bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a PDGF‐like mitoattractant produced by NIH/3T3 cells after transformation with SV40
Authors: Bleiberg Ilan , Harvey Anita K. , Smale Georgeann , Grotendorst Gary R.
Published: February 2005

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from non‐transformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I‐PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI < 7.0) of MW < 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40‐transformed NIH/3T3 cells by supplying the cells with their own PDGF‐like growth factor.
Superinduction by cycloheximide of mitogen‐induced secreted proteins produced by Balb/c 3T3 cells
Authors: Hamilton Richard T. , Nilsen‐Hamilton Marit , Adams Greg
Published: February 2005

We describe here some of the characteristics of the regulation of a group of secretory proteins whose secreted levels rise within 2–4 h of adding fibroblast growth factor (FGF), epidermal growth factor (EGF), or serum to quiescent Balb/c 3T3 cells. The levels of these secretory proteins are regulated similarly to the interferons. When cycloheximide is present during the induction period, the amounts of [35S]methionine incorporated into five of these proteins that we have called “superinducible proteins” (SIPs) is increased 2–5 fold. Superinduction of the SIPs is seen also in response to polyribol‐polyriboC, the classical inducer of interferons. None of the SIPs, however, are immunoprecipitated by anti‐β‐interferon antibody. Induction and superinduction of the SIPs is inhibited by actinomycin D. Superinduction occurs at concentrations of cycloheximide that inhibit protein synthesis by at least 85%. The SIPs are not major intracellular proteins; they are barely detectible in cellular peptide growth factor to stimulate DNA synthesis; EGF, FGF, and serum induce the SIPs, whereas insulin does not, and insulin alone weakly stimulates DNA synthesis in these cells. Because FGF, EGF, and serum cause the SIPs to be produced at concentrations of cycloheximide that inhibite 85% of bulk protein and DNA synthesis, it follows that the SIPs are produced directly from the action of the growth factor and not as a consequence of increased growth. Although probably not interferons, in analogy to the lymphokines, the SIPs could be a set of autocrine or paracrine factors that rapidly convey the growth of differentiation signal between cells.
The differentiation of avian skeletal muscle in culture: Changes in responsiveness of adenylyl cyclase to prostaglandin E1 and adrenergic agonists
Authors: Curtis David H. , Zalin Rosalind J.
Published: February 2005

The responsiveness of adenylyl cyclase during avian myogenesis in vitro has been examined. Measurements of cyclic AMP generation in intact cells revealed that the precursor myoblast is highly responsive to prostaglandin E1 (11‐fold maximum stimulation): whereas its response to isoproterenol is much smaller (2‐fold). From the onset of terminal differentiation, responsiveness to the β‐adrenergic agonist increases progressively, reaching a 5.5‐fold maximum response by 96 hr of culture. In contrast, there is little change in the cell population's responsiveness to prostaglandin E1. The rise in catecholamine responsiveness is consistent with previously reported increases in β‐receptors accompanying differentiation. DL‐propanolol blocks the response of myoblasts and myotubes to 10−6 M isoproterenol with the same half maximal inhibition value of 1 × 10−8 mol. The results also suggest a change in the adrenergic character of the receptors and/or coupling to adenylyl cyclase as myoblasts differentiate. First the α‐adrenergic antagonist phentolamine (10−7−10−4 mol) inhibits the myoblast's response but enhances that of the myotube. Second, the potency ratios for the responses to isoproterenol, epinephrine, and norepinephrine shift from 1.1:1.0:1.0 in the myoblast to 3.3:2.1:1.0 in the myotube. The findings are discussed with reference to (1) the role of prostaglandins in the positive control of muscle differentiation and (2) the changes in the catecholamine‐responsive adenylyl cyclase system as an aspect of the expression of the muscle phenotype.
Changes in self‐renewal potential of human leukemic cells (K562): A bidirectional stochastic process
Authors: von Melchner Harald , Höffken Klaus
Published: February 2005

Daughter cells arising from a single cell division in the leukemic cell line K562 have equivalent self‐renewal potential with respect to their ability to form clones in semisolid medium. However, individual cells isolated from these clones in sequence have vastly different abilities in their self‐renewal potentials. Thus, cells originating from a clone with any particular self‐renewal potential exhibit the full range of self‐renewal potentials–‐from highly renewing to none renewing, cells. These results show that self‐renewal potential in the K562 cell line is a random, reversible and partially noninherited characteristic. It is suggested that the stochastic variability of the intraclonal self‐renewal potential of K562 progeny cells either reflects the initial expression of a differentiation program or the expression of the predeterministic portion of the normal myelopoietic differentiation pathway.
Serum‐independent regulation of initiation of DNA synthesis relating to temperature‐sensitive defect in rat 3Y1tsD123 fibroblasts and its compensation by simian virus 40
Authors: Zaitsu Hirokazu , Kimura Genki
Published: February 2005

Randomly proliferating 3Y1stD123 cells are arrested in G1 phase within 24 h after a shift up to 39.8°C (temperature arrest), yet the density‐arrested cells (prepared at 33.8°C) enter S phase at 39.8°C with serum stimulation, with or without preexposure to 39.8°C for 24 h (Zaitsu and Kimura 1984a). When the density‐arrested 3Y1tsD123 cells were preexposed to 39.8°C for 96 h, they lost the ability to enter S phase at 39.8°C by serum stimulation and required a longer lag time to enter S phase at 33.8°C by serum stimulation than did the cells not preexposed to 39.8°C. Simian virus 40 induced cellular DNA synthesis at 39.8°C in the density‐arrested 3Y1tsD123 preexposed to 39.8°C for 96 h. In the absence of serum after a shift down to 33.8°C, the temperature‐arrested 3Y1stD123 cells entered S phase and then divided once. We postulate from these results that (1) that ts defect in 3Y1tsD123 is involved in a serum‐independent process. Once this process is accomplished, its accomplishment is invalidated slowly with preexposure to 39.8°C. This and the serum‐dependent processes occur in parallel but not necessarily simultaneously. The accomplishment of both (all) processes is required for the initiation of S phase. The density‐arrested 3Y1tsD123 cells have accomplished the serum‐independent process related to the ts defect, but have not accomplished serum‐dependent processes. In case of the temperature‐arrested 3Y1tsD123 cells, the reverse holds true. The lag time for entry into S phase depends on the preparedness for the initiation of DNA synthesis (on the extent of accomplishment of each of all processes required for entry into S phase). (2) To induce cellular DNA synthesis, simian virus 40 stimulates directly the serum‐independent process. However, we do not rule out the possibility that simian virus 40 stimulates serum‐dependent processes simultaneously.
Oxonol dyes as monitors of membrane potential: The effect of viruses and toxins on the plasma membrane potential of animal cells in monolayer culture and in suspension
Authors: Bashford C. Lindsay , Alder Glenn M. , Gray Michael A. , Micklem Kingsley J. , Taylor C. Christopher , Turek Paul J. , Pasternak Charles A.
Published: February 2005

Optical indicators of the cationic, cyanine and anionic oxonol classes were used to evaluate the plasma membrane potential of animal cells in suspension and in monolayer culture. The optical signals were calibrated by using diffusion potential either of K+ (in the presence of valinomycin) or of H+ (in the presence of carbonyl cyanide p‐trifluoromethoxyphenylhydrazone; FCCP); both classes of dye gave similar values of plasma membrane potential, in the range −40 to −90 mV for different cell types. Addition of haemolytic Sendai virus or Staphylococcus aureus α‐toxin depolarizes cells and causes them to leak monovalent cations; these effects are antagonized by extracellular Ca2+. Cells infected with vesicular stomatitis or Semliki Forest virus become depolarized during an infectious cycle; infection with other viruses was without affect on plasma membrane potential.
1α, 25‐Dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line
Authors: Franceschi Renny T. , James Wilbur M. , Zerlauth Gerold
Published: February 2005

The ability of the hormonally active vitamin D metabolite, lα,25‐dihydroxyvitamin D3, to affect cell growth, morphology and fibronectin production has been examined using the MG‐63 human osteosarcoma cell line. Hormone treatment reduced cell growth rate, saturation density and [3H]thymidine incorporation. Inhibition was specific for lα,25‐dihydroxyvitamin D3 relative to other vitamin D metabolites (lα,25‐dihydroxyvitamin D3 > 25‐dihydroxyvitamin D3 > 24R,25‐dihydroxyvitamin D3D3), antagonized by high concentrations of serum and readily reversed by removal of lα,25‐dihydroxyvitamin D3 from the culture medium. Hormone treatment also increased cell associated alkaline phosphatase activity up to twofold and altered morphology such that treated cells were more spread out on the culture dish and contained more cytoplasmic processes. Significantly, lα,25‐dihydroxyvitamin D3 increased cellular and medium concentrations of fibronectin, a glycoprotein known to be involved in cellular adhesiveness. MG‐63 cells contain a specific lα,25‐dihydroxyvitamin D3 receptor which may mediate these responses.
Characterization of mouse lymphoma cells with altered nucleoside transport
Authors: Cohen Amos , Leung Changyee , Thompson Ellen
Published: February 2005

A mutant clone (NT‐1) of a T‐cell lymphoma was selected for its ability to grow in HAT medium (hypoxanthine, aminopterin and thymidine) in the presence of the nucleoside transport inhibitor P‐nitrobenzyl‐6‐mercaptoinosine (NBMI). NT‐1 cells contain half the number of NBMI binding sites present on the parental S49 cells and are partially able to transport nucleosides in the presence of the transport inhibitor (NBMI). These observations suggest that the mutant cells are heterozygous for nucleoside transport proteins and contain two types of transport proteins: the first protein can both bind and is inhibited by NBMI similar to the wild type phenotype, and the second is an altered protein. The altered transport protein apparently lost its NBMI binding sites without a parallel loss of nucleoside transport ability suggesting that the nucleoside transported sites are separate from the binding sites of the transport inhibitor.
The influence of cell population density and serum on the (Na+K)‐ATPase of 3T3 cells
Authors: McCaldin Brian , Tenang Effendi M.
Published: February 2005

The (Na+K)‐ATPase of 3T3 cells has been measured as a function of culture density and the type of serum in which the cells were grown. When 3T3 cells were grown in medium containing 10% newborn calf serum their (Na+K)‐ATPase activity increased as the culture density increased and the cells became quiescent at densities of about 17.5 × 104 cells/cm2. When these cells are subcultured into medium containing 10% foetal bovine serum the activity of the enzyme decreases as the culture density increases and the cells attain final densities of about 5 × 104 cells/cm2.
Differential expression of two linked selection genes (HSVI‐tk and Eco.gpt) in transformed teratocarcinoma and in L cells
Authors: Lasserre C. , Thillet J. , Besnard C. , Bucchini D. , Pictet R. , Jami J.
Published: February 2005

Upon transfection of (TK−)F9 teratocarcinoma stem‐cells and (TK−)L fibroblasts with a plasmid carrying two selection genes, Eco.gpt and HSVI‐tk, selection for gpt gene yielded ten times fewer colonies than selection for tk. Only the transformed clones selected for gpt had measurable xanthine guanine phosphoribosyltransferase (XGPRT) activity (Jami et al., 1983). Eco.gpt coding for XGPRT was under the control of simian virus 40 (SV40) early genes' regulating sequences (SV‐gpt). In the present study, it was verified that the low efficiency of gpt selection in mouse cells was not due to the eucaryotic controlling sequences added to the bacterial gene. The transformed clones selected for tk that had no XGPRT activity possessed at least one uninterrupted copy of the composite SV‐gpt gene and as many copies of the transforming plasmid as the cells selected for gpt expression. In a further test, the gpt gene was placed under the control of tk‐regulating sequences and inserted with the tk gene in the same vector. Under these conditions, expression of XGPRT in the transformed clones selected for tk was improved, even though relative selection for gpt remained low.
Similar action of platelet‐derived growth factor and epidermal growth factor in the prereplicative phase of human fibroblasts suggests a common intracellular pathway
Authors: Westermark Bengt , Heldin Carl‐Henrik
Published: February 2005

We have investigated the effects of platelet‐derived growth factor (PDGF) and epidermal growth factor (EGF) in the prereplicative phase of human foreskin fibroblasts cultured under defined conditions in serum‐free MCDB 105 medium. Specific antisera against PDGF and EGF were used to inhibit the stimulation after certain incubation times. It was found that PDGF or EGF had to be present during the major part of the G0G1 phase (> 8 h) in order to cause any appreciable commitment to DNA synthesis; half maximal stimulations were obtained after 9 h and 11 h of incubat ons with PDGF and EGF, respectively. When tested during a suboptimal period of time (6 h), neither an increase in concentration of PDGF or EGF, nor the addition of both growth factors simulataneously caused any appreciable stimulation of DNA synthesis. However, a suboptimal pulse of PDGF, followed by a suboptimal pulse of EGF, or vice versa, led to commitment to DNA synthesis. This finding indicates that PDGF and EGF, at least in part, induce similar intracellular events that transmit the mitogenic signal.
Development of heart cells in culture: Studies using an affinity purified antibody to a myosin light chain
Authors: Klein Irwin , Daood Monica , Whiteside Theresa
Published: February 2005

Cultured neonatal rat heart cells can be used to study the factors that regulate cardiac contractility and myocyte development in vitro. An antibody to the 26,000 dalton light chain of myosin (MLC1), has been produced and purified on a Sepharose 4B affinity column prepared with rat heart myosin. When primary cultures of myocytes are studied by indirect immunofluorescence using the antibody a predictable pattern of myofibrillar structure is observed to develop over 72h. This myosin cytoskeleton is highly organized and the myosin fibrils exhibit cross striations. The antibody does not stain non‐muscle heart cells and there is no evidence for myocyte division in culture. The qualitative immunofluorescent pattern of myosin organization in the same in both spontaneously beating and in non‐contracting cells.
Biotin and choline replace the growth requirement of Madin‐Darby canine kidney cells for high‐density lipoproteins
Authors: Cohen David C. , Gospodarowicz Denis
Published: February 2005

MDCK cells maintained on extracellular matrix (ECM)‐coated dishes and exposed to Dulbecco's modified Eagle's medium (DME) supplemented with transferrin and either high‐density lipoproteins (HDLs) or phosphatidyl choline (PC) liposomes have a growth rate and final cell density similar to those of cultures exposed to serum‐supplemented DME. When MDCK cells are exposed to a medium consisting of a mixture (1:1) of DME and F12 medium (D/F), the addition of transferrin (10 μg/ml) alone supports cell growth and the presence of HDLs or PC liposomes is no longer required. MDCK cells exposed to D/F medium supplemented with transferrin can be passaged for more than 50 generations in total absence of serum. The F12 components that support growth in the absence of HDLs or PC liposomes are biotin (which is absent in DME) and choline (which is present in insufficient concentration in DME). Supplementation of DME with transferrin, biotin (3.6 ng/ml), and choline (10 μg/ml) allows optimal growth of MDCK cells and permits serial propagation through more than 50 generations. The growth requirement of MDCK cells for HDLs or PC liposomes can therefore be replaced by adequate concentrations of biotin and choline. The widely observed fact that a combination of DME/F12 medium is more effective than DME alone in supporting cell growth may be due in part to the lack of biotin and suboptimal choline concentration in DME.
Temperature effects upon the time course of contraction and reextension in Stentor coeruleus
Authors: Nielsen Lis Engdahl , Rotne Jens
Published: February 2005

A double‐flash microphotographic technique has been used to follow the variation with temperature of the following kinetic parameters related to the contraction and reextension of the ciliate Stentor coeruleus, namely the rate of contraction, the initiation time before contraction, the rate of reextension and the initiation time before reextension, all described by first order kinetics. Activation enthalpies, entropies and free energies related to the above mentioned parameters were calculated from the variation of the rate constants with temperature. The enthalpies and entropies appear to be of minor interest compared to the free energies. For the contraction and the initiation of contraction the ΔG‡ values obtained were 14 and 15 kcal/mole, respectively, while the reextension and the initiation of reextension both were represented by a value of ΔG‡ about 19 kcal/mole. These results are compared to activation parameters for different motile systems and for the formation and breakdown of ATP‐myosin complexes. A model for the contraction and reextension processes is proposed in accordance with the results measured.
Transcription of ribosomal RNA is differentially controlled in resting and growing BALB/c 3T3 cells
Authors: Perrone‐Bizzozero Nora , Iapalucci‐Espinoza Silvia , Medrano Estela E. , Franze‐Fernández María T.
Published: February 2005

Shortly after serum‐deprived BALB/c 3T3 fibroblasts are stimulated to grow in medium containing 10% calf serum, the RNA polymerase I activity in permeabilized cells shows a two‐fold increase over the values observed in either serum‐deprived or density‐inhibited resting cells. Inhibition of protein synthesis by pactamycin or cycloheximide specifically reduces the enhanced RNA polymerase I activity in serum‐stimulated cultures without affecting the values in resting cells. On the other hand, inhibition of rRNA processing by the nucleoside analogs 5‐fluoruridine and toyocamycin decreases the rate of 45S rRNA transcription in serum‐stimulated cells but has no effect on the values found in resting cultures. These data suggest that the regulation of rRNA transcription occurs by two different mechanisms, depending on the growth state of the cell. One mechanism, in serum‐stimulated cells, is dependent on a continuous protein synthesis and a correct 45S rRNA processing; the other, in resting cells, is independent of these two parameters.
An analysis of multiple mechanisms of adenosine toxicity in baby hamster kidney cells
Authors: Archer Sherry , Juranka Peter F. , Ho Jeok H. , Chan Voon‐Loong
Published: February 2005

Analysis of the response of baby hamster kidney cells to adenosine in the presence of the adenosine deaminase inhibitor erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine has revealed two distinct mechanisms of toxicity. The first is apparent at low concentrations of adenosine (< 5 μM) and is dependent upon the presence of a functional adenosine kinase. The initial toxicity is abolished by uridine, is unrelated to the inhibition of ribonucleotide reductase, and is accompanied by a decrease in the size of the pyrimidine nucleotide pool. Toxicity at higher concentrations of adenosine is adenosine kinase independent and is potentiated by homocysteine thiolactone. An elevation in the intracellular level of S‐adenosylhomocysteine, which was observed following treatment with higher concentrations of adenosine (> 10 μM), is believed to mediate toxicity at these levels. Interestingly, BHK cells were resistant to intermediate levels of adenosine. The mechanism of resistance is currently unknown, but appears unrelated to a lack of inhibition of aenosine deaminase. It is proposed that substrate inhibition of adenosine kinase may be a determinant of this property.
Characterization of neutrophil alkaline phosphatase‐inducing factor (NAP‐IF)
Authors: Sato Noriharu , Asano Shigetaka , Mori Mayumi , Ueyama Yoshito , Sugano Kentaro , Ohsawa Nakaaki , Takaku Fumimaro
Published: February 2005

A factor, termed neutrophil alkaline phosphatase‐inducing factor (NAP‐IF), that has the capacity to increase the NAP activity of granulocytes was characterized by using two samples: cystic fluid (CF) and conditioned medium of a tumor cell line (T3M5). The molecular weight of NAP‐IF was shown to be between 13,000 and 45,000, and its isoelectric point was between 5.5 and 6.2. It was sensitive to heat and proteolytic enzymes, but was resistant to DNase and RNase, suggesting that NAP‐IF is an acidic protein or glycoprotein. These characteristics of NAP‐IF seem to be similar to those of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) that is also present in the CF. NAP‐IF rich fractions obtained by isoelectric focusing from CF were also found to be rich in a subclass of GM‐CSF: granulocyte‐CSF (G‐CSF). Furthermore, a high correlation was noted between the activities of G‐CSF and NAP‐IF (γ = 0.798, P < 0.005). These results suggest that the two activities, i.e., G‐CSF and NAP‐IF, may be attributable to an identical macromolecule.
Investigation of adenylate energy charge, phosphorylation potential, and ATP concentration in cells stressed with starvation and heat
Authors: Calderwood Stuart K. , Bump Edward A. , Stevenson Mary Ann , Van Kersen Ine , Hahn George M.
Published: February 2005

We have attempted to determine the appropriate parameter of energy status assoiciated with the survival of CHO fibroblasts under starvation conditions. Survival correlated well with adenylate energy charge (EC) but not so well with the phosphorylation potential or ATP concentration. Starved cells exhibited the capacity to resist (transiently) decreases in both EC and survival. A fall in EC was associated with decreased survival. Using this correlation, we subsequently investigated whether killing after thermal stress occurred by a mechanism analogous to starvation, perhaps due to inhibition of energy yielding pathways. This hypothesis proved to be false; over 99% of cells were killed before a decrease was observed in any of the parameters of energy status. Cells were, however, sensitized to heat under nutritionally deprived conditions, a finding which may be significant for tumor treatment by heat in vivo.
F.9 Embryonal carcinoma cell calcitonin autocrine system: Correlation between immunoreactive calcitonin secretion and calcitonin receptor number
Authors: Binet Elisabeth , Laurent Pascale , Evain‐Brion Daniele
Published: February 2005

Mouse teratocarcinoma cells in culture offer an in vitro system to study the initial steps of embryogenesis. It has been suggested that, at such early stages, cell functions are regulated by an autocrine process in which embryonic cells produce factors that in turn act on themselves. F.9 cells possess specific membrane receptors for calcitonin (CT) (120 fmol/mg of protein, Ka, = 3.5 × 108 M−1). These cells produce CT detected by heterologous radioimmunossay in serum‐free culture‐conditioned medium (75 pg/107 cells/12 h). When F.9 cells are incubated in serum‐free medium, CT binding and secretion concomitantly drop by 50% within the first 2 h, then increase progressively to an upper plateau after the sixth hour. Preincubation with 10−6 M CT leads to disappearance of CT receptors and CT secretion in the culture medium up to 6 h. Avoiding accumulation of CT in the medium by a continuous flow rate for 6 h leads to a progressive decrease of CT receptors. In addition, retinoic acid treatment of cells induces a parallel progressive decrease of CT receptor number and of total CT synthesis. These results suggest a reciprocal regulation of CT receptors and CT secretion, or a close relationship between their regulations.
Altered degradation of epidermal growth factor in a diphtheria toxin‐resistant clone of KB cells
Authors: Haigler Harry T. , Wiley H. Steven , Moehring Joan M. , Moehring Thomas J.
Published: February 2005

We have investigated the cellular fate of epidermal growth factor (EGF) in KB cells and a variant, KB‐R2A, that was isolated based on its resistance to diphtheria toxin and subsequently was shown to be resistant to infection by RNA viruses (Moehring and Moehring, 1972, Infect. Immunity. 6:487–492). Both cell lines bind 125I‐EGF and internalize the cell‐bound hormone at the same rate. However, when the degradation of internalized 125I‐EGF was measured by the release of low molecular weight (mw) hydrolysis products into the medium, the toxin‐resistant KB‐R2A cells degraded the hormone at a drastically reduced rate; 50% and 3% of the cell‐bound 125I‐EGF was degraded and released by 80 min in the KB and KB‐R2A cells, respectively. To investigate the fate of cell‐associated EGF prior to release into the medium, the radioactivity in extracts of cells labeled with 125I‐EGF was fractionated by native gel electrophoresis. In KB cells three peaks of radioactivity other than native 125I‐EGF were resolved. Time course and subcellular fractionation studies showed that the first processed product appeared while the hormone was located in the endocytic vesicles and the appearance of the other two peaks correlated with the arrival of the hormone in the lysosomal compartment. KB‐R2A cells also produced the first intermediate but they produced only very low amounts of the other two peaks. These studies show that endocytic vesicles in both cell lines contain enzymes capable of processing EGF prior to the arrival of the hormone in the lysosomes and show that the KB‐R2A cells have a lesion that prevents the complete degradation of the hormone. We propose that the KB‐R2A cell line has a defective mechanism for the intracellular processing of a number of ligands that are internalized by the process of receptor‐mediated endocytosis and that this defect is located beyond the initial endocytic step.
cAMP‐induced phenotypic reversion of adhesion, aggregation, and endocytosis in adhesion‐defective CHO cell variants
Authors: Cheung E. , Juliano R. L.
Published: February 2005

ADvF11 cells are a CHO adhesion variant which, unlike wild type (WT) cells, are not able to adhere to fibronectin (Fn) coated substrata or to be aggregated by Fn‐beads. However, ADvF11 cells bind Fn‐beads to the same extent as WT cells, thus suggesting that the defect(s) associated with ADvF11 cells are distal to the initial receptor‐ligand binding event (Cheung and Juliano, Exp. Cell Res. 152:127, 1984). In this communication we report that cAMP analogs such as dibutyryl‐cAMP (dbcAMP) and 8‐bromo‐cAMP are able to correct defect(s) associated with ADvF11 cells enabling them to adhere to Fn‐coated dishes and to aggregate in the presence of Fn‐beads. However, only approximately 40% of ADvF11 cells were found to be responsive to dbcAMP suggesting heterogeneity in the cell population with respect to dbcAMP sensitivity. Further analysis of this partial response led us to isolate a subclone of ADvF11 cells, F11CA11, which is highly responsive to dbcAMP treatment. Induction of Fn‐mediated cell adhesion and aggregation in F11CA11 by dbcAMP is both time and dose dependent. Optimal responses were obtained after overnight incubation in alpha‐MEM containing, 1% fetal calf serum, 4% bovine serum albumin, 0.5 mM dbcAMP and 0.2 mM methyl‐isobutyl‐xanthine (MIX), a phosphodiesterase inhibitor. Under these conditions, 70–80% of F11CA11 cells were found to be adherent, compared to 5–7% of untreated F11CA11 cells and 95–100% of WT cells. Aggregation of dbcAMP‐MIX treated F11CA11 cells induced by Fn‐beads also approached that of WT cells. In addition, treatment with dbcAMP‐MIX markedly increased the ability of F11CA11 cells to internalize Fn‐beads. The maintenance of the adherent phenotype required the constant presence of dbcAMP‐MIX. Removal of dbcAMP‐MIX from the incubation medium resulted in return to the original nonadhesive phenotype. Thus, elevation of cAMP levels can dramatically modify the behavior of F11CA11 cells with respect to fibronectin mediated adhesion, aggregation and endocytosis, in effect causing a phenotypic reversion of all three parameters to wild type status. This suggests that the mechanisms for adhesion, aggregation and endocytosis may each involve regulation by cyclic AMP‐protein kinase systems.
Modulation of cell attachment to culture support by pH, fibronectin, hemin, and cobalt protoporphyrin
Authors: Imbenotte José , Verger Claude , Sassa Shigeru
Published: February 2005

When chick embryo fibroblasts were seeded in the presence of minimum essential medium supplemented with 1% (v/v) horse serum, the rate of cell attachment, after 1 hr incubation, was less than 5% at pH 6.5 and about 50% and 80% at pH 7.5 and pH 8.3, respectively. If, however, cultures were pretreated with fibronectin, a substance that promotes cell adhesion, a high rate of cell attachment was also observed at pH 6.5. Two other compounds of totally different chemical nature, cobalt‐protoporphyrin (CoPP) and hemin, also enhanced cell attachment at pH 6.5. CoPP was shown to increase the synthesis of proteins, but it did not affect the intracellular heme content of cells incubated at pH 6.5. The possibility that CoPP, and presumably also hemin, induce cell attachment by promoting the synthesis of a fibronectin like protein is discussed.
Effects of interaction between estradiol‐17β and progesterone on the proliferation of cloned breast tumor cells (MCF‐7 and T47D)
Authors: Schatz Robert W. , Soto Ana M. , Sonnenschein Carlos
Published: February 2005

We have previously reported that the proliferation of cloned MCF‐7 and T47D human mammary tumor cells can be inhibited by increasing concentrations of charcoal‐dextran stripped female human serum (CDFHS). The maximal proliferation rate was restored by the addition of 3 × 10−11 M estradiol‐17β to the culture media. These observations suggest that the proliferation of T47D and MCF‐7 cells is regulated by a blood‐borne inhibitor whose effects are neutralized by estrogens. In the present report we explore the possibility that progesterone alters the estrogenic response. MCF‐7 cells were grown in DME containing 2–40% CDFHS. Progesterone, at 3 × 10−7 M to 3 × 10−12 M, had no effect on the yield of MCF‐7 or T47D cells that were cultured in the presence or absence of estradiol‐17β.
Platelet‐derived growth factor stimulates rapid polyphosphoinositide breakdown in fetal human fibroblasts
Authors: Chu Shu‐Heh W. , Hoban Carolyn J. , Owen Albert J. , Geyer Robert P.
Published: February 2005

The addition of human platelet‐derived growth factor (PDGF) to confluent, quiescent cultures of human diploid fibroblasts induced the rapid breakdown of cellular polyphosphoinositides. The levels of 32P‐labeled phosphatidylinositol 4,5‐bisphosphate (PIP2), phosphatidylinositol 4‐phosphate (PIP), and phosphatidylinositol (PI) decreased by 30 to 40% within 1 min after exposure of the cells to PDGF. The levels of PIP and PIP2 returned to their initial values within 3 and 10 min, respectively, after PDGF addition. The level of PI continued to increase after it had returned to control values and was up threefold within 30 min after PDGF addition. In cells prelabeled with myo‐[3H]inositol PDGF caused an eightfold increase in the levels of inositol trisphosphate (IP3) within 2 min. Lesser increases, twofold and 1.3‐fold, respectively, were seen in levels of inositol bisphosphate (IP2) and inositol monophosphate (IP). Within 10 min after PDGF addition the levels of all three inositol phosphates had decreased to control values. The levels of IP3 measured 2 min after PDGF addition depended on the PDGF concentration and were maximal at 5–10 ng/ml of PDGF. Similar concentrations of PDGF stimulate maximal cell growth and DNA synthesis in these cells.
Insulin receptor capping and its correlation with calmodulin‐dependent myosin light chain kinase
Authors: Majerick Mary H. , Bourguignon Lilly Y. W.
Published: February 2005

Both fluorescence microscopy and fluorometric analysis techniques have been used to characterize insulin receptor capping in IM‐9 human lymphoblastoid cells. Morphologically, insulin caps appear similar to lectin or antiimmuno‐globulin‐induced caps displaying a preferential accumulation of actin, myosin, and actin‐binding protein directly underneath the cap structure. Using the fluorescent calcium indicator quin2 we have detected no change in the calcium activity following insulin stimulation. However, in the presence of a number of calmodulin inhibitors, such as W‐5, W‐7, W‐12, and trifluoperazine (TFP), insulin capping is significantly inhibited, which implies that a calmodulin‐regulated process is involved. Using double immuonofluorescence microscopy, we have found that the calmodulin‐dependent myosin light chain kinase (MLCK) is concentrated directly beneath insulin caps. Upon treatment with trifluoperazine (TFP), the redistribution of both MLCK and insulin receptors are inhibited concomitantly. Our data indicate that the calmodulin‐dependent myosin light chain kinase may be directly responsible for the activation of actomyosin‐mediated contractility during insulin receptor capping.
The in vitro autocrine secretion of CSFs alone does not account for the longterm growth of murine myeloid leukemic cells in suspension cultures
Authors: Fichelson Serge , Heard Jean‐Michel , Levy Jean‐Paul
Published: February 2005

To investigate the mechanisms supporting the in vitro longterm growth of murine leukemic myeloblastic cells, factor‐dependent and autonomous myeloblastic cells have been examined for their CSF responsiveness, CSF secretion, and autostimulation of growth. Purified CSF‐1, GM‐CSF, and IL‐3 stimulated cloning and proliferation of both autonomous (ACL) and factor‐dependent cell lines (FDCL), and were unable to induce differentiation of these cells. Sensitivities to CSFs were similar for ACL, FDCL and normal bone marrow cells. Most of the cell lines secreted CSFs, stimulating colony formation from normal bone marrow cells in bilayer agar cultures. The number of induced granulo‐macrophagic colonies was similar in the presence of either ACL or FDCL. The ability to stimulate their own proliferation was similar for both ACL and FDCL in a longterm suspension culture assay as well as in a shortterm cloning assay. These results strongly suggest that the autocrine secretion of CSF‐related activities are not sufficient to fully account for the in vitro longterm proliferation of virus‐transformed myelomonocytic cells.
A self‐renewing, bipotential erythroid/mast cell progenitor in continuous cultures of normal murine bone marrow
Authors: Wendling Françoise , Shreeve Mona , McLeod David , Axelrad Arthur
Published: February 2005

We have established permanent lines of nonadherent cells from fresh normal mouse bone marrow in media containing pokeweed mitogen‐stimulated spleen cell conditioned medium (PWSCM). These lines continuously produced erythropoietic progenitor cells (detected by their ability to form erythroid bursts in semi‐solid medium containing erythropoietin) together with cells having characteristics of the mast cell lineage (as demonstrated by metachromatic staining with toluidine blue, histamine content and membrane receptors for IgE). Sixteen such cell lines have been established in sixteen attempts. Cloning experiments were carried out to determine the nature of the progenitor cell(s) responsible for the permanence of these cultures. When cells were cultured in methylcellulose medium containing PWSCM, colonies were observed which reached macroscopic size after 4 weeks of incubation. Replating of individual primary colonies resulted in secondary colony formation, indicating the presence of progenitor cells with self‐renewal potential. Forty‐seven primary colonies were picked and their cells were suspended in liquid culture medium containing PWSCM. Of these, twenty‐one could be expanded to establish permanently growing sublines. Sixteen of these sublines were found to be composed of both erythroid progenitors and mast cells. In five sublines only mast cells could be seen; none of the sublines appeared to be purely erythroid. Karyotypic analysis of mast cells and of erythroid cells of seven sublines derived from individual colonies which arose in cocultures of male and female cells revealed that the mast cells and erythroid cells were both of the same sex in each of the seven sublines; this demonstrates the single cell origin of each colony and of the two lineages derived from it. We conclude that these nonadherent, factor‐dependent cell lines are maintained by self‐renewal and differentiation of bipotential progenitor cells apparently restricted to the erythroid and mast cell lineages.
SV40‐transformed human fibroblasts: Evidence for cellular aging in precrisis cells
Authors: Stein Gretchen H.
Published: February 2005

Precrisis SV40‐transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40‐transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: (1) an age‐related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen‐deprived at the end of lifespan; and (2) the ability of the cells to enter a viable, G1‐arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that precrisis SV40‐transformed HDF retain the first process described above, but lack the second process. It is shown that SV40‐transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40‐transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that precrisis SV40‐transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1‐arrested senescent state because they have lost their ability to enter a viable, G1‐arrested state in response to mitogen deprivation.
Norepinephrine and epidermal growth factor: Dynamics of their interaction in the stimulation of hepatocyte DNA synthesis
Authors: Cruise Jennifer L. , Michalopoulos George
Published: February 2005

Primary cultures of adult rat hepatocytes are stimulated to enter DNA synthesis by norepinephrine (NE). This stimulation is maximal if the hepatocytes are incubated with NE for more than 12 hr, beginning no later than 2–4 hr after the cells are first plated. After 24 hr in culture, hepatocytes are unresponsive to NE stimulation. A strong synerergistic interaction between NE and epidermal growth factor (EGF) may be observed in cultures incubated with both EGF and NE, or pretreated with NE, then exposed to EGF. This interaction may be related to the finding that NE, in similarity with other factors that enhance EGF stimulation, reduces binding at the EGF receptor during the first 24 hr in culture.
The selective induction of a small number of proteins during G1 transit results from the mitogenic action of pp60v‐src in tsASV‐infected rat cells
Authors: Durkin Jon P. , Whitfield J. F.
Published: February 2005

Since the mitogenic/oncogenic pp60v‐src product of the avian sarcoma virus (ASV) mutant, tsLA23, is abnormally thermolabile, tsLA23‐NRK cells were phenotypically nontransformed at 40°C and were consequently rendered quiescent by serum deprivation at this temperature. These serum‐deprived cells were stimulated to transit G1 either as transformed cells by simply dropping the temperature to a pp60v‐src‐activating 36°C, or as nontransformed cells by adding serum at 40°C. Serum stimulation rapidly increased total protein synthesis in these cells and over 100 changes in cellular proteins (resolved by two‐dimensional gel electrophoresis) occurred during G1 transit. By contrast, pp60v‐src‐activation did not increase total protein synthesis and only six proteins (18.5–44 kD) were clearly seen to appear or increase when quiescent cells were stimulated to transit G1 by activating pp60v‐src. Three of these six pp60v‐src‐ induced proteins also appeared or accumulated during the G1transit of serum‐stimulated cells. The appearance and/or accumulation of the six proteins and the subsequent initiation of DNA replication may have resulted from pp60v‐src stimulating only a small number of critical cellular genes because both the protein changes and DNA replication were completely suppressed by the transcription inhibitor actinomycin D.
Two classes of primitive pluripotent hemopoietic progenitor cells: Separation by adherence
Authors: Kerk David K. , Henry Elizabeth A. , Eaves Allen C. , Eaves Connie J.
Published: February 2005

Methylcellulose cultures containing mouse marrow cells at low densities and partially purified preparations of erythropoietin and Interleukin‐3 were scored after 2 weeks for the presence of macroscopic multilineage colonies (from “primary” CFU‐macro GEMM). Whole cultures were then harvested and replated to assess the number of “secondary” CFU‐macro GEMM produced, but not detected, during the primary culture period. In such experiments adherent marrow cells yielded significantly higher numbers of secondary CFU‐macro GEMM than did either fresh or nonadherent marrow cells. Removal of macroscopic colonies prior to replating showed that most secondary CFU‐macro GEMM were not derived from primary CFU‐macro GEMM. In vivo studies also revealed a differential effect of adherence separation on the frequency of day 10 CFU‐S, which decreased, by comparison to cells capable of long‐term repopulation, which increased. Primary adherent CFU‐macro GEMM from 5‐fluorouracil (5‐FU) treated mice showed an 18‐fold higher self‐renewal capacity than their counterparts in normal marrow. Nevertheless the majority of secondary CFU‐macro GEMM obtained from primary cultures of adherent 5‐FU cells were again not derived from primary CFU‐macro GEMM. Cells capable of immediately generating large multilineage colonies thus appear to represent an intermediate compartment of pluripotent progenitors whose self‐renewal properties, may, however, vary over a considerable range. Our results further suggest that these progenitors are derived ultimately from a more primitive adherent cell whose tendency to begin to divide in vitro is low and whose presence correlates with cells capable of long‐term myeloid repopulation in vivo.
Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF
Authors: McGrath Michael , Palmer Sarah , Nandi Satyabrata
Published: February 2005

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum‐free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three‐to four‐fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period.

Isolated mammary organoids produced a ‘stellate’ type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse ‘basket’ type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a ‘mixed’ type colony was observed which contained both the large and small epithelial cell types.

Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha‐lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.
Cell cycle phase‐dependent induction of ornithine decarboxylase‐antizyme
Authors: Linden Margareta , Anehus Siw , Långström Eva , Baldetorp Bo , Heby Olle
Published: February 2005

The activites of ornithine decarboxylase (ODC) and ODC inhibitory protein (ODC‐antizyme) were studied in Ehrlich ascites tumor cells, separated according to their position in the cell cycle by centrifugal elutriation. Release and/or synthesis of ODC‐antizyme was induced by putrescine treatment. Each mouse received an intraperitoneal injection of 25 μmoles of putrescine at 0, 1, 2, and 3 hr after tumor transplantation. Tumor cells obtained from putrescine‐treated and control mice at 4 hr after transplantation were separated into fractions representing all phases of the cell cycle. The cell cycle distribution of the tumor cells in each fraction was determined by flow cytometry.

In control tumor cells the ODC activity exhibited two maxima; inlate‐G1/early‐S and in late‐S/G2. A marked decrease in ODC activity was observed in mid‐S phase. This decrease coincided with maximum ODC‐antizyme activity (revealed by putrescine treatment), suggesting that ODC‐antizyme is involved in the regulation of ODC activity during the cell cycle.