ARTICLE |
Correspondence to: Buran KurdiHaidar, UCSD Cancer Center, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0058..
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Summary |
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Human ATPase (hASNA-I) is a novel human gene recently cloned on the basis of homology to the arsA gene of bacteria. Its protein product is an ATPase that is free in the cytoplasm and bound in the perinuclear area and nucleolus in human cells. We prepared the hASNA-I-specific 5G8 monoclonal antibody and used it to investigate the expression of hASNA-I in normal human tissues and breast cancers. hASNA-I was detected immunohistochemically only in the epithelial cells of the liver, kidney, and stomach wall, in the adrenal medulla, in the islet cells of the pancreas, in the red pulp of the spleen, and in cardiac and skeletal muscle. No staining was observed in the uterus, testis, lung, thyroid, cerebellum, and large intestine. Although no staining was also observed in normal breast tissue, all four cases of breast fibroadenomas and all 15 cases of either primary or metastatic breast carcinoma demonstrated increased staining. No embryological or functional common denominator is readily apparent. However, the increased expression in malignant breast cells is of particular interest with respect to the use of this antibody for screening of cytological specimens. (J Histochem Cytochem 46:12431248, 1998)
Key Words: ATPase, hASNA-I, arsenite, nucleocytoplasmic transport, breast epithelium, breast carcinoma, immunohistochemical, distribution
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Introduction |
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Resistance to toxic metalloids in bacteria is mediated by plasmid-borne, multicomponent, ATP-dependent efflux systems (
The ArsA protein is a member of a superfamily of ATP binding proteins with a nucleotide binding motif distinct from that of other ATPases (
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Materials and Methods |
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Immunohistochemistry
A multitissue block, containing a variety of normal formalin-fixed and paraffin-embedded tissue sections containing five normal sections of each tissue type, was purchased from Dako (Carpinteria, CA). A tissue block containing samples of normal breast, breast fibroadenomas, and primary and metastatic infiltrating ductal carcinomas was obtained from H. Batifora (City of Hope, CA). In addition, sections from four normal breasts and four infiltrating ductal breast carcinomas were obtained from the Tissue Collection and Distribution core laboratory at the UCSD Cancer Center. This study was conducted using 5G8, an IgM anti-hASNA-I mouse monoclonal antibody that was obtained from hybridoma tissue culture medium. This antibody was previously shown to detect a single protein band in human cell lysates by Western blotting, and its further characterization has been reported (
Control Cell Lines
A subline of the human malignant melanoma T289 cells (
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Results |
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Immunohistochemical Staining of Cells Overproducing hASNA-I
The 5G8 anti-hASNA-I mouse monoclonal antibody was used to stain formalin-fixed, paraffin-embedded T289/E and T289/A cells (Figure 1). No significant staining was observed in T289/E cells expressing a basal level of hASNA-I with either 5G8 or the control purified mouse IgM (Figure 1A and Figure 1C, respectively). In T289/A cells that were molecularly engineered to overproduce hASNA-I, only background staining was observed with the control IgM antibody (Figure 1B), whereas strong immunostaining was observed with 5G8 (Figure 1D). This result indicated that 5G8 is suitable for immunohistochemical analysis of hASNA-I expression in formalin-fixed, paraffin-embedded tissues.
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Immunohistochemical Staining of Normal Human Tissues
The 5G8 antibody was used to immunohistochemically detect hASNA-I in a variety of paraffin-embedded human tissues. Expression of hASNA-I was not ubiquitous but instead was limited to specific tissues and to limited types of cells in the tissues. hASNA-I-immunoreactive tissues included liver, pancreas, stomach, spleen, kidney, heart, and skeletal muscle, as well as the adrenal gland. No detectable hASNA-I immunoreactivity was found in breast, cerebellum, thyroid, uterine epithelium, testis, lung, or in the large or small intestine (data not shown). Figure 2A shows negative staining of colon epithelium. In the adrenal gland (Figure 2B), the chromaffin cells of the medulla were distinctly positive and no staining was observed in any of the cortical layers. In the kidney, the proximal and distal tubules and the epithelial cells of Bowman's capsule in the cortex were hASNA-I positive (Figure 2C). In the stomach, no staining was detected at the luminal edge of the glands but the epithelial cells lining the neck and crypt were positive, with an apparent differential staining between the parietal and chief cells (Figure 2D). In the spleen, only the red pulp stained for hASNA-I. No staining was detected in the white pulp (Figure 2E). The identity of the hASNA-I-immunoreactive cells in the red pulp of the spleen could not be further ascertained using these sections. In cardiac muscle, both the fibers and the intercalated discs were strongly positive (Figure 2F), and so were the fibers of skeletal muscle (data not shown). In the pancreas, staining was limited to the islets of Langerhans, and the ß-cells stained more intensely than the -cells (Figure 2H). All hepatocytes were hASNA-I-positive, although differences in staining intensity were observed among individual cells (Figure 2G). In all tissues in which staining was detected, the hASNA-I was cytoplasmic.
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Immunohistochemical Staining of Breast Tissues
As a first step towards determining how hASNA-I is altered by malignant transformation, we examined multiple breast tissue sections from four normal breasts, four breast fibroadenomas, 13 infiltrating duct carcinomas in the breast, and metastatic deposits of two other infiltrating duct carcinomas. No immunostaining for hASNA-I was found in the duct or lobular epithelium of any of the normal breast control sections (Figure 3A and Figure 3B). Detectable hASNA-I immunostaining was observed in all four of the fibroadenomas examined, in both the epithelial and myoepithelial cells. There was some variability in staining intensity; sections of two representative fibroadenomas are shown in Figure 3C and Figure 3D. All of the carcinomas stained positively for hASNA-I; representative sections are shown in Figure 3E and Figure 3F. The two metastatic carcinoma deposits examined were strongly hASNA-I-immunoreactive (Figure 3G and Figure 3H). Thus, all breast tissue sections containing either adenomatous or carcinomatous cells demonstrated elevated levels of hASNA-I detectable by immunocytochemistry using the anti-hASNA-I mouse monolonal 5G8 antibody.
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Discussion |
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hASNA-I is a novel human ATPase whose cDNA we isolated as a homologue of the E. coli ArsA, which in bacteria regulates the transport of arsenite and related metalloids. Insight into the role of hASNA-I in the physiology of human cells was derived from a number of findings relating to the ubiquitous expression of its mRNA (
Immunohistochemical staining of normal human tissues was carried out using the specific anti-hASNA-I monoclonal antibody to further elucidate the role it plays in different human organs. This study indicated that different tissues vary in their level of detectable hASNA-I and that staining of immunopositive tissues is cell type-specific. In addition, individual immunopositive cells also differed in their level of hASNA-I.
We have previously reported the presence of hASNA-I mRNA, determined by Northern blot analysis, in a variety of human tissues including heart, brain, lung, liver, skeletal muscle, kidney, and pancreas (
Immunohistochemical staining showed that hASNA-I is selectively present in specific cell types for which no embryological or functional common denominator is readily apparent. Among endocrine cells, staining was observed in the chromaffin cells of the adrenal medulla and the islet cells of the pancreas. Specific epithelial cells in the glands of the stomach wall and kidney demonstrated strong staining, whereas other parts of the same epithelial structures showed undetectable staining for hASNA-I. Localization to the intercalated disks in cardiac muscle was particularly striking.
The finding that hASNA-I levels are markedly increased in fibroadenomatous and carcinomatous lesions of the breast is of potential interest for the cytological detection of breast cancer. A more thorough examination of the various subtypes of breast malignancies and of nipple and needle aspiration cytology specimens from these cases is needed. It will also be of interest to determine the molecular mechanisms that underlie this overexpression of hASNA-I and how they are linked to the transformation process.
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Acknowledgments |
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Supported in part by Grants CA69004 from the National Institute of Health and 2RB-0125 from the California Breast Cancer Research Program.
We thank M.A. Lawrence for technical assistance.
Received for publication February 10, 1998; accepted July 8, 1998.
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