bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation between the proliferative response to granulocyte colony‐stimulating factor and the positivity of transferrin receptor in acute myeloblastic leukemia cells
Authors: Motoji Toshiko , Mineshima Michio , Watanabe Masahiko , Takanashi Minoko , Masuda Michihiko , Oshimi Kazuo , Mizoguchi Hideaki
Published: February 2005

The use of granulocyte colony‐stimulating factor (G‐CSF) after chemotherapy for acute myeloblastic leukemia (AML) has been reported. However, there is a drawback in that G‐CSF may stimulate the proliferation of AML progenitors. To determine the parameter(s) indicative of responsiveness of AML blasts to G‐CSF, various surface phenotypes of blasts were examined in relation to the blast colony formation stimulated by G‐CSF in 39 AML patients. A correlation was found only with transferrin receptor positivity among the various phenotypes studied. The population mean of percentages of transferrin receptor‐positive blasts in the group responding to G‐CSF in vitro was significantly higher than that of blasts in the group not responding to G‐CSF. A further correlation was found between transferrin receptor positivity and the number of G‐CSF receptors on the blasts; that is, blasts expressing more G‐CSF receptors have greater transferrin receptor positivity. In our previous study, we observed that blasts with a large number of G‐CSF receptors produce more colonies in response to G‐CSF. These results indicated that blasts expressing more transferrin eceptors have a large number of G‐CSF receptors and may show more active proliferation in response to G‐CSF. Therefore, the proliferative response of blasts to G‐CSF can be predicted by examining transferrin receptor positivity. The clinical use of G‐CSF in AML patients may be recommended when the patient's blasts have a low level of transferrin receptor expression. The measurement of transferrin receptors on blasts, instead of the rather complicated G‐CSF receptor determination, would be a useful indicator for the safer application of G‐CSF in AML patients.
Erythroid lineage‐specific activity in conditioned medium derived from cloned human marrow stromal cells (CFU‐RF)
Authors: Aye M. T. , Izaguirre C. A.
Published: February 2005

Using long‐term culture techniques, it has been shown that stromal cells in the marrow microenvironment are essential for the continued production and self‐renewal of hematopoietic stem cells. We previously reported the development of a methylcellulose colony assay for a population of marrow stromal progenitors called CFU‐RF. In this paper, a method is described for subculturing cells from individual CFU‐RF‐derived colonies to allow conditioned medium production (StCM). StCM, prepared in this way, was found to possess an erythroid lineage‐specific activity that stimulated the formation of macroscopic erythroid colonies in cultures containing erythropoietin (epo). Using dose‐response curves, the KG1 colony assay, and antibody neutralization, it was shown that the activity could not be attributed to interleukin 3 (IL3) or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). However, it was further shown that a monolayer of stromal cells, which had earlier been producing the erythroid activity, could be stimulated by IL1 to produce granulocytic colony‐stimulating activity, but only as long as IL1 was present in the culture medium. These findings indicate a mechanism whereby the same stromal population could be modulated to promote growth and differentiation of different hematopoietic lineages.
Simultaneous occurrence of myelomonocytic leukemia and multiple myeloma: Involvement of common leukemic progenitors and their developmental abnormality of “lineage infidelity”
Authors: Akashi Koichi , Harada Mine , Shibuya Tsunefumi , Fukagawa Koji , Kimura Nobuhiro , Sagawa Kimitaka , Yoshikai Yasunobu , Teshima Takanori , Kikuchi Masahiro , Niho Yoshiyuki
Published: February 2005

We investigated the origin of leukemic progenitors in a case of the simultaneous occurrence of myelomonocytic leukemia and multiple myeloma (IgG‐K). At presentation, myeloperoxidase and nonspecific esterase‐positive myelomono‐cytic cells had proliferated up to 12.2 x 109/liter in the peripheral blood. Bone marrow cell differentials revealed the coexistence of myelomonocytic cells (30%) and atypical plasmacytoid cells (26%). Myelomonocytic cells in peripheral blood expressed both myeloid antigens (CD11b, CD13, CD14, CD15, CD33) and T/B‐lymphoid antigens (CD2, CD4, CD5, CD7, CD10, PCA‐1). Bone marrow mononuclear cells (BMMC) could be divided into PCA‐1 strongly positive and PCA‐1 weakly positive populations, which were considered to represent myeloma cells and myelomonocytic cells, respectively; the former were CD2‐positive (CD2+), CD14−, and CD15−, whereas the latter were CD2+, CD14+, and CD15+. Immunohistochemical analysis revealed that, in addition to plasmacy‐toid cells, a minority of myelomonocytic cells showed a positive reaction for IgG staining, and production of IgG was observed in the culture supernatant of CD14+ myelomonocytic cells in peripheral blood. Southern blot analysis revealed the presence of two identical rearrangement bands of immunoglobulin heavy chain gene in both BMMC containing myeloma cells and myelomonocytic cells and CD14+ myelomonocytic cells in peripheral blood. In a long‐term methylcellulose assay, peripheral blood mononuclear cells produced large compact colonies consisting of macrophages and IgG+ plasmacytoid cells (Mϕ/P colonies), while BMMC produced a different type of colonies consisting of CD14+ myelomono‐blasts, macrophages, and IgG+ plasma cells (Mb/Mϕ/P colonies) in addition to Mϕ/P colonies. Recloning experiments showed that primary Mb/Mϕ/P colonies gave rise to both secondary Mϕ/P and Mb/Mϕ/P colonies. These observations strongly suggest that common leukemic progenitors provide both myeloma and myelomonocytic leukemia cells, and the mechanism of “lineage infidelity” is probably involved in the development of their “bilineal” differentiation.
Modulation of drug resistance in homoharringtonine‐resistant C‐1300 neuroblastoma cells with cyclosporine A and dipyridamole
Authors: Tebbi Cameron K. , Chervinsky David , Baker Raymond M.
Published: February 2005

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C‐1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross‐resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P‐glycoprotein, parallel to the graded development of resistance with a 29‐fold elevation in the highest resistant cells. High‐performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. Cyclos‐porine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.
Dietary fish oils modify adipocyte structure and function
Authors: Parrish Christopher C. , Pathy Diane A. , Parkes Joel G. , Angel Aubie
Published: February 2005

Dietary fish oils, enriched with w‐3 fatty acids (e.g., MaxEPA fish oil), inhibit lipogenesis and have a marked hypotriglyceridemic effect in man and experimental animals. Dietary w‐3 fatty acids also reduce adipose tissue trophic growth in rats. To understand the metabolic basis for this, we measured the effect of fish oil feeding upon rat plasma triglyceride concentration, fat pad mass, fat cell size, fat cell lipolysis, as well as lipoprotein binding to adipocyte plasma membranes. In adolescent (250 g) male Wistar rats fed 20% (w/w) fish oil supplemented diets for 3 weeks, plasma triglyceride levels and epididymal and perirenal fat pad mass were significantly (P<0.005) reduced compared to pair‐fed controls given 20% lard diets. These differences in fat pad mass between the diets were greater than differences in whole animal mass or in the mass of livers, testes, kidneys, spleens, or hearts. Isoproternol‐stimulated lipolvsis was significantly (P < 0.005) higher in fish oil fed rats than in pair‐fed controls. In young (100 g) rats plasma triglyceride levels were 10 times lower in the fish oil fed group after 5 weeks as compared to the lard‐fed controls. This was accompanied by a reduction in epididymal and perirenal fat pad mass as well as a 2‐3‐fold decrease in adipocyte volumes; there was no significant difference between the two groups in fat cell number in each region. Plasma membranes of epididymal adipocytes from fish oil fed rats bound significantly (P < 0.001) less HDL1 than the lard‐fed rats, possibly as a result of a reduction in fat cell size and/or alteration of plasma membrane structure. Thus in both young and old rats, the reduction in plasma triglyceride concentration in conjunction with increased hormone‐stimulated lipolysis may explain in part the selective reduction in adipose tissue trophic growth accompanying fish oil consumption.
Transforming growth factor beta inhibits plasminogen activator (PA) activity and stimulates production of urokinase‐type PA, PA inhibitor‐1 mRNA, and protein in rat osteoblast‐like cells
Authors: Allan Elizabeth H. , Zeheb Ron , Gelehrter Thomas D. , Heaton Joanne H. , Fukumoto Seiji , Yee John A. , Martin T. John
Published: February 2005

Transforming growth factor(β)(TGFβ) treatment of rat osteoblast‐rich calvarial cells or of the clonal osteogenic sarcoma cells, UMR 106‐01, resulted in dosedependent inhibition of plasminogen activator (PA) activity, and increased production of 3.2 kb mRNA and protein for PA inhibitor‐1 (PAI‐1). Although tissue‐type PA(tPA) protein was not measured, TGF(β)did not influence production of mRNA for tPA. Production of 2.3 kb mRNA for urokinase‐type PA (uPA) was also increased by TGF(β)in a dose‐dependent manner. The effects of TGFβ on synthesis of mRNA for PAI‐1 and uPA were maintained when protein synthesis was inhibited, and were abolished by inhibition of RNA synthesis. Although uPA had not been detected previously as a product of rat osteoblasts, treatment of lysates of osteoblast‐like cells with plasmin yielded a band of PA activity on reverse fibrin autography, corresponding to a low Mr form of uPA. Untreated conditioned media from normal osteoblasts or UMR 106‐01 cells contained no significant TGFβ activity, but activity could be detected in acidified medium. Treatment of conditioned media with plasmin resulted in activation of approximately 50% of the TGFβ detectable in acidified media. The results identify several effects of TGFβ on the PA‐PA inhibitor system in osteoblasts. Net regulation of tPA activity through the stimulatory actions of several calciotropic hormones and the promotion of PAI‐1 formation by TGFβ could determine the amount of osteoblast‐derived TGFβ activated locally in bone. Stimulation of osteoblast production of mRNA for uPA could reflect effects on the synthesis of sc‐uPA, a precursor for the active form of the enzyme.
Human keratinocyte growth‐promoting activity on the surface of fibroblasts
Authors: Yaeger Peter C. , Stiles Charles D. , Rollins Barrett J.
Published: February 2005

To proliferate in serum‐containing medium, normal human keratinocytes must be co‐cultured with fibroblast feeder cells. Conditioned medium from feeder cell cultures cannot substitute for the cells themselves. We tested the hypothesis that fibroblasts display a keratinocyte growth‐promoting activity on their outer cell surface. The results of our investigation showed that (1) glutaraldehyde‐fixed fibroblast feeder cells promote keratinocyte growth, (2) the growth‐promoting effect requires contact between fixed fibroblasts and keratinocytes, and (3) feeder activity is highly enriched within the plasma membrane fraction of fibroblasts. We conclude that at least part of the fibroblast “feeder” activity involves a keratinocyte growth‐promoting factor which is bound to the outer surface of fibroblast plasma membranes.
Inhibition of phorbol ester‐induced monocytic differentiation by dexamethasone is associated with down‐regulation of c‐fos and c‐jun (AP‐1)
Authors: Hass Ralf , Brach Marion , Kharbanda Surender , Giese Günter , Traub Peter , Kufe Donald
Published: February 2005

Previous studies have shown that treatment of human myeloid leukemia cells with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) is associated with induction of monocytic differentiation and expression of the c‐jun and c‐fos early response genes. The present work demonstrates that the glucocorticoid dexamethasone inhibits TPA‐induced increases in c‐jun and c‐fos mRNA levels in U‐937 leukemia cells. These findings were associated with a block in appearance of the monocytic phenotype, including inhibition of TPA‐induced increases in lamin A, lamin C, and vimentin transcripts. Other studies have demonstrated that TPA‐induced monocytic differentiation and expression of the c‐jun and c‐fos genes in myeloid leukemia cells are regulated by protein kinase C (PKC). The finding that dexamethasone has no effect on TPA‐induced activation of PKC suggests that this glucocorticoid inhibits signals downstream or parallel to this enzyme. Nuclear run‐on assays demonstrate that: (1) induction of c‐jun and c‐fos expression by TPA is regulated by transcriptional mechanisms, (2) TPA‐induced expression of c‐jun and c‐fos does not require protein synthesis, and (3) TPA‐induced expression of both genes is inhibited at the transcriptional level by dexamethasone. To further define the effects of dexamethasone at the molecular level, we prepared a series of deleted c‐jun promoter fragments linked to the chloramphenicol acetyltransferase (CAT) gene. Increases in CAT activity during transient expression of these constructs in TPA‐treated U‐937 cells could be assigned to the region (−97 to −20) of the promoter that contains the AP‐1 binding site. This induction of CAT activity was sensitive to dexamethasone. These findings suggest that dexamethasone down‐regulates TPA‐induced transcription of the c‐jun gene during monocytic differentiation by inhibiting activation of the AP‐1 site.
Enhanced sulfated‐proteoglycan core protein synthesis by incubation of rabbit chondrocytes with recombinant transforming growth factor‐β1
Authors: Malemud Charles J. , Killeen Wendy , Hering Thomas M. , Purchio Anthony F.
Published: February 2005

Rabbit articular chondrocytes were incubated with recombinant transforming‐growth‐factor‐β 1, (rhTGF‐β1) and its effect on newly synthesized proteoglycan measured. rhTGF‐β1 stimulated proteoglycan synthesis at a concentration as low as 5 ng/ml without further increase in radiosulfate incorporation up to 50 ng/ml. The quantitative increase in radiosulfate incorporation in rh‐TGF‐β1‐treated chondrocytes was greater in the cell‐associated culture compartment than in the medium compartment. rhTGF‐β1 promoted an increased proteoglycan retention in the cell‐associated compartment as evidenced by an increase in the t 1/2 retention from 8 h to 11 h. Specific enhanced synthesis of [35S]‐methionine‐labeled core proteins was seen in rh‐TGF‐β1‐treated chondrocytes. rh‐TGF‐β1 increased the synthesis of the 2 core proteins derived from hydrodynamically large proteoglycans. They possessed apparent molecular weights of > 480 kD and 390 kD after 3‐5% acrylamide gel electrophoresis. A compartmental analysis revealed that the cell‐associated culture compartment contained only the larger of the 2 core proteins derived from large proteoglycans. Two other core proteins with apparent molecular weights 52 kD and 46 kD were also stimulated by rhTGF‐β1. These results indicated that TGF‐β probably plays a significant role in stimulating proteoglycan core protein synthesis in articular chondrocytes and therefore may be an important growth factor in the restoration of cartilage extracellular matrix after injury.
Il‐1β and prostaglandins regulate integrin mRNA expression
Authors: Milam Stephen B. , Magnuson Victoria L. , Steffensen Bjorn , Chen Dali , Klebe Robert J.
Published: February 2005

The purpose of this study was to examine the effects of IL‐1β on integrin expression in MG‐63 human osteosarcoma cells. Human recombinant IL‐1β (rIL‐1β) produced significant increases in both α2‐ and α5‐subunit mRNA levels, as well as a smaller increase in αv‐subunit mRNA. In contrast, IL‐1β decreased α4‐subunit mRNA levels by approximately 30% relative to untreated controls. These findings suggest that human IL‐1β differentially regulates expression of integrins. When cultures were treated with both IL‐1β and the cyclooxygenase inhibitor, indomethacin, the expression of α2‐, α5‐, and αv‐subunit mRNA levels were dramatically increased relative to untreated controls; co‐treatment with 0.5 mM prostaglandin E2 (PGE2) partially reversed this effect. Indomethacin alone did not affect integrin mRNA levels.

Treatment with IL‐1β or IL‐1β + indomethacin also induced significant changes in MG‐63 morphology (i.e., increased cell elongation) and increased the ability of cells to contract collagen gels. PGE2 reversed the above effects on cell morphology and gel contraction.

These findings indicate that (a) IL‐1β differentially regulates the expression of integrins and (b) that PGE2, which is induced by IL‐1β, may provide a negative feedback loop which counteracts the stimulatory effect of IL‐1β on integrin gene expression. It is suggested that products of inflammation may affect cell behavior by differentially regulating the expression of various integrins.
Differential binding, biological and biochemical actions of recombinant PDGF AA, AB, and BB molecules on connective tissue cells
Authors: Grotendorst Gary R. , Igarashi Atsuyuki , Larson Ronald , Soma Yoshinao , Charette Marc
Published: February 2005

We have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF‐AB and PDGF‐BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF‐AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF‐AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF‐AA were absent. PDGF‐AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti‐Type α and β PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF α and β receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of β receptors. These results indicate that cells possessing high levels of PDGF β receptors only are capable of responding equally well to either PDGF AB or BB.
Sphingosine reverses growth inhibition caused by activation of protein kinase c in vascular smooth muscle cells
Authors: Weiss Robert H. , Huang Chou‐Long , Ives Harlan E.
Published: February 2005

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1, 2‐dioctanoyl‐snglycerol (DiC8), when added 2 h after α‐thrombin, reverses by 95% the induction of DNA synthesis in VSM cells by α‐thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N‐acetylsphingosine and C11‐sphingosine were used to investigate this phenomenon further. Neither phorbol 12‐myristate 13‐acetate (PMA;200 ng/ml) nor sphingosine (up to 10 μM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by α‐thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose‐dependent manner with complete reversal at 10 μM. Neither N‐acetylsphingosine nor C11‐sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III‐S phosphorylation. Protein kinase C activity was inhibited 50% by 300 μM sphingosine, but 15% by similar concentrations of N‐acetylsphingosine and C11‐sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at > 5 μM), but not N‐acetylsphingosine or C11‐sphingosine, blocked [3H]phorbol dibutyrate binding in a dose‐ and time‐dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.
Regulation of glucose uptake by stressed cells
Authors: Pasternak C. A. , Aiyathurai J. E. J. , Makinde V. , Davies A. , Baldwin S. A. , Konieczko E. M. , Widnell C. C.
Published: February 2005

Lactate production by BHK cells is stimulated by arsenite, azide, or by infection with Semliki Forest virus (SFV). In the case of arsenite or SFV infection, the increase correlates approximately with the increase in glucose transport as measured by uptake of [3H] deoxy glucose (dGlc); in the case of azide, the increase in lactate production exceeds that of glucose transport. Hence glucose utilization by BHK cells and its stimulation by anaerobic and other types of cellular stress is controlled at least in part at the level of glucose transport. The glucose uptake by BHK cells is also stimulated by serum and by glucose deprivation. In these circumstances, as with arsenite, stimulation is reversible, with t1/2 of 1–2 hours; stimulation is compatible with a translocation of the glucose transporter protein between an intracellular site and the plasma membrane (shown here for serum and previously for arsenite). The surface binding and rate of internalization of [125I]‐labelled tranferrin and [125l] α2‐macroglobulin was studied to determine whether changes in glucose transport are accompanied by changes in the surface concentration or rate of internalization of membrane proteins. The findings indicate that changes in glucose transport do not reflect a consistent and general redistribution of membrane receptors. Taken together, the results are compatible with the proposal that BHK cells exposed to stimuli like insulin or serum, or to stresses like arsenite, azide, SFV infection or deprivation of glucose, respond in the same manner: namely, by an increased capacity to transport glucose brought about by reversible and specific translocation of the transporter protein from an (inactive) intracellular site to the plasma membrane.
Impaired secretion and increased insolubilization of IgE‐receptor complexes in mycophenolic acid‐treated (guanine nucleotide‐depleted) RBL‐2H3 mast cells
Authors: Wilson Bridget Smith , Seagrave Jeanclare , Oliver Janet M.
Published: February 2005

In RBL‐2H3 rat leukemic mast cells, cross‐linking anti‐DNP IgE‐receptor complexes with multivalent antigen (DNP‐BSA) activates a signal transduction pathway leading to Ca2+ influx and secretion. Cross‐linking IgE‐receptor complexes also stimulates a pathway that inactivates (desensitizes) receptors;this pathway becomes important at high concentrations of cross‐linking antigen. Recent evidence that antigen‐induced secretion is impaired by mycophenolic acid (MPA), an inhibitor of guanine nucleotide synthesis de novo, has implicated a GTP‐binding protein (G protein) in the signaling pathway. Other recent studies have indicated that the conversion of cross‐linked receptors to a detergent‐insoluble (cytoskeleton‐associated) form at high antigen concentrations is correlated with the loss of signaling activity. Here we show that secretion elicited by an optimal concentration of antigen (0.05 μg/ml DNP‐BSA) is only inhibited by about 25% in guanine nucleotide‐depleted cells, whereas secretion elicited by 5 μg/ml DNP‐BSA, a concentration in the range that causes the high‐dose inhibition of secretion, is inhibited by more than 60%. We also show that IgE‐receptor complexes are insolubilized in response to 5 but not 0.05 μg/ml DNP‐BSA in both control and guanine nucleotide‐depleted cells. Importantly, the extent of insol‐ubilization elicited by 5 μg/ml DNP‐BSA is increased by more than 60% in the guanine nucleotide‐depleted samples. These results raise the possibility that guanine nucleotide depletion reduces the secretory response to high antigen concentrations in two ways: by inhibiting the G protein‐coupled signaling pathway and by increasing the availability of receptors to the pathway leading to receptor insolubilization and inactivation.
Differential protective effects of O‐phenanthroline and catalase on H2O2‐induced DNA damage and inhibition of protein synthesis in endothelial cells
Authors: Jornot Lan , Petersen Hilke , Junod Alain F.
Published: February 2005

The respective roles of H2O2 and ·OH radicals was assessed from the protective effects of catalase and the iron chelator o‐phenanthroline on (1) the inhibition of protein synthesis, and (2) DNA damage and the related events (activation of the DNA repairing enzyme poly(ADP) ribose polymerase with the associated depletion of NAD and ATP stores) in cultured endothelial cells exposed to the enzyme reaction hypoxanthine‐xanthine oxidase (HX‐XO) or pure H2O2. Catalase added in the extracellular phase completely prevented all of these oxidant‐induced changes. O‐phenanthroline afforded a complete protective effect against DNA strand breakage and the associated activation of the enzyme poly(ADP) ribose polymerase. By contrast, iron chelation was only partially effective in maintaining the cellular NAD and ATP contents, as well as the protein synthetic activity. In addition, the ATP depletion following oxidant injury was much more profound than NAD depletion. These results indicate that: (1) ·OH radical was most likely the ultimate O2 species responsible for DNA damage and activation of poly‐(ADP) ribose polymerase; (2) both H2O2 and ·OH radicals were involved in the other cytotoxic effects (inhibition of protein synthesis and reduction of NAD and ATP stores); and (3) NAD and ATP depletion did not result solely from activation of poly(ADP) ribose polymerase, but other mechanisms are likely to be involved. These observations are also compatible with the existence of a compartmentalized intracellular iron pool.
Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: Further evidence for a “calcium receptor”
Authors: Zaidi Mone , Kerby Julie , Huang Christopher L.‐H. , Alam A. S. M. Towhidul , Rathod Hersha , Chambers Timothy J. , Moonga Baljit S.
Published: February 2005

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation‐sensitive “calcium receptor,” as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 μM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 μM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]e). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane “calcium receptor” that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.
Depolarization of the membrane potential decreases the ATP‐induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores in rat thyroid FRTL‐5 cells
Authors: Törnquist Kid
Published: February 2005

The aim of the present study was to investigate the effect of membrane depolarization on ATP‐induced changes in intracellular Ca2+ ([Ca2+],) and the refilling of intracellular Ca2+ stores in thyroid follicular FRTL‐5 cells. Depolarizing the cells with 50 mM K+, an amount sufficient to almost totally depolarize the cells as determined by bisoxonol, significantly reduced the ATP‐induced uptake of 45 Ca2+. This effect was not dependent on an enhanced efflux of Ca2+, as no difference in the ATP‐induced efflux of45Ca2+ was obtained between control cells and depolarized cells. The ATP‐induced transient increase in [Ca2+]Į in Fura‐2 loaded cells was not altered by depolarization, whereas the ATP‐induced plateau in [Ca2+]Į was decreased compared with control cells. Furthermore, in cells stimulated with ATP in a Ca2+‐free buffer, readdition of Ca2+ after the termination of the ATP response induced a decreased response in [Ca2+]Į in depolarized cells. Refilling of intracellular Ca2+ stores was investigated by tirst stimulating the cells with noradrenalin (NA). The effect of NA was then terminated with prazosin, and the cells restimulated with ATP. In cells depolarized with high K+ the response to ATP was decreased compared with that seen in control cells. The results thus suggest that both the ATP‐induced influx of extracellular Ca2+ and the refilling of intracellular Ca2+ stores is decreased in depolarized FRTL‐5 cells.
Decreased transforming growth factor‐(β) response and binding in insulin‐independent teratoma‐derived cell lines with increased tumorigenic properties
Authors: Serrero Ginette , Zhou Jian , Mills Dianne , LePak Nancy
Published: February 2005

The mouse C3H teratoma‐derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin‐independent cell line celled 1246‐3A was isolated. It was found that, in contrast to 1246 cells, 1246‐3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 106 cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor (α)‐ and (β)‐like polypeptides which stimulate their proliferation. Highly tumorigenic insulin‐independent cell lines able to give rise to tumor when injected at a density of 104 cells/mouse were isolated by using. an in vitro‐in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF‐(β)1, The binding of TGF‐(β)1, to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF‐(β)1, to the cells. However, the decreased number of cell surface TGF‐(β)1, binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF‐(β) protein by the tumorigenic cells, as all of TGF‐(β) produced by the cells was in a latent form. Affinity cross‐linking experiments indicated that the 1246 cell line displayed several TGF‐(β) cross‐linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross‐linked bands, particularly of the 92 and 70 kDa species.
The effects of chronic exposure to supraphysiological concentrations of 3,5,3′ triiodo‐L‐thyronine (T3) on cultured GC cells
Authors: Reynolds Anne Marie , Surks Martin I. , Shapiro Lawrence E.
Published: February 2005

We have characterized the effects of supraphysiological concentrations of T3 on GC cells, a cultured cell line in which physiologic concentrations of T3 regulate cell growth, protein content, and growth hormone (GH) production. GC cells were exposed to 3 times (1.0 nM) and 80 times (25.0 nM) the physiologic concentration of T3 (0.3 nM) for either 4 d or for greater than 3 months. Both short and prolonged exposure to supranormal T3 concentrations supported maximal cell growth rate and induced significant increases in total protein (P < 0.025) and GH production (P < 0.01) per celi when compared to measurements in control GC cells. In addition, exposure to 1.0 nM and 25.0 nM T3 for greater than 3 months enhanced the toxicity of heat shock in a manner similar to previously described effects on GC cells due to T3 exposure of shorter duration. Thus, initial responses to raised T3 concentrations in cultured GC cells persisted without alteration when hormone exposure was prolonged for greater than 3 months.
Effects of growth factors, hormones, bacterial lipopolysaccharides, and lipotechoic acids on the clonal growth of normal ureteral epithelial cells in serum‐free culture
Authors: Wille John J. , Park Jong , Elgavish Ada
Published: February 2005

In vitro tissue culture techniques were employed to study the effects of bacterial endotoxins on the growth of normal epithelial cells from the human ureter (NHU). Primary cultures of NHU cells were initiated from explant outgrowth cultures of human ureteral tissue and cultured on collagen gel in F‐12* medium containing 1% fetal calf serum (FCS). Optimal clonal growth of secondary cultures of NHU cells seeded at relatively low seeding cell densities, directly on plastic dishes, was achieved in F‐12* medium containing bovine pituitary extract (0.5% BPE) and 0.05% BSA. Results indicated that insulin in the F‐12* medium could be replaced by three orders of magnitude less IGF‐1. Further clonal growth experiments demonstrated that PGE1 is growth stimulatory and can replace BPE as a growth factor requirement. This finding was in agreement with the fact that BPE growth requirement could be replaced by cholera toxin or dibutyryl cAMP. These results suggested that both BPE and cholera toxin operated by activation of a cAMP‐dependent mitogenic pathway. Seven gram‐negative bacterial lipopolysaccharides (LPS) and three gram‐positive bacterial lipotechoic acids (LT) were tested for their effects on NHU clonal growth. Three out of the five LPS derived from Escherichia coli (strains 055:B5, 0128:B12, and 0127:B8), LPS from Klebsiella pneumoniae, and LPS from Pseudomonas aeruginosa all showed significant growth inhibitory effects at minimally effective doses ranging from 5 to 25 μg/ml. LPS derived from E. coli strain (0111:B4) had no growth effects at the highest concentration tested (100 μg/ml). In contrast, LT derived from Streptococcus pyogenes, S. faecalis, Staphylococcus aureas, and Bacillus subtilis all markedly enhanced clonal growth at concentrations ranging from 1 μg/ml < [LT] < 50 μg/ml. LT from Strep. pyogenes was inhibitory to clonal growth at 100 μg/ml. The growth inhibitory effects of LPS were shown to be sensitive to the presence of hydrocortisone in the growth medium, indicating that LPS effects on growth are mediated via the arachidonic acid cascade. We speculate that these results indicate a link between the susceptibility of uroepithelial tissue to the pathogenic microflora seen in urinary tract diseases and the differential sensitivity of proliferation‐competent uroepithelial cells to growth inhibition by LPS produced by gram‐negative bacteria. However, further studies with uropathogenic serotypes will be necessary to corroborate this possibility. The growth‐stimulating activity of LTs produced by gram‐positive bacteria may be due to their ability to bind to cell‐associated fibronectin and to activate the fibronectin receptor as part of ligand receptor–induced mitogenic transmembrane signalling pathway.
Low‐amplitude, low‐frequency electric field‐stimulated bone cell proliferation may in part be mediated by increased IGF‐II release
Authors: Fitzsimmons Robert J. , Strong Donna D. , Mohan Subburaman , Baylink David J.
Published: February 2005

We have developed an in vitro model incorporating a low‐amplitude (10−7 V/cm), low frequency (f < 100 Hz), capacitively coupled electric field in order to study the mechanism through which an electric field may increase bone cell proliferation. Utilizing this model we have previously shown that electric field‐stimulated bone cell proliferation was dependent on release of mitogen activity into the culture medium from exposed cells. The current studies were intended to characterize this mitogen activity. In these studies we found that electric field‐stimulated human bone cell proliferation was associated with increased IGF‐II mRNA accumulation and IGF‐II secretion suggesting that IGF‐II may in part mediate the increase in bone cell proliferation following electric field exposure.
Density‐dependent regulation of cell surface γ‐glutamyl transpeptidase in cultured glial cells
Authors: Morgenstern Kurt , Hanson‐Painton Olivia , Wang Bao‐Le , De Bault Lawrence
Published: February 2005

A decline in cell surface γ‐glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation. To elucidate the underlying factor(s) mediating γ‐glutamyl transpeptidase down‐regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower‐density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at postconfluency. The γ‐glutamyl transpeptidase down‐regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down‐regulation of γ‐glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low‐density cultures with conditioned medium, use of matrix‐coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that downregulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that γ‐glutamyl transpeptidase down‐regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of γ‐glutamyl transpeptidase demonstrated that γ‐glutamyl transpeptidase antigen levels decrease as C6 cell density increases. These results led us to propose that cell‐cell contact stimulates the disappearance of γ‐glutamyl transpeptidase from the surface of cultured C6 glial cells.
Derivatized dextrans mimic heparin as stabilizers, potentiators, and protectors of acidic or basic FGF
Authors: Tardieu Michele , Gamby Chantal , Avramoglou Thierry , Jozefonvicz Jacqueline , Barritault Denis
Published: February 2005

Acidic and basic fibroblast growth factors (aFGF and bFGF) belong to a family of structurally related polypeptides characterized by a high affinity for heparin. a and bFGF display mitogenic activity for many cell types. Biological activity is strongly potentiated by heparin which stabilizes their molecular conformation by preventing physicochemical or enzymatic degradation.

In our previous study we have shown that a water‐soluble derivatized dextran named DDE, containing 82.2% methyl carboxylic acid groups, 6.1% benzyl‐amide, and 5.6% sulfonate with a specific anticoagulant activity equivalent to heparin of 0.5 IU/mg could potentiate the mitogenic activity of aFGF on CCL39 cells. Optimal concentrations for maximal potentiation of 400 μg/ml and 20 μg/ml were obtained respectively for DDE and heparin. In the present report, we have uncovered the fact that several carboxymethyl benzylamide sulfonate dextrans differing in degree and positioning of the substituent groups can mimic heparin in regard to the protection, stabilization, and potentiating effects with aFGF or bFGF.

Our data establishes that the dextran derivatives studied can act as potentiating agents for FGFs. Native dextran (DDA) had no effect. Dextran derivatives can also protect aFGF and bFGF from heat as well as from pH denaturation, and against trypsic and chymotrypsic degradation. The dextran derivative DDI (82% methylcarboxylic acid, 23% benzylamide, 13% sulfonate) was studied in greater detail and exhibited a greater protection for bFGF and a lesser protecting effect for aFGF than heparin. Derivatized dextrans which have very weak anticoagulant activity are of great interest as alternatives to heparin for use as stabilizers, potentiators, protectants, and slow‐release matrices for FGFs in pharmaceutical formulations.
Regulation of prostaglandin H synthase activity in dog urothelial cells
Authors: Zenser Terry V. , Thomas Dori J. , Jacob Ammini K. , Davis Bernard B.
Published: February 2005

TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0–2 hr) increase in PGE2 synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase‐mediated pathway. In this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 μM) to media. PGE2 synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24‐hr incubation with 0.1 μM TPA or 1.0 μM A23187, arachidonic acid elicited significantly more PGE2, synthesis in agonist‐treated cells than it did in control cells in primary culture. Microsomes from 24‐hr TPA‐treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE2 content of overnight media was approximately 10‐fold greater in TPA‐treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0–2 hr) response. TPA at 0.1 μM was a maximum effective dose for both responses. The 24‐hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA‐mediated increase in PGE2 synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells.
Transforming growth factor β1 is a powerful modulator of platelet‐derived growth factor action in vascular smooth muscle cells
Authors: Janat M. Fouad , Liau Gene
Published: February 2005

We have studied the effect of transforming growth factor β1 (TGF‐β1) on vascular smooth muscle cell (SMC) mitogenesis and expression of thrombospondin and other growth related genes. We found that TGF‐β1 treatment of vascular SMC induced a prolonged increase in steady‐state mRNA levels of thrombospondin as well as α1 (lV) collagen. The increase began at approximately 2 h, peaked by 24 h, and remained considerably elevated 48 h after growth factor addition. There was a corresponding increase in thrombospondin protein as well as increased expression of several other secreted polypeptides. The increase in thrombospondin contrasted sharply with that observed for platelet‐derived growth factor (PDGF) which induced a rapid and transient increase in thrombospondin mRNA level. Although TGF‐β1 was able to directly enhance expression of thrombospondin as well as the growth‐related genes c‐fos and c‐myc, and induced c‐fos expression with identical kinetics as PDGF, it was unable to elicit [3H]thymidine incorporation into DNA in three independent smooth muscle cell strains. However, TGF‐β1 was able to strongly increase the mitogenic response of SMC to PDGF. Addition of both TGF‐β1 and PDGF to SMC also caused a synergistic increase in the expression of thrombospondin as well as c‐myc. Interestingly, in one other smooth muscle cell strain, a weak and delayed mitogenic response to TGF‐β1 alone was observed. Our results strongly suggest that induction of throm‐bospondin expression by TGF‐β1 and by PDGF occurs by distinct mechanisms. In addition, that TGF‐β1 can enhance PDGF‐induced mitogenesis may be due to the ability of TGF‐β1 to directly induce the expression of thrombospondin, c‐fos, c‐myc, and the PDGF β‐receptor.
Parathyroid hormone stimulates protein kinase C but not adenylate cyclase in mouse epidermal keratinocytes
Authors: Whitfield James F. , Chakravarthy Balu R. , Durkin Jon P. , Isaacs Richard J. , Jouishomme Hervé , Sikorska Marianna , Williams Ross E. , Rixon Raymond H.
Published: February 2005

Intact human parathyroid hormone, hPTH [1–84], and the hPTH [1–34] fragment stimulated membrane‐associated protein kinase C (PKC) activity in immortalized (but still differentiation‐competent) murine BALB/MK‐2 skin keratinocytes. Unexpectedly, the hormone and its fragment did not stimulate adenylate cyclase. The failure of PTH to stimulate adenylate cyclase activity was not due to the lack of a functioning receptor‐cyclase coupling mechanism because the cells were stimulated to synthesize cyclic adenosine monophosphate (cyclic AMP) by the β‐adrenergic drug isoproterenol. Thus, skin keratinocytes seem to have an unconventional PTH receptor that is coupled to a PKC‐activating mechanism but not to adenylate cyclase. These observations suggest that normal and neoplastic skin keratinocytes respond to the PTH‐related peptide that they make and secrete.