Detection of human somatic cell structural gene mutations by two‐dimensional electrophoresis

Hanash, Samir M.; Chu, Ernest H. Y.; Kuick, Rork; Skolnick, M.; Neel, James V.; Strahler, John R.; Pivirotto, S.; Niezgoda, W.

doi:10.1002/prot.340020103

Cited by 9 publications

(3 citation statements)

References 10 publications

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“…Cells to be studied by 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE) were thoroughly washed to remove serum proteins, then used immediately or radioiodinated with I2'I using the lactoperoxidase technique (26). First and second dimensions were run as described (27), and spots analyzed for position and intensity on a Masscomp image analyzer computer system (BioImage, Ann Arbor, MI) using a 1,024 x 1,024-pixel format (160 plpixel) (28). To detect radioiodinated polypeptides, the gels were dried and sealed in light-tight cassettes with x-ray film (XAR-5; Eastman-Kodak, Rochester, NY) and Cronex enhancing screens (DuPont, Wilmington, DE).…”

Section: Methodsmentioning

confidence: 99%

Phenotypic and functional similarities between 5‐azacytidine‐treated t cells and a t cell subset in patients with active systemic lupus erythematosus

Richardson

Strahler

Pivirotto

et al. 1992

Arthritis & Rheumatism

164

159

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Objective. Antigen-specific CD4+ T cells treated with DNA methylation inhibitors become autoreactive, suggesting a novel mechanism for autoimmunity. To test whether this mechanism might be involved in systemic lupus erythematosus (SLE), phenotypic markers for the autoreactive cells were sought.Methods. Cloned normal T cells were treated with the DNA methylation inhibitor 5-azacytidine (5-azaC) and studied for altered gene expression. T cells from patients with active SLE were then studied for a similar change in gene expression, and cells expressing the marker were tested for autoreactivity.Results. 5-azaC-treated normal T cells had increased CDlla (leukocyte function-associated antigen la) expression relative to other membrane molecules. A T cell subset with similar CDlla expression was found in patients with active SLE. This subset contained cells that spontaneously lysed autologous macrophages, with a specificity similar to that of 5-azaC-treated cells. Conclusion. The model of 5-azaC-induced autoreactivity may have relevance to SLE.Antigen-specific CD4+ T cell clones respond to autologous macrophages (Mgi) without antigen, following treatment with DNA methylation inhibitors such as 5-azacytidine (5-azaC). The response is inhibited by monoclonal antibodies (MAb) against CD3 and class I1 major histocompatibility complex (MHC) determinants, and occurs with autologous but not allogeneic Mgi (1,2), suggesting that the cells are responding to autologous class I1 MHC determinants via a pathway involving the antigedMHC receptor. Similar concentrations of 5-azaC alter gene expression in a wide range of cell types, including T cells (3-3, and the autoreactivity may be caused by changes in the expression of as-yet-unidentified genes involved in T cell activation.5-azaC-induced autoreactivity may have relevance to autoimmunity. Recent experiments have shown that murine T cells can become autoreactive following 5-azaC treatment, responding to and, intriguingly, lysing, syngeneic Mgi. Adoptive transfer of murine 5-azaC-treated cells induces an immune complex glomerulonephritis and anti-DNA antibodies in nonirradiated syngeneic mice (6). These findings suggest a novel model for systemic lupus erythematosus (SLE), in which T cell DNA hypomethylation could alter expression of crucial genes, resulting in T cell autoreactivity, and the autoreactive T cells could then produce a lupus-like illness. Reports that lupusinducing drugs inhibit T cell DNA methylation and induce autoreactivity (7) support this concept. Moreover, recent evidence indicates that T cell DNA methylation is impaired in patients with active SLE (8).

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Section: Methodsmentioning

confidence: 99%

Phenotypic and functional similarities between 5‐azacytidine‐treated t cells and a t cell subset in patients with active systemic lupus erythematosus

Richardson

Strahler

Pivirotto

et al. 1992

Arthritis & Rheumatism

164

159

View full text Add to dashboard Cite

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“…The molecular changes in BAs include gene amplification of proto‐oncogenes such as K‐ras , ErbB‐2 , EGFR , β ‐catenin , Cyclin D and c‐Met and inactivation of tumor‐suppressor genes such as p53 , p16 , p21 , p27 , APC and DCC . Protein studies of clinical tumor samples have led to the identification of cancer‐specific protein markers that provide the basis for developing new diagnostic and prognostic approaches 21–23. Over the past decade, advances in mass spectrometry (MS) and bioinformatics have improved our ability to discriminate cancer‐specific peptides.…”

Section: Introductionmentioning

confidence: 99%

Alterations in Barrett's‐related adenocarcinomas: A proteomic approach

Peng

Sheta²,

Powell

et al. 2007

Intl Journal of Cancer

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In this study, we applied high-resolution, two-dimensional, gel electrophoresis and matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry analysis (MALDI TOF MS) to identify novel proteins that are involved in Barrett's tumorigenesis. We analyzed 12 primary tissue samples that included 8 Barrett's-related adenocarcinomas (BA) and 4 normal mucosae samples. Twenty-three spots were consistently altered (≥2-fold) in at least half of the tumors when compared with all normal samples and thus subjected to further analysis. The MALDI TOF MS analysis demonstrated biologically interesting upregulated proteins such as ErbB3, Dr5 and Cyclin D1 as well as several members of the zinc finger proteins (Znf146, Znf212 and Znf363). Examples of downregulated proteins included Lgi1 and Klf6. We selected four proteins (ErbB3, Dr5, Znf146 and Lgi1) that are novel for BAs for validation using quantitative real-time reverse-transcription PCR on 39 BA tissue samples when compared with normal samples. We demonstrated mRNA upregulation of ERBB3 (51.3%), DR5 (41%) and ZNF146 (30.7%) and downregulation of LGI1 (100%) in BA. We have further validated the protein overexpression of ErbB3, Dr5 and Znf146, using immunohistochemical (IHC) analysis on a tissue microarray that contained 75 BAs and normal gastric and esophageal mucosae samples. BA tissue samples demonstrated overexpression of ErbB3 (42%), Dr5 (90%) and Znf146 (30%) when compared with normal tissues. In conclusion, we have identified and validated several novel proteins that are involved in Barrett's carcinogenesis. ' 2007 Wiley-Liss, Inc.

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“…N-Ethyl-N-nitrosourea has been shown to be a very potent mutagen in the mouse specific-locus test, leading to its use as a model chemical mutagen (3). To determine the usefulness and limitations of two-dimensional PAGE for mutation studies, we have used a human diploid lymphoblastoid cell line (TK-6) mutagenized with N-ethyl-N-nitrosourea (4). Evidence is presented for the nonrandom distribution of structural gene mutations among the 263 scored loci encoding proteins.…”

mentioning

confidence: 99%

Nonrandom distribution of structural mutants in ethylnitrosourea-treated cultured human lymphoblastoid cells.

Hanash

Boehnke

Chu

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Two-dimensional polyacrylamide gel electrophoresis has been used to detect somatic cell gene mutations altering protein structure, following ethylnitrosourea treatment of cultured human lymphoblastoid cells. A total of 267 polypeptides encoded by 263 loci were scored in a series of 1143 lymphoblastoid clones. Sixty-five electrophoretic mutants were detected at a total of 49 loci. Sixteen of the 65 mutations were phenotypically repeat mutations, occurring at 11 loci. Furthermore, structural mutations occurred more frequently at loci known to be polymorphic. These results provide evidence that the mutations that are detectable at the protein level by two-dimensional polyacrylamide gel electrophoresis do not occur at random and that their frequency is greater among polymorphic loci.The question of how random mutational events are distributed throughout the genome of higher eukaryotes is not only of broad evolutionary interest but also has practical relevance in experimental mammals and humans; also of broad interest is the question of what are the potential errors in extrapolating from mutation rates at a relatively few loci to the entire genome? Because two-dimensional polyacrylamide gel electrophoresis permits the clear visualization of hundreds of cellular polypeptides (1), to monitor human populations for changing mutation rates, we have been exploring the potential of two-dimensional PAGE coupled with silver staining for polypeptide localization (2). N-Ethyl-N-nitrosourea has been shown to be a very potent mutagen in the mouse specific-locus test, leading to its use as a model chemical mutagen (3). To determine the usefulness and limitations of two-dimensional PAGE for mutation studies, we have used a human diploid lymphoblastoid cell line (TK-6) mutagenized with N-ethyl-N-nitrosourea (4). Evidence is presented for the nonrandom distribution of structural gene mutations among the 263 scored loci encoding proteins. MATERIALS AND METHODSExperimental Design. The human lymphoblastoid cell line TK-6 was kindly provided by William Thilly (5). A subclone with a high plating efficiency of -70% was isolated. The subclone could be maintained in exponenti gowth fpr Etn indefinite period as a proliferating mass culture. The experimental design consisted of treating a mass culture with either N-ethyl-N-nitrosourea at 50 ,g/ml for 40 min or five successive daily exposures to the mutagen at 10 ,ug/ml. The former treatment resulted in 95% cell killing, and the latter resulted in 60% cell killing. Single-cell clones were isolated at various times following treatment and individually cultured, until a population size of 8-12 x 106 cells was obtained. Harvested cells were either pelleted for two-dimensional PAGE analysis or stored frozen in liquid nitrogen for subsequent propagation and repeated analysis. Control clones were isolated from an untreated mass culture under conditions comparable to those for treated clones.Two-Dimensional Electrophoresis. Cell pellets were solubilized by addition of 40 A.l of a lysis buffer ...

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Detection of human somatic cell structural gene mutations by two‐dimensional electrophoresis

Cited by 9 publications

References 10 publications

Phenotypic and functional similarities between 5‐azacytidine‐treated t cells and a t cell subset in patients with active systemic lupus erythematosus

Phenotypic and functional similarities between 5‐azacytidine‐treated t cells and a t cell subset in patients with active systemic lupus erythematosus

Alterations in Barrett's‐related adenocarcinomas: A proteomic approach

Nonrandom distribution of structural mutants in ethylnitrosourea-treated cultured human lymphoblastoid cells.

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