction of c‐fos and PDGF expression under pulsatile as compared to steady flow indicates of c‐fos and PDGF expression under pulsatile as compared to steady flow indicates that a complex, flow‐mediated regulatory mechanism of gene expression exists in HUVEC. The increased expression of these proto‐oncogenes mediated by flow may be important in regulating long‐term cellular responses. © 1993 Wiley‐Liss, Inc.
Involvement of the Golgi region in the intracellular trafficking of cholera toxin
Authors: Nambiar Madhusoodana P. , Oda Tatsuya , Chen Chaohua , Kuwazuru Yasuo , Wu Henry C.
Published: February 2005

The intracellular pathway following receptor‐mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)‐elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID50 value similar to the LD50 of BFA in Y1 adrenal cells. Binding and internalization of [125I]‐cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA‐sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK1 cells, of which the Golgi structure was BFA‐resistant. These results strongly suggest that a BFA‐sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley‐Liss, Inc.
Nuclear protein redistribution in heat‐shocked cells
Authors: Warters Raymond L. , Chu G. L. , Wong R. S. L. , Lyons B. W. , Dewey W. C.
Published: February 2005

An increase was observed in the total protein mass of nuclei isolated from Chinese hamster ovary cells heated at 45°C or 45.5°C. An increase in the fractional recovery of DNA polymerase α and β, and of DNA topoisomerase activity coincided with this increase in the protein mass of nuclei from heated cells. Nuclear protein mass which was soluble in 2.0 M NaCl decreased 0.5 fold, while DNA‐associated and nuclear matrix‐associated protein mass increased 2.2 and 3.4 fold, respectively. The results indicate that the increase in nuclear protein mass observed in nuclei from heated cells is due in part to an increased binding, or precipitation, of nuclear proteins onto the cell's DNA and nuclear matrix. © 1993 Wiley‐Liss, Inc.
Heat shock alters centrosome organization leading to mitotic dysfunction and cell death
Authors: Vidair C. A. , Doxsey S. J. , Dewey W. C.
Published: February 2005

To identify the cellular target(s) responsible for thermal killing in the G1 phase of the cell cycle, synchronous cultures of Chinese hamster ovary cells (CHO) were heat shocked and studied for one cell cycle by time‐lapse videomicroscopy and immunocytochemistry. At the first mitosis post‐heating, the fraction of cells giving rise to multinucleated progeny approximately equaled the nonclonogenic fraction. In addition, the cells yielding multincleated progeny were delayed in prophase‐metaphase relative to the cells yielding two uninucleated progeny (clonogenic cells). To study the basis for the delay in prophase‐metaphase and subsequent formation of multinucleated cells, cells in mitosis were examined by immunofluorescence for spindle abnormalities. Multipolar mitotic spindles and chromosome misalignment were induced by heat. All multiple spindle poles induced by heat stained for pericentriolar material (PCM), the microtubule nucleating material of centrosomes. Heated cells in mitosis also contained additional foci of PCM which were not associated with the spindle. Cells made thermotolerant by a nonlethal heat shock were resistant to both thermal killing and the induction of multiple foci of PCM. Quantitative analysis revealed a good correlation between the fraction of cells with multipolar spindles, the fraction with more than two foci of PCM, and the nonclonogenic fraction. These data indicate that heat‐induced alterations to the PCM of centrosomes resulted in multipolar mitotic spindles, delay in prophase‐metaphase, and formation of multinucleated cells which were nonclonogenic. These results identify the centrosome as a G1 target for cell killing. © 1993 Wiley‐Liss, Inc.
Regulation of neutrophil interleukin 8 gene expression and protein secretion by LPS, TNF‐α, and IL‐1β
Authors: Fujishima Seitaro , Hoffman Andrew R. , Vu Thanh , Kim K. Jin , Zheng Hui , Daniel David , Kim Youngmee , Wallace Ellen F. , Larrick James W. , Raffin Thomas A.
Published: February 2005

Neutrophils are possibly involved in the pathogenesis of various lung diseases through the release of numerous mediators. In the present study, we studied the regulation of IL‐8 gene induction and protein secretion in human blood neutrophils. Northern blot analysis revealed that LPS increased IL‐8 mRNA levels in neutrophils, with a maximal fivefold increase by 2 h. IL‐8 mRNA levels returned to baseline value within 12 h. In contrast, LPS‐stimulated monocytes demonstrated a sustained increase of IL‐8 mRNA levels for more than 24 h. TNF‐α, IL‐1β, and phorbol myristate acetate also increased IL‐8 mRNA levels in neutrophils. Immunohistochemical analysis confirmed that IL‐8 was localized within stimulated neutrophils. IL‐8 secretion by neutrophils and monocytes was quantified using a specific ELISA for IL‐8. Resting neutrophils secreted minimal IL‐8 activity. However when cells were stimualted with LPS, TNF‐α, or IL‐1bT, neutrophils secreted IL‐8. IL‐8 secretion was most marked during the first 2 h after stimulation and decreased thereafter. In contrast, monocytes maintained a high rate of IL‐8 secretion over 12 h. Although a single monocyte secreted 70‐fold more IL‐8 than did a single neutrophil after 4 h of incubation, the high abundance of neutrophils in peripheral blood made the neutrophil‐secreted IL‐8 more significant. During the first 2 h, neutrophils secreted ∼40% of the IL‐8 released by monocytes in the same volume of blood. This ratio decreased to 9% after 12 h. Neutrophil‐secreted IL‐8 may play an autocrine or paracrine role during the initial stage of inflammation. © 1993 Wiley‐Liss, Inc.