bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple pathways are involved in protection of MCF‐7 cells against death due to protein synthesis inhibition
Authors: Geier Avraham , Weiss Chana , Beery Rachel , Haimsohn Michal , Hemi Rina , Malik Zvi , Karasik Avraham
Published: February 2005

Previously we have shown that IGF‐1 protected MCF‐7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). In the present study we investigated the ability of protein kinase C activator 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), the protein kinase A activator 8‐bromoadenosine 3′5′‐cyclic monophosphate (Br‐cAMP), and the enzyme inhibitor aurintricarboxylic acid (ATA) to protect MCF‐7 cells against death, due to a continuous presence of CHX. Cell death was evaluated after 48 h of incubation by several techniques (trypan blue staining, release of lactic dehydrogenase, cellular ATP content, transmission electron microscopy, and DNA fragmentation). Apoptosis which terminates in necrosis, characterized this mode of cell death. TPA and ATA at optimal concentrations of 40 ng/ml and 100 μg/ml, respectively, reduced cell death to the control level (without CHX), while Br‐cAMP at an optimal concentration of 650 μg/ml reduced cell death only partially. IGF‐1, TPA, and ATA, which stimulated protein synthesis in the control MCF‐7 cells, had no effect on protein synthesis in the CHX‐treated cells, indicating that the survival effect is not due to new protein synthesis. The protein kinase C inhibitor staurosporine blocked the survival effect of TPA and IGF‐1 in a dose‐dependent manner, however did not affect the survival effect of ATA. The tyrosine kinase inhibitor genistein blocked the survival effect of IGF‐1, but not that of TPA and ATA. Our results provide evidence for several distinctive pathways, the activation of which protects MCF‐7 cells against death, due to protein synthesis inhibition. © 1995 Wiley‐Liss, Inc.
Specific binding of leukemia inhibitory factor to murine myoblasts in culture
Authors: Bower J. , Vakakis N. , Nicola N. A. , Austin L.
Published: February 2005

Leukemia inhibitory factor (LIF) is a member of the cytokine family of growth factors. It has been shown to exert a variety of actions on a diverse range of cell types, including neuronal, bone, and hemopoietic cells (Hilton, 1992, Trends Biochem. Sci., 17:72–76). In many of these cell types, studies have indicated the presence of specific receptors for LIF (Godard et al., 1982, J. Biol. Chem., 267:3214–3222; Hilton et al., 1988, Proc. Natl. Acad. Sci. USA, 85:5971–5975; Hilton and Nicola, 1992, J. Biol. Chem., 267:10238–10247.). The mechanism by which these receptors act is believed to involve tyrosine phosphorylation and the signal transducing receptor component gp130. We have previously shown that LIF is capable of inducing both human and murine myoblasts to proliferate in culture (Austin et al., 1992, J. Neurol. Sci., 112:185–191). We now report that LIF binds specifically to receptors on the surface of myoblasts, with an equilibrium dissociation constant of 400 pM and the number of receptors per cell varies with cell density. Binding competition studies showed that LIF binding to these receptor sites was not competed for by a number of other growth factors which stimulate myoblast proliferation including basic fibroblast growth factor (bFGF), transforming growth factor‐α (TGFα), insulin‐like growth factor 1 (IGF‐1), and interleukin‐6 (IL‐6). There was a time and concentration‐dependent down‐regulation of receptor numbers following preincubation of myoblasts with LIF. The processing of these receptors subsequent to binding, involves as a first step, internalization and degradation by the myoblast. LIF appeared to stimulate myoblast proliferation rather than cell survival. © 1995 Wiley‐Liss, Inc.
Regulation of multidrug resistance gene mdr1b/mdr1 expression in isolated mouse uterine epithelial cells
Authors: Kuo M. Tien , Julian Joanne , Husain Farah , Song Renduo , Carson Daniel D.
Published: February 2005

The mammalian uterine epithelium (UE) undergoes drastic physiological and morphological changes during pregnancy. Steady‐state levels of murine mdr1b mRNA, transcribed from a multidrug resistance gene encoding a membrane protein which functions as a transporter of lipophilic cytotoxic agents, are low in nonpregnant, cycling UE, but drastically increase (about 1,500‐ to 2,000‐fold) at day 8 of gestation. At day 16 of gestation, levels of mdr1b mRNA are 2,500‐ to 3,000‐fold higher than those in the cycling UE cells. Levels of mdr1b mRNA were elevated to levels comparable to those observed during pregnancy, in the UE of ovariectomized mice following 5–8 days of estrogen and progesterone administration. Withdrawal of these hormones resulted in a drastic reduction of mdr1b mRNA within 36 hr. These results suggested that steroid hormones alone can account for increased mdr1b mRNA expression and do not require the presence of other placenta/embryo‐derived factors. Moreover, the hormonal effect on uterine mdr1b mRNA biosynthesis during pregnancy apparently is a delayed phenomenon. Nuclear run‐on assays demonstrated that the rate of mdr1b transcription in UE cells prepared from 15‐day pregnant mice (d‐15 UE cells) was about two‐ to three‐fold higher than that in nonpregnant UE cells. This increased transcription rate alone cannot account for mdr1b mRNA accumulation during pregnancy. mdr1b mRNA expression was investigated in primary cultures of d‐15 UE cells. mdr1b mRNA levels decayed by 50% within 3‐4 hr of culture and reached a steady‐state 0.5‐2% of initial levels by 24 hr. The rate of mdr1b mRNA decay in primary d‐15 UE cells was decreased by treatment with α‐amanitin or cycloheximide, suggesting that the decay pathway requires both transcription and de novo protein synthesis. Our results suggest that multiple mechanisms are involved in the maintenance of the high levels of mdr1b mRNA in pregnant UE cells. Furthermore, these data suggest that increased mRNA stability may contribute to the accumulation of mdr1b transcript during pregnancy. © 1995 Wiley‐Liss, Inc.
Hepatocyte growth factor is involved in formation of osteoclast‐like cells mediated by clonal stromal cells (MC3T3‐G2/PA6)
Authors: Sato Takuya , Hakeda Yoshiyuki , Yamaguchi Yuji , Mano Hiroshi , Tezuka Ken‐Ichi , Matsumoto Kunio , Nakamura Toshikazu , Mori Yoshihisa , Yoshizawa Kenji , Sumitani Koji , Kodama Hiroaki , Kumegawa Masayoshi
Published: February 2005

Osteoclast formation from hemopoietic precursors has been shown to require the support of stromal cells in bone tissue. In this study, we demonstrated that hepatocyte growth factor (HGF) is one of the stromal cell‐derived molecules responsible for osteoclast‐like cell formation. For our experimens, we used a coculture system for osteoclastic cell formation and activation in which hemopoietic blast cells are cocultured with calvaria‐derived stromal MC3T3‐G2/PA6 (PA6) cells on dentine slices in the presence of 1, 25‐dihydroxyvitamin D3 [1,25(OH)2D3]. Addition of anti‐HGF neutralizing IgG to the cocultures inhibited the formation of osteoclastic cells and their dentine‐recorbing activity. We detected a single 6.0‐kb transcript for HGF in PA6 cells, and also recognized immunoreactive Mr. 81,000 and 88,000 forms of HGF in conditioned medium (CM) from PA6 cell cultures, the level of which reached 6 ng/ml. Both the CM and HGF stimulated the proliferation of blast cells synergistically with granulocyte‐macrophage colony‐stimulating factor, resulting in an increased number of osteoclast precursors that respond to 1,25(OH)2D3 that are tartrate‐resistant acid phosphatase‐positive multinucleate cells in stromal cell‐free blast cell cultures in plastic wells. The effect of the CM was diminished by the addition of anti‐HGF IgG. However, neither the CM nor HGF stimulated the formation of osteoclastic cells and pits on dentine slices in the absence of PA6 cells. These results suggest that although HGF cannot completely replace stromal cells, it is one of the paracrine mediators produced by stromal cells that act on proliferation of osteoclastic cell precursors. © 1995 Wiley‐Liss, Inc.
Transforming growth factor‐β1 modulates adenylyl cyclase signaling elements and epidermal growth factor signaling in cardiomyocytes
Authors: Nair Bipin G. , Yu Yiming , Rashed Hani M. , Sun Hui , Patel Tarun B.
Published: February 2005

Studies presented in this report were designed to investigate the effects of transforming growth factor‐b̃1 (TGF‐b̃1) on epidermal growth factor (EGF)‐ mediated stimulation of cAMP accumulation in cardiac myocytes and elucidate the mechanism(s) involved in this modulation. TGF‐b̃1 (20 pM) treatment of cardiac myocytes, in a time‐dependent manner, decreased the ability of EGF (100 nM) to increase cAMP accumulation. Significant attenuation of EGF‐elicited cAMP accumulation was observed 2 h after exposure to TGF‐b̃1 and 18 h after addition of TGF‐b̃1, the ability of EGF to increase cAMP accumulation was completely obliterated. TGF‐β1 neither decreased immunoprecipitable EGF receptors in membranes from cardiomyocytes nor altered the specific binding of [125I] EGF to cardiomyocyte membranes. However, TGF‐b̃1 decreased the ability of EGF to phosphorylate membrane proteins on tyrosine residues. TGF‐b̃1 treatment of cardiomyocytes also decreased the ability of forskolin to augment cAMP accumulation in intact cells and stimulate adenylyl cyclase activity. Similarly, in membranes of TGF‐b̃1‐treated cells, neither isoproterenol nor EGF stimulated adenylyl cyclase activity. Interestingly, as assessed by the ability of A1F4− to stimulate adenylyl cyclase, TGF‐b̃1 did not alter the coupling between Gs and catalytic subunits. Likewise, TGF‐b̃1 did not alter the functional activity of the inhibitory regulatory element of the system, Gi. Western analysis of cellular proteins revealed that TGF‐b̃1 did not alter the amounts of Gsα, Giα2, and Giα3. We conclude that TGF‐b̃1 attenuates EGF‐elicited cAMP accumulation in cardiomyocytes, in part, by decreasing the EGF receptor kinase function and that TGF‐b̃1‐mediated alterations in the activity of adenylyl cyclase catalytic subunit also contribute toward the regulation of adenylyl cyclase by various agonists. © 1995 Wiley‐Liss, Inc.
Repression of the Id2 (inhibitor of differentiation) gene promoter during exit from the cell cycle
Authors: Biggs Joseph R. , Zhang Yong , Murphy Elizabeth V.
Published: February 2005

The Id2 gene is one of several “Id‐like” genes which encode helix‐loop‐helix proteins which dimerize with basic helix‐loop‐helix proteins and inhibit binding to the DNA enhancer element known as an E box. By repressing the DNA binding activity of basic helix‐loop‐helix proteins, Id proteins inhibit transcription of tissue‐specific genes in myoblasts, hematopoietic precursor cells, and other types of undifferentiated cells. Serum starvation results in the disappearance of Id gene transcripts in most types of cultured cells, and often induces differentiation of these cells. In order to gain some insight into this process, we have analyzed Id2 promoter function in the glioma cell line U87Y. We have isolated 300 base pairs of Id2 promoter sequence which is sufficient to repress the activity of a reporter gene in serum‐starved U87Y cells, but induces the activity of the reporter gene when the cells are stimulated with fresh serum. Two regions within this 300 base pair sequence contain repressor elements; deletion of either region results in increased promoter activity. Both repressor regions serve as binding sites for a protein in extracts from serum‐starved U87Y cells but not in serum‐stimulated cells. © 1995 Wiley‐Liss, Inc.
Preferential induction of apoptosis in mouse CD4+CD8+αβTCRIoCD3εIo thymocytes by zinc
Authors: Telford William G. , Fraker Pamela J.
Published: February 2005

High concentrations of zinc salts (500 m̈M and greater) are known to inhibit apoptosis in a variety of systems. However, closer examination of dose effects revealed that lower concentrations of zinc (80‐200 m̈M) could induce apoptosis in approximately 30‐40% of mouse thymocytes following 8 h incubation. The ability of zinc to cause thymocyte apoptosis was detected flow‐cytometrically by reductions in propidium iodide DNA fluorescence and forward scatter, both quantitative indicators of apoptotic death. Zinc induced both internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis as determined by gel electrophoresis and electron microscopy, respectively. In addition, transcriptional and translational inhibitors prevented zinc‐induced apoptosis, indicating a requirement for de novo mRNA and protein synthesis, another characteristic of apoptotic death. Fluorescent immunophenotype‐specific apoptotic analysis indicated that zinc‐induced apoptosis occurred primarily in the less mature CD4+CD8+αb̃TCRIoCD3εIo thymocyte subset, with lower amounts of death occurring in the other subsets. This lineage specificity was shared with glucocorticoid‐induced apoptosis. Taken together, these results indicate that zinc induces true apopotitic death in mouse thymocytes and suggests a role for zinc in the regulation of apoptosis. © 1995 Wiley‐Liss, Inc.
Different regulatory pathways involved in ATP‐stimulated chloride secretion in rat epididymal epithelium
Authors: Chan H. C. , Zhou W. L. , Fu W. O. , Ko W. H. , Wong P. Y. D.
Published: February 2005

The regulatory pathways involved in the ATP‐stimulated CI− secretion across rat epididymal epithelium were investigated by the short‐circuit current (ISC) technique. Biphasic characteristic was observed in the ISC responded to ATP (0.01‐10 m̈M). Inhibitor of P1 receptor, 8‐phenyltheophylline (up to 100 m̈M), did not have any effect on both phases of the ATP‐stimulated ISC. The order of potency for stimulation of the two phases in ISC was ATP>ADP> AMP, adenosine, consistent with the presence of P2‐purinoceptors. CI− channel blocker, disulfonic acid stilbene (DIDS, 300 m̈M), only inhibited the first peak of the ATP‐stimulated ISC while diphenylamine‐dicarboxylic acid (DPC, 1 mM) reduced both, indicating the involvement of different conductance pathways. DIDS was found to have an inhibitory effect on Ca2+‐activated ISC (induced by ionomycin, 10 m̈M) but not cAMP‐activated ISC (induced by forskolin, 1 m̈M) which could only be blocked by DPC. Both peaks of the ATP‐activated ISC could be significantly inhibited by pretreatment with a Ca2+‐chelating agent, BAPTA‐AM (50 m̈M). An increase in cellular cAMP content upon stimulation of ATP was measured by radioimmunoassay. No significant increase in cAMP production was observed in cells stimulated with adenosine. The ATP‐induced cAMP increase was prevented by pretreatment with BAPTA‐AM (100 m̈M) indicating that cAMP production upon ATP stimulation was secondary to an increase in intracellular Ca2+ concentration. These results indicate that the ATP‐stimulated CI− secretion could be mediated by Ca2+ and cAMP‐dependent regulatory pathways giving rise to the biphasic nature of the ATP‐induced ISC. © 1995 Wiley‐Liss, Inc.
Developmental changes in phosphorylation of the transcription factor CREB in the embryonic murine palate
Authors: Weston Wayde M. , Greene Robert M.
Published: February 2005

Cyclic AMP, via activation of cAMP‐dependent protein kinase (PKA) and subsequent protein phosphorylation, regulates a number of cellular and tissue responses that are critical to normal development of the mammalian palate. The present study examines the expression, distribution, and phosphorylation in the developing murine palate of a substrate for PKA known as the cAMP‐response element binding protein (CREB). This 43 × 103 Mr protein functions as a regulator of cAMP‐inducible gene expression. CREB is expressed constituitively throughout the palatal morphogenetic period and is ubiquitously distributed throughout palatal tissue. Immunofluorescent staining of palatal cells and tissues with an anti‐CREB antibody revealed CREB to be localized to cell nuclei. Western blot analysis of extracts of staged palatal shelves with an antibody specific for phospho‐ser 133‐CREB demonstrated a steady increase in CREB phosphorylation at this residue during palate development. These observations show a temporal correlation with expression levels of cAMP‐regulated genes in palate cells. The data indicate that CREB activity in the developing palate is most likely to be regulated at the level of protein phosphorylation as opposed to changes in levels of CREB protein expression. © 1995 Wiley‐Liss, Inc.
Effects of dexamethasone, heat shock, and serum responses on the inhibition of hsc70 synthesis by antisense RNA in NIH 3T3 cells
Authors: Li Ting , Hightower Lawrence E.
Published: February 2005

A dexamethasone (Dex)‐inducible antisense RNA expression vector was constructed that contains the 5′‐untranslated region and one third of the coding sequence for the bovine hsc70 protein. This vector was used to transfect NIH 3T3 cells from which clonal cell lines expressing hsc70 antisense RNA were developed. Quantitative Northern blot analysis with strand‐specific probes was used to demonstrate the Dex‐inducible accumulation of hsc70 antisense RNA in proliferating cell cultures and the inhibition of hsc70 RNA levels. Surprisingly, antisense RNA was either much less effective in reducing the amounts of hsc70 RNA in Dex‐treated cultures than in untreated controls or cells compensated by producing more hsc70 RNA in response to increasing amounts of antisense RNA. Hsc70 protein synthesis did not decrease in either Dex‐treated or untreated cultures: it actually increased, again suggesting the activation of a compensatory response. In Dex‐treated cultures subjected to heat shock, hsc70 antisense RNA blocked the induction of hsp70, indicating that newly synthesized RNA was targeted effectively before it became translationally active. To test this hypothesis further, Dex‐treated cultures were made quiescent by serum deprivation and then restimulated with serum, which causes a burst of RNA and protein synthesis. Consistent with this hypothesis, increased synthesis of hsc70 was blocked in serum‐stimulated cultures expressing antisense RNA. © 1995 Wiley‐Liss, Inc.
Vasculotropin/Vascular endothelial growth factor is an autocrine growth factor for human retinal pigment epithelial cells cultued in vitro
Authors: Guerrin Marina , Moukadiri Hafida , Chollet Pascale , Moro Fraņoise , Dutt Kamla , Malecaze Fraņois , Plouët Jean
Published: February 2005

Vasculotropin (VAS), also called vascular endothelial growth factor (VEGF) or vascular permeability factor, is a secreted growth factor whose target cell specificity has been reported as restricted to vascular endothelium. Its effects are mediated by at least two distinct membrane‐spanning tyrosine kinase receptors, KDR and flt‐1, the expression of which also seems restricted to vascular endothelium. We describe here that cultured human retinal pigment epithelial (HRPE) cells express both KDR and flt‐1 receptors, bind VAS/VEGF on two high affinity sites (apparent Kd of 9 and 210 pM corresponding to 940 and 18,800 sites per cell) and proliferate or migrate upon recombinant VAS/VEGF addition. HRPE cells also express the mRNA corresponding to the 121 and 165 amino acid forms of VAS/VEGF. HRPE cells release in their own culture medium and store in their extracellular matrix self‐mitogenic and chemoattractant factors indistinguishable from 121 and 165 VAS/VEGF isoforms. The autocrine role of VAS/VEGF was confirmed by the inhibition of these bioactivities by neutralizing specific anti‐VAS/VEGF antibodies. © 1995 Wiley‐Liss, Inc.
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

Genes 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

Previous 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

A 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

We 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

The 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

This 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

Cell 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

Recent 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

PDGF‐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

Recent 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

Recently, 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.
Basic fibroblast growth factor and epidermal growth factor downmodulate the growth of hematopoietic cells in long‐term stromal cultures
Authors: Dooley Douglas C. , Oppenlander Barbara K. , Spurgin Pam , Mead Jay H. , Novak F. Patrick , Plunkett Michael , Beckstead Jay , Heinrich Michael C.
Published: February 2005

The bone marrow microenvironment consists of stromal cells and extracellular matrix components which act in concert to regulate the growth and differentiation of hematopoietic stem cells. There is little understanding of the mechanisms which modulate the regulatory role of stromal cells. This study examined the hypothesis that mesenchymal growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) modulate stromal cell activities and thereby influence the course of hematopoiesis. Both bFGF and EGF were potent mitogens for marrow stroma. However, both factors proved to be inhibitory to hematopoiesis in primary log‐term marrow cultures. Inhibition was also observed when hematopoietic cells and bFGF or EGF were added to subconfluent irradiated stromal layers, demonstrating that the decline of hematopoiesis was not due to overgrowth of the stromal layer. Loss of hematopoietic support in bFGF and EGF was dose‐dependent. Removal of bFGF and EGF permitted stromal layers to regain their normal capacity to support hematopoiesis. In stroma‐free long‐term cultures, neither factor affected CFU‐GM expansion. Basic FGF slightly enhanced granulocyte‐macrophage colony forming unit (CFU‐GM) cloning efficiency in short‐term agarose culture. Basic FGF did not reduce the levels of interleukin‐6 (IL‐6), GM‐CSF, or G‐CSF released by steady state or IL‐1‐stimulated stroma. Similarly, the constitutive levels of steel factor (SF) mRNA and protein were not affected by bFGF. Basic FGF did not alter the level of TGF‐β1 in stromal cultures. We conclude that bFGF and EGF can act as indirect negative modulators of hematopoietic growth in stromal cultures. The actual mediators of regulation, whether bound or soluble, remain to be identified. © 1995 Wiley‐Liss, Inc.
Interferon‐γ and tumor necrosis factor‐α suppress both early and late stages of hematopoiesis and induce programmed cell death
Authors: Selleri Carmine , Sato Tadatsugu , Anderson Stacie , Young Nel S. , Maciejewski Jaroslaw P.
Published: February 2005

Increased expression of interferon gamma (IFN‐γ) and tumor necrosis factor alpha (TNF‐α) in bone marrow failure disorders suggests a possible pathophysiologic role of these cytokines in disease. In this study, we tested the action of TNF‐α and IFN‐γ on phenotypically and functionally defined stages of hematopoietic development using highly purified progenitor cell populations assayed in standardized culture systems. We hypothesized that the inhibitory effects of IFN‐γ and TNF‐α might be related to the induction of programmed cell death. In methylcellulose colony assays, IFN‐γ and TNF‐α inhibited the growth of early hematopoietic cells, including committed CD34+ CD38+ progenitor cells and phenotypically less mature CD34+CD38− cells, with 50% decreased colony formation occurring in the range of 750‐1,000 U/ml of IFN‐γ and 10–15 ng/ml of TNF‐α. More potent suppressive effects were observed in cultures supplemented with the combination of both cytokines than in cultures treated with IFN‐γ or TNF‐α alone. When used at these concentrations, IFN‐γ and TNF‐α inhibited growth of CD34+‐enriched long‐term culture‐initiating cells by 88% and 68%, respectively. IFN‐γ and TNF‐α triggered apoptosis of total bone marrow and CD34+ cells, recognized by the presence of a characteristic pattern of DNA degradation after low molecular weight DNA extraction, and by detection of apoptotic cells by the in situ terminal deoxynucleotidyl transferase assay. We speculate that chronic exposure of hematopoietic tissue to TNF‐α and IFN‐γ in vivo may result in broad depletion of the stem and progenitor cell pools. Death of these cells due to apotosis rather than transient inhibition of proliferation may be responsible for long‐lasting hematologic consequences. © 1995 Wiley‐Liss Inc.