Interleukin-6 (IL-6) is one of the major mediators of inflammation, and its expression is inducible by the other inflammatory lymphokines, interleukin-1 (IL-1) and tumor necrosis factor a (TNF-a). We demonstrate that a common IL-6 promoter element, termed inflammatory lymphokine-responsive element (ILRE), is important for induction of IL-6 gene expression by IL-1 and TNF-a despite possible differences in the mechanisms of action of these lymphokines. Remarkably, the ILRE sequence, located between -73 to -63 relative to the mRNA cap site, is highly homologous to NF-KB transcription factor-binding motifs and binds an IL-1-TNF-a-inducible nuclear factor; the sequence specificities, binding characteristics, and subcellular vocalizations of this factor are indistinguishable from those of NF-KB. In addition, mutations of the ILRE sequence which impair the binding of this nuclear factor abolished the induction of IL-6 gene expression by IL-1 and TNF-a in vivo. These results indicate that a nuclear factor indistinguishable from NF-KB is involved in the transcriptional activation of the IL-6 gene by IL-1 and TNF-a.

These findings indicate that the expression of HO-1 in the ischemic kidney may be critical in the recovery of renal cell function in this animal model. These findings also suggest that H0-1 induction may play an important role in conferring protection on renal cells from oxidative damage caused by heme.

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further de®ne the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines diering only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM 7/7 DT40 cells displayed retarded cellular proliferation, defective G 2 /M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM 7/7 DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but signi®cant reduction in targeted integration frequency was observed in ATM 7/7 DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM de®cient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.

Reductive metabolism of carbon tetrachloride (CCl(4)) is thought to cause lipid peroxidation which results in hepatic injury. Heme oxygenase-1 (HO-1) (EC 1.14.99.3), the rate-limiting enzyme in heme catabolism, is known to be induced by oxidative stress and to confer protection against oxidative tissue injuries. In this study, we examined the role of HO-1 induction in a rat model of CCl(4)-induced acute liver injury. CCl(4) treatment (1 mL/kg, intraperitoneally) produced severe hepatic injury in rats as revealed by significant increases in serum alanine transaminase (ALT) (EC 2.6.1.2) activity and hepatic malondialdehyde (MDA) content, severe liver cell injury, and increases in hepatic tumor necrosis factor-alpha (TNF-alpha) mRNA expression and DNA binding activity of nuclear factor-kappa B (NF-kappa B). Following CCl(4) treatment, hepatic HO-1 expression was markedly increased both at transcriptional and protein levels in hepatocytes, especially around the central vein. HO-1 induction was mediated in part through a rapid increase in microsomal free heme concentration presumably derived from hepatic cytochrome P450. Inhibition of HO activity by tin-mesoporphyrin (Sn-MP), which resulted in a sustained increase in microsomal free heme concentration, exacerbated liver injury, as judged by the sustained increase in serum ALT activity, extensive hepatocytes injuries, a more pronounced expression of hepatic TNF-alpha mRNA and an enhanced NF-kappa B activation. These findings indicate that induction of HO-1 is an adaptive response to CCl(4) treatment, and it may be critical in the recovery of hepatocytes from injury. Our findings also suggest that HO-1 induction may play an important role in conferring protection on hepatocytes from oxidative damage caused by free heme.

Background:Cholangiocarcinoma (CC) is a highly malignant carcinoma. We attempted to clarify the prognostic significance of c-Met overexpression and its association with clinicopathological factors in patients with CC.Patients and methods:One hundred and eleven patients with intrahepatic CC (IHCC) and 136 patients with extrahepatic CC (EHCC) who had undergone curative surgery were divided immunohistologically into c-Methigh and c-Metlow groups. Clinicopathological factors and outcomes were compared between the groups. c-Met and epidermal growth factor receptor (EGFR) expression was also examined in 10 CC cell lines.Results:The positivity of c-Met was 45.0% in IHCC and 68.4% in EHCC; c-Methigh expression was demonstrated in 11.7% of IHCC and 16.2% of EHCC. c-Methigh expression was significantly correlated with the 5-year survival rate for CC overall (P=0.0046) and for IHCC (P=0.0013), histopathological classification in EHCC, and for EGFR overexpression in both IHCC and EHCC. Coexpression and coactivation of c-Met and EGFR were also observed in CC cell lines. Multivariate analysis revealed that c-Methigh expression was an independent predictor of poor overall and disease-free survival in patients with IHCC.Conclusions:c-Met overexpression is associated with EGFR expression and is a poor prognostic factor in CC.

Carbon monoxide (CO) provides protection against oxidative stress via anti-inflammatory and cytoprotective actions. In this study, we tested the hypothesis that a low concentration of exogenous (inhaled) CO would protect transplanted lung grafts from cold ischemia-reperfusion injury via a mechanism involving the mitogen-activated protein kinase (MAPK) signaling pathway. Lewis rats underwent orthotopic syngeneic or allogeneic left lung transplantation with 6 h of cold static preservation. Exposure of donors and recipients (1 h before and then continuously post-transplant) to 250 ppm CO resulted in significant improvement in gas exchange, reduced leukocyte sequestration, preservation of parenchymal and endothelial cell ultrastructure and reduced inflammation compared to animals exposed to air. The beneficial effects of CO were associated with p38 MAPK phosphorylation and were significantly prevented by treatment with a p38 MAPK inhibitor, suggesting that CO's efficacy is at least partially mediated by activation of p38 MAPK. Furthermore, CO markedly suppressed inflammatory events in the contralateral naïve lung. This study demonstrates that perioperative exposure of donors and recipients to CO at a low concentration can impart potent anti-inflammatory and cytoprotective effects in a clinically relevant model of lung transplantation and support further evaluation for potential clinical use.

p53 is recruited in response to DNA-damaging genotoxic stress and plays an important role in maintaining the integrity of the genome. We show that exposure of cells to various genotoxic agents, including anticancer drugs such as mitomycin and 5-fluorouracil, results in an increase in p53 mRNA levels and in p53 promoter activation, indicating that the p53 genotoxic stress response is partly regulated at the transcriptional level. The results of the p53 promoter analysis show that a novel p53 promoter element, termed a p53 core promoter element (from ؊70 to ؊46), is essential for basal p53 promoter activity and p53 promoter activation induced by genotoxic agents such as anticancer drugs and UV. Although a B motif partially overlaps with this element and those genotoxic agents activate NF-B, it does not play a major role in p53 genotoxic stress response: NF-B p65 expression did not induce significant p53 promoter activation, and NF-B inhibitors (N-acetyl cysteine and IB␣) did not inhibit genotoxic stress-inducible p53 promoter activation. Finally, we characterized nuclear factors, the binding of which to the p53 core promoter element is essential for basal p53 promoter activity and p53 promoter activation induced by genotoxic agents.The p53 gene is one of the most commonly mutated genes identified in various types of human tumors, and the results of numerous studies suggest that the inactivation or abnormality of p53 is a critical step leading to neoplastic transformation (for reviews, see references 22, 31, and 51). Although the normal function of p53 is not still completely defined, the results of recent studies suggest that p53 controls a cell cycle checkpoint that is important for maintaining the integrity of the genome (19,29). Loss of p53 functions thus results in an enhanced frequency of genomic rearrangements or genomic instability (32, 54) and eliminates the growth arrest response or programmed cell death (apoptosis) induced by DNA-damaging genotoxic insults (24,28). In accordance with this function, p53 is recruited in response to various DNA-damaging agents such as UV, ␥-irradiation, and anticancer drugs (15,24,34) and functions, as a transcription factor with sequence-specific DNA binding and transcriptional activating activities (12,13,16,25,26,41,50), to induce expression of various cellular genes involved in cell cycle control, such as a gene for p21, a cyclin-Cdk inhibitor which blocks cell cycle progression (11). Other candidate cellular target genes for p53 include genes for Gadd45, which stimulates DNA repair in association with proliferating cell nuclear antigens (47); Bcl-2 family members which play critical roles in apoptosis (36); a tumor angiogenesis inhibitor which is presumably involved in tumor progression or metastasis (7); and cyclin G, the function of which is not clear at present (40). Importantly, the p53 mutants found in various tumors are defective in sequence-specific transactivation (26,50).While p53 gene expression is highly inducible with several stimuli, such as serum stimulat...

Renal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 microM). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury.