ARTICLE |
Correspondence to: Philip G. Board, Molecular Genetics Group, John Curtin Schl. of Medical Research, Australian National University, PO Box 334, Canberra ACT 2601, Australia. E-mail: Philip.Board@anu.edu.au
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Summary |
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Omega class glutathione transferase (GSTO) has been recently described in a number of mammalian species. We used immunohistochemistry to determine the cellular and tissue distribution of GSTO1-1 in humans. Expression of GSTO1-1 was abundant in a wide range of normal tissues, particularly liver, macrophages, glial cells, and endocrine cells. We also found nuclear staining in several types of cells, including glial cells, myoepithelial cells of the breast, neuroendocrine cells of colon, fetal myocytes, hepatocytes, biliary epithelium, ductal epithelium of the pancreas, Hoffbauer cells of the placenta, and follicular and C-cells of the thyroid. These observations and the known activity of GSTO1-1 suggest biological functions that are not shared with other GSTs. (J Histochem Cytochem 49:983987, 2001)
Key Words: glutathione transferase, omega, GSTO, immunohistochemistry
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Introduction |
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Glutathione S-transferases (GSTs) are a family of enzymes that detoxify xenobiotics, primarily by catalysis of the nucleophilic attack of reduced glutathione on electrophilic compounds. In humans, the cytosolic GSTs can be divided into six major classes: alpha, mu, pi, sigma, theta, and zeta. These isoenzymes are found in a range of tissues, and their expression has significant biological and clinical implications, including drug resistance and carcinogenesis (
Recently, a new human GST isoenzyme was identified by analysis of the Expressed Sequence Tag (EST) database (-helices (
To learn more about the role and significance of the omega class GSTs we investigated the expression of GSTO1-1 using immunohistochemistry in a wide range of normal human tissues.
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Materials and Methods |
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Polyclonal antiserum was prepared in rabbits by immunization with recombinant human GSTO1-1 that had been expressed in E. coli and purified by means of an N-terminal His tag (
Archival paraffin blocks of surgical pathology specimens or postmortem tissue specimens from the Department of Anatomical Pathology at the Canberra Hospital were used to obtain tissue sections for immunohistochemistry. In all cases the tissue had been fixed in 10% neutral phosphate-buffered formalin before processing and embedding in paraffin. A pathologist (JED) examined the hematoxylin and eosin sections to exclude light microscopic pathology. At least three samples of each site from different subjects were selected.
Sections were cut at 3 µm onto slides coated with 3-aminopropyltriethoxy-silane (Sigma; St Louis, MO). Sections were deparaffinized and endogenous peroxidase blocked with 3% hydrogen peroxide in PBS. Nonspecific binding was blocked by incubating sections with 10% normal goat serum in PBS for 20 min at room temperature (RT). Sections were then incubated in rabbit anti-human GSTO1-1 (1:300) in antibody diluent (1% bovine serum albumin, 0.05% sodium azide in PBS) overnight at RT in a moist box. Pre-immune rabbit serum (1:300) and antibody diluent served as negative controls. After 24 hr, sections were washed (three times for 5 min) in PBS and incubated with biotinylated goat antiserum to rabbit IgG (1:1000 in PBS) (Vector Laboratories; Burlingame, CA) for 1 hr at RT. Sections were washed with PBS and streptavidin (Vector Laboratories) labeled with horseradish peroxidase (1:1500 in PBS). Peroxidase activity was developed using 3,3'-diaminobenzidine. Sections were counterstained with hematoxylin, then dehydrated, cleared, and mounted. Sections were viewed by a pathologist and photographed.
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Results |
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The distribution and localization of GSTO1-1 in human normal tissues are summarized in Table 1. Cytoplasmic staining of GSTO1-1 was observed in most tissues. Of note, there was staining in endocrine cells (e.g., thyroid (Fig 1), pancreas, Leydig cells of testis, neuroendocrine cells of the colon, stromal cells of ovary), glial cells (Fig 2), macrophages (Fig 3), hepatocytes (Fig 4), and epithelial cells in some but not all tissues.
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In many tissues, GSTO1-1 was seen in only one cell type (Table 1). For example, GSTO was expressed in neuroendocrine cells in the colon, macrophages in lymph nodes, tubular cells of kidney, and glial cells of the brain. In the skin and lung (Fig 3), staining was seen only in macrophages. GSTO1-1 was not detected in endothelial cells, smooth muscle cells, or lymphocytes in any organ examined.
Nuclear staining was seen in glial cells, myoepithelial cells of the breast, neuroendocrine cells of colon, fetal myocytes, hepatocytes, biliary epithelium, ductal epithelium of the pancreas, Hoffbauer cells of the placenta, and follicular and C-cells of the thyroid (Table 1).
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Discussion |
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Recently,
The potential nuclear activity of GSTO1-1 is unknown. A mouse GST called p28 with 72% sequence identity with GSTO1 and clearly a member of the omega class has been identified in a radiation-resistant cell line (
An additional advantage of immunohistochemical techniques over Northern blotting analysis and EST screening is the ability to determine which cells in each tissue express GSTO. For example, we found specific expression in neuroendocrine cells of the colon, macrophages in lymph nodes, Hoffbauer and cytotrophoblastic cells in the placenta, and glial cells in the brain. In the skin and lung, expression of GSTO1-1 was seen only in macrophages. It is likely that expression of GSTO in lung and skin detected by Northern blotting and EST database screening (
The widespread distribution of GSTO1-1 suggests that it has important biological functions. It is possible that the glutathione-dependent dehydroascorbate reductase and thiol transferase activities of GSTO1-1 may be generally required for normal cellular processes (
In conclusion, GSTO1-1, like other GSTs, has a distinct pattern of tissue expression. In particular, we noted expression in nuclei of many tissues. These observations, coupled with prior understanding of the unique activity of GSTO1-1, suggest biological functions not shared with other GSTs.
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Acknowledgments |
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Supported in part by the Private Practice Trust Fund of the Canberra Hospital.
Received for publication December 14, 2000; accepted February 21, 2001.
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Literature Cited |
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