bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of dexamethasone‐inducible tyrosine aminotransferase activity in WB‐F344 rat liver epithelial stemlike cells cultured in the presence of sodium butyrate
Authors: Coleman William B. , Smith Gary J. , Grisham Joe W.
Published: February 2005

Sodium butyrate acts as a differentiation‐promoting agent for a wide variety of cell types, including some tumor cell lines. In this study, we examined the effects of sodium butyrate (SB) on the functional differentiation of cultured WB‐F344 rat liver epithelial stemlike cells. Treatment of WB‐F344 cells with 3.75 mM SB resulted in an inhibition of cellular proliferation, alterations to normal cellular morphology (increased cell size and decreased nuclear/cytoplasmic ratio), and significant increases in cellular protein synthesis. The SB‐mediated changes in cell morphology, proliferative status, and protein catabolism were accompanied by development of dexamethasone‐inducible tyrosine aminotransferase (TAT) enzyme activity. Culture of WB‐F344 cells in growth medium containing SB and dexamethasone (DEX; 1 × 10‐6 M) resulted in greater than sevenfold increase in the basal TAT activity compared with control cultures. An additional sixfold increase in TAT activity was observed when cells cultured in medium containing SB and DEX were exposed to 1 × 10‐7 M DEX during the last 24 hours of culture. The DEX‐inducible TAT activity developed by SB‐treated WB‐F344 cells responded to the modulating effects of insulin and L‐tyrosine in a manner that closely resembled that reported for cultured hepatocytes and hepatoma cell lines. These studies show that treatment of WB‐F344 rat liver epithelial stemlike cells with the differentiation‐promoting agent SB in vitro leads to expression of the differentiation‐specific hepatocyte enzyme TAT. © 1994 Wiley‐Liss, Inc.
Control of AKR fibroblast phenotype by fibronectin: Regulation of cell‐surface fibronectin binding receptor by fibronectin
Authors: Huang Shuang , Varani James , Chakrabarty Subhas
Published: February 2005

Results of previous studies show that the expression of fibronectin and its cell‐surface fibronectin binding receptor is coregulated in 3‐methylchloranthrene transformation of normal AKR‐2B cells to form AKR‐MCA cells and in N, N,‐dimethylformamide (DMF) induction of differentiation of transformed AKR‐MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the corgulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR‐2B cells, and on the responses of transformed AKR‐MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR‐2B cells down‐modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down‐modulation of cell‐surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down‐modulation of the expression of α5β1 integrins. Expression of antisense fibronectin RNA in AKR‐MCA cells down‐modulated the ability of DMF to restore normal fibronectin production, cell‐surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell‐surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell‐surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell‐surface fibronectin binding receptor. © 1994 Wiley‐Liss, Inc.
Inhibition of mechanical strain‐induced fetal rat lung cell proliferation by gadolinium, a stretch‐activated channel blocker
Authors: Liu Mingyao , Xu Jing , Tanswell A. Keith , Post Martin
Published: February 2005

Normal growth of the fetal lung is dependent upon fetal breathing movements. We have previously demonsrated that mechanical strain, simulating fetal breathing movements, stimulated DNA synthesis and cell division by reaggregated alveolar‐like structures of fetal rat lung cells. Herein, we report that both intracellular and extracellular calcium modulate strain‐induced proliferative activity. Strain‐induced cell proliferation was inhibited by BAPTA/AM, an intracellular calcium chelator. The intracellular calcium modulators, cyclopiazonic acid and 2,5‐di‐(tert‐butyl)‐1, 4‐benzohydroquinone, increased DNA synthesis of unstrained cultures and partially reduced strain‐induced cell growth activity. A similar effect was noted with the calcium ionophore A23187. Extracellular Ca2+ increased DNA synthesis in unstrained cultures in a concentration‐dependent fashion. The stimulatory effect of strain on DNA synthesis was also dependent on the calcium concentration in the medium. Furthermore, strain‐enhanced DNA synthesis was inhibited by the presence of a divalent ion chelator, EGTA, in the medium. Mechanical strain increased 45Ca2+ influx within 1 min after the onset strain. This rapid entry of calcium was not affected by calcium channel blockers, such as verapamil or Ni2+. Calcium channel blockers verapamil, nifedipine, Ni2+, Co2+, or La3+ also did not inhibit strain‐induced cell growth activity. In contrast, gadolinium, a stretch‐activated channel blocker, inhibited strain‐induced 45Ca2+ influx and suppressed strain‐enhanced DNA synthesis. We conclude that the entry of calcium into cells through stretch‐activated ion channels plays a critical role in strain‐induced fetal lung cell proliferation. © 1994 Wiley‐Liss, Inc.
Differential effects of cytotactin/tenascin fusion proteins on intracellular pH and cell morphology
Authors: Krushel Leslie A. , Prieto Anne L. , Edelman Gerald M. , Crossin Kathryn L.
Published: February 2005

Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytototactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD‐containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pHi are correlated with the phenomenon of counteradhesion whereas the ability to increase pHi is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pHi. © 1994 Wiley‐Liss, Inc.
Overexpression of plasminogen activator inhibitor type‐1 in senescent fibroblasts from normal subjects and those with Werner syndrome
Authors: Goldstein Samuel , Moerman Elena J. , Fujii Satoshi , Sobel Burton E.
Published: February 2005

We previously reported that plasminogen activator inhibitor type‐1 (PAI‐1) mRNA was present at higher steady‐state levels in prematurely senescent fibroblasts derived from a subject with Werner syndrome (WS) compared to early passage (EP) fibroblasts from an age‐matched normal subject (Murano et al., 1991, Mol. Cell. Biol. 11:3905–3914). To explore the generality of this phenomenon with respect to chronological age of donor (in vivo aging) and the late‐passage (LP) or senescent phase of the fibroblast replicative lifespan, we assayed PAI‐1 mRNA in cells and PAI‐1 antigen in medium conditioned by 20 normal fibroblast strains at EP and LP and six WS strains during their curtailed replicative lifespans. The lowest accumulations of PAI‐1 were found in medium conditioned by fetal and newborn cells with a shallow but progressive rise seen in postnatal cells from normal donors of increasing chronological age. With few exceptions, normal LP fibroblasts showed increased PAI‐1 accumulations in medium compared to their EP counterparts. Conditioned medium from four of the six WS strains showed PAI‐1 accumulations that were significantly higher than the media of any normal controls at EP and LP. PAI‐1 mRNA levels were generally commensurate with the cumulative amount of PAI‐1 in the medium but the frequent exceptions indicate that translational and post‐translational mechanisms also regulate PAI‐1 output. The augmentation in PAI‐1 output of fibroblasts as a direct function of chronological age and during in vitro senescence suggests that PAI‐1 may play an important role in the reduced capacity for wound healing and the increasing tendency to thrombogenesis and atherogenesis seen during biological aging and in particular in persons with Werner syndrome. © 1994 Wiley‐Liss, Inc.
Glutathione stimulates A549 cell proliferation in glutamine‐deficient culture: The effect of glutamate supplementation
Authors: Kang Y. James , Feng Yi , Hatcher Emiko L.
Published: February 2005

Extracellular glutathione (GSH) is degraded by an external cell‐surface enzyme, γ‐glutamyltranspeptidase (γ‐GT). The products are transported into cells to participate in important cellular processes. In the present study, we tested the hypothesis that extracellular GSH is a source of glutamic acid for cells that express γ‐GT. Under a glutamine‐deficient culture condition, the extracellular GSH‐supplemented glutamic acid would enhance intracellular glutamine synthesis, thereby stimulating cell proliferation. Human lung carcinoma A549 cells were cultured in glutamine‐deficient Dulbecco's modified Eagle medium, and they did not proliferate unless glutamine was supplemented. Extracellular GSH, however, provoked a partial proliferation. The GSH effect correlated with a high level of γ‐GT activity and an increased intracellular level of glutamic acid. A constituent amino acid of GSH, glutamic acid but not cysteine, produced the same growth‐stimulatory effect as GSH. Furthermore, neither oxothiazolidine‐4‐carboxylate (OTC), a celluar cysteine‐delivery compound, nor cysteinylglycine, a dipeptide released from the γ‐GT reaction, stimulated cell proliferation. Moreover, buthionine sulfoximine (BSO), a selective inhibitor of γ‐glutamylcysteine synthetase, enhanced the GSH growth stimulatory effect, suggesting that increased cellular GSH synthesis does not correlate with cell growth stimulation. The results obtained demonstrated that glutamine is required for A549 cell proliferation and exogenous GSH partially substitutes for the growth stimulatory action of glutamine. It also suggests that the glutamic acid rather than the cysteine released from the GSH is responsible for the cell proliferation. © 1994 Wiley‐Liss, Inc.
Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias
Authors: Stenberg Paula E. , McDonald Ted P. , Jackson Carl W.
Published: February 2005

Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May‐Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar rat bone marrow was fixed and processed for transmission electron microscopy after VCR administration alone, after 5‐fluorouracil (5‐FU) administration alone, or after 5‐FU followed by intravenous injection of 0.1–1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5‐FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5‐FU caused formation of large membrane complexes in the cytoplasm of Wistar rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5‐FU and VCR, and was maximal (∼two‐third of megakaryocytes) at VCR dosages of 0.75–1.0 mg/kg. The majorityof megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted α‐granule proteins, and an increased incidence of emperipolesis. Rats administered 5‐FU alone did not demonstrate these abnormalities, with the exception of an increase in dense compartments. Platelets from rats treated with VCR aloene or 5‐FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface‐connected canalicular system (SCCS). The membrane complexes in platelets of 5‐FU‐, VCR‐treated Wistar rats as well as untreated Wistar Furth rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5‐FU‐, VCR‐treated rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte α‐granules. © 1995 Wiley‐Liss, Inc.
Reglation of autocrine gastrin expression by the TGFα autocrine loop
Authors: Howell Gillian M. , Ziober Barry L. , Humphrey Lisa E. , Willson James K. V. , Sun Luzhe , Lynch Mark , Brattain Michael G.
Published: February 2005

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686–2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15–21), might be controlled by autocrine TGFα in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utlizie TGFα. The FET cell line requires exogenous TGFα/EGF for optimal growth and has a classical TGFα autocrine loop which is disrupted by TGFα or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGFα/EGF and exhibits a nonclassical TGFα autocrine loop which is not disrupted by neutralizing antibodies to either TGFα itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGFα autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGFα mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGFα reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGFα loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops. © 1995 Wiley‐Liss, Inc.
Regulation of c‐fos expression in senescing Werner syndrome fibroblasts differs from that observed in senescing fibroblasts from normal donors
Authors: Oshima Junko , Campisi Judith , Tannock T. Charles A. , Martin George M.
Published: February 2005

The Werner syndrome (WS) is a segmental progeroid syndrome caused by a recessive mutation (WRN) mapped to 8p12. The replicative life spans of somatic cells cultured from WS patients are substantially reduced compared to age‐matched controls. Certain molecular concomitants of the replicative decline of normal fibroblast cultures have recently been defined, and it appears that multiple changes in gene expression accompany normal cell senescence. If the mechanisms by which WS cells exit the cell cycle were entirely comparable, the molecular markers of senescence should be identical in normal and WS cells. We find that this is not the case. The constitutive expression of statin, a nuclear protein associated with the nonproliferating state, was comparably expressed in normal and WS senescent cells. Likewise, the steady state levels of p53, a protein known to be involved in the G1 checkpoint of the cell cycle, were similar in early‐passage fibroblasts from normal and WS subjects. The levels of p53 were not increased in senescent fibroblasts, whether derived from normal or WS subjects. By contrast, the inducibility of mRNA and protein expression of the c‐fos protooncogene is preserved in late‐passage WS cells. This is in contrast to what is observed in late‐passage fibroblasts from normal subjects. Additional genotypes will have to be examined, however, to determine the specificity of this new aspect of the WS phenotype. © 1995 Wiley‐Liss, Inc.
Betamethasone activation of CTP: Cholinephosphate cytidylyltransferase is mediated by fatty acids
Authors: Mallampalli Rama K. , Salome Ronald G. , Li Chu Han , van Rollins Mike , Hunninghake Gary W.
Published: February 2005

The purpose of the present study was to determine the mechanisms by which glucocorticoids increase the activity of CTP: cholinephosphate cytidylyltransferase, a key enzyme required for the synthesis of surfactant phosphatidylcholine. Lung cytidylyltransferase exists as an inactive, light form low in lipids (L‐form) and an active, heavy form high in lipid content (H‐form). In vivo, fatty acids stimulate and aggregate the inactive L‐form to the active H‐form. In vivo, betamethasone increases the amount of H‐form while decreasing the amount of L‐form in fetal lung. There is also a coordinate increase in total free fatty acids in the H‐form. In the present study, we used gas chromatography–mass spectrometry to measure the fatty acid species associated with the H‐forms in fetal rat lung after the mothers were treated with betamethasone (1 mg/kg). In vivo, betamethasone increased the total amount of free fatty acids associated with the H‐form by 62%. Further, the hormone selectively increased the mass of myristic and oleic acids in H‐form by 52 and 82%, respectively. However, betamethasone produced the greatest increase in the amount of H‐form linoleic acid, which increased fourfold relative to control. In vitro, each of the fatty acids increased L‐form activity in a dose‐dependent manner; however, linoleic acid was the most potent. Linoleic and oleic acids also effectively increased L‐form aggregations. These observations suggest that in vivo glucocorticoids elevate the level of specific fatty acids which convert cytidylyltransferase to the active form. © 1995 Wiley‐Liss, Inc.
Laminin decreases PRL and IGFBP‐1 expression during in vitro decidualization of human endometrial stromal cells
Authors: Brar A. K. , Frank G. R. , Richards R. G. , Meyer A. J. , Kessler C. A. , Cedars M. I. , Klein D. J. , Handwerger S.
Published: February 2005

The expression of laminin, a major constituent of endometrial cell basement membranes, is increased during differentiation of human endometrial stromal cells (decidualization). To determine whether laminin plays a role in decidualization, we studied the effects of laminin substrate on the synthesis and release of prolactin (PRL) and insulin‐like growth factor binding protein‐1 (IGFBP‐1), two major secretory proteins of decidualized stromal cells. Endometrial stromal cells were plated on laminin as well as several other extracellular matrix (ECM) proteins (types 1 and IV collagen or fibronectin) and on plastic, and cultured in media containing medroxyprogesterone acetate (MPA) and estradiol. Cells cultured on plastic or ECM proteins displayed similar morphological changes indicative of decidualization. However, the release of PRL and IGFBP‐1 from cells cultured on plastic and ECM proteins (types 1 and IV collagen and fibronection) was approximately 2.1‐fold and 2.8‐fold greater respectively, than from cells cultured on laminin. The decrease in PRL and IGFBP‐1 expression in cells cultured on laminin was not due to differences in initial cell attachment efficiency or final DNA content. In addition, laminin had no effect on the content of laminin protein or fibronectin mRNA levels, indicating that the effects of laminin on PRL and IGFBP‐1 were specific. PGE2 stimulated the release of PRL and IGFBP‐1 from cells cultured on laminin to levels comparable to those from cells cultured on plastic or other ECM proteins. This indicates that the decrease in PRL and IGFBP‐1 release by laminin was not due to a generalized unresponsiveness. In contrast to the effects of laminin during decidualization, PRL expression was not altered by laminin in terminally differentiated decidual cells isolated at term. Our results support a role for laminin in selectively regulating PRL and IGFBP‐1 gene expression during in vitro decidualization of human endometrial stromal cells. © 1995 Wiley‐Liss, Inc.
Role of intracellular free Ca(II) and Zn(II) in dexamethasone‐induced apoptosis and dexamethasone resistance in human leukemic CEM cell lines
Authors: Adebodun Foluso , Post Jan F. M.
Published: February 2005

The levels of intracellular free Ca(II) and Zn(II) during dexamethasone (dex)‐induced apoptosis in CEM cell lines were determined by 19F nuclear magnetic resonance (NMR), using the fluorinated intracellular chelator 1,2‐bis‐(2‐amino‐5‐fluorophenoxy)ethane‐N, N, N′, N′‐tetraacetic acid (5‐FBAPTA). The effects of these divalent metal ions on growth rate and DNA degradation were evaluated. Measurements were done on one dex‐sensitive (CEM‐C7) and three different dex‐resistant variants (CEM‐C1, CEM‐4R4, and CEM‐ICR27). Dex caused a continuous increase in the Ca(II) level in dex‐sensitive CEM‐C7 cells, while in CEM‐C1 cells dex caused an initial increase in the Ca(II) level which in ≈︁36 h was restored to its normal value. The intracellular Ca(II) level in CEM‐4R4 cells was not significantly affected by dex, while that of CEM‐ICR27 cells decreased after dex incubation. Only the dex‐sensitive CEM‐C7 cells showed dex‐induced DNA degradation. An intracellular free Zn(II) level of ≈︁1 nM was measured for the dex‐resistant CEM‐C1 cells. No detectable level of intracellular Zn(II) was found in the other cell lines. Incubation with <100 μM Zn(II) did not inhibit dex‐induced apoptosis in CEM‐C7 cells (e.g., DNA degradation). Treatment with ≈︁250 μM Zn(II) caused significant decrease in growth rate in all cell lines and prevented dex‐induced DNA degradation in CEM‐C7 cells. A calibrated amount of Ca(II) ionophore (A23187), used to increase Ca(II) concentrations up to the dex‐induced levels, did not induce DNA degradation in CEM‐C7 or CEM‐C1 cells. While elevation of intracellular Ca(II) by itself is not sufficient to initiate apoptosis in CEM‐C7 cells, the results reported here suggest that Ca(II) is involved in the killing mechanism as a secondary factor. The combination of dex and ionophore caused significant DNA degradation in CEM‐C1 cells, which normally showed resistance to each compound individually. The combination of dex and the Zn(II) chelator phenanthroline also caused extensive DNA degradation in the normally dex‐resistant CEM‐C1 cells, suggesting that Zn(II) plays a role in the dex resistance of these cells. © 1995 Wiley‐Liss, Inc.
Phenotypic features of breast cancer cells overexpressing ornithine‐decarboxylase
Authors: Manni Andrea , Wechter Rita , Wei Lisa , Heitjan Daniel , Demers Laurence
Published: February 2005

Polyamines (PA) have been shown to be critical mediators of estradiol‐induced breast cancer cell proliferation. This finding suggests that constitutive activation of the PA pathway may promote tumor progression, possibly leading to hormone independence. To test this hypothesis, we transfected hormone‐responsive MCF‐7 breast cancer cells with a complementary DNA coding for ornithine‐decarboxylase (ODC), the first rate‐limiting enzyme in PA biosynthesis. Marked ODC over‐expression observed in stably transfected clones was associated with a selective increase in cellular putrescine content, while spermidine and spermine levels were not altered. ODC‐overexpressing MCF‐7 cells were resistant to the antiproliferative effects of low but not high concentrations of the enzyme inhibitor, α‐difluoromethylornithine. In agreement with our hypothesis, sensitivity to the growth‐promoting action of estradiol was reduced by approximately one third (P < 0.001) in ODC‐overexpressing MCF‐7 cells compared with vector‐only transfected clones. Basal growth under anchorage‐dependent conditions was only marginally increased by ODC overexpression (P = 0.048), while clonogenicity in soft agar was actually reduced. These data suggest that activation of PA biosynthesis may contribute in part to the acquisition of estrogen independence by breast cancer cells. Since only putrescine content was increased as a result of ODC overexpression, these data may underestimate the overall influence of the PA pathway on breast cancer phenotype. © 1995 Wiley‐Liss, Inc.
Mechanical strain increases endothelin‐1 gene expression via protein kinase C pathway in human endothelial cells
Authors: Wang Danny Ling , Wung Being‐Sun , Peng Yi‐Ching , Wang Jaang Jung
Published: February 2005

Vascular endothelial cells (ECs) are constantly subjected to mechanical strain due to relaxation and contraction of vessel walls. The effects of cyclical strain on endothelin‐1 (Et‐1) secretion and Et‐1 mRNA levels in human umbilical vein ECs were examined. Cultured ECs grown on a flexible membrane base were deformed by negative pressure (16 kPa at 60 cycles/min). Cells subjected to strain showed increased Et‐1 secretion (0.54 ng/hr/106 cells) compared with unstrained control cells (0.22 ng/hr/106 cells). Northern blot analysis of cells strained for 2 hours or longer demonstrated a sustained elevated Et‐1 mRNA level at more than double the level in unstrained controls. This strain‐induced ET‐1 mRNA level returned to its basal level 2 hours after the release of strain. Cells treated with actinomycin D before or during strain treatment showed no strain‐induced gene expression. Pretreatment of ECs with a protein kinase C (PKC) inhibitor, Calphostin C, strongly inhibited the strain‐induced Et‐1 gene expression. Pretreatment of ECs with cAMP or cGMP‐dependent protein kinase inhibitors (KT5720 or KT5823) only partially inhibited the increased Et‐1 mRNA levels in strain‐treated cells. EGTA strongly inhibited the Et‐1 gene expression. The intracellular calcium chelator BAPTA/AM also showed an inhibitory effect on Et‐1 mRNA levels. We conclude that mechanical strain can stimulate the secretion of Et‐1 from ECs by increasing Et‐1 mRNA levels via transcription, and that this gene induction is mediated predominantly via the PKC pathway and requires extracellular Ca2+. This strain‐induced Et‐1 gene expression in ECs may contribute to the regulation of vascular tone and structure in normal and pathological states of the cardiovascular system. © 1995 Wiley‐Liss, Inc.
Differential effects of a heparin antagonist (hexadimethrine) or chlorate on amphiregulin, basic fibroblast growth factor, and heparin‐binding EGF‐like growth factor activity
Authors: Cook Paul W. , Ashton Nina M. , Karkaria Cyrus E. , Siess Don C. , Shipley Gary D.
Published: February 2005

Amphiregulin (AR) and heparin‐binding EGF‐like growth factor (HB‐EGF) are two recently identified members of the EGF family. Both AR and HB‐EGF share with EGF the ability to interact with the type‐1 EGF receptor; however, AR and HB‐EGF differ from EGF in that both of these mitogens bind to heparin while EGF does not. To determine whether interactions with heparin‐like molecules on the cell surface influence binding of AR and HB‐EGF with EGF receptors and the subsequent mitogenic activity exerted by these growth factors, murine AKR‐2B and Balb/MK‐2 cells were treated with either an inhibitor of proteoglycan sulfation (chlorate) or a heparin antagonist (hexadimethrine). As expected, neither treatment significantly altered the specific binding of 125I‐EGF on AKR‐2B cells. Interestingly, treatment with either chlorate or hexadimethrine inhibited the ability of AR to compete with 125I‐EGF for cell surface binding and also attenuated AR‐mediated DNA synthesis. Thus, as has been suggested for other heparin‐binding growth factors such as basic fibroblast growth factor (bFGF), the interaction of AR with an EGF‐binding receptor appears to be facilitated by interaction with cell‐associated sulfated glycosami‐noglycans or proteoglycans. Unexpectedly, however, neither chlorate nor hexadimethrine treatment caused an inhibition of HB‐EGF‐induced mitogenic activity. Chlorate treatment did not significantly alter the ability of HB‐EGF to compete with 125I‐EGF for cell surface binding sites, however, heparin and hexadimethrine reduced the ability of HB‐EGF to compete for 125I‐EGF binding. These results suggest that, in AKR‐2B cells, HB‐EGF may mediate its mitogenic response at least in part through a receptor which appears to be selective for HB‐EGF and permits HB‐EGF‐mediated mitogenic responses in the presence of hexadimethrine or heparin. Finally, hexadimethrine inhibited the specific binding and mitogenic activity of bFGF, suggesting that this cationic polymer can function as an antagonist of heparin‐binding mitogens other than AR. © 1995 Wiley‐Liss, Inc.
Patterns of cyclic AMP‐dependent protein kinase gene expression during ontogeny of the murine palate
Authors: Greene Robert M. , Lloyd Martha R. , Uberti Michelle , Nugent Paul , Pisano M. Michele
Published: February 2005

Normal growth and differentiation of embryonic palatal tissue depends on regulated levels of intracellular cAMP. Cyclic AMP‐dependent protein kinases (PKA) act to mediate the biological activities of cAMP. PKA isozyme protein profiles demonstrate a clear pattern of temporal alterations in embryonic palatal tissue during its development. In order to ascertain the molecular basis for changing PKA isozyme profiles during palatal ontogeny, the spatial and temporal expression of mRNAs for regulatory (Rlα, Rllα, and Rllβ) and catalytic (Cα) subunits of PKA was examined. RNA extracted, from murine embryonic palatal tissue (days 12–14 of gestation) was examined by Northern blot analysis. Significant levels of constitutively expressed Rlα and Cα mRNA were seen on all days of gestation examined. Rlα transcripts were substantially less abundant in palate mesenchymal cells in vitro than in palatal tissue in vivo. Levels of Rllα and Rllβ mRNA were highest on gestational day (GD) 12, a period characterized by pronounced palatal tissue growth. In addition, patterns of tissue distribution of Rllβ, not previously described, were examined in the developing embryonic palate. A dramatic developmental shift in tissue distribution of Rllβ was seen. The isozyme was evenly distributed between palatal epithelial and mesenchymal cells on GD 12 but by GD 14, Rllβ was predominantly localized to palatal epithelial cells. Direct activation of adenylate cyclase with forskolin in murine embryonic palate mesenchymal (MEPM) cells resulted in an increase in Rllα mRNA levels but had no effect on steady state levels of Rllβ or Cα mRNA. In addition, elevation of intracellular levels of cAMP resulted in a shift in the transcriptional profile of Rlα mRNAs. Results of this study document specific patterns of expression for the genes encoding the various cAMP‐dependent protein kinase regulatory and Cα subunits in murine embryonic palatal tissue. In addition, we have demonstrated adaptational changes of this kinase in MEPM cells in response to conditions of increased intracellular levels of cAMP. © 1995 Wiley‐Liss, Inc.
Iron and copper requirements for proliferation and differentiation of a human promyelocytic leukemia cell line (HL‐60)
Authors: Sergeant Susan , Johnson W. Thomas
Published: February 2005

Trace mineral deficiencies tend to have profound effects on the integrity of formed blood elements. Anemia and neutropenia are commonly seen in copper (Cu) deficiency. We therefore developed a serum‐free medium to examine the trace mineral requirements, in particular iron and Cu, for proliferation and retinoic acid (RA)‐induced differentiation of HL‐60 cells. This defined medium (DFM) was composed of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with insulin and human apo‐transferrin (each at 5 μg/ml) and 1.4 μM FeSO4. The iron concentration range for optimal cellular proliferation was narrow (2–3 μM). HL‐60 cells could be maintained in DFM for 15 passages with a doubling time of 38–40 hr. The Cu content of IMDM was very low. Thus, by the fourth passage in DFM, the activity of cuproenzymes (cytochrome c oxidase, CCO; and copperzinc superoxide dismutase, CuZnSOD) began to decline. Supplementation of DFM with CuSO4 (50 nM) restored enzyme activities. Treatment of cells with a Cu chelator (tetrathiomolybdate, 1 μM) rapidly reduced the activities of both CCO and CuZnSOD. Over the Cu concentration range examined (5–350 nM), Cu supplementation had little effect on HL‐60 proliferation. Cell retained the ability to differentiate along the granulocytic pathway when treated with RA, but seemed to be less sensitive to the inducing agent except at the highest concentration tested (1 μM). This decreased sensitivity to RA did not seem to be related to the Cu status of the cells but rather to the absence of a component of serum. Indeed, cells grown in DFM regained their sensitivity to RA when allowed to differentiate in IMDM with 5% serum. These data indicate that the processes of growth and terminal differentiation in HL‐60 cells are not greatly influenced by Cu. Thus, it seems likely that the insult resulting in neutropenia which is associated with Cu deficiency may occur earlier than the promyelocytic stage. However, the possibility that the mechanisms contributing to neutropenia may be unrelated to primary defects in the biochemistry of neutrophil maturation cannot be ruled out. © 1995 Wiley‐Liss, Inc.
Antiprogestins inhibit growth and stimulate differentiation in the normal mammary gland
Authors: Li Minglin , Spitzer Eva , Zschiesche Wolfgang , Binas Bert , Parczyk Karsten , Grosse Richard
Published: February 2005

Antiprogestins possess a potent antitumor activity in hormone‐dependent experimental breast cancer models. Though the underlying mechanism is not clear, induction of functional differentiation seems to be a major event. This study attempts to test directly for antiproliferative and differentiation promoting activities of antiprogestins on the normal mammary gland. To this end, whole organ cultures of mammary glands from estradiol/progesterone‐primed virgin mice maintained in a serum‐free medium with aldosterone, prolactin, insulin, and hydrocortisone were exposed to the antiprogestin ZK114043. A 4‐day treatment of organ cultures led to a strong inhibition of epithelial DNA synthesis. In parallel, ZK114043 caused alveolar cells to acquire a more differentiated phenotype distinguished by secretory active alveoli composed of single cell layers with increased fat droplet accumulation and enhanced expression of the milk proteins b̃‐casein and whey acidic protein (WAP). Particularly strong effects were found on the expression of mammary‐derived growth inhibitor (MDGI). Both half‐maximal inhibition of epithelial DNA synthesis and stimulation of MDGI mRNA expression were found at about 5 ng/ml of ZK114043. Presence in the medium of 5 m̈g/ml hydrocortisone rendered antiglucocorticoid effects of ZK114043 highly unlikely. Furthermore, prevention of action of ZK114043 by the progesterone agonist R5020 and ZK114043 stimulated expression of b̃‐casein and MDGI mRNA in cultured glands of 10‐week‐old unprimed virgin mice suggest a progesterone receptor‐mediated mechanism of antiprogestin action. Two other antiprogestins, Mifepristone and Onapristone, likewise stimulated MDGI expression. The data provide direct evidence that antiprogestins act like a differentiation factor in the normal mammary gland. © 1995 Wiley‐Liss, Inc.
Bone marrow stromal cells selectively stimulate the rapid expansion of lineage‐restricted myeloid progenitors
Authors: Kameoka Jun‐Ichi , Yanai Nobuaki , Obinata Masuo
Published: February 2005

Bone marrow stromal cells serve hematopoietic microenvironments where different blood cells are controlled in their growth and differentiation. To characterize functions of stromal cells, 33 bone marrow stromal cells including preadipocytes, endothelial cells, and fibroblasts were established from transgenic mice harboring temperature‐sensitive SV40 T‐antigen gene and their selective stimulatory abilities to support large colony formation of lineage‐specific hematopoietic progenitor cells (erythroid, monocyte/macrophage, granulocyte, and monocyte‐granulocyte) were examined. Among established stromal cells, 27 clones showed erythropoietic stimulatory activity in the presence of erythropoietin. On myeloid progenitors, the stromal cells showed lineage‐restricted stimulatory activity and a reciprocal relationship was observed between granulocyte formation and macrophage formation, but these activities were not dependent on the amount of produced colony‐stimulating factors (CSFs). Our present study with many stromal cells established from bone marrow indicated that each stromal cell in the bone marrow may provide the preferable microenvironment for a rapid expansion of the lineage‐restricted progenitor cells in combination with CSFs. © 1995 Wiley‐Liss, Inc.
Regulation of insulin‐like growth factor (IGF) binding protein‐2 synthesis and degradation by platelet‐derived growth factor and the IGFs is enhanced by serum deprivation in vascular smooth muscle cells
Authors: Cohick Wendie S. , Gockerman Amy , Clemmons David R.
Published: February 2005

Growth factors such as platelet‐derived growth factor (PDGF) and insulin‐like growth factor (IGF‐1) stimulate proliferation and migration of vascular smooth muscle cells (SMC). IGF‐l bioactivity is modulated by high‐affinity binding proteins (IGFBP) which are important regulators of these processes. Procine vascular SMC synthesize IGFBP‐2 and IGFBP‐4 in vitro. In the present study, levels of IGFBP‐2 in conditioned media (CM) were increased approximately 1.6 to 2.2‐fold when cells were exposed to PDGF (20 ng.ml) or insulin (5 μg/ml) for 24 hr following a 24 hr incubation in serum‐free media, or following a 72 hr exposure to either growth factor. Similar increases in IGFBP‐2 mRNA levels were observed. Exposure of cells to PDGF for 24 hr without prior serum deprivation resulted in smaller (47 ± 11%) increases in IGFBP‐2 protein levels but failed to alter mRNA levels. IGF‐1, FGF‐b̃ and EGF failed to increase IGFBP‐2 using either experimental paradigm. In contrast, IGFBP‐2 protein levels were consistently decreased (75 ± 14%) after 72 hr of exposure to IGF‐II without corresponding decreases in IGFBP‐2 mRNA levels. Immunoprecipitation of [35S] methionine‐labeled IGFBP‐2 indicated that this decrease was not due to a decrease in synthesis of IGFBP‐2. Immunoblot analysis of CM from cells treated with IGF‐II indicated that the decrease in intact protein corresponded with an increase in two non‐IGF binding IGFBP‐2 fragments of 22 and 14 kD. Increased abundance of these fragements was also observed following IGF‐I exposure, although corresponding decreases in intact IGFBP‐2 were not usually observed. The relative abundance of these fragments did not appear to be affected by treatment with PDGF or insulin. In contrast to IGFBP‐2, regulation of the levels of IGFBP‐4 in CM did not appear to be altered by serum deprivation. Insulin consistently increased IGFBP‐4 mRNA and protein levels under all situations. PDGF tended to increase IGFBP‐4 protein levels, although this effect was less consistent and not as great as the increase observe with insulin. Treatment with IGF‐I or ‐ll consistently decreased IGFBP‐4 levels in CM but tended to increase their mRNA levels under all situations. These data indicate that insulin, PDGF, and the IGFs regulate both IGFBP‐2 and IGFBP‐4. While PDGF and insulin stimulate IGFBP‐2 and 4 synthesis, the IGFs appear to activate protease(s) which regulate IGFBP‐2 and ‐4 levels post‐translationally. The regulation of IGFBP‐2 levels by each of these mechanisms appears to be amplified by serum deprivation, but this is not observed with IGFBP‐4. © 1995 Wiley‐Liss, Inc.
Transforming growth factor‐beta isoform expression in insulin‐like growth factor stimulated myogenesis
Authors: Bosche William J. , Ewton Daina Z. , Florini James R.
Published: February 2005

Transforming growth factor betas (TGF‐b̃s) are the defining members of a superfamily of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF‐b̃s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF‐b̃s have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation. Insulin‐like growth factors (IGFs) have also been shown to be secreted by myoblasts and to induce myogenesis. This study characterizes the effects of IGF treatment on the expression and secretion of TGF‐b̃s in the IGF‐sensitive L6A1 myoblast line. IGF downregulated the expression of TGF‐b̃3 in a concentration‐dependent manner at 24 and 48 hours; TGF‐b̃1 was not sensitive to IGF treatment at 24 hours but was downregulated by IGFs at 48 hours. This downregulation was mediated by the type I IGF receptor and modulated by IGF binding proteins secreted by the myoblasts. Some reexpression of TGF‐b̃1 and TGF‐b̃3 mRNAs was observed after extensive morphological differentiation had occurred. These results support the hypothesis that IGFs act through the IGF type I receptor as part of a concerted mechanism to modulate expression of the TGF‐b̃ genes, as part of a coordinated set of changes associated with terminal myogenic differentiation. © 1995 Wiley‐Liss, Inc.
Characterization of a signal generated by oxidation of protein thiols that activates the heat shock transcription factor
Authors: Freeman Michael L. , Borrelli Michael J. , Syed Khalid , Senisterra Guillermo , Stafford Diane M. , Lepock James R.
Published: February 2005

The diazenecarbonyl derivative, diamide, was used to produce nonnative protein disulfides in Chinese hamster ovary cells in order to characterize the events that occur during thiol oxidation‐induced denaturation that trigger induction of Hsp 70. We limit the term protein denaturation to a process involving a conformational rearrangement by which the ordered native structure of a protein changes to a more disordered structure. Protein thiol oxidation resulted inimmediate destabilization of proteins, as assessed by differential scanning calorimetry (DSC). The DSC profile indicated both a decrease in the onset temperature for detection of denaturation and destabilization of a class of proteins with an average transition temperature (Tm) of 60°C. Concomitant with destabilization was an increase in proteins associated with isolated nuclei. Thiol oxidation also induced heat shock transcription factor (HSF) binding activity, however, this was nearly undetectable immediately following diamide treatment: maximum activation occurred 3 hr following exposure. In contrast, heat shock denatured thermolabile proteins which exhibited a Tm of 48°C. Heat shock also resulted in a rapid increase in proteins associated with isolated nuclei and produced immediated and maximum activation of HSF binding. The accumulation of Hsp and Hsc 70 mRNA following thiol oxidation reflected the delay in HSF binding. Acquisition of HSF binding activity occurred immediately if diamide‐treated cells were subsequently exposed to a heat shock, indicating that HSF was not inactivated by the diamide treatment. Ostensibly, the cellular system for detecting denatured/abnormal proteins failed to immediately recognize the signal generated by thiol oxidation. These results suggest that at least two processes are involved in the induction of Hsp 70 by nonnative disulfide bond formation: destabilization of protein structure resulting in denaturation and recognition of denatured protein. © 1995 Wiley‐Liss, Inc.