Immunohistochemical Analysis of the Distribution of the Human ATPase (hASNA-I) in Normal Tissues and Its Overexpression in Breast Adenomas and Carcinomas

Volume 46(11): 1243–1248, 1998 The Journal of Histochemistry & Cytochemistry ht t p://w w w .jhc.org A RTI CLE Immunohistochemical Analysis of the Distribution of the Human ATPase (hASNA-I) in Normal Tissues and Its Overexpression in Breast Adenomas and Carcinomas Buran Kurdi–Haidar, Dennis Heat h, Pet er Naredi, Nissi Varki, and St even B. How ell Depart ment of M edicine and t he UCSD Cancer Cent er, Universit y of Calif ornia at San Diego, La Jolla, Calif ornia (BK–H,DH, NV,SBH), and Depart ment of Surgery I, Universit y Hopist al, Umea, Sw eden (PN) Human ATPase (hASNA-I) is a novel human gene recent ly cloned on t he basis of homology t o t he arsA gene of bact eria. It s prot ein product is an ATPase t hat is f ree in t he cyt oplasm and bound in t he perinuclear area and nucleolus in human cells. We prepared t he hASNA-I-specif ic 5G8 monoclonal ant ibody and used it t o invest igat e t he expression of hASNA-I in normal human t issues and breast cancers. hASNA-I w as det ect ed immunohist ochemically only in t he epit helial cells of t he liver, kidney, and st omach w all, in t he adrenal medulla, in t he islet cells of t he pancreas, in t he red pulp of t he spleen, and in cardiac and skelet al muscle. No st aining w as observed in t he ut erus, t est is, lung, t hyroid, cerebellum, and large int est ine. Alt hough no st aining w as also observed in normal breast t issue, all f our cases of breast f ibroadenomas and all 15 cases of eit her primary or met ast at ic breast carcinoma demonst rat ed increased st aining. No embryological or f unct ional common denominat or is readily apparent . How ever, t he increased expression in malignant breast cells is of part icular int erest w it h respect t o t he use of t his ant ibody f or screening of cyt ological specimens. (J Histochem Cytochem 46:1243–1248, 1998) SU M M A RY RESI STAN C E TO TO XI C M ETALLO I D S in bacteria is mediated by plasmid-borne, multicomponent, ATP-dependent efflux systems (Kaur and Rosen 1992b; Silver and Ji 1994). The well-characterized ars operon (Rosen et al. 1990) mediates resistance to arsenite, arsenate, and antimonite in E. coli and contains two regulatory (arsR and arsD) and three structural genes (arsA, B, C) (Kaur and Rosen 1992a; Broer et al. 1993). The arsA gene codes for an oxyanion ATPase that associates with the protein encoded for by the arsB, the putative channelforming transmembrane protein. Together, the two proteins transport arsenite and antimonite out of the cells across the plasma membrane. The ArsA protein is a member of a superfamily of ATP binding proteins with a nucleotide binding motif distinct from that of other ATPases (Koonin 1993). In the course of our studies of resistance to toxic metal Correspondence to: Buran Kurdi–H aidar, PhD, UCSD Cancer Center, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0058. Received for publication February 10, 1998; accepted July 8, 1998 (8A4599). © The Hist ochemical Societ y, Inc. KEY W O RD S ATPase hASNA-I arsenite nucleocytoplasmic transport breast epithelium breast carcinoma immunohistochemical distribution in human cells, we cloned the human arsenite-stimulated ATPase (hASN A-I) cDN A based on DN A sequence homology to the distinct nucleotide binding motif (Kurdi–H aidar et al. 1996). The human hASN A-I is an ATPase. H owever, although it is stimulated by arsenite, unlike its bacterial counterpart it has significant basal ATPase activity even in the absence of oxyanions (Kurdi–H aidar et al. unpublished observations). Structural and biochemical characteristics of hASN A-I suggest that it is functionally different from the ArsA protein of E. coli. To investigate the role of hASN A-I in the physiology of human cells, we produced the specific anti-hASN A-I mouse monoclonal antibody 5G8. Immunocytochemical and Western blot analysis of subcellular fractions identified a soluble pool of hASN A-I in the cytoplasm and bound pools at the nuclear membrane and in the nucleolus in human cells (Kurdi–H aidar et al. in press). This distribution suggested that hASN A-I plays a role in nucleocytoplasmic transport in human cells. We report here an immunohistochemical study of the expression of hASN A-I in normal human tissues and the discovery that hASN A-I levels are 0022-1554/98/$3.30 Downloaded from jhc.sagepub.com by guest on February 5, 2016 1243 1244 Kurdi–H aidar, H eath, N aredi, V ark i, H ow ell Figure 1 Immunohist ochemical det ect ion of overexpression of hASNA-I in human cells. Cont rol T289/E cells st ained w it h purif ied mouse IgM (A ) and 5G8 (C). hASNA-I-overexpressing T289/A cells st ained w it h purif ied mouse IgM (B) and 5G8 (D). Bars ⫽ 100 ␮m. markedly increased in breast fibroadenomas and carcinomas. M aterials and M ethods Immunohist ochemist ry 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 H ope, 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 (Kurdi–Haidar et al. in press). The immunohistochemical staining of the formalin-fixed, paraffin-embedded sections was enhanced by heating in a microwave oven for 5 min on high power in 10 mM citrate, pH 6.0 (Shi et al. 1991) to unmask the hASN A-I epitope recognized by the monoclonal 5G8 antibody. Sections were washed and nonspecific binding was Figure 2 Immunohist ochemical det ect ion of hASNA-I in normal human t issues. Immunonegat ive cells of colon ( A ). Immunoposit ive cells show n are chromaf f in cells of t he adrenal medulla (B), epit helial cells of Bow man’s capsule and of proximal and dist al t ubules of t he kidney (C), epit helial cells lining t he neck and crypt of t he glands of t he st omach w all (D ), red pulp of t he spleen (E), f ibers and int ercalat ed disks of cardiac muscle (F), hepat ocyt es of liver (G), and islet cells of t he pancreas (H ). Bars ⫽ 100 ␮m. Downloaded from jhc.sagepub.com by guest on February 5, 2016 Immunohist ochemical Dist ribut ion of hASNA-I Downloaded from jhc.sagepub.com by guest on February 5, 2016 1245 1246 Kurdi–H aidar, H eath, N aredi, V ark i, H ow ell blocked with 10% horse serum in Tris-buffered saline (TBS) (50 mM Tris-H Cl, 0.9% N aCl, pH 8.0) for 20 min, then incubated overnight at 4C with the primary 5G8 antibody at a 1 ⬊100 dilution in the blocking solution, followed by three 10-min washes in TBS. The mouse monoclonal 5G8 antibody was then localized using the labeled strepavidin–biotin system provided in the alkaline phosphatase Dako LSAB2 Kit. Incubation with the anti-mouse biotinylated “link” antibody was carried out for 10 min and was followed by three 5-min washes in TBS. A second 10-min incubation in alkaline phosphatase-conjugated strepavidin was also followed by three 5-min washes in TBS. Alkaline phosphatase was detected using reagents of the alkaline phosphatase substrate Kit III purchased from Vector Laboratories (Burlingame, CA), in the presence of levamisole, an inhibitor of most forms of alkaline phosphatase other than the conjugate intestinal isoenzyme (Vector Laboratories). Nuclei were counterstained with nuclear Fast Red, purchased from Vector Laboratories, and slides were mounted in H emo-D Cytoseal 60 mount medium obtained from VWR (San Diego, CA). Cont rol Cell Lines A subline of the human malignant melanoma T289 cells (Taetle et al. 1987) overproducing the hASNA-I protein (T289/A) and its empty vector-infected counterpart (T289/E) were generated by retroviral infection using vectors LAPONL (Kurdi–Haidar et al. unpublished results) and LPONL (Yee et al. 1994), respectively. Both sublines were maintained in the presence of 0.4 mg/ml G418 in culture medium (Kurdi–H aidar et al. in press). Tissue culture cell pellets were washed with PBS, fixed in 10% buffered formalin, and paraffin-embedded using standard protocols. Sections were stained either with 5G8 or with control purified mouse IgM (PharM ingen; San Diego, CA) at a concentration of 0.05 ␮g/ml. Results Immunohist ochemical St aining of Cells Overproducing hASNA-I The 5G8 anti-hASN A-I mouse monoclonal antibody was used to stain formalin-fixed, paraffin-embedded T289/E and T289/A cells (Figure 1). N o significant staining was observed in T289/E cells expressing a basal level of hASN A-I with either 5G8 or the control purified mouse IgM (Figures 1A and 1C, respectively). In T289/A cells that were molecularly engineered to overproduce hASN A-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. Immunohist ochemical St aining of Normal Human Tissues The 5G8 antibody was used to immunohistochemically detect hASN A-I in a variety of paraffin-embedded human tissues. Expression of hASN A-I was not ubiquitous but instead was limited to specific tissues and to limited types of cells in the tissues. hASN A-Iimmunoreactive tissues included liver, pancreas, stomach, spleen, kidney, heart, and skeletal muscle, as well as the adrenal gland. N o detectable hASN A-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 hASN A-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 hASN A-I. N o staining was detected in the white pulp (Figure 2E). The identity of the hASN A-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 ␤-cells stained more intensely than the ␣-cells (Figure 2H ). All hepatocytes were hASN A-I-positive, although differences in staining intensity were observed among individual cells (Figure 2G). In all tissues in which staining was detected, the hASN A-I was cytoplasmic. Immunohist ochemical St aining of Breast Tissues As a first step towards determining how hASN A-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. N o immunostaining for hASN A-I was found in the duct or lobular epithelium of any of the normal breast control sections (Figures 3A and 3B). Detectable hASN A-I immunostaining was observed in all four of the fibroadenomas examined, Figure 3 Immunohist ochemical det ect ion of hASNA-I in human breast . Immunonegat ive cells of normal breast (A,B). Immunoposit ive cells show n are (C,D) epit helium and myoepit helium of f ibroadenomat ous breast , ( E,F) primary breast carcinoma cells, and ( G,H) met ast at ic breast carcinoma cells. Each panel represent s immunost aining of an independent sample. Bars ⫽ 100 ␮m. Downloaded from jhc.sagepub.com by guest on February 5, 2016 Immunohist ochemical Dist ribut ion of hASNA-I Downloaded from jhc.sagepub.com by guest on February 5, 2016 1247 1248 Kurdi–H aidar, H eath, N aredi, V ark i, H ow ell in both the epithelial and myoepithelial cells. There was some variability in staining intensity; sections of two representative fibroadenomas are shown in Figures 3C and 3D. All of the carcinomas stained positively for hASN A-I; representative sections are shown in Figures 3E and 3F. The two metastatic carcinoma deposits examined were strongly hASN A-I-immunoreactive (Figures 3G and 3H ). Thus, all breast tissue sections containing either adenomatous or carcinomatous cells demonstrated elevated levels of hASN A-I detectable by immunocytochemistry using the antihASN A-I mouse monolonal 5G8 antibody. Discussion 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 (Kurdi–Haidar et al. 1996), its distinct biochemical characteristics, and its subcellular localization. We have already established that hASNA-I forms complexes whose size is consistent with tetramers (Kurdi–Haidar et al. unpublished observations), and that it is subcellularly distributed in the cytoplasm, nuclear membrane, and nucleolus (Kurdi–H aidar et al. in press). The fact that it is not found in the plasma membrane suggests that hASNA-I is a paralogue rather than an orthologue of ArsA and that it probably plays a different role in human cells than does the ArsA protein in bacteria. Immunohistochemical staining of normal human tissues was carried out using the specific anti-hASN A-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 hASN A-I and that staining of immunopositive tissues is cell type-specific. In addition, individual immunopositive cells also differed in their level of hASN A-I. We have previously reported the presence of hASNA-I mRN A, determined by N orthern blot analysis, in a variety of human tissues including heart, brain, lung, liver, skeletal muscle, kidney, and pancreas (Kurdi–H aidar et al. 1996). Concordant distribution was found by immunohistochemistry, with at least some cells that stained positively for hASN A-I with 5G8 in heart, liver, kidney, and pancreas. The apparent lack of concordance in lung is unexplained, although bronchi and vascular structures larger than arterioles were not present in the sections examined in this study. Additional portions of brain will need to be examined immunohistochemically to determine the source of hASN A-I message in this organ. 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 hASN A-I. Localization to the intercalated disks in cardiac muscle was particularly striking. The finding that hASN A-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 hASN A-I and how they are linked to the transformation process. Acknow ledgment s Supported in part by Grants CA69004 from the N ational Institute of H ealth and 2RB-0125 from the California Breast Cancer Research Program. We thank M .A. Lawrence for technical assistance. Literature Cited Broer S, Ji G, Broer A, Silver S (1993) Arsenic efflux governed by the arsenic resistance determinant of Staphylococcus aureus plasmid pI258. J Bacteriol 175:3480–3485 Kaur P, Rosen BP (1992a) M utagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase. J Biol Chem 267:19272–19277 Kaur P, Rosen BP (1992b) Plasmid-encoded resistance to arsenic and antimony. Plasmid 27:29–40 Koonin EV (1993) A superfamily of ATPase with diverse functions containing either classical or deviant ATP-binding motif. J M ol Biol 229:1165–1174 Kurdi–H aidar B, Aebi S, H eath D, Enns RE, N aredi P, H om DK, H owell SB (1996) Isolation of the ATP-binding human homolog of the arsA component of the bacterial arsenite transporter. 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