bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma membrane fluidity gradients of human peripheral blood leukocytes
Authors: Collins James M. , Grogan W. McLean , Scott Robert B.
Published: February 2005

The shape of the fluidity gradient of the outer hemileaflet of the plasma membrane of normal, living, human white blood cells was determined using a series of n‐(9‐anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 11, 12, and 16, to establish a baseline for future studies on the consequences of various pathological states. Fluorescence uptake and steady‐state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, with the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence was, for the n‐(9‐anthroyioxy) series, in the order 2 > 3 > 6 > 7 > 9 > 11 =12 = 16 for neutrophils, lymphocytes, and monocytes. Anisotropy values were constant from 5 to 30 min after addition of the various probes. The orders of the anisotropy magnitudes, indicative of the shapes of the fluidity gradient, were, for neutrophils, 6 > 7 > 9 > 2 = 3 = 11 = 12 > 16, and for lymphocytes, 7 > 6 > 9 > 11 > 2 = 3 > 11 = 12 > 16, and for monocytes, 9 > 7 > 6 > 11 > 2 = 3 > 12 > 16. The kinetics of anisotropy from 1 to 5 min after addition of the probes differed for each of the three cell types. Probes with an n‐value less than or equal to the maxima (n = 6, neutrophils; n = 7, lymphocytes; n = 9, monocytes) rapidly (1.2 min) reached equilibrium, whereas those probes with n‐values greater than the maxima took progressively longer times to equilibrate as n increased. This behavior is consistent with the existence of an energy barrier just below the approximate region sensed by the probes, which would correspond to just below 6AS for neutrophils, 7AS for lymphocytes, and 9AS for monocytes.
Ras and neu oncogenes reverse serum inhibition and epidermal growth factor dependence of serum‐free mouse embryo cells
Authors: Shirahata Sanetaka , Rawson Cathleen , Loo Deryk , Chang Yung‐Jin , Barnes David
Published: February 2005

Serum‐free mouse embryo cells, cultured in basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibronectin, and high‐density lipoprotein, do not exhibit growth crisis, lack detectable chromosomal aberrations, are nontumorigenic in vivo, are dependent on epidermal growth factor for survival, and are growth inhibited by serum or platelet‐free plasma. These cells after transfection with the human Ha‐ras or rat neu oncogenes no longer required epidermal growth factor for survival, were tumorigenic in vivo, and also proliferated in serum‐containing medium. Autocrine activity capable of replacing epidermal growth factor was detected in conditioned medium from ras‐transformed cultures, but little such activity was detected in medium from neu‐transformed cultures. In addition, the capability of ras or neu‐transformed cells to grow in serum‐containing medium could not be mimicked in untransformed cells by the addition of growth factors or conditioned medium from transformed cells. These results suggest that the known structural similarity of the neu gene product to the EGF receptor is also reflected in a functional similarity by which the mutationally activated neu protein can replace the ligand‐activated EGF receptor. These results also suggest that the ability of ras‐and neutransformed cells to escape the effect of the inhibitory serum activity is a nonautocrine property distinct from the acquisition of EGF autonomy.
Role of messenger RNA subcellular localization in the posttranscriptional regulation of human histone gene expression
Authors: Zambetti G. , Stein J. , Stein G.
Published: February 2005

Histone mRNAs are naturally localized on non‐membrane‐bound polysomes and selectively destabilized during inhibition of DNA replication. Targeting histone mRNA to membrane‐bound polysomes, by incorporating sequences coding for a signal peptide into the message, results in the stabilization of the histone fusion mRNA when DNA synthesis is interrupted (Zambetti et al.: Proceedings of the National Academy of Sciences of the United States of America 84:2683–2687, 1987). A single nucleotide substitution that abolishes the synthesis of the signal peptide results in the localization of the histone fusion mRNA on non‐membrane‐bound polysomes to the same extent as endogenous histone mRNA and fully restores the coupling of histone fusion mRNA stability to DNA replication. Signal peptide‐histone fusion mRNAs containing two point mutations that result in the incorporation of two positively charged amino acids into the hydrophobic domain of the signal peptide are partially retained on non‐membrane‐bound polysomes and are partially destabilized during inhibition of DNA synthesis. These data indicate that the degree to which the signal peptide‐histone fusion mRNAs are associated with non‐membrane‐bound polysomes is correlated with the extent to which the mRNAs are degraded during inhibition of DNA synthesis. These results suggest that the subcellular location of histone mRNA plays an important role in the posttranscriptional regulation of histone gene expression.
Effects of β‐all‐trans retinoic acid on growth, proliferation, and cell death in a multicellular tumor spheroid model for squamous carcinomas
Authors: Sacks Peter G. , Oke Victor , Vasey Tracey , Calkins Dennise P. , Terry Nicholas H. A.
Published: February 2005

The growth of multicellular tumor spheroids, MTSs, from squamous carcinoma line MDA 886Ln was inhibited by β‐all‐trans retinoic acid (RA). Inhibition occurred within 3 to 5 days of treatment, and MTS size then remained static for up to 2 weeks. Although their growth stopped, 10‐day‐treated MTSs incorporated [3H]thymidine into trichloroacetic acid‐precipitable material, and the [3H]‐thymidine labeling index, determined by autoradiography, was equivalent between control and RA‐treated MTSs. Bivariate flow cytometric analysis of bro‐modeoxyuridine‐labeled MTSs showed equivalent S phase progression of labeled cells over an 8‐hour chase. MTS growth stasis was not related to RA‐induced cell cycle effects. Monitoring of MTSs for cell sloughing showed no significant cell shedding that could account for stasis. Quantitation of cell number and DNA content per MTS showed an RA‐induced decrease. This was confirmed by histological analysis, which demonstrated the temporal appearance of accellular areas. MTS growth statis is thus related to an RA‐induced cell loss in this MTS model for squamous carcinomas.
Altered regulation of platelet‐derived growth factor A‐chain and c‐fos gene expression in senescent progeria fibroblasts
Authors: Winkles Jeffrey A. , O'Connor Mary L. , Friesel Robert
Published: February 2005

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet‐derived growth factor (PDGF) A‐chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson‐Gilford (progeria) syndrome fibroblasts, AG3513, consdtutively expresses PDGF A‐chain mRNA and PDGF‐AA homodimers. The PDGF A‐chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I‐PDGF‐BB, which binds with high affinity to both A‐ and B‐type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF‐stimulated AG3513 progeria fibroblasts, c‐fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A‐chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogeninducible gene expression in normal cells.
Regulation of the Na+‐dependent and the Na+‐independent polyamine transporters in renal epithelial cells (LLC‐PK1)
Authors: Parys Jan B. , De Smedt Humbert , van Dan Bosch Ludo , Borghgraef Roger , Geuns Jan
Published: February 2005

We have studied the regulation of the Na+‐dependent and Na+‐independent polyamine transport pathways in the renal LLC‐PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL‐2‐difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth‐promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na+‐dependent than for the Na+‐independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth‐promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na+‐dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 < 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non‐metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na+‐dependent pathway against the polyamine‐in‐duced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine‐induced decay. These observations suggest that this polyamine‐induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine‐induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na+‐dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na+‐deperdent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a Physiological role for these transporters, such as an involvement in urinary polyamine disposal.
Clonal variation of adenylyl cyclase activity in a rat tumor cell line caused by change in G protein‐catalytic unit interaction
Authors: Kawabata Masahiro , Yoshikura Hiroshi , Horio Keizo , Fujiwara Kenji , Iwamoto Aikichi
Published: February 2005

Two subclones of the rat XC cell line characterized by different morphology exhibited quite different adenylyl cyclase responses upon various stimulations. Upon treatment with cholera toxin, clone RK1 accumulated a high level of intracellular cAMP thereby changing its polygonal morphology to an elongated morphology, while the other clone, LK1, with a fibroblastic morphology, failed to increase the intracellular cAMP and remained morphologically unchanged. When membrane fractions derived from these two clones were stimulated with 10 μM forskolin, 10 μM GTPλS, or 10 mM NaF, five‐ to 20‐fold more cAMP was accumulated in RK1‐derived membranes than in LK1‐derived membranes. With the same membrane fractions, upon treatment with Mn++, which directly stimulates the catalytic unit, a high level of cAMP was accumulated both in RK1 and LK1, indicating that the catalytic function inducible by Mn++ was similar in both clones. There was no significant difference in the level of expression of G protein α2, αi (at least αi1 and αi2), and β subunits between LK1 and RK1. Cholateextracts of the membrane protein of LK1 and RK1 reconstituted the adenylyl cyclase activity of the cyc variant of S49 lymphoma cells to the same level. Therefore, it is inferred that the defect in LK1 resides in the interaction of stimulatory G proteins and the actual catalyst.
Transforming growth factor type β1 modulates the effects of basic fibroblast growth factor on growth and phenotypic expression of rat astroblasts in vitro
Authors: Labourdette Gerard , Janet Thierry , Laeng Pascal , Perraud Frederic , Pettmann Brigitte , Lawrence David
Published: February 2005

In a search of the growth factors possibly involved in brain ontogenesis we have examined the effects of transforming growth factor β1 (TGF‐β1) on the growth and phenotypic expression of rat astroblasts in primary culture. Along TGF‐β1 elicited only a slight negative effect on the growth of these cells. However, this factor was found to modulate the mitogenic effects of other growth factors. On quiescent cells it potentiates the mitogenic effect of basic fibroblast growth factor (bFGF) but not that of other growth factors, namely, epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), and thrombin. TGF‐β1 did not modulate significantly the stimulatory effect of these growth factors on the activity of the enzyme glutamine synthetase (GS); but kinetic studies showed that TGF‐β1 delays the stimulation of GS activity. DNA synthesis monitored by the incorporation of [125I]iododeoxyuridine (125I‐dUrd) was maximum after 24–30 h of treatment with bFGF. With bFGF plus TGF‐β1 the maximum was shifted to 30–36 h. This shift is compatible with the idea that TGF‐β1 induces responsiveness in some cells which are otherwise unresponsive to the mitogenic action of bFGF, and that this induction requires some time. This hypothesis is sustained by the observation that in cells treated for only 12 h with bFGF, the treatment with TGF‐β1 for the same 12 h or for longer time did not stimulate significantly the cell growth. Stimulation occurred only when the bFGF treatment was continued after 12 h. Potentiation of the mitogenic effect of bFGF and shift of the maximum 125I‐dUrd incorporation towards 24 h was seen with cells pretreated with TGF‐β1. This potentiation effect decreased with increasing time between the two treatments. The potentiation effect of TGF‐β1 is not mediated by an induction of new bFGF membrane receptors as seen by binding studies.
Dissociation of proto‐oncogene induction from growth response in normal human fibroblasts
Authors: Yaar Mina , Peacocke Monica , Cohen Michael S. , Gilchrest Barbara A.
Published: February 2005

Proto‐oncogenes are cellular homologues of viral oncogenes that are known to be associated with regulation of growth and differentiation. The c‐myc and c‐fos proto‐oncogenes have been extensively studied by using established cell lines but to a lesser extent by using normal cells. Using physiologic growth modulators, we have shown that mitotic stimulation of normal human dermal fibroblasts is associated with induction of c‐myc and c‐fos, but that growth inhibition of these cells is not necessarily accompanied by their down‐regulation. When treated with both serum and interferon‐alpha during quiescence, fibroblasts were delayed in their progress into the S‐phase of the cell cycle as compared to cells treated with serum alone and displayed substantial growth inhibition as measured by cell number at the end of 1 week. However, this growth inhibition was not preceded by down‐regulation or delay in induction of c‐myc and c‐fos mRNA. The above studies suggest that in normal fibroblasts growth inhibition is not necessarily dependent on down‐regulation of transcription of either c‐myc or c‐fos and that interferon may act to inhibit cell growth either through a post‐transcriptional effect on cellular proto‐oncogenes necessary for cell proliferation or through induction of other, as yet unrecognized gene(s) associated with growth arrest.
Mitogenic effect of transforming growth factor β1 on human fibroblasts involves the induction of platelet‐derived growth factor α receptors
Authors: Ishikawa Osamu , LeRoy E. Carwile , Trojanowska Maria
Published: February 2005

Platelet‐derived growth factor (PDGF) and transforming growth factor β (TGF‐β), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF‐β on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF‐β1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF‐β1 potentiates DNA synthesis in PDGF‐stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF‐β enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF‐AA and 125I PDGF‐BB, consistent with the increased expression of the α type PDGF receptor. The induction of PDGF α receptor subunits by TGF‐β may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.
Decreased L system amino acid transport and decreased gamma‐glutamyl transpeptidase are independent processes in human chronic lymphocytic leukemia B‐lymphocytes
Authors: Woodlock Timothy J. , Brown Richard , Mani Matthew , Pompeo Lisa , Hoffman Howard , Segel George B. , Silber Robert
Published: February 2005

The‐L system of amino acid transport is markedly diminished in chronic lymphocytic leukemia (CLL) B‐lymphocytes, with a maximal velocity less than 15% that of normal B‐lymphocytes. Another membrane‐associated function, the activity of the ectoenzyme, gamma‐glutamyl transpeptidase (GGT), is diminished in CLL B‐cells to 30% that of normal B‐cells. In addition to its transpeptidase activity, a role for GGT has been postulated in the transport of amino acids. In the present report, the possible relationship of these two physiologic functions in CLL B‐cells was studied. The L system transport defect in CLL is restored by phorbol ester‐induced cell maturation; following incubation with 0.15 μM tetradecanoyl phorbol acetate (TPA) for 17 hours, the L system initial velocity showed a 20‐fold increase. In contrast, there was no significant effect on GGT activity with cell maturation. Furthermore, an antibody which diminished GGT activity by 50% in lymphoid cells did not inhibit L system transport. Thus, the impaired L system amino acid transport and GGT activity appear to be independent processes in CLL B‐cells.
Polarized membrane expression of brush‐border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone
Authors: Ronco P. , Antoine M. , Baudouin B. , Geniteau‐legendre M. , Lelongt B. , Chatelet F. , Verroust P. , Vandewalle A.
Published: February 2005

To analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases [neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)] in primary cultures of renal proximal tubule cells grown in various culture media. The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness. In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)‐supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains. By contrast, in serum‐free hormonally defined medium (DM: insulin, 5 μg/ml; dexamethasone, 5 × 10−8 M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule. When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments. This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde‐fixed cells but does not change the total enzymatic activity. This suggests the presence in tubular cells of a dexamethasone‐dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the apical domain.
Identification and partial characterization of genes that are transactivated by different pathways in quiescent mouse cells stimulated with serum
Authors: Boeggeman E. , Masibay A. S. , Qasba P. K. , Sreevalsan T.
Published: February 2005

We have identified four cDNA clones, cl‐1, cl‐5, cl‐15, and cl‐16, that represent genes induced by serum in resting mouse 3T3 cells. Partial sequence analysis of the four cDNAs indicated that cl‐15 corresponds to the mouse beta‐actin gene. Comparison of the DNA sequences of the other three clones with the sequence data bank (Genbank) showed little homology to other known DNA sequences and thus represent novel genes. The level of the mRNAs corresponding to the four genes began to increase in resting cells following serum stimulation, reached a peak between 5 h and 8 h and then started to decline. Inhibitors of transcription diminished the induction of the mRNAs corresponding to the four genes. Cycloheximide and anisomycin hadlittle effect on the induction of beta actin mRNA while the induction of the other three genes was suppressed by the same inhibitors. 12‐O‐Tetradecanoylphorbol‐13‐acetate and the calcium ionophore A23187 enhanced the expression of the cl‐16 mRNA while epidermal growth factor, fibroblast growth factor, or insulin enhanced the expression of cl‐1‐ and cl‐5‐specific transcripts. The level of beta‐actin mRNA was elevated in resting cells by epidermal growth factor and 12‐O‐tetradecanoylphorbol‐13‐acetate and to a lesser extent by fibroblast growth factor, insulin, and dibutyryl cyclic AMP‐elevating agents. Pertussis toxin, an inhibitor of the action ofG proteins, did not significantly suppress the activation of the four genes by serum. However, 2‐aminopurine, a protein kinase inhibitor, suppressed the induction of the four transcripts in serum‐stimulated cells. The possible pathways involved in the activation of these genes in resting cells are discussed.
Correlation between redistribution of a 26 kDa protein and development of chronic thermotolerance in various mammalian cell lines
Authors: Lee Yong J. , Hou Zi‐Zheng , Curetty Lindali , Borrelli Michael J. , Corry Peter M.
Published: February 2005

Previous studies suggested that a 26 kDa protein might play an important role in protein synthesis‐independent thermotolerance development in CHO cells. To determine if this phenomenon was universal, four mammalian cell lines, viz., CHO, HA‐1, murine Swiss 3T3, and human HeLa, were studied. Cells were heated at 42°C, and the level of 26 kDa protein in the nucleus was measured, together with clonogenic survival and protein synthesis. The results demonstrated that (1) the 26 kDa protein was present in the four different cell lines, and (2) the level of the 26 kDa protein in their nuclei was decreased by 30–70% after heating at 42°C for 1 hr. However, restoration of this protein occurred along with development of chronic thermotolerance. The protein synthesis inhibitor cycloheximide (10 μg/ml) neither inhibited the development of chronic thermotolerance nor affected the restoration of the 26 kDa protein in the nucleus. In fact, this drug protected cells from hyperthermic killing and heat‐induced reduction of 26 kDa protein in the nucleus. Heat sensitizers, quercetin (0.1 mM), 3,3′‐dipentyloxacarbocyanine iodide (DiOC5[3]: 5 μg/ml), and stepdown heating (45°C‐10 min→42°C), potentiated hyperthermic killing and inhibited or delayed the restoration of the 26 kDa protein to the nucleus. These results support a correlated, perhaps causal relationship between the restoration of the 26 kDa protein and chronic thermotolerance development in four different mammalian cell lines.
Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content
Authors: Kavanagh Terrance J. , Grossmann Angelika , Jaecks Erik P. , Jinneman John C. , Eaton David L. , Martin George M. , Rabinovitch Peter S.
Published: February 2005

Glutathione (GSH) is important in defense against oxygen free radical damage, in detoxification of xenobiotics, and in mitogenesis. The reducing conditions provided by low molecular weight thiols such as 2‐mercaptoethanol (ME) have been shown to promote the growth of lymphocytes in culture. We wished to determine the effects of 2‐ME on GSH content, and to determine to what extent GSH status affected lymphocyte proliferation. GSH content was quantitated in human peripheral blood lymphocytes (PBL) using a flow cytometric assay with monochlorobimane. This analysis was performed on PBL as well as on the CD4+ T‐cell subset, as identified with fluorescent anti‐CD4 monoclonal antibodies (mAb). Cells were viably sorted on the basis of their GSH content, and incubated for 3 days with mitogenic concentrations of PHA (for PBL) or anti‐CD3 mAb (for CD4+ cells) in the presence of bromodeoxyuridine (BrdU). BrdU/Hoechst cell cycle analysis was then performed on these cells. High GSH sorted cells had a higher percentage of cells capable of entering the cell cycle than low GSH sorted cells. This data indicates that some of the heterogeneity in proliferative capacity within PBL in culture is directly or indirectly related to GSH content. Incubation of cells in 2‐ME prevented the loss of GSH that occurs when cells are cultured. 2‐ME improved the proliferative capacity of unsorted cells, and of cells sorted for high and low GSH. Acridine orange staining of anti‐CD3 mAb stimulated cells sorted for high and low GSH indicated that an early event in cell activation was affected by GSH content.
Inhibition of late endosome‐lysosome fusion: Studies on the mechanism by which isotonic‐K+ buffers alter intracellular ligand movement
Authors: Ward Diane Mcvey , Hackenyos David P. , Davis‐Kaplan Sandra , Kaplan Jerry
Published: February 2005

Incubation of alveolar macrophages or hepatocytes in media in which Na+ is replaced by K+ (‘isotonic‐K buffer’) inhibited the movement of internalized ligand from late endosomes to lysosomes (Ward et al.: journal of Cell Biology 110:1013–1022, 1990). In this study we investigate the mechanism responsible for the isotonic‐K+ block in movement of ligand from late endosomes to lysosomes. We observed that iso‐K+ inhibition of endosome‐lysosome fusion is not unique to alveolar macrophages or hepatocytes but can be seen in a variety of cell types including J774 and Hela cells. The inhibition in intracellular ligand movement was time dependent with the maximum change occurring after 60 minutes. Once established the inhibition resulted in a prolonged and apparently permanent decrease in vesicle movement. Cells were able to recover from the effects of iso‐K+ buffers over a time course of 5–10 minutes when placed back in Na+‐containing media. The effect of iso‐K+ buffers was independent of intracel‐lular pH changes and appeared to involve cell swelling. When cells were incubated in iso‐K+ buffers under conditions in which cell volume changes were reduced, intracellular ligand movement approached normal levels. Such conditions included replacing Cl− with the less permeant anion gluconate, and by addition of sucrose to isotonic‐K+ buffers. Analysis of the mechanism by which changes in cell volume could alter intracellular movement ruled out changes in cyclic nucleotides. Ca2+, or microtubules. These results suggest that changes in cell shape or volume can alter intracellular transport systems by novel routes.
Suppression of programmed death and G1 arrest in B‐cell hybridomas by interleukin‐6 is not accompanied by altered expression of immediate early response genes
Authors: Sabourin Luc A. , Hawley Robert G.
Published: February 2005

The murine B‐cell hybridoma B9 requires interleukin‐6 (IL‐6) for its survival and proliferation in vitro. We show here that withdrawal of IL‐6 from B9 cultures results in programmed death, concomitant with arrest of the cells in the G1 phase of the cell cycle. Unlike several other systems that undergo programmed cell death, no induction of transcripts corresponding to the testosterone‐repressed message‐2 or transglutaminase genes is observed during this process. Upon readdition of IL‐6 to G1‐arrested B9 cells, viability is maintained and entry into S phase occurs after a lag period of 10 to 12 hr. Northern blot analysis showed that the immediate‐early mRNAs normally induced shortly after growth factor stimulation in quiescent fibroblasts (c‐fos, c‐jun, Egr‐1, c‐myc, JE, and KC), and other growth‐related genes (2F1, c‐Ha‐ras, and p53), are either not induced or remain unchanged during G1 to S phase progression. A correlation was found, however, between the temporal pattern of expression of several G1/S phase genes (dihy‐drofolate reductase, thymidine kinase, transferrin receptor, and histone H3) and DNA synthesis. These results demonstrate that IL‐6‐induced viability and growth of hybridoma (and, presumably, plasmacytoma) cells is mediated via novel signal transduction pathways.
Analysis of the dominance of mutations in cAMP‐binding sites of murine type I cAMP‐dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells
Authors: Shuntoh Hisato , Steinberg Robert A.
Published: February 2005

Structural lesions in cAMP‐binding sites of regulartory (R) subunit of cAMP‐dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide‐depencient kinase activation in preparations from cells that were hemizygous or heterozygous for mutant RI subunit expression. No wild‐type kinase activation was observed in extracts from heterozygous mutant cells. This “dominance” was investigated by characterizing expression of wild‐type and mutant RI subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild‐type RI subunits and 3, subunits with a lesion (Glu200) tnat inactivate:. cAMP‐binding site A. By both studies of cAMP dissociation and two‐dimensional gel analysis, wild‐type R subunits comprised about 35% of total RI subunits in heterozygous mutants. Synthesis of wild‐type and mutant RI subunits was equivalent, but wild‐type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel RI subunit‐containingspeciesfromheterozygousmutantpreparationswhoseelution behavior suggested a trimeric kinase consisting of an RI subunit dimer and one catalytic (C) subunit. Wild‐type RI subunit was found only in dimer and “trimer” peaks; the tetrameric kinase peak contained only mutant RI subunit. It is concluded that C subunit binds preferentially to mutant RI subunit in heterozygous cells forming either tetrameric kinase with mutant RI subunit homodimers or trimeric kinase with RI subunit heterodimers. This preferential binding results both suppression of wild‐type kinase activation and differential stabilization of mutant RI subunits.
Serum factor revealing two distant phases of negative proliferation control in mitogen‐stimulated normal fibroblasts
Authors: Böhmer Ralph M.
Published: February 2005

When quiescent normal skin fibroblasts are stimulated by mitogens such as epidermal growth factor (EGF), the proportion of cells entering a division cycle decreases with increasing cell density. The presence of a synthetic double‐stranded RNA (poly I:C) enhances this density‐related restriction. Fetal calf serum (FCS) as well as human serum (HS) and human platelet‐poor plasma (HPPP) completely abrogate the inhibiting effect of cell density on EGF mitogenicity, both in the presence and absence of poly I:C. HS and HPPP are up to ten times more potent than FCS in overcoming density‐related restriction of EGF mitogenicity in human skin fibroblasts, whereas the mitogenic potencies of FCS, HS and HPPP in the absence of EGF are identical. Thus the mitogenic activity of FCS, HS and HPPP and their ability to overcome the density‐related restriction of EGF‐induced proliferation may be due to different molecules. Addition of FCS or HS at various times after EGF exposure reveals two distinct control points within the prerepli‐cative phase: one within the first 2 hours and the other between 10 and 20 hours after the beginning of EGF exposure. Thus, the interactions of EGF, serum, and poly I:C reveal the kinetics of a cell density‐related mechanism of negative proliferation control.
Expression of multiple proteins structurally related to gamma‐glutamyl transpeptidase in non‐neoplastic adult rat hepatocytes in vivo and in culture
Authors: Mathis Georg A. , Wyss Peter A. , Schuetz Erin G. , Hughey Rebecca P. , Sirica Alphonse E.
Published: February 2005

Freshly isolated hepatocytes from normal adult rat liver do not express measurable gamma‐glutamyl transpeptipdase (GGT) mRNA in contrast to the significant GGT mRNA levels expressed by normal adult rat kidney and hyperplastic bile ductular tissue from bile duct‐ligated rats. However, the induction of GG activity in rat hepatocytes by two‐thirds hepatectomy was accompanied by the appearance of a high level of GGT mRNA. We are now able to demonstrate that normal adult rat hepatocytes express 5 protein bands which cross‐react with 2 different anti‐rat kidney GGT antisera. The apparent molecular weights were 26.9, 58.0, 63.9, 73.5, and 83.4 kDa, respectively. Expression of the 26.9‐ and 58.0‐kDa proteins strikingly parallels the pattern of induction of GGT enzymatic activity. This suggests that these 2 proteins correspond to the active dimeric enzyme previously described in kidney and neoplastic hepatocellular tissue. In normal hepatocytes, the 73.5‐kDa protein represents 50% of the total GGT‐immunoreactive protein, contrast to kidney, where this band contains less than 4% of the GGT protein. The kinetics of expression of the 73.5‐kDa protein upon induction of GGT activity in hepatocytes, as well as in culture turnover studies, suggests that this protein is a precursor form of the active enzyme, such as the described 78/79‐kDa single‐chain glycoprotein propeptide of GGT. It appears that in normal hepato‐cytes, this precursor is not processed to the same extent as in kidney or in hyperplastic bile ductular tissue.
Interactions of dimethyl sulfoxide and granulocyte‐macrophage colony‐stimulating factor on the cell cycle kinetics and phosphoproteins of G1‐enriched HL‐60 cells: Evidence of early effects on lamin B phosphorylation
Authors: Brennan J. K. , Lee K. S. , Frazel M. A. , Keng P. C. , Young D. A.
Published: February 2005

We have found that GM‐CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL‐60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL‐60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombiniant human GM‐CSF and/or 0–1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]‐orthophosphate‐labeled cell proteins separated by giant 2‐D gel electrophoresis. Results were correlated with (1) intracellular pH, determined by measurement of BCECF fluorescence; (2) [32P]‐orthophosphate uptake; (3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide‐stained cells; and (4) growth, determined by cell volume and concentration. GM‐CSF stimulated and DMSO inhibited the GM‐CSF‐stimulated phosphorylation of 1 protein (∼65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1 not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15–30 minutes), cell volume change (16–24 hr), and cell concentration increase (28–32 hr). GM‐CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synch onization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM‐CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM‐CSF‐DMSO antagonism is exerted through the same mitogenic pathway, that a major locus of cytokinetic effect is on G1 to S transit, and that nuclear envelope protein phosphorylation is an important early event.
Retinoic acid priming potentiates the induction of urokinase‐type plasminogen activator by cyclic adenosine monophosphate in mouse mammary carcinoma cells
Authors: Mira‐Y‐Lopez Rafael
Published: February 2005

Interactive regulation of gene expression by retinoic acid (RA) and cyclic adenosine monophosphate (cAMP) in mammary tumor cells was explored using Shionogi mouse mammary carcinoma cells (SC115) as a model and urokinase‐type plasminogen activator (uPA) as a target gene product. Twenty‐four hour treatment of SC115 cells with 100 nM RA, 1 mM 8‐bromo‐cAMP (BrcAMP), and 100 nM RA + 1 mM BrcAMP resulted in extracellular uPA activity increases of 1.4‐fold, sevenfold, and 20‐fold, respectively. These effects were dose‐dependent with regard to both interacting members. Similar responses were obtained if 1 nM cholera toxin or 10 μM forskolin was used instead of the cAMP analog. Retinoids lacking the carboxylic acid function were inactive. The changes in uPA activity were accompanied by similar changes in uPA antigen concentration, as seen via Western blot analysis, and uPA mRNA abundance, as seen via Northern blot analysis. Actinomycin D, an inhibitor of RNA synthesis, blocked uPA stimulation by BrcAMP, suggesting that mRNA levels were transcriptionally regulated. The effect of BrcAMP on extracellular uPA activity was first evident at 2 h and peaked at 6 h; the effect of RA alone and the synergistic response to joint treatment, however, followed a slower time course, requiring at least 12 h for initial expression and increasing gradually with time up to at least 48 h. Priming with RA for 48 h followed by extensive washing of the cells resulted in a threefold enhancement of the stimulatory effect of BrcAMP on uPA. Experiments utilizing the casein/plasminogen overlay method for the detection of uPA secretion by single cells showed that the enhanced response to BrcAMP was due to an increased rate of uPA secretion per cell rather than to an increased fraction of uPA‐secreting cells. Initial investigation of the mechanism of RA potentiation of cAMP responsiveness showed that RA did not alter cellular cAMP levels or total cAMP‐dependent protein kinase A activity. Finally, the tumor promoter phorbol myristate acetate, an activator of protein kinase C, also increased SC115 cell uPA activity and synergized with RA. This raised the possibility that the enhancement of cAMP responsiveness by RA was indirectly mediated via an effect on protein kinase C. Experiments with protein kinase C‐depleted cells, however, showed that this was not the case. In conclusion, RA treatment of SC115 cells potentiates the effect of cAMP on uPA expression at the single cell level via a partially irreversible mechanism independent of protein kinase C. The molecular target of RA and whether SC115 cell differentiation underlies the effect of RA remain to be established.
cAMP promotes branching of laminin‐induced neuronal processes
Authors: Weeks Benjamin S. , Papadopoulos Vassilios , Dym Martin , Kleinman Hynda K.
Published: February 2005

Laminin is a potent stimulator ofneurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium‐proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108‐15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin‐induced neurites. A four‐fold increase in the number of branches per neurite and a two‐to‐three‐fold increase in the number of neurites per cell were observed in both NG108‐15 and PC12 cells cultured on laminin when either 8‐BrcAMP or forskolin was added. The cAMP‐induced branching was also observed when PC12 cells were cultured on a laminin‐derived synthetic peptide (PA22‐2), which contains the neurite‐promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22‐2 were axonal, not dendritic, and the cAMP‐induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin‐mediated neurite outgrowth, but cAMP can modulate the effects of laminin.
Role of TGFα and its receptor in proliferation of immortalized rat tracheal epithelial cells: Studies with tyrphostin and TGFα antisera
Authors: Ferriola Patrice C. , Earp H. Shelton , Di Augustine Richard , Nettesheim Paul
Published: February 2005

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGFα and do so regardless of the mechanism by which they were transformed. In order to determine whether TGFα is an autocrine growth regulator of immortalized RTE ‐cells, we have examined the function of TGFα/EGF receptors and the growth requirements for TGFα in these cells. The level of immunoprecipitated TGFα/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGFα/EGF receptors. Scatchard analysis of TGFα/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high‐affinity (Kd = 0.4nM) and low‐affinity (Kd = 9.8nM) binding sites. A tyrphostin TGFα/EGF receptor tyrosine kinase inhibitor decreased in a dose‐dependent manner the proliferation as well as EGF‐induced autophosphorylation of the TGFα/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF‐dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGFα antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGFα and its receptor.
Role of putrescine in interleukin 1β production in human histiocytic lymphoma cell line U937
Authors: Tahara Hideki , Otani Shuzo , Matsui‐Yuasa Isao , Koyama Hidenori , Nishizawa Yoshiki , Morisawa Seiji , Morii Hirotoshi
Published: February 2005

Treatment with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) and incubation with lipopolysaccharide (LPS) induces Interleukin 1β (IL‐1β) production in the histiocytic lymphoma cell line U937. Here we investigated the effect of treatment with both TPA and 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3) on LPS‐induced IL‐1β production in U937 cells. To clarify the mechanism of IL‐1β production, the possible role of polyamines in this process was examined. Combined treatment with TPA and 1,25(OH)2D3 for 72 h followed by incubation with LPS for 24 h caused synergistic induction of both IL‐1β release and mRNA expression. On the other hand, TPA increased the numbers of vitamin D3 receptors, which may be one mechanism of this synergistic induction. Ornithine decarboxylase (ODC), a rate‐limiting enzyme for polyamine biosynthesis, was also induced by these compounds biphasically: the first peak of ODC activity was observed at 4 h of the incubation with the two compounds and the second peak was at 4 h after the addition of LPS. To find whether these peaks were related to IL‐1β production, DL‐α‐difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, was added together with TPA and 1,25(OH)2D3. DFMO decreased the cellular levels of putrescine and spermidine and suppressed IL‐1β release and IL‐1β mRNA expression by 65%. Exogenous putrescine, but not spermidine, abrogated these kinds of inhibition. Similar results were obtained with DFMO and the polyamines during the differentiation of the cells up to the monocyte or macrophage stage. These results thus suggest that changes in either of these intracellular polyamines, especially putrescine, help to regulate the differentiation of U937 cells, resulting in partial control of the regulation of IL‐1β production.
A toxin‐resistant mouse L‐cell mutant defective in protein transport along the secretory pathway
Authors: Laurie S. M. , Robbins A. R.
Published: February 2005

Using methods designed for isolation of mutants defective in receptor‐mediated endocytosis, a novel L‐cell mutant was obtained that exhibits resistance to three different protein toxins as well as alterations in secretion. This rrutant, LEFIC, is resistant to modeccin, Pseudomonas exotoxin, and ricin. Thesi toxins, which enter the cytoplasm via receptor‐mediated endocytosis, are thought to penetrate into cells at the level of late endosomes or the trans Golgi network. Early endosomal acidification appears to be normal in the mutant based on its accumulation of iron from transferrin and its sensitivity to diphtheria toxin A chain‐transferrin conjugate. Within the secretory pathway twodelays in transport ofvesicular stomatitis virus (VSV) G protein wereobserved in LEFIC: a 20–30 min delay in acquisition of Endo H resistance and a 1–2 hr delay in appearance of newly synthesized G protein on the cell surface. Movement of endogenous proteins along the secretory pathway was also affected in LEFIC. Fibronectin secretion was delayed by 15 min, and membrane proteins were delayed in arrival at the cell surface. The phenotype of LEFIC is consistent with a defect in a component or compartment shared by both the late endocytic and constitutive secretorypathways.