bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of calcium in the initiation of superoxide release from alveolar macrophages
Authors: Holian Andrij , Daniele Ronald P.
Published: February 2005

The role of calcium in the release of superoxide anion (O) was examined in alveolar macrophages after stimulation with the Soluble stimuli: concanavalin A (Con A), N‐formyl methionyl phenylalanine (FMP), and the calcium ionophore, A23187. The release of O by Con A was unaffected over a wide range of extracellular calcium concentrations (20 μM to 3 mM), whereas increasing the extracellular calcium above 2 mM inhibited FMP‐stimulated O release. In contrast, A23187 did not stimulate O release in calcium‐free medium (≤ 30 μM). The addition of EGTA (50 μM) to calcium‐free medium had no effect on Con A stimulation of O release or FMP‐stimulated O release. These results suggest that, for the three soluble stimuli, there are different roles for Ca+2 in the activation and transmission of stimulatory signals across the cell membrane.

Con A‐ or FMP‐stimulated calcium efflux from calcium‐loaded cells in either calcium‐free medium or 0.5 mM calcium‐containing medium. In calcium‐free medium, FMP transiently retarded 45Ca+2 uptake, while in 0.5 mM calcium‐containing medium, FMP transiently stimulated 45Ca+2 uptake. For either Con A or FMP, calcium efflux preceded O release by 30–45 sec. Quinine, an agent that blocks membrane hyperpolarization in macrophages, completely blocked O release by concanavalin A or FMP and inhibited 45Ca+2 efflux by 50% or more for both agents. These results support the hypothesis that redistribution of cellular Ca+2 is one of the initial steps leading to the release of O.
Control of melanin synthesis and secretion by B16/C3 melanoma cells
Authors: Laskin Jeffrey D. , Piccinini Linda , Engelhardt D. L. , Weinstein I. Bernard
Published: February 2005

In culture, B16/C3 murine melanoma cells grown in the presence of serum undergo melanogenesis at a specific time after plating. At this time, melanin is synthesized intracellularly and then secreted into the extracellular culture fluid. We have found that melanin secretion is dependent on the presence of serum in the growth medium. When confluent cultures are deprived of serum, that is, refed with serum‐free medium, cells remain viable but do not undergo melanogenesis. Addition of serum‐free medium supplemented with either melanocyte‐stimulating hormone (MSH) or dibutyryl cAMP induced melanogenesis in these cells but did not result in melanin secretion. Furthermore, when B16/C3 cells are grown in serum‐free, hormone‐supplemented medium, they also undergo melanogenesis but fail to release melanin. The addition of serum, however, to B16/C3 cells induced to undergo melanogenesis with MSH, dibutyryl cAMP, or hormone‐supplemented medium promotes melanin secretion. Fractionation studies hence revealed that serum contains specific factors capable of inducing melanin secretion. These results demonstrate that factors that regulate melanin synthesis are distinct from those that induce cells to release melanin into their extracellular environment. Furthermore, the ability to induce melanogenesis with single factors will permit us to study the precise sequence of events leading to differentiation in B16/C3 cells under chemically defined conditions.
Cell cycle perturbations in normal and transformed fibroblasts caused by detachment from the substratum
Authors: Campisi Judith , Medrano Estela E.
Published: February 2005

Exponentially growing, anchorage‐dependent fibroblasts were impeded in their progress through the cell cycle as a result of brief trypsinization from the substratum followed by replating. Untransformed mouse (3T3, clone A31), hamster (CHEF/18‐1) and human (FS2) fibroblasts were partially inhibited from entering the DNA synthetic (S) phase of the cell cycle for 8 or 12 hours after detachment, even though the cells reattached within an hour of replating and attained a spread morphology 5 or 8 hours later. The decline in the proportion of cells in S phase was accompanied accumulation of cells in G1 as measured by autoradiography and flow microfluorimetry. Cells removed from the substratum by EDTA alone showed identical disturbances of exponential growth. These cell cycle perturbations could be a result of the detachment per se, as opposed to the rounded morphology. Synchronized A31 cells, exposed to colcemid or cytochalasin B for two hours, were not delayed in their entry into S, whereas trypsinization delayed S phase entry by 4 to 5 hours. These drugs disrupt the cytoskeleton without causing detachment. Isotope incorporation experiments revealed no decreases in the rates of protein or RNA synthesis following replating. However, exponentially growing A31 cells, treated for 2 hours with an inhibitor of protein synthesis behaved similarly to those briefly detached from their substratum: 7 hours after treatment, there were fewer cells in S and more cells in G1 relative to untreated cells. Brief treatment with an inhibitor of hn‐RNA synthesis did not alter the cell cycle distribution of these fibroblasts. Three tumorogenic A31 derivatives were less affected by brief detachment from the substratum than were the untransformed cells. The derivative exhibiting the least in vitro growth control (an SV‐40 transformant) showed the least sensitivity to trypsinization, while that derivative having the most in vitro growth control (a Moloney sarcoma virus transformant) was most sensitive. A chemically [benzo(a)pyrene] transformed derivative gave intermediate results with respect to both growth control and sensitivity to detachment from the substratum. The results suggest that as yet unidentified protein(s) necessary for the normal transit through G1 may be quite sensitive to the presence of an anchoring substratum.
Mitogenic proteins of the 3T3 plasma membrane
Authors: Lieberman Michael A.
Published: February 2005

A mitogenic factor from 3T3 plasma membranes has been identified and partially characterized. The factor appears to be a peripheral membrane protein that can be released by mild trypsin, chymotrypsin, or plasmin treatment. This component is sensitive to heat and acid, and has a molecular weight in the range of 150,000–200,000 daltons as determined by gel filtration. A similar mitogenic activity has also been found on the membranes of both SV‐40‐transformed 3T3 cells and human fibroblasts. The factor appears to be distinct from all previously described mitogenic components.
Synthesis of cathepsin B by cells derived from the HL60 promyelocytic leukaemia cell line
Authors: Burnett David , Crocker John , Vaughan Andrew T. M.
Published: February 2005

Cells of the human promyelocytic HL60 line were induced to differentiate into granulocyte‐like cells with dimethylsulphoxide (DMSO) or macrophage‐like cells with 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA). The synthesis of Cathepsin B by these cells was studied by immunoperoxidase staining and assay of cell lysates using the fluorimetric substrate benzoyloxycarbonyl‐phenylanalyl‐arginine‐4‐methyl‐7‐coumarylamide. Only 2–5% of the uninduced HL60 cells and DMSO‐induced cells were immunohistochemically positive for Cathepsin B, compared with over 80% of the TPA‐induced cells. Cathepsin B activity was lowest in the lysates of uninduced HL60s. DMSO‐induced cells contained 1.5–2‐fold the enzyme activity of HL60s and TPA‐induced cell lysates demonstrated 5–14‐fold the activity of uninduced HL60s. Induction of Cathepsin B synthesis was therefore associated with differentiation of the promyelocytes into cells of the monocyte/macrophage type, but not granulocyte‐like cells. Cathepsin B was located immunohistochemically in human palatine tonsils. The enzyme was only demonstrated within macrophages in these tissues. Cathepsin B may therefore be a useful immunohistochemical marker for malignant and nonmalignant cells of the monocyte/macrophage lineage.
Synergistic killing of Hela cells by hydroxyurea and caffeine
Authors: Beetham Karen L. , Busse Paul M. , Tolmach L. J.
Published: February 2005

Synchronous populations of HeLa S3 cells suffer synergistic killing during S phase in the presence of 0.5–5 mM hydroxyurea together with 5–10 mM caffeine. Both the rate and the extent of killing are greater than expected for independent action of the two drugs. Only simultaneous treatment is effective. The dependence of the synergistic killing on cell age resembles the age dependence for killing by hydroxyurea alone (>3 mM), but not that by high concentrations of caffeine. In addition, rapid killing occurs if caffeine is added to cultures that have been incubated in the presence of hydroxyurea from early G1 and are blocked at the beginning of S, although such cells are killed only slowly on continued incubation in ⩾ 10 mM hydroxyurea alone. Furthermore, cells that are incubated with the two drugs from early G1 begin to undergo synergistic killing at about 12 h after mitotic collection, but they do not commence DNA replication for another 2–3 h if the drugs are removed. It is concluded that cells that have reached a point in the cycle identical with or close to the end of G1 are sensitive to the combination whether or not they are able to synthesize DNA, and whether or not they are sensitive to hydroxyurea alone. A tentative model is proposed: hydroxyurea is postulated to kill cells by interacting with sites of replication in DNA, and the synergism is attributed to the extra replication points that caffeine is known to induce.
The study of human myeloid differentiation using bromodeoxyuridine (BrdU)
Authors: Keoffler H. Phillip , Yen James , Carlson John
Published: February 2005

Little is known concerning the mechanism of myeloid differentiation. A human promyelocytic cell line (HL‐60) differentiates to granulocytes or macrophage‐like cells when cultured with a variety of agents. How these agents trigger myeloid differentiation is not understood. This study shows that 1.0–10.0 μg/ml bromodeoxyuridine (BrdU) induced myeloid differentiation of HL‐60 in liquid culture. After 7 days, BrdU (3.0 μg/ml) produced only moderate inhibition of HL‐60 growth, but induced myeloid maturation with 40% of the cells becoming morphologically more mature; 41% developed the ability to reduce nitroblue tetrazolium (NBT); 19% phagocytized Candida albicans; and 18% developed Fc receptors. The action of BrdU was mimicked by 5‐iododeoxyuridine. Thymidine (Td) (1‐ to 10‐fold excess) competitively inhibited incorporation of [3H]BrdU into DNA of HL‐60 and inhibited the triggering of HL‐60 differentiation by BrdU. The BrdU‐induced maturation of HL‐60 correlated with the incorporation of BrdU into DNA of HL‐60. DNA buoyant density studies showed that about 46% of the Td was replaced by BrdU in each DNA strand of HL‐60 as the cells differentiated in culture containing 3 μg/ml BrdU for 7 days. We established 20 thymidine kinase (TK)‐deficient HL‐60 clones. The HL‐60 TK‐deficient cells were unable to phosphorylate Td, to incorporate either [3H]Td or [3H]BrdU or differentiate in the presence of BrdU (1‐1000 μg/ml). The HL‐60 TK‐deficient cells retained the ability to differentiate in the presence of other HL‐60 inducers. Taken together, the studies suggest myeloid differentiation of HL‐60 is triggered because of incorporation of BrdU into DNA of the cells.
Insulin‐like growth factor I/somatomedin‐C (IGF‐I/SM‐C) and glucocorticoids synergistically regulate mitosis in competent human fibroblasts
Authors: Conover Cheryl A. , Dollar Laura A. , Hintz Raymond L. , Rosenfeld Ron G.
Published: February 2005

In serum‐free medium, insulin‐like growth factor‐I/somatomedin‐C (IGF‐I/SM‐C) was weakly mitogenic for adult human fibroblasts in culture. However, in the presence of 0.5% human hypopituitary serum (HHS), which by itself had little effect, there was a marked dose‐dependent response to IGF‐I/SM‐C with a 10‐ to 20‐fold increase in [3H]thymidine incorporation at 25 ng/ml IFG‐I/SM‐C. With the further addition of dexamethasone or hydrocortisone to the combination of IGF‐I/SM‐C + 0.5% HHS, there was a dramatic synergistic effect resulting in a 60‐ to 70‐fold increase in [3H]thymidine incorporation. This stimulation was two times greater than that seen with 20% FCS. In contrast, glucocorticoids had no effect in serum‐free medium or with HHS alone. These [3H]thymidine incorporation results were clearly supported by cell replication studies. Dose‐response curves for 125I IGF‐I/SM‐C binding and IGF‐I/SM‐C stimulation of [3H]thymidine incorporation were similar with 1/2 maximal effects for both at 5 ng/ml. However, the striking synergism seen with glucocorticoids occurred in the absence of any glucocorticoid‐induced change in IGF‐I/SM‐C binding, indicating that the interaction of IGF‐I/SM‐C and glucocorticoids occurs at a postreceptor level. These data demonstrate that in the presence of a low concentration of HHS, IGF‐I/SM‐C and glucocorticoids stimulate complete cell cycle traverse and replication of human fibroblasts.
Calcium uptake by isolated rat intestinal cells
Authors: Bronner Felix , Pansu Danielle , Bosshard Arlette , Lipton Jeffrey H.
Published: February 2005

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time‐ and concentration‐dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D‐deficient animals took up less calcium than cells from vitamin D‐replete cells. In vivo vitamin D repletion with 1,25‐dihydroxyvitamin D3 raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D‐deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.
Slow internalization of human chorionic gonadotropin by cultured granulosa cells
Authors: Robinson Margaret S. , Rhodes James A. , Albertini David F.
Published: February 2005

Kinetic studies were performed on two‐day cultures of rat ovarian granulosa cells to follow the fate of surface‐bound 125l‐labeled human chorionic gonadotropin (125l‐hCG). Low pH was used to release hCG from its surface receptor, allowing us to distinguish between surface‐bound and internalized hormone. Because our results indicated that hormone is lost from the cell surface by dissociation as well as internalization, equations were derived to determine independent rate constants for each process. We calculate that if hormone binding were irreversible, the t1/2 for internalization would be 8.5 hours. Morphometric studies on the uptake of horseradish peroxidase indicate that the t1/2 for internalization of bulk membrane in granulosa cells is 55 to 77 minutes. Thus, the rate of uptake of surface‐bound hCG appears to be seven to nine times slower than the rate of uptake of bulk plasma membrane, which suggests that the LH/hCG receptor may be selectively excluded from the endocytic vesicles of granulosa cells.
Biosynthesis of small nuclear RNAs in human cells
Authors: Chandrasekharappa Settara C. , Smith James H. , Eliceiri George L.
Published: February 2005

We have examined some aspects of the biosynthesis of human small nuclear RNAs (snRNAs). The sensitivity of U5 and U4 snRNA synthesis to α‐amanitin in whole cells suggests that RNA polymerase II is involved in the synthesis of these RNA species, in addition to that of U1, U2, and U3 snRNA. Two RNA bands were detected, whose properties are compatible with being U3 and U4 RNA precursors. The cytoplasmic U1 RNA precursor (pU1) was retained by an anti‐RNP antibody column, while the cytoplasmic precursors to U1 and U2 (pU2) RNA were immunoprecipitated by monoclonal anti‐Sm antibodies. Therefore, soon after their transcription, these cytoplasmic RNA precursors assemble with the polypeptides which bear the RNP (pU1) and Sm (pU1 and pU2) antigenic determinants. It has been shown before that, shortly after protein synthesis is interrupted, the apparent cytoplasmic→nuclear transition of newly made U2 RNA is inhibited, while U2 RNA transcription is not. The present data indicate that the trimming of the U2 RNA precursor to mature U2 RNA is not affected early after suppression of protein synthesis.
The uncoupling of macromolecular synthesis from cell division in SV3T3 cells by glucocorticoids: The imposition of a G2 block
Authors: Das Hasi R. , Lavin Mary , Sicuso Anthony , Young Delano V.
Published: February 2005

Through a receptor‐mediated process glucocorticosteroids block cell division by 20–45 hours in SV40‐transformed 3T3 (SV3T3) mouse fibroblasts growing in a low calf serum (0.30% v/v) medium containing biotin. However, the rate of DNA synthesis, determined at various times after dexamethasone addition by the incorporation of radioactive thymidine into acid‐insoluble material, is not inhibited by this steroid as late as 66 hours. A modest decrease is observable by 91 hours. There is also no reduction in the uptake of exogenous thymidine into acid‐soluble cellular pools. Similarly, RNA synthesis and the uptake of radioactive uridine are not affected by the glucocorticoid up to 69 hours. Measurements of the amounts of cellular DNA (by the fluorescent dye, 4′,6‐diamidino‐2‐phenylindole) and protein revealed that both macromolecules are present in elevated quantities in steroid‐treated cells. (The constancy of the protein content in the nonproliferative stage suggests that protein synthesis and degradation are occurring at equal rates.) If the steroid is removed and fresh 10% calf serum medium added, cell division commences (even if nearly 90% of protein synthesis is inhibited by cycloheximide) as early as 45 minutes later such that by 2 hours the viable cell count increases by as much as 70%. Since the growth curve after recovery resembles a step function, it appears that the cells are partially synchronized by the glucocorticoid. These results demonstrate that the glucocorticoid cytostatic effect in SV3T3 cells is the result of a block not in G1, as previously thought, but in G2.
Demonstration of a late amiloride‐sensitive event as a necessary step in initiation of DNA synthesis by thrombin
Authors: Stiernberg Janet , Labelle Edward P. , Carney Darrell H.
Published: February 2005

Amiloride, a Na+ influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast‐like cells. Long exoosures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin‐stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin‐initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride‐sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 μM) during a 12‐hr exposure to thrombin did not block thrombin‐initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride. 86Rb+ transport studies demonstrated that in this system ouabain‐sensitive K+ uptake via the Na, K‐ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride‐sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin‐stimulated Na+ transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8–12 hr after thrombin addition maximally inhibited thrombin‐stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin‐initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8–12 hr after thrombin addition.
Loss of α2‐macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides
Authors: Dipaola Mario , Keith Charles H. , Feldman David , Tycko Benjamin , Maxfield Frederick R.
Published: February 2005

We have found that certain naphthalenesulfonamides [e.g., N‐6(‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell‐surface receptors for α2‐macroglobulin and epidermal growthfactor (EGF) in fibroblasts. The loss of α2‐macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for α2‐macroglobulin, and the EGF receptors do not reappear at the surface when W‐7 isremoved. Receptor loss was measured as a change in the capacity for binding iodinated ligands;nochange in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W‐7 did not induce a loss of cell surface β2‐microglobulin, a membrane protein which is excluded from coated pits and which isnot internalized, indicating that the effect of W‐7 was specific for membrane receptorsandnot a result of bulk depletion of plasma membrane. The loss of α2‐macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W‐7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsiblefor the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: (1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; (2) the microinjection of calmodulin into cells did not reverse the effects of W‐7; using pressure microinjection, we introduced up to a 100‐fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rho‐damine‐labeled α2‐macroglobulln, we saw that the W‐7 induced inhibition of receptor‐mediated endocytosis was the same in injected and uninjected cells; (3) we injected calcineurin, a calmodulin‐binding protein, into cells (1‐3 pg/ cell) and observed no effect on the receptor‐mediated endocytosis of rhoda‐mine‐labeled α2‐macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.
Induction of ornithine decarboxylase, RNA, and protein synthesis in macrophage cell lines stimulated by immunoadjuvants
Authors: Prosser Frederick H. , Schmidt Christopher J. , Nichols Scott V. , Nichols William K.
Published: February 2005

Early biochemical changes associated with adjuvant stimulation of macrophage protein synthesis were studied using two murine macrophage cell lines, PU5‐1.8 and J774.1. An induction of ornithine decarboxylase (ODC) was detected 2 hours after exposure of PU5‐1.8 and J774.1 cells to two crude immunoadjuvants, BCG cell walls (BCGcw) and lipopolysaccharides from Escherichia coli (LPS). The chemically defined immunoadjuvant glycopeptide, N‐acetyl‐muramyl‐L‐alanyl‐D‐isoglutamine (MDPL) also promoted an increase in ODC activity at 2 hours that was maximal after 4 hours, while little or no effect was observed with the D‐alanyl analog (MDPD) that is devoid of adjuvant activity. The increase in ODC activity promoted by BCGcw in PU5‐1.8 and J774.1 cells returned toward control levels by 6 to 8 hours. BCGcw also stimulated RNA and protein synthesis which remained elevated for at least 24 hours and was associated with a decrease in DNA synthesis and cell proliferation. ODC induction by BCGcw and MDPL was enhanced by the addition of PGE2 in both cell lines. Indomethacin slightly depressed the magnitude of ODC stimulation by BCGcw in J774.1 cells but failed to alter the response of PU5‐1.8 cells. Additional observations indicated that the induction of ODC by BCGcw in both cell lines was preceded by an activation of cyclic AMP‐dependent protein kinase. These observations suggest that a cyclic AMP‐mediated induction of ODC may be an early biochemical marker of adjuvant stimulation in macrophages.
Regulation of production of a platelet‐derived growth factor‐like protein by cultured bovine aortic endothelial cells
Authors: Fox Paul L. , Dicorleto Paul E.
Published: February 2005

Platelet‐derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF‐like protein are blood platelets, several transformed cell lines, and cultured endothelial cells (EC). We have examined the regulation of production of a PDGF‐like protein in cultures of bovine aortic EC using a specific radioreceptor assay for PDGF. EC constitutively secreted PDGF‐like protein into serum‐containing or serum‐free medium. The rate of production of PDGF‐like protein was constant for at least 3 weeks and was not due to release of an internal store, since cell lysis by repeated freeze/thaw cycles did not relase significant amounts of the protein. Synthesis of PDGF‐like protein was sensitive to changes in the pH of the media and was maximal at pH 8.5. Production of PDGF‐like protein was independent of EC growth rate: rapidly dividing cells and confluent, quiescent cells produced equal amounts per cell. However, sparse, quiescent EC produced more PDGF‐like protein per cell than did confluent, quiescent cells. Several phorbol esters stimulated production of PDGF‐like protein. At a concentration of 10−6 M, a twofold stimulation was observed upon addition of the tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and nearly a fourfold stimulation upon addition of the nonpromoting analog, methyl TPA. Incubation of EC with endotoxin (10 μ/ml) resulted in a twofold stimulation of PDGF‐like protein production. In all experiments with endotoxin and phorbol esters, an increase in the production of PDGF‐like protein was accompanied by morphological changes in the EC cultures. The cells appeared elongated and fibroblastic and exhibited low viability. A mathematical model was developed in which PDGF‐like protein production was shown to consist of two separate components—production at a constant rate by healthy cells and a large burst of synthesis and secretion by dying cells. These results suggest that injurious agents may be capable of stimulating production of a growth factor by the endothelium.
Characterization of the metabolic forms of 6‐thioguanine responsible for cytotoxicity and induction of differentiation of HL‐60 acute promyelocytic leukemia cells
Authors: Ishiguro Kimiko , Schwartz Edward L. , Sartorelli Alan C.
Published: February 2005

HL‐60 human acute promyelocytic leukemia cells that lack hypoxanthineguanine phosphoribosyltransferase (HGPRT) activity have been developed by mutagenization and selection. These cells exhibited markedly decreased sensitivity to the cytotoxic action of 6‐thioguanine (TG) and, in contrast to parental HL‐60 cells, had the capacity to undergo terminal granulocytic differentiation after treatment with this purine antimetabolite. Analysis of extracellular and intracellular metabolites of TG revealed negligible metabolism of TG in these HGPRT− HL‐60 cells. These findings are consistent with the concept that inhibition of cellular replication requires generation of analog nucleotide and suggest that TG itself is capable of initiation of differentiation. 6‐Thioguanosine (TGuo) had limited activity, while β‐2′‐deoxythioguanosine (dTGuo) was inactive, as an inducer of maturation of HGPRT− HL‐60 cells. These cells converted relatively large amounts of the nucleosides to the free base TG; the simultaneous exposure of cells to 8‐aminoguanosine (AGuo), an inhibitor of purine nucleoside phosphorylase activity, decreased the degradation of TGuo and dTGuo to TG and promoted the intracellular accumulation of TG nucleotides, presumably through the action of nucleoside kinase activities. In a double mutant deficient in both HGPRT and deoxycytidine kinase (DCK) activities, dTGuo was devoid of cytotoxicity and was an effective inducer of maturation. The potency of dTGuo as an inducer in this system was not significantly affected by the presence of AGuo. These results suggested that dTGuo itself was also an active initiator of maturation. Thus, induction of differentiation appeared to be due to the free base, TG, as well as its deoxynucleoside form, dTGuo, whereas the formation of TG nucleotides appeared to antagonize maturation and produce cytotoxicity.
Density‐associated loss of functional receptors for somatomedin‐C/insulinlike growth factor I (SM‐C/IGF‐I) on cultured human fibroblast monolayers
Authors: Rosenfeld Ron G. , Dollar Laura A. , Conover Cheryl A.
Published: February 2005

The mitogenic activity of somatomedin‐C/insulinlike growth factor‐I (SM‐C/IGF‐I) appears to be greatly influenced by cell culture conditions, especially the presence of other growth factors and nutrients in the culture medium. To investigate the effect of cell density on SM‐C/IGF‐I activity, we have evaluated SM‐C/IGF‐I binding and stimulation of DNA synthesis and cell replication as a function of cell density in cultured human fibroblast monolayers. At fibroblast concentrations of 2.7 × 105 and 1.48 × 106 cells per 60‐mm dish, specific binding of [125I]SM‐C/IGF‐I per 106 cells was 170% higher in sparse than dense monolayers (9.3% vs. 3.4%). Increased binding in sparse monolayers was attributable to approximately twice as many receptors in sparse as in dense cells (31,000 vs. 16,000 sites per cell), as well as to a modest increase in the affinity constant. Similarly, half‐maximal stimulation of [methyl‐3H]thymidine incorporation was achieved at SM‐C/IGF‐I concentrations of 2.5 ng/ml in sparse cells but required 20 ng/ml in dense cells. Although this required only 45% occupancy of membrane receptors on sparse cells, and almost 80% occupancy on dense cells, the total number of occupied receptors was similar in both sparse and dense cells (approximately 13,000 receptors/cell for half‐maximal stimulation). The presence of increased numbers of “functional receptors” on sparse fibroblasts thus results in enhanced sensitivity to SM‐C/IGF‐I stimulation of DNA synthesis and cell replication. Progressive decreases in the number of functional receptors, secondary to cell crowding, may contribute to density‐dependent inhibition of fibroblast growth.
Treatment with α‐difluoromethylornithine plus a spermidine analog leads to spermine depletion and growth inhibition in cultured L1210 leukemia cells
Authors: Casero Robert A. , Bergeron Raymond J. , Porter Carl W.
Published: February 2005

Of the three biological polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), the relevance of Spm to cell proliferation has yet to be defined because of our general inability to deplete it selectively in intact cells. In the present study, Spm depletion was accomplished by treating cultured L1210 cells for 96 hr with α‐difluoromethylornithine (DFMO) and an analog of Spd such as aminopropylcadaverine. N4‐methylSpd, N4‐ethylSpd, or homoSpd. DFMO, a specific inhibitor of ornithine decarboxylase, halts continued polyamine biosynthesis and the Spd analog serves as a functional substitute for Spd. Thus, while the Spd analog fulfills the role(s) of Spd in cell proliferation, Spm becomes steadily depleted. In cells treated with DFMO plus the analog, aminopropylcadaverine, Spm pools decline steadily and growth inhibition occurs after 48 hr (when Spm pools decline to 60% of control). By 96 hr, Spm is ∼ 15% of control and growth is < 30%. Prevention studies with exogenous polyamines confirm a causai relationship between Spm depletion and growth inhibition. The critical levels of polyamines for cell proliferation to take place were found to be 30% of control for Spd and 60% for Spm. The use of DFMO plus a Spd analog is proposed as a system for studying the cellular consequences of Spm depletion. Spd depletion can be achieved for comparison purposes by treating cells with DFMO alone.
Urokinase‐type plasminogen activator and its inhibitor secreted by cultured human monocyte‐macrophages
Authors: Saksela Olli , Hovi Tapani , Vaheri Antti
Published: February 2005

Human blood monocytes in culture differentiate to macrophagelike cells within 1 week. Coinciding with this morphological transition the cells started releasing increasing amounts of the serine proteinase plasminogen activator (PA; Mr 56,000) of the urokinase (u‐PA) type and the proteinase inhibitor alpha‐2‐macroglobulin (α2M). Unlike the cell‐associated PA activity, which was also readily detected in fresh monocytes, the activity secreted into the serum‐free culture medium could be measured only after treatment of the samples with sodium dodecyl sulphate. Heat or acid treatment of the medium was not sufficient to reveal the PA activity, suggesting that, apart from α2M, another PA‐inhibiting substance was present in the culture medium. The inhibitor (Mr 65,000) was found to be synthesized by macrophages and specifically inhibited u‐PA activity but not tissue‐type PA (t‐PA) or plasmin activity. Dexamethasone decreased the secretion of PA by differentiated macrophages without affecting the production of α2M or the PA inhibitor. Dexamethasone also inhibited the morphological differentiation of the cells when added to the monocyte‐phase cells.
Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase
Authors: Burstein Samuel A. , Boyd Cheryl N. , Dale George L.
Published: February 2005

A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP‐treated horse serum ± pokeweed mitogen‐stimulated spleen cell conditioned medium (PWM‐SCM) in 96‐well microplates. Following the culture period, Triton X‐100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE‐positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.
EGF induces receptor down‐regulation with no receptor recycling in KB cells
Authors: Lyall Raymond M. , Pastan Ira , Willingham Mark C.
Published: February 2005

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down‐regulate their specific plasma membrane receptors. Using both 125I‐EGF and a monoclonal antibody against the EGF‐receptor (EGF‐R1), we studied the down‐regulation of the EGF‐receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37°C with EGF rapidly decreases the number of plasma membrane EGF‐receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF‐receptor in KB cells and that the major portion of internalized EGF‐receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF‐receptor is markedly different from that of receptors not subject to down‐regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.
Bacterial lipopolysaccharide induction of ornithine decarboxylase in the macrophage‐like cell line RAW264: Requirement of an inducible soluble factor
Authors: Taffet Steven M. , Haddox Mari K.
Published: February 2005

Ornithine decarboxylase (ODC, EC 4.1.1.17) activity is induced in the RAW264 macrophage‐like cell line by bacterial lipopolysacharide (LPS). As little as 0.1 ng/ml LPS promoted an increase in ODC activity, while maximal ODC activity (30‐fold above control) was induced with 1.0 μg/ml LPS. An increase in ODC activity was detectable within 90 min of LPS addition. The LPS‐induced increase in ODC activity was prevented by inhibitors of protein and RNA synthesis. The induction of the enzyme by LPS was not dependent on prostaglandin production. However, PGE2(1 μg/ml) and 8‐bromo‐cyclic AMP (1mM), neither of which had an effect on ODC activity when added alone, each acted synergistically to enhance the LPS induction of ODC activity. Enzyme induction was not associated with an alteration in Km for ornithine, which remained constant at 0.04 mM. The extent of the increase in ODC in response to LPS increased with increasing cellular density. This relationship was dependent not on absolute cell density of the monolayer but on the cell number in relation to medium volume, and this dependence could be extrapolated to the origin. Addition of conditioned media from LPS‐stimulated but not unstimulated cultures enhanced the ODC increase in sparsely plated cultures in response to a maximal concentration of LPS. The addition of polymyxin B, a reagent that blocks the effects of LPS, including the increase in ODC activity, did not totally inhibit the conditioned medium stimulation. This data indicates that two signals, LPS and a LPS‐induced mediator, are involved in the induction of ODC activity in RAW264 cells.
Neutrophil degranulation: Evidence pertaining to its mediation by the combined effects of leukotriene B4, platelet‐activating factor, and 5‐HETE
Authors: O'Flaherty Joseph T.
Published: February 2005

Stimulus‐activated polymorphonuclear neutrophils (PMN) produce leukotriene B4 (LTB4), 5‐hydroxyeicosatetraenoate (5‐HETE), and platelet‐activating factor (PAF). Each of these lipids promotes PMN degranulation; in combination they have additive and potentiating effects that result in prominent degranulation responses at relatively low concentrations. Thus, the combined interactions of LTB4, 5‐HETE, and PAF may mediate responses in PMN activated by other stimuli. This possibility was examined by measuring the responses of PMN made insensitive to one or more of these lipids. Cells were pretreated with LTB4, 5‐HETE, and/or PAF for 8 min; exposed for 2 min to cytochalasin B (which is required for lipid‐induced degranulation); and then challenged. PMN challenged with only buffer released minimal amounts of granulebound enzymes. Furthermore, the lipid‐pretreated cells were hyporesponsive to challenge with (1) various combinations of these same lipids or (2) ionophore A23187. The relative potencies of the lipids in producing hyporesponsiveness to themselves or A23187 were: 5‐HETE < PAF ≤ LTB4 < PAF + LTB4 < PAF + LTB4 + 5‐HETE. For both types of challenge, reduced responsiveness occurred in cells pretreated with > 0.1 nM LTB4 and/or > 0.2 nM PAF, persisted in cells washed after lipid pretreatment, and did not develop in cells pretreated with various combinations of bioinactive structural analogues of the lipids. Thus, PAF, LTB4, and 5‐HETE interacted to desensitize PMN, and the degranulating actions of A23187 required cells that were fully responsive to each of the three lipids. This supports the concept that the lipids act together in mediating certain of the ionophore's effects. However, lipid‐desensitized PMN degranulated fully when challenged with C5a, a formylated oligopeptide, or phorbol myristate acetate. Degranulation responses, therefore, may proceed through various pathways, only some of which involve the lipid products studied here.
Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): Partial chemical and biological characterization
Authors: Gospodarowicz Denis , Massoglia Sharon , Cheng Jannie , Lui Ge‐Ming , Böhlen Peter
Published: February 2005

Bovine pituitary fibroblast growth factor has been purified 222,000‐fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single‐chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half‐maximal stimulation at 23–40 pg/ml (1.5–2.6 pM) and saturation between 140 and 280 pg/ml (9.3–18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.
Activation of collagen synthesis in primary culture of rat liver parenchymal cells (hepatocytes)
Authors: Hata Ryu‐Ichiro , Ninomiya Yoshifumi , Sano Junjiro , Konomi Hiroshi , Hori Hisae , Sunada Hironobu , Tanaka Shizuko , Kabuki Keiichiro , Nagai Yutaka , Tsukada Yutaka
Published: February 2005

An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high‐molecular‐weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (αA and βB) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. (a) The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. (b) The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. (c) Immuno‐electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day‐8 hepatocytes in primary culture.
The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain
Authors: Wilkins L. , Gilchrest B. A. , Szabo G. , Weinstein R. , Maciag T.
Published: February 2005

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10−9 M), hydrocortisone, (5 × 10−5 M), insulin (10 μg/ml), transferrin (10 μg/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10−10 M), and bovine brain extract (150 μg/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor‐supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]‐Dopa incorporation. The growth factor‐supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline‐labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co‐cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.
Membrane phospholipid asymmetry as a factor in erythrocyte‐endothelial cell interactions
Authors: Schlegel Robert A. , Prendergast Thomas W. , Williamson Patrick
Published: February 2005

The tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.
Antagonistic effects of insulin and dexamethasone on glucose‐regulated and heat shock protein synthesis
Authors: Kasambalides Efthimios J. , Lanks Karl W.
Published: February 2005

The present study extends our previous observation (Kasambalides and Lanks, J. Cell. Physiol., 114:93–98, 1983), that dexamethasone inhibits the alterations in heat shock protein (HSP) and glucose‐regulated protein (GRP) synthesis caused by glucose deprivation. We now show that insulin, even in the presence of high extracellular glucose concentrations, will induce 95K and 82K GRP synthesis while suppressing 85K and 69K HSP synthesis. Heat shock of insulin‐treated cultures causes induction of the 82K GRP rather than the 85K and 69K HSP's. All of the insulin effects are antagonized by dexamethasone. These data suggest that the changes in GRP and HSP synthesis induced by glucose deprivation and heat shock, respectively, may reflect the operation of a normal physiological mechanism that regulates glucose metabolism.