bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractA 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

AbstractHuman 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

AbstractStimulation 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

AbstractMouse (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

AbstractSerum 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

AbstractAfter 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

AbstractCells 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

AbstractThe 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

AbstractPlasma 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

AbstractReceptor‐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

AbstractCyclic 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

AbstractThe 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.
Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin‐like growth factor I/somatomedin‐C
Authors: Strain Alastair J. , Hill David J. , Swenne Ingemar , Milner R. D. G. 
Published: February 2005

AbstractHepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8–16 weeks gestation obtained following prostaglandin‐induced pregnancy terminations. They were maintained on collagencoated tissue culture dishes in selective arginine‐free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H‐thymidine. The labeling index of hepatocytes was consistently enhanced by 25–250 ng/ml human placental lactogen (HPL), 25–250 ng/ml human growth hormone (HGH), 10–50 ng/ml insulin‐like growth factor l/somatomedin‐C (IGF l/Sm‐C), and 10% dialyzed fetal calf serum, reaching a maximum of three‐ to four‐fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF l/Sm‐C. Although this proportion did not change following treatment with HGH and HPL, IGF l/Sm‐C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF l/Sm‐C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF l/Sm‐C.