Overexpression of NP95 mRNA by tumor promoters in the promotion phase of a two-stage BALB/3T3 cell transformation assay.
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We studied altered gene expressions in BALB/3T3 cells treated by different tumor promoters in the promotion phase of a transformation assay, an in vitro model of a two-stage carcinogenicity test, using fluorescent mRNA differential display analysis. Expression of the NP95 gene, which was previously found to be the gene of a murine nuclear protein associated with cell proliferation, was increased in the cultures treated by 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, and orthovanadate. The upregulation of NP95 mRNA was confirmed by reverse transcription-PCR, and Northern blot. TPA, okadaic acid, and orthovanadate enhanced cell proliferation as measured by a 5-bromo-2'-deoxyuridine incorporation assay. The expression level of NP95 mRNA was not affected by the treatment with typical carcinogens benzo[a]pyrene and 3-methylcholanthrene at concentrations at which they act as initiators of cell transformation. These facts may imply that the enhancement of cell transformation by these tumor promoters is due, at least in part, to the acceleration of cell proliferation. NP95 mRNA was also increased in the transformed BALB/3T3 cells. Overexpression of NP95 may also participate in the maintenance of the transformed phenotype. (+info)
The angiogenic and lymphangiogenic vascular endothelial growth factor (VEGF)-D is the only member of the VEGF family that is not induced by hypoxia or by serum factors, but its induction is mediated by direct cell-cell contact. Here we show that VEGF-D mRNA is down-modulated either by beta-catenin mobilization from the cell membrane, by activation of the Wnt signaling pathway, or by transfection with the beta-catenin stable mutant. Down-modulation of beta-catenin by means of RNA interference showed an increase of VEGF-D mRNA steady state in fibroblasts. The beta-catenin-dependent decrease of VEGF-D mRNA is indirect and mainly due to reduced VEGF-D mRNA stability, as demonstrated by experiments of mRNA decay in the presence of transcription or translation inhibitors. By transient transfection of chimeric constructs carrying fusion of VEGF-D sequences under the control of the cytomegalovirus early promoter, we demonstrated that beta-catenin negative regulation is on the VEGF-D mRNA 3'-untranslated region. We mapped the VEGF-D mRNA-destabilizing element to a sequence, conserved between mouse and human VEGF-D, which contains an AU-rich element of group I. These results unveiled a new regulatory pathway for VEGF-D, which explains, at least in part, VEGF-D regulation in tumor progression. (+info)
Importance of amino acids of the central portion of the second intracellular loop of the gastrin-releasing Peptide receptor for phospholipase C activation, internalization, and chronic down-regulation.
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Little is known about the function of the central portion of the second intracellular loop (i2 loop) of peptide receptors in activation of downstream pathways and receptor modulatory processes such as receptor internalization or chronic down-regulation (DR). Recent data suggest a role for i2 loop hydrophobic amino acids in these processes. We used site-directed mutagenesis to address these issues with the gastrin-releasing peptide receptor (GRP-R). Each i2 loop residue from 142 to 148 was mutated and the receptors were expressed in Balb 3T3 cells. Two mutants showed a minimal (<2-fold) decrease in affinity. Five mutants showed decreased efficacy for activating phospholipase C (PLC). Two double mutants (IM143.147AA and VM144.147AA) showed a minimal decrease in affinity but had a decreased ability to fully activate PLC. Only the IM double mutation had decreased maximal internalization, whereas the R145A single mutant showed an increase, suggesting a tonic inhibitory role for Arg-145 in internalization. Three single and both double mutants showed decreases in receptor DR. There was a weak correlation between the extent of GRP-R internalization and the maximal PLC activation, whereas changes in the maximal PLC activation were significantly (p = 0.008) coupled to receptor DR. This study shows that amino acids of the i2 loop of the GRP-R are important in activation of PLC, internalization and down-regulation, but not for affinity. Our results support the proposal that internalization and chronic down-regulation have differing dependence on PLC and are largely independent processes, because some mutants showed no changes in internalization, but significant alterations in down-regulation. (+info)
Synergistic regulation of the acute phase protein SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines.
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SIP24/24p3 is a secreted murine acute phase protein which has been speculated to play an anti-inflammatory role in vivo. Recently SIP24/24p3 has been found to be able to specifically induce apoptosis in leukocytes. By using (35)S metabolic labeling method, we studied the regulation of SIP24/24p3 by glucocorticoid and pro-inflammatory cytokines IL-6 and TNF-alpha in cultured Balb/c 3T3 and BNL cells. The following results were observed: (1) dexamethasone induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells, the induction was more significant in BNL cells; (2) dexamethasone and IL-6 synergistically induced the expression of SIP24/24p3 in both Balb/c 3T3 and BNL cells; (3) in Balb/c 3T3 cells dexamethasone and TNF-alpha acted synergistically to induce the expression of SIP24/24p3, whereas in BNL cells dexamethasone and TNF-alpha induced the expression of SIP24/24p3 in an additive manner; (4) dexamethasone and IL-6/TNF-alpha acted synergistically in Balb/c 3T3 cells and additively in BNL cells to induce the expression of SIP24/24p3. The inducibility of SIP24/24p3 by multiple factors will help to explain its highly specific expression in vivo. The difference in the expression patterns of SIP24/24p3 in different cell types is also suggestive to its expression and regulation in hepatic and extrahepatic tissues. Finally, the fact that SIP24/24p3 protein can be induced by both pro-inflammatory as well as anti-inflammatory factors is indicative of the important role of SIP24/24p3 in the entire acute phase response process. (+info)
Influence of type I collagen surface density on fibroblast spreading, motility, and contractility.
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We examine the relationships of three variables (projected area, migration speed, and traction force) at various type I collagen surface densities in a population of fibroblasts. We observe that cell area is initially an increasing function of ligand density, but that above a certain transition level, increases in surface collagen cause cell area to decline. The threshold collagen density that separates these two qualitatively different regimes, approximately 160 molecules/ microm(2), is approximately equal to the cell surface density of integrin molecules. These results suggest a model in which collagen density induces a qualitative transition in the fundamental way that fibroblasts interact with the substrate. At low density, the availability of collagen binding sites is limiting and the cells simply try to flatten as much as possible by pulling on the few available sites as hard as they can. The force per bond under these conditions approaches 100 pN, approximately equal to the force required for rupture of integrin-peptide bonds. In contrast, at high collagen density adhesion, traction force and motility are limited by the availability of free integrins on the cell surface since so many of these receptors are bound to the surface ligand and the force per bond is very low. (+info)
Efficient replication of full-length murine leukemia viruses modified at the dimer initiation site regions.
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Retroviruses encapsidate two copies of full-length viral RNA molecules linked together as a dimeric genome. RNA stem loop structures harboring palindromic (or "kissing") loop sequences constitute important cis-elements for viral dimerization known as dimer initiation sites (DIS). In murine leukemia virus (MLV), a 10-mer and a 16-mer palindrome (DIS-1 and DIS-2, respectively) located in the viral leader region mediate dimerization in vitro and affect dimer stability of vector RNA in vivo. We have investigated the effect on viral replication of introducing deletions or nucleotide substitutions within these palindromes in a full-length MLV genome. Our results demonstrate that viruses modified at the dimer initiation site regions are viable and show wild-type levels of RNA encapsidation. One mutant lacking the DIS-1 palindrome was severely impaired and displayed an increased cellular ratio of spliced versus genomic RNA that most likely contributes to the inefficient replication. The implications for development of DIS-modified retrovirus-based vectors are discussed. (+info)
AG490 inhibits G1-S traverse in BALB/c-3T3 cells following either mitogenic stimulation or exogenous expression of E2F-1.
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AG490, a member of the tryphostin family of protein kinase inhibitors, repressed G(0)-G(1) traverse in BALB/c-3T3 cells. While the early induction of STAT activity was repressed by AG490, extracellular signal-regulated kinase (ERK) activation was unaffected and a pattern of gene expression suggested that cells exited G(0) in the presence of the inhibitor. Although AG490 did not alter the induction of cyclin D1 protein, neither cyclin D1- nor cyclin D3-associated kinase activity was observed in growth-inhibited cells. Surprisingly, p130 was partially phosphorylated, and E2F3A protein was expressed in mitogen-stimulated AG490-treated cells despite the lack of cyclin D-associated kinase activity. These data suggest that AG490 inhibits a cellular pathway required for mid-G(0)-G(1) traverse that is located after the induction of early processes potentially mediated by E2F (although independent of cyclin D-associated kinase activity) but before the late G(1) increase in E2F-dependent transcription. Infection of AG490-treated cells with an E2F-1 adenovirus caused the induction of cyclin A, but could not overcome the drug-induced cell cycle arrest that was coincident with the repression of cyclin-dependent kinase 2 (cdk2)-associated kinase activation. We conclude that cdk2-associated kinase activity is modulated by a cellular process repressed by AG490. Furthermore, this cdk2-associated kinase activity is required for G(0)-G(1) traverse in some role other than the regulation of E2F-dependent transcription. (+info)
Antisense thymidylate synthase electrogene transfer to increase uptake of radiolabeled iododeoxyuridine in a murine model.
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In vitro and in vivo experiments from our laboratory and others have suggested that the combination of thymidylate synthase (TS) inhibitor and radiolabeled iododeoxyuridine (IdUrd) is synergistic. Efficacy is limited by drug resistance, which is often mediated by TS overexpression. We designed an in vivo electrogene transfer (EGT) model for delivering antisense TS plasmid (ATS) into tumor to increase the subsequent efficacy of (131)I-IdUrd therapy. METHODS: Plasmid complementary to nucleotide 531-710 in the coding region of the mouse TS (mTS) mRNA was constructed. TS activity and (131)I-IdUrd DNA incorporation were determined 48 h after in vitro EGT of ATS to CT26 cells. In vivo therapeutic effect and radioactivity retained in tumor after various combinations of EGT ATS, 5-fluorouracil (5-FU), and continuous infusion of (131)I-IdUrd by osmotic minipump were determined. RESULTS: A reduction of TS activity was achieved after in vitro EGT ATS. Flow cytometry analysis indicated that ATS-treated cells were arrested at S phase. In the in vivo tumor model, the combination of EGT ATS and 5-FU was able to partially overcome 5-FU drug resistance. Sixty percent of tumors can be eradicated by the combination of EGT ATS, 5-FU, and infusion of (131)I-IdUrd. The tumors treated by EGT ATS had higher radioactivity retained 1 wk after (131)I-IdUrd therapy than after EGT of control plasmid. CONCLUSION: In situ EGT ATS can downregulate TS and increase the therapeutic effect of radiolabeled IdUrd therapy. The combination of EGT ATS, 5-FU, and (131)I-IdUrd may result in tumor eradication. (+info)