ABSTRACT
O6-methylguanine produced in DNA by the action of simple alkylating agents, such as N-methyl-N-nitrosourea (MNU), causes base-mispairing during DNA replication, thus leading to mutations and cancer. To prevent such outcomes, the cells carrying O6-methylguanine undergo apoptosis in a mismatch repair protein-dependent manner. We previously identified MAPO1 as one of the components required for the induction of apoptosis triggered by O6-methylguanine. MAPO1, also known as FNIP2 and FNIPL, forms a complex with AMP-activated protein kinase (AMPK) and folliculin (FLCN), which is encoded by the BHD tumor suppressor gene. We describe here the involvement of the AMPK-MAPO1-FLCN complex in the signaling pathway of apoptosis induced by O6-methylguanine. By the introduction of siRNAs specific for these genes, the transition of cells to a population with sub-G1 DNA content following MNU treatment was significantly suppressed. After MNU exposure, phosphorylation of AMPKα occurred in an MLH1-dependent manner, and this activation of AMPK was not observed in cells in which the expression of either the Mapo1 or the Flcn gene was downregulated. When cells were treated with AICA-ribose (AICAR), a specific activator of AMPK, activation of AMPK was also observed in a MAPO1- and FLCN-dependent manner, thus leading to cell death which was accompanied by the depolarization of the mitochondrial membrane, a hallmark of the apoptosis induction. It is therefore likely that MAPO1, in its association with FLCN, may regulate the activation of AMPK to control the induction of apoptosis triggered by O6-methylguanine.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Apoptosis Regulatory Proteins/metabolism , Apoptosis , Base Pair Mismatch , Carrier Proteins/metabolism , DNA/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Proteins/metabolism , AMP-Activated Protein Kinases/antagonists & inhibitors , Alkylation/drug effects , Animals , Apoptosis/drug effects , Base Pair Mismatch/drug effects , Cell Line , Enzyme Activation/drug effects , Gene Knockdown Techniques , Methylnitrosourea , Mice , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/metabolismABSTRACT
To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-alpha and IFN-gamma or stimulation with LPS demonstrated a time- and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and +128 bp was found to mediate IFN-gamma, TNF-alpha, and LPS responsiveness as well as the synergistic effect of IFN-gamma/TNF-alpha on hBf promoter activity. Site-directed mutagenesis of a IFN-gamma-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-gamma responsiveness. Mutagenesis of a nuclear factor (NF)-kappaB cis element at -466 to -456 bp abrogated TNF-alpha and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-gamma, TNF-alpha, and LPS is through GAS and NF-kappaB cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway.
Subject(s)
Complement Factor B/genetics , Macrophages/immunology , Monocytes/immunology , Protein C/metabolism , Sepsis/immunology , Sepsis/physiopathology , Base Sequence , Complement Factor B/immunology , Complement Factor B/metabolism , Complement Pathway, Alternative/immunology , Humans , Interferon-gamma/immunology , Interferon-gamma/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Molecular Sequence Data , Monocytes/metabolism , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/immunology , Protein C/immunology , RNA, Messenger/metabolism , Sepsis/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/immunology , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Heparin is used as an interdialytic locking solution for hemodialysis (HD) central venous catheters (CVCs). The purpose of this study was to compare effectiveness of two heparin concentrations (10,000 and 1,000 U/mL) in preventing catheter malfunction. We compared two time periods: a 6-month period with heparin 10,000 U/mL and a 3-month period with heparin 1,000 U/mL. Adults on HD using a CVC (tunneled or untunneled) in Calgary, Alberta, were included. The primary outcome was catheter malfunction. A total of 139 and 134 patients in the heparin 10,000 and 1,000 U/mL periods, respectively, were included. The crude rate of catheter malfunction, per 1,000 HD sessions, was similar for heparin 10,000 (7.6; 95% CI, 5.3 to 10.8) and 1,000 (6.7; 95% CI, 4.3 to 10.3) U/mL periods, respectively (p = 0.76). After adjusting for CVC characteristics and use of recombinant tissue plasminogen activator (rt-PA), there was no association between heparin concentration and CVC malfunction (hazard ratio, 0.77; 95% CI, 0.37 to 1.61). In conclusion, the use of a lower concentration of heparin was not associated with an increased risk of catheter malfunction but may be associated with greater rt-PA use. The association between heparin concentration and rt-PA use requires further study.
