bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance
Authors: Black Adrian R. , Subjeck John R.
Published: February 2005

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total KNA, and rRNA synthesis. Increasing exposure of CHO cells to 45°C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotol‐erance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance.

The involvement of rRNA synthesis was further investigated by using actinomy‐cin D at 0.1 μg ml−1, a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was 3‐fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cell following heat shock.
Continuous maintenance of transformed fibroblasts under reduced serum conditions: Utility as a model system for investigating growth factor‐specific effects in nonquiescent cells
Authors: Mulder Kathleen M. , Brattain Michael G.
Published: February 2005

We report the continuous growth maintenance of untransformed and chemically transformed fibroblasts (AKR‐2B, AKR‐MCA cells) in low concentrations of serum (0.1% FBS). The cell lines established (AKR‐0.1F, MCA‐0.1F) proliferated at rates comparable to cells maintained under high serum conditions (10% FBS). Complete removal of serum from the cells did not induce quiescence. The MCA‐0.1F cells were more similar to the untransformed AKR‐2B fibroblasts in their morphology, saturation density, inability to form colonies under anchorage‐independent conditions, steady‐state level of c‐myc expression, and kinetics of induction of c‐myc in response to specific growth factors. This report demonstrates the utility of this cell line as anonquiescent model system for investigating growth factor‐specific effects in serum‐free, cycling cells. Addition of transforming growth factor‐β (TGF‐β) (5 ng/ml) to proliferating MCA‐0.IF cells, in the absence of any serum, induced a multilayered growth pattern at confluency, similar to that of AKR‐MCA cells maintained in 10% FBS. Other growth factors tested did not elicit this effect. The induction of this growth pattern by TGF‐β was associated with a sustained induction of the c‐myc proto‐oncogene at con‐fluency, but not with a restoration of anchorage‐independent growth. The data suggest that TGF‐β may play a role in the up‐regulation of c‐myc at confluency previously described for AKR‐MCA cells maintained in 10% serum.
Effects of N,N‐dimethylformamide and extracellular matrix on transforming growth factor‐β binding to a human colon carcinoma cell line
Authors: Levine Alan E. , Black Bradley , Brattain Michael G.
Published: February 2005

Alterations in the binding of transforming growth factor‐β (TGF‐β) to the MOSER human colon carcinoma cell line caused by N, N‐dimethylformamide (DMF) or extracellular matrix (ECM) were examined. DMF induced a more differentiated phenotype in the MOSER cells and resulted in a twofold increase in TGF‐β binding to the cells. This was due to an increase in receptor number with no significant alteration in the KD. The extent of increased TGF‐β binding was dependent on the dose and time of exposure to DMF. Upon removal of DMF, the receptor level returned to that of untreated cells within 6 hr. The binding of TGF‐β1 and TGF‐β2 to the cells was increased equally. Despite this increase in TGF‐β binding in the presence of DMF, the sensitivity of the MOSER cells to the growth inhibitory effects of TGF‐β was unaltered. Growth of the MOSER cells on ECM derived from a well‐differentiated colon cell line increased the TGF‐β receptor number twofold without altering the KD. No change was observed if the MOSER cells were grown on ECM derived from a poorly differentiated cell line. While no alteration in sensitivity to TGF‐β was observed on cells grown in the presence of DMF, MOSER cells grown on the ECM derived from well‐differentiated colon carcinoma cell lines were twofold more sensitive to the growth inhibitory effects of TGF‐β. These results indicated that growth conditions which resulted in a more differentiated phenotype resulted in an increase in the cellular receptors for TGF‐β.
Stimulation by parathyroid hormone of calcium absorption in confluent Madin‐Darby canine kidney cells
Authors: Kennedy Susan M. , Flanagan Joyce L. , Mills John W. , Friedman Peter A.
Published: February 2005

Parathyroid hormone (PTH) increases renal calcium absorption exclusively in cortical thick limbs and distal tubules. Lack of sufficient tissue has precluded detalled biochemical study of the mechanisms responsible for the hypercalcemic effect of PTH. Therefore, we assessed PTH action on calcium transport in Madin‐Darby canine kidney (MDCK) cells, a cell line expressing distal characteristics, to determine its suitability as a model for analyzing PTH action. Calcium transport across MDCK cells grown to confluence on porous filters was measured at 37°C in Ussing chambers. Mucosal‐to‐serosal calcium fluxes (JCa, mol/min cm−2 × 10−9) were measured with 45Ca at −3, −1, 5, 10, and 20 min; agonist was added at 0 min. Basal JCa averaged 0.98. PTH at 0.2 μM increased JCa by 12% (P 0.05) and 1 μM PTH by 70% (P0.01). Calcitonin (1 μM) had no effect on JCa. The fact that high concentrations of dibutyryl cAMP (1 mM) and forskolin (10 μM) increased JCa by only 37% and 22%, respectively, suggested that cAMP‐independent mechanisms may participate in PTH‐stimulated JCa. Therefore we examined the effect of other putative second messengers. In the presence of 2 mM external [Ca], 10 nM A23187 increased JCa by 88%, and 10 μM A23187 increased JCa by 121%. Addition of 10 μM phorbol 12‐myristate 13‐acetate (PMA) increased JCa by 60%. We conclude that: (1) PTH specifically stimulates unidirectional calcium absorption in MDCK cells; (2) both adenylate cyclasecoupled and calcium‐coupled receptors may participate in signaling the response to PTH; and (3) confluent MDCK cells represent a useful experimental model for elucidating the biochemical mechanisms involved in the renal hypercalcemic action of PTH.
Dimethylsulfoxide modifies the sensitivity of BALB/c‐3T3 cells to the activation of Na+/H+ exchange by phorbol esters
Authors: Martinez Raul , Gillies Robert J.
Published: February 2005

In a companion paper (Gillies et al.: J. Cell. Physiol. 139:124–129, 1989) we show that phorbol esters (PEs) are unable to stimulate Na+/H+ exchange in BALB/C‐3T3 cells under a wide variety of conditions. The Na+/H+ exchangers of a number other cell types are also not responsive to PEs yet have been rendered responsive by treatment with agents such as dimethylsulfoxide (DMSO). We undertook the present study to evaluate whether or not the treatment of BALB/c‐3T3 cells with DMSO will induce modifications in the sensitivity of these cells to activation by PEs. The present study indicates that a 3–5 day exposure of BALB/c‐3T3 cells to 1.25% DMSO leads to changes in the sensitivity of these cells to the activation of Na+/H+ exchanger by PEs. These changes in sensitivity were apparent at day 3 and maximal at day 5. Non‐tumor‐promoting analogues of PEs do not activate Na+/H+ exchange, suggesting that the effect is mediated through kinase C. Sphingosine prevents PE‐, but not serum‐induced alkalinization. However, the half‐time of the intracellular pH (pHin) response to serum was increased by sphingosine, suggesting that kinase C participates in, but is not required for the serum induced activation. Since DMSO does not induce any apparent morphological change, the change in sensitivity of Na+/H+ exchange to PEs is not likely to be related to differentiation, but may be associated with structural changes in the Na+/H+ exchanger and/or changes in isoforms of kinase C which recognize the exchanger as a substrate.
Expression of asparagine independence in variants of ICR 2A haploid frog cells
Authors: Hepfer Carol Ely , Little Wayne A. , Hengst Jamie A. , Freed Jerome J.
Published: February 2005

Properties of the change from asparagine dependence (asn‐) to independence (asn+) were investigated in the androgenetic haploid frog cell line ICR 2A. Two types of asn+ variants arose spontaneously during culture. Glutamine‐dependent asparagine synthetase (AS) activity, found to be deficient in asn‐ cells, was repressed by asparagine in one type of variant and expressed constitutively in the other. No quantitative differences in AS‐specific DNA sequences or changes in ploidy were evident between asn+ and asn− cells. The asn‐ frequency in ICR 2A populations, not dramatically influenced by chemical mutagens, was increased 130‐fold by exposure to 5‐azacytidine. The methylation of CCCG sequences at the 5′ end of the AS structural gene was found to be reduced equally in both types of asn+ variant. These results indicate that decreased DNA methylation is essential but not necessarily sufficient for the expression of AS activity in this frog cell system.
Inhibition of DNA synthesis and cell division by a cell surface sialoglycopeptide
Authors: Fattaey Heideh , Johnson Terry C. , Chou Hsin‐Hwei
Published: February 2005

We have isolated and purified a cell surface sialoglycopeptide (SGP) from bovine cerebral cortex cells that previously was shown to be a potent inhibitor of cellular protein synthesis. The following studies were carried out to characterize the potential ability of the SGP to inhibit DNA synthesis and to arrest cell division. Treatment of exponentially proliferating Swiss 3T3 cells with the SGP inhibitor resulted in a marked inhibition of thymidine incorporation within 24 h. When the SGP was removed from inhibited cultures, a sharp rise in 3H‐thymidine incorporation followed within 3–4 h that peaked well above that measured in exponentially growing cultures, suggesting that the inhibitory action of the SGP was reversible and that a significant proportion of the arrested cells was synchronized in the mitotic cycle. In addition to DNA synthesis, the inhibitory action of the SGP was monitored by direct measurement of cell number. Consistent with the thymidine incorporation data, the SGP completely inhibited 3T3 cell division 20 h after its addition to exponentially growing cultures. Upon reversal there was a delay of 15 h before cell division resumed, when the arrested cells quickly doubled. Most, if not all, of the growth‐arrested cells appeared to have been synchronized by the SGP. The SGP inhibited DNA synthesis in a surprisingly wide variety of target cells, and the relative degree of their sensitivity to the inhibitor was remarkably similar. Cells sensitive to the SGP ranged from vertebrate to invertebrate cells, fibroblast and epitheliallike cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines.
Superoxide dismutase induces differentiation of friend erythroleukemia cells
Authors: Beckman Barbara S. , Balin Arthur K. , Allen R. G.
Published: February 2005

Friend erythroleukemia cells (FELC) served as a model system for cell differentiation because these cells can be triggered to differentiate by a variety of chemical agents. Treatment with the classical inducer of differentiation, hexamethylene bisacetamide (HMBA), stimulated superoxide dismutase (SOD) activity, which increased in parallel with HMBA‐induced differentiation. Furthermore, FELC were shown to differentiate in response to the addition of liposomes containing SOD. Oxidative treatment with Iiposomes containing D‐amino acid oxidase or xanthine oxidase, cumene peroxide, or potassium superoxide also induced differentiation, whereas antioxidants such as a‐tocopherol, butylated hydroxytoluene, or p‐carotene did not induce differentiation. Also, HMBA induction of differentiation was suppressed by treatment with antioxidants.
Environment‐dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor‐beta
Authors: Rollins Barrett J. , O'Connell Therese M. , Bennett Gregory , Burton L. Eugene , Stiles Charles D. , Rheinwald James G.
Published: February 2005

Transforming growth factor‐beta (TGF‐β) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF‐β, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF‐β was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF‐β. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30‐ to 100‐fold higher concentrations of TGF‐β were required to achieve comparable growth inbibition. This differential sensitivity occurred despite the fact that in both culture systems TGF‐β in the culture medium had a half‐life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF‐β proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF‐β‐coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF‐β within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF‐β to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.
Effects of interleukin‐1 on fibroblast extracellular matrix, using a 3‐dimensional culture system
Authors: Qwarnströ E. E. , MacFarlane S. A. , Page R. C.
Published: February 2005

This study describes the alterations induced by Interleukin‐1α and ‐β (IL‐1α and IL‐1β) on fibroblast‐synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen‐coated Terasaki wells for 3 or 6–9 weeks to create 3‐dimensional cell‐containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL‐1α or IL‐1β (10−9 M) at 37°C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL‐1α and IL‐1β induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross‐sectional area and an increased density of the matrices. Three‐week matrices were reduced 26% and 45% by using IL‐1α and IL‐1β, respectively. Comparable values obtained by using 6‐week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL‐β than with IL‐1α. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3‐week matrix following IL‐1β incubation. There was also a decrease in collagen type I staining of 9‐week matrices treated with IL‐1α or IL‐1β. These studies show that IL‐1 has an effect on fibroblast‐synthesized extracellular matrix and indicate that the effects of IL‐1α and IL‐1β may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.
Modulation of endothelial fibronectin synthesisby polymorphonuclear granulocytes
Authors: Macarak Edward J. , Gorfien Stephen , MacGregor Rob Roy
Published: February 2005

The attachment of polymorphonuclear granulocytes (PMNs) to vascular endothe‐lial cells occurs continually in normal tissues; however, knowledge of the factors that control leukocyte margination is incomplete. In the present study, we used cell cultures of pulmonary artery endothelium to study their interaction with PMNs. Endothelial cells were seeded in Costar 24‐well plates following which PMNs were inoculated onto the endothelial monolayers and incubated for 2 to 20 hours. During this period, fibronectin synthesis by endothelial cells was estimated by ELISA. In wells to which PMNs had been added, supernatant fibronectin concentration was increased at all time points during the 20 hour incubation. At 20 hours, supernatants from wells to which PMNs had been added contained approximately 2 1/2 times the control level of fibronectin. Since the amount of fibronectin, as determined by ELISA, adsorbed onto the added PMNs was negligible, these data suggest that PMNs can modulate the synthesis of fibronectin by pulmonary artery cells. Pulse labeling experiments and measurements of endothelial intracellular fibronectin also suggest this possibility. The endothelial response does not appear to be owing to nonspecific physical interaction since similarly sized polystyrene beads did not cause any change in supernatant fibronectin levels while glutaraldehyde‐fixed PMNs caused only a 20–25% increase in fibronectin levels.
Lipopolysaccharide‐induced expression of the competence gene KC in vascular endothelial cells is mediated through protein kinase C
Authors: Shen Xin‐Yi , Hamilton Thomas A. , Dicorleto Paul E.
Published: February 2005

The KC gene is a cell cycle‐dependent competence gene originally identified in platelet‐derived growth factor‐stimulated BALB/c‐3T3 cells. This gene is also induced in murine peritoneal macrophages in response to activation stimuli. We have examined the expression of the KC gene in cultured porcine aortic endothelial cells following treatment with bacterial lipopolysaccharide (LPS) as a first step in defining the early molecular events involved in endothelial cell stimulation by physiologically relevant modulators. LPS markedly elevated the steady‐state level of KC mRNA in confluent endothelial cells; maximum induction of KC occurred in the cells following exposure to 10 ng/ml LPS for 2 h. LPS did not increase the growth fraction of the cells, nor was the KC mRNA level changed in dense endothelial cells stimulated to enter the cell cycle with epidermal growth factor. However, KC mRNA expression was elevated by addition of serum to starved, subconfluent endothelial cell cultures. Treatment of endothelial cells with phorbol myristate acetate (PMA) and 1‐oleoyl‐2‐acetyl‐glycerol (OAG) also induced KC gene expression. A maximum response was obtained with 10 nM PMA, the effect decreasing with higher levels of the phorbol ester. The calcium ionophore A23187 exhibited little stimulatory activity alone; however, the ionophore did cause a doubling in the PMA‐stimulated KC expression. The increased expression of KC induced by LPS and PMA was inhibited by the presence of 1‐(5‐isoquinoline‐sulfonyl)‐2‐methylpiperazine (H7), a protein kinase C inhibitor, but not by HA1004 (an H7 analogue with little protein kinase C inhibitory activity). No cytotoxicity was observed in inhibitor or LPS‐treated endothelial cell cultures. These results demonstrate that KC gene expression is stimulated by LPS in vascular endothelial cells in a proliferation‐independent process. Second, unlike LPS‐induced KC expression in macrophages and platelet‐derived growth factor‐induced KC expression in 3T3 cells, LPS induction of KC in endothelial cells appears to require activation of protein kinase C.
Effect of mitogen and lymphokine stimulation on proteoglycan synthesis by lymphocytes
Authors: Bartold P. Mark , Haynes David R. , Vernon‐Roberts Barrie
Published: February 2005

The ability of mouse thymocytes and peripheral blood lymphocytes from rats to synthesize and secrete proteoglycans in the presence of a variety of mitogens and lymphokines was studied in vitro, and it was confirmed that such lymphocytes synthesize and secrete significant quantities of proteoglycans. Mitogenic stimulation of the cells with phytohaemagglutanin (PHA) induced a fourfold increase in proteoglycan synthesis; stimulation with interleukin‐1 stimulated proteoglycan synthesis up to fivefold. Proteoglycan synthesis could also be stimulated by culturing the cells in the presence of interleukin‐2. To determine if this response was related to cell proliferation, the cells were cultured in the presence of PHA and either cyclosporine or prostaglandin E2 two agents that inhibit lymphocyte proliferation. Under these conditions, proteoglycan synthesis remained elevated, indicating that this effect may be independent of cell proliferation. Chemical analysis of the proteoglycans indicated them to be composed of chondroitin sulfate and heparan sulfate. Their molecular size was small compared with cartilage proteoglycans but similar to the small dermatan sulfate proteoglycans synthesized by fibroblasts. On the basis of molecular size, three proteoglycan populations were identified, and their relative proportions were altered by mitogenic stimulation of the cells. Taken together, these findings imply that proteoglycan synthesis is intimately associated with lymphocyte activation and may be related to cellular function in immune responses.
Protein kinase C modulates effects of prostanoids on cyclic adenosine monophosphate in guinea pig chief cells
Authors: Raufman Jean‐Pierre , Cosowsky Laurey
Published: February 2005

In the course of examining the role of protein kinase C in signal transduction in dispersed chief cells from guinea pig stomach, we observed that phorbol esters inhibit prostaglandin (PG)‐stimulated increases in cyclic adenosine monophos‐phate (cAMP). Phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C, decreased maximal levels of PGE2‐stimulated cAMP by 40%. This dose‐dependent effect was observed within 30 sec and was maximal by 1 min of incubation at 37°C. Phorbols that do not activate protein kinase C did not have this effect. Adding H7, a protein kinase C inhibitor, abolished the inhibitory effects of PMA. Adding phosphodiesterase inhibitors did not alter the inhibitory effects of PMA, indicating that these effects are not caused by activation of cyclic nucleotide phosphodiesterases. PMA did not alter the increase in cellular cAMP caused by cholera toxin, forskolin, secretin, or vasoactive intestinal peptide. Hence the site of these prostanoid‐specific actions of protein kinase C does not appear to be stimulatory or inhibitory guanine nucleotide binding proteins or the catalytic component of the adenylyl cyclase system. In dispersed chief cells, activation of protein kinase C may inhibit prostanoid‐induced stimulation of the adenylyl cyclase system by a direct effect on prostaglandin receptors.
Down‐regulation of proteolytic activity in 12‐O‐tetradecanoyl‐phorbol‐13‐acetate‐induced k562 leukemia cell cultures: Depletion of active urokinase by excess type 1 plasminogen activator inhibitor
Authors: Alitalo Riitta , Andersson Leif C. , Tapiovaara Hannele , Sistonen Lea , Vaheri Antti , Stephens Ross
Published: February 2005

The human chronic myeloid leukemia cell line K562 acquires several megakaryoblastoid features when cultured in the presence of the tumor promoter 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). We observed strongly increased secretion of several proteins into the culture media of K562 cells within a few hours of TPA treatment. Two of the major secreted polypeptides were identified by immunoprecipitation from media of metabolically labeled cultures as the tissue inhibitor of metalloproteinases (TIMP) and the type 1 plasminogen activator inhibitor (PAI‐1). Maximal amounts of PAI‐1 mRNA and secretion of PAI‐1 polypeptides were observed after 24 hr of TPA treatment and PAI‐1 persisted at elevated levels for several days. The induction of PAI‐1 mRNA was dependent on de novo protein synthesis. Uninduced and induced cells secreted urokinase plasminogen activator in its single‐chain proenzyme form (pro‐u‐PA), which was cleaved extracellularly to the active two‐chain form as shown by pulse‐chase labeling experiments. Upon TPA induction, the secretion of u‐PA polypeptides increased severalfold, and there was a transient accumulation of pro‐u‐PA in the culture medium. However, this did not lead to increased u‐PA activity in the cultures, since active u‐PA was removed by complex formation with the large excess of coinduced PAI‐1. Induction of u‐PA mRNA was biphasic: The first peak of about tenfold increase in steady‐state u‐PA mRNA at 3 hr was followed by a steep decline to the baseline level at 12 hr, and a second, slower accumulation of u‐PA mRNA occurred over the next few days. The biphasic accumulation of u‐PA mRNA was also reflected in u‐PA protein synthesis. We conclude that concerted changes in favor of a nonproteolytic extracellular environment occur in TPA‐induced K562 cultures undergoing megakaryoblastoid differentiation. These changes include excessive secretion of TIMP and inhibition of the induced u‐PA by the simultaneous accumulation of PAI‐1.
TGF‐β stimulates primary human skin fibroblast DNA synthesis via an autocrine production of PDGF‐related peptides
Authors: Soma Yoshinao , Grotendorst Gary R.
Published: February 2005

Transforming growth factor‐β (TGF‐β) stimulates DNA synthesis in human foreskin fibroblasts after a prolonged lag period as compared with other growth factors. The mechanism of induction of DNA synthesis appears to be dependent on the synthesis and secretion of PDGF‐related proteins as antibodies which are specific for PDGF can block the TGF‐β‐induced DNA synthesis. Other growth factors such as PDGF, EGF, or FGF do not induce the synthesis of these PDGF‐related proteins. Additionally, TGF‐β treatment of human foreskin fibroblasts induces the expression of the PDGF A‐chain gene but not the B‐chain gene. This phenomenon appears to function in vivo, as subcutaneous injection of TGF‐β in rat skin induces the expression of the PDGF A‐chain gene. These data suggest that TGF‐β may stimulate the growth of fibroblastic cells via an autocrine production of PDGF‐related proteins.
Electric stimulation of human fibroblasts causes an increase in Ca2+ influx and the exposure of additional insulin receptors
Authors: Bourguignon Gerard J. , Jy Wenche , Bourguignon Lilly Y. W.
Published: February 2005

Previously we reported that treating human fibroblasts in cell culture with high‐voltage, pulsed galvanic stimulation (HVPGS) can significantly increase cellular protein and DNA synthesis (Bourguignon and Bourguignon: FASEB J., 1:398–402, 1987). In this study we have identified two of the early cellular events which occur following exposure to HVPGS: (1) an increase in Ca2+ uptake from the external medium and (2) an increase in the number of insulin receptors on the fibroblast cell surface. The increase in Ca2+ uptake begins within the first minute of electric stimulation while increased insulin binding is not detected until the second minute of stimulation. The HVPGS‐induced increase in insulin binding can be inhibited by bepridil, a specific Ca2+ channel blocker, suggesting that the Ca2+ influx is required for the exposure of additional insulin receptors on the cell surface.

Furthermore, we have determined that the addition of insulin to electrically stimulated cultures results in (1) an immediate, second increase in Ca2+ uptake and (2) significant increases in both protein and DNA synthesis compared to cells which were not stimulated. All three of these insulin‐dependent effects are also inhibited by bepridil.

Based on these results, we propose that HVPGS initially triggers the opening of voltage‐sensitive calcium channels in the fibroblast plasma membrane. The increased level of intracellular Ca2+ then induces the exposure of additional insulin receptors on the cell surface. If insulin is available to bind the additional receptors, the fibroblasts will significantly increase both protein and DNA synthesis.
Protamine selectively inhibits collagen synthesis by human intestinal smooth muscle cells and other mesenchymal cells
Authors: Perr Hilary A. , Drucker David E. M. , Cochran David L. , Diegelmann Robert F. , Lindblad William J. , Graham Martin F.
Published: February 2005

Collagen synthesis is a major function of human intestinal smooth muscle (HISM) cells and contributes to intestinal fibrosis in chronic inflammatory bowel disease. As an extension of previous in vitro studies of the role of heparin in regulating HISM cell proliferation and collagen synthesis, the effect of protamine sulfate was studied. Protamine decreased collagen production by 50% in confluent and proliferating cultures. This effect was concentration‐dependent and was selective for collagen in that neither noncollagen production nor DNA accumulation in the culture plates was affected. Other human mesenchymal cells which produce collagen, such as dermal fibroblasts and aortic smooth muscle cells, responded to protamine in a similar fashion. Protamine has a strong cationic charge and is rich in lysine and arginine. To determine which of these properties was important in decreasing collagen production, the effect of protamine was compared to that of other polyionic compounds. Poly‐L‐lysine decreased collagen production to a lesser degree than protamine. Poly‐L‐arginine was toxic to the cells. Poly‐L‐glu‐tamic acid, which has an opposite charge to protamine, had no effect. These findings suggest that both the number and the arrangement of lysyl residues, in addition to positive charge, are important. Binding assays demonstrated that protamine did not inhibit collagen production by binding to ascorbate in the culture medium. Electrophoretic separation and chromatography of collagen types expressed following protamine treatment showed that the ratio of type I to type III collagen remained 2:1. This observation suggests that suppression of collagen production is not specific to a particular collagen type. The selective inhibition of collagen production by protamine provides an important tool to study the regulation of collagen production in human cells and may also provide potential therapy of fibrotic disorders.
Evidence that alterations in small molecule permeability are involved in the Clostridium perfringens type a enterotoxin‐lnduced inhibition of macromolecular synthesis in vero cells
Authors: Hulkower Keren I. , Wnek Andrew P. , McClane Bruce A.
Published: February 2005

The mechanism by which Clostridium perfringens enterotoxin (CPE) simultaneously inhibits RNA, DNA, and protein synthesis is unknown. In the current study the possible involvement of small molecule permeability alterations in CPE‐induced inhibition of macromolecular synthesis was examined. Vero cells CPE‐treated in minimal essential medium (MEM) completely ceased net precursor incorporation into RNA and protein within 15 minutes of CPE treatment. However, RNA and protein synthesis continued for at least 30 minutes in Vero cells CPE‐treated in buffer (ICIB) approximating intracellular concentrations of most ions. Addition of intracellular concentrations of amino acids to ICIB (ICIB‐AA) caused a further small but detectable increase in protein synthesis in CPE‐treated cells. ICIB did not affect CPE‐specific binding levels or rates. Similar small molecule permeability changes (i.e., 86Rb‐release) were observed in cells CPE‐treated in either ICIB or in Hanks' balanced salt solution. Collectively these findings suggest that CPE‐treatment of cells in ICIB‐AA ameliorates CPE‐induced changes in intracellular concentrations of ions and amino acids and permits the continuation of RNA and protein synthesis. These results are consistent with and support the hypothesis that permeability alterations for small molecules are involved in the CPE‐induced inhibition of precursor incorporation into macromolecules in Vero cells.
ATP‐resistant variants of transformed mouse fibroblasts
Authors: Belzer Ilana , Friedberg Ilan
Published: February 2005

Addition of ATP to cultures of transformed mouse fibroblasts, 3T6 cells, resulted in cell growth inhibition, whereas the growth of the non‐transformed counterparts, 3T3 cells, was only slightly affected. The inhibition was found to be specific for adenine nucleotides, and concentration dependent. At relatively low concentrations (e.g., 0.1 mM) the effect of ATP was cytostatic, whereas at higher concentrations (e.g., 1.0 mM) a cytotoxic effect was exerted. ATP‐resistant variants of 3T6 cells were selected by exposure of cultures to gradually elevated concentrations of ATP. The variants were found to resemble the non‐transformed counterparts, 3T3 cells, more than the 3T6 parent cells, by the following criteria: ATP‐induced alte rations in the membrane potential, changes in membrane permeability, cell growth inhibition, and colony formation on soft agar. The data indicate that long exposure of the transformed cells to external ATP results in redifferentiation and reduction in their tumorigenicity.
Retinoic acid modulates dome formation by MDCK cells in defined medium
Authors: Taub Mary
Published: February 2005

Retinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K‐1). After 4–5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7days with 3 × 10−7 M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 × 10−3 μ M2 in area, whereas in control medium the majority of the domes were between 0 and 9 × 10−3 μm2 in area. The dependence of the retinoic acid effect on prostaglandin E1 (PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K‐1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1‐independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K‐1 lacking PGE1 This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1‐independent MDCK cells. Dibutyryl cAMP‐resistant MDCK cells, which normally do not form domes in medium K‐1, were also studied. Remarkably, the dibutyryl cAMP‐resistant MDCK cells were observed to form domes at a significant frequency when medium K‐1 was supplemented with retinoic acid. However in medium K‐1 lacking PGE1,an effect of retinoic acid on dome formation by dibutyryl cAMP‐resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP‐resistant MDCK cells to form domes in medium K‐1 has previously been attributed to their decreased cAMP‐dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP‐dependent protein kinase or activation of a separate pathway.
Diacylglycerol and calcium induce rapid enhancement of A‐system amino acid transport by independent mechanisms in human T‐lymphocytes
Authors: Woodlock Timothy J. , Segel George B. , Lichtman Marshall A.
Published: February 2005

Sn‐1,2‐diacylglycerols (DAG) and ionized‐free calcium can act as intracellular second messengers for cell activation. Traditionally, T‐lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T‐lymphocytes demonstrate rapid enhancement of A‐(alanine‐favoring) system amino acid uptake when treated with DAG or ionomycin. A 30–40% increase in the initial velocity of uptake (vi) of the synthetic A‐system specific amino acid, methylamino‐isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The vi was enhanced 60% from 12 to 19 μmol/liter cell water per min after 30 min exposure of T‐cells to optimal concentrations of dioctanoylglycerol (30 μM), oleoylacetylglycerol (30 μM), or ionomycin (5 μM) (P < .01 for each agent). A 50‐fold excess of non‐radioactive MeAIB inhibited 80% of [14C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier‐mediated on treatment with these agents. Cycloheximide, 100 μg/ml, inhibited protein synthesis but did not block the A‐system amino acid transport enhancement induced by DAG or ionomycin. The DAG‐induced increase in the vi was blocked 40% with 100 μM H‐7, an inhibitor of protein kinase C. H‐7 treatment did not inhibit the ionomycin‐induced A‐system enhancement. A marked increase in cytoplasmic free calcium was measured when T‐lymphocytes were exposed to ionomycin but not on DAG exposure, and the A‐system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A‐system amino acid uptake in T‐lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A‐system effect of similar magnitude independent of protein kinase C by an increase in cytoplasmic calcium.
Functional receptors for nerve growth factor on Ewing's sarcoma and Wilm's tumor cells
Authors: Thomson Timothy M. , Pellicer Angel , Greene Lloyd A.
Published: February 2005

Quantification of changes in levels of c‐fos RNA was used as an indicator of the presence of functional responses to nerve growth factor in several human nonneuronal cell lines which have previously been shown to express high levels of NGF receptors. Four Ewing's sarcomas, one Wilm's tumor, and one melanoma were examined. Of these cell lines, the Ewing's sarcoma IARC‐EW1 showed greatly increased levels (10–20‐fold) of c‐fos RNA after 1 hour of exposure to NGF. Except for the melanoma line, the other tumor lines exhibited small, but reproducible, elevation of c‐fos RNA expression. In IARC‐EW1 cells, this induction was analyzed for kinetics, dose‐response, and suppression by selective inhibitors of NGF action. The results indicate that these cells bear high‐affinity receptors for NGF, which utilize signal pathways similar to NGF receptors on PC12 cells. Thus, we report new types of cells with functional responses to NGF and indicate that these may constitute a new model which will usefully complement those presently used for studying the mechanism of action of NGF.
Transforming growth factors beta slow down cell‐cycle progression in a murine interleukin‐2 dependent T‐cell line
Authors: Stoeck Michael , Miescher Sylvia , MacDonald H. Robson , Von Fliedner Vladimir
Published: February 2005

Transforming growth factors beta (TGF‐β) inhibit the growth of a variety of cell types, including lymphocytes. The immunosuppressive effects of TGF‐β have been attributed to the interference of these molecules with the interleukin‐2 (IL2)‐driven component of lymphocyte proliferation. In order to elucidate in more detail the effects of TGF‐β on IL‐2‐induced proliferation, we investigated the effects of porcine transforming growth factor beta 1 and 2 (pTGF‐β1 and 2) on the IL‐2‐driven proliferation of a murine IL‐2‐dependent T‐lymphocyte line (CTLL). The results showed that pTGF‐β1 and 2 decreased 3H‐thymidine incorporation in CTLL cells in a dose‐dependent fashion (maximum decrease of 75–85%). Combined‐time kinetic analysis of the effects of pTGF‐β on 3H‐thymidine incorporation, cell growth, and cell‐cycle distribution (monitored as DNA content distribution) revealed that, in the first 48 h of culture, pTGF‐β1 increased the doubling time from 11.4 to 19.2 h without significantly affecting the cell‐cycle distribution of CTLL cells. After 96 h of culture in the presence of pTGF‐β1, cells started to accumulate in G0/G1, although at this time point 30% of the pTGF‐β1‐treated cells were still in S‐G2/M. Furthermore, during the first 48 h, neither the expression of the 55 kd chain of the IL‐2 receptor (IL‐2R) nor the expression of the transferrin receptor (TfR) was affected by TGF‐β. After 72 h of culture in the presence of pTGF‐β1, the expression of the IL‐2R and TfR was decreased. The data suggest that in CTLL cells TGF‐β initially slows the progression of cells in all phases of cell cycle. In addition, the initial TGF‐β‐mediated decrease of IL‐2‐induced 3H‐thymidine incorporation and cell proliferation in CTLL cells is not due primarily to downregulation of the IL‐2R and/or TfR.
Transformed cells overexpress a cytosolic nucleic acid binding protein
Authors: Stoler Daniel L. , Manly Kenneth F.
Published: February 2005

A protein blotting technique was used to identify a 57,000 dalton cytosolic nucleic‐acid‐binding protein found in neoplastically transformed cell lines. Specifically, greater amounts of this protein were found in Kirsten Murine Sarcoma Virus‐, Simian Virus 40‐, and methylcholanthrene‐transformed Balb 3T3 cells than in comparable untransformed cells. An analogous protein was identified in other transformed mammalian cells. Increased levels of the DNA binding protein in sarcoma virus transformants were shown to be dependent on the continued maintenance of the transformed phenotype. The properties of this protein are compared to those of other previously reported nucleic acid binding proteins.
The Na+, K+, 2Cl−‐cotransport system in HeLa cells and HeLa cell mutants exhibiting an altered efflux pathway
Authors: Kort Jens J. , Koch Gebhard
Published: February 2005

We have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2Cl−‐cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LK1) exhibited alterations in the Na+, K+, 2Cl−‐cotransport system. LK, cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, bumetanide‐sensitive potassium influx, measured by 86Rb+ uptake, was increased in the LK1, cells (increase in vmax). Km values of the cotransporter in HeLa cells and LK1 mutants revealed similar properties for 86Rb+ and 22Na+ uptake. In addition, (3H)‐bumetanide binding studies were carried out on intact HeLa cells; 1.7 pmol/mg protein (3H)‐bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding sites/cell. LK1cells present, 1.44 pmol/mg protein, specifically bound (3H)‐bumetanide and, respectively, 137,000 binding sites/cell. The LK1 cells also exhibited an increase in the number of (3H)‐ouabain binding sites as well as an increase in the activity of the Na+, K+‐ATPase, expressed as a function of ouabain‐sensitive86 Rb+ uptake. Furthermore, LK1cells were different in the concentrations of intracellular Na+ (↑)and K+ (↓) when compared to the HeLa parental cells. When grown in low K+ medium (0.2 mM K+), protein content and cell volume were increased in the LK1 cells, while the DNA content was not significantly different between both cell lines.
Expression of Na,K‐ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells
Authors: Martin‐Vasallo Pablo , Ghosh Sikha , Coca‐Prados Miguel
Published: February 2005

We have analyzed the expression of Na, K‐ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non‐pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na, K‐ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na, K‐ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na, K‐ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na, K‐ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A × 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na, K‐ATPase alpha subunit isoforms as compared to cultured cells.
Effects of oxygen‐free radicals on proliferation kinetics of cultured rabbit articular chondrocytes
Authors: Vincent FrançOise , Brun Hervé , Clain Elisabeth , Ronot Xavier , Adolphe Monique
Published: February 2005

It can be postulated that among the factors implicated in cartilaginous lesions, oxygen‐derived free radicals seem to have a prominent part. To investigate this hypothesis, rabbit articular chondrocyte cultures have been exposed to oxygenderived reactive species generated by the hypoxanthine–xanthine oxydase system. We observed a dose‐dependent decrease of cellular growth. In order to explain this result, cell cycle progression and binucleate cell fractions have been studied. A greater number of binucleate cells and an increase in cell volume were observed. Flow cytometry analysis revealed a perturbation in cell cycle progression leading to a significant increase in the proportion of cells in G2 phase and an important augmentation in cell protein content confirmed by biochemical assays. This model shows which type of alteration can be induced by oxygen‐derived free radicals in vitro. In addition, we deem this model to be useful for studying degenerative processes and for screening drugs that can scavenge oxygen‐free radicals.