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L Warren

Publications and source records attributed to L Warren.

At least 37 records · Page 2Linked to original sources
PubMed1995

The structure of P-glycoprotein and the secretion of lysosomal enzymes in multidrug-resistant cells.

We have previously demonstrated that multidrug-resistant cells have a lower content of lysosomal enzymes, a consequence of an increased rate of secretion. The question was therefore to know whether an intact functional P-glycoprotein was necessary for expression of this property. Control NIH3T3 and mdr1-gene-transfected cells (pHaMDR1) were used together with 2 variants either lacking 23 amino acids at the carboxyl terminal (pHaMDRC 23) or in which 4 extra amino acids are inserted (pHaMDRBL2). Transfected and variant cells exhibited reduced uptake of [3H]-vinblastine and [3H]-daunomycin, a finding consistent with their drug resistance. By contrast, only pHaMDR1 cells had a reduced level of N-acetyl glucosaminidase that paralleled an increased rate of secretion of the same enzyme. The mutant cells secreted lysosomal enzyme at the same rate and had the same intracellular lysosomal enzyme content as NIH3T3 cells. Abnormal behavior of lysosomal enzymes in multidrug-resistant cells therefore seemed to require an intact P-glycoprotein molecule. Although sequestration in lysosomes and then secretion of drugs may possibly contribute to protection, it would not be an essential component of multidrug resistance.

3T3 Cells↗
PubMed1992

Increased accumulation of drugs in multidrug-resistant cells induced by liposomes.

A multidrug-resistant cell such as the human lymphoblastic leukemic cell CEM/VLB100 accumulates far less vinblastine (VLB) than its drug-sensitive parent, CEM. When CEM/VLB100 cells are exposed to liposomes consisting of the phospholipids cardiolipin, dioleoylphosphatidic acid, or phosphatidylinositol bearing unsaturated fatty acids and then tested for uptake of VLB, accumulation of drug rapidly rises to levels approaching those of CEM cells, which are relatively unaffected by the liposome treatment. The liposomes are not carriers of entrapped drug. Phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine are inactive, and the addition of cholesterol to liposomes inhibits uptake. Exposure of cells to liposomes does not appear to alter the efflux of drugs. We suggest that the liposomal lipids, introduced into the plasma membranes of CEM/VLB100 cells, change their properties so that accumulation of drugs by cells is largely restored. The cytotoxicity of VLB in CEM/VLB100 cells is increased approximately 10-fold by cardiolipin liposomes.

Biological Transport↗
PubMed1991

Secretion of lysosomal enzymes by drug-sensitive and multiple drug-resistant cells.

The multiple drug-resistant human lymphoblastic leukemic cell, CEM/VLB100, in which P-glycoprotein (P-170) is overexpressed, has a lowered content of lysosomal enzymes, such as N-acetylglucosaminidase and beta-galactosidase, and the relative rates of secretion of these enzymes are significantly greater than those of its drug-sensitive counterpart, CEM. The ability of CEM/VLB100 cells to accumulate [3H]vinblastine ([3H]-VLB) is also greatly reduced. Multiple drug-resistant cells whose mode of resistance is not associated with P-170 do not have reduced enzyme content, and their rate of secretion is the same as that of their drug-sensitive parents. Linkage of drug and enzyme elimination is suggested by the observation that verapamil inhibits both the efflux of [3H]VLB and the secretion of lysosomal enzymes in CEM/VLB100 cells; the content of both [3H]VLB and enzyme increases in these cells when chronically exposed to verapamil. Further, both secretion of N-acetylglucosaminidase and efflux of [3H]VLB by CEM/VLB100 cells are enhanced by the addition of NaCl to the suspending, sucrose-containing medium. When cells have taken up [3H]VLB and are then fractionated by means of a Percoll centrifugation gradient, the distribution of drug among the various populations of vesicles is similar to that of N-acetylglucosaminidase. Losses of both enzyme and drug take place from these vesicular populations to varying degrees, when CEM/VLB100 cells are induced to secrete. It is proposed that, in a multiple drug-resistant cell such as CEM/VLB100, the presence of P-170 in the plasma membrane may, in some indirect manner, lead to increased exocytosis of lysosomal enzyme, ultimately resulting in a significant depletion of enzyme. Further, a toxic, cationic drug such as vinblastine, accumulating in lysosomes and acidic vesicles, is also eliminated from the cell by exocytosis. This pathway may supplement the known, major mode of efflux directly involving P-170.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Cytokine activity in bovine mammary gland secretions during the periparturient period.

The presence of cytokine activity in periparturient bovine mammary secretions was evaluated. Mammary secretions were modified for use in biological assays for interleukin-2 (IL-2) like and antiviral activity. The level of IL-2 like activity in mammary gland secretions was lower during the last week of gestation when compared to levels detected approximately two weeks prepartum. Antiviral titers gradually increased as parturition approached. Results from Western blots indicated that the antiviral activity observed in prepartum secretions may be due to tumor necrosis factor (TNF). Interferons (IFN) were not detected in the colostrum samples.

Animals↗
PubMed1990

Stimulation of lysosomal enzyme secretion by growth factors.

Levels of extracellular lysosomal enzymes are relatively high in tumors and especially so at their periphery. By degrading the intercellular matrix, these and other nonlysosomal enzymes could facilitate invasion and metastasis by tumor cells. Using a rapid assay, we have shown that cells transformed by a variety of agents can be stimulated in culture by several growth factors to secrete lysosomal enzymes. These factors have little or no stimulatory activity on their nontransformed counterparts. The basal rate of secretion of N-acetylglucosaminidase (NAGA) and the efficiency of the stimulus are greater in transformed cells in log phase of growth. These observations suggest that altered or increased responsiveness to paracrine and autocrine growth factors not only may be responsible for the persistent division of malignant cells but also may promote their invasiveness.

Acetylglucosaminidase↗
PubMed1989

Stimulated secretion of lysosomal enzymes by cells in culture.

F9 mouse teratocarcinoma and PyS-2 cells in culture incubated with monovalent cations in buffered sucrose solution (0.25 M) can secrete as much as 40% of their total lysosomal enzymes into the medium within 30 min. Longer incubation does not lead to further loss of enzyme, suggesting that only a certain fraction of lysosomes is capable of discharge. The simultaneous presence of sucrose and cation, each at the respective optimal concentrations of 0.25 and 0.15 M, is required for lysosomal discharge (i.e. twice isoosmolarity). The cells remain fully viable. Sodium ions are more effective than lithium and potassium ions, whereas amines and divalent cations are less effective. Other sugars including glucose can replace sucrose to varying extents. Secretion is accompanied by a rapid short-lived rise in the level of cAMP. Forskolin as well as agents that activate G protein such as cholera toxin, AlF4-, and vanadate ions also increase the rate of secretion. Sucrose-Na+ stimulation takes place independently of changes in influx or efflux of calcium ions or changes in the levels of extracellular or free intracellular calcium ions. Neomycin, an inhibitor of phospholipase C, has little effect on secretion. Our results suggest that the secretion observed is mediated by a cAMP-dependent mechanism involving G proteins. Calcium ions and phospholipase C appear to play little or no part in the activation process.

Acetylglucosaminidase↗
PubMed1989

Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybridomas.

A panel of 10 hybridomas was assembled to assess the influence of various genetic and biological factors upon glycosylation of secreted monoclonal IgG. After exhaustive Pronase digestion of IgG, glycopeptides were characterized chromatographically by apparent size, charge, and concanavalin A (Con A)-Sepharose and Lens culinaris (LcH)-agarose affinity. Six glycosylation phenotypes were found to be common among all clones studied. Despite this phenotypic heterogeneity in glycosylation of IgG, considerable similarity exists between different clones. In particular, virtually all IgG glycopeptides bear a core fucose residue. Second, the majority of the glycosylation repertoire is comprised of two phenotypes, characterized by glycopeptides which differ in affinity for Con A-Sepharose. Of these, the predominantly expressed phenotype is the same for all clones. The carbohydrate structure derived from this phenotype, elucidated by 500-MHz 1H NMR spectroscopy, is (Formula: see text). Significant variability between different hybridomas exists in the relative expression of the two major phenotypes. Other differences between clones may reflect the expression of an additional site which is glycosylated differently. However, there is no apparent correlation of phenotype with either the hybridoma's parentage or the serologically defined polypeptide structure of the IgG which it secretes. In addition to clonal variability, other sources of variability in phenotypic expression were identified. A generational variability is apparent upon continuous culturing of the same hybridoma. Also, differences in culture medium pH or proliferative state of the cells may have a modest influence upon the glycosylation phenotype.

Animals↗
PubMed1989

Antibody-dependent cytotoxicity mediated by natural killer cells is enhanced by castanospermine-induced alterations of IgG glycosylation.

Inhibitors of glycosylation and carbohydrate processing were used to probe the functional consequences of specific, differential alterations in glycosylation of monoclonal IgG secreted by hybridoma clones. Neither the absence of glycosylation nor the presence of atypical oligosaccharides significantly influenced binding of the monoclonal antibody to the cell surface antigen recognized. However, lymphocyte-mediated antibody-dependent cytotoxicity was enhanced significantly, as compared to native (unmodified) IgG-sensitized target cells, when target cells were sensitized with IgG bearing the atypical oligosaccharides induced metabolically by castanospermine, N-methyldeoxynojirimycin, deoxymannojirimycin or monesin, but not by swainsonine. The enhanced cytotoxicity was mediated by natural killer cells but not by monocytes or interferon-activated polymorphonuclear leukocytes. By contrast, antibody-dependent cytotoxicity mediated by activated polymorphonuclear leukocytes against target cells sensitized with the IgG glycosylation phenotypes induced by swainsonine and tunicamycin, but not by castanospermine, was decreased in comparison to cytotoxicity against target cells sensitized with native IgG. The enhanced lymphocyte-mediated cytotoxicity was Fc receptor-dependent. A panel of monoclonal antibodies directed against different human tumor target cells was used to demonstrate that the castanospermine-induced IgG phenotype generally enhanced antibody-dependent tumoricidal activity mediated by natural killer cells. However, differences in lymphocyte response to an alteration in IgG glycosylation were observed.

Alkaloids↗
PubMed1988

Analysis of IgG glycopeptides by alkaline borate gel filtration chromatography.

A protocol for the characterization of IgG glycopeptides is described. Central to this scheme is the novel application of an alkaline borate buffer to gel filtration chromatography. The use of this buffer significantly enhances the resolution of glycopeptides. Furthermore, it results in the separation of a unique size class of glycopeptides derived from IgG secreted by murine hybridomas. Although predominantly neutral, these glycopeptides differ both qualitatively and quantitatively by lectin affinity chromatography from the other glycopeptides which are presumably derived from the Fc portion of IgG.

Borates↗
PubMed1986

Sialic acid lyase in human promyelocytic leukemic cells (HL-60) during phorbol-ester-induced differentiation.

There is a marked increase in the activity of sialic acid lyase (N-acetylneuraminate lyase; EC 4.1.3.3; also known as sialic acid aldolase) in HL-60 cells induced to differentiate into macrophages by the phorbol ester, tetradecanoylphorbol 12-myristate 13 acetate (TPA). Exposure of HL-60 cells to retinoic acid, butyric acid or dimethyl sulfoxide has little or no effect. The level of the enzyme remains unaltered in HL-60 cells grown in the presence of an inactive analog of TPA, nor does it change in variants of HL-60 cells resistant to TPA.

Butyrates↗
PubMed1986

Transforming gene in human atherosclerotic plaque DNA.

The monoclonal hypothesis equates atherosclerotic plaques with benign smooth muscle cell tumors and proposes that plaques can arise via mutational or viral events. Here, we provide direct evidence that molecular events, heretofore associated only with tumor cells, are common to plaque cells as well. Three distinct groups of human coronary artery plaque (hCAP) DNA samples transfected into NIH 3T3 cells gave rise to transformed foci. DNA samples from a panel of normal noncancerous human tissues, including coronary artery, were negative in the assay. Southern-blotted focus DNA yielded positive signals when hybridized to the 32P-labeled nick-translated repetitive human "Alu" DNA sequence. The DNA from cloned foci was used successfully in a second round of transfection. Focus DNA hybridized to nick-translated v-Ki-ras, v-Ha-ras, or N-ras probes failed to detect human fragments of these genes. Primary focus cells from each of five clones elicited tumors after injection into nude mice (6/42). Several distinct high molecular weight (greater than 6.6 kilobases) bands were detected after BamHI-digested tumor DNA was hybridized to Alu. Preliminary characterization of these hCAP DNA-associated tumors indicates that they are similar to the fibrosarcomas that arise after injection of ras-transformed cells into nude mice. We propose that transforming genes in plaque cells behave in a manner analogous to the way in which oncogenes behave in cancer cells.

Animals↗