bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat‐induced transcription from RNA polymerases II and III and HSF binding activity are co‐ordinately regulated by the products of the heat shock genes
Authors: Price Brendan D. , Calderwood Stuart K.
Published: February 2005

Heat shock leads to co‐ordinate increases in transcription of a family of heat shock genes, including the mouse hsp70.1 and B2 genes. Activation of the heat shock transcription factor (HSF) by heat shock stimulates transcription of the murine hsp70.1 gene (by RNA polymerase II). B2 genes are short, repetitive sequences whose transcription (by RNA polymerase III) are also increased after heat shock. We have studied whether heat‐induced transcription is auto‐regulated by the products of the heat shock genes. The results indicate: (1) after an initial heat shock, transcription of the heat shock genes by RNA polymerases II and III becomes desensitized to further heat shock, and the heat‐induced DNA binding activity of the HSF is lost, (2) if accumulation of heat shock gene products is inhibited, the desensitizing effect of a prior heat shock is removed, and (3) transcription of the hsp70. 1 and the B2 gene apparently involves different mechanisms, with hsp70.1 employing the HSF and the B2 gene using a separate, heat‐activated transcriptional mechanism. However, the level of transcription from the hsp70.1 and B2 genes and the stability of their respective RNAs are co‐ordinately regulated by the level of heat shock protein in the cell. The data indicate that auto‐regulation of the level of mouse heat shock gene products is mediated by RNA polymerase II transcripts but that the regulatory mechanism can control transcription from RNA polymerase III genes as well. © 1992 Wiley‐Liss, Inc.
Lactoferrin binding sites and nuclear localization in K562(S) cells
Authors: Garré Cecilia , Bianchi‐Scarrà Giovanna , Sirito Mario , Musso Marco , Ravazzolo Roberto
Published: February 2005

Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated.

In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA‐linked protein in nuclear extracts. © 1992 Wiley‐Liss, Inc.
Effect of membrane phosphatidylethanolamine‐deficiency/phosphatidylcholine‐excess on the metabolism of phosphatidylcholine and phosphatidylethanolamine
Authors: Fisk Harold A. , Kano‐Sueoka Tamiko
Published: February 2005

Cells of epithelial origin generally require ethanolamine (Etn) to grow in defined culture medium. When such cells are grown without Etn, the membrane phospholipid composition changes drastically, becoming phosphatidylethanolamine (PE)‐deficient due to a reduced de novo rate of PE synthesis, and growth stops. We have hypothesized that the cessation of growth occurs because this membrane phospholipid environment is no longer suitable for membrane‐associated functions. Phospholipid has long been known to play a role in the transduction of some signals across membranes. In addition to the well‐known phosphatidylinositol cycles, hydrolysis of phosphatidylcholine (PC) and PE has recently been shown to play a central role in signal transduction. Using an Etn‐requiring rat mammary cell line 64‐24, we have studied the metabolism of PC and PE in response to the phorbol ester phorbol 12, 13‐dibutyrate (PDBu) under conditions where cells have either normal or PE‐deficient membrane phospholipid. In cells having normal membrane phospholipid, the synthesis of PC was stimulated by PDBu (∼fourfold), as was the degradation of PC and PE (by twofold and fourfold, respectively). Product analysis suggested that PDBu stimulated hydrolysis of PC by both phospholipases C and D (PLC and PLD), and of PE by PLD. However, in PE‐deficient cells, neither lipid synthesis or degradation were significantly stimulated by PDBu. Analysis of the CDP‐choline pathway of PC sythesis indicated that the regulatory enzyme, CTP: phosphorylcholine cytidylyltransferase, was stimulated about twofold by PDBu in cells having normal membrane, but not in PE‐deficient cells. These results indicate that the membrane phospholipid environment profoundly affects phospholipid metabolism, which no doubt influences cell growth and regulation. © 1992 Wiley‐Liss, Inc.
Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle
Authors: Terada Naohiro , Lucas Joseph J. , Szepesi Agota , Franklin Richard A. , Domenico Joanne , Gelfand Erwin W.
Published: February 2005

The effects of rapamycin (RAP) on cell cycle progression of human T cells stimulated with PHA were examined. Cell cycle analysis showed that the RNA content of cells stimulated with PHA in the presence of RAP was similar to that of control T cells stimulated with PHA for 12–24 hr in the absence of the drug. This level was substantially higher than that seen in cells stimulated in the presence of cyclosporin A (CsA), an immunosuppressant known to block cell cycle progression at an early point in the cycle. However, the point in the cell cycle at which RAP acted appeared to be well before the G1/S transition, which occurs about 30–36 hr after stimulation with PHA. In an attempt to further localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary were examined, including p110Rb phosphorylation, which occurred at least 6 hr prior to DNA synthesis, p34cdc2 synthesis, and cyclin A synthesis. In control cultures, p110Rb phosphorylation was detected within 24 hr of PHA stimulation; p34cdc2 and cyclin A synthesis were detected within 30 hr. Addition of RAP to the cultures inhibited each of these events. In contrast, early events, including c‐fos, IL‐2, and IL‐4 mRNAs expression, and IL‐2 receptor (p55) expression, were only marginally affected, if at all, in PHA‐stimulated T cells. Furthermore, the inhibition of cell proliferation by RAP could not be overcome by addition of exogenous IL‐2. These results indicate that RAP blocks cell cycle progression of activated T cells after IL‐2/IL‐2 receptor interaction but prior to p110Rb phosphorylation and other key regulatory events signaling G1/S transition. © 1993 Wiley‐Liss, Inc.
Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes
Authors: Pillai Sreekumar , Menon Gopinathan K. , Bikle Daniel D. , Elias Peter M.
Published: February 2005

Calcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle‐bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo‐1 and ion‐capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80–120 nM, whereas Cai of differentiated keratinocytes was 200–300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60–160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium‐containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium‐containing precipitates appeared as fine‐tocoarse electron‐dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle‐bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures. © 1993 Wiley‐Liss, Inc.
Major influence of cell differentiation status on characteristics of proteoglycans synthesized by cultured rabbit renal proximal tubule cells: Role of insulin and dexamethasone
Authors: Lelongt B. , Vandewalle A. , Brenchley P. E. C. , Baudouin B. , Géniteau‐Legendre M. , Verroust P. J. , Ronco P. M.
Published: February 2005

To analyze the influence of epithelial cell differentiation and the effects of hormones on the characteristics of cell‐associated and secreted proteoglycans (PGs), we studied their distribution, synthesis, and biochemical features in a model of renal proximal tubule cells in primary culture in which cell differentiation could be controlled by medium composition. In cells cultured in serum‐free, hormonally defined medium supplemented with insulin and dexamethasone that exhibited a high degree of morphological and functional proximal differentiation (Ronco et al., 1990), cell‐associated PGs were similar to those extracted in vivo by their size estimated by Sepharose CL‐6B chromatography (Kav = 0.27, vs. 0.26), composition (heparan‐sulfate), and localization in a continuous basal layer of extra‐cellular matrix (ECM). In contrast, major quantitative and qualitative anomalies of cell‐associated PGs were observed in poorly differentiated cells grown in 1% fetal calf serum‐supplemented medium (FCS). PGs alterations included: (1) reduced and irregular expression of PGs at the cell basal pole, (2) a 2.8‐fold decrease in [35S]‐incorporation into cell‐associated PGs, (3) a 3.1‐fold increase in trypsin‐releasable PGs, and (4) the emergence of a high MW PG composed exclusively of chondroitin‐sulfate (CS) (Kav = 0.09 on Sepharose CL‐6B) as well as of putative free CS‐glycosaminoglycan (GAG) chains (Kav = 0.49 on Sepharose CL‐6B). The same alterations were identified in the basal defined medium devoid of hormones but were partially or totally abolished by addition of insulin and dexamethasone, respectively. At variance with cell‐associated PGs, production and biochemical features of secreted PGs were not influenced by cell differentiation status and medium composition. © 1993 Wiley‐Liss, Inc.
Sequestration and secretion of insulin‐like growth factor‐I by bovine aortic endothelial cells
Authors: Gajdusek Corinne M. , Luo Zhengyu , Mayberg Marc R.
Published: February 2005

Endothelial cells elaborate growth promoting activities in culture medium that support limited smooth muscle cell and fibroblast growth in vitro in the absence of serum. We investigated whether insulin‐like growth factor‐I (IGF‐I) was synthesized and secreted by bovine aortic endothelial cells in vitro. Subconfluent endothelial cell cultures in serum‐free medium secreted severalfold higher IGF‐I levels than confluent cultures by acid‐sizing chromatography and IGF‐I radioimmunoassay. The IGF‐I secretory level was not sustained during a second serum‐free incubation. In contrast, secretion of IGF binding proteins persisted and was maintained at constant levels throughout the same observation periods. Analysis of poly (A+)RNA by northern blots revealed hybridization of an IGF‐I cDNA to a 7.5‐ to 7.0‐kb transcript and superinduction of the 7.5–7.0‐kb mRNA by the translational inhibitor, cyclohexamide. However, no endogenously labeled IGF‐I was detected in conditioned media after incubation of cultures with [35S]cysteine or [3H]leucine. When cultures were incubated in the presence of serum supplemented with IGF‐I, subconfluent cultures sequestered and released more IGF‐I than confluent cultures. We concluded that the majority of IGF‐I secreted in vitro was sequestered from serum. © 1993 Wiley‐Liss, Inc.
Transduced endothelial cells expressing high levels of tissue plasminogen activator have an unaltered phenotype in vitro
Authors: Jaklitsch Michael T. , Biro Sadatoshi , Casscells Ward , Dichek David A.
Published: February 2005

Elevated cellular plasminogen activator activity has been associated with significant alterations in the in vitro phenotype of both malignant cell lines and nonmalignant endothelial cells. To examine the role of elevated cellular plasminogen activator activity in the production of altered endothelial cell behavior, bovine coronary artery endothelial cells were transduced with a retroviral vector expressing large amounts of tissue plasminogen activator. Cells transduced with the tissue plasminogen activator vector were compared with both untransduced cells and cells transduced with a control vector in a series of in vitro assays of cellular attachment, proliferation, migration, and invasion. The morphology of the 2 transduced populations was unchanged. There was a small decrease (5–15%) in the horizontal migration rate of both transduced cell populations, as compared with that of untransduced cells. No significant differences were detected among the three cell populations in any of the other assays. We conclude that expression of high levels of tissue plasminogen activator does not specifically affect endothelial cell phenotype in vitro. These data lend support to the hypothesis that elevated plasminogen activator activity is necessary but not sufficient to produce alterations in endothelial cell behavior. © 1993 Wiley‐Liss, Inc.
Altered calcium homeostasis and cell injury in silica‐exposed alveolar macrophages
Authors: Rojanasakul Yongyut , Wang Liying , Malanga Carl J. , Ma Jane Y. C. , Banks Daniel E. , Ma Joseph K. H.
Published: February 2005

There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca2+]i. However, the mechanism of silica‐ induced cytotoxicity relative to [Ca2+]i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 μm in diameter) and the fluctuation in their [Ca2+]i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura‐2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca2+]i in a dose‐dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca2+]o markedly inhibited the [Ca2+]i rise (≈90%), suggesting that Ca2+ influx from extracellular source is a major mechanism for silica‐induced [Ca2+]i rise. When used at low doses but sufficient to cause a transient [Ca2+]i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca2+]o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca2+ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca2+ influx is inhibited by the Ca2+ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca2+ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica‐induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica‐induced [Ca2+]i rise was not due to the impairment of Ca2+‐pumps, which restricts Ca2+ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca2+]i rise. Taken together, these results suggest that the elevation of [Ca2+]i caused by silica is due mainly to Ca2+ influx through plasma membrane Ca2+ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca2+ leakage during phagocytosis was attributed to the silica‐induced [Ca2+]i rise. © 1993 Wiley‐Liss, Inc.
Relationship between c‐Kit expression and proliferation in acute myeloblastic leukemia cell lines
Authors: Tohda S. , Yang G. S. , Ashman L. K. , McCulloch E. A. , Minden M. D.
Published: February 2005

We have proposed that the transmembrane receptor encoded by the c‐Kit protooncogene and its ligand play an important role in regulating the proliferation of blasts cells in acute myeloblastic leukemia (AML). To test this hypothesis, immunobeads were used to separate blasts from three Kit‐expression positive cell lines into strongly Kit‐protein positive and weakly Kit‐protein positive fractions. The strongly positive fraction had greater proliferative potential than the weakly positive fraction as assessed both by colony‐formation in methylcellulose and growth of clonogenic cells in suspension. The reproducibility of the percentage of each blast population found in the strongly and weakly positive fractions provided evidence that Kit‐protein expression is regulated. Kinetic experiments provided evidence for reversible transitions between strong and weak Kit protein expression. Thus regulated expression of the Kit receptor may be a mechanism for controlling blast cell growth in culture. © 1993 Wiley‐Liss, Inc.
K+ fluxes mediated by Na+‐K+‐Cl− cotransport and Na+‐K+‐ATPase pumps in renal tubule cell lines transformed by wild‐type and temperature‐sensitive strains of simian virus 40
Authors: Vandewalle A. , Vuillemin T. , Teulon J. , Baudouin B. , Wahbe F. , Bens M. , Cassingéna R. , Ronco P.
Published: February 2005

The relative contributions of Na+‐+‐pumps and Na+‐+‐− contransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild‐type or a temperature‐sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na+‐+‐mediated component represented 74% and 44–48% of total Rb+ influx into PC.RC and SV40‐transformed cells, respectively. Proliferating transformed cells showed substantial ouabain‐resistant bumetanide‐sensitive (Or‐Bs) Rb+ influx (41–45% of total) which indicated the presence of Na+‐+‐− contransport. The Or‐Bs component of Rb+ influx was greatly reduced when temperature‐sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33°C) to the restrictive temperature (39.5°C) to arrest cell growth. The ouabain‐sensitive Rb+ influx mediated by the Na+‐+‐, the total and amiloride‐sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or‐Bs influx was obtained when renal tubule cells transformed by the wild‐type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113–125, 1992). Reinitiation of cell growth by removal of TEA or return to 33°C of the temperature‐sensitive cells restored the Or‐Bs component of Rb influx. Taken together, these results indicate that the Na+‐+‐− cotransport activity is critically dependent on cell growth con ditions. © 1993 Wiley‐Liss, Inc.
Stimulation of DNA synthesis and protooncogene expression in primary rat hepatocytes in long‐term DMSO culture
Authors: Serra Rosa , Isom Harriet C.
Published: February 2005

We have previously described the use of a chemically defined medium (CDM) supplemented with epidermal growth factor (EGF) and dimethylsulfoxide (DMSO) to maintain long‐term cultures of rat hepatocytes in a highly differentiated state. In this study, conditions necessary to stimulate high levels of DNA synthesis in hepatocytes in long‐term DMSO culture were defined. Hepatocytes were maintained in culture for 20 days in CDM containing DMSO and EGF, insulin, and glucagon. EGF, insulin, and glucagon were then removed for 7 days. Readdition of EGF, insulin, and glucagon at day 27 (shiftup) was accompanied by a three‐ to sixfold increase in labeling index. If DMSO or dexamethasone (dex) + DMSO were removed at time of shiftup, the labeling index increased by 18‐ to 54‐fold. TGFβ inhibited DNA synthesis stimulated by EGF shiftup, TGFα shiftup, or EGF shiftup in combination with removal of dex + DMSO. Stimulation of DNA synthesis was accompanied by a specific, sequential induction of protooncogene mRNA levels; c‐fos mRNA was induced 23‐fold at 0.5 h after readdition of EGF; c‐myc mRNA was induced three‐ to fourfold by 0.5 h; TGFα mRNA was induced sevenfold by 8 h; K‐ras mRNA was induced fourfold by 26 h. Changes in protooncogene expression paralleled changes seen in regenerating liver. When DMSO was removed for greater than 48 h, the cells flattened and spread out, chords of cells were no longer well defined, albumin mRNA levels decreased, and fibronectin, β1 integrin, and TGFβ transcripts increased. © 1993 Wiley‐Liss, Inc.
Long‐term culture of disaggregated rat osteoclasts: Inhibition of bone resorption and reduction of osteoclast‐like cell number by calcitonin and PTHrP[107‐139]
Authors: Fenton A. J. , Martin T. J. , Nicholson G. C.
Published: February 2005

The islated osteoclast bone resorption assay has proved to be a useful means of examining the response of mammalian and avian osteoclasts to a variety of stimuli. The assay has traditionally been performed over a period of 24 hours. By extending the duration of the osteoclast bone resorption assay, we have been able to assess the long‐term effects of carboxyl‐terminal parathyroid hormone‐related protein (hPTHrP[107‐139]), salmon calcitonin (sCT) and hPTH[1‐34] on bone resorption and TRACP‐positive osteoclast‐like cell number. We found that, in control cultures over a period of up to 144 hours, the osteoclast‐like cells not only remained viable but their numbers also increased. The number of mononucleated and multinucleated osteoclast‐like cells doubled in the first 48 hours before stabilizing over the remainder of the incubation period. Osteoblasts also proliferated, resulting in a resorption response to hPTH [1‐34] being evident from 48 hours onward. hPTHrP[107‐139] persistently inhibited basal and PTH‐stimulated bone resorption for at least 96–144 hours. Where as “escape” from the inhibijtory effect of sCT was seen after 48–72 hours. Decreased numbers of bothe mononucleated and multinucleated TRACP‐positive osteoclast‐like cells were seen by 48 hours in cultures treated with sCT. In contrast, hPTHrP[107‐139] reduced the number of mononuclear TRACP‐positive cells with only a late effect on multinucleated cells. Furthermore, the incsreased number of osteoclast‐like cells seen in response to hPTH[1‐34] was inhibited by carboxyl‐terminal PTHrP. In summary, this study indicates that the extended bone resorption assay system is a complex one where bothe osteoclastic resorption and osteoclast maturation are evident. Using this syste, we have shown that hPTHrP[107‐139] acts as a potent long‐term inhibitor of osteoclastic bone resorption, without evidence of escape from its effect. Its action to reduce the number of mononucleated osteoclst‐like cells suggests that it affects several aspects of osteoclast activity. © 1993 Wiley‐Liss, Inc.
Cyclic AMP‐induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms
Authors: Chen Jan‐Kan , Li Shing‐Rong , Tsai Ray Jui‐Fung
Published: February 2005

The contraction of collagen lattices made with forskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracelluar cyclic AMP raising drugs including cholera toxin (CT), forskolin, and dibutyryl‐cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor‐β (TGF‐β). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin‐induced intracellular cyclic AMP increase was attenuated by TGF‐β, but not by aFGF and insulin, when added simultaneously. Thus, TGF‐β may attenuate CT's inhibition on collagen lattice contraction by attenuating CTinduced intracellualr cyclic AMP increse, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMPindependent mechannism. © 1993 Wiley‐Liss, Inc.
Differential effects of transforming growth factor‐β1 and bone morphogenetic protein 4 on gene expression and differentiated function of preosteoblasts
Authors: Zhou Hong , Hammonds R. Glenn , Findlay David M. , Martin T. John , Ng Kong Wah
Published: February 2005

Transforming growth factor‐β (TGF‐β) and bone morphogenetic protein 4 (BMP 4) are both able, under certain circumstances, to induce endochondral bone formation in vivo. This study compared the effects of TGF‐β and BMP 4 on the gene expression of a retinoic acid (RA) responsive rat clonal preosteoblast cell line, UMR 201, as well as the way in which these proteins interact with RA in these cells. Both similarities as well as differences between the effects and mechanism of action of TGF‐β1 and BMP 4 were demonstrated. TGF‐β1 (0.1 ng/ml) strongly induced matrix gla protein (MGP) mRNA and increased the steady state osteonectin (ON) mRNA level. Cotreatment with TGF‐β1 and RA did not result in a further increase in MGP mRNA expression. In contrast, BMP 4 alone had no influence on MGP or ON mRNA expression but it significantly enhanced the RA induction of MGP mRNA. Pro‐α(1) (l) collagen mRNA was increased by TGF‐β1 (1 ng/ml) and BMP 4 (50 ng/ml). The addition of either TGF‐β1 or BMP 4 together with RA resulted in a further increase in pro‐α1(l) collagen mRNA levels. Both RA and TGF‐β1, but not BMP 4, increased the transcriptional rate of the pro‐α 1(l) collagen gene. TGF‐β1 reduced the constitutive as well as RA‐induced expression of osteopontin (OP) mRNA while BMP 4 reduced only the constitutive expression of OP mRNA. RA increased the transcriptional rate of the OP gene. Since the responses of UMR 201 cells to these structurally related factors were not identical, the results lend support to the concept that the coordinated expression of members of the TGF‐β1 superfamily may be necessary to control the progression of specific cell types through their differentiation pathways. © 1993 Wiley‐Liss, Inc.
Secretion of α‐amylase in human parotid gland epithelial cell culture
Authors: Chopra Dharam P. , Xue‐Hu Iris C.
Published: February 2005

The secretions of the salivary gland system are essential for the maintenance of oral health. The nature of cell‐specific secretions of the various glands and their regulation is not completely understood. The objective of this study was to establish epithelial cell cultures from the human parotid gland that exhibit the tissue‐specific function of α‐amylase secretion. A specimen of normal human parotid gland was obtained at surgery and used to obtain primary cultures by the explant/outgrowth procedure. The cultures were maintained in keratinocyte basal medium, supplemented with insulin (5 μg/ml), EGF (10 ng/ml), hydrocortisone (0.5 μg/ml), bovine pituitary extract (25 μg/ml), and antibiotics. The cultures were passaged using 0.125% trypsin to dissociate the cells. Phase contrast and ultrastructural observations showed that the cells were polygonal and exhibited desmosomes. Their cytoplasm contained tonofilament bundles and abundant rough endoplasmic reticulum and Golgi complexes. Immunofluorescence studies showed that all cells were positive for cytokeratins. Immunoblot analysis revealed keratins with molecular weights of 58, 56, 52, 50, 48, 46, and 40 KD, which are characteristic of secretory epithelia. The cells have been passaged 35 times so far, undergoing a cumulative 120–140 population doublings. The serially passaged epithelial cell cultures produced and secreted α‐amylase, a major component of parotid gland acinar cell secretion. The β‐adrenergic agonist, isoproterenol (ISP), stimulated α‐amylase secretion, which was accompanied by increased intracellular concentrations of cAMP. ISP‐induced stimulation of amylase and cAMP was blocked by the β‐adrenergic antagonist, propranolol. Further, dibutryl cAMP also enhanced the secretion of amylase. Thus we have established a long‐term epithelial cell culture model of human parotid gland epithelial cells that exhibits differentiated function and retains the intact β‐adrenergic receptor system. © 1993 Wiley‐Liss, Inc.
Serum‐stimulated cell cycle progression and stress protein synthesis in C3H10T1/2 fibroblasts treated with sodium arsenite
Authors: Van Wijk Roeland , Welters Marij , Souren Jan E. M. , Ovelgonne Hans , Wiegant Fred A. C.
Published: February 2005

In this work, we demonstrate that a nonlethal dose of arsenite administered to quiescent C3H10T1/2 fibroblasts can enhance the mitogenic effect of suboptimal concentrations of serum. The mitogenic effect was dependent on the serum concentration and on the time interval between the administration of arsenite and that of serum. This suggests that mitogen sensitivity changes in time after arsenite treatment. It is shown that the concentrations of arsenite that enhance the mitogenic effect of serum also increase the mRNA levels of c‐fos, HSP68, and HSP84 and induce the specific synthesis of Heat Shock Proteins (HSPs). The physiological significance of this phenomenon is most likely to counteract the long‐term toxic effect of arsenite by early induction of compensation for cell loss. © 1993 Wiley‐Liss, Inc.
CSF‐1 control of C‐FMS expression in normal human bone marrow progenitors
Authors: Panterne Béatrice , Zhou Yi‐Quing , Hatzfeld Jacques , Li Ma‐Lin , Levesque Jean‐Pierre , Clark Steven C. , Hatzfeld Antoinette
Published: February 2005

We have previously shown (Zhou et al: Blood, 72:1870, 1988) that IL3, added with low concentrations of CSF‐1 (1 ng/ml) to normal human CD34+ enriched cells, promoted the development of various types of colonies including those containing immature monocytes. However, when high concentrations of CSF‐1 (20 ng/ml) were added alone or together with IL3, smaller colonies with mature macrophages were found. Here we show by in situ hybridization that IL3 allows the development, from CD34+ cells, of a subpopulation of immature progenitors which express the CSF‐1 receptor (c‐fms) mRNA. The expression of c‐FMS protein was also substantiated by immunocytochemical studies using anti‐c‐fms antibody. The percentage of c‐fms positive cells peaked at day 7 and began to decrease thereafter. When anti‐CSF‐1 antibodies were included in the culture, the decrease in c‐fms mRNA after day 7 was abrogated. This indicated that endogenous CSF‐1 was produced as CD34+ cells developed into monocytes or progenitors of monocytes and that CSF‐1 modulates c‐fms expression. We further demonstrated that when a high dose of CSF‐1 (20 ng/ml) was added at day 7 to IL3‐stimulated CD34+ cells, a rapid down‐regulation of c‐fms mRNA and protein was seen. No down‐regulation was observed with low concentration of CSF‐1 (1 ng/ml). The possibility that different concentrations of CSF‐1 could modulate the development of monocytic progenitors is discussed. © 1993 Wiley‐Liss, Inc.
IPG (inositolphosphate glycan) as a cellular signal for TGF‐β1 modulation of chondrocyte cell cycle
Authors: Vivien Denis , Petitfrère Emmanuelle , Martiny Laurent , Sartelet Hervè , Galéra Philippe , Haye Bernard , Pujol Jean‐Pierre
Published: February 2005

The knowledge of transforming growth factor (TGF)‐β receptors has greatly progressed in the recent years. TGF‐β receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF‐β to its signaling receptors. In addition, four other proteins that bind TGF‐β1 or TGF‐β2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF‐β receptors remain an enigma. TGF‐β family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF‐β effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF‐β1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF‐β1. Using [3H]‐inositol labeling and Triton X‐114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI‐PLC) or by exposure to TGF‐β, supporting that a PI‐anchored molecule gives rise to IPG by TGF‐β‐induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF‐β action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF‐β on RAC growth. © 1993 Wiley‐Liss, Inc.
Hydrogen peroxide‐induced oxidative stress to the mammalian heart‐muscle cell (cardiomyocyte): Nonperoxidative purine and pyrimidine nucleotide depletion
Authors: Janero David R. , Hreniuk David , Sharif Haamid M.
Published: February 2005

Hydrogen peroxide (H2O2) overload may contribute to cardiac ischemia‐reperfusion injury. We report utilization of a previously described cardiomyocyte model (J. Cell. Physiol., 149:347, 1991) to assess the effect of H2O2‐induced oxidative stress on heart‐muscle purine and pyrimidine nucleotides and high‐energy phosphates (ATP, phosphocreatine). Oxidative stress induced by bolus H2O2 elicited the loss of cardiomyocyte purine and pyrimidine nucleotides, leading to eventual de‐energization upon total ATP and phosphocreatine depletion. The rate and extent of ATP and phosphocreatine loss were dependent on the degree of oxidative stress within the range of 50 μM to 1.0 mM H2O2. At the highest H2O2 concentration, 5 min was sufficient to elicit appreciable cardiomyocyte highenergy phosphate loss, the extent of which could be limited by prompt elimination of H2O2 from the culture medium. Only H2O2 dismutation completely prevented ATP loss during H2O2‐induced oxidative stress, whereas various freeradical scavengers and metal chelators afforded no significant ATP preservation. Exogenously‐supplied catabolic substrates and glycolytic or tricarboxylic acidcycle intermediates did not ameliorate the observed ATP and phosphocreatine depletion, suggesting that cardiomyocyte de‐energization during H2O2‐induced oxidative stress reflected defects in substrate utilization/energy conservation. Compromise of cardiomyocyte nucleotide and phosphocreatine pools during H2O2‐induced oxidative stress was completely dissociated from membrane peroxidative damage and maintenance of cell integrity. Cardiomyocyte de‐energization in response to H2O2 overload may constitute a distinct nonperoxidative mode of injury by which cardiomyocyte energy balance could be chronically compromised in the post‐ischemic heart. © 1993 Wiley‐Liss, Inc.
1,25‐dihydroxy vitamin D3 and 12‐O‐tetradecanoyl phorbol‐13‐acetate synergistically induce monocytic cell differentiation: FOS and RB expression
Authors: Yen Andrew , Coles Mark , Varvayanis Susi
Published: February 2005

1,25‐dihydroxy vitamin D3 and 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA) interact synergistically to induce monocytic differentiation of U937 histiocytic lymphoma cells. Addition of TPA causes an otherwise ineffective dose of 1,25‐dihydroxy vitamin D3 to induce differentiation. The induced differentiation depends on the simultaneous (vs. sequential) presence of both agents. The kinetics of induced differentiation are consistent with a G1 specific cellular response to initiate the metabolic cascade culminating in cell differentiation. The induced differentiation occurs with down‐regulation of c‐fos protein and an accompanying up‐regulation of RB protein expression, consistent with a possible need for up‐regulated RB expression to maintain a given differentiated phenotype and suppress transcriptional activators that might typically be associated with proliferation. © 1993 Wiley‐Liss, Inc.
Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro
Authors: Schnaper H. William , Grant Derrick S. , Stetler‐Stevenson William G. , Fridman Rafael , D'Orazi Gabriella , Murphy Anne N. , Bird Robert E. , Hoythya Matti , Fuerst Thomas R. , French Deborah L. , Quigley James P. , Kleinman Hynda K.
Published: February 2005

It has been proposed that proteases are important in endothelial cell behavior. We examined the contribution of the gelatinase/type IV collagenase system in an in vitro model of endothelial differentiation. Human umbilical vein endothelial cells rapidly align and form networks of tubes when cultured on a basement membrane preparation, Matrigel. Zymograms of culture supernates demonstrate a 72‐kD and a 92‐kD gelatinase activity; the cells produce most of the 72‐kD gelatinase, whereas the 92‐kD activity is derived entirely from the Matrigel. Addition of antibodies against type IV gelatinase/collagenase decreases the area of the tube network. Both tissue inhibitors of metalloproteinases, TIMP‐1 and TIMP‐2, similarly decrease tube formation when added to cultures. Conversely, exogenous recombinant 72‐kD gelatinase increases tube‐forming activity. The effects of the anti‐gelatinase antibodies and the TIMPs are not additive. Inhibition by either antibodies or TIMPs is greatest when they are added at culture initiation, suggesting that the protease activity is important in the early steps of morphogenesis. However, culture of the cells on Matrigel does not increase early expression of mRNA for the 72‐kD gelatinase. Expression of message for the enzyme actually decreases during the course of the assay, while transcription of mRNA for TIMPs increases, further supporting the concept that collagenases facilitate an early event in tube formation. These data demonstrate that gelatinase/type IV collagenase activity is important in endothelial cell morphogenesis on Matrigel, and suggest a role for collagenases in formation of new capillaries in vivo. © 1993 Wiley‐Liss, Inc.
Accumulation of insulin‐like growth factor binding protein‐3 in conditioned medium of human fibroblasts increases with chronologic age of donor and senescence in vitro
Authors: Goldstein Samuel , Moerman Elena J. , Baxter Robert C.
Published: February 2005

We have found that insulin‐like growth factor binding protein‐3 (IGFBP‐3) accumulates to higher levels in medium conditioned by a strain of normal fibroblasts at late passage (LP) and a strain derived from subjects with Werner syndrome (WS) of premature aging, compared to medium conditioned by the same normal cells at early passage (EP) (Goldstein et al., Proc. Natl. Acad. Sci. USA, 88:9680–9684, 1991). To explore the generality of this phenomenon with respect to chronological age of donor (in vivo aging) and LP (in vitro senescence) we assayed IGFBP‐3 in medium conditioned by 18 normal fibroblast strains at EP and LP and two WS strains at the midpoint of their curtailed replicative lifespans and assessed IGFBP‐3 mRNA levels in cells by Northern analysis. The lowest accumulations of IGFBP‐3 were found in medium conditioned by fetal cells with progressively increasing amounts postnatally; direct correlations between IGFBP‐3 levels and donor age were seen in EP cells 3 days after subculture (during logarithmic growth) r = 0.80, P < 0.001, and 7 days after subculture (at confluence) r = 0.77, P < 0.001. With two exceptions, conditioned medium of cell strains accumulated more IGFBP‐3 at LP; IGFBP‐3 levels correlated with chronological age after 3 days, r = 0.50, P = 0.05, and after 7 days, r = 0.75, P < 0.001. IGFBP‐3 content of WS culture medium fell within the range of LP normal cells. Cumulative IGFBP‐3 levels were inversely proportional to the thymidine labeling index, a measure of proliferative vigor. With some exceptions IGFBP‐3 mRNA levels were commensurate with the amount of IGFBP‐3 accumulated in the medium, suggesting that distal translational and posttranslational mechanisms also regulate IGFBP‐3 production in some strains. The trend toward augmented IGFBP‐3 output of fibroblasts as a direct function of chronological age and in vitro senescence and as an inverse function of proliferative vigor is consistent with the known inhibitory effect of excess IGFBP‐3 on IGF‐mediated DNA synthesis and the reduced regenerative potential that is evident during biological aging in vivo. © 1993 Wiley‐Liss, Inc.
Effect of pH and ATP on the equilibrium density of lysosomes
Authors: Mayorga Luis S. , De Veca Mercedes G. , Colombo Maria I. , Bertini Francisco
Published: February 2005

Lysosomes are membrane bound structures that accumulate and hydrolyze material internalized by the endocytic pathway. A very conspicuous property of this subcellular compartment is its relatively high equilibrium density. The actual mechanism that regulates lysosomal density is poorly understood. In an attempt to gain knowledge on the factors that regulate lysosomal density we have assessed the equilibrium density of lysosomal markers after in vitro incubation of a lysosome‐enriched subcellular fraction. Incubation at pH 6 for 10 min at 37°C causes a density shift of several lysosomal markers to light density regions of Percoll gradients. Addition of ATP was able to prevent the acid‐induced density shift. Pretreatment of the vesicles with N‐ethylmaleimide (NEM) or trypsin inhibited the effect of ATP. Working in intact cells, ATP depletion, a condition that causes cytoplasmic acidification, also decreases lysosomal density. The results indicate that at low pH lysosomal density is preserved by an active process that requires ATP and membrane associated proteins. © 1993 Wiley‐Liss, Inc.
Microtubules are involved in transport of macromolecules by vesicles in cultured bovine aortic endothelial cells
Authors: Liu Shu Ming , Magnusson Karl‐Eric , Sundqvist Tommy
Published: February 2005

The macromolecular transport in bovine aortic endothelial monolayers, cultured in vitro, was studied by fluorescence microscopy, confocal laser scanning microscopy, and transmission electron microscopy. A fluid‐phase endocytic tracer, fluorescein isothiocyanate dextran 70 kD (FITC‐dextran 70), was found to be transported into and out of endothelial cells via vesicles arranged as chains stretching between the luminal surface and the cell interior and also from cell interior to the abluminal surface. The endocytic activity was reduced by colchicine, which disrupts microtubules, and increased during treatment with cytochalasin B, which blocks microfilament polymerization. These findings indicate that microtubules are required for fluid‐phase endocytosis and that microfilaments hinder this process. © 1993 Wiley‐Liss, Inc.
Modulation of the immunophenotype of ovarian cancer cells by N,N‐dimethylformamide and transforming growth factor‐β1
Authors: Grunt Thomas W. , Oeller Helga , Somay Canatay , Dittrich Evelyn , Fazeny Barbara , Mannhalter Christine , Dittrich Christian
Published: February 2005

Exposure of HOC‐7 ovarian adenocarcinoma cells to regulators of cell differentiation caused inducer‐dependent alterations of the antigenic pattern of the cells. Immunocytochemistry revealed that N,N‐dimethylformamide (DMF) elevated the membrane staining for epidermal growth factor (EGF)‐receptor and for desmoplakins I and II. DMF also stimulated cytoplasmic and surface labeling for CA 125 and the deposition of fibronectin into the extracellular matrix. Stimulation of fibronectin was also seen after addition of transforming growth factor (TGF)‐β1. These responses were quantified using a fixed‐cell, enzyme‐linked immunosorbent assay (ELISA) and revealed that DMF dose‐dependently induced expression of EGF‐receptor, CA 125, fibronectin, and desmoplakins I and II. TGF‐β1 stimulated fibronectin and desmoplakins I and II only. Production of EGF and TGF‐α was not affected by these inducers. Immunocytochemistry, ELISA and Western blotting showed that both inducers caused down‐regulation of myc oncoproteins. DMF was more effective in changing the immunophenotype of HOC‐7 cells than TGF‐β1. Desmoplakins I and II demonstrated elevated epithelial differentiation, whereas fibronectin indicated stimulation of extracellular matrix formation. Elevated EGF‐receptor could not compensate for the growth inhibition induced by DMF. The expression of myc oncoproteins was inversely related to cell proliferation. CA 125, however, seems to be unrelated to cell growth. © 1993 Wiley‐Liss, Inc.
Intracellular calcium oscillations induced in a T‐cell line by a weak 50 Hz magnetic field
Authors: Lindströum Ewa , Lindströum Per , Berglund André , Mild Kiell Hansson , Lundgren Erik
Published: February 2005

Applied weak magnetic fields have been shown to affect cellular activity on several levels, but the mechanisms involved remain elusive. We have decided to study an early signal transduction event in the human T cell line Jurkat; oscillations of free [Ca2+]i, of the type seen by crosslinking the CD3 complex. Cells were exposed to a 50 Hz, 0.1 mT, sinusoidal magnetic field while intracellular free calcium was measured in individual cells, using fura‐2 as a probe. An acute response was observed with oscillatory increases in [Ca2+]i, which subsided when the field was turned off. The effect of the magnetic field on [Ca2+]i was comparable to that achieved by an anti‐CD3 monoclonal antibody. © 1993 Wiley‐Liss, Inc.