bluuebunny/crossref_metadata_2025 huggingface dataset:
Recovery of HeLa cell population growth after treatment with 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB)
Authors: Tamm Igor
Published: February 2005

Dose‐response curves for the inhibition of heterogeneous nuclear RNA (hnRNA) synthesis in HeLa cells by 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB; 5–100 μM; 30 min) are biphasic and indicate the existence of two subpopulations of hnRNA molecules, one highly sensitive and the other completely resistant, as previously reported for molecules >1,000 nucleoides long (Tamm et al., 1976; Sehgal et al., 1976a). In the short‐term experiments, the drug‐sensitive synthesis of hnRNA was inhibited 50% at a DRB concentration of ∼7 μM, and 70% at 20 μM, whereas drug‐resistant synthesis, which comprises ∼20% of total, continued at DRB concentrations as high as 100 μM. After 24 hr of DRB treatment in medium containing 5% fetal calf serum, the increase in cell number in the exponentially growing population was inhibited by only 42% at 20 μM DRB, and the formation of colonies of ≥ ten cells was not decreased. DRB at 40 μM concentration decreased population growth by 76% and colony formation by 63%. Treatment with 60 μM DRB was sufficient to prevent a net increase in cell number and to reduce colony formation by 78%. After termination of treatment, the time required for the surviving population to begin rapid proliferation was directly related to the concentration of DRB used to treat cells and to the duration of treatment. After 24‐hr treatment with 40 μM DRB, cultures recovered within 1 day, whereas after 60 μM DRB, 3–4 days were required. After 40‐hr treatment with 60 μM DRB, 5–6 days were required for recovery, and after 80 μM DRB, 9–11 days. During the “dormant” period the cell number ranged from 15 to 60% of the initial number and was fairly stable for given conditions. After the “dormant” period, recovery was rapid. The population growth rate in cultures undergoing treatment with DRB is directly related to serum concentration; however, the recovery rate during the post‐treatment period is unaffeced by serum concentration.
Regulation of prostaglandin production and ectoenzyme activities in cultured aortic endothelial cells
Authors: Chesterman Colin N. , Ager Ann , Gordon John L.
Published: February 2005

Prostaglandin production, angiotensin‐converting enzyme, and 5′‐nucleotidase were measured in porcine aortic endothelial cells in situ (with a multiwell template on an opened aorta), in primary culture and in subcultures. Changes during culture were monitored and the effects of culture conditions were investigated by growing cells on a biological matrix or on plastic, by adding different sera to the growth medium, and by harvesting cells enzymically or mechanically. Prostacyclin production by endothelium in primary culture is highest immediately after cell isolation and subsequently declines; this pattern is repeated each time the cells are subcultured. The level at which production stabilises is ∼ 200 pgċ106 cells−1ċh−1. Detaching cells by physical means stimulates production much more than enzymic dispersion; the type of serum or the presence of a biological matrix does not alter prostaglandin production. The relative amount of prostaglandin E produced increases with time, from ∼20% of the prostacyclin production shortly after isolation to > 100% in subcultured cells. None of the culture conditions that we tested altered this trend. Angiotensin‐converting enzyme activity decreases during primary culture, but activity can be sustained by including homologous serum (from whole blood or from platelet‐free plasma) in the culture medium. The method of harvesting cells, or the presence of a matrix, did not affect enzyme activity. 5′‐Nucleotidase also declines during culture, with a progressive decrease in both Km and Vmax from template to primary culture to subcultures. None of the variations in culture conditions prevented this change. Ectoadenosine‐deaminase activity, not detectable in cultured cells, can be measured in the template. Part of this activity was released by the vascular wall and could be due to plasma diffusing from the interstitial space.
Growth of the mitochondrial inner membrane in synchronous cultures of Tetrahymena pyriformis: An examination of phospholipid accumulation
Authors: Hemmingsen Sean M. , Young Paul G.
Published: February 2005

Based on morphological evidence, mitochondrial inner membrane growth has been reported to be discontinuous in heat shock‐synchronized Tetrahymena pyriformis. As a biochemical measure of membrane growth under these conditions, we have examined phospholipid accumulation in the cell. No marked modulation of the accumulation of any of the major phospholipids could be detected through the cell cycle. At least 89% of the cardiolipin in the cells is restricted to the mitochondria, and we have used it as a marker for the growth of the mitochondrial inner membrane. During the heat shock synchrony, cardiolipin accumulates uniformly in parallel with the exponential rate of increase of total cellular phospholipids. These results suggest that at least the phospholipid component of all membrane systems in the cell grow continuously and uniformly. Additionally, we have shown that the total phospholipid content of Tetrahymena increases by a factor of 2.4 per generation following a series of heat shocks. No such net overaccumulation is observed for protein content.
The regulation of macrophage protein turnover by a colony stimulating factor (CSF‐1)
Authors: Tushinski R. J. , Stanley E. R.
Published: February 2005

CSF‐1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF‐1. Removal of CSF‐1 (∼0.4 nM) from exponentially growing BMM cultured in 15% fetal calf serum containing medium decreases the rate of DNA synthesis by more than 100‐fold. Addition of CSF‐1 to these cells causes them to resume DNA synthesis within 12 h. More immediate effects of CSF‐1 were observed on BMM protein metabolism. BMM cultured for 24 h in the absence of CSF‐1 reduce their protein synthetic rate by 50–60%. The protein synthetic rate commences to decrease at 2–3 h after CSF‐1 removal. Readdition of CSF‐1 to BMM previously incubated in its absence causes a return to the protein synthetic rate of exponentially growing cells within 2 h. In the presence of CSF‐1, BMM synthesize protein at a rate of ∼8.7%/h and degrade it at a rate of ∼0.9%/h. Removal of CSF‐1 results in a decrease in the protein synthetic rate to ∼3.4%/h and an increase in the rate of protein degradation to ∼3.4%/h. The rate of protein synthesis by BMM increases linearly with CSF‐1 concentration over the range of concentrations stimulating both survival and proliferation, while the rate of protein degradation decreases exponentially over the range of concentrations stimulating survival without proliferation. Therefore, it appears that the stimulation of the rate of protein synthesis and inhibition of the rate of protein degradation are two distinct effects of CSF‐1, both part of the pleiotropic response to this growth factor. The inhibition of the rate of protein degradation by CSF‐1 may be most significant for its survival inducing effect.
Degradation of microinjected proteins: Effects of lysosomotropic agents and inhibitors of autophagy
Authors: Rote Kevin V. , Rechsteiner Martin
Published: February 2005

HeLa cells, injected with radioiodinated proteins by fusion with RBC ghosts, were exposed to inhibitors of lysosomal proteolysis and autophagy. The degradation of injected [125I]bovine serum albumin (BSA) was unaffected by chloroquine, NH4Cl, nocodazole, colcemid, puromycin, cycloheximide, or enucleation. Although degradation of [125I]lactate dehydrogenase (LDH) and [125I]pyruvate kinase (PK) was inhibited one‐third by chloroquine or ammonia, their degradation was unaffected by the other compounds. In contrast, enhanced degradation of 125I‐PK resulting from depriving injected HeLa cells of amino acids and serum was inhibited 70% by colcemid and abolished by chloroquine or ammonia. Similarly, degradation of [14C]sucrose‐labeled BSA‐polylysine conjugates that entered HeLa cells by endocytosis was inhibited as much as 80% by chloroquine and ammonia.

Sensitivity of both enhanced proteolysis and degradation of exogenous proteins to ammonia or chloroquine indicates they are effective inhibitors of lysosomal proteolysis in HeLa cells. Failure of ammonia or chloroquine to inhibit degradation of injected 125I‐BSA and the modest inhibition of degradation of injected 125I‐LDH or 125I‐PK indicates that virtually all BSA molecules and most PK or LDH molecules are degraded by a nonlysosomal proteolytic system. Components of this degradative system are present in vast excess or are long lived, since inhibition of protein synthesis for 20 hr had no effect on the degradation of injected proteins.
Susceptibility to merocyanine 540‐mediated photosensitization: A differentiation marker on murine hematopoietic progenitor cells
Authors: Meagher Richard C. , Sieber Fritz , Spivak Jerry L.
Published: February 2005

Merocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane‐bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540‐mediated photo‐sensitization. Late erythroid progenitors (CFU‐E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU‐E), megakaryocyte progenitors (CFU‐Meg), day 7‐spleen colony forming cells (day 7‐CFU‐S), granulocyte/macrophage progenitors (CFU‐GM), and day 11‐spleen colony forming cells (day 11‐CFU‐S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies (“clusters”). We conclude that sensitivity to MC 540‐mediated photosensitization is developmentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late‐appearing spleen colonies are the progeny of two distinct classes of progenitor cells.
The respiration‐linked limiting step of tumor cell transition from the non‐cycling to the cycling state: Its inhibition by oxidizable substrates and its relationships to purine metabolism
Authors: Olivotto M. , Caldini R. , Chevanne M. , Cipolleschi M. G.
Published: February 2005

The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G1‐S transition of these cells, unaffected by 2,4‐dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G1‐S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration‐linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration‐linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.
Growth‐stimulating effect of catecholamines on rat aortic smooth muscle cells in culture
Authors: Blaes Nelly , Boissel Jean‐Pierre
Published: February 2005

The effect of epinephrine was tested on the proliferation of rat arterial smooth muscle cells (SMC) in secondary cultures. Epinephrine added daily to the culture medium caused a striking stimulation of growth. The effect increased with time and was dose‐dependent. Maximal stimulation was observed at a concentration of 1010−5 M and after 72 hours. At higher concentrations (10−3 M) epinephrine exhibited toxic effects on SMC. When SMC were maintained quiescent by deprivation of serum, the subsequent addition of epinephrine required serum to significantly enhance growth. This growth stimulation increased with serum concentration (from 0.1 % to 10%). All the adrenergic agonists tested were found to stimulate SMC growth, with an activity classified by decreasing order as follows: norepinephrine > epinephrine > isoproterenol. Finally, this mitogenic response of SMC to catecholamines was specific since it could be blocked by adrenergic blocking agents, phentolamine being more efficient than propranolol in that connection. The results suggest that epinephrine and other catecholamines may act as growth factors for aortic SMC, at least in rat, mostly through adrenoreceptors.
Effect of 5‐azacytidine treatment on mouse embryonal carcinoma cells
Authors: Cremisi Chantal
Published: February 2005

Several properties of embryonal carcinoma (EC) cell lines, such as multipotent PCC4‐aza‐1 cells and nullipolent F9 cells originating from murine teratocarcinoma cells, were examined after treatment with 5‐azacytidine, which produces undermethylated DNA. Drug‐treated PCC4‐aza‐1 cells exhibited morphological changes and differentiated, whereas azacytidine‐treated F9 cells displayed no detectable morphological change. After treatment with 5 azacytidine, PCC4‐aza‐1 cells, whether or not they differentiated, as well as F9 cells, became permissive for polyoma even though both cell types are usually resistant to polyoma. In contrast, only the differentiated azacytidine‐treated PCC4‐aza‐1 cells became sensitive to SV40 infection, i.e., synthesized T antigen, despite the resistance normally shown by such cells to this viral infection. In some PCC4‐aza‐1 and F9 cells, drug treatment induced expression of H2 antigen but did not derepress plasminogen activator synthesis. These results suggest that undermethylation of certain cellular genes in PCC4‐aza‐1 and F9 cells is correlated with the establishment of Py permissivity, SV40 sensitivity, H2 antigen expression, and the triggering of a differentiation process. The relationship between the expression of these characters and differentiation is discussed.
Proliferative effects of purified granulocyte colony‐stimulating factor (G‐CSF) on normal mouse hemopoietic cells
Authors: Metcalf D. , Nicola N. A.
Published: February 2005

When granulocyte colony‐stimulating factor (G‐CSF), purified to homogeneity from mouse lung‐conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G‐CSF, a small proportion of macrophage and granulocyte‐macrophage colonies also developed. G‐CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence‐activated cell sorter, indicating that G‐CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G‐CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G‐CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte‐macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G‐CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G‐CSF was mixed with purified granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or macrophage colony‐stimulating factor (M‐CSF), the combination stimulated the formation by adult marrow cells of more granulocyte‐macrophage colonies than either stimulus alone and an overall size increase in all colonies. G‐CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM‐CSF.
Isozymes of creatine kinase in mammalian cell culures
Authors: Van Brussel Elizabeth , Yang Jing J. , Seraydarian Maria W.
Published: February 2005

Previous studies on the energy metabolism of rat myocardial cells in culture supported the hypothesis that the creatine‐phosphocreatine–creatine kinase system plays an important role in the intracellular transport of energy from the mitochondria to the myofibrils and in the regulation of energy production coupled to energy utilization in this model system. Effective functional compartmentation of ATP could result from the binding of creatine kinase to cellular organelles (e.g., myofibrils and mitochondria) such that high energy charge at the myofibrils is maintained by the reverse creatine kinase reaction, while phosphocreatine is synthesized mainly at the mitochondria in the forward creatine kinase reaction. It was, therefore, essential to demonstrate the presence of mitochondrial creatine kinase in the cultured myocardial cells to support this hypothesis, particularly since the mitochondrial creatine kinase was reportedly absent in fetal hearts. Using electrophoresis on cellulose acetate strips, the mitochondrial creatine kinase isozyme, as well as MM, MB, and BB isozymes, have now been demonstrated in myocardial cultures derived from neonatal rats. The mitochondrial creatine kinase increased with age in culture and with age of animal from which the culture is derived. Furthermore, the addition of creatine to culture media stimulates its synthesis. The mitochondrial creatine kinase isozyme was not detected in nonmuscle cells in culture derived from the neonatal rat hearts, nor in L6 muscle cell line. Phosphocreatine was present in all cells, but the regulation of energy metabolism and energy shuttle by creatine‐phosphocreatine–creatine kinase could be operative only in the cells where the mitochondrial creatine kinase is present. This regulatory mechanism provides for an efficient system concomitant with the continuous energy demand of the myocardium; it is not ubiquitous and its development in myocardial cells seems to be triggered postnatally.
Induction of 86Rb Fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes
Authors: Grinstein S. , Cohen S. , Sarkadi B. , Rothstein A.
Published: February 2005

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low‐K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+ ‐ and volume‐induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane‐rich vesicle fraction, enriched over 11‐fold in 5′‐nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside‐out and trapped 86Rb in an osmotically active compartment of ∼1.3 μl/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP‐dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume‐ and Ca2+‐induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.
Cell shape and growth regulation in skeletal muscle: Exogenous versus endogenous factors
Authors: Vandenburgh Herman H.
Published: February 2005

Passive stretch (10–12%) of tissue‐cultured avian skeletal myotubes in serum‐containing medium stimulates myotube growth in a manner analogous to hormonal stimulation of adult muscle. The resulting increase in myotube length is accompanied by marked reduction in the number of surface microvilli seen by scanning electron microscopy. We investigated the possible involvement of exogenous growth factors in the transduction of stretch‐induced alterations in cell shape into the concurring biochemical changes that are associated with cell growth. We show that the acute stimulation of myotube amino acid transport and protein synthesis by stretch are independent of serum growth factors in the culture medium by evidence obtained from serum dose‐response experiments. The myotubes synthesize and secrete high molecular weight factors into their culture medium, which regulates myotube amino acid transport and protein synthesis. Stretch of the myotubes did not alter the appearance of these factors in the culture medium. The initial growth‐related biochemical alterations induced by myotube stretch in vitro thus depend only on events intrinsic to the cells. However, subsequent stretch‐induced growth of the myotubes occurs only in serum‐containing medium. There are both serum‐independent and serum‐dependent steps in the transduction of the stretch stimulus into myotube growth.
Reduced free‐methionine in methionine‐dependent SV40‐transformed human fibroblasts synthesizing apparently normal amounts of methionine
Authors: Stern Peter H. , Mecham James O. , Wallace C. Douglas , Hoffman Robert M.
Published: February 2005

Many different types of cancer cells have been shown to be methionine‐dependent. These cells, unlike normal cells, grow poorly or not at all when methionine is replaced by its immediate precursor homocysteine in the growth medium (Met−Hcy+ medium). We have previously shown that apparently normal total amounts of methionine are synthesized by methionine‐dependent SV40‐transformed human fibroblasts. However, methionine‐dependent cells in Met−Hcy+ medium accumulate reduced amounts of S‐adenosylmethionine (AdoMet) and elevated amounts of S‐adenosylhomo‐cysteine (AdoHcy) that together probably limit growth. In this report, we demonstrate that the amount of free methionine is low in methionine‐dependent SV40‐transformed human fibroblasts in Met−Hcy+ medium compared to normal human diploid fibroblasts. In contrast, in Met+Hcy− medium, the amount of free methionine is comparable in both cell types. The deficient pool of free methionine in methionine‐dependent cells in Met−Hcy+ medium allows only low amounts of AdoMet to be formed. However, large amounts of the biosynthesized methionine are channeled into protein synthesis. Possible mechanisms are discussed to explain this cancer‐associated metabolic defect.
Anchorage dependence and Ca2+ requirement are independently modulated by hydrocortisone hormone in rat C6 glioma cells
Authors: Armelin Mari C. S. , Armelin Hugo A.
Published: February 2005

We had previously shown that hydrocortisone (Hy), a glucocorticoid hormone, regulates the expression of the transformed phenotype of rat C6 glioma cells. The hormone reversibly renders C6 cells dependent on anchorage and high Ca2+ concentration for growth. We had also isolated Hyresistant C6 variants in agarose suspension cultures. Here we report that Hyresistant variants selected in high (1.8mM) Ca2+ medium become growth‐arrested in low (30 μM) Ca2+ medium upon hormone treatment. We conclude that Hy‐induced anchorage dependence and Hy‐induced high Ca2+ requirement for growth of C6 glioma cells, are two independent phenotypes.
Distribution and mobility of concanavalin a receptors on isolated guinea pig epidermal cells at various stages of differentiation
Authors: Takigawa Masahiro , Danno Kiichiro , Furukawa Fukumi
Published: February 2005

Trypsinized guinea pig epidermal cells were separated by velocity sedimentation at unit gravity. Based on the relationship between cell size and both morphological and functional aspects of differentiation, the cells were classified as lower (a diameter <12.5 μM), middle (a diameter between 12.5 and 15 μM), and upper (a diameter >15 μM) epidermal cells. Fresh cells exposed to rhodaminated concanavalin A (Con A) were sedimented and reacted with fluoresceinated anti‐Con A serum to distinguish cell surface Con A from intracellular lectin. Labeling at 4°C resulted in a uniform surface distribution of Con A irrespective of cell size. After a 1‐hr incubation of Con A‐labeled cells in lectin‐free medium at 37°C, lower epidermal cells and approximately half of middle epidermal cells internalized Con A/receptor complexes by endocytosis while lectin remained diffusely on the remainder of middle epidermal cells and upper epidermal cells. By electron microscopy, ferritin‐Con A was clustered on surface areas and invaginations of the plasma membrane before being endocytosed. We concluded that the differentiation of epidermal cells was accompanied by progressive decrease in endocytosis and, most probably, mobility of Con A receptors.
Transferrin receptor expression during exponential and plateau phase growth of human tumour cells in culture
Authors: Musgrove E. , Rugg C. , Taylor I. , Hedley D.
Published: February 2005

Transferrin receptor expression by the human tumour cell lines CCRF‐CEM leukaemia and PMC‐22B melanoma was studied, measuring the specific binding of fluorescein isothiocyanate (FITC)‐labelled transferrin using a fluorescence‐activated cell sorter. By measuring the fluorescence of cells stained at subsaturating concentrations of conjugate it was possible to calculate the average numbers of receptors per cell and the binding affinity by Scatchard analysis. These values (1.9 × 105 binding sites/cell, KA 1.2 × 109 M−1 for CCRF‐CEM during exponential growth and 6.9 × 104 binding sites/cell, KA 1.4 × 10−9 M−1 for PMC‐22B) are in close agreement with previously published data obtained using radiolabelled transferrin. The present method, however, allowed the transferrin receptor expression of individual cells within a population to be measured and thus it has been possible to test the hypothesis that transferrin receptor is a marker for cycling cells. Frequency‐distribution histograms of transferrin receptor showed a wide range of values for both cell lines during exponential growth. When the extreme ranges were sorted and the cells examined for cellular DNA content it was found that those with the highest transferrin receptor expression were enriched with cells in S, G2, and M phases of the cell cycle, whereas those with low transferrin receptor expression were mainly in G1. However, two‐parameter‐correlated dot plots of transferrin receptor expression versus DNA content showed there was considerable overlap between the ranges of receptor expression for the different cell cycle compartments. Using a stathmokinetic method we have measured the proportion of quiescent cells in fed plateau phase cultures. Transferrin receptor expression was downgraded under these growth conditions but, contrary to expectation, the decline affected the population uniformly, without the emergence of a distinct, transferrin receptor‐negative subpopulation corresponding to the increasing proportion of quiescent cells. Thus, although transferrin receptor expression bears some relation to cell cycle phase and reflects the proliferative activity of populations of cells, it is incapable of identifying individual cells which are out of cycle.
In vitro characteristics of the lipid‐filled interstitial cell associated with postnatal lung growth: Evidence for fibroblast heterogeneity
Authors: Maksvytis Harvey J. , Niles Richard M. , Simanovsky Leonid , Minassian I. A. , Richardson Laura L. , Hamosh Margit , Hamosh Paul , Brody Jerome S.
Published: February 2005

This study explores the in vitro modulation of the lipid‐filled phenotype of the lipid interstitial cell (LIC) isolated from the developing rat lung. Isolated LIC lose their cytoplasmic lipid droplets when cultured in fetal bovine serum (FBS) but retain their potential for lipid storage, since they rapidly reaccumulate lipid when subcultured in neonatal rat serum (NRS) and to a lesser extent in adult rat serum (ARS). The return of LIC to a lipid‐filled state may not represent cell differentiation, since it occurs in the presence of bromodeoxy‐uridine. NRS contains twice the free fatty acids (FFA) of FBS and ARS, and doubling the FFA concentration of FBS and ARS increases LIC storage lipids. Serum triglyceride (TG) is 10 times higher in ARS and 23 times higher in NRS than in FBS. Since LIC lipoprotein lipase (LPL) activity is in the range of 3T3‐L1 adipocytes (0.56 vs. 1.72 units/mg DNA), the LIC has the potential of incorporating serum lipoprotein‐triglyceride. The LPL activity of LIC is 9–12 times that of fetal and adult rat lung fibroblasts and 50 times that of human lung, trachea, or skin fibroblasts; LIC are probably a source of endothelial LPL in the developing lung. The response of LIC and ARLF cyclic‐AMP to hormones known to influence lipid synthesis or degradation showed that: only LIC responded to glucagon; prostagladin E1 was a more potent stimulus to LIC; isoproterenol was a more potent stimulus to ARLF; and neither cell responded to ACTH. The unique nature of LIC tends to support further the concept of fibroblast heterogeneity within tissues.
Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: Role of receptor and postreceptor steps
Authors: Collins Mary K. L. , Rozengurt Enrique
Published: February 2005

Prolonged treatment of Swiss 3T3 cells with phorbol 12,13 dibutyrate (PDB) rendered the cells refractory to subsequent mitogenic stimulation by both PDB and vasopressin. In contrast, the cells retained full responsiveness to a wide variety of other mitogens. An early response to vasopressin and phorbol esters, inhibition of (125l)‐labeled epidermal growth factor [(125l)‐EGF] binding, was also substantially decreased in PDB pretreated cells. The cross desensitization was not produced by vasopressin; this ligand induced homologous but not heterologous desensitization. Exposure of Swiss 3T3 cells to PDB caused a down regulation of (3H)‐PDB receptors but did not reduce the binding of vasopressin to refractory cells. The time‐course (t1/2 = 7 h) and dependence on PDB concentration (half maximal at 20 nM) for this phorbol ester receptor loss paralleled the induction of the mitogenic desensitizations to both PDB and vasopressin. However, the time‐course of recovery revealed an important dissociation between receptor presence and mitogenic response.

When Swiss 3T3 cultures, which had been pretreated with PDB, were washed to remove this ligand and incubated in its absence for 24 h, both (3H)‐PDB receptors and PDB or vasopressin inhibition of (125l)‐EGF binding were almost completely restored to control levels. However the homologous and heterologous mitogenic desensitizations showed a very different reversal time. After a 24‐h recovery period PDB‐treated refractory cells were still unable to synthesize DNA in response to PDB or vasopressin. The mitogenic desensitizations were however completely reversible; after a 48‐h incubation in the absence of PDB the cells responded fully to the mitogenic actions of PDB or vasopressin. This finding suggests that a further postreceptor step was also desensitized by prolonged PDB treatment. The presence of a low level of cycloheximide during the PDB pretreatment blocked induction of this postreceptor refractoriness. We propose that this refractory postreceptor step selectively blocks both PDB and vasopressin stimulation of DNA synthesis and may represent the point at which the mitogenic pathways of phorbol esters and vasopressin converge.
Interaction of T lymphocytes and macrophages with cultured vascular endothelial cells: Attachment, invasion, and subsequent degradation of the subendothelial extracellular matrix
Authors: Savion N. , Vlodavsky I. , Fuks Z.
Published: February 2005

Circulating macrophages and T lymphocytes can invade the vascular endothelium and migrate from the circulatory system to an extravascular compartment such as inflammatory organs. In an in vitro model system we have examined the capacity of murine T lymphocytes and peritoneal macrophages to attach and invade a confluent vascular endothelial cell monolayer and to degrade sulfated proteoglycans in the subendothelial extracellular matrix.

Concanavalin A and antigen‐specific (egg albumin) activated T lymphocytes labeled with [3H]thymidine attached to the apical surface of the vascular endothelium in a time‐dependent manner. A subsequent invasion of the endothelial cell monolayer was observed by scanning electron microscopy. Both activated T lymphocytes and murine macrophages degraded the [35S]O4=‐containing fragments in a process which required cell‐matrix contact but was not dependent on serum proteases.

Sulfated glycosaminoglycan chains produced from matrix proteoglycans by treatment with papain or alkaline borohydride were 3–4 times larger than the cell‐mediated degradation fragments. This suggests that both macrophages and T lymphocytes elaborate upon stimulation an endoglicosidase capable of cleaving glycosaminoglycans specifically and releasing heparan sulfate‐rich fragments. The ability of activated cells of the immune system to attach and invade the vascular endothelium and to degrade sulfated proteoglycans is very similar to that reported for highly metastatic tumor cells.
An examination of the efficiency of glucose and glutamine as energy sources for cultured chick pigment epithelial cells
Authors: Barbehenn E. K. , Masterson E. , Koh Shay‐Whey , Passonneau J. V. , Chader G. J.
Published: February 2005

Embryonic chick pigment epithelial cells in culture require glucose as their major energy source for long‐term growth, pigment formation, and colony organization. Cell number increases with glucose concentration at least up to 5.0 mM. Cells can be grown with glutamine as the major energy source but produce comparable cell numbers for only the first 3 days in culture, after which they cease growing. However, they are able to metabolize glutamine at a two to sixfoid higher rate than cells grown in the presence of glucose as measured by CO2 release and by incorporation into protein. In cells grown in the presence of both glucose and glutamine, basal ATP levels were 31.1 nmoles/mg protein; P‐creatine averaged 15.2 nmoles/mg protein and showed marked variability between experimental groups. During starvation, P‐creatine levels fell while ATP levels remained relatively constant. Glucose was required for the recovery of P‐creatine to prestarvation levels when measured 5 min after refeeding. Because of these marked changes in P‐creatine concentration as a function of nutritional status, the ATP/P‐creatine ratio becomes a useful measure of the energy state of the cell.
Compound 48/80 impairs cytokinesis in murine leukemiccells
Authors: Darzynkiewicz Zbigniew , Carter Sally
Published: February 2005

Compound 48/80 (poly‐p‐methoxyphenethylmethylamine), an agent commonly used to trigger degranulation of mast cells, at concentrations of 5–20 μg/ml suppresses the proliferation of L1210 and Friend leukemic cells in vitro, inducing the formation of giant cells, which are polykaryons. Both the proportion of polykaryons in cultures and their size (which reflects the number of nuclei per polykaryon) increase during growth in the presence of 48/80 up to 48 hr; thereafter, the cells lose viability. A predominant number of nuclei in these polykaryons contain a 4C, or higher DNA content. The data indicate that compound 48/80 impairs the cleavage (cytokinesis) and perhaps mitotic processes. Mechanisms by which compound 48/80 induces the described effects are unknown but may be related to the polycationic nature of the polymer and its interaction with the cell membrane. Certain attributes of compound 48/80 suggest that this or similar polymers may have value as research tools for the study of regulatory mechanisms involved in cell division.
Calmodulin antagonists sensitize cells to pseudomonas toxin
Authors: Sundan Anders , Sandvig Kirsten , Olsnes Sjur
Published: February 2005

L cells and mouse 3T3 cells, which are very sensitive to Pseudomonas aeruginosa exotoxin A (PEA), were protected with weak bases and low concentrations of monensin. BHK cells and a number of other cell lines which are much less sensitive to PEA were much less protected under these conditions. Trifluoperazine, dansylcadaverine, and several other calmodulin antagonists strongly sensitized BHK cells to the toxin whereas they did not affect the sensitivity of the mouse 3T3 and L cells. The sensitization of the BHK cells was counteracted by treatment with weak bases or low concentrations of monensin. Calmodulin antagonists also sensitized cells to toxin which had become inaccessible to antitoxin, indicating that the effect of the calmodulin antagonists is exerted on a process taking place after the toxin is endocytosed.
Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts
Authors: Stieber Anna , Gonatas Jacqueline O. , Gonatas Nicholas K.
Published: February 2005

A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4°C of the conjugate was observed over the entire plasma membrane, including “coated” and smooth pits. Endocytosis of HRP and the WGA‐HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.
Arrest states in a set of mutants of rat 3Y1 cells temperature‐sensitive for entering S phase
Authors: Zaitsu Hirokazu , Kimura Genki
Published: February 2005

Four temperature‐sensitive (ts) mutants of rat 3Y1 cells (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested at 39.8°C mainly with a 2N DNA content (temperature‐arrested cells). The states of these cells were compared with findings in case of cells arrested at 33.8°C at saturation density (density‐arrested cells), with regard to the ability to enter S phase after release from arrest or after serum stimulation at 39.8°C. With the 3Y1tsD123, the ts defect is an event which seems essential for the initiation of S phase and occurs after mitosis but not after release from the density arrest. The defect in 3Y1tsF121 related to the efficiency of utilization of serum component(s). In case of 3Y1tsG125, the state of temperature arrest appeared to locate between the state of density arrest and the beginning of S phase. There was no significant difference between the density‐ and the temperature‐arrested cells, in case of 3Y1tsH203.
Collagen synthesis by cloned pulmonary artery endothelial cells
Authors: Macarak Edward J.
Published: February 2005

Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U‐14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)‐cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak I contained a collagen which contained approximately 6% of the 3‐hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of α1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to α‐chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin‐resistant components had mobilities slower than that of α(III) and were not disuifide‐bonded.
Partial characterization of a hepatocyte growth factor from rat platelets
Authors: Russell William E. , McGowan Joan A. , Bucher Nancy L. R.
Published: February 2005

Rat serum has been shown to stimulate DNA synthesis in primary cultures of adult rat hepatocytes 2–3 times more potently than serum from several other mammalian sources, including humans. Parallel to its stimulation of thymidine incorporation into DNA, rat serum increased the total DNA content of the hepatocyte cultures over time, and also increased the frequency of nuclear labeling and mitosis. Moreover, normal rat serum, derived from whole blood (NRS), stimulated DNA synthesis in hepatocytes twice as effectively as platelet‐poor rat serum, derived from plasma (ppNRS). Addition of a rat platelet lysate (RPL) to ppNRS restored the activity to equal that of NRS. The avid binding of the active principle to CM Sephadex and its sensitivity to trypsin digestion suggest that it is a cationic polypeptide with an apparent molecular weight of about 65,000, as determined by gel filtration. It was inactivated by reduction of disulfide bonds, or by exposure to pH below 5.5, to NaCl concentration below 0.05 M, to 65°C for 30 min, or to 100°C for 10 min. Although it resembles the human platelet‐derived mitogen platelet‐derived growth factor (PDGF) in several of its properties, it differs in others. Hence the hepatocyte growth factor from rat platelets, which accounts for 50% of the DNA synthesis‐stimulatory activity of rat serum, appears to be a distinct entity.
Cellular responses to external ATP which precede an increase in nucleotide permeability in transformed cells
Authors: Weisman Gary A. , De Barun K. , Friedberg Ilan , Pritchard Robert S. , Heppel Leon A.
Published: February 2005

Transformed mouse fibroblasts, such as 3T6, exhibit an increase in plasma membrane permeability to nucleotides and other normally impermeant molecules when incubated with external ATP in an alkaline medium low in divalent cations. Increased nucleotide permeability, induced by external ATP, occurs after a 3‐ to 5‐min lag period. Prior to this event, there is a dramatic Na+ influx and K+ efflux, a significant reduction in the levels of intracellular ATP and organic phosphates, and a reduction in the plasma membrane potential. Accordingly, we postulate that these cellular responses to external ATP play a role in the efflux of nucleotides.

Ouabain, a specific inhibitor of the plasma membrane (Na+, K+)‐ATPase, acts together with low concentrations of external ATP to increase nucleotide permeability in 3T6 cells. This effect occurs at concentrations of ouabain and ATP which alone do not increase nucleotide permeability. In addition, ouabain and low concentrations of ATP alone have little effect on the level of intracellular ATP. This is in contrast to energy inhibitors and uncouplers which appear to enhance nuclectide permeability by lowering the intracellular ATP concentration. Ouabain alone causes a threefold increase in intracellular Na+ levels and a similar reduction in intracellular K+ levels under our experimental conditions, supporting the idea that ion fluxes are involved in the mechanism of permeabilization.
Regulation of mitotic activity and the cell cycle in primary chick muscle cells by neurotransferrin
Authors: Popiela Heinz , Taylor Daniel , Ellis Stanley , Beach Robert , Festoff Barry
Published: February 2005

We previously demonstrated that neurotransferrin (NTF), a transferrin extracted from adult chicken peripheral nerves, promotes growth of primary chick muscle cells in the absence of embryo extract. NTF was shown to stimulate DNA synthesis and cell proliferation. In the present study, we demonstrate that NTF is a mitogen using two independent methods; counts of orcein‐stained mitotic figures and analysis of cell cycle kinetics with a fluorescence‐activated cell sorter. In low‐density cultures mitotic activity increases with increasing doses of NTF followed by a plateau at concentrations greater than 6 μg/ml. Residual, embryonic mitotic activity progressively declines with time after plating muscle cells in the absence of NTF. Absence of NTF for 2 days causes cells to lose irreversibly their myogenic potential. In the presence of NTF, mitotic activity increases for 2 days followed by a decline concurrent with myoblast fusion and formation of myotubes. Cell cycle analysis showed that NTF addition causes cell populations to shift from Gt to S and G2 + M within 18.5 hr. Muscle cells, plated at high densities in the absence of NTF, show mitotic activities similar to those plated at low densities in the presence of NTF. Addition of NTF to high‐density cultures is ineffective in stimulating mitosis. These studies show that at typical cell plating densities, NTF is a required mitogen for primary chick muscle cell cultures.
Different growth factors stimulate cell division of rat mammary epithelial, myoepithelial, and stromal cell lines in culture
Authors: Smith J. A. , Winslow D. P. , Rudland P. S.
Published: February 2005

A number of single‐cell‐cloned cell lines have been used to examine the growth‐promoting effects of putative mammotrophic agents on the various cell types in normal and neoplastic rat mammary glands. A partially purified novel pituitary‐derived growth factor stimulates only cuboidal epithelial cells to divide whereas fibroblast growth factor (FGF) stimulates the growth of stromal and myoepithelial‐like cells. Epidermal growth factor (EGF) has a widespread but variable growth‐stimulating action, but prolactin and growth hormone are essentially inactive when added alone at a concentration of 5 μg/ml. Phosphoethanolamine stimulates the growth of one epithelial cell line and a derivative myoepithelial‐like cell line, but is inactive on the other cell lines tested. The use of defined cloned cell lines provides a direct and reproducible assay for the identification and purification of inducers of mammary growth.