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PubMed1993

[The modifications of mutagenesis and anticarcinogenesis].

The problems of anticarcinogenesis are discussed in the context of mechanistic approaches and the well-known mechanisms of multistage carcinogenesis. Various events at extra- and intracellular levels are involved in mutagenesis and carcinogenesis. The most promising candidates for suppression of chemical carcinogenesis are some nitrosation modifiers, dietary sorbents, metabolic activation modulators, reactive metabolite trappers, repair inducers, gene expression inhibitors, etc. Short-term tests for mutagenicity are more preferable for screening potential anticarcinogenic agents. The bioassays for anticarcinogenesis should be assessed for sensitivity, specificity and predictability and the genetic profiles of the antimutagenic activity of the modifiers analysed.

Animals↗

PubMed2006

Presenilin function in Caenorhabditis elegans.

The genome of the nematode Caenorhabditis elegans contains homologs of several genes associated with familial Alzheimer's disease in humans. apl-1 encodes a transmembrane protein belonging to the amyloid precursor protein family, sel-12 and hop-1 are the two somatically expressed presenilin genes that resemble PS1 and PS2 on both a structural and a functional level. Mutations in the sel-12-encoded presenilin gene cause defective Notch/lin-12 signaling and result in reduced egg-laying, caused by cell specification and cell attachment defects. spr-1, spr-3, spr-4 and spr-5 were identified as the suppressors of the egg-laying defect of presenilin/sel-12 loss of function mutants in genetic suppressor screens. The corresponding proteins are C. elegans homologs of human REST, CoREST and LSD1, respectively. REST/NSRF (Re1 silencing transcription factor/neural-restrictive silencing factor) is a transcriptional repressor that blocks the expression of neuronal genes in non-neuronal tissues in vertebrates. CoREST is a conserved histone deacetylase and demethylase-containing co-repressor complex possessing a potential chromatin-modifying activity. It is recruited to the promoter via REST-mediated DNA binding. LSD1 is a flavin-dependent demethylase of histone H3. Mutations in spr-1, spr-3, spr-4 and spr-5 genes suppress the egg-laying phenotype of sel-12 loss of function mutants by derepressing the expression of the second C. elegans presenilin gene, hop-1.

Alzheimer Disease↗

PubMed1995

Effects of retroviral-mediated tissue plasminogen activator gene transfer and expression on adherence and proliferation of canine endothelial cells seeded onto expanded polytetrafluoroethylene.

PURPOSE: Seeding prosthetic arterial grafts with genetically modified endothelial cells (ECs) has the potential to substantially improve graft function. However, preliminary applications suggest that grafts seeded with retrovirally transduced ECs yield a significantly lower percent surface coverage than those seeded with nontransduced ECs. The objective of this study was to test the hypothesis that canine ECs transduced with the human tissue plasminogen activator (tPA) gene would have a lower rate of adherence to pretreated expanded polytetrafluoroethylene (ePTFE) both in vitro and in vivo and that they would proliferate at a slower rate on pretreated ePTFE in vitro. METHODS: Early passage ECs derived from canine external jugular vein were transduced with the retroviral MFG vector containing the gene for human tPA. ECs exposed to media alone served as controls. Iodine 125-labeled ECs were seeded in vitro onto ePTFE graft segments pretreated with canine whole blood, fibronectin (50 micrograms/ml), or media alone, and the percent of ECs adherent at 1 hour were determined (n = 3). Additional tPA-transduced and -nontransduced ECs were grown for 10 days on either fibronectin (50 micrograms/ml)-pretreated ePTFE wafers or tissue culture plastic pretreated with gelatin (1%) or fibronectin (50 micrograms/ml), and the EC proliferation rates were determined (n = 3). Furthermore, 125I-labeled ECs were seeded onto fibronectin (50 micrograms/ml)-pretreated ePTFE graft segments implanted as carotid and femoral artery interposition grafts (n = 3). The grafts were harvested after 1 hour, and the percent of ECs adherent was determined. RESULTS: Human tPA was detected by immunohistochemical staining in 61% +/- 5% of the transduced ECs and was expressed at 35.4 +/- 12.9 ng/hr/10(6) cells. Fibronectin and whole blood pretreatment of the ePTFE grafts led to greater EC adherence in vitro than did media alone (90.9% +/- 5.3% vs 77.8% +/- 5.8% vs 4.7% +/- 1.1%, p < or = 0.05). No significant difference in the rates of adherence or proliferation was seen in vitro between the transduced and nontransduced ECs. No significant difference in proliferation was found for the transduced ECs on the three matrices tested in vitro. In contrast, adherence of the transduced ECs in vivo was significantly lower than that of nontransduced ECs (64.7% +/- 2.1% vs 73.7% +/- 4.1%, p < or = 0.05) 1 hour after implantation. CONCLUSIONS: Lower rates of surface endothelialization by genetically modified ECs in vivo do not appear to be due to an impaired capacity to initially adhere or proliferate on the synthetic graft but may result from decreased adherence after exposure to in vivo hemodynamic forces.

Animals↗

PubMed2006

Genome-wide isolation of resistance gene analogs in maize (Zea mays L.).

Conserved domains or motifs shared by most known resistance (R) genes have been extensively exploited to identify unknown R-gene analogs (RGAs). In an attempt to isolate all potential RGAs from the maize genome, we adopted the following three methods: modified amplified fragment length polymorphism (AFLP), modified rapid amplification of cDNA ends (RACE), and data mining. The first two methods involved PCR-based isolations of RGAs with degenerate primers designed based on the conserved NBS domain; while the third method involved mining of RGAs from the maize EST database using full-length R-gene sequences. A total of 23 and 12 RGAs were obtained from the modified AFLP and RACE methods, respectively; while, as many as 109 unigenes and 77 singletons with high homology to known R-genes were recovered via data-mining. Moreover, R-gene-like ESTs (or RGAs) identified from the data-mining method could cover all RACE-derived RGAs and nearly half AFLP-derived RGAs. Totally, the three methods resulted in 199 non-redundant RGAs. Of them, at least 186 were derived from putative expressed R-genes. RGA-tagged markers were developed for 55 unique RGAs, including 16 STS and 39 CAPS markers.

Base Sequence↗

PubMed1994

Gene transfer into hematopoietic stem cells: long-term maintenance of in vitro activated progenitors without marrow ablation.

Adoptive transfer of genetically modified somatic cells will play an increasingly important role in the management of a wide spectrum of human diseases. Among the most appealing somatic cells as potential gene transfer vehicles are hematopoietic cells, because of their wide distribution and their well-characterized capacities for proliferation, differentiation, and self-renewal. Genes can be readily transferred into short-lived and lineage-restricted hematopoietic cells, but there remains a need to develop reliable methods for gene transfer into hematopoietic stem cells in large animals. In this work, we used a gene transfer approach in which hematopoietic cells in long-term marrow cultures were exposed to the replication-defective retrovirus N2, bearing the reporter gene neo, on multiple occasions during 21 days of culture. Genetically marked cultured autologous cells were infused into 18 canine recipients in the absence of marrow-ablative conditioning. neo was detected by Southern blotting and/or the polymerase chain reaction in the marrow, blood, marrow-derived granulocyte/macrophage and erythroid progenitors, and cultured T cells in dogs after infusion. In most dogs, the proportion of long-term marrow culture cells contributing to hematopoiesis rose during the first 3 months after infusion and peaked within the first 6. The maximal levels attained were between 10% and 30% G418-resistant (neo-positive) granulocyte/macrophage progenitors. At 12 months, five dogs maintained greater than 10% G418-resistant progenitors, and for two of them this level exceeded 20%. Two dogs had greater than 5% G418-resistant hematopoietic progenitors at 24 months after infusion. Our data suggest that very primitive hematopoietic progenitors are maintained in long-term marrow cultures, where they can be triggered into entering the cell cycle. In vivo, these activated cells likely continue normal programs of proliferation, differentiation, and self-renewal. Their progeny can be maintained at clinically relevant levels for up to 2 years without the requirement that endogenous hematopoiesis be suppressed through chemo- or radiotherapy prior to adoptive transfer. Long-term marrow culture cells may thus be ideal targets for gene therapy involving adoptive transfer of transduced hematopoietic cells.

Animals↗

PubMedDate not supplied

Recloning of SV40 early gene transfected human endothelial cells repeatedly recovers subpopulations with low passage characteristics and morphologies.

Cultured human umbilical vein endothelial cells (HUVECs) are a valuable model for investigation of endothelial functions, but they enter senescence at low passage. Transfection of early passage HUVECs with the early genes of SV40 greatly extends the replicative potential of these cells, but eventually results in marked changes in growth, morphology, and biochemistry. Here we report a modified approach that appears to have overcome the problem of late passage decline after transfection. Plasmid pX-8 containing the SV40 early genes was transfected into passage four HUVECs. At passage five, these transfectants were cloned by limiting dilution and selected on the basis of both morphological and biochemical resemblance to their untransfected counterparts. Two clones that expressed factor VIII and in which the basal and the tumor necrosis factor-alpha inducible levels of interleukin 6 and endothelial adhesion molecules were normal were chosen. Vimentin and fibronectin distribution in these clones resembled untransfected cells. At passage 25, growth pattern changes were becoming evident, but recloning these late passage clones recovered numerous subclones of normal, cobblestone appearance. Two of these were further characterized and found to resemble their original parental clone by all of the biochemical criteria listed above. These subclones appeared to transform more rapidly than the parental clone, but repeated subcloning again rescued clones with normal morphologies and normal biochemical characteristics. We conclude that periodic recloning may indefinitely perpetuate lines that are useful equivalents of their original counterparts.

Cell Adhesion Molecules↗

PubMed1999

[Food additives and genetically modified food--a risk for allergic patients?].

Adverse reactions to food and food additives must be classified according to pathogenic criteria. It is necessary to strictly differentiate between an allergy, triggered by a substance-specific immunological mechanism, and an intolerance, in which no specific immune reaction can be established. In contrast to views expressed in the media, by laymen and patients, adverse reactions to additives are less frequent than is believed. Due to frequently "alternative" methods of examination, an allergy to food additives is often wrongly blamed as the cause of a wide variety of symptoms and illness. Diagnosing an allergy or intolerance to additives normally involves carrying out double-blind, placebo-controlled oral provocation tests with food additives. Allergic reactions to food additives occur particularly against additives which are organic in origin. In principle, it is possible that during the manufacture of genetically modified plants and food, proteins are transferred which potentially create allergies. However, legislation exists both in the USA (Federal Drug Administration, FDA) and in Switzerland (Ordinance on the approval process for GM food, GM food additives and GM accessory agents for processing) which require a careful analysis before a genetically modified product is launched, particularly where foreign genes are introduced. Products containing genetically modified organisms (GMO) as additives must be declared. In addition, the source of the foreign protein must be identified. The "Round-up ready" (RR) soya flour introduced in Switzerland is no different from natural soya flour in terms of its allergenic potential. Genetically modified food can be a blessing for allergic individuals if gene technology were to succeed in removing the allergen (e.g. such possibilities exist for rice). The same caution shown towards genetically modified food might also be advisable for foreign food in our diet. Luckily, the immune system of the digestive tract in healthy people tolerates foreign antigens. Food allergies in adults occur mainly among those allergic to pollen.

Adult↗

PubMed2006

Concise review: epigenetic mechanisms contribute to pluripotency and cell lineage determination of embryonic stem cells.

Epigenetic mechanisms, such as histone modifications and DNA methylation, have been shown to play a key role in the regulation of gene transcription. Results of recent studies indicate that a novel "bivalent" chromatin structure marks key developmental genes in embryonic stem cells (ESCs), wherein a number of untranscribed lineage-control genes, such as Sox1, Nkx2-2, Msx1, Irx3, and Pax3, are epigenetically modified with a unique combination of activating and repressive histone modifications that prime them for potential activation (or repression) upon cell lineage induction and differentiation. However, results of these studies also showed that a subset of lineage-control genes, such as Myf5 and Mash1, were not marked by these histone modifications, suggesting that distinct epigenetic mechanisms might exist for lineage-control genes in ESCs. In this review article, we summarize evidence regarding possible mechanisms that control these unique histone modifications at lineage-control gene loci in ESCs and consider their possible contribution to ESC pluripotency. In addition, we propose a novel "histone modification pulsing" model wherein individual pluripotent stem cells within the inner cell mass of blastocysts undergo transient asynchronous histone modifications at these developmental gene loci, thereby conferring differential responsiveness to environmental cues and morphogenic gradients important for cell lineage determination. Finally, we consider how these rapid histone modification exchanges become progressively more stable as ESCs undergo differentiation and maturation into specialized cell lineages.

Animals↗

PubMed2005

High susceptibility of human c-Ha-ras proto-oncogene transgenic rats to carcinogenesis: a cancer-prone animal model.

Transgenic animals carrying human c-Ha-ras proto-oncogene, v-Ha-ras transgenic mice, pim-1 transgenic mice and several knockout mice deficient of tumor suppressor genes, such as p53, have been shown to exhibit increased carcinogen susceptibility. As a result, studies into practical application and medium-term screening of environmental carcinogens are under way. Given the advantages of rat models characterized by larger organ size, abundant information regarding preneoplasias and virus-free constitution, we have concentrated on the generation of transgenic rats bearing copies of the human c-Ha-ras proto-oncogene and shown the Hras128 strain to be extremely sensitive to the induction of mammary carcinomas, and to a lesser extent, lesions in the urinary bladder, esophagus and skin. In most, if not all, the mammary cancers mutations of the transgene but not the endogenous H-ras gene are present, appearing to occur early in the process of tumorigenesis, which involves proliferation of cells in TEB and intraductal hyperplasia before carcinomas arise. Preliminary findings suggest that this is independent of endogenous ovarian hormones, although inhibited by soy isoflavones and promoted by atrazine and nonylphenols. Although further studies of the mechanisms are clearly necessary, the model appears to have great potential for screening purposes, not only for modifiers active in the breast, but also other organs where tumors characterized by ras gene mutations develop.

Animals↗

PubMed2001

Gene therapy in vascular medicine: recent advances and future perspectives.

Gene therapy is emerging as a potential strategy for the treatment of cardiovascular diseases, such as restenosis after angioplasty, vascular bypass graft occlusion, and transplant coronary vasculopathy, for which no known effective therapy exists. The first human trial in cardiovascular disease was started in 1994 to treat peripheral vascular disease using vascular endothelial growth factor. In addition, therapeutic angiogenesis using the vascular endothelial growth factor gene was applied in the treatment of ischemic heart disease. The results from these clinical trials seem to exceed expectation. Improvement of clinical symptoms in peripheral arterial disease and ischemic heart disease has been reported. At least five different potent angiogenic growth factors have been tested in clinical trials to treat peripheral arterial disease or ischemic heart disease. In addition, another strategy for combating disease processes, to target the transcriptional process, has been tested in a human trial. Transfection of cis-element double-stranded oligodeoxynucleotides is an especially powerful tool in a new class of antigen strategies for gene therapy. Transfection of double-stranded oligodeoxynucleotides corresponding to the cis sequence will result in the attenuation of the authentic cis-trans interaction, leading to the removal of trans-factors from the endogenous cis-elements, with subsequent modulation of gene expression. Genetically modified vein grafts transfected with a decoy against E2F, an essential transcription factor in cell cycle progression, revealed apparent long-term potency in human patients. This review focuses on the future potential of gene therapy for the treatment of cardiovascular disease.

Angioplasty↗

PubMed2025

EP300-mediated lactylation leads to ulcerative colitis via CD86-positive plasmacytoid dendritic cells: A Mendelian randomization and mediation analysis.

This study explores the potential mechanism between lactylation and ulcerative colitis (UC) using two-sample Mendelian randomization and multi-omics analysis. This study employed expression quantitative trait loci and protein quantitative trait loci as exposures, with UC from the Finnish database as the outcome, to conduct Mendelian randomization analysis on lactylation-related target genes, aiming to investigate the causal relationships between these exposures and the outcome. Sensitivity and pleiotropy tests, combined with colocalization analysis, are performed to identify the best target genes and ensure the robustness of the results. Finally, immune cells are included for mediation analysis between lactylation and UC to explore potential mechanisms of action. Through Mendelian randomization analysis combined with sensitivity and pleiotropy tests, 2 lactylation target genes were found to have a significant causal relationship with UC. Subsequent colocalization analysis confirmed EP300 as a potential gene target. After including immune cells in the mediation analysis, it was discovered that there is a potential mechanism involving EP300, CD86+ plasmacytoid dendritic cells (pDCs), and UC. There is a significant causal relationship between lactylation and UC. Furthermore, the lactylation-modified gene EP300 may lead to UC occurrence by regulating CD86+ pDCs.

Humans↗

PubMed2003

Long-lasting gene expression by particle-mediated intramuscular transfection modified with bupivacaine: combinatorial gene therapy with IL-12 and IL-18 cDNA against rat sarcoma at a distant site.

The immune response is modulated by genetic adjuvants using plasmid vectors expressing cytokines. Skeletal muscle can express a foreign gene intramuscularly administered via a needle injection, and the potential of muscle as a target tissue for somatic gene therapy in treating cancer has been explored. In the present study, we investigated the efficacy of particle-mediated intramuscular transfection modified with a local anesthetic agent, bupivacaine, on luciferase and green fluorescent protein. The results indicate that these proteins are more efficiently expressed and persist longer in muscle modified in this way compared with the needle-injection method. Using an established rat sarcoma model, particle-mediated intramuscular gene-gun therapy with a combination of IL-12 and IL-18 cDNA was conducted. Growth of the distant sarcoma was significantly inhibited by particle-mediated intramuscular combination gene therapy, and the survival rate was also improved. Furthermore, the combination gene-gun therapy maintained significant levels of interferon-gamma and induced a high activity of tumor-specific cytotoxic T lymphocytes. These results suggest that the sustained local delivery of IL-12 and IL-18 cDNA using intramuscular gene-gun therapy modified with bupivacaine can induce long-term antitumor immunity, and can provide the great advantage of inhibiting the disseminated tumor.

Anesthetics, Local↗

PubMed1999

Present and potential future use of gene therapy for the treatment of non-insulin dependent diabetes mellitus (Review).

This review describes the latest approaches towards using gene therapy as a treatment for non-insulin dependent diabetes mellitus (NIDDM; Type 2 diabetes). We examine attempts to directly deliver the insulin gene to non-beta-cells, to improve insulin secretion from existing beta-cells and to develop ex vivo approaches to implanting genetically modified cells. Future research into the pathology of non-insulin dependent diabetes, combined with the latest developments in gene delivery systems, may potentially make gene therapy an attractive alternative NIDDM treatment in the future.

Adult↗

PubMed2000

Genetic regulation of anti-erythrocyte autoantibodies and splenomegaly in autoimmune hemolytic anemia-prone new zealand black mice.

New Zealand Black (NZB) mice spontaneously produce anti-erythrocyte autoantibodies (AEA) in association with splenomegaly, thus serving as a model for autoimmune hemolytic anemia. Although these autoimmune traits are inherited as a dominant fashion, expression in F(1) hybrids of NZB and most non-New Zealand strains is suppressed due to the contribution of wild-type modifying genes present in the latter strains. Using chromosomal microsatellite markers in the (C57BL/6 x NZB)F(1) x NZB backcross progeny, we mapped C57BL/6 modifying loci for AEA production and splenomegaly. Generation of AEA was found to be down-regulated by a combined effect of two major independently segregating dominant alleles-one linked to D7MIT30 on chromosome 7 and the other linked to D10MIT42 on chromosome 10. Splenomegaly was modified mainly by a single C57BL/6 allele linked to D4MIT58 on chromosome 4. Thus, the autoimmune hemolytic anemia in the NZB strain is under multigenic control and a combined action of not only susceptibility but also modifying alleles with suppressive activities affects the outcome of disease features in the progeny. There are potentially important candidate genes which may be linked to the regulation of AEA and splenomegaly.

Anemia, Hemolytic, Autoimmune↗

PubMed1997

LTW4 protein on mouse chromosome 1 is a member of a family of antioxidant proteins.

Based on its map position, polymorphism pattern, and expression in the kidney, the gene encoding liver 20,000-30,000 MW protein 4 (LTW4) can be considered a potential candidate for the Jckm2 modifying locus, which mediates the severity of polycystic kidney disease in the juvenile cystic kidney mouse. Using two-dimensional gel electrophoresis, we identified variants of a 26-kDa polypeptide that differed in their isoelectric points between the C57BL/6J and the DBA/2J inbred strains in a pattern similar to that originally described for LTW4 protein. N-terminal amino acid sequence was obtained by microsequencing analysis, and full-length clones were obtained by RT-PCR amplification and characterized. The map position of the cloned gene was determined and corresponded to that previously described for Ltw4. The gene has homology to a class of proteins characterized as thiol-specific antioxidants that are protective against damage caused by oxidative stress. The murine MER5 gene is also a member of this gene family and has recently been renamed Antioxidant protein 1 (Aop1), based on its functional characterization. We therefore propose that the gene encoding LTW4 be called Aop2.

Amino Acid Sequence↗

PubMed2005

Fine-tuning in Ca2+ homeostasis underlies progression of cardiomyopathy in myocytes derived from genetically modified embryonic stem cells.

Mutations of genes encoding contractile proteins are responsible for familial hypertrophic cardiomyopathies. Understanding the process of differentiation of cardiomyocytes carrying a mutated protein is a crucial step towards potential treatments of inherited cardiac disorders. Embryonic Stem (ES) cells which faithfully recapitulate in vitro the process of cardiac cell differentiation can be genetically modified to incorporate a mutation mimicking a cardiomyopathy. ES cell lines engineered to express a wild-type (MLC2vGFP) or a mutated form (R58QMLC2vGFP) of ventricular myosin light chain 2 (MLC2v) fused to GFP were differentiated into cardiomyocytes within embryoid bodies (EBs). Visualization of GFP combined with sarcomeric actinin immunofluorescence of EBs revealed that mutated MLC2v dramatically prevented myofibrillogenesis. Cardiomyocytes expressing wild-type MLC2v featured spontaneous Ca(2+) spiking, but not those harboring the mutation. Expression of cardiac transcription factors Mef2c, GATAs, myocardin and Nkx2.5 was not affected by cell expression of mutated MLC2v. A dramatic decrease in expression of mRNAs encoding alpha-actin, MLC2a and MLC2v was observed in R58QMLC2vGFP EBs. This event was attributed to a failure of Mef2c to translocate into the nucleus, a Ca(2+)-dependent process. Expression in mutated cells of a constitutively active Ca(2+)- and calmodulin-dependent kinase II or treating EBs with ionomycin fully restored translocation of Mef2c into the nucleus and expression of mRNAs encoding sarcomeric proteins partially rescued contractile activity of EBs. Alteration of Ca(2+) homeostasis in mutated cardioblasts affects the transcriptional program of cardiac cell differentiation leading to a defect in myofibrillogenesis, and, in turn, in contractility. Genetically modified ES cells provide a unique cell model to determine abnormalities in Ca(2+) homeostasis underlying progression of human cardiomyopathies.

Calcium↗

PubMed1999

Expression of mutated hepatitis B virus X genes in human hepatocellular carcinomas.

To explore the role of hepatitis B virus (HBV) X protein in liver carcinogenesis, independently from its role in viral replication, we have analyzed X gene structure and expression in tumorous and non-tumorous tissues obtained from 9 hepatitis B surface antigen (HBsAg)-negative, HBV DNA-positive patients. HBV replication was undetectable in tumorous tissues. HBV X gene was truncated at its 3' end in 5 of 9 tumorous tissues and 1 of 8 non-tumorous livers. Sequence analysis performed on uninterrupted X genes from 3 tumors and 3 surrounding non-tumorous tissues showed a high rate of mutations, selectively in the tumorous livers. In 1 of the 3 tumors, a frameshift mutation induced a new stop at codon 129. HBV RNAs were tested by reverse transcriptase-polymerase chain reaction (RT-PCR) with surface (S), core (C) and X specific primers. X, but not S and C, RNA expression was found in 6 of 8 tumors and in 6 of 7 non-tumorous tissues. This finding was consistent with immunohistochemical detection of X, but not S and C, antigens in all tumors also expressing X RNA. Our results provide evidence for selective expression of HBV X, but not S and C, RNA and protein in the tumorous and non-tumorous tissue of HBsAg-negative, HBV DNA-positive patients. It also shows that the structure of the X gene is modified (interrupted or highly mutated) in the majority of tumorous livers. Taken together, our findings are consistent with a potential role of mutated X proteins in HBV-related liver oncogenesis.

Adolescent↗

PubMed2004

Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (i) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.

Adaptor Proteins, Signal Transducing↗