Effect of O6-benzylguanine on nitrogen mustard-induced toxicity, apoptosis, and mutagenicity in Chinese hamster ovary cells.

O6-Benzylguanine (BG) inactivates O6-alkylguanine-DNA alkyltransferase (AGT), resulting in an increase in the sensitivity of cells to the toxic effects of O6-alkylating agents. BG significantly enhances the cytotoxicity and decreases the mutagenicity of nitrogen mustards [i.e., phosphoramide mustard (PM), melphalan, and chlorambucil], a group of alkylating agents not known to produce O6-adducts in DNA. The enhancement is observed in cells irrespective of AGT activity. Exposure of Chinese hamster ovary cells to 100 microM BG results in enhancement in the cytotoxicity of PM (300 microM), chlorambucil (40 microM), and melphalan (10 microM) by 9-, 7-, and 18-fold, respectively. In contrast, mutation frequency after treatment with 300 microM PM is decreased from 259 mutants/10(6) cells to 22 mutants/10(6) cells when cells are pretreated with BG. The enhancement of toxicity of these bis-alkylating agents appears to involve cross-link formation, because neither cytotoxicity nor mutagenicity of a monoalkylating PM analogue is significantly altered when combined with BG. Enhanced cytotoxicity and decreased mutagenicity is concomitant with a dramatic increase in the number of cells undergoing apoptosis when BG is combined with PM, melphalan, or chlorambucil at 72-94 h after treatment. Cell cycle analysis demonstrates that BG alone or combined with nitrogen mustards arrests cells in G1 phase of the cell cycle. At 16 h after treatment, 11 and 57% of cells treated with PM alone or with BG plus PM are in G1 phase, respectively. Our data suggest that treatment with BG causes G1 arrest and drives noncycling cells treated with nitrogen mustards into apoptosis, thus protecting against mutagenic DNA damage introduced by nitrogen mustards.

Convergence of redox-sensitive and mitogen-activated protein kinase signaling pathways in tumor necrosis factor-alpha-mediated monocyte chemoattractant protein-1 induction in vascular smooth muscle cells.

Monocyte chemoattractant protein-1 (MCP-1) is an important component of the inflammatory response of the vessel wall and has been shown to be regulated by cytokines, such as tumor necrosis factor-alpha (TNF-alpha). However, the precise signaling pathways leading to MCP-1 induction have not been fully elucidated in vascular smooth muscle cells (VSMCs). Cytokine signal transduction involves protein kinases as well as reactive oxygen species (ROS). The relation between these 2 factors is not clear. In this study, we show that TNF-alpha induces a parallel phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs. Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of MCP-1 induction (43+/-10% inhibition). Incubation of VSMCs with multiple antioxidants (diphenylene iodonium, liposomal superoxide dismutase, catalase, N-acetylcysteine, dimethylthiourea, and pyrrolidine dithiocarbamate) had no effect on TNF-alpha-mediated MCP-1 upregulation. However, simultaneous blockade of the ERK1/2 and ROS pathways by using PD098059 combined with diphenylene iodonium or N-acetylcysteine potently enhanced the ability of MAPK kinase inhibitors to abrogate MCP-1 mRNA expression (100+/-2% inhibition). Thus, parallel ROS-dependent and ERK1/2-dependent pathways converge to regulate TNF-alpha-induced MCP-1 gene expression in VSMCs. These data unmask a complex but organized integration of ROS and protein kinases that mediates cytokine-induced vascular inflammatory gene expression.

Cell transformation by the superoxide-generating oxidase Mox1.

Reactive oxygen species (ROS) generated in some non-phagocytic cells are implicated in mitogenic signalling and cancer. Many cancer cells show increased production of ROS, and normal cells exposed to hydrogen peroxide or superoxide show increased proliferation and express growth-related genes. ROS are generated in response to growth factors, and may affect cell growth, for example in vascular smooth-muscle cells. Increased ROS in Ras-transformed fibroblasts correlates with increased mitogenic rate. Here we describe the cloning of mox1, which encodes a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase of phagocytes, gp91phox. mox1 messenger RNA is expressed in colon, prostate, uterus and vascular smooth muscle, but not in peripheral blood leukocytes. In smooth-muscle cells, platelet-derived growth factor induces mox1 mRNA production, while antisense mox1 mRNA decreases superoxide generation and serum-stimulated growth. Overexpression of mox1 in NIH3T3 cells increases superoxide generation and cell growth. Cells expressing mox1 have a transformed appearance, show anchorage-independent growth and produce tumours in athymic mice. These data link ROS production by Mox1 to growth control in non-phagocytic cells.

Successful acyclovir treatment of herpes simplex type 2 hepatitis in a patient with systemic lupus erythematosus: a case report and meta analysis.

Herpes simplex virus (HSV) hepatitis is a rare complication of HSV infection with a high reported mortality rate in untreated patients. The authors present a case of HSV hepatitis in a 26-year-old female with focal proliferative lupus nephropathy who was status post one cycle of pulse high-dose (1 gm/ m2) cyclophosphamide. Treatment with parenteral acyclovir was successful. A meta analysis of well-documented cases of HSV hepatitis treated with acyclovir, excluding those that omit initial serum concentrations of hepatic transaminases, suggests that the early administration of parenteral acyclovir may have been instrumental in the achievement of a successful outcome, and that a patient's serum levels of hepatic transaminases at the time of treatment initiation may predict outcome. This is the first reported case of successful parenteral acyclovir treatment of HSV hepatitis in a patient with lupus nephritis who has recently undergone cyclophosphamide immunosuppression, and includes a meta analysis to examine the hypothesis that initial markers of hepatic injury may predict outcome of acyclovir treatment.

OXBOX and REBOX, overlapping promoter elements of the mitochondrial F0F1-ATP synthase beta subunit gene. OXBOX/REBOX in the ATPsyn beta promoter.

Three positive (PR1-3) and one negative (NR1) transcriptional control domain have been tentatively mapped in the promoter of the human F0F1-ATP synthase beta subunit gene (ATPsyn beta) in the context of expression in myogenic cells. Lipofection of promoter-chloramphenicol acetyltransferase fusion constructs into C2C12 myogenic cells revealed that two of the three positive domains (PR1 and PR2) function in both myoblasts and myotubes, whereas the third positive domain (PR3) and the sole negative domain (NR1) seem to function only in myotubes. PR1 contains a cluster of four CCAAT cis-elements, PR2 is a small 44-base pair region containing an SP1-like motif, and PR3 is a region previously shown to be recognized by both OXBOX- and REBOX-binding factors. By site-directed polymerase chain reaction linker mutations, the activity of the OXBOX/REBOX cis-element in myoblasts is shown to be masked by flanking sequences in PR3. The negative domain, NR1, is located between 300 and 1,000 base pairs upstream from the OXBOX/REBOX elements in a region containing multiple Alu repeats. Mobility gel shift analysis of DNA-protein complexes using competitor DNAs verified the involvement of both OXBOX- and REBOX-binding factors in PR3. Similar experiments show SP1-specific binding at PR2. These data with observations of OXBOX and REBOX-specific binding of an OXBOX/REBOX-like region within the conserved sequence block C of the human mitochondrial DNA D-loop sequence are consistent with the idea that OXBOX- and REBOX DNA-binding factors coordinate the expression of mitochondrial energy genes in highly oxidative tissues by working with well characterized general transcription factors such as SP1 and CCAAT DNA-binding proteins, which exist in the nucleus, and MTF, which exists in the mitochondrion.

Genetic mapping of human heart-skeletal muscle adenine nucleotide translocator and its relationship to the facioscapulohumeral muscular dystrophy locus.

The mitochondrial heart-skeletal muscle adenine nucleotide translocator (ANT1) was regionally mapped to 4q35-qter using somatic cell hybrids containing deleted chromosome 4. The regional location was further refined through family studies using ANT1 intron and promoter nucleotide polymorphisms recognized by the restriction endonucleases MboII, NdeI, and HaeIII. Two alleles were found, each at a frequency of 0.5. The ANT1 locus was found to be closely linked to D4S139, D4S171, and the dominant skeletal muscle disease locus facioscapulohumeral muscular dystrophy (FSHD). A crossover that separated D4S171 and ANT1 from D4S139 was found. Since previous studies have established the chromosome 4 map order as centromere-D4S171-D4S139-FSHD, it was concluded that ANT1 is located on the side of D4S139, that is opposite from FSHD. This conclusion was confirmed by sequencing the exons and analyzing the transcripts of ANT1 from several FSHD patients and finding no evidence of aberration.

Transcriptional control of nuclear genes for the mitochondrial muscle ADP/ATP translocator and the ATP synthase beta subunit. Multiple factors interact with the OXBOX/REBOX promoter sequences.

The OXBOX promoter regions of the genes for the muscle-specific adenine nucleotide translocator (ANT1) and the beta subunit of the ATPsynthase (ATPsyn beta) have been implicated in the increased transcription of these nuclear-encoded oxidative phosphorylation (OXPHOS) genes in heart and skeletal muscle. DNA binding, electrophoretic mobility shift (gel-shift) assays now reveal that the OXBOX region has two unique but overlapping elements, the 13-base pair (bp) OXBOX and an 8-bp REBOX. The OXBOX binding factors are found only in myogenic cell lines, whereas the REBOX factors are ubiquitous. Methylation interference experiments have defined the boundaries of the OXBOX and REBOX elements, confirmed that the OXBOX factors are muscle-specific, and shown that the OXBOX and REBOX factors do not bind concurrently. The binding of the REBOX factors was found to be sensitive to NADH and thyroxine, suggesting that it may modulate OXPHOS gene expression in response to environmental and hormonal changes. Hence, the OXBOX/REBOX complex provides one mechanism by which mammalian energy metabolism can be adapted to developmental and environmental demands.

Differential expression of adenine nucleotide translocator isoforms in mammalian tissues and during muscle cell differentiation.

The adenine nucleotide translocator (ANT) catalyzes the exchange of ADP and ATP across the mitochondrial internal membrane. Its three isoforms, ANT1, ANT2, and ANT3 are coded by differentially regulated nuclear genes. The patterns of expression of these genes in human, bovine, and mouse tissue are similar. ANT1 is highly expressed in heart and skeletal muscle and is induced during myoblast differentiation. It is coordinately regulated with the nuclear gene for the mitochondrial ATP synthase beta subunit, with which it shares the positive muscle cis element, the OXBOX. ANT2 is either absent or weakly expressed in all tissues. ANT3 is ubiquitously expressed in all tissues, and its transcript level is proportional to the level of oxidative metabolism. The tissue-specific expression of the ANT gene family thus provides insight into the molecular basis of the differential reliance of mammalian tissues on oxidative phosphorylation.