bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha1‐adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: Evidence for compartmentalization of quin‐2 and fura‐2
Authors: Goligorsky Michael S. , Hruska Keith A. , Loftus David J. , Elson Elliot L.
Published: February 2005

This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to α1‐adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen‐coated polycarbonate membranes, or on collagen‐coated glass coverslips. Quin‐2 and fura‐2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin‐2, and 122 nM, as determined using fura‐2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10−5 M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin‐2 and fura‐2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180–300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin‐2 or fura‐2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell‐impermeant fura‐2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid‐phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+‐indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells “scrape‐loaded” with fura‐2 revealed a four‐fold increase in [Ca2+]i concentration following stimulation with phenylephrine.
Stimulation of sulfated‐proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes
Authors: Malemud Charles J. , Mills Teresa M. , Shuckett Rhonda , Papay Robert S.
Published: February 2005

Forskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell‐associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell‐associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL‐2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin‐treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin‐stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H‐glucosamine‐labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin‐treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans.
α‐Thrombin‐induced inositol phosphate formation in go‐arrested and cycling hamster lung fibroblasts: Evidence for a protein kinase C‐mediated desensitization response
Authors: L'Allemain Gilles , Paris Sonia , Magnaldo Isabelle , Pouysségur Jacques
Published: February 2005

In resting Chinese hamster fibroblasts (CCL39) α‐thrombin rapidly induces the breakdown, of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li+, is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris‐ and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C‐mediated generation of inositol trisphosphate from phosphatidylinositol 4,5‐bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10−4‐10 U/ml. Thrombin‐induced phosphoinositide hydrolysis rapidly desensitizes (t1/2 < 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0‐arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin‐induced inositol phosphate formation. Conversely, growth factor deprivation of CCL39 cells results in a progressive increase of thrombin‐induced phosphoinositide hydrolysis, from the very low level of exponentially growing cells to the maximal level of G0‐arrested cells. This “up regulation” was found maximal in A51, a very well growth‐arrested CCL39 derivative, and reduced or virtually abolished in two tumoral and growth factor‐relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in variants selected for autonomous growth.
Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein‐deficient serum
Authors: Chen Jan‐Kan , Hoshi Hiroyoshi , McClure Don B. , McKeehan Wallace L.
Published: February 2005

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein‐deficient serum was dependent on either high‐ or low‐density lipoprotein. High‐ and low‐density lipoprotein stimulated cell growth by three‐ and five‐fold, respectively, over a 6‐day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 μg/ml of high‐ and low‐density lipoproteins, respectively. No correlation between the activation of 3‐hydroxyl‐3‐methylglutaryl coenzyme A reductase activity and lipoprotein‐stimulated cell proliferation was observed. Lipid‐free total apolipoproteins or apolipoprotein C peptides from high‐density lipoprotein were partially effective and together with oleic acid effectively replaced native high‐density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high‐density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high‐ and low‐density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.
The role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium
Authors: Krall J. Frederick , Morin Anne
Published: February 2005

Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45Ca2+ from the medium. Ca2+ uptake by these cells was stimulated by the addition to the medium of 8‐bromo‐cGMP but not by 8‐bromo‐cAMP. Ca2+ uptake was also stimulated by carbachol and by the nitro‐vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor‐mediated functions that reflected some neurotransmitter‐sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor.

The cells were also sensitive to the β‐adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca2+ uptake. Carbachol failed to inhibit isoproterenol‐dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine‐sensitive properties of uterine smooth muscle may be retained in cell culture.
System a transport activity in normal rat hepatocytes and transformed liver cells: Substrate protection from inactivation by sulfhydryl‐modifying reagents
Authors: Chiles Thomas C. , Kilberg Michael S.
Published: February 2005

The transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein‐modifying reagents, including the sulfhydryl‐preferring reagents N‐ethylmaleimide (NEM) and p‐chloromercuribenzene sulfonate (PCMBS). Uptake of 2‐aminoisobutyric acid (AIB), a specific probe for hepatic System A‐mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non‐substrates produced little or no protection. Amino acid‐dependent protection was stereospecific and system‐specific. L‐norleucine competitively inhibited AIB uptake (Ki = 1.9 ± 0.1 mM) in H4 cells and also protected System A from PCMBS‐dependent inactivation (half‐maximal protection occurred at an amino acid concentration of 0.6 + 0.1 mM). N‐bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition.
Platelet‐derived growth factor stimulation of [3H]‐glucosamine incorporation in density‐arrested BALB/c‐3T3 cells
Authors: Harrington Maureen A. , Wharton Walker , Pledger W. J.
Published: February 2005

G0/G1 traverse in density‐arrested BALB/c‐3T3 cells is controlled by multiple serum‐derived growth factors. Platelet‐derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. We have identified the stimulation of a biochemical process specific to competence formation in BALB/c‐3T3 cells. PDGF treated BALB/c‐3T3 cells incorporated 5–10‐fold more [3H]‐glucosamine (GlcN) into acid‐insoluble material as compared to plateletpoor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density‐arrested BALB/c‐3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cellcycle traverse. Stimulation of [3H]‐GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]‐GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.
Phosphorylation of regulatory subunit of type I cyclic AMP‐dependent protein kinase: Biphasic effects of cyclic AMP in intact S49 mouse lymphoma cells
Authors: Russell Joanne L. , Steinberg Robert A.
Published: February 2005

Intact S49 mouse lymphoma cells were used as a model system to study the effects of cyclic AMP (cAMP) and its analogs on the phosphorylation of regulatory (R) subunit of type I cAMP‐dependent protein kinase. Phosphorylation of R subunit was negligible in mutants deficient in adenylate cyclase; low levels of cAMP analogs, however, stimulated R subunit phosphorylation in these cells to rates comparable to those in wild‐type cells. In both wild‐type and adenylate cyclase‐deficient cells, R subunit phosphorylation was inhibited by a variety of N6‐substituted derivatives of cAMP; C‐8‐substituted derivatives were generally poor inhibitors. Two derivatives that were inactive as kinase activators (N6‐carbamoylmethyl‐5′‐AMP and 2′‐deoxy‐N6‐monobutyryl‐cAMP) were also ineffective as inhibitors of R subunit phosphorylation. Preferential inhibition by N6‐modified cAMP analogs could not be ascribed simply to selectivity for the more aminoterminal (site I) of the two cAMP‐binding sites in R subunit: Analog concentrations required for inhibition of R subunit phosphorylation were always higher than those required for activation of endogenous kinase; 8‐piperidino‐cAMP, a C‐8‐substituted derivative that is selective for cAMP‐binding site I, was relatively ineffective as an inhibitor; and, although thresholds for activation of endogenous kinase by site I‐selective analogs could be reduced markedly by coincubation with low levels of site II‐selective analogs, no such synergism was observed for the inhibitory effect. The uncoupling of cyclic nucleotide effects on R subunit phosphorylation from activation of endogenous protein kinase suggests that, in intact cells, activation of cAMP‐dependent protein kinase requires more than one and fewer than four molecules of cyclic nucleotide.
Internalisation and recycling of the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor on a murine myelomonocytic leukemia
Authors: Walker Francesca , Burgess Antony W.
Published: February 2005

Radioiodinated granulocyte‐macrophage colony‐stimulating factor (125I‐GM‐CSF) binds to specific receptors (molecular weight approximately 50,000 daltons) on the murine myeiomonocytic leukemia, WEHI‐38D+. At 4°C 125I‐GM‐CSF remains on the surface of the cells and can be eluted by washing the cells with acidified isotonic buffer. When the cells are warmed to 37°C, the 125I‐GM‐CSF is internalized rapidly (t1/2: 7 min). The internalisation appears to be entirely receptor mediated and is independent of energy sources inhibited by sodium azide. This GM‐CSF‐mediated internalisation is not due to a general increase in the turnover of cell surface molecules as the specific binding of 25I‐transferrin is not affected by incubation of WEHI‐3BD+ cells with GM‐CSF. The initial 125I released when the cells are warmed to 37°C appears to be intact 125I‐GM‐CSF; however, after 2 h 80% of the 125I released was not precipitable with trichloroacetic acid and presumably represented degraded 125I‐GM‐CSF. Ammonium chloride or monensin reduced the release of 125I‐GM‐CSF from the cells, suggesting that the receptor‐bound ligand was processed through the lysosomes. A cor siderable proportion of the internalised GM‐CSF receptors were recycled to the surface and were available for ligand binding. Synthesis of new GM‐CSF receptors contributed to the re‐expression of GM‐CSF receptors after down‐regulation and it is possible that the GM‐CSF enhances the synthesis of its own receptors.
Enucleation of normal and transformed cells
Authors: Degaetano Douglas , Schindler Melvin
Published: February 2005

A quantitative analysis based on cenrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3‐K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3‐K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well‐formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2‐deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directdly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.
Effect of recombinant tumor necrosis factor on HL‐60 cells: Cell‐cycle specificity and synergism with actinomycin D
Authors: Darzynkiewicz Zbigniew , Carter Sally P. , Old Lloyd J.
Published: February 2005

The tumor necrosis factor (TNF) exhibits a multitude of activities depending on the type of target cells. We characterized the cytostatic and cytotoxic effects of recombinant TNF, alone and in combination with actinomycin D (AMD), on the human leukemic cell line HL‐60. Because HL‐60 cells, when triggered to monocytic differentiation by phorbol esters, are known to produce and secrete TNF, their sensitivity to the factor could indicate an autocrine function of TNF in this cell system. Indeed, HL‐60 cells were affected by TNF; their doubling time was increased by about 50% and progression through the cell cycle was perturbed. Initially, (up to 8 h) TNF induced a temporary arrest in G2 while later (24–48 h) it delayed progression through the G1 phase. Also, a transient increase in RNA content peaking at 6–8 h was apparent. The cytotoxicity of TNF alone was low. Thus, TNF may be involved in the regulation of the cell cycle of HL‐60 cells during early stages of their differentiation.

The cytotoxicity of TNF was markedly potentiated in the presence of AMD; the effect was AMD but not TNF concentration‐dependent. Whereas at 20 and 50 ng/ml of AMD alone nonviable cells did not exceed 20% during the first 24 h of treatment, their proportion increased to 80 and 90%, respectively, in the presence of TNF. The most sensitive were cells in the S phase of the cell cycle. The observed synergistic effect of TNF and AMD does not appear to be caused by the action of TNF increasing the permeability of the cell membrane to AMD. The results indicate that HL‐60 cells, ordinarily resistant to the cytotoxic action of TNF, can be rendered sensitive by treatment with AMD. This implies that a combination of TNF and AMD may be considered in oncology for treatment of tumors otherwise nonresponding to TNF alone.
Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: Evidence for a role of protein kinase C
Authors: Muir Jane G. , Murray Andrew W.
Published: February 2005

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (Pl), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimualtion was blocked by the bombesin receptor antagonist, [D‐Arg1, D‐Pro2, D‐Trp7,9, Leu11] substance P, indicating that the effects on both Pl and PC were mediated through the same receptor. The tumor‐promoting phorbol ester 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacylglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol‐12, 13‐dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylyltransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.
Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles
Authors: Sullivan Peter C. , Ferris Andrea L. , Storrie Brian
Published: February 2005

We investigated the effects of reduced temperature, the pH elevators NH4 CI, monensin, and HEPES (N‐2‐hydroxy‐ethylpiperazine‐N'‐2‐ethanesulfonic acid) buffer, as well as the metabolic poisons NaF/KCN on transport of the fluid phase pinocytic marker, horseradish peroxidase (HRP), to lysosomes in Chinese hamster ovary (CHO) cells. In cell fractionation experiments, these agents appeared to block HRP transit at specific point(s) from “early” to “late” (i.e., low to high density) prelysosomal vesicles and lysosomes. Reduced temperature (17°C) most strongly inhibited HRP transport from low density, early endosomes to lysosomes. In long‐term HRP uptakes at 17°C, marked peroxidase accumulation occurred both in early endosomes and in lysosomes. Loss (reversible pinocytosis) of HRP from “very early” endosomes occurred at 17°C. All three pH elevators including the common media supplement HEPES buffer inhibited transit of internalized HRP into lysosomes. For all three pH elevators, inhibition was most pronounced at the “early” endosome stage. The respiratory inhibitors NaF/KCN also inhibited transport most strongly at the early endosome stage. Together these results suggest that “early” steps in the endocytic transport of HRP are the most sensitive and that the conditions tested may exert direct effects on the processing of endocytic vesicles.
The role of aggregation in embryonal carcinoma cell differentiation
Authors: Smith Steven C. , Reuhl Kenneth R. , Craig Jane , McBurney Michael W.
Published: February 2005

Cultures of the P19 line of embryonal carcinoma cells differentiate into various cell types including cardiac muscle when aggregated and exposed to medium containing 1% dimethylsulfoxide (DMSO). DMSO‐treated aggregates became completely covered with an epithelial cell type 3 to 4 days following drug exposure. This epithelial cell was tentatively identified as primitive extraembryonic endoderm by its ultrastructural appearance and its possession of cytokeratin intermediate filaments. Muscle cells developed within the interior of DMSO‐treated aggregates. They first became apparent 5 to 6 days after DMSO exposure and were characterized by the presence of striated muscle‐specific myosin, immature myofibrils, and intercalated discs. We determined the proportion of cells developing into epithelium and muscle in aggregates of various sizes and showed that the proportion of epithelium was highest in small aggregates whereas muscle cells developed only in aggregates of relatively large size. The muscle was usually associated with necrotic areas which developed within the interior of large aggregates. Our results suggest that cardiac muscle differentiation in the aggregates requires both the DMSO‐induced formation of an epithelial cell coat and one other condition which may be the proximity to necrotic areas.
Enhanced synthesis of a Mr = 55,000 dalton peptide by senescent human fibroblasts
Authors: Sottile Jane , Hoyle Marian , Millis Albert J. T.
Published: February 2005

The secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40–50% lifespan completed), late passage (> 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55–60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.
Bombesin induction of c‐fos and c‐myc proto‐oncogenes in Swiss 3T3 cells: Significance for the mitogenic response
Authors: Rozengurt Enrique , Sinnett‐Smith James W.
Published: February 2005

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c‐fos and c‐myc mRNAs. Enhanced expression of c‐fos (122 ± 14‐fold) occurred within minutes of peptide addition followed by increased expression of c‐myc (82 ± 16‐fold). The concentrations of peptide required for half‐maximal increase in the levels of c‐fos and c‐myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D‐Arg1, D‐Pro2, D‐Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin‐stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c‐fos and c‐myc mRNA levels promoted by bombesin. Down‐regulation of protein kinase C by long‐term exposure to phorbol esters prevented c‐fos and c‐myc induction by bombesin. This and other results indicate that the induction of these proto‐oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c‐fos and c‐myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c‐fos and c‐myc mRNA levels. This apparent dissociation of mitogenesis from proto‐oncogene induction was even more dramatic in 3T3 cells with down‐regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c‐fos and c‐myc mRNA levels at comparable concentrations. Thus, the induction of c‐fos and c‐myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto‐oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.
Effect of acute serum depletion on Na+ ‐K+ homeostasis in cultured human skin fibroblasts
Authors: Hopp L. , Kuriyama S. , Kino M. , Aviv A.
Published: February 2005

In order to elucidate changes in cell transport behavior of cultured human skin fibroblasts in response to acute serum depletion, we performed uptake and washout of 22Na+ and 86Rb+ as well as measurements of the intracellular Na+ and K+ levels in the presence and absence of ouabain. Pronounced and lasting increase in cellular Na+ and decrease in K+ were observed after removal of fetal bovine serum (FBS) from the medium. The sum of the Na+ and K+ contents (nEq/105 cells) was lower in FBS‐free medium (mean ± SD; 17.3 ± 2.2) than in FBS‐containing medium (26.2 ± 3.8; P < .02). Simultaneously, a decrease in cellular water volume was detected in the FBS‐free medium. The cation uptake and washout data suggest that FBS removal primarily renders the cells more permeable to Na+ and K+ with a secondary stimulation of the ouabain‐sensitive Na+ extrusion mechanism. FBS at a concentration of 0.2% prevented approximately 50% of the maximal increase in the 86Rb+ washout rate constant associated with FBS depletion. Ouabain (2 μM) produced an increase in the 86Rb+ washout rate constant. This effect was substantially larger in cells subjected to medium without FBS (from 0.0303 to 0.2500 min−1) than in fibroblasts incubated in medium with FBS (from 0.0107 to 0.0487 min−1). The cellular K+ content was drastically reduced by ouabain to a level not different in medium with or without FBS (33.9 ± 4.5 to 1.75 ± 0.38 and 16.7 ± 1.4 to 1.4 ± 0.13 nEq/105 cells, respectively). The 22Na+ washout data exhibited a three‐exponential pattern. Analytical solutions of the washout data by means of two models (serial and parallel) with three compartments showed that FBS depletion resulted in increase of the size of all three compartments. It is concluded that in cultured human skin fibroblasts, FBS is essential to the maintenance of a normal Na+ and K+ homeoatasis. The removal of FBS results in dramatic permutation of this homeostasis that develops within minutes and lasts for hours.
Down‐modulation of EGF receptors in cells transformed by the src oncogene
Authors: Wasilenko William J. , Shawver Laura K. , Weber Michael J.
Published: February 2005

The effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat‐1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I‐EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src‐transformed cells did not appear to be due to secreted transforming growth factor‐α (TGF‐α), since such factors were undetectable in culture fluids from the src‐transformed cells. By several criteria of transformation, an EGF‐receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down‐modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.
Calcium and calmodulin in cellular intoxication with Clostridium difficile toxin B
Authors: Caspar Maria , Florin Inger , Thelestam Monica
Published: February 2005

In cultured human lung fibroblasts treated with Clostridium difficile toxin B, the development of the cytopathogenic effect was inhibited by the proton ionophore monensin but was not affected by some other ionophores. The calcium channel blockers verapamil and LaCl3 protected the cells against intoxication, as did the calmodulin antagonists trifluoperazine, amitriptylin, R 24571, and dansylcadaverine. Since these agents could not prevent intoxication when added after toxin internalization was completed, we suggest that calmodulin and uptake of extracellular calcium are needed for the internalization but not for the cytosolic action of the toxin.
Colcemid effects on B16 melanoma cell progression and aberrant mitotic division
Authors: Bhuyan B. K. , Adams E. G. , Badiner G. J. , Trzos R. J.
Published: February 2005

Mitotic cells selectively harvested after several h of colcemid treatment are routinely used to obtain synchronized cell cultures. DNA flow cytometry shows that when colcemid‐treated B16 mitotic cells divide, they give rise to daughter cells in G1, some of which contain abnormal amounts of DNA. Two subpopulations appear to exist, one having a DNA content distribution expected of G1 cells, another having a mean DNA content about 0.8 of expected and an SD of DNA content more than 5 times expected. The effect was dependent on dose and duration of exposure to colcemid. Colcemid was more cytotoxic to cells in G2 + M than to G1 + S phase cells, and it slowed the progression of G1 cells to S. These effects of colcemid were much greater in aneuploid B16 melanoma cells than in pseudodiploid Chinese hamster ovary (CHO) cells.
Iron‐transferrin‐induced increase in protein kinase C activity in CCRF‐CEM cells
Authors: Phillips Jerry L. , Boldt David H. , Harper Jamie
Published: February 2005

Iron transferrin has been found to induce a mean 10‐fold increase in the activity of protein kinase C in CCRF‐CEM cells. This increase was not detectable up to 45 min after treatment of cells with iron transferrin, although after 60 min, a maximal increase in enzyme activity was observed. Similarly, iron transferrin at concentrations of 0.1–0.5 μg/ml did not alter protein kinase C activity, while concentrations of iron transferrin of 1–100 μg/ml induced a maximal increase in enzyme activity. Apotransferrin and iron in the form of ferric citrate, as well as complexes of transferin with copper, nickel, zinc, manganese, and cobalt did not increase protein kinase C activity. Additionally, CCRF‐CEM cells pretreated with either actinomycin D or cycloheximide and then incubated with iron transferrin did not exhibit increased enzyme activity. Treatment with iron transferrin was found to have no effect on protein kinase C activity in normal human peripheral blood lymphocytes and in HL60, Daudi, and U937 cells. However, normal lymphocytes stimulated with phytohemagglutinin for 48 hr exhibited a 2‐fold increase in protein kinase C activity following treatment with iron transferrin. These results indicate a specific effect of iron transferrin on protein kinase C activity in CCRF‐CEM cells and in mitogen‐stimulated human lymphocytes that may occur through increased synthesis of the enzyme.
Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+‐activated K+ channels
Authors: Arcangeli Annarosa , Ricupero Letizia , Olivotto M.
Published: February 2005

The role of the plasma membrane potential (Δψp) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. Δψp was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22‐hour depolarization phase (from −28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl−, the relative contribution of these ions to Δψp was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4‐acetoamide‐4′‐isothiocyanostilbene‐2,2′‐disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to Δψp appeared negligible, the potential being essentially generated by K+ and Cl− fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (Pk) mediated by Ca2+‐activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen‐bis‐acetamide [HMBA], diazepam), although they did not modify the ionic basis of Δψp, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+‐activated K+ channels, suggesting that a properly modulated Δψp depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.
Changes in protein kinase C and cAMP‐dependent kinase in lymphocytes after treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate or concanavalin A: Quantitation of activities with an in situ gel assay
Authors: Grove Deborah S. , Mastro Andrea M.
Published: February 2005

Primary lymphocytes can be stimulated to proliferate by mitogenic lectins such as concanavalin A (Con A). While the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) alone is not mitogenic for these cells, it can enhance the response to Con A. Previously, protein kinases and phosphorylation have been reported to be important in lymphocyte proliferation. More recently TPA has been found to bind and activate protein kinase C. Therefore, we examined kinase activity in lymphocytes stimulated with the complete mitogen Con A and the comitogen TPA. In order to monitor more than one kinase we used an in situ gel assay and developed the system to compare both protein kinase C and cAMP‐dependent kinases. When total cell extracts were assayed in the presence of histone five major bands of activity were detected by autoradiography of the gel. The bands corresponding to protein kinase C and to cAMP‐dependent kinases were identified by partial purification of the enzymes, by binding of [20‐3H(N)]7‐phorbol‐12, 13‐dibutyrate (3H‐PDBU), and by photoaffinity labelling with 8‐azidoadenosine‐3′:5′‐cyclic monophosphate (8‐N3‐[32P]cAMP). Differential extraction of cell lysate allowed comparison of soluble and particulate kinases.

We found that when the preparations from either TPA‐ or Con A‐treated lymphocytes were assayed, protein kinase C activity increased three‐ to four‐fold in the particulate fraction within 5 min after treatment. A concurrent decrease of 30–50% occurred in the cytosol. In contrast, cytosolic cAMP‐dependent protein kinase II increased 1.4‐fold in the same period with Con A. PKI and PKII showed the most significant changes after 24 h of stimulation by Con A when the activity of the holoenzyme decreased to half that of the unstimulated cells. Therefore, although TPA and Con A separately can affect protein kinase C this alone is not sufficient for proliferation to occur.
Cytogenetic evaluation of human endothelial cell cultures
Authors: Nichols Warren W. , Buynak Eugene B. , Bradt Carole , Hill Rosina , Aronson Margaret , Jarrell Bruce E. , Mueller Stephen N. , Levine Elliot M.
Published: February 2005

Cytogenetic evaluation of serially subcultivated human endothelial cells revealed significant differences between cultures derived from fetal umbilical cords and cultures derived from various vessel sites in adults. A rapid increase in the prevalence of polyploid cells, to levels of 100% in many cases, was detected in human umbilical vein endothelial cell cultures but not in endothelial cell cultures from adult vessels. Because the development of polyploidy has been viewed as one signpost of in vitro senescence, it may be that these in vitro observations of high levels of polyploidy are a reflection of the fact that umbilical tissue is at the end of its in vivo developmental lifespan when studied. Consistent karyotypic alterations also were observed in two clones from adult human abdominal aorta, even though these cultures exhibited low percentages of polyploid cells. Cultures of one clone exhibited a trisomy of chromosome 11, on which there are at least three onc gene loci, and a deletion of chromosome 13 through band q14. A loss of band 13q14 is a prezygotic chromosomal lesion known to predispose to retinoblastoma. In the other clone, two cell populations were observed, and each displayed a chromosomal abnormality A trisomy of the long arm of chromosome 2 was noted in one cell population via a marker chromosome involving 2 and 14. The other cell population exhibited an abnormality of chromosome 2. Neither of these karyotypic alterations was detected in the parent culture from which the clones were derived. The results reported in this study have both practical and theoretical implications. The high incidence of polyploidy in serially cultivated umbilical cultures as well as the occurrence of chromosomal changes in umbilical and aortic cultures testify to the need for cytogenetic monitoring of cell cultures even though they are derived from presumably normal tissue. Cytogenetic changes in the endothelium may be important in atherogenesis and other pathologic states. The conversion of diploid endothelial cells into polyploid endothelial cells may provide a convenient model cell system for studying mechanisms of the development of polyploidy in cells and their relationship to in vitro senscence.
Membrane blebbing is associated with Ca2+‐activated hyperpolarizations induced by serum and α2‐macroglobulin
Authors: Dixon S. J. , Pitaru S. , Bhargava U. , Aubin J. E.
Published: February 2005

We have reported previously that serum and α2‐macroglobulin (α2M) induce Ca2+‐activated hyperpolarizations in the membrane potential of a clonal rat osteosarcoma cell line (ROS 17/2) (Dixon and Aubin, J. Cell. Physiol., 132:215–225, 1987). In this report, we describe morphological changes that accompany these hyperpolarizations. Both cell surface blebbing (zeiosis) and transient hyperpolarizations were induced by application of 10% fetal bovine serum (FBS) or α2M; neither was induced by serum‐free medium, a suspension of latex beads, or purified bovine serum albumin. Following a brief application of FBS or α2M at time 0, electrical activity typically occurred between 7–40 s and was always followed by blebbing activity that began at 30 s and persisted for 3–5 min. In contrast, continuous exposure to FBS resulted in the persistence of both blebbing activity and transient hyperpolarizations for periods of at least several hours. Scanning electron microscopy (SEM) revealed that the blebs appeared concomitantly with the disappearance of microvilli and the appearance of surface pits that measured 100–300 nm in diameter. Coated pits and vesicles, similar in size to the pits observed by SEM, were observed using transmission electron microscopy (TEM). By TEM, blebs were found to contain few organelles other than centrally located free ribosomes. Fluorescence microscopy of nitrobenzooxadizole‐phallacidin‐labeled cells indicated that blebs contained filamentous actin and that microfilament bundles remained primarily on the substratum side of blebbed cells. We propose that blebbing results from a dynamic local reorganization of microfilaments initiated by ligand‐induced transient increases in intracellular Ca2+.
Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots
Authors: Knox Peter , Crooks Sheila , Scaife Michael C. , Patel Sandhiya
Published: February 2005

Human diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM ϵ‐aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen‐depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis.
Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors
Authors: Montesano R. , Pepper M. S. , Vassalli J.‐D. , Orci L.
Published: February 2005

We have previously shown that the tumor promoter 4β‐phorbol 12‐myristate 13‐acetate (PMA) induces capillary endothelial cells grown to confluency on the surface of three‐dimensional collagen gels to invade the underlying matrix and to form capillarylike tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell, 42:469–477, 1985). Since angiogenesis frequently occurs within a fibrin‐rich extracellular matrix, we have examined the ability of PMA‐treated endothelial cells to invade fibrin gels. Control endothelial cells grown on fibrin gels formed a confluent monolayer on the gel surface and did not invade the underlying matrix. Treatment of the cultures with PMA resulted in a progressive lysis of the substrate without invasion of the fibrin matrix. However, if the cells were treated with PMA either in the presence of fibrinolytic inhibitors (Trasylol, ϵ‐aminocaproic acid) or in the absence of detectable plasminogen, dissolution of the substrate was prevented, and the endothelial cells invaded the fibrin gel, forming vessellike tubular structures similar to those previously observed with collagen gels. These results demonstrate that the invasive and morphogenetic events induced by PMA do not necessarily require an interaction between endothelial cells and collagen fibrils but can also occur with other biologically relevant substrata. They also suggest (1) that invasion may occur via a plasmin‐independent mechanism and (2) that in vivo, neutralization of excess proteolytic activity may play an important permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.