1 In the present study we investigated a possible role for the p38 mitogen-activated protein (MAP) kinase pathway in mediating nuclear factor-kappa B (NF-kB) transcriptional activity in the erythroleukaemic cell line TF-1. 2 TF-1 cells stimulated with the phosphatase inhibitor okadaic acid (OA) demonstrated enhanced NF-kB and GAL4p65-regulated transcriptional activity which was associated with elevated p38 phosphorylation. However, pretreatment with the p38 MAPK speci®c inhibitor SB203580 (1 mM) or overexpression of kinase-de®cient mutants of MKK3 or MKK6 did not aect OA-enhanced NF-kB transcriptional potency, as determined in transient transfection assays. In fact, 5 and 10 mM SB203580 enhanced rather than inhibited NF-kB-mediated promoter activity by 2 fold, which was independent of phosphorylation of the p65 subunit. 3 The SB203580-mediated increase in NF-kB transcriptional activity was associated with enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK), but not p38 kinase. 4 Overexpression of kinase-de®cient mutants belonging to the ERK1/2, JNK, and p38 pathways showed that only dominant-negative Raf-1 abrogated SB203580-enhanced NF-kB activity. This would implicate the involvement of the ERK1/2 pathway in the enhancing eects of SB203580 on NF-kB-mediated gene transcription. 5 This study demonstrates that the p38 MAP kinase pathway is not involved in the OA-induced activation of NF-kB. SB203580 at higher concentrations activates the ERK pathway, which subsequently enhances NF-kB transcriptional activity.
The inducible nitric oxide synthase (iNOS) promoter contains nuclear factor B (NF-B) binding sites. NF-B activation is determined, in part, by the intracellular redox status. The aim of this study was to determine the importance of the cellular glutathione status in relation to NF-B activation and iNOS expression in hepatocytes in vivo and in vitro. For in vivo experiments, rats were injected with endotoxin and sacrificed 6 hours later. Glutathione was depleted by diethylmaleate. For in vitro experiments, cultured hepatocytes from untreated rats were exposed to a cytokine mixture. Glutathione levels were depleted by diethylmaleate and restored by N-acetylcysteine. Nitric oxide radicals (NO) are synthesized by the enzyme nitric oxide synthase (NOS). Three isoforms of this enzyme encoded by distinct genes are known. 1,2 The constitutive isoforms are neuronal NOS (type I) and endothelial NOS (type III). Neuronal NOS is involved in neurotransmission, whereas NO derived from endothelial NOS has antithrombotic and vasorelaxing properties. Inducible NOS (type II, iNOS) is not expressed under normal conditions, but is induced by cytokines and endotoxin (lipopolysaccharide [LPS]) in various cell types including hepatocytes, macrophages, smooth muscle cells, and chondrocytes. 3 iNOS-derived NO is an important component of the nonspecific host defense against invading microbial agents. 4 It has been shown that mice lacking a functional iNOS gene are more susceptible to infection with Staphylococcus aureus 5 and Leishmania major. 6 Furthermore, inhibition of viral replication by NO is shown by many laboratories by using NO donors, inhibiting NO production by NOS-inhibitors, or by using iNOS knockout mice. 7 To induce iNOS messenger RNA (mRNA), activation of the transcription factor nuclear factor-B (NF-B) is essential 8-12 although probably not sufficient 13,14 for full iNOS induction. To induce gene transcription, NF-B must be translocated from the cytoplasm to the nucleus. 15 Nuclear translocation of NF-B is triggered by changes in the redox state. [16][17][18] Therefore, the intracellular glutathione (GSH) status may be a key determinant for the capacity of cells to express iNOS. Indeed, it has been shown that GSH depletion prevents iNOS induction and/or NO production in response to cytokines in cultured rat hepatocytes, 19,20 in the J774 macrophage cell line, 21 and in cultured macrophages. 22 To our knowledge, neither the relevance of the intracellular GSH status for the induction of iNOS in the liver in vivo nor the contribution of NF-B in the GSH-dependent iNOS expression has been reported.The aim of this study was to investigate the importance of the intracellular GSH status for the capacity of hepatocytes and inflammatory cells to express iNOS in response to endotoxin and/or cytokines in vivo and in vitro. MATERIALS AND METHODS Animals and Experimental Design in Vivo.Specified pathogen-free male Wistar rats (200-250 g) were purchased from Harlan-CPB, Zeist, the Netherlands. They were kept under routine laboratory
Cytokines and growth factors induce activation of the family of signal transducers and activators of transcription (Stats) that directly activate gene expression. Recently, constitutively activated Stat1, Stat3, and Stat5 were identified in nuclear extracts of acute myeloid leukemia (AML) patients, suggesting involvement of constitutive Stat activity in the events of leukemogenesis. In the present study, blasts of nine AML cases were investigated for the constitutive binding activity of the recently identified transcription factor LIL-Stat (LPS- and IL-1-inducible Stat). Band-shift assays were performed using the LPS-and IL-1-responsive element (LILRE) oligonucleotide, a gamma interferon activation site-like site that is present in the human IL-1β promoter. Constitutive LIL-Stat binding activity was observed in three leukemic cell lines and in seven out of nine AML cases. Transient transfection studies with a reporter plasmid containing three sequential LIL-Stat binding sites showed distinct transcriptional activity of LIL-Stat only in those AML blasts that constitutively expressed LIL-Stat. In CD34+ cells LIL-Stat also constitutively bound to its consensus sequence. However, when these cells were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and stem cell factor (SCF) for differentiation along the monocytic lineage, the LIL-Stat binding activity disappeared totally. In agreement with these findings neither mature monocytes nor granulocytes showed constitutive or inducible LIL-Stat binding activity. We conclude that the LIL-Stat transcription factor is constitutively activated in undifferentiated and leukemic hematopoietic cells, but not in mature cells. This may suggest a role for this transcription factor in the process of differentiation. © 1998 by The American Society of Hematology.
In acute myelogenous leukemia (AML) and adult T-cell leukemia, it has been demonstrated that the transcription factor LIL-STAT is constitutively activated. To identify and characterize this unknown LIL-STAT protein, electrophoretic mobility shift assay (EMSA) and oligoprecipitation assays were performed by using lipopolysaccharide/interleukin-1 (IL- 1 IntroductionThe signal transducers and activators of transcription (STAT) proteins are a family of transcription factors involved in cytokine and growth factor signaling and are critical for the proliferation and differentiation of hematopoietic cells. The DNA-binding target of STAT dimers upon receptor activation is the consensus interferon ␥ (IFN-␥) activation site (GAS), which was initially reported to bind the IFN-␥-induced STAT1. So far, 7 different STAT genes have been identified. 1 Recently, a novel GAS-binding STAT factor was characterized with unique binding activity for a lipopolysaccharide (LPS)/ interleukin-1 (IL-1)-responsive element (LILRE) sequence motif of 8 nucleotides, TTCCTGAGA. LILRE is located within the enhancer region of the pro-IL-1 gene. Activation of this LIL-STAT factor was mediated by LPS, IL-1, and IL-6 stimulation in different cell lines and could be shifted in electrophoretic mobility shift assay (EMSA) by antibodies directed against the N-terminus of STAT1, but not by those specific for the C-terminus of STAT1. In addition, no cross-competition existed with the other GAS-binding oligonucleotides. 2 Spontaneous expression of LIL-STAT has been demonstrated in acute myeloid leukemia (AML) cells and adult T-cell leukemia. 3,4 In addition, it was shown that the expression was dependent on the stage of differentiation. In primitive hematopoietic CD34 ϩ cells, LIL-STAT expression was present, but it was absent in differentiated monocytes and granulocytes. 3 However, it is unclear whether LIL-STAT is a novel STAT protein separate from the well-defined STAT1 to STAT6 proteins.STAT activation can be detected by elevated DNA-binding activity, as measured in EMSAs using an oligonucleotide probe corresponding to specific GAS-binding sites. STAT1 homodimers bind to the GAS site of the Fc␥RI gene, but not STAT3 homodimers. 5 In contrast, both STAT1 and STAT3 bind to the high-affinity synthetic derivative of the c-fos promoter m67 hSIE (mutated human sis-inducible element) 6 and S1-S3 (consensus binding sequence for STAT1 or STAT3). 7,8 In the present study, we demonstrate by EMSA and oligoprecipitation assays that binding of ␣ and  isoforms of STAT1 and STAT3 to the LILRE DNA motif is stimulus dependent and differs between monocytic leukemic cells and monocytes. Study design Cell culture, AML cells, cytokines, and antibodiesHuman monocytic leukemic cells, namely THP-1 and AML cells, were isolated as described before. 3 Nuclear proteins were extracted from leukemic cells and monocytes 9 after 10 minutes of incubation with 10 ng/mL recombinant human IFN-␥ (Endogen, Woburn, MA) or 10 ng/mL IL-6. 10 Peripheral blood cells or bone marrow cells we...
In the present study, cisplatin (cDDP) and carboplatin (CBDCA) were combined in different in vitro and in vivo assays to determine whether combined cDDP and CBDCA treatment would eventually lead to a better antitumor response. Co-incubation of CC531 cells with cDDP and CBDCA led to higher intracellular Pt concentrations (30.5 +/- 3.4 ng Pt/10(6) cells) than did cDDP (16.9 +/- 9.4 ng Pt/10(6) cells) or CBDCA (1.28 +/- 0.72 ng Pt/10(6) cells) incubation alone. In survival assays an additive cell kill was seen after combined treatment with cDDP and CBDCA. DNA binding experiments using isolated salmon-sperm DNA exposed to the drugs separately or in combination were in agreement with the survival studies (for cDDP a binding of 12.42 micrograms Pt/mg DNA; for CBDCA, 0.49 microgram Pt/mg DNA; and for combined CBDCA and cDDP, 12.9 micrograms Pt/mg DNA at 76 h). Toxicity studies in rats treated with cDDP plus CBDCA required a dose reduction for cDDP amounting to 20% of the MTD, whereas the CBDCA dose could be maintained. Pharmacokinetics studies showed higher AUCs and t1/2 beta in plasma as well as the peritoneal cavity after combined treatment with cDDP and CBDCA (both given i.p.) or following cDDP given i.p. and CBDCA given i.v. Pt concentrations in peritoneal tumors corresponded with these observations, with higher Pt concentrations following combined treatment than after single-agent injection. In addition, combined administration of cDDP i.p. and CBDCA i.v. led to higher Pt concentrations in peritoneal tumors than did administration of both drugs i.p. (3.93 +/- 0.9 vs 2.76 +/- 0.2 mg Pt/g tissue). The higher Pt concentrations in the peritoneal tumors after combined treatment was associated with a significantly better antitumor response in comparison with that observed after single-agent treatment (a growth delay of 30.2 +/- 5.6 days for cDDP i.p. plus CBDCA i.v. vs 16.1 +/- 5.4 days for cDDP alone and 10.8 +/- 4.2 days for CBDCA alone).
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