bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective induction of cell cycle regulatory genes cdk1 (p34cdc2), cyclins a/b, and the tumor suppressor gene RB in transformed cells by okadaic acid
Authors: You Jinsong , Bird R. Curtis 
Published: February 2005

AbstractGenes encoding cdk1 (p34cdc2), cyclin A, cyclin B, and the tumor suppressor gene Rb are fundamental regulators of cell cycle progression which associate as a complex with the transcription factor E2F. Expression of many of these proteins has previously been shown to be repressed by okadaic acid, a specific inhibitor of protein phosphatases 1/2A (PP1/PP2A), resulting in growth arrest in nontransformed but immortilized cells. We have investigated levels of mRNA encoding cdk1 (p34cdc2) cyclin A, cyclin B, Rb, GAPDH, c‐myc, and histone H4 genes for sensitivity to okadaic acid in HeLa cells to determine if transformation altered their regulation. Serum starvation slowed growth and diminished mRNA levels for all genes tested except c‐myc and GAPDH. When starved cells were subsequently exposed to 19 nM okadaic acid or refed 10% serum, mRNA levels of cyclin A, cyclin B, cdk1, and Rb dramatically increased while mRNA levels of cyclin A, cyclin B, cdk1, and Rb dramatically increased while mRNA levels for c‐myc and GAPDH were largely unaffected. Histone H4 mRNA levels and the rate of DNA synthesis were greatly enhanced by serum addition but not affected apreciably by okadaic acid. Okadaic acid was also effective in blocking proliferation of exponentially growing HeLa cells at G2/M and S phase. Despite the cell cycle phase‐specific block, elevated mRNA levels for cdk1, cyclin A, cyclin B, Rb, and suppression of H4 mRNA levels were detected and persisted for at least 12 hr following okadaic acid removal. The results demonstrate that cell cycle progression is blocked and several cell cycle regulatory genes, encoding transcription factor E2F‐associated proteins, experience elevation of mRNA levels through mechanisms sensitive to okadaic acid likely through a PP1/PP2A‐sensitive mechanism. Data from transformed cells contrast with data from immortalized but nontransformed cells in which okadaic acid also blocks cell cycle progression during G2/M phase but suppresses expression of these genes. Such contrasts may be correlated with reduced growth factor dependence and transformation. © 1995 Wiley‐Liss, Inc.
EGF‐Induced increase in diacylglycerol, choline release, and DNA synthesis is extracellular calcium dependent
Authors: Dean Nicholas M. , Boynton Alton L. 
Published: February 2005

AbstractPrevious studies have demonstrated a strict extracellular Ca2+ dependence for the G0 to G1 and G1 to S transition in growth factor‐treated T51B rat liver cells that is associated with increased levels of protein kinase C activity. Consequently, we have examined these cells for changes in phospholipid‐derived second messengers in response to epidermal growth factor (EGF) and thrombin in order to determine which signals are generated during the initiation of the G0 to G1 transition. Thrombin is coupled to a phosphoinositide hydrolyzing phospholipase C, as we have found a rapid Ca2+‐independent increase in the levels of inositol 1,4,5‐trisphosphate (Ins[1,4,5]P3), inositol 1,4‐bisphosphate (Ins[1,4]P2), and inositol 4‐monophosphate (Ins[4]P), as well as a concomitant, transient elevation in diacylglycerol. No changes in either intracellular or extracellular choline metabolites, or an increase in DNA synthesis, were found in response to thrombin. By contrast, treatment of T51B cells with EGF results in a slower, more prolonged extracellular Ca2+‐dependent increase in both [3H]‐glycerol radiolabeled diacylglycerol, and diacylglycerol mass, an increase in choline release into the extracellular medium, and eventually a substantial DNA synthesis. We were, however, unable to detect any changes in phosphatidylinositol (Ptdlns) turnover, either by accumulation of inositol phosphates or by changes in phospholipids in response to EGF. These results indicate that DNA synthesis can readily occur in the absence of stimulated Ptdlns turnover, and that Ptdlns turnover is not sufficient in itself or necessary to induce DNA synthesis and is not necessary for a Ca2+‐dependent increase in diacylglycerol. Moreover, we have demonstrated that the extracellular Ca2+‐dependent increase in diacylglycerol levels in response to EGF is associated with an increase in extracellular choline release, which is indicative of an activation of a phosphatidylcholine‐linked phospholipase D. These results suggest that diacylglycerol sources other than Ptdlns's may be important in the extracellular Ca2+‐dependent regulation of EGF‐mediated cell replication. © 1995 Wiley‐Liss, Inc.
PC12‐E2 cells: A stable variant with altered responses to growth factor stimulation
Authors: Wu Yvonne Y. , Bradshaw Ralph A. 
Published: February 2005

AbstractA variant cell line, designated E2, characterized by more rapid responses to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) and markedly more robust responses to interleukin‐6 and 8‐Br‐cAMP, has been subcloned from the rat PC12 cell line. The enhanced responsiveness to NGF in E2 cells is not due to receptor overexpression as judged by TrkA protein levels and tyrosine kinase activity, but may be associated with the increased and prolonged tyrosine phosphorylation of ERK1 (extracellular signal regulated kinase 1) and ERK2. The rapid morphological differentiation induced by different growth factors in E2 cells is constitutively express some differentiation‐associated molecules that allow direct entry into the neuronal program. © 1995 Wiley‐Liss, Inc.
Lymphocyte transendothelial migration toward smooth muscle cells in interleukin‐1 β‐stimulated co‐cultures is related to fibronectin interactions with α4β1 and α5β1 integrins
Authors: Molossi Silvana , Elices Mariano , Arrhenius Thomas , Rabinovitch Marlene 
Published: February 2005

AbstractWe previously reported infiltration of immune‐inflammatory cells in coronary arteries from cardiac allografts, associated with increased endothelial and smooth muscle cell fibronectin synthesis regulated by interleukin (IL)‐1b̃. We now investigate, using a porcine endothelial‐smooth muscle cell co‐culture system, whether IL‐1b̃‐stimulated fibronectin production is functionally important in lymphocyte transendothelial migration. Lymphocytes were harvested from porcine peripheral blood and, in the unactivated state or following activation with phorbol myristic acetate (PMA) and IL‐2, were characterized by fluorescence‐activated cell sorter (FACS) analysis and added to a confluent endothelial monolayer on the upper chamber of a transwell system. Endothelial cells, as well as smooth muscle cells (in the bottom of the chamber), were stimulated with IL‐1b̃. Then transendothelial lymphocyte migration was determined in the presence of CS1 and RGD (fibronectin) peptides, blocking α4b̃1 and α5b̃1 integrin receptors on lymphocyte surfaces, respectively. A 55‐70% inhibition of lymphocyte migration was observed when compared to control peptides. The combination of CS1 and RGD peptides did not significantly enhance the inhibitory effect of either peptide alone. A similar decrease in lymphocyte transendothelial migration toward smooth muscle cells was documented using a monoclonal antibody to cellular fibronectin. Furthermore, using smooth muscle cell conditioned medium; we reproduced the enhanced transendothelial lymphocyte migration as well as the inhibition with blocking peptides or fibronectin antibodies. Our data suggest that cytokine‐mediated fibronectin synthesis in vascular cells recruits inflammatory cells through interactions of specific peptides with cell surface α4b̃1 α5b̃1 integrins. © 1995 Wiley‐Liss, Inc.
Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan‐containing coats around normal human mesothelial cells
Authors: Heldin Paraskevi , Suzuki Masanobu , Teder Priit , Pertoft Håkan 
Published: February 2005

AbstractThe composition and permeability of the pericellular coat surrounding normal human mesothelial (NHM) cells have been studied in vitro. NHM cells were grown in the presence of 3H‐glucosamine and the amount of label recovered in hyaluronan and chondroitin sulfate was determined after selective enzymatic digestion of the polysaccharides in medium, pericellular, and intracellular pools. For comparison a similar analysis was carried out on mesothelioma cells (Mero‐14). Of the labeled polysaccharides in the medium and pericellular pools of NHM cells about 80–90% could be ascribed to hyaluronan and only 3–5% to chondrojtin sulfate. In contrast, Mero‐14 synthesized only minute amounts of hyaluronan whereas chondroitin sulfate corresponded to 61% of the total glycosaminoglycans in the culture. The results exclude a structure of the pericellular layer of NHM cells similar to the hyaluronan‐proteoglycan aggregates found in cartilage. The permeability of the pericellular layer was tested by the exclusion of polystyrene microspheres and bacteria of diameter 0.1–3.0 μm, as well as erythrocytes of diameter 7 μm. While the erythrocytes were excluded the smaller particles penetrated the coat. By adding 0.5 mg/ml of aggregating cartilage proteoglycan to the medium particles of 0.3 μm or larger were also excluded. Thus exogenous proteoglycans can reinforce the structure of the pericellular layer. © 1995 Wiley‐Liss Inc.
Serglycin and betaglycan proteoglycans are expressed in the megakaryocytic cell line CHRF 288‐11 and normal human megakaryocytes
Authors: Schick Barbara P. , Jacoby Jennifer A. 
Published: February 2005

AbstractThis study has characterized the proteoglycans from the megakaryocytic tumor cell line CHRF 288‐11 and the effect of the differentiation‐inducing agents phorbol‐12‐myristate‐13‐acetate (PMA) and dimethylsulfoxide (DMSO) on proteoglycan synthesis in these cells. There appeared to be two classes of proteoglycans. One, serglycin, was recognized to have a core protein of 31 kDa, an overall molecular mass of 200–300 kDa, and glycosaminoglycan chains of mean size <25 kDa. The size of this proteoglycan was increased by both PMA and DMSO. Synthesis was increased by PMA and reduced by DMSO. mRNA for serglycin was increased at 24 to 72 hr following PMA treatment. In addition, the cells contained a core protein triplet at 96, 110, and 120 kDa, and the medium only the bands at 96 and 110 kDa, suggesting the presence of betaglycan. Synthesis of this proteoglycan was enhanced by PMA. This proteoglycan had an overall size of 130–150 kDa on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in control cells, but in the presence of PMA, a component >250 kDa was present. Probes for Northern blot analysis were prepared by polymerase chain reaction (PCR) based on the sequences of human serglycin and betaglycan. The serglycin probe recognized a 1.4 kb band, and the betaglycan probe recognized a 4.1 kb band, on blots prepared from RNA from CHRF cells and cultured normal human megakaryocytes. Both proteoglycans in their intact form adhered to peptides derived from fibronectin and collagen, but the free GAGs released by alkaline borohydride digestion did not adhere. Synthesis of two proteoglycans appears to be a part of the differentiation process of megakaryocytic tumor cells and normal megakaryocytes. © 1995 Wiley‐Liss Inc.
Concanavalin A induced apoptosis in fibroblasts: The role of cell surface carbohydrates in lectin mediated cytotoxicity
Authors: Kulkarni G. V. , McCulloch C. A. G. 
Published: February 2005

AbstractCell surface carbohydrates play important regulatory roles during development and in tissue homeostasis by mediating cellular signalling. Concanavalin A (con A) cross links mannose residues on cell surfaces. We used con A to examine the role of cell surface carbohydrates in apoptosis. Balb/c 3T3 (3T3) and diploid human gingival fibroblasts (HGF) incubated with con A (50 μmg/mL) exhibited rounding, reduction of cell size, loss of cytoskeletal definition, nuclear condensation, and ultrastructural changes consistent with apoptotic cell death. However oligonucleosomal DNA fragmentation was not observed. The sugar methyl‐α‐D‐mannopyranoside (MADM) competes for binding sites with con A but failed to induce‐apoptosis. Cell survival assays after con A treatment demonstrated a concentration dependent (5–500 μg/mL) loss of cell viability in 3T3 and HGF cells which was blocked by MADM. The relationship between cell surface binding of con A and cell death was studied in 3T3 cells labelled with varying concentrations of FITC‐con A. Flow cytometry and cell survival analyses revealed that smaller cells with lower total cell surface FITC‐con A binding sites were ∼8 times more susceptible to cell death than larger cells with higher number of binding sites. This suggests a positive relationship between cell size, number of con A binding sites, and cell death. Flow cytometric cell cycle analysis of HGF cells showed a 46% reduction in the proportion of G1 phase cells but there was little change in the relative proportions of S and G2+M phase cells. 3H‐thymidine labelling of con A treated cells showed a five‐fold decrease (chi sq; P < 0.05) in the percentage of labelled cells, indicating blockade of cell cycle transit into S. Thus, cell death occurred predominantly in G1, possibly due to inhibition of protein synthesis which in turn prevented entry of cells into S phase. 35S‐methionine uptake in con A treated cells was significantly reduced (∼42% con A 50 μg/mL; ∼67% con A 500 μg/mL; P < 0.05) compared to untreated controls indicating inhibition of de novo protein synthesis. SDS‐PAGE of total cellular proteins confirmed reduction of predominantly lower molecular mass proteins after con A treatment. Con A treatment (50 μg/mL) induced a 25% reduction of free intracellular calcium ion concentration over 30 min suggesting that calcium dependent enzymes and translational mechanisms may be inhibited. Collectively, these results indicate that con A binding of cell surface carbohydrates can induce apoptotic cell death in fibroblastic cells due in part to protein synthesis inhibition. © 1995 Wiley‐Liss Inc.
Transforming growth factor‐β receptor expression on endothelial cells: Heterogeneity of type III receptor expression
Authors: Morello Jean Pierre , Plamondon Josée , Meyrick Barbara , Hoover Richard , O'Connor‐McCourt Maureen D. 
Published: February 2005

AbstractRecent studies of whole animal response have defined a role for circulating TGF‐β in the preservation and stabilization of microvascular endothelial function (Lefer et al. [1993] Proc. Natl. Acad. Sci. U.S.A., 90:1018–1022; Pfister et al. [1992] J. Exp. Med., 176:265–269). In order to determine which TGF‐β receptor types are responsible for this endothelial cell responsiveness, we used an affinity‐labeling technique with 125I‐TGF‐β1 and ‐β2 to characterize TGF‐β receptors on five different endothelial cell cultures: early passage bovine lung and rat epididymal fat pad microvascular endothelial cells (BLMEC and REEC), established endothelial cell lines from bovine adrenal medulla capillaries (EJG), fetal bovine heart (FBHE), and bovine pulmonary artery (CPAE). Since it is known that endothelial cells from different parts of the vasculature vary with respect to cell surface antigen expression (McCarthy et al. [1991] Trends Pharmacol. Sci., 12:462–467; Augustin et al. [1994] Bioessays, 16:901–906), it is important to compare TGF‐β receptor expression on microvascular and macrovascular endothelial cells. We observed 85 kDa and 200–400 kDa labeled receptor bands and analyzed their relationship to the cloned Type II and III receptors using peptide antibodies. We used dithiothreitol and phosphoinositol‐phospholipase C pretreatments to establish whether the 65 kDa labeled band which we observed corresponded to the Type I receptor or a glycophosphotidylinositol‐linked binding protein. The results demonstrated that microvascular but not macrovascular endothelial cells express high levels of the Type III receptor. This differential expression of the Type III receptor indicates that distinct anatomical segments of the vasculature have distinct TGF‐β receptor profiles. The presence of the Type III receptor on micro‐but not macrovascular endothelial cells may account for the reportedly different potency of TGF‐β1 and TGF‐β2 on these two endothelial cell types. Analysis of the 85 kDa and 65 kDa affinity‐labeled bands revealed that all the endothelial cells express the Type II receptor and a band consistent with the presence of a dithiothreitol‐sensitive Type I receptor. Two isoform‐specific phosphoinositol‐phospholipase C releasable TGF‐β binding proteins were also detected: a 60 kDa protein on one micro‐ (EJG) and one macro‐ (FBHE) vascular endothelial cell line and a 150/180 kDa protein on the macrovascular cell lines (FBHE and CPAE). These studies emphasize the heterogeneous nature of endothelial cells and underline the importance of using microvascular endothelial cells when examining TGF‐β responses related to microvascular function. © 1995 Wiley‐Liss Inc.
Platelet‐derived growth factor‐B increases colon cancer cell growth in vivo by a paracrine effect
Authors: Hsu Stephen , Huang Fei , Friedman Eileen 
Published: February 2005

AbstractPDGF‐B released from colon tumor cells regulated tumor growth in athymic mice in a paracrine manner by inducing blood vessel formation. A positive correlation was found between expression of PDGF B‐chain in cells grown in vitro and the number of factor VIII‐positive blood vessels in tumors induced by three classes of colon carcinoma cell lines. Elevated expression of PDGF‐B was also correlated with tumor size. Each cell line had the same mutations in the colon cancer genes APC, DCC, and p53 and had wild type c‐K‐ras genes (Huang et al. [1994] Oncogene, 9:3701–3706.) eliminating the possibility that any differences in tumor blood vessel formation were due to mutations and/or deletions in these genes. Colon carcinoma cells released biologically active PDGF capable of stimulating the growth of NIH3T3 cells, which was inhibited by neutralizing antisera to PDGF‐AB chains. An inverse correlation was found between induction of factor VIII‐positive blood vessels and expression of vascular endothelial growth factor (VEGF), while no correlation was seen with expression of either TGFα or k‐FGF. Basic fibroblast growth factor (FGF) expression was not detected in these tumor cells. TGFβ1 was capable of inducing PDGF‐B expression in the undifferentiated U9 colon carcinoma cell line, but this sensitivity was not seen in differentiated cells. In contrast, TGFβ1 inhibited VEGF expression in both undifferentiated cells and differentiated colon cancer cells. Thus, TGFβ1 has two roles in the growth of undifferentiated U9 colon carcinoma cells in vivo: direct stimulation of cell proliferation as we have showed in earlier studies, and an increase in angiogenesis by inducing PDGF‐B. © 1995 Wiley‐Liss, Inc.
Induction of P‐Glycorprotein mRNA by protein synthesis inhibition is not controlled by a transcriptional repressor protein in rat and human liver cells
Authors: Schuetz John D. , Strom Stephen C. , Schuetz Erin G. 
Published: February 2005

AbstractRecent studies have suggested that a labile transcriptional repressor protein is important in the regulation of pgp mRNA expression. However, cycloheximide (CHX) the protein synthesis inhibitor used, can increase mRNAs by either stabilizing the mRNA transcript or directly activating gene transcription. To determine whether CHX posttranscriptionally increased pgp mRNA, we compared the effect of CHX, which inhibits protein synthesis by stabilizing polysomes, with puromycin (PURO), which inhibits protein synthesis by polysome destabilization. In rat hepatocytes, CHX induced pgp2 mRNA, and the increase was proportional to the degree of protein synthesis inhibition. In contrast, despite almost complete inhibition of protein synthesis, PURO did not induce pgp2 mRNA. Further studies demonstrated that PURO pretreatment could block pgp2 mRNA induction by CHX. Likewise, in cultures of primary human hepatocytes CHX, but not PURO, induced MDR1 mRNA. A polymerase chain reaction assay was developed to assess whether CHX treatment altered the length of the 3′‐untranslated region (UTR) of pgp2. CHX treatment time dependently increased the length of the pgp2 3′‐UTR. To determine whether CHX acts as a transcriptional agonist, we performed nuclear run‐off analysis and found no increase in pgp2 gene transcription compared to untreated control. Further, transcription studies were performed by transiently transfecting HepG2 cells with plasmids containing 5′ segments of human MDR1 fused with the reporter chloramphenicol acetyltransferase (CAT). These plasmids were not transcriptionally activated by CHX. In summary, our results cast doubt on the existence of a labile transcriptional repressor protein for pgp. Furthermore, these are the first studies to demonstrate that polysomal destabilization by PURO can block CHX induction of pgp. © 1995 Wiley‐Liss, Inc.
Expression and regulation of retinoid X Receptors in B16 melanoma cells
Authors: Desai Dinakar S. , Niles Richard M. 
Published: February 2005

AbstractRecently, a new subfamily of nuclear retinoid receptors that is distinct from that of RARs has been identified and named Retinoid X receptors (RXRs). These receptors specifically bind 9‐cis‐retinoic acid (9cisRA), but not all‐trans‐retinoic acid (ATRA). We determined which RXR subtypes were expressed in B16 mouse melanoma cells and then studied the effect of ATRA, 8‐bromo‐cyclic AMP (8BrcA), and phorbol dibutyrate (PDB) on RXR mRNA levels. ATRA induces differentiation in these cells while 8BrcA and PDB antagonize the RA‐induced differentiation of B16 melanoma cells. Northern analysis demonstrated the expression of RXRα and RXRβ mRNA in B16 cells, but RXRγ was not detectable. Further analysis using RT‐PCR also failed to detect RXRγ in these cells. Longterm RA treatment decreased the expression of RXRα, but not RXRβ mRNAs. PDB did not alter the expression of either RXR mRNAs, however, 8BrcA treatment resulted in a time dependent decrease in the amount of RXRβ, but not RXRα mRNA. Inhibition of protein synthesis by cycloheximide resulted in a large increase in RXRα and RXRβ mRNA levels. This effect of cycloheximide was time and concentration dependent with maximal stimulation of RXRα and RXRβ mRNAs occurring at 4 h of treatment. Inhibition of transcription with actinomycin D completely abolished the cycloheximide‐induced increase of RXRβ. In contrast to its effect on other genes, such as immediate response genes, cycloheximide treatment did not increase the half‐life of RXRβ mRNA. Nucclear run‐on assays showed that cycloheximide treatment of intact B16 melanoma cells stimulated the transcription rate of RXRβ, but not RXRα. These results suggest the presence of an unstable transcription factor that negatively regulates the expression of RXRβ in B16, melanoma cells. In addition, since RXRβ is the predominant isotype in B16 cells, 8BrcA may, at least partially, inhibit RA‐induced differentiation through down‐regulation of this RXR. © 1995 Wiley‐Liss, Inc.