bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth inhibitory activity in extracts from human fibroblasts
Authors: Macieira‐Coelho Alvaro , Söderberg Anita
Published: February 2005

Indirect evidence for the presence of a growth inhibitor in normal human fibroblasts has been obtained previously; the inhibitory activity has been found associated with crude cell extracts, but the molecule responsible for the growth inhibition has never been isolated. We have isolated a glycopeptide fraction from human fibroblast cultures, whose synthesis decreases when the cells are stimulated into the division cycle. It was separated by electric charge, lectin affinity, and molecular mass. When added to quiescent cells simultaneously with a growth stimulus, the glycopeptide reduces DNA synthesis activity. The relationship of the kinetics of the synthesis of the glycopeptide with the cell division cycle and its molecular weight are different from what has been described so far for other growth regulators. The decreased synthesis of this inhibitor, induced by growth factors, seems to be one of the requirements for the initiation of the division cycle by human fibroblasts. This response to growth factors was stable during the lifespan of the fibroblast population and became less pronounced only in cells at the end of their replicative potential. © 1993 Wiley‐Liss, Inc.
Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon‐γ and interleukin‐1α
Authors: Quintavalla J. C. , Berg R. A. , Beavis A. J. , Piccoli S. P. , Rediske J. J. , Kurkinen M. , Patrick R. A. , Robertson F. M.
Published: February 2005

Articular chondrocytes from rheumatoid joints have been shown to express class II major histocompatibility (MHC) antigens that were correlated with the presence of interferon‐gamma (IFN‐γ) in the inflamed joint. Chondrocytes expressing MHC antigens function as antigens function as antigen presenting cells and thus stimulate lymphocyte proliferation. These responses suggest a powerful role for the IFN‐γ stimulation of chondrocytes. The present studies were designed to examine the functional role of chondrocytes exposed to IFN‐γ during cartilage degradation that occurs in synovial disease. Destruction of cartilage in arthritis is partially attributable to metalloproteinases released by the chondrocytes in response to interleukin‐1 (IL‐1). Bovine articular chondrocytes treated with interleukin‐1 alpha (IL‐1α) produced enhanced levels of stromelysin mRNA, however, Northern blots could not determine the percentage of cells responding. Exposure of bovine articular chondrocytes to IFN‐γ induced the expression of bovine HLA‐DR (boHLA‐DR) antigen in 50% of the cells. Using a modified cell sorting technique, chondrocytes that expressed class II MHC antigens produced two fold greater stromelysin mRNA than chondrocytes that did not express this antigen. In contrast, collagen type II mRNA levels were similar in chondrocytes, regardless of the expression of class II MHC antigens. In situ hybridization studies showed that less than half of all cartilage chondrocytes were induced to synthesize stromelysin mRNA. These observations suggest that IFN‐γ stimulates specific subpopulations of chondrocytes to be functionally active in inflammation‐induced metalloprotease secretion. © 1993 Wiley‐Liss, Inc.
Increase in epidermal growth factor receptor and its messenger ribonucleic acid levels with differentiation of human trophoblast cells in culture
Authors: Alsat Eliane , Haziza Jocelyne , Evain‐Brion Danièle
Published: February 2005

Epidermal growth factor receptor (EGFR) expression was studied during the differentiation of human trophoblast cells in culture. In vitro, intravillous mononuclear cytotrophoblasts aggregate and fuse within 24 h to form a syncytium. This morphological differentiation was associated with a significant twofold increase in specific 125I‐EGF binding capacity (P < 0.01). Scatchard analyses showed an apparent rise in the number of high‐affinity binding sites (0.33 ± 0.04 and 0.63 ± 0.07 pmol/mg protein at 24 and 48 h, respectively), with no change in their affinity (1.34 and 1.42 × 10−10 mol/L). Affinity labeling of 125I‐EGF in cultured trophoblast cells followed by SDS‐PAGE and autoradiography revealed a band of 175 KDa corresponding to EGFR, the intensity of which increased with the time in culture. EGF‐dependent phosphorylation of membrane proteins from cultured trophoblast cells revealed major phosphorylated proteins of 170 KDa (EGFR) and 35 KDa, which were both increased at 48 h, indicating a rise in EGFR‐kinase activity during syncytium formation. Northern blot analysis of EGFR‐mRNA, followed by hybridization with a 32P‐cDNA probe for EGFR, revealed an increase in EGFR gene expression in syncytiotrophoblasts, as compared to cytotrophoblasts. Thus, the increase in bioactive EGFR observed during the differentiation of trophoblast cells was due to an increase in their synthesis. Cultured trophoblast cells are therefore a good model of spontaneous up‐regulation of EGFR expression with cell differentiation. © 1993 Wiley‐Liss, Inc.
Content of nonhistone protein in nuclei after hyperthermic treatment
Authors: Chu G. L. , Ross G. , Wong R. S. L. , Warters R. , Dewey W. C.
Published: February 2005

When nuclei were isolated from Chinese hamster ovary cells after being heated, there was a large increase in the amount of 3H‐tryptophan labeled nonhistone protein in the nucleus relative to the whole cell. After 15 min or 30 min of heating at 45.5°C, the nuclear nonhistone protein content increased by 1.6 or 1.8, respectively. In contrast, when the nuclear nonhistone protein content was determined in the intact cell by using autoradiography to quantify 3H‐tryptophan labeled protein in the nucleus and cytoplasm in sections of fixed cells, the nuclear nonhistone protein content increased by only 1.14 or 1.28 for 15 or 30 min at 45.5°C, respectively. Therefore, heat does not induce a massive movement of cytoplasmic protein into the nucleus. © 1993 Wiley‐Liss, Inc.
Polyamine regulation of Na+/glucose symporter expression in LLC‐PK1 cells
Authors: Peng Hua , Lever Julia E.
Published: February 2005

Addition of polyamines or their analogs to newly confluent LLC‐PK1 cells resulted in down‐regulation of Na+‐glucose transport (symport) activity. Polyamines prevented the induction of this symporter by the differentiation inducer hexamethylene bisacetamide (HMBA) but did not influence induction by the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX). Partial depletion of endogenous polyamines after addition of α ‐difluoromethylornithine (DFMO) resulted in a 4 to 5‐fold increase in symporter expression. Symporter induction by either HMBA or DFMO was inhibited by the protein kinase inhibitor H‐7 but H‐7 did not affect symporter induction by IBMX. Changes in symporter activity were accompanied by changes in levels of the 75 kD symporter subunit detected by Western blot. Cultures exposed to HMBA exhibited reduced levels of ornithine decarboxylase activity. Our results suggest that induction of symporter expression by HMBA may be mediated in part by its effects on polymine metabolism, and point to parallel roles of polyamines and cyclic AMP in regulating the expression of this physiologically important renal transport system. © 1993 Wiley‐Liss, Inc.
Structural reaction pattern of hepatocytes following exposure to hypotonicity
Authors: Pfaller Walter , Willinger Christian , Stoll Barbara , Hallbrucker Christian , Lang Florian , Häussinger Dieter
Published: February 2005

Isolated rat hepatocytes were exposed to hypotonic media (225 mosmol/l) for 5 and 15 min and processed for a quantitative electron microscopic stereologic analysis. Within 5 min of hypotonicity, the hepatocyte volume increased by 25% and thereatter displayed a volume regulatory decrease leading to mean cellular volume, which was 16% above that of controls. Stereologic analysis of the major subcellular compartment, the cytosol, showed an identical change as the whole cell. In contrast to that, the mitochondrial compartment increased in volume by 30% within the first 5 min of exposure and returned by regulatory volume decrease back to values of the isotonic controls after 15 min of hypotonicity. In contrast, hypotonicity (220 mosmol/l)‐stimulation of flux through mitochondrial glutaminase and the glycine cleavage enzyme complex, as assessed by 14CO2 production from [1‐14C]glutamine or [1‐14C]glycine in isolated perfused rat liver persisted throughout a 15‐min period of hypotonic exposure. Thus hypotonicity‐induced alterations of mitochondrial metabolism apparently do not parallel the time course of mitochondrial volume changes. This suggests that persistent mitochondrial swelling is not required for functional alterations, but that the latter may be triggered by the initial swelling of mitochondria. Hypotonic exposure did not alter the nuclear volume of isolated hepatocytes. Cell membrane surface nearly doubled after 5 min of hypotonic exposure, but returned within 15 min of exposure to values observed in normotonic media. This may reflect the participation of exocytosis in hepatocyte volume regulation. © 1993 Wiley‐Liss, Inc.
Regulation of matrix synthesis rates by the ionic and osmotic environment of articular chondrocytes
Authors: Urban J. P. G. , Hall A. C. , Gehl K. A.
Published: February 2005

Chondrocytes in cartilage are embedded in a matrix containing a high concentration of proteoglycans and hence of fixed negative charges. Their extracellular ionic environment is thus different from that of most cells, with extracellular Na+ being 250–350 mM and extracellular osmolality 350–450 mOsm. When chondrocytes are isolated from the matrix and incubated in standard culture medium (DMEM; osmolality 250–280 mOsm), their extracellular environment changes sharply. We incubated isolated bovine articular chondrocytes and cartilage slices in DMEM whose osmolity was altered over the range 250–450 mOsm by Na+ or sucrose addition. 35S‐sulphate and 3H‐proline incorporation rates were at a maximum when the extracellular osmolality was 350–400 mOsm for both freshly isolated chondrocytes and for chondrocytes in cartilage. The incorporation rate per cell of isolated chondrocytes was only 10% that of chondrocytes in situ both 4 and 24 hours after isolation. For freshly isolated chondrocytes, the rate increased 30–50% in DMEM to which NaCl or sucrose had been added to the increase osmolality. In chondrocytes incubated overnight in DMEM, the rate was greatest in DMEM of normal osmolality and fell from the maximum in proportion to the change in osmolality. The effects of surcrose addition on incorporation rates were similar but not identical to those of Na+ addition. Changes in cell volume might be linked to changes in synthesis rates since the cell volume of chondrocytes (measured by Coulter‐counter) increased 30–40% when the cells are removed from their in situ environment into DMEM. Synthesis rates can thus be partly regulated by changes in extracellular osmolality, which in cartilage is controlled by proteoglycan concentration. This provides a mechanism by which the chondrocytes can rapidly respond to changes in extracellular matrix composition. © 1993 Wiley‐Liss, Inc.
Effect of cell age and phenylhydrazine on the cation transport properties of rabbit erythrocytes
Authors: Brugnara Carlo , De Franceschi Lucia
Published: February 2005

We studied the effect of cell age on the cation transport systems of rabbit erythrocytes by increasing the proportion of circulating young erythrocytes with either repeated bleeding or with phenylhydrazine (PHZ) treatment. We found that when the reticulocyte content of rabbit blood is increased by bleeding (from 1 to 40–50% of the circulating red cells), the response of the various transport pathways differs. The largest increase (fivefold) was found in the activity of K‐CI contransport which peaked 3 days after the last bleeding. The Na‐K pump activity peaked at a similar time, but the % increase was twofold less than the K‐CI contransport. There was very small increase in the activity of the Na‐Li exchange, whereas the Na‐H exchange reached peak values 10 days after the last bleeding (twofold increase), when activities of K‐Cl contransport and Na‐K pump had returned to almost normal levels. In vivo PHZ treatment resulted in anemia and marked reticulocytosis (80–90% of circulating cells). Transport rates were markedly increased (Na‐K pump 9.6‐fold, Na‐H exchange 6.8‐fold, Na‐Li exchange 2.75‐fold; K‐CI contransport: 10–20‐fold). When blood from PHZ‐treated rabbits was incubated in vitro for 24–48 hour, red cell volume and K content decreased. This process was associated with a 70% reduction in the activity of the K‐CI contransport after 24 hours and a 90% reduction after 48 hours. The activity of the other systems also declined and approached baseline values after 48 hours. Loss of transport activity was not affected by 10 μM E‐64, whereas 10 mM methylamine reduced the inactivation of the Na‐H exchange and of the Na‐Li exchange. PHZ treatment of rabbit red cells in vitro resulted in marked increase of the K‐CI contransport and inhibition of Na‐K pump, Na‐H exchange, and Na‐Li exchange. These effects were abolished by DTT, with the exception of the Na‐K pump inhibition, which was DTT insensitive. Thus both cell age and oxidative damage are important determinants of cation transport in rabbit red cells. © 1993 Wiley‐Liss, Inc.
Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells
Authors: Dorheim Marie‐Annette , Sullivan Mary , Dandapani Viji , Wu Xiying , Hudson Jane , Segarini Patricia R. , Rosen David M. , Aulthouse Amy L. , Gimble Jeffrey M.
Published: February 2005

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7–10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose‐dependent decrease in enzyme activity. The steady‐state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte‐induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated. © 1993 Wiley‐Liss, Inc.
Angiotensin II‐induced vascular smooth muscle cell hypertrophy: PDGF A‐chain mediates the increase in cell size
Authors: Berk Bradford C. , Rao Gadiparthi N.
Published: February 2005

We report here that angiotensin II‐mediated hypertrophy of vascular smooth muscle cells (VSMC) exhibits PDGF A‐chain‐and ‐pathways. Secretion of PDGF A‐chain is required for the increase in cell size, but not for the increase in protein synthesis. Angiotensin II stimulates a hypertrophic growth response in VSMC characterized by increases in cell size and protein synthesis, but not cell number. Because angiotensin II‐stimulated VSMC hypertrophy has been associated with increased PDGF A‐chain expression, we studied its role in the hypertrophic response by inhibiting PDGF A‐chain expression with hydrocortisone or anti‐PDGF antibody. Hydrocortisone (1 μM for 48 h) inhibited basal protein synthesis by 47%, but angiotensin II‐stimulated protein synthesis was enhanced (111% increase after hydrocortisone treatment vs. 25% increase in control). In contrast, hypertrophy, as measured by cell size, was completely inhibited. Although hydrocortisone had no effect on early growth signals stimulated by angiotensin II (e.g., activation of protein kinase C, stimulation of Na+/H+ exchange, and c‐fos and c‐myc expression), it significantly decreased angiotensin II‐stimulated secretion of PDGF‐like material into the medium from 0.4 to 0.1 ng/ml/24 h (p < 0.01). However, the time course for PDGF secretion (maximal at 16–24 h) was significantly slower than the time course for angiotensin II‐stimulated protein synthesis (maximal at 4–12 h). To block the action of PDGF A‐chain selectively, VSMC were treated with anti‐PDGF A‐chain antibody. The antibody completely inhibited the angiotensin II‐stimulated increase in cell size, but it had no significant effect on protein synthesis at early times (<8 h). These findings demonstrate two pathways involved in angiotensin II‐stimulated VSMC hypertrophy: an increase in cell size dependent on PDGF A‐chain and an increase in protein synthesis independent of PDGF A‐chain. © 1993 Wiley‐Liss, Inc.
Platelet‐activating factor secreted by DDAVP‐treated monocytes mediates von willebrand factor release from endothelial cells
Authors: Hashemi S. , Palmer D. S. , Aye M. T. , Ganz P. R.
Published: February 2005

We have previously shown that although DDAVP (1‐deamino‐8‐D‐arginine vasopressin), a synthetic analogue of the natural hormone arginine vasopressin, does not directly promote release of vWf from human umbilical vein endothelial cells (ECs), enhanced release does occur when ECs were exposed to either monocytes or to supernatants recovered from DDAVP‐treated monocytes. In the present study, we have found that exposure of monocytes to DDAVP did not increase secretion of interleukins (IL)‐1β, IL‐6, IL‐8, tumor necrosis factor (TNF‐α), growth factors G‐CSF (granulocyte‐), GM‐CSF (granulocyte, monocyte‐colony stimulating factor), prostaglandins (PG) E2, PGF2α, or PGI2 or purine nucleotides such as ATP and ADP. However, increased levels of platelet‐activating factor (PAF) were secreted by DDAVP‐treated monocytes in a time‐ and dose‐dependent manner that positively correlated with the enhancement in vWf release from ECs. Moreover, this effect could also be elicited when lipid extracts of these supernatants or purified PAF were added directly to ECs. This response could be inhibited with (±)‐trans‐2,5‐Bis(3,4,5‐trimethoxyphenyl)‐1,3‐dioxolane, a specific PAF receptor antagonist, when the ECs were exposed to supernatants from DDAVP‐treated monocytes or to pure PAF. The present data indicate that enhanced secretion of PAF from monocytes is one mechanism whereby DDAVP can provoke release of vWf from ECs. © 1993 Wiley‐Liss, Inc.
Low oxygen stimulates proliferation of fibroblasts seeded as single cells
Authors: Falanga Vincent , Kirsner Robert S.
Published: February 2005

In standard tissue culture conditions (20% oxygen), single human dermal fibroblasts (one cell per well) do not proliferate. We now report that low oxygen tension is a potent stimulus for the proliferation and expansion of human adult and neonatal dermal fibroblasts seeded as single cells. This preferential single‐cell proliferation in low oxygen is shown to be also a feature of human lung and dermal rodent fibroblasts, but not of human fibrosarcoma and immortalized 3T3 cells, which proliferate without difficulty in standard oxygen conditions. It is suggested that single‐cell proliferation and its dramatic stimulation in low oxygen may represent a fundamental biologic process with an opportunity to better understand mammalian cell growth regulation. © 1993 Wiley‐Liss, Inc.
Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation
Authors: Tilly Jonathan L. , Hsueh Aaron J. W.
Published: February 2005

A method combining the advantages of electrophoretic DNA fractionation and autoradiography is described for the qualitative and quantitative analysis of internucleosomal DNA fragmentation that occurs during apoptosis, or “programmed cell death”. This procedure utilizes terminal transferase enzyme to uniformly add one molecule of [α 32P]‐to the 3′‐of DNA fragments. Following gel electrophoresis and autoradiographic analysis, the total amount of radiolabel incorporated into the low molecular weight DNA fraction can be quantitated and used to estimate the degree of apoptotic DNA fragmentation in any given sample. This method requires as little as 15 ng of total cellular DNA and increases the sensitivity of apoptotic DNA detection by at least 100‐fold over the widely used ethidium bromide staining method. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells. © 1993 Wiley‐Liss, Inc.
Adhesiveness and proliferation of epithelial cells are differentially modulated by activation and inhibition of protein kinase C in a substratum‐dependent manner
Authors: Zhou You , Dziak Ewa , Opas Michal
Published: February 2005

In the present study, we have examined the regulation of attachment, onset of proliferation and the subsequent growth, in vitro, of chick retinal pigmented epithelial (RPE) cells as a function of the nature of the substratum and of either the activation or inhibition of protein kinase C (PKC). The RPE cells have an adhesive preference for protein carpets which contain laminin. This preference disappears gradually with time in culture. The adhesion of RPE cells to fibronectin is shown to be a receptor‐mediated process which involves the RGD recognition signal. This study also demonstrates that a PKC activator, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), affects RPE cell adhesion in a substratum‐dependent manner. Exposure of RPE cells to TPA lowers the cell attachment efficacy to ECM protein substrata but does not affect cell attachment to plastic. The onset of cell proliferation is accelerated by TPA on all of the substrata tested. The minimal duration of an effective TPA pulse exerting a long‐lasting influence on RPE cell proliferation is between 1.5 and 3.5 hr. Stimulation of cell proliferation by TPA in long‐term cultures is independent of the nature of the growth substratum. The acceleration of the onset of cell proliferation by TPA is sensitive to 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7), an inhibitor of conventional PKC, and thus appears to be dependent on the activation of conventional PKC. H7 also affects cell‐cell contacts, causing an alteration in the shape (“squaring”) of RPE cells packed into large colonies. Conversely, the effects of TPA on both the attachment and the long‐term proliferation of RPE cells are not dependent a conventional PKC isotype, since H7 cannot abolish the influence of TPA on either process. We conclude that the effect of TPA on long‐term proliferation of RPE cells is either dependent on a novel PKC isotype or independent of PKC. © 1993 Wiley‐Liss, Inc.
Synergistic effects of cytokine and hyperthermia on cytotoxicity in HT‐29 cells are not mediated by alteration of induced protein levels
Authors: Lee Yong J. , Hou Zizheng , Curetty Lindali , Cho Joong M. , Corry Peter M.
Published: February 2005

In this study, we investigated the mechanism of synergistic effects of cytokine and hyperthermia on cytotoxicity in HT‐29. When cells were heated at 42°C in the presence of recombinant human tumor necrosis factor (rhTNF‐α), recombinant interferon‐gamma (rhIFN‐γ), or in a combination of both, a synergistic increase in the cytotoxic effects of the respective drugs was observed. We hypothesized that alteration of cytokine or heat‐induced polypeptides synthesis was responsible for a synergistic interaction between heat and cytokine.

Five heat shock proteins (HSPs, Mr 110,000, 100,000, 90,000, 70,000, and 28,000) were preferentially synthesized during chronic heating at 42°C. In contrast, the synthesis of two proteins (Mr 60,000 and 29,000) was induced by treatment with rhIFN‐γ (1,000 U/ml). Although the combination of chronic hyperthermia (42°C) with TNF‐α, IFN‐γ, or TNF‐α + IFN‐γ increased cytotoxicity, alteration/induction of polypeptides was not correlated with the synergistical cytotoxic effects of cytokine and heat. Thus, the synergistic effects of cytokine and hyperthermia are not mediated through an induction of polypeptides. © 1993 Wiley‐Liss, Inc.
Effects of epidermal growth factor, estrogen, and progestin on DNA synthesis in mammary cells in vivo are determined by the developmental state of the gland
Authors: Haslam Sandra Z. , Counterman Laura J. , Nummy Katherine A.
Published: February 2005

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are involved in the growth and development of the normal mammary gland. While studies have been carried out to investigate the in vivo effects of EGF in the immature mammary gland, nothing is known about the growth effects of EGF or its potential interactions with E and/or P in the adult mammary gland. The present studies were undertaken to investigate the effects of EGF, E, and P on mammary cell proliferation in immature, peripubertal vs. adult, sexually mature mice. We have found that EGF promotes epithelial and stromal cell proliferation in both the immature and adult mammary glands. In the immature gland, the end bud epithelium is most responsive to the proliferative effects of EGF and there is no apparent interaction between EGF, E, and/or P. In contrast, in the mature gland EGF adds to the proliferative effects of E+P in the ductal epithelium resulting in more extensive ductal sidebranching. Thus these results demonstrate that the developmental state of the mammary gland determines the nature and extent of the interactions between EGF, E, and P in growth and development. © 1993 Wiley‐Liss, Inc.
Cell density, negative proliferation control, and phosphorylation of retinoblastoma protein
Authors: Böhmer Ralph M.
Published: February 2005

Cell density negative control (CDNC) of normal human fibroblast proliferation occurs after stimulation by mitogens with different signal transduction mechanism. Delayed exposure to agents that interfere with CDNC, such as doublestranded RNA and vanadate, reveals the existence of a biochemical event, involved in CDNC, that occurs 5–8 hr after the beginning of mitogenic stimulation. This is earlier than the point of “mitogenic commitment,” defined by the duration of mitogen exposure required for cell cycle entry (8–18 hr). Phosphorylation of the retinoblastoma gene product (pRB) begins 8–10 hr after mitogen stimulation and is nearly complete at 18 hr, just as the first cells enter S‐phase. CDNC prevents pRB phosphorylation. Interferon β delays pRB phosphorylation by up to 20 hr but has little effect on the timing of mitogenic commitment. Thus mitogenic commitment is located in time between CDNC and pRB phosphorylation. When agents that cause a release from CDNC are applied to dense, negatively controlled cultures after 18 hr of EGF stimulation, pRB phosporylation occurs 6–8 hr after release. This suggests that the negatively controlled cells process the mitogenic signal but accumulate at a restriction point. The relatively early timing of CDNC‐related events in the prereplicative phase raises the possibility that pRB phosphorylation is a consequence rather than a prerequisite for release from cell density negative control. © 1993 Wiley‐Liss, Inc.
Dissociation of early and late markers of murine myeloid differentiation by interferon‐gamma and interleukin‐6
Authors: Ruhl Stefan , Pluznik Dov H.
Published: February 2005

Murine myeloid leukemia M1 cells undergo terminal differentiation to mature macrophages after stimulation with interleukin‐6 (IL‐6). This process can be monitored by measuring the expression of early markers such as the high affinity receptor for monomeric IgG2a (FcγRI) and la antigen followed by late markers such as lysozyme production and finally morphological changes from blast cells to mature macrophages. The same early markers that are expressed on M1 cells after induction with IL‐6 are also expressed on monocytic cells after activation with interferon‐γ (IFNγ). We used IL‐6 and IFNγ to investigate whether the early stages of M1 cell differentiation could be accomplished without commitment of the cells to terminal differentiation. Cytofluorometry shows that the expression of the same early differentiation markers (FcγRI and la antigen) that are inducible by IL‐6 on M1 cells can be induced by IFNγ as well. However, stimulation with IFNγ, in contrast to IL‐6, does not induce the late differentiation markers such as lysozyme production, phagocytic activity, and morphological changes. Northern analysis supports these findings in that expression of FcγRI mRNA is induced by either cytokine, whereas expression of mRNA for lysozyme is inducible by IL‐6 only. Nuclear run‐on analysis reveals that the changes in steady state mRNA levels of both FcγRI and lysozyme are regulated by a transcriptional mechanism. These data suggest that early stages in the process of myeloid differentiation can be separately induced by IFNγ and thus are independent from the later events induced by IL‐6. © 1993 Wiley‐Liss, Inc.
Epidermal growth factor receptors lose ligand binding ability as WI‐38 cells progress from short‐term to long‐term quiescence
Authors: Donigan Anne M. , Cavalli R. Christopher , Pena Angel A. , Savage C. Richard , Soprano Dianne Robert , Soprano Kenneth J.
Published: February 2005

WI‐38 cells, density arrested for short periods of time, can be stimulated to re‐enter the cell cycle by epidermal growth factor (EGF) alone. However, cells density arrested for longer periods have a prolonged prereplicative phase when serum stimulated and cannot be stimulated by EGF alone. Radio‐ligand binding studies performed on WI‐38 cells showed that actively growing cells bind [125I]EGF at relatively low levels that increase to a maximum as the cells become contact inhibited. As the cells enter a state of deeper quiescence, EGF binding falls to one‐third to one‐fifth the short‐term growth arrested levels, remaining constant thereafter. The EGF‐receptor complexes internalize more slowly in long‐term growth arrested cells, and the rate of ligand association to the receptor is lower than short‐term growth arrested cells. The amount of EGF receptor protein in lysates of equal numbers of both short‐ and long‐term quiescent cells remains the same. These results suggest that the failure of long‐term growth arrested cells to respond to EGF is not due to dramatic changes in the amount of receptor protein during prolonged quiescence but more likely to an alteration in the ability of these receptors to bind ligand and/or activate the EGF signal transduction pathway. © 1993 Wiley‐Liss, Inc.
Regulation of malic enzyme gene expression by nutrients, hormones, and growth factors in fetal hepatocyte primary cultures
Authors: Molero Cristina , Benito Manuel , Lorenzo Margarita
Published: February 2005

The culture of fetal hepatocytes for 64 h in medium supplemented with 5 mM glucose, T3, insulin, and dexamethasone resulted in the coordinate precocious expression of malic enzyme mRNA, protein, and specific activity. T3 was the main inducer; meanwhile, insulin exerted a small synergistic effect when added with T3. Dexamethasone had a potentiation effect on the T3 response of malic enzyme mRNA expression regardless of the presence of insulin. This effect of dexamethasone on T3 response of malic enzyme mRNA expression was time (64 h) and glucose dependent. Glucagon, and to a greater degree dibutyryl‐cAMP, repressed malic enzyme mRNA as well as protein expression by T3 and dexamethasone, in the absence of insulin. Glucose and other carbon sources such as lactate‐pyruvate or dihydroxyacetone induced the abundance of malic enzyme mRNA in the absence of hormones. Insulin and T3 produced a high accumulation of malic enzyme mRNA in lactate‐pyruvate medium, this effect being decreased by dexamethasone. EGF supressed the induction produced by T3 and dexamethasone on malic enzyme mRNA, while the expression of β‐actin mRNA remained essentially unmodified. © 1993 Wiley‐Liss, Inc.
Inducers of the heat shock response stimulate phospholipase C and phospholipase A2 activity in mammalian cells
Authors: Calderwood Stuart K. , Stevenson Mary Ann
Published: February 2005

Although the mammalian heat shock response has been well characterized, the processes that mediate the induction of the response and the regulation of heat shock protein function are not completely understood. We have investigated the potential role in heat‐shocked cells of phosphoinositide‐specific phospholipase C (PLC), a membrane enzyme activity involved in transmembrane signal transduction. Our studies indicate that heat shock activates PLC in each of seven cell lines, including cells of human, rat, mouse, and hamster origin. Heat shock produced increases in inositol phosphate concentrations comparable in magnitude to those achieved after simulation with growth factors, indicating that heat shock might initiate transmembrane signaling cascades of potential importance in cellular regulation. Common cellular responses to heat and growth factors also included feedback modulation of PLC by its products and the parallel stimulation of phospholipase A2 activity. In addition to heat shocki, other agents that induce the stress response stimulated PLC activity. The data indicate a close correlation between expression of the mammalian heat shock response and stimulation of PLC activity and indicate a possible role for this enzyme activity in the regulation of some aspects of the stress response. © 1993 Wiley‐Liss, Inc.
Expression and loss of the transferrin receptor in growing and differentiating HD3 cells
Authors: Grdisa Mira , Mathew Anu , Johnstone R. M.
Published: February 2005

During induced differentiation and maturation of HD3 cells (a chicken erythroblast cell line infected with a temperature‐sensitive mutant of the avian erythroblastosis virus), the levels of transferrin receptor (TFR) and nucleoside transporter increase. Both these activities increase before elevated levels of hemoglobin are detected. Shortly after induction, as cellular TFR levels rise, a native‐size TFR is detected in the cell‐free culture medium, associated with an exosome fraction (100,000 xg pellet). Nucleoside transporter (measured as NBMPR‐binding activity) is not increased in this pellet with induction. Previous studies have suggested that exosome formation in peripheral reticulocytes may be a significant route for loss of specific membrane proteins (Johnstone et al., 1991). Although the present experiments in HD3 cells do not address the quantitative importance of exosome formation, the studies suggest that exosome formation is an early event in commitment to the red cell lineage and is not a phenomenon restricted to the terminal stages of red cell maturation. © 1993 Wiley‐Liss, Inc.
Expression of basic fibroblast growth factor and its receptor in an invasive bladder carcinoma cell line
Authors: Allen Lynn E. , Maher Pamela A.
Published: February 2005

Basic fibroblast growth factor (bFGF) is a multifunctional growth factor that can stimulate cell proliferation, production of proteases, and angiogenesis. Loss of mechanisms that regulate bFGF activity could result in tumor development. To test this idea, cells derived from an invasive bladder carcinoma (EJ) were compared with cells derived from a noninvasive bladder carcinoma (RT4) for the expression of bFGF and high and low affinity FGF receptors. bFGF was produced by the invasive EJ cells but not by the noninvasive RT4 cells, suggesting that bFGF could act in an autocrine fashion in the EJ cells to promote their invasion and growth in the surrounding tissue. The two cells lines also showed differences in FGF receptor expression. The EJ cells expressed both high and low affinity FGF receptors as determined by Scatchard analysis, whereas the RT4 cells expressed only high affinity receptors. The high affinity receptors on the RT4 cells expressed recognized by an antibody to known FGF receptors. Furthermore, in contrast to the EJ cells, bFGF did not induce protein tyrosine phosphorylation in the RT4 cells. Thus these data suggest that the invasive potential of bladder carcinoma cells may be regulated by the expression of both bFGF and its receptors. © 1993 Wiley‐Liss, Inc.
Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor‐βs
Authors: Rundhaug Joyce E. , Nettesheim Paul
Published: February 2005

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor‐β (TGFβ) and to be growth inhibited by exogenous TGFβ. The purpose of the present studies was to determine whether the endogenous TGFβ(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGFβ1 and TGFβ2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8‐and 3‐fold increases in colony formation and cell number, respectively, above control IgG‐treated cultures. Antibody dose‐response experiments revealed that TGFβ2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGFβs were acting locally. Anti‐TGFβ1 treatment of cultures at various stages of growth resulted in 1.2–1.7‐fold increases in DNA synthesis above controls, whereas anti‐TGFβ2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7‐ and 2.5‐fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGFβs inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4–70‐fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGFβ1 and TGFβ2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of endogenous TGFβs. © 1993 Wiley‐Liss, Inc.
CSF‐1 stimulates nucleoside transport in S1 macrophages
Authors: Meckling‐Gill Kelly A. , Guilbert Larry , Cass Carol E.
Published: February 2005

We have examined nucleoside transport (NT) in a cell line derived from primary day 7 murine bone marrow macrophages (S1 macrophages) in response to the macrophage growth factor, colony‐stimulating factor 1 (CSF‐1). Adenosine and uridine transport in quiescent S1 macrophages occurred primarily by two facilitated diffusional routes, one that was sensitive and one that was relatively resistant to the inhibitor nitrobenzylthioinosine (NBMPR). Addition of CSF‐1 to quiescent cultures resulted in increased adenosine and uridine transport with biphasic kinetics with respect to the cell cycle. Basal NT activity was elevated (about twofold) within 15 min of CSF‐1 addition, returned to near basal levels by 1 h, and then increased again (three‐ to fourfold) 8–12 h later, returning again to basal levels by 48 h post CSF‐1 stimulation. We propose that the large increase in NT activity at 8–12 h corresponded with the time when cultures synchronously began to enter the S phase of the cell cycle. In addition to these changes in the absolute rates, the proportions of NBMPR‐sensitive and NBMPR‐insensitive transport also change after CSF‐1 addition. Quiescent cultures exhibited primarily NBMPR‐insensitve transport while logrithmically growing cultures exhibited primarily NBMPR‐sensitive nucleoside transport activity. The increase in the NBMPR‐sensitive component of the transport process paralleled a similar increase in the number of high‐affinity NBMPR binding sites, suggesting that the mechanism for upregulating NBMPR‐sensitive NT activity involves increases in the number of NBMPR‐sensitive transporter sites. Interestingly, we were unable to detect Na+‐dependent concentrative uptake of adenosine, uridine, or formycin‐B either in the S1 macrophage cell line or in primary (day 7) murine macrophages. Thus these bone marrow derived macrophages did not display the characteristically large Na+‐dependent transport systems observed by others in peritoneal macrophages, implying that these two populations of macrophages are, indeed, functionally distinct. © 1993 Wiley‐Liss, Inc.
Isoprenoid regulation of cell growth: Identification of mevalonate‐labelled compounds inducing DNA synthesis in human breast cancer cells depleted of serum and mevalonate
Authors: Wejde Johan , Carlberg Magdalena , Hjertman Magnus , Larsson Olle
Published: February 2005

Growth arrest induced by serum depletion and/or treatment with mevinolin (an inhibitor of mevalonate synthesis) in the human breast cancer cell line Hs578T was overcome by exogenous mevalonate, indicating that some product or metabolite of mevalonate may be involved in the mediation of serum‐regulated growth of these cells. In the search for such compounds we first tested a variety of known end products of mevalonate with respect to their ability to counteract the inhibition of DNA synthesis caused by serum‐free medium and mevinolin. Thereby high doses (10 μg/ml) of dolichol‐20 were found to cause a partial counteraction. After straight‐phase HPLC purification of endogenous lipids, isolated from 3H‐ or 14C‐mevalonate‐labelled Hs578T cultures, we found that non‐sterol lipids co‐eluting with dolichols efficiently induced DNA synthesis. After further purification with reverse‐phase HPLC it was confirmed that virtually all of this effect was achieved by compound(s) (seen as a single UV and radioactive peak) co‐eluting with dolichol‐20. Nanogram doses, at most, of this (these) compound(s) elicited a substantial stimulation of DNA synthesis. The lipid(s) also counteracted the inhibition by mevinolin of N‐linked glycosylation, indicating that it (they) also interfere(s) with this processing. Since treatment with tunicamycin (an inhibitor of N‐linked glycosylation) abolished this growth‐stimulative effect, N‐linked glycosylation seems to be a necessary event in the processes leading to lipid‐induced initiation of DNA synthesis.
Paradoxical biological effects of overexpressed insulin‐like growth factor‐1 receptors in chinese hamster ovary cells
Authors: Kato Hisanori , Faria Teresa N. , Stannard Bethel , Levy‐Toledano Rachel , Taylor Simeon I. , Roberts Charles T. , Leroith Derek
Published: February 2005

One major approach to the study of growth factor receptor action has been to overexpress wild‐type or mutant receptors in cultured cells and to evaluate biological responses to exogenous ligand. Studies of this type with insulin and insulin‐like growth factor‐I (IGF‐I) receptors often use Chinese hamster ovary (CHO) cells. We have compared the effect of receptor overexpression in CHO cells and in NIH‐3T3 fibroblasts in order to assess the suitability of CHO cells for studies of this nature and the contribution of cell type‐specific factors to those responses generally assayed. Overexpression of IGF‐I receptors in NIH‐3T3 cells resulted in increased sensitivity and maximal responsiveness of thymidine incorporation, 2‐deoxyglucose uptake, and phosphatidylinositol‐3 (PI3) kinase activation to IGF‐I stimulation. In CHO cells, on the other hand, overexpression of either IGF‐I or insulin receptors increased the sensitivity of thymidine incorporation to ligand, but maximal responsiveness was unchanged or decreased. Overexpression of the insulin receptor increased sensitivity of glucose uptake and the maximal response of PI3 kinase activation to insulin. Overexpression of the IGF‐I receptor did not affect sensitivity or maximal responsiveness of glucose uptake or PI3 kinase activation to IGF‐I. These data suggest that IGF‐I and insulin signal pathways may differ in CHO cells, and that there may even be divergent IGF‐I signaling pathways for short vs. long‐term effects. Whether this is a result of differences in the number of endogenous receptors, hybrid receptor formation, or defects in post‐receptor signaling, the use of CHO cells to assess receptor function must be approached with caution. © Wiley‐Liss, Inc.
Effect of thermotolerance on heat‐induced excess nuclear‐associated proteins
Authors: Borrelli Michael J. , Stafford Diane M. , Rausch Cynthia M. , Lee Yong J. , Corry Peter M.
Published: February 2005

Earlier studies reported that thermotolerance had two effects on the heat‐induced increase in nuclear‐associated proteins (NAPs); reduction in NAP levels immediately following hyperthermia and facilitation of NAP recovery to control levels. It has also been demonstrated that there are two phases of thermotolerance; one that requires newly synthesized proteins (protein synthesis dependent thermotolerance; PSDT), and another that does not (protein synthesis independent thermotolerance; PSIT). This study was designed to determine if these two phases of thermotolerance affected NAP binding in a similar or different manner. The results demonstrated that protein synthesis during thermotolerance development was not required to reduce NAP levels measured immediately following hyperthermia, but was required to facilitate NAP recovery to control levels following hyperthermia. Reducing NAP levels was the predominant mechanism by which thermotolerance protected cells from this lesion at 43.0°C while facilitated NAP recovery predominated in protecting against exposure to 45.5°C. The facilitated recovery of NAPs required only proteins synthesized following thermotolerance induction and prior to the second heat challenge. Proteins synthesized following the second heat challenge were not requisite. Finally, the processes that facilitate NAP recovery were inhibited at 3°C, suggesting that they are enzymatically mediated. © 1993 Wiley‐Liss, Inc.
Plasma membrane lipid packing and leukocyte function‐associated antigen‐1‐dependent aggregation of lymphocytes
Authors: Smith Douglas M. , Williamson Patrick L. , Schlegel Robert A.
Published: February 2005

A simple model system for study of adhesion mediated by leukocyte function‐associated antigen‐1 (LFA‐1) is aggregation of lymphocytes stimulated in vitro. Although aggregation is blocked by monoclonal antibodies to LFA‐1, not all lymphocytes expressing LFA‐1 aggregate, indicating that LFA‐1 is necessary but not sufficient for aggregation. To investigate whether the lipid bilayer plays a role in the functional activation of LFA‐1, human peripheral blood lymphocytes and murine splenic lymphocytes were stimulated in culture, and measurements made of aggregation vs. packing of plasma membrane lipids. Progression of cells into aggregates was paralleled by a decrease in lipid packing of the population as a whole, as monitored by increased staining with the fluorescent probe merocyanine 540. Cells from aggregates stained more intensely than nonaggregated cells from the same population, indicating that aggregates are preferentially formed from cells in the population with the loosest packed membrane. In contrast, aggregated cells were found to express equivalent or even lower amounts of LFA‐1 than nonaggregated cells. Looser lipid packing is therefore associated with the development of LFA‐1‐dependent aggregation, and might be involved in the functional activation of this cell adhesion molecule. © 1993 Wiley‐Liss, Inc.