Stable antisense RNA expression neutralizes the activity of low-density lipoprotein receptor-related protein and promotes urokinase accumulation in the medium of an astrocytic tumor cell line.

Low-density lipoprotein receptor-related protein (LRP) binds and internalizes multiple ligands that are structurally and functionally diverse. However, the effects of LRP on cellular phenotype remain unclear. To study LRP in human astrocytic tumor cells, we designed LRP antisense RNA expression constructs in which the antisense cDNA fragment was expressed under the control of the cytomegalovirus (CMV) promoter. U-1242 MG astrocytic tumor cells were transfected with the antisense constructs and cloned from single cells to yield multiple cell lines with decreased LRP expression. Further studies were performed with two cell lines in which LRP antigen was completely eliminated (L(alpha)42) or substantially decreased (Lalpha47), as determined by Western blot analysis. Untransfected U-1242 MG cells and cells that were stably transfected with empty vector (pBK-CMV) bound activated alpha2-macroglobulin (alpha2M) in a specific and saturable manner. The Bmax was about 5000 receptors/cell. Lalpha42 cells did not bind alpha2M, and binding was decreased by >60% in Lalpha47 cells. Lalpha42 and Lalpha47 cells also demonstrated reduced susceptibility to the cytotoxin, Pseudomonas exotoxin A, and accumulated greatly increased levels of urokinase-type plasminogen activator (uPA) in conditioned medium. The accumulation of uPA demonstrates a major role for LRP in the catabolism of this protein in astrocytic tumor cells. The LRP-deficient cell lines, developed using antisense technology, represent a new model system for studying LRP function in astrocytes.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Effects of bacterial lipopolysaccharide on the hydrolysis of phosphatidylinositol-4,5-bisphosphate in murine peritoneal macrophages

LPS and lipid A initiated enhanced hydrolysis of PIP2 in macrophages. When murine peritoneal macrophages were labeled with [2-3H]myoinositol and stimulated with either LPS or lipid A, a rapid (within 10 sec) rise in Ins(1,4,5)P3 was observed. The breakdown pattern of Ins(1,4,5)P3 was complex; this included breakdown of Ins(1,4,5)P3 and formation of Ins(1,3,4,5)P4 (approximately 10 to 30 sec), and ultimately formation of Ins(1,3,4)P3 (approximately 60 sec). Within 10 sec after treatment, LPS caused an average increase of about fourfold to fivefold in Ins(1,4,5)P3, which declined over 5 min. When the total isomers of InsP3 were measured, levels rose about twofold in response to LPS or to lipid A and remained elevated for as long as 5 min. Lipid A, in the concentration range of 0.1 to 10 micrograms/ml, induced elevated intracellular levels of Ca2+ as quantified by fluorescence with Quin 2 or with Fura 2. When single, adherent Fura 2-loaded macrophages were treated with lipid A, basal levels of calcium rose over 10 sec from approximately 55 nM to almost 600 nM. LPS, paradoxically, did not cause such substantial increases in intracellular calcium (i.e., increases of approximately 26 nM) when judged by Fura 2 fluorescence. LPS treatment led to enhanced phosphorylation of a characteristic set of proteins, similar to those induced by stimulating protein kinase C (PKC) with phorbol myristate acetate as previously reported. The enhanced phosphorylation of pp28, pp33, and pp67 in macrophages was evident by 15 min and optimal by 30 min. Taken together, these observations indicate that LPS and lipid A cause increased breakdown of phosphatidylinositol 4,5-bisphosphate, which led to enhanced intracellular levels of calcium and also to enhanced protein phosphorylation, presumably mediated by PKC. The data thus suggest that one major intracellular signal transduction mechanism, initiated by LPS and lipid A in macrophages, is the rapid breakdown of PIP2.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.     

Selectivity and stereospecificity of the reactions of dichlorodiammineplatinum(II) with three purified plasma proteins

The reactions of cis- and trans-dichlorodiammineplatinum(II) (cis- and trans-DDP) with albumin and two plasma proteinase inhibitors were compared. Reaction with alpha 2-macroglobulin (alpha 2M) resulted in subunit crosslinking and loss of proteinase binding activity. The reaction also modified a receptor recognition site present on each alpha 2M subunit. While more trans-DDP was incorporated into alpha 2M than cis-DDP, cis-DDP was more effective at blocking receptor recognition, alpha 1-proteinase inhibitor was also inactivated by reaction with either cis- or trans-DDP. These reactions resulted in binding of platinum to methionine-358 at the reactive center of this inhibitor. Trans-DDP, however, was less selective and also bound to the single cysteine residue (Cys-232) of alpha 1PI. Reaction of albumin with cis-DDP resulted in incorporation of about 1 mol platinum per mol protein, and this platinum modified the single cysteine (Cys-34) in the molecule. Albumin incorporated twice as much trans-DDP, but the binding did not involve cysteine-34. In general, reactions of cis-DDP with proteins appear to be more selective than those observed for modification with the trans isomer.     

cis-dichlorodiammineplatinum (II) as a selective modifier of the oxidation-sensitive reactive-center methionine in alpha 1-antitrypsin

Methionine 358 in the plasma protein alpha 1-antitrypsin (alpha 1AT) is an oxidation-sensitive reactive-center residue critical for proteinase-inhibitory activity. Reaction of alpha 1AT with 20 microM to 1.67 mM cis-dichlorodiammineplatinum (II) (cis-DDP) or trans-DDP afforded concentration-dependent loss of trypsin-inhibitory activity. This effect, studied by gel electrophoresis and activity assays, is essentially independent of pH over the range 4.9-8.6. Binding assays showed covalent incorporation of 1 mol of cis-DDP into each mol of alpha 1AT. cis-DDP protected a single methionine residue from oxidation and made alpha 1AT resistant to degradation by papain, which cleaves alpha 1AT at Met358. These findings strongly suggest that cis-DDP inactivates alpha 1AT by binding exclusively to its reactive-center methionine. alpha 1AT bound twice as much platinum when reacted with trans-DDP. Because carboxamidomethylated alpha 1AT incorporated nearly 1 mol of both cis- and trans-DDP, the trans isomer apparently binds to both the reactive-center methionine and to the single cysteine residue of alpha 1AT. Because of its greater selectivity, cis-DDP is the superior reagent for modification of the alpha 1AT reactive-center methionine.     

Proteomic Analysis Reveals a Role for Bcl2-associated Athanogene 3 and Major Vault Protein in Resistance to Apoptosis in Senescent Cells by Regulating ERK1/2 Activation

Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer.Cellular senescence plays an important role in determining the response of tumors to cancer therapy (1). Senescence is regulated by the p53 and p16-pRB tumor suppressor pathways and characterized by irreversible cell cycle arrest and expression of the lysosomal protein, senescence associated beta galactosidase (SA-β-gal)¹. Additional characteristics of senescent cells include the presence of senescence-associated heterochromatic foci, and a senescence associated secretory phenotype (SASP) (2). Because of the SASP of senescent cells, therapy-induced senescence (TIS) may be harmful in cancer and the quantitative elimination of senescent cells could prove to be therapeutically beneficial. A recent study demonstrated that pharmacologically targeting the metabolic pathways of TIS in vivo prompted tumor regression and improved treatment outcomes (3).A characteristic of senescent cells is their ability to resist apoptosis although the responsible mechanism is poorly understood. Impairment of apoptosis in senescent cells is associated with a poor outcome in cancer (4). Manipulation of the apoptotic machinery may serve as a therapeutic means of eliminating senescent cells with harmful SASP. It has been proposed that in senescent cells, p53 may preferentially activate genes that arrest proliferation, rather than those that facilitate apoptosis. Alternatively, resistance to apoptosis may be caused by altered expression of proteins that inhibit, promote, or mediate apoptotic cell death, such as Bcl2.Bcl2 associated athanogene 3 (Bag3) is a member of the BAG family of chaperones that interacts with the ATPase domain of heat shock protein-70 (Hsp70). In addition to its BAG domain, Bag3 contains a WW domain and a proline-rich (PXXP) repeat, which mediates binding to partners other than Hsp70. Bag3 is expressed in response to cellular stress under the induction of HSF1 and is known to suppress apoptosis and regulate autophagy (5–6). Suppression of apoptosis may be partially explained by the ability of Bag3 to protect Bcl2 family members against proteasomal degradation (7). In normal cells, Bag3 is constitutively expressed in only a few cell types, including cardiomyocytes (8). Bag3 is overexpressed in leukemia and several solid tumors where it has been reported to sustain cell survival, induce resistance to therapy, and promote metastasis. The pleiotropic functions of Bag3 may reflect its ability to assemble scaffolding complexes, which participate in multiple signal transduction pathways (9).In this study, we describe a role for Bag3 in regulating cancer chemotherapy induced senescence in breast cancer cell. Using a quantitative SILAC approach, we show that Bag3 is up-regulated in TIS. Mass spectrometry analysis reveals that Bag3 binds to the Major Vault Protein (MVP) complex, a protein complex strongly associated with chemotherapy resistance. We also show that Bag3 and MVP contribute to apoptosis resistance by regulating ERK1/2 signaling in senescent MCF7 and ZR751 cells.     

Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression

Tissue-type plasminogen activator (tPA), a serine protease well known for generating plasmin, has been demonstrated to induce matrix metalloproteinase-9 (MMP-9) gene expression and protein secretion in renal interstitial fibroblasts. However, exactly how tPA transduces its signal into the nucleus to control gene expression is unknown. This study investigated the mechanism by which tPA induces MMP-9 gene expression. Both wild-type and non-enzymatic mutant tPA were found to induce MMP-9 expression in rat kidney interstitial fibroblasts (NRK-49F), indicating that the actions of tPA are independent of its proteolytic activity. tPA bound to the low density lipoprotein receptor-related protein-1 (LRP-1) in NRK-49F cells, and this binding was competitively abrogated by the LRP-1 antagonist, the receptor-associated protein. In mouse embryonic fibroblasts (PEA-13) lacking LRP-1, tPA failed to induce MMP-9 expression. Furthermore, tPA induced rapid tyrosine phosphorylation on the beta subunit of LRP-1, which was followed by the activation of Mek1 and its downstream Erk-1 and -2. Blockade of Erk-1/2 activation by the Mek1 inhibitor abolished MMP-9 induction by tPA in NRK-49F cells. Conversely, overexpression of constitutively activated Mek1 induced Erk-1/2 phosphorylation and MMP-9 expression. In mouse obstructed kidney, tPA, LRP-1, and MMP-9 were concomitantly induced in the renal interstitium. Collectively, these results suggest that besides its classical proteolytic activity, tPA acts as a cytokine that binds to the cell membrane receptor LRP-1, induces its tyrosine phosphorylation, and triggers intracellular signal transduction, thereby inducing specific gene expression in renal interstitial fibroblasts.     

<Emphasis Type="Italic">α</Emphasis>-Amylase inhibitor-1 gene from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> expressed in <Emphasis Type="Italic">Coffea arabica</Emphasis>plants inhibits α-amylases from the coffee berry borer pest

Background  

Coffee is an important crop and is crucial to the economy of many developing countries, generating around US70 billion per year. There are 115 species in the < i > Coffea < /i > genus, but only two, < i > C. arabica < /i > and < i > C. canephora < /i > , are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer ( < i > Hypotheneumus hampei < /i > ), is responsible for worldwide annual losses of around US70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.