bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential effects of superoxide, hydrogen peroxide, and hydroxyl radical on intracellular calcium in human endothelial cells
Authors: Dreher D. , Junod A. F.
Published: February 2005

Changes in intracellular Ca2+ homeostasis are thought to contribute to cell dysfunction in oxidative stress. The hypoxanthine‐xanthine oxidase system (X‐XO) mobilizes Ca2+ from intracellular stores and induces a marked rise in cytosolic calcium in different cell types. To identify the reactive O2 species involved in the disruption of calcium homeostasis by X‐XO, we studied the effect of X‐XO on [Ca2+]i by spectrofluorimetry with fura‐2 in human umbilical vein endothelial cells (HUVEC). The [Ca2+]i response to X‐XO was essentially diminished by superoxide dismutase (SOD) (200 U/ml) and catalase (CAT) (200 U/ml), which scavenge the superoxide anion, O2−, or H2O2, respectively. The [Ca2+]i increase stimulated by 10 nmol H2O2/ml/min, generated from the glucose‐glucose oxidase system, or 10 μM H2O2, given as bolus, was about a third of that induced by X‐XO (10 nmol O2−/ml/min) but was comparable to that induced by X‐XO in the presence of SOD. The X‐XO—stimulated [Ca2+]i increase was significantly reduced by 100 μM o‐phenanthroline, which inhibits the iron‐catalysed formation of the hydroxyl radical. On the other hand, the [Ca2+]i response to low dose X‐XO (1 nmol O2−/ml/min) was markedly enhanced in the presence of 1 μM H2O2, which itself had no effect on [Ca2+]i. More than 50% of this synergistic effect was prevented by o‐phenanthroline. These results indicate that the effect of X‐XO on calcium homeostasis appears to result from an interaction of O2− and H2O2, which could be explained by the formation of the hydroxyl radical. © 1995 Wiley‐Liss, Inc.
Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells
Authors: Graham Martin F. , Willey Amy , Adams John , Yager Dorne , Diegelmann Robert F.
Published: February 2005

The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L‐[5‐3H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polycrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate‐replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady‐state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two‐ to fourfold. L‐ascorbate phosphate did not increase the activity of L‐ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady‐state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L‐ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L‐ascorbate‐phosphate is not an adequate substitute for L‐ascorbate in this cell line. © 1995 Wiley‐Liss, Inc.
Activation of protein kinase C inhibits ATP‐induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways
Authors: Gallinaro Brian J. , Reimer Wesley J. , Dixon S. Jeffrey
Published: February 2005

Extracellular ATP elicits transient elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in osteoblasts through interaction with more than one subtype of cell surface P2‐purinoceptor. Elevation of [Ca2+]i arises, at least in part, by release of Ca2+ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca2+]i of indo‐1‐loaded UMR‐106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca2+, ATP (100 μM) induced transient elevation of [Ca2+]i to a peak 57 ± 7 nM above basal levels (31 ± 2 nM, means ± S. E. M., n = 25). Exposure of cells to the PKC activator 12‐O‐tetradecanoyl‐β‐phorbol 13‐acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 ± 4 nM (n = 11), without altering basal [Ca2+]i. Inhibition was half‐maximal at approximately 1 nM TPA. The Ca2+ response to ATP was also inhibited by the PKC activators 1,2‐dioctanoyl‐sn‐glycerol or 4β‐phorbol 12, 13‐dibutyrate, but not by the control compounds 4α‐phorbol or 4α‐phorbol 12, 13‐didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H‐7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca2+]i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P2Y‐receptor agonists, ADP and 2‐methylthioATP, than the response to the P2U‐receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides. © 1995 Wiley‐Liss, Inc.
Quantitative export of FGF‐2 occurs through an alternative, energy‐dependent, non‐ER/Golgi pathway
Authors: Florkiewicz Robert Z. , Majack Richard A. , Buechler Robbie D. , Florkiewicz Elin
Published: February 2005

Although basic fibroblast growth factor (bFGF/FGF‐2) is found outside cells, it lacks a conventional signal peptide sequence; the mechanism underlying its export from cells is therefore unknown. Using a transient COS‐1 cell expression system, we have identified a novel membrane‐associated transport pathway that mediates export of FGF‐2. This export pathway is specific for the 18‐kD isoform of FGF‐2, is resistant to the anti‐Golgi effects of Brefeldin A, and is energy‐dependent. In FGF‐2‐transfected COS‐1 cells, this ER/Golgi‐independent pathway appears to be constitutively active and functions to quantitatively export metabolically‐labeled 18‐kD FGF‐2. Co‐transfection and co‐immunoprecipitation experiments, using a vector encoding the cytoplasmic protein neomycin phosphotransferase, further demonstrated the selectivity of this export pathway for FGF‐2. When neomycin phosphotransferase was appended to the COOH‐terminus of 18‐kD FGF‐2, the chimera was exported. However, the transmembrane anchor sequence of the integral membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) blocked export. The chimeric protein localized to the plasma membrane with its FGF‐2 domain extracellular and remained cell‐associated following alkaline carbonate extraction. Taken together, the data suggest that FGF‐2 is “exported” from cells via a unique cellular pathway, which is clearly distinct from classical signal peptide‐mediated secretion. This model system provides a basis for the development and testing of therapeutic agents which may block FGF‐2 export. Such an intervention may be of considerable use for the treatment of angiogenesis‐dependent diseases involving FGF‐2. © 1995 Wiley‐Liss, Inc.
Effect of phosphotyrosyl‐IRS‐1 level and insulin receptor tyrosine kinase activity on insulin‐stimulated phosphatidylinositol 3, MAP, and S6 kinase activities
Authors: Wilden Peter A. , Broadway Dale E.
Published: February 2005

The role of tyrosine phosphorylation of the insulin receptor substrate 1 (IRS‐1) was studied utilizing parental CHO cells or CHO cells that overexpress IRS‐1, the insulin receptor, or both IRS‐1 and the insulin receptor. Insulin stimulation of these four cell lines led to progressive levels of IRS‐1 tyrosine phosphorylation of one, two, four, and tenfold. Maximal insulin‐stimulated IRS‐1 associated Ptdlns 3′‐kinase activit in these cells was 1‐, 1.5‐, 3‐, and 3‐fold, while insulin sensitivity, as determined by ED50, was 1‐, 2.5‐, 10‐, and 10‐fold. Both sensitivity and maximal response paralleled the increased level of phosphotyrosyl‐IRS‐1; however, the increased level of phosphotyrosyl‐IRS‐1 seen in CHO/IR/IRS‐1 cells did not further increase these responses. Likewise, maximal insulin‐stimulated MAP kinase activity in these cell lines increased in parallel with IRS‐1 tyrosine phosphorylation except in the CHO/IR/IRS‐1 cell lines with activity levels of one‐, five‐, nine‐, and ninefold. However, insulin sensitivity of the MAP and S6 kinases and maximal insulin‐stimulated S6 kinase activity was not changed by a twofold increase in phosphotyrosyl‐IRS‐1, but an increase was observed with insulin‐stimulated receptor autophosphorylation and kinase activity in CHO/IR cells which led to a tenfold increase in insulin receptor autophosphorylation and a fourfold increase in IRS‐1 tyrosine phosphorylation. Thus, these three kinase activities may be differentially coupled to the activation of the insulin receptor kinase activity via IRS‐1 and other possible cellular substrates. © 1995 Wiley‐Liss, Inc.
Inhibition of heme synthesis induces apoptosis in human erythroid progenitor cells
Authors: Muta Koichiro , Krantz Sanford B.
Published: February 2005

Heme synthesis by erythroid progenitor cells is maintained by erythropoietin (EP), insulin‐like growth factor‐I (IGF‐I), and stem cell factor (SCF), and without these growth factors apoptosis (programmed cell death) occurs. To clarify the possible interaction between heme synthesis and programmed cell death of human erythroid progenitor cells, the effect of specific inhibition of heme synthesis on apoptosis of highly purified human erythroid colony forming cells (ECFC) was studied. When the amount of uncleaved DNA was determined as a measure of apoptosis, the heme synthesis inhibitors, succinylacetone (SA) (0.1 mmol/L) or isonicotinic acid hydrazide (INH) (10 mmol/L), significantly decreased the amount of uncleaved DNA (P < 0.01) in the presence of erythropoietin (EP). Addition of recombinant heavy‐chain ferritin (rHF) (10 nmol/L), or deprivation of transferrin from the culture medium, which decreased heme synthesis, also reduced the amount of uncleaved DNA (P < 0.01). The production of apoptosis by diverse inhibitors of heme synthesis was in each case reversed by the addition of hemin (0.1 mmol/L) and did not occur with HL‐60 cells. When the colony‐forming capacity of ECFC was determined by plasma clot assay, SA, INH, or rHF reduced the number of CFU‐E (P < 0.01), and the effect of SA was reversed by hemin. The addition of SA did not alter the c‐myc response of ECFC to EP. These data indicate that inhibition of heme synthesis induces apoptosis of human erythroid progenitor cells, in a manner independent of an early c‐myc response, and suggest that the presence of apoptosis in ineffective erythropoiesis may be secondary to impaired heme synthesis. © 1995 Wiley‐Liss, Inc.
Substance P‐related antagonists inhibit vasopressin and bombesin but not 5′‐3‐O‐(thio) triphosphate‐stimulated inositol phosphate production in swiss 3T3 cells
Authors: Seckl Michael J. , Newman Richard H. , Freemont Paul S. , Rozengurt Enrique
Published: February 2005

The substance P (SP) analogues [DArg1, DPhe5, DTrp7,9, Leu11] SP (AntD) and [Arg6, DTrp7,9, MePhe8] SP (6–11) (AntG) inhibit the action of many different neuropeptides including SP. These analogues might be useful in the treatment of small cell lung cancer but their mechanism of action is unclear. Here, we analyzed the effect of AntD and AntG on neuropeptide vs. guanosine 5′‐3‐O‐(thio) triphosphate (GTPγS) stimulated inositol phosphate generation in permeabilized Swiss 3T3 cells. AntD inhibited vasopressin and bombesin stimulated inositol phosphate formation (IC50 of 0.75 μM and 2 μM, respectively). Similarly, AntG inhibited vasopressin‐stimulated inositol phosphate generation with an IC50 of 1 μM. Strikingly, neither AntD up to 10 μM nor AntG up to 20 μM was able to inhibit GTPγS‐stimulated inositol phosphate generation. Dose‐response curves of neuropeptide‐induced inositol phosphate generation were dramatically displaced to the right by either 10 μM AntD or 20 μM AntG. However, neither antagonist affected the dose response of GTPγS‐stimulated inositol phosphate generation. Furthermore, 20 μM AntD had no effect on AIF−4‐induced inositol phosphates in COS‐1 cells transfected with Gαq. AntD inhibited [3H]vasopressin binding competitively in intact Swiss 3T3 cells and both AntD and AntG inhibited [3H]vasopressin binding in Swiss 3T3 and rat liver membranes. Scatchard analysis revealed that AntD inhibited vasopressin binding by reducing receptor affinity without affecting receptor number in both intact and membrane preparations of Swiss 3T3 cells. The results strongly suggest that SP analogues AntD and AntG block the action of the Ca2+ mobilizing neuropeptides at the receptor level, rather than inhibiting G protein‐stimulated inositol phosphate production. © 1995 Wiley‐Liss, Inc.
Okadaic acid co‐induces vimentin expression and cell cycle arrest in MPC‐11 mouse plasmacytoma cells
Authors: Giese G. , Wiegers W. , Kubbies M. , Scherbarth A. , Traub P.
Published: February 2005

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC‐11 mouse plasmacytoma cells was compared with that of the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Cell cycle progression of asynchronously grown MPC‐11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA‐caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid‐treated relative to TPA‐treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 × 10−9M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC‐11 cells by okadaic acid. © 1995 Wiley‐Liss, Inc.
Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells
Authors: Bellone Graziella , Geuna Massimo , Carbone Anna , Silvestri Stefania , Foa Robin , Emanuelli Giorgio , Matera Lina
Published: February 2005

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl‐binding receptors (Prl‐R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage‐specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)‐3, granulocyte‐macrophage colony stimulating factor (GM‐CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit‐granulocyte (CFU‐G) and burst forming unit‐erythroid (BFU‐E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl‐binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR‐7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR‐7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl‐R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage‐specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl. and hemopoietic factors. © 1995 Wiley‐Liss, Inc.
Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin‐like growth factor‐1
Authors: Vardy Daniel A. , Kari Csaba , Lazarus Gerald S. , Jensen Pamela J. , Zilberstein Asher , Plowman Gregory D. , Rodeck Ulrich
Published: February 2005

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin‐like growth factor (IGF)‐1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF‐1/insulin induced production of keratinocyte‐derived autocrine growth factors that bind to the EGF receptor. Increased steady‐state mRNA levels for transforming growth factor alpha (TGF‐α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF‐1. IGF‐1 also induced production and secretion of TGF‐α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF‐1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF‐1 requires activation of an EGF receptor‐mediated autocrine loop. © 1995 Wiley‐Liss, Inc.
Hydrocortisone and progesterone regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture
Authors: Darcy Kathleen M. , Shoemaker Suzanne F. , Lee Ping‐Ping H. , Ganis Barbara A. , Ip Margot M.
Published: February 2005

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum‐free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1–10 μg/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01–1 μg/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 μg/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross‐talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells. © 1995 Wiley‐Liss, Inc.
RB phosphorylation in sodium butyrate‐resistant HL‐60 cells: Cross‐resistance to retinoic acid but not vitamin D3
Authors: Yen Andrew , Varvayanis Susi
Published: February 2005

To examine the potential coupling between inducible cellular changes in RB (retinoblastoma) tumor suppressor protein phosphorylation and ability to G0 growth arrest and differentiate, HL‐60 promyelocytic leukemia cells were cultured in incremental sodium butyrate (NaB) concentrations and thereby made resistant to the growth inhibitory effects of sodium butyrate, which normally induces G0 arrest and monocytic differentiation in wild type HL‐60 cells. The resistant cells were also unable to differentiate in response to NaB, indicating that a regulatory function controlling both G0 growth arrest and differentiation had been affected. The induced resistance was not genetic in origin since the cells regained the ability to G0 arrest and differentiate after being recultured in medium free of sodium butyrate for only three days. The resistant cells had similar cell cycle phase durations as the original wild type cells. The resistant cells retained the ability to both G0 arrest and differentiate in response to 1,25‐dihydroxy vitamin D3 (VD3), normally an inducer of G0 arrest and monocytic differentiation in wild type cells. However, they were cross‐resistant to retinoic acid (RA), another ligand for the same steroid thyroid hormone receptor family, which induces G0 arrest and myeloid differentiation in wild type cells. The ability to G0 arrest and phenotypically differentiate in response to RA were both grossly impaired. Unlike wild type cells which undergo early down‐regulation and then hypophosphorylation of the RB protein when induced to differentiate, in resistant cells, hypophosphorylation of RB in response to NaB was grossly retarded. These changes in RB protein occurred faster when the cells were treated with VD3. In contrast, the changes in RB phosphorylation occurred significantly slower when the cells were treated with RA. The results suggest a coupling between the ability to G0 growth arrest and phenotypically convert and the ability to hypophosphorylate RB. © 1995 Wiley‐Liss, Inc.
Multifunctional phosphatidic acid signaling in mammary epithelial cells: Stimulation of phosphoinositide hydrolysis and conversion to diglyceride
Authors: Imagawa Walter , Bandyopadhyay Gautam , Nandi Satyabrata
Published: February 2005

We have shown previously that phosphatidic acid esterified to polyunsaturated fatty acids is mitogenic for primary cultures of mouse mammary epithelial cells embedded within collagen gels. We hypothesized that this mitogenic competence resulted from the ability of this phospholipid to activate multiple signal transduction pathways in mammary epithelium. A closer examination of this hypothesis was undertaken by examining the effect of exogenous phosphatidic acid on phosphoinositide (PI) hydrolysis and its intracellular metabolism to diglyceride, an activator of protein kinase C. For assays of phosphoinositide‐specific phospholipase C activation, mammary epithelial cells from virgin Balb/c mice were isolated by collagenase dissociation of mammary glands and cultured on the surface of Type I collagen‐coated culture dishes. Phosphatidic acid (PA) stimulated a sustained increase in inositol phosphates and caused inositol phospholipid depletion when added to cells in which inositol phospholipids were prelabeled with 3H‐myoinositol. This effect was specific for PA among phospholipids tested. Neither lineoleic acid, that can be released from PA, nor prostaglandin E2 affected PI hydrolysis. When mammary epithelial cells were cultured inside collagen gels in the presence of exogenous PA or phosphatidylcholine (PC) radiolabeled with 3H‐glycerol, PA was found to persist intracellularly and be dephosphorylated to diglyceride (an activator of protein kinase C) to a greater extent than PC, a nonmitogenic phospholipid. In contrast to PA, epidermal growth factor (EGF) only slightly stimulated PI hydrolysis, showing that these two different growth‐promoting factors do not actively couple to the same signal transduction pathways in mammary epithelial cells. These results show that PA may activate multiple pathways in mammary epithelial cells either directly or via its metabolism to diglyceride. © 1995 Wiley‐Liss, Inc.
Analysis of sequence‐specific binding activity of cis‐elements in human thymidine kinase gene promoter during G1/S phase transition
Authors: Good Lifeng , Chen Jie , Chen Kuang Yu
Published: February 2005

Expression of thymidine kinase (TK) gene in normal human diploid cells is both cell cycle and age dependent and appears to be transcriptionally regulated. Several studies have indicated that the G1/S control sequence may reside within the region of about 130 bp upstream of the transcription initiation site. We have previously shown that a trans‐acting factor, CBP/tk (CCAAT binding protein for TK gene), binds to either one of the two inverted CCAAT boxes in a cell cycle‐ and age‐dependent manner (Pang and Chen, 1993, J. Biol. Chem., 268:2909–2916). An upstream 25 bp fragment (−109/−84), containing both Yi‐like and E2F‐like binding sites, has recently been proposed to be essential for the G1/S regulation of human TK gene. To assess the contribution of various cis‐elements in human TK promoter to the G1/S regulation, we have examined the binding activity of these cis‐elements in the nuclear extracts derived from human IMR‐90 cells at low passage number. Our results indicated that no binding activity could be detected using either the 25 bp fragment (−109/−94) or the authentic Yi sequence. However, Yi binding activity was observed in SV‐40 transformed IMR‐90 cells. In contrast, the 28 bp fragment (−91/−64) that contains the distal inverted CCAAT box exhibited a strong binding in serum‐stimulated young IMR‐90 cells. The binding of CBP/tk to the 28 bp fragment was abolished by a single base mutation in the CCAAT box. The CBP/tk binding of the 28 bp fragment could not be displaced by either the 25 bp fragment or the authentic Yi element. A deletion of the 5′‐flanking region of the 28 bp fragment up to 5 bases also abolished the binding activity. The CBP/tk binding in IMR‐90 cells was supershifted by antiserum against NF‐Ya, but not by antiserum made against p107, pRb, cyclin A, p33cdk2, or p34cdc2. Taken together, our results suggest that the G1/S regulatory cis‐element in human TK promoter may be confined only to CBP/tk binding sites. © 1995 Wiley‐Liss, Inc.
Alterations in the molecular response to DNA damage during cellular aging of cultured fibroblasts: Reduced AP‐1 activation and collagenase gene expression
Authors: Choi Augustine M. K. , Pignolo Robert J. , Rhys Colette M. J. Ap , Cristofalo Vincent J. , Holbrook Nikki J.
Published: February 2005

Transcriptional activation of c‐fos in response to both serum stimulation and DNA damage requires the serum response element. The inability of in vitro aged or senescent fibroblasts to proliferate in response to serum has been shown to be associated with repressed c‐fos expression and reduced AP‐1 binding activity. In contrast, we have observed similar levels of c‐fos mRNA and protein expression in young (early passage) and old (late passage) cells following their treatment with ultraviolet (UV) irradiation or methyl methanesulfonate (MMS). Thus, the early events in the signal transduction pathway leading to transcriptional activation of c‐fos following DNA damage are distinct from those mediating the gene's expression in response to mitogenic stimulation. Despite normal levels of c‐fos expression, we observed a reduced level of AP‐1 binding activity in old cells relative to young cells treated with UV irradiation or MMS. Reduced AP‐1 binding activity is associated with reduced expression of the AP‐1‐dependent gene, collagenase, in old cells treated with DNA damaging agents. Since other DNA damage‐inducible genes also contain an AP‐1 regulatory element presumed to play a role in their expression, reduced AP‐1 binding activity is likely to have a major impact on the old cell's ability to respond appropriately to DNA damage. © 1995 Wiley‐Liss, Inc.
THP‐1 macrophage membrane‐bound plasmin activity is up‐regulated by transforming growth factor‐β1 via increased expression of urokinase and the urokinase receptor
Authors: Falcone Domenick J. , McCaffrey Timothy A. , Mathew Jean , McAdam Kimberly , Borth Wolfgang
Published: February 2005

Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen. The regulation of these receptors is important for several macrophage functions. In these studies, the effect of transforming growth factor‐b̃1 (TGF‐b̃1) on uPA, uPA receptor, and plasminogen receptor expression by human THP‐1 macrophage was examined. TGF‐b̃1 induction of uPA expression by THP‐1 cells was differentiation dependent. Suspension and adherent cultures expressed similar constitutive levels of uPA. Exposure of adherent cells to TGF‐b̃1 led to a dose‐ and time‐dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA. In contrast, uPA expression by suspension cultures was unresponsive to TGF‐b̃1. The differential response exhibited by suspension and adherent THP‐1 cells may reflect differences in their expression of TGF‐b̃1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors. TGF‐b̃1‐induced alterations in uPA receptor expression by adherent THP‐1 cells were examined by quantitating membrane‐bound uPA activity. Membrane‐bound uPA activity increased three‐fold when cells were incubated with TGF‐b̃1. The increase in membrane‐uPA activity expressed by TGF‐b̃1‐treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF‐b̃1 primed cells with exogenous uPA did not lead to an increase in membrane‐bound uPA activity. Furthermore, immunoreactive uPA receptor was increased in TGF‐b̃1‐treated cells. Following incubation with plasminogen, membrane‐bound plasmin activity increased three‐fold in TGF‐b̃1‐treated cells. However, no change in immunoreactive membrane‐bound plasmin(ogen) was observed. In addition, binding of 125I‐Lys‐plasminogen to THP‐1 cells was not affected by TGF‐b̃1 treatment. We conclude that TGF‐b̃1 stimulates membrane‐bound plasmin activity, without affecting plasminogen receptor expression, through the up‐regulation of uPA and the uPA receptor expression. © 1995 Wiley‐Liss, Inc.
Kinetics of 125I‐PDGF binding and down‐regulation of PDGF receptor in human arterial smooth muscle cell strains during cellular senescence in vitro
Authors: Aoyagi Masaru , Fukai Naomi , Ogami Kazuo , Yamamoto Mari , Yamamoto Kiyotaka
Published: February 2005

Platelet‐derived growth factor (PDGF) is one of the major mitogens in serum to stimulate replication of human smooth muscle cells (SMCs) in culture. Previous studies using human fibroblasts failed to demonstrate changes in the receptor systems for growth factors during cellular senescence. We investigated the kinetics of 125I‐PDGF(‐BB) binding and down‐regulation of the PDGF receptor in three human arterial SMC strains during cellular aging. The number of specific 125I‐PDGF binding sites per cell increased slightly at a population doubling level (PDL) of 60%–80% of life span and then decreased at the PDL above 90%. The number of receptors per cell‐surface area decreased with increasing in vitro age. The apparent Kd for the 125I‐PDGF binding decreased with in vitro senescence. The internalization and degradation of 125I‐PDGF per receptor were significantly reduced in senescent SMCs than young cells. Furthermore, down‐regulation of the PDGF receptor was significantly greater in sensescent SMCs than young cells. Immunoblot studies demonstrated that changes in b̃‐subunit of the PDGF receptor accounted for those in the studies using 125I‐PDGF and that tyrosine phosphorylation of the PDGF receptor was significantly greater in young SMCs than aged cells. Our results suggest that age‐related changes in the receptor systems for PDGF may be important contributors to the failure of DNA synthesis in senescent SMCs. © 1995 Wiley‐Liss, Inc.
On the mechanisms of the growth‐promoting effect of prostaglandins in hepatocytes: The relationship between stimulation of DNA synthesis and signaling mediated by adenylyl cyclase and phosphoinositide‐specific phospholipase C
Authors: Refsnes Magne , Dajani Olav F. , Sandnes Dagny , Thoresen G. Hege , Røttingen John‐Arne , Iversen Jens‐Gustav , Christoffersen Thoralf
Published: February 2005

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide‐specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum‐free medium in the presence of EGF and insulin. PGE2 or PGF2α (added 0‐3 h after plating) enhanced the incorporation of [3H]‐thymidine into DNA (measured at 50 h); at 100 γM the stimulation was about threefold. PGI2 and PGD2 also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1‐10 nM), a potent activator of hepatic adenylyl cyclase, or 8‐bromo‐cAMP (0.1‐10 γM) enhanced the DNA synthesis. When 8‐bromo‐cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE2 and 8‐bromo‐cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE2, PGF2α, PGI2, and PGD2 increased intracellular inositol‐1,4,5‐trisphosphate (InsP3), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca2+, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 γM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE2 on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE2 on glucagon‐induced cAMP accumulation did not abolish the ability of PGE2 to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide‐specific phospholipase C in the growth‐promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism. © 1995 Wiley‐Liss, Inc.
Endothelin‐I induces gene expression through stimulation of endothelin type a receptors in normal rat kidney cells
Authors: Yumet Gladys , Chin Michael H. , Carey Brian , Soprano Kenneth J. , Lipson Kenneth E.
Published: February 2005

RNA blots of total cellular RNA isolated from quiescent and endothelin (ET‐1)‐stimulated normal rat kidney (NRK) cells demonstrated that ET‐1 induced the expression of c‐jun, jun B, and c‐fos mRNA in a time‐dependent manner with maximal expression of mRNA by 1 hr after the addition of ET‐1. Five hundred picomolal ET‐1 was sufficient to induce maximal mRNA expression. These data agreed with saturation experiments which demonstrated that maximal binding of [125I]ET‐1 was achieved at concentrations greater than 100 pM. The Kd and Bmax values for [125I]ET‐1 binding to NRK membranes were 20.5 pM and 22.2 fmol/mg protein, respectively. Competition experiments for the binding of [125I]ET‐1 to NRK membranes demonstrated that ET‐1 was a more potent inhibitor (Ki = 0.047 nM) than ET‐3 (Ki = 10.8 nM). No specific binding of [125I]ET‐3 (40 or 500 pM) to NRK membranes could be observed. The expression of c‐jun, jun B, and c‐fos mRNA was inhibited by the endothelin type A receptor (ET)‐selective antagonist, BQ‐123. Thus, these data demonstrate that ET‐1 mediates the expression of immediate response gene mRNA in NRK cells via the ETA receptor. ET‐1 stimulation of NRK cells also upregulated EGF receptors, providing a possible mechanism for ET‐1 complementation of epidermal growth factor (EGF) mitogenicity in NRK cells. © 1995 Wiley‐Liss, Inc.
Increased expression of a high molecular weight (130 KD) protein kinase C isoform in a differentiation‐defective ras‐transfected keratinocyte line
Authors: Rosales J. L. , Isseroff R. R.
Published: February 2005

The role of ras on protein kinase C (PKC) signaling was examined in two keratinocyte cell lines. Increasing the level of extracellular calcium from 0.15 mM to 1.0 mM induces some features of differentiation in the spontaneously immortalized HaCaT line, but fails to do so in a c‐H‐ras‐transfected subline (ras‐HaCaT). Raising extracellular calcium also induced a transient increase in membrane‐associated PKC activity 5 min after calcium addition, in HaCaT, but not in the ras‐HaCaT cells. Partial purification of PKC from the membrane/particulate fraction revealed the major isoform expressed in HaCaT to be an 80 KD species recognized by the anti‐PKCα antibody. In ras‐HaCaT, the major expressed isoform is a 130 KD species recognized by the PKCb̃ antibody. The kinase activity of the partially purified high molecular weight PKC is phospholipid dependent but calcium independent. Further evaluation of PKC in the HaCaT and ras‐HaCaT membrane/particulate cell fraction by immunoblotting using affinity‐purified antibodies against PKCα, b̃, δ, ε and ζ revealed a 130 KD band reacting with the PKCδ antibody. Increased expression of this high molecular weight protein was observed in ras‐HaCaT. Immunoprecipitation of PKC in ras‐HaCaT using the PKCδ antibody also revealed a 130 KD species. Analysis of the PKCδ immunoprecipitate demonstrated a phospholipid, but not calcium‐dependent kinase which autophosphorylated. These results suggest that the 130 KD protein may be a novel (calcium‐independent) PKC (nPKC) isoform and increased expression in the rastransfected HaCaT may be a consequence of oncogenic ras expression. This 130 KD species may also play a role in the ras‐associated inhibition of differentiation in HaCaT. © 1995 Wiley‐Liss, Inc.
Laminin responsiveness is associated with changes in fibroblast morphology, motility, and anchorage‐independent growth: Cell system for examining the interaction between laminin and EGF signaling pathways
Authors: Lin Meei‐Lih , Bertics Paul J.
Published: February 2005

Laminin can influence the adhesion, differentiation, and motility of motility of several cell types, including epithelial and neural cells. In addition, laminin, which contains an epidermal growth factor (EGF)‐like motif, can stimulate DNA synthesis in fibroblasts possessing the EGF receptor, but laminin does not compete for EGF binding. To further investigate laminin action in fibroblasts, and the relationship between laminin and EGF receptor function, we have developed a system wherein cells containing laminin‐binding activity were cloned from a mouse fibroblast cell line (B82L‐wt) that cannot adhere to laminin but that have been transfected with the wild‐type human EGF receptor. Although only the isolated clones can efficiently attach to laminin‐coated plates, all the cells can adhere to plastic, fibronectin, and collagen I, and all exhibit comparable levels of cell surface‐associated laminin. Ligand‐binding assays showed that the cells with laminin attachement activity possess high‐affinity EGF binding (Kd ∼ 0.4 nM), and all express a similar level of the human EGF receptor. However, when compared to the B82L‐wt cells, the cells with laminin‐binding activity exhibit altered morphology, anchorage‐independent growth, and motility. Specifically, the morphology of the fibroblasts possessing laminin binding activity appears more elongated and they spread more‐extensively on plastic plates. Analysis of their growth in soft agar revealed that the clones have a 2‐5‐fold increase in colony formation in comparison to the B82L‐wt cells. The cells possessing laminin attachment ability also exhibit laminin‐induced motility, and this movement is directional (chemotaxis) rather than random (chemokinesis), indicating functional laminin receptors and signaling pathways. To examine the specific laminin receptors involved in these effects, the influence of anti‐integrin subunit antibodies on cell adhesion and migration was evaluated. These studies showed that an anti‐α6 integrin antibody can completely inhibit the clonal cells' attachment and migration to laminin, and anti‐α6 immunoblots revealed that only the clones express measurable levels of α6. These data indicate that α6‐containing integrins contribute to the lamininmediated attachment and motility of these clones and that this system may also influence the morphology and anchorage‐independent growth of these fibroblasts. In addition, these cells provide a unique system for examining the interaction between EGF and laminin receptor action. © 1995 Wiley‐Liss, Inc.
Adaptation of ehrlich ascites carcinoma cells to energy deprivation in vivo can be associated with heat shock protein accumulation
Authors: Kabakov A. E. , Molotkov A. O. , Budagova K. R. , Makarova Yu M. , Mosin A. F. , Gabai V. L.
Published: February 2005

Tumor adaptation to chronic energy starvation in vivo was studied on Ehrlich ascites carcinoma (EAC) cells. EAC cells were isolated from mice and incubated in a glucose‐free medium containing blocators of mitochondrial ATP generation (rotenone, 2,4‐dinitrophenol, or oligomycin). ATP level in the treated cells decreased to 3–4% of the initial during 30 min of the incubation. The aggregation of cytoskeletal proteins, blebbing, and necrotic death within 2–3 h were observed in ATP‐depleted EAC which were isolated and treated in the exponential phase of growth (5 days after inoculation), whereas stationary EAC (8 days after inoculation) were considerably more resistant to ATP depletion, and actin aggregation as well as bleb formation were suppressed in these cells despite the ATP loss. In contrast to the exponentially growing cells, thermotolerance and unexpected expression of inducible HSP68 and HSP27 as well as an elevated level of HSP90 were found in stationary EAC. Since the stationary cells had decreased content of ATP, ATP/ADP ratio, and energy charge, we suggest that this energy dysbalance may be conducive to HSP induction within the ascites tumor in vivo, and, at the same time, EAC cells with elevated content of HSPs acquire resistance to chronic energy starvation occurring in late stages of the tumor growth. © 1995 Wiley‐Liss Inc.
Lithium‐stimulated proliferation and alteration of phosphoinositide metabolites in MCF‐7 human breast cancer cells
Authors: Welshons Wade V. , Engler Kathleen S. , Taylor Julia A. , Grady Leigh H. , Curran Edward M.
Published: February 2005

Lithium, which is used to treat bipolar psychiatric disorders, can stimulate proliferation of a number of cells in tissue culture. Proliferation of MCF‐7 human breast cancer cells, which also respond to EGF and estrogens, was stimulated by LiCl (1–5 mM) within the concentration range that is encountered during human therapy with lithium. Stimulation of growth was specific for lithium; rubidium, potassium, and sodium showed no such effect. In the presence of antiestrogen, lithium stimulated the growth of hormone‐dependent breast cancer cells MCF‐7, ZR‐75‐1, and T47D but not hormone‐independent MDA‐MB‐231 cells or an estrogen‐independent clone of MCF‐7 cells. Lithium‐stimulated proliferation was limited by cytotoxicity which could be moderated by added potassium chloride (5–20 mM) in the medium. Each of the mitogens lithium, 17β‐estradiol, and EGF increased the rate of uptake of myo‐inositol into MCF‐7 cells. Whether normalized to inositol lipids, to protein, or to DNA, steady‐state levels of inositol phosphates were elevated by each of the mitogens including lithium, which inhibits the breakdown of inositol phosphates in the phosphoinositide signaling pathway. These data indicate that therapeutic concentrations of lithium can stimulate the proliferation of human breast cancer cells by a mechanism that may involve the phosphoinositide pathway. © 1995 Wiley‐Liss Inc.
Influences of silicates and carnitine‐silicate mixtures on the inhibition of aggregation of erythrocytes elicited by the presence of fibrinogen
Authors: Ramsohoye Pamela , Fritz Irving B.
Published: February 2005

Carnitine and acylcarnitine derivatives have been reported to inhibit cell aggregation (Fritz and Burdzy, 1989, J. Cell. Physiol., 140:18–28). A follow‐up of these observations showed that whereas the previously described effects of long‐chain acylcarnitines were well replicated, those of carnitine on erythrocytes showed marked variability. The latter phenomenon was traced to the presence of silicates in carnitine solutions derived from the use of sodium hydroxide solutions stored in glass containers for the neutralization of carnitine. The present experiments have led to the discovery that oligomeric forms of silicates are powerful inhibitors of red blood cell aggregation which otherwise occurs in the presence of fibrinogen alone. The active form(s) of silicates in this assay, which appear to be generated by polymerization of silicates in metasilicate solutions on neutralization, are unstable and therefore transient under usual conditions. We estimate that the active oligomeric forms contain between 4 to 18 silicon atoms per molecule. When maintained at ‐ 18°C in the presence of carnitine, but not in its absence, the active forms of oligomeric silicates remained stable for months, judging from their ability to inhibit cell aggregation. We conclude that carnitine stabilized the oligomeric form(s) of silicate, or that the species stabilized is an oligomeric silicate‐carnitine complex. Comparable concentrations of choline, deoxycarnitine, or γ‐aminobutyrate were less effective in stabilizing the active silicate oligomers. The active forms of the silicate oligomers had K'i values of about 10 μM, calculated as the monomeric form, in inhibiting red blood cell aggregation. The data indicate that free carnitine does not directly inhibit erythrocyte inhibition, as previously interpreted, whereas long‐chain acylcarnitine derivatives are active in the absence of silicates. Possible mechanism of actions of silicate oligomers on membranes are discussed. © 1995 Wiley‐Liss Inc.