bluuebunny/crossref_metadata_2025 huggingface dataset:
Bi‐functional action of transforming growth factor‐β on DNA synthesis in early passage human fetal fibroblasts
Authors: Hill D. J. , Strain A. J. , Elstow S. F. , Swenne I. , Milner R. D. G.
Published: February 2005

We investigated the influence of transforming growth factor‐β (TGF‐β) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H] thymidine and cell replication. In serum‐free medium, without additional peptide growth factors, TGF‐β had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF‐β exhibited a bi‐functional action on the cells. A dose‐dependent stimulation of [3H] thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF‐β caused a dose‐related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin‐C/insulin‐like growth factor I, epidermal growth factor, or platelet‐derived growth factor, their actions were potentiated by the presence of TGF‐β on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses wieghing more than 100 g. Similar results were found for changes in cell number in response to TGF‐β when stimulated by SM‐C/IGF I. The ability of TGF‐β to modulate [3H] thymidine incorporation did not involve a change in the time required for growth‐restricted cells to enter the S phase of the replication cycle. These data suggest that TGF‐β may exert either a growth‐promoting or growth‐inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.
Isolation and partial characterization of endothelial cell extracellular complexes
Authors: Hatcher Victor B. , Fadl‐Allah Nora , Levitt Mindy A. , Brown Andrea , Margossian Sarkis S. , Gordon Portia B.
Published: February 2005

Human endothelial cells release components into the growth medium that stimulate cell‐substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell‐substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS‐polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large‐molecular‐weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell‐substratum adhesion and components that suppress cell‐substratum adhesion.
Purified HDL‐apolipoproteins, A‐I and C‐III, substitute for HDL in promoting the growth of SV40‐transformed REF52 cells in serum‐free medium
Authors: Chen Jan‐Kan , Labrake‐Farmer Sharon , McClure Don B.
Published: February 2005

The lipid‐free apolipoproteins of human high density lipoprotein (HDL) have been assayed for their ability to substitute for native HDL in promoting the growth of a SV40‐transformed REF52 cell line in serum‐free medium. Total HDL‐apolipoproteins (apoHDL) were found to mimic almost exactly the growth promoting effects of whole HDL. The apoHDL‐associated growth promoting activity eluted from a Sephacryl S‐200 column in two separate fractions coin‐ciding with the protein peaks of apolipoprotein A‐I and the C group of apolipoproteins. These two fractions, designated S‐II and S‐IV, respectively, acted additively in promoting WT1A cell growth when tested at saturating concentrations. The active component in the S‐II fraction maximally stimulated WT1A cell growth at 40–60 μg/ml and was identified as apolipoprotein A‐1 by NaDodSO4 polyacrylamide gel electrophoresis and affinity chromatography on anti‐(apoA‐I). The active component in the S‐IV fraction was maximally active at 1–2 μg/ml and was identified as apolipoprotein C‐III by DEAE ion exchange high pressure liquid chromatography and polyacrylamide gel electrophoresis (at pH 8.3) in 6 M urea. These results indicate that the growth promoting effect of HDL on WT1A cells is mediated via the HDL‐apolipoproteins, A‐I and C‐III, and that the mechanism responsible does not necessarily involve their participation in the uptake (or utilization) of HDL‐associated lipids.
Transport and metabolism of adenosine in human erthrocytes: Effect of transport inhibitors and regulation by phosphate
Authors: Plagemann Peter G. W.
Published: February 2005

Rapid kinetic techniques were applied to determine the effect of transport inhibitors on the transport and metabolism of adenosine in human red cells. Dipyridamole inhibited the equilibrium exchange of 500 μM adenosine by deoxcoformycin‐treated cells in a similar concentration dependent manner as the equilibrium exchange and zero‐trans influx of uridine with 50% inhibition being observed at about 20 nM. lntracellular phosphorylation of adenosine at an extracellular concentration of 5 μM pM was inhibited only by dipyridamole concentrations ≥ 100nM, which inhibited transport about 95%. Lower concentrations of dipyridamole actually stimulated adenosine phosphorylation, because the reduced influx of adenosine lessened substrate inhibition of adenosine kinase. When the cells were not treated with deoxycoformycin, > 95% of the adenosine entering the cells at a concentration of 100μM became deaminated. A 95–98% inhibition of adenosine transport by treatment with dipyridamole, dilazep, or nitrobenzylthioinosine inhibited its deamintion practically completely, whereas adenosine phosphorylation was inhibited only 50‐85%. Whether adenosine entering the cells is phosphorylated or deaminated is strictly based on the kinetic properties of the responsible enzymes, substrate inhibition of adenosine kinase, and the absolute intracellular steady state concentration of adenosine attained. The latter approaches the extracellular concentration of adenosine, since transport is not rate limiting, except when modulated by transport inhibitors.

In spite of the extensive adenosine deamination in cells incubated with 100 7μM adenosine, little IMP accumulated intracellularly when the medium phosphate concentration was 1 mM, but IMP formation increased progressively with increase in phosphate Concentration to 80 mM. The intracellular phosphoribosylation of adenine and hypoxanthine were similarly dependent on phosphate concentration.

The results indicate that adenosine is the main purine source for erythrocytes and is very efficiently taken up and converted to nucleotides under physiological conditions, whereas hypoxanthine and adenine are not significantly salvaged. Hypoxanthine resulting from nucleotide turnover in these cells is expected to be primiarily released from the cells. Adenosine was also phosphorolyzed in human red cells presumably by 5′‐methyltioadenosine phosphorylase, but this reaction seems without physiological significance as it occurs only at high adenosine and phosphate concentrations and if deamination is inhibited.
Prostaglandin E2 synthesis in the inner medullary collecting duct of the rat: Implications for vasopressin‐dependent cyclic AMP formation
Authors: Jackson Brian A.
Published: February 2005

The effects of osmolality on prostaglandin E2 (PGE2) biosynthetic capacity and the interaction between endogenous PGE2 synthesis and vasopressin (AVP)‐dependent cyclic AMP generation were examined in papillary collecting ducts (PCD) microdissected from collagenase‐digested rat kidneys. Increasing medium osmolality with NaCl:urea (1:2 molar ratio) progressively increased PGE2 synthesis in PCD up to 1,500 mOsm. Addition of NaCl:urea or NaCI alone were equally effective in stimulating PGE2 biosynthetic capacity in PCD. In contrast, addition of urea alone had a much smaller stimulatory effect on PGE2 synthesis. Inhibition of endogenous PGE2 synthesis with naproxen (10−5M) suppressed AVP‐dependent cAMP formation in PCD when incubated in 300 mOsm medium but had no effect when incubated in 1,500 mOsm medium. Addition of 2.5×10−5 M PGE2 also suppressed AVP‐dependent cAMP formation in PCD only when incubated in 300 mOsm medium. The present study suggests that the PCD is a site of active PGE2 synthesis that is modulated by osmolality. Our results do not support the concept that endogenous PGE2 antagonizes vasopressin action via inhibition of AVP‐dependent cAMP formation.
Growth inhibition of 3T3 fibroblasts by lysosomotropic amines: Correlation with effects on intravesicular pH but not vacuolation
Authors: Cain Cynthia Corley , Murphy Robert F.
Published: February 2005

The effects of five lysosomotropic amines on the growth of Swiss 3T3 fibroblasts were measured and compared with effects on intravesicular pH. Tributylamine and benzylamine, amines that affect intravesicular pH without causing vacuolation, were found to inhibit cell growth to a similar extent as vacuologenic amines previously tested. Excellent correlation between the half‐maximal concentrations for the growth and pH effects were found for tributylamine, benzylamine, chloroquine, and ammonium chloride. The results suggest that growth inhibition by these amines is a direct result of their effects on pH and not due to other effects (such as vacuolation). In contrast, a 100‐fold difference in the half‐maximal concentrations was found for methylamine, suggesting that methylamine inhibits growth by a mechanism unrelated to pH.
Tyrosine phosphorylation in cells treated with transforming growth factor‐β
Authors: Libby James , Martinez Ricardo , Weber Michael J.
Published: February 2005

Cells stimulated with epidermal growth factor (EGF) or any one of a diverse group of other mitogenic agents display an increased tyrosine phosphorylation of a pair of 42,000 Mr proteins. Transforming Growth Factor‐β (TGF‐β) is able to potentiate the mitogenic effects of Epidermal Growth Factor on some fibroblastic cells (such as the NRK‐49F cell line) and, in addition, permits the anchorage‐independent growth of these cells. In this study we asked whether these growth‐regulatory actions of Transforming Growth Factor‐β are associated with changes in tyrosine phosphorylation of cellular proteins, in particular the 42,000 Mr proteins. We found no effect of Transforming Growth Factor‐β on the extent or time‐course of tyrosine phosphorylation, either by itself or in combination with Epidermal Growth Factor. Since the tyrosine phosphorylation of the 42,000 Mr proteins is stimulated both by receptors with tyrosine kinase activity and by diacylglycerol analogs (but not by Transforming Growth Factor‐β), we suggest that the activity of the receptor for Transforming Growth Factor‐β is linked neither to tyrosine phosphorylation nor to phosphatidyl inositol turnover.
Murine basophil—mast differentiation: Toward optimal conditions for selective growth and maturation of basophil—mast or allied cells
Authors: Lanotte M. , Arock M. , Lacaze N. , Guy‐Grand D.
Published: February 2005

Recent investigations revealed that basophil‐mast cells were related to the hemopoietic system. Strikingly, murine bone marrow showed a singular paucity in cells with basophil‐mast features; moreover in clonogenic assays (methylcellulose, agarose) bone marrow was found to be manifestly poor in basophil‐mast progenitor cells. Our work brought to light several new facts concerning the culture and differentiation of this cell type: 1° pure and mixed mast clones can be derived in large numbers from bone marrow, provided progenitors are cultured in collagen matrix. Up to 1,382 hemopoietic clones were analysed in situ after staining: 30% contained mast cells (34 per 105 cells), thus the basophil‐mast lineage was one of the most frequent. We concluded that other cloning media were noticeably nonoptimal for the growth and/or maturation of mast cells. We suggested that collagen and the molecular edifices derived from it, both found in variable amounts in the natural mast environments, should play essential roles in mast phenotype expression. 2° Cholera toxin (CT) selectively eradicated nonmast progenies: mast progenitors and mast progenies were resistant. In this way, pure and rapidly expanding mast cell clones were obtained at a frequency never reported before. CT possibly acts both directly, as a stimulator of mast cell proliferation, or indirectly on marrow subpopulations which repress basophil‐mast cell growth and maturation. In vitro culture conditions, specifically designed for basophil‐mast lineage, should prove of interest in the search for an unifying hypothesis concerning the multiple forms of mast cells found in various tissues.
Enhanced synthesis of the glucose/calcium‐regulated proteins in a hamster cell mutant deficient in transfer of oligosaccharide core to polypeptides
Authors: Lee Amy S. , Wells Steven , Kim Kyu Seong , Scheffler Immo E.
Published: February 2005

The properties of two Chinese hamster temperature‐sensitive mutants, K12 and H3.5, were examined. Both mutants originated from the same parental cell line, Wg1A, and were isolated as cell cycle mutants arrested in G1. Previously, we had been shown that the H3.5 ts mutation affected the transfer of the oligosaccharide from the lipid carrier to the nascent polypeptide and that the K12 ts mutation regulated the transcription of two glucose/calcium‐regulated genes. We report here that these two mutants exhibit almost identical phenotypes at the biochemical level. Furthermore, a genetic complementation test demonstrates that the two ts lesions must be closely related, or even identical. Our results suggest that a specific defect in glycosylation may result in the overproduction of the glucose/calcium‐regulated proteins and is capable of activating the promoter of the major glucose‐regulated gene.
Correlation between plasma membrane surface area and transferrin secretion rate in isolated hepatocytes
Authors: Pechinot D. , Foucrier J. , Feldmann G.
Published: February 2005

It is generally considered that in exocytosis the size of the secreting cells does not increase when the membranes of exocytosis vesicles fuse with the plasma membrane. As the factors involved in the regulation of this phenomenon are poorly understood, we thought it worthwhile to investigate the relationship between the plasma membrane surface area and secretory activity. Isolated rat hepatocytes were prepared by liver collagenase perfusion. Secretion of the plasma protein, transferrin (Tf) was detected at the single cell level with specific anti‐rat transferrin antibodies using the reverse hemolytic plaque test. Hepatocyte surface and hemolytic ring surface areas were calculated from diameters of hepatocyte and hemolytic plaque measured after 5h of incubation. A highly significant correlation was established between the plaque‐forming hepatocyte surface areas and the corresponding hemolytic surface areas. This result was confirmed using an automatic image analysis method. Two‐month‐old rats were compared to 4‐month‐old rats. We observed that the ratio of the quantity of transferrin secreted by hepatocytes to the hepatocyte surface area was constant for a given incubation time, whatever the size of the hepatocytes. These results suggest that the plasma membrane surface area of hepatocytes may constitute a limiting factor in Tf secretion.
Properties of colony‐stimulating factors produced by macrophage cell lines and hybrid cells
Authors: Ohki Kazunori , Nagayama Ariaki
Published: February 2005

Colony‐stimulating factors (CSFs) produced by two simian virus 40(SV40) transformed macrophage cell lines ( BAM1 and BAM3 ), and three hybrids (HM3‐11, HM3‐12, and HM3‐14) derived from fusion between BAM3 and a Chinese hamster cell line (hs222‐16) were examined. HM3‐11 and HM3‐14 produce two molecular species of CSF, which are not found in the conditionec media from cultures of BAM1 and BAM3 or lipopolysaccharide (LPS), phorbolmyristate‐acetate (PMA), and zymosan‐stimulated BAM3. HM3‐12, which is classified into another group in terms of CSF secretion, does not produce these two CSFs. On the basis of various criteria, one of these CSF species ( peak 1‐CSF ) was characterized as a macrophage‐colony‐stimulating factor (M‐CSF). The other CSF (peak 2‐CSF) induced a group of bone marrow cells in granulocytes and macrophages as well as growth of a mast cell line, IC2. This CSF has an apparent molecular weight of 18,000, estimated by SDS‐polyacrylamide gel electrophoresis. Unlike interleukin 3 (IL3) from WEHI‐3 cells, the growth factor activity of peak 2‐CSF binds to DEAE‐Sephacel. Thus, peak 2‐CSF is similar to a granuclocyte‐macrophage colony‐stimulating factor (GM‐CSF) rather than to IL3. The anti L cell CSF serum does not inhibit the CSF activity in Chinese hamster fibroblast conditioned medium, and the IC2 cells do not respond to Chinese hamster lung conditioned medium (CHLCM), suggesting that peak 1‐ and peak 2‐CSF are of mouse origin.
Altered type I protein kinase in adhesion defective CHO cell variants
Authors: Cheung E. , Brown P. J. , Juliano R. L.
Published: February 2005

ADv cells are Chinese hamster ovary (CHO) cell variants which cannot adhere to fibronectin coated substrata (Harper amp; Juliano: J. Cell Biol., 1980; Nature 1981a, b). We have shown that the defect in some clones of ADv cells is distal to the initial interaction between fibronectin and its cell surface receptors (Cheung and Juliano: Exp. Cell Res., 1984), and that it extends to fibronectin mediated aggregation and endocytosis. The adhesion defect in some ADv clones can be corrected by raising intracellular cAMP levels (Cheung & Juliano: J. Cell Physiol., 1985). Here we examine the protein kinase activities and phosphorylation patterns in an adhesion defective variant clone ADv F11CA11. Analysis of the cAMP dependent protein kinase activity (cAdPK) in crude extracts of F11CA11 cells shows an apparent increase in K (activation) as compared to wild type (WT) CHO cell extracts. Further, the DE‐52 cellulose chromatography profile of cAdPK in the F11CA11 variant is markedly different from WT in that the type I cAdPK peak elicited by 1 μM cAMP is essentially missing in F11CA11, while the type II cAdPK peak is similar to that in WT. Raising the cAMP level to 100 μM elicits a type I peak in F11CA11 with about 45% of the activity of the WT peak. Binding studies with 3H‐cAMP reveal that the type I peak in F11CA11 has a Kd of 1.7 times; 10−8 M as compared to 2.0 × 10−9 M for WT, whereas the type II peak Kd is approximately 1 × 10−9 M for both WT and F11CA11. Two‐dimensional polyacrylamide gel analysis of 32Pi labeled WT cells and F11CA11 cells with or without cAMP treatment reveals the presence of a protein(s) of 50 kilodaltons which is phosphorylated in WT cells and in cAMP treated F11CA11 cells but not in untreated F11CA11 cells. These findings, coupled with our previous observations, strongly indicate taht the adhesion defect in ADvF11CA11 cells is associated with an altered type I cAdPK having lower affinity for cAMP. At normal cellular cAMP levels this enzyme fails to phosphorylate one or more critical protein substrates; however, by raising internal cAMP levels, the defect can be overcome. Thus type I cAdPK seems to play an important role in the regulation of fibronectin mediated cell adhesion, cell aggregation, and endocytosis.
Carbonic anhydrase cytochemistry in mitochondria‐rich cells of salamander larvae gill epithelium as related to age and H+ and Na+ concentrations
Authors: Lewinson D. , Rosenberg M. , Goldenberg S. , Warburg M. R.
Published: February 2005

Carbonic anhydrase (CAH) was localized in the mitochondria‐rich cells (MRC) of 1‐week‐old salamander larvae gill epithelium, in both MRC and pavement cells of 6‐week‐old larvae, and in regenerated stems of previously amputated gills. CAH activity of the MRC was measured quantitatively using a microscope densitometric technique. Changes in CAH activity per cell and changes in the numbers of CAH‐positive MRC were followed under different H+ and Na+ concentrations at the two age groups. CAH activity per cell increased with age, whereas the numbers of CAH‐positive MRC dropped. CAH activity per cell in the 1‐week‐old age group reached maximal values at pH 7.4 and stayed relatively high in the more alkaline media. Moderate increases of Na+ concentrations had small but significant effects on increasing CAH activity of gill MRC. When taking into consideration not only the changes in cellular activity but also the changes in the number of CAH‐positive cells under the different acclimation media, an activity index (ICAH) was calculated. Thus, the ICAH in the 1‐week‐old was found to be dependent on the decline of ambient H+ concentrations (expressed as increasing pH), reaching maximal effect at pH 8.0. On the other hand, raising the Na+ concentrations of the acclimation media to 110 and 220 mOsm/liter caused a maximal inhibition of tissue CAH activity as expressed by ICAH. In conclusion, it is suggested that salamander larvae gill MRC take part in the adaptation of the larvae to changing H+ concentrations of their milieu rather than in their adaptation to changes in its osmolality.
Conditional responsiveness of a chemically transformed cell line to growth factor stimulated DNA synthesis
Authors: Whipple A. P. , Furlanetto R. W. , Scher C. D.
Published: February 2005

BP3T3, a clonal benzo(a)pyrene‐transformed BALB/c‐3T3 cell line, is conditionally responsive to growth factor stimulation. Density arrested cell populations deprived of growth factors by pretreatment with 0.5% platelet‐poor plasma synthesized DNA both in response to ng/ml concentrations of PDGF, EGF, and somatomedin C, and in response to insulin, plasma, and serum. The above agents acted singly to induce DNA synthesis, but synergism is suggested because a higher percentage of cells were stimulated to enter the S phase when the growth factors were added in combination. Desensitization to growth factors occurred when cultures were pretreated with the high concentration of growth factors present in 10% serum (or plasma). In desensitized cultures none of the above agents, added singly or in combination, stimulated DNA synthesis. This effect appears to be global because pretreatment with one growth factor (e.g., insulin) inhibited the action of another (e.g., PDGF). Cell density appears to play a critical role in regulating DNA synthesis. Unlike nontransformed BALB/c‐3T3 cells whose density is regulated by the serum concentration, the density of BP3T3 cells reached a plateau when cultures were grown in a serum (or plasma) concentration of 3% or greater. Such density arrested cultures were growth factor unresponsive; however, the cells rapidly responded to growth factors by synthesizing DNA and replicating when reseeded at a lower cell density. Thus the growth of BP3T3 cells is regulated by both growth factors and cell density.
Requirement of prostaglandin F2α in 17β‐estradiol stimulation of DNA synthesis in rabbit endometrial cultures
Authors: Orlicky D. J. , Lieberman R. , Williams C. , Gerschenson L. E.
Published: February 2005

We have hypothesized that two of the endogenously synthsized endometrial prostaglandins (PGs), prostaglandin F2α (PGF2α), and prostaglandin E1 (PGE1), play a regulatory role in growth control of the rabbit endometrium. PGF2α increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used to examine the possible role of these PGs in the mechanism of action of 17β‐estradiol on DNA synthesis. Towards this end, binding, second messenger and DNA synthesis experiments were performed. 17β‐estradiol stimulation restulted in a time dependent (optimal: approximately 6 h) and 17β‐estradiol concentration dependent (optimal: approximately 10−7 M 17β‐estradiol in phenol red‐containing medium) increase in [3H]PGF2α binding. Scatchard type analysis of the binding data revealed an increase in receptor number while the receptor affinity for [3H]PGF2α remained the same as in the control treated cultures. This 17β‐estradiol stimulated increase in PGF2α receptor allowed a suboptimal concentration of PGF2α (10−9M) to increase intracellular levels of inositol polyphosphates, while by itself this concentration of PGF2α caused no significant change in intracellular inositol polyphosphate levels. 17β‐estradiol, alone among the several studied steroid hormones, could increase [3H]PGF2α binding. Proliferation studies revealed that, in these primary cultures of rabbit endometrium, 17β‐estradiol could increase DNA synthesis but not in the presence of indomethacin, unless PGF2α was addd to the medium at a concentration (10−9M) near or above what is normally accumulated in the medium by these cultures. In the absence of 17β‐estradiol stimulation, addition of these same low concentrations of PGF2α had no effect on DNA synthesis. Apparently, through its effect on the PGF2α receptor, 17β‐estradiol enhances the PGF2α stimulated DNA synthesis response approximately 100 fold. The DNA synthesis inducd by 17β‐estradiol can be inhibited by PGE1, as can PGF2α‐induced DNA synthesis. We propose that 17β‐estradiol may be mediating its mitogenic effect through an alteration of the prostglandin agonist:antagonist control of proliferation in rabbit endometrial cultures. In additon we suggest that, if 17β‐estradiol acts to increase PGF2α receptors as part of its mode of action, this may be of importance in other tissues possessing both prostaglandin and 17β‐estradiol receptos.
Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha‐transforming growth factors in ras‐transformed mouse mammary epithelial cells
Authors: Salomon David S. , Perroteau Isabelle , Kidwell William R. , Tam James , Derynck Rik
Published: February 2005

A mouse mammary epithelial cell line, NMuMG, exhibits a low capacity to grow in semisolid medium as colonies and it is not tumorigenic in nude mice. In contrast, NMuMG cells which have been transformed by an activated c‐Harvey ras proto‐oncogene, NMuMG/rasH, or by the polyoma middle T‐transforming gene, NMuMG/pyt, are able to grow in soft agar and, when injected into nude mice, produce undifferentiated carcinomas. Human epidermal growth factor (EGF) or human alpha‐transforming growth factor (α TGF) can stimulate, in a dose‐dependent fashion, the anchorage‐independent growth of NMuMG and NMuMG/pyt cells in soft agar but fail to enhance the anchorage‐independent growth of the NMuMGrasH cells. Likewise, human EGF or human αTGF is also able to stimulate the anchorage‐dependent growth of normal NMuMG cells and NMuMG/pyt cells in a serum‐free medium supplemented with insulin, transferrin, fetuin, and laminin, or in medium containing low concentrations of serum, wheres these same growth factors under comparable culture conditions have little or no effect upon the anchorage‐dependent growth of the ras‐transformed NMuMG‐rasH cells. The biological refractoriness of the NMuMG/rasH cells to human EGF or human α TGF is reflected by a reduction in the total number of cell surface receptors for EGF and by an absence of a high‐affinity population of binding sites for mouse [125l]EGF on these cells as compared to the NMuMG or NMuMG/pyt cells. In addition, concentrated conditioned medium (CM) obtained from NMuMG/rasH and NMuMG/pyt cells contains a relatively higher amount of biologically active TGFs than CM obtained from comparably treated NMuMG cells as measured by the ability to induce the anchorage‐independent growth of normal rat kidney cells in soft agar. The higher levels of biologically active TGFs found in the CM from the transformed cells relative to the NMuMG cells is paralleled by a corresponding increase in the CM from these cells in the amount of immunoreactive αTGF, by an increase in the amount of EGF receptor‐competing activity, and by an increase in the levels of αTGF mRNA in the NMuMG/rasH cells. These results demonstrate that mammary epithelial cells which have been transformed by an activated ras proto‐oncogene, but not by the polymoa middle T‐transforming gene, become unresponsive to exogenous EGF or αTGF. The growth refractoriness of the NMuMG/rasH cells to exogenous EGF may be due to the reduction in the number and affinity of EGF receptors on these cells and because of an increased capacity of these cells to synthesize and secrete their own αTGFs.
Alpha and beta subunits of the LFA‐1 membrane molecule are involved in human monocyte–endothelial cell adhesion
Authors: Mentzer Steven J. , Crimmins Mary A. V. , Burakoff Steven J. , Faller Douglas V.
Published: February 2005

Human monocyte adhesion to vascular endothelium is an important transitional event in mononuclear phagocyte development. The molecular mechanism involved in monocyte adhesion to endothelial cells was studied using purified human monocytes and a panel of monoclonal antibodies (MAb). The purified human monocytes were phenotypically characterized and expressed relatively low levels of HLA class II antigens. The monocytes were labeled with Indium‐111 to provide high specific activity and a sensitive measure of adhesion. Using this radionuclide adhesion assay, monocytes demonstrated consistent and reproducible adhesion to a confluent monolayer of human umbilical vein‐derived endothelial cells. To identify the cell surface molecules involved in human monocyte–endothelial cell adhesion, 15 MAb to 11 monocyte surface structures were used to attempt to inhibit adhesion. MAb recognizing 10 monocyte cell surface molecules did not inhibit adhesion. In contrast, MAb recognizing the alpha and beta subunits of LFA‐1 (lymphocyte function‐associated) significantly inhibited monocyte adhesion to endothelial cells. Monocyte adhesion was comparably inhibited by F(ab')2 and intact MAb. Significant inhibition was observed at 5μg/ml of anti‐LFA‐1 MAb. These results indicate that the alpha and beta subunits of the LFA‐1 membrane molecule are involved in human monocyte–endothelial cell adhesions.
Effects of serum and conditioned medium on protein degradation, migration of nonhistone proteins to the nucleus, and DNA synthesis in transformed cells
Authors: Polet Herman , Swager Jeffrey S.
Published: February 2005

Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [3H] NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5–6.5, showed that [3H] NHP fractions with high degradation rates in resting cells corresponded to the [3H] NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H] NHP are linked. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear.
Effect of extracellular hemin on hemoglobin and ferritin content of erythroleukemia cells
Authors: Fibach E. , Konijn A. M. , Bauminger R. E. , Ofer S. , Rachmilewitz E. A.
Published: February 2005

Mouse (MEL) and human (K‐562) erythroleukemia cell lines can be induced to undergo erythroid differentiation, including hemoglobin (Hb) synthesis, by extra cellular hemin. In order to study the effect of extracellular hemin on intracellular ferritin and Hb content, we have used Mossabauer spectroscopy to measure the amount of 57Fe incorporated into ferritin or Hb and a fluorescent enzyme‐linked immunosorbent assay (ELISA) to measure the ferritin protein content. When K‐562 cells were cultured in the presence of a 57Fe source either as transferrin or citrate, in the absence of a differentiation inducer, all the intracellular 57Fe was detected in ferritin. When the cells were cultured in the presence of 57Fe‐hemin, 57Fe was found in both ferritin and Hb. 57Fe in ferritin increased rapidly, and after 2 days it reached a plateau at 5 × 10−14g/cell. 57Fe in Hb increased linearly with time and reached the same value after 12 days. Addition of other iron sources such as iron‐saturated transferrin, iron citrate, or iron ammonium citrate caused a much lower increase in ferritin protein content as compared to hemin. When K‐562 cells were induced by 57Fe‐hemin in the presence of 56Fe‐transferrin, 57Fe was found to be incorporated in equal amounts into both ferritin and Hb. However, when the cells were induced by 56Fe‐hemin in the presence of 57Fe‐transferrin, 57Fe was incorporated only into ferritin, but not into Hb, which contained 56Fe iron. These results indicate that in K‐562 cells, when hemin is present in the culture medium it is preferentially incorporated into Hb, regardless of the availability of other extra‐or intracellular iron sources such as transferrin in ferritin. In MEL cells induced to differentiate by dimethylsulfoxide (DMSO) a different pattern of iron incorporation was observed; 57Fe from both transferrin and hemin was found to incorporate in ferritin as well as in Hb.
Fusion‐mediated microinjection of active amine and diamine oxidases into cultured cells: Effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells
Authors: Bachrach Uriel , Ash Issar , Abu‐Elheiga Lufti , Hershkovitz Maty , Loyter Abraham
Published: February 2005

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.
Inhibition by cytochalasin B of DNA synthesis in a thermosensitive anchorage‐independent growth mutant
Authors: Miyashita Kiichi
Published: February 2005

After a shift from a nonpermissive to a permissive temperature, synchronized DNA synthesis and cell division were observed in a cold‐sensitive anchorageindependent growth mutant (cs‐17–25) of Chinese hamster lung cells in Methocel culture. Only 15 min exposure to the permissive temperature was sufficient for induction of DNA synthesis in the cells. A low dose of actinomycin D (0.02 μg/ml) or cytochalasin B (5 μg/ml) was able to inhibit the DNA synthesis when added at an early period after the temperature shift. The inhibitory effects of actinomycin D and cytochalasin B on RNA and protein synthesis were very similar at both temperatures. The degree of multinucleation caused by cytochalasin B was altered quickly depending on the incubation temperature. These results suggest that stimulation of this mutant involves an obligatory actin‐mediated step closely correlated to transcription of early mRNAs.
Kidney proximal tubular cells isolated by collagenase perfusion grow in defined media in the absence of growth factors
Authors: Rosenberg Mark R. , Michalopoulos George
Published: February 2005

Cells from kidney proximal tubules have been successfully isolated, characterized, and cultured from male Fischer 344 rats between 150–400 g using a two‐step collagenase perfusion. The cells undergo high levels of DNA synthesis and mitosis in both serum free media (with an without hormone supplementation) and media containing 10% fetal bovine serum. Confluent monolayers were observed between 5 to 7 days after seeding 2 × 105 cell/35mm collagen‐coated plate. Approximately 50% of the total kidney and 70% of the cortex was isolated using this technique. The viability of the isolated tubules was 75 ± 8% and the estimated number of viable cells was 12 ± 3 × 106 cells. At the time of isolation greater than 90% of the isolated tubules and cells were positive for gamma glutamyltransferase (GGT), periodic acid‐schiff (PAS), and glucose‐6‐phosphatase (G‐6‐Pase). Both GGT and G‐6‐Pase decreased rapidly during the first 3 days in primary culture as assessed by histochemistry. Ultrastructurally the isolates consisted of cells with numerous microvilli and mitochondria. The size and number of microvilli decrease rapidly in primary culture. The morphologic and biochemical evidence suggests that the primary isolates and cultures are proximal tubular in origin.
Transferrin receptor numbers and transferrin and iron uptake in cultured chick muscle cells at different stages of development
Authors: Sorokin L. M. , Morgan E. H. , Yeoh G. C. T.
Published: February 2005

The mechanism of iron uptake and the changes which occur during cellular development of muscle cells were investigated using primary cultures of chick embryo breast muscle. Replicating presumptive myoblasts were examined in exponential growth and after growth had plateaued. These were compared to the terminally differentiated cell type, the myotube. All cells, regardless of the state of growth or differentiation, had specific receptors for transferrin. Presumptive myoblasts in exponential growth had more transferrin receptors (3.78 ± 0.24 × 1010 receptors/μg DNA) than when division had ceased (1.70 ± 0.14 × 1010 receptors/μg DNA), while myotubes had 3.80 ± 0.26 × 1010 receptors/μg DNA. Iron uptake occurred by receptor‐mediated endocytosis of transferrin. While iron was accumulated by the cells, apotransferrin was released in an undegraded form. There was a close correlation between the molar rates of endocytosis of transferrin and iron. Maximum rates of iron uptake were significantly higher in myotubes than in presumptive myoblasts in either exponential growth or after growth had plateaued. There were two rates of exocytosis of transferrin, implying the existence of two intracellular pathways for transferrin. These experiments demonstrate that iron uptake by muscle cells in culture occurs by receptor‐mediated endocytosis of transferrin and that transferrin receptor numbers and the kinetics of transferrin and iron uptake vary with development of the cells.
Evidence for the recycling nature of the fibronectin receptor of macrophages
Authors: Molnar Janos , Hoekstra Steven , Ku Cecilia S.‐L. , van Alten Pierson
Published: February 2005

Plasma fibronectin (pFN) has been shown to mediate phagocytosis of several types of artificial particles and tissue debris by macrophages. In the present investigation some of the dynamic aspects of this receptor‐mediated cellular process have been studied. Plasma fibronectin did not bind specifically to fibronectin (FN)‐receptors of rat peritoneal macrophages at either 4°C or 37°C. On the other hand, pFN aggregated on the surface of gelatin‐coated latex beads (gLtx) and 125I‐labeled pFN covalently coupled to latex beads (pFN*‐Ltx) bound strongly to macrophages at both temperatures. Both of these particles were also internalized at 37°C. Treatment of macrophages by chymotrypsin, thermolysin, or trypsin in a protein‐free tissue culture medium did not affect either of the above reactions; however, pronase treatment strongly reduced both the binding and internalization of the pFN‐coated particles. The pronase‐treated macrophage monolayers in time regained their ability to bind and internalize pFN‐gLtx* when incubated in fresh tissue culture medium. Such recovery, however, did not take place when the medium contained cycloheximide. On the other hand, phagocytosis of pFN‐gLtx* was not affected directly by cycloheximide with untreated macrophages; this suggests that the FN‐receptor recycles during sustained phagocytosis. This assumption was substantiated by the observations that some of the established lysosomotropic amines—i.e., chloroquine, dansylcadaverine, and dimethyldansylcadaverine—caused total inhibition of internalization without affecting the binding of particles to macrophages. Furthermore, chloroquine protected the FN‐receptors against destruction by pronase. Together these results suggest that macrophage receptors for FN are protein, present both on the cell surface and intracellularly, and recycle between the plasma membrane and intracellular sites during phagocytosis.
Phospholipid and arachidonic acid metabolism in zymosan‐stimulated human monocytes: Modulation by cAMP
Authors: Godfrey Richard W. , Manzi Rosalie M. , Gennaro Doris E. , Hoffstein Sylvia T.
Published: February 2005

Receptor‐ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]‐arachidonic acid ([3H]‐AA)‐labeled human monocytes released 25 ± 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 × 10−4 M isobutylmethylxanthine (IBMX) or 1 × 10−3 M dibutyrl cyclic AMP (d‐cAMP) inhibited total [3H]‐AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]‐AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d‐cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan‐treated cells indicated that 5‐HETE was the predominant metabolite generated from [3H]‐AA, and its production was depressed during treatment with d‐cAMP. Phospholipase activity in human monocyte homogenates was not effected by d‐cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.
Cyclic AMP‐mediated suppression of normal and neoplastic B cell proliferation is associated with regulation of myc and Ha‐ras protooncogenes
Authors: Blomhoff Heidi Kiil , Smeland Erlend B. , Beiske Klaus , Blomhoff Rune , Ruud Erik , Bjøro Trine , Pfeifer‐Ohlsson Susan , Watt Rosemary , Funderud Steinar , Godal Tore , Ohlsson Rolf
Published: February 2005

Cyclic AMP functions as a negative regulator of cell proliferation in a variety of cell systems. We show here that the proliferation of normal and neoplastic B cells can be inhibited by high intracellular levels of cAMP. Thus forskolin treatment of the neoplastic B precursor cell line Reh induced a rapid increase in the cAMP level, which was followed by an accumulation of cells in the GO/G1 phase of the cell cycle over a period of 2–3 days. Similar inhibition of Reh cell proliferation after 3 days was observed whether forskolin was present continuously or only during the first 5 hr. Both c‐myc and c‐Ha‐ras protein levels were transiently down‐regulated at 4 hr of forskolin treatment, suggesting that these protooncogenes play a role in the process leading to cAMP‐mediated growth cessation. Northern‐blot analysis showed that the steadystate levels of c‐myc RNA rapidly declined in all phases of the cell cycle, to return to control levels within a time period of 24 hr. In contrast, the c‐Ha‐ras mRNA level was steadily maintained. Thus the expression of c‐myc and c‐Ha‐ras protein was regulated at different metabolic levels. The reduced proliferative capacity of the B precursor cell line in the presence of forskolin was not linked to induced differentiation. This was judged from the lack of appearance of three different B cell differentiation markers; cytoplasmic immunoglobulin heavy chain and two antigens recognized by the monoclonal antibodies B1 (CD20) and HH1 (CD37). We also showed that forskolin partially inhibited the proliferation of normal B lymphocytes stimulated by anti‐immunoglobulins (anti‐μ) and B cell growth factor (BCGF). The burst of c‐myc mRNA during activation of normal B cells was also reduced by forskolin.
Macrophage cell lines transformed by the malignant histiocytosis sarcoma virus: Increase of CSF receptors suggests a model for transformation
Authors: Klingler K. , Johnson G. R. , Walker F. , Nicola N. A. , Decker T. , Ostertag W.
Published: February 2005

The malignant histiocytosis sarcoma virus (MHSV) contains Ha‐v‐ras‐related oncogenic sequences and rapidly transforms myeloid cells in vivo and in vitro. Myeloid cell lines can be derived which do not require growth factor for continued proliferation. We initiated this work to define the process of transformation leading to autonomy of cell growth in transformed myeloid cells. Five established cell lines were examined. All express macrophagespecific cell‐surface antigens and exhibit several other properties typical for mature macrophages. Growth properties, growth factor release, and growth factor receptor presentation were examined: Release of growth factors is not a consistent feature. All cell lines show cell‐density‐independent colony formation and do not release self‐stimulating factors, thus excluding autocrine stimulation as a model leading to transformation. All cell lines express unusually high levels of granulocyte‐macrophage (GM)‐and multi‐CSF receptors and, except for one M‐CSF receptors. The high increase in GM‐CSF and other growth factor receptors may be causally related to the transformed state of the cells. MHSV can be used as a tool to easily derive cell lines of the macrophage pathway as a model to study myeloid transformation, differentiation, and macrophage function.