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Biomedical subjects

J L Andres

Publications and source records attributed to J L Andres.

13 recordsLinked to original sources
PubMed1998

Regulation of BRCA1 and BRCA2 expression in human breast cancer cells by DNA-damaging agents.

Germline mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 have been linked to the development of breast cancer, ovarian cancer, and other malignancies. Recent studies suggest that the BRCA1 and BRCA2 gene products may function in the sensing and/or repair of DNA damage. To investigate this possibility, we determined the effects of various DNA-damaging agents and other cytotoxic agents on the mRNA levels of BRCA1 and BRCA2 in the MCF-7 and other human breast cancer cell lines. We found that several agents, including adriamycin (a DNA intercalator and inhibitor of topoisomerase II), camptothecin (a topoisomerase I inhibitor), and ultraviolet radiation induced significant decreases in BRCA1 and BRCA2 mRNA levels. Decreased levels of BRCA1 and BRCA2 mRNAs were observed within 6-12 h after treatment with adriamycin and persisted for at least 72 h. Adriamycin also induced decreases in BRCA1 protein levels; but these decreases required several days. U.V. radiation induced dose-dependent down-regulation of BRCA1 and BRCA2 mRNAs, with significant decreases in both mRNAs at doses as low as 2.5 J/m2, a dose that yielded very little cytotoxicity. Adriamycin-induced down-regulation of BRCA1 and BRCA2 mRNAs was first observed at doses that yielded relatively little cytotoxicity and little or no apoptotic DNA fragmentation. Adriamycin and U.V. radiation induced distinct dose- and time-dependent alterations in the cell cycle distribution; but these alterations did not correlate well with corresponding changes in BRCA1 and BRCA2 mRNA levels. However, the adriamycin-induced reduction in BRCA1 and BRCA2 mRNA levels was correlated with p53 functional status. MCF-7 cells transfected with a dominant negative mutant p53 (143 val-->ala) required at least tenfold higher doses of adriamycin to down-regulate BRCA1 and BRCA2 mRNAs than did parental MCF-7 cells or control-transfected MCF-7 clones. These results suggest that BRCA1 and BRCA2 may play roles in the cellular response to DNA-damaging agents and that there may be a p53-sensitive component to the regulation of BRCA1 and BRCA2 mRNA expression.

Antineoplastic Agents↗
PubMed1994

Dexamethasone enhancement of betaglycan (TGF-beta type III receptor) gene expression in osteoblast-like cells.

Betaglycan (type III transforming growth factor-beta (TGF-beta) receptor) is a cell surface heparan/chondroitin sulfate proteoglycan that binds TGF-beta via its core protein and is abundantly expressed in osteoblastic cells. A previous report (Centrella et al., Mol. Cell. Biol. 11, 4490-4496, 1991) showed post-translational enhancement by glucocorticoid of TGF-beta binding to betaglycan. Upon the availability of the betaglycan cDNA, we investigated the effects of a glucocorticoid analogue, dexamethasone, on the regulation of betaglycan expression in osteoblast-like cells. Betaglycan mRNA was expressed as an approximately 6-kb band in MC3T3-E1 cells. The betaglycan mRNA level was enhanced severalfold by dexamethasone in these cells. The effect of dexamethasone on the betaglycan mRNA level was observed within 9 h and was sustained at least up to 48 h. The dexamethasone effect was dose-dependent, with a saturation concentration at 10(-7) M. Among the steroid hormones examined, dexamethasone exhibited the most potent effect on betaglycan mRNA expression, while retinoic acid also enhanced it moderately. Dexamethasone enhancement of betaglycan mRNA expression was blocked by actinomycin D, but it was not blocked by cycloheximide. Cross-linking experiments showed that dexamethasone treatment increased the binding of radiolabeled TGF-beta 1 to betaglycan, but did not affect binding to the type II receptor. A similar dexamethasone enhancement of betaglycan mRNA expression was also observed in a preosteoblast-like cell line, RCT1. These results suggest that dexamethasone enhances betaglycan expression at least in part via transcriptional events in osteoblasts and this would be one of the target points of glucocorticoid regulation of bone metabolism.

Animals↗
PubMed1994

Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites.

Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membrane anchorage is required. Soluble betaglycan can bind TGF-beta but does not enhance binding to membrane receptors. In fact, recombinant soluble betaglycan acts as potent inhibitor of TGF-beta binding to membrane receptors and blocks TGF-beta action, this effect being particularly pronounced with the TGF-beta 2 isoform. The results suggest that release of betaglycan into the medium converts this enhancer of TGF-beta action into a TGF-beta antagonist.

Animals↗
PubMed1992

Binding of two growth factor families to separate domains of the proteoglycan betaglycan.

Cell surface proteoglycans help present some polypeptide growth factors such as basic fibroblast growth factor (bFGF) to their receptors and may act as reservoirs for others such as transforming growth factor-beta (TGF-beta). Betaglycan, a cell surface heparan sulfate/chondroitin sulfate proteoglycan that binds TGF-beta via its core protein, is shown here to bind bFGF via its heparan sulfate chains. We investigated the potential for regulation of betaglycan by its ligands in osteoblasts, a system in which bFGF and TGF-beta have complementary effects. We report here that the apparent molecular mass of betaglycan from an osteoblast-enriched primary culture of fetal rat calvaria is decreased in response to bFGF, as detected by an increased electrophoretic migration of betaglycan. The betaglycan forms expressed in bFGF-treated osteoblasts have a reduced content of heparan sulfate GAGs, without detectable changes in the content of chondroitin sulfate GAGs or the size of the core protein. bFGF did not affect the overall population of cell-surface-associated proteins identified by sulfate labeling, which contained primarily heparan sulfate, and had only small effects on the major secreted proteoglycans, which were, by contrast, chondroitin sulfate proteoglycans. The effect of bFGF on betaglycan is therefore a selective one. These results suggest that cells can interact with members of the TGF-beta and FGF families through separate domains of the same membrane proteoglycan, and can selectively regulate the bFGF-binding carbohydrate chains of this proteoglycan in response to bFGF.

3T3 Cells↗
PubMed1991

Purification of the transforming growth factor-beta (TGF-beta) binding proteoglycan betaglycan.

We report the purification of betaglycan, a low-abundance membrane proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). Betaglycan solubilized from rat embryo membrane preparations was purified to near-homogeneity by sequential chromatography through DEAE-Trisacryl, wheat germ lectin-Sepharose, and TGF-beta 1-agarose. Purified betaglycan has properties similar to betaglycan affinity-labeled in intact cells: it binds TGF-beta 1 and TGF-beta 2 with KD approximately 0.2 nM, contains heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains and N-linked glycans attached to a 110-kDa core protein, and can spontaneously associate with phosphatidylcholine liposomes. The betaglycan core obtained by enzymatic removal of the GAG chains has high affinity for TGF-beta and associates with artificial liposomes, indicating that the core protein binds TGF-beta and anchors to membranes independently of the GAG chains present on the native protein or of any ancillary protein.

Animals↗
PubMed1991

Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system.

We describe the primary structure of rat betaglycan, a polymorphic membrane-anchored proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). As deduced from its cDNA sequence, the 853 amino acid core protein of betaglycan has an extracellular domain with clustered sites for potential attachment of glycosaminoglycan chains. These chains are dispensable for TGF-beta binding to the core protein. The transmembrane region and the short cytoplasmic tail of betaglycan are very similar to these regions in human endoglin, an endothelial cell membrane glycoprotein involved in intercellular recognition. The ectodomain of betaglycan can be released as a soluble proteoglycan; a potential cleavage site near the transmembrane region is identical to the highly regulated cleavage site of the membrane-anchored transforming growth factor-alpha precursor. The unique features of betaglycan suggest important roles in cell interaction with TGF-beta.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of a novel protein phosphatase highly specific for ribosomal protein S6.

Ribosomal protein S6 is the principal phosphoprotein of the eucaryotic ribosome that becomes multiply phosphorylated on serine residues in response to a wide variety of mitogenic stimuli. In this paper the principal protein phosphatases able to dephosphorylate S6 were characterized in Xenopus laevis ovary and eggs. Two enzymes termed peak I and peak II were found to account for most S6 phosphatase activity in both oocytes and eggs. The peak I enzyme had an apparent Mr of 200,000 on gel filtration, dephosphorylated the beta subunit of phosphorylase kinase and phosphorylase a, and was inhibited by inhibitor 1 and inhibitor 2, suggesting it was similar to protein phosphatase 1. The peak II enzyme was purified over 12,000-fold and had an apparent Mr = 55,000 on glycerol gradient centrifugation. This phosphatase could dephosphorylate all sites in S6 but was unable to dephosphorylate phosphorylase a or phosphorylase kinase. However, it was inhibited by nanomolar concentrations of inhibitor 1 and inhibitor 2. These results indicate the peak II enzyme represents a new class of highly specific protein phosphatase and suggest that inhibition of dephosphorylation in cellular extracts by inhibitor 1 and inhibitor 2 is not a sufficient criterion for implicating protein phosphatase 1 in a cellular process.

Animals↗
PubMed1989

Membrane-anchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-beta.

Transforming growth factors beta 1 and beta 2 bind with high affinity to the core protein of a 250-350-kD cell surface proteoglycan. This proteoglycan (formerly referred to as the type III TGF-beta receptor) coexists in many cells with the receptor implicated in TGF-beta signal transduction (type I TGF-beta receptor), but its function is not known. We report here that soluble TGF-beta-binding proteoglycans are released by several cell types into the culture media, and can be found in serum and extracellular matrices. As has been shown for the membrane-bound form, the soluble proteoglycans have a heterogeneous core protein of 100-120 kD that carries chondroitin sulfate and/or heparan sulfate glycosaminoglycan chains and a small amount of N-linked carbohydrate. The membrane-bound form of this proteoglycan is hydrophobic and associates with liposomes, whereas the soluble forms lack a membrane anchor and do not associate with liposomes. Differences in the electrophoretic migration of the soluble and membrane forms of this proteoglycan suggest additional structural differences in their core proteins and glycosaminoglycan chains. These soluble and membrane-bound proteoglycans, for which we propose the name "betaglycans," might play distinct roles in pericellular retention, delivery, or clearance of activated TGF-beta.

Animals↗
PubMed1988

The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor.

The transforming growth factor-beta (TGF-beta) receptor type III is a low abundance cell surface component that binds TGF-beta 1 and TGF-beta 2 with high affinity and specificity, and is present in many mammalian and avian cell types. Type III TGF-beta receptors affinity-labeled with 125I-TGF-beta migrate in sodium dodecyl sulfate-polyacrylamide electrophoresis gels as diffuse species of 250-350 kDa. Here we show that type III receptors deglycosylated by the action of trifluoromethanesulfonic acid yield affinity-labeled receptor cores of 110-130 kDa. This marked decrease in molecular weight is also achieved by combined treatment of type III receptors with heparitinase and chondroitinase ABC. Digestion of receptor-linked glycosaminoglycans by treatment of intact cell monolayers with heparitinase and chondroitinase does not prevent TGF-beta binding to the type III receptor core polypeptide and does not release the receptor polypeptide from the membrane. The type III TGF-beta receptor binds tightly to DEAE-Sephacel and coelutes with cellular proteoglycans at a characteristically high salt concentration. Thus, the type III TGF-beta receptor has the properties of a membrane proteoglycan that carries heparan and chondroitin sulfate glycosaminoglycan chains. The binding site for TGF-beta appears to reside in the 100-120-kDa core polypeptide of this receptor. The type III receptor is highly sensitive to cleavage by trypsin. Trypsin action releases the glycosaminoglycan-containing domain of the receptor leaving a 60-kDa membrane-associated domain that contains the cross-linked ligand. A model for the domain structure of the TGF-beta receptor type III is proposed based on these results.

Affinity Labels↗
PubMed1987

Identification of protein phosphatases 1 and 2B as ribosomal protein S6 phosphatases in vitro and in vivo.

Protein phosphatases 1 and 2B from rabbit skeletal muscle were found to catalyze the dephosphorylation of ribosomal protein S6 in vitro. Phosphorylation of protein phosphatase-1 by the transforming protein of Rous sarcoma virus, pp60v-src, abolished S6 dephosphorylation by the purified enzyme. Analysis of the dephosphorylation of phosphorylase a and phosphorylase kinase in Xenopus oocyte extracts and after microinjection indicated the presence of oocyte enzymes similar to protein phosphatases-1 and -2B. Studies with 32P-labeled 40 S ribosomal subunits suggested that these enzymes were functioning as S6 phosphatases in oocytes. These findings support the hypothesis that regulation of protein phosphatase activity may be involved in the increase in S6 phosphorylation observed after mitogenic stimulation.

Animals↗
PubMed1984

Structure elucidation of platelet activating factor derived from human neutrophils.

Platelet activating factor (PAF) synthesized by human neutrophils challenged by opsonized zymosan or calcium ionophore was isolated from cells and buffer using Bligh and Dyer extraction following the addition of tracer amounts of tritiated-PAF. The extract was subjected to TLC separation of phospholipid classes, followed by reverse phase HPLC for molecular species separation. All fractions were measured for radioactivity, biological activity and fast atom bombardment mass spectrometry. While the radioactive tracer PAF could be separated into three molecular species, PAF biological activity eluted as a single component which was characterized as 1-O-hexadecyl-2-acetyl-glycero-3-phosphocholine. The lack of molecular species heterogeneity of PAF produced in response to stimuli implies a higher degree of control of biosynthesis than previously suspected.

Adult↗
PubMed1983

Toxicity of 3,3',4,4'- and 2,2',5,5'-tetrabromobiphenyl: correlation of activity with aryl hydrocarbon hydroxylase induction and lack of protection by antioxidants.

3,3',4,4'-Tetrabromobiphenyl is a minor component of commercial polybrominated biphenyl (PBB) mixture fireMaster BP-6 and is a potent inducer of aryl hydrocarbon hydroxylase (AHH). A single ip dose of 3,3',4,4'-tetrabromobiphenyl (150 mumol/kg) caused significant reduction in the growth rate in the immature male Wistar rat, as well as pale enlarged livers and marked reduction in thymus size. Under light microscopy, hepatocytes were enlarged and vacuolated. The vacuoles, which were most prominent in the midzonal region of the lobule, corresponded to fat droplets in oil-red-O-stained sections. The thymus, especially the cortex, was markedly depleted of lymphocytes. Neither the reduced growth, altered organ weights nor the histopathology was reversed for the duration of the study by the coadministration of the antioxidants butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), or vitamin E. Vitamin E did, however counter the negative effect of 3,3',4,4'-tetrabromobiphenyl on growth during the first 5 d of the study. 2,2',5,5'-Tetrabromobiphenyl, also a minor component of fireMaster BP-6, is a weak phenobarbital-type inducer of cytochrome P-450. When administered at the same dose, 2,2',5,5'-tetrabromobiphenyl did not elicit any observed toxic effects. These data confirm the correlation between AHH induction and toxicity for these PBBs and suggest that 3,3',4,4'-tetrabromobiphenyl may significantly contribute to the toxicity of fireMaster BP-6. Although there is evidence that polychlorinated biphenyls, and perhaps 2,3,7,8-tetrachlorodibenzo-p-dioxin, exert certain toxic effects via a lipid peroxidation mechanism, the toxic changes measured during this study were not reversed by the administration of the antioxidants.

Animals↗