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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Expression of Plasma Glutathione Peroxidase in Human Liver in Addition to Kidney, Heart, Lung, and Breast in Humans and Rodents By Fong-Fong Chu, Robert Steven Esworthy, James H. Doroshow, Khiem Doan, and Xian-Fang Liu We analyzed the expression of plasma glutathione peroxidase (GSHPx-P) messenger RNA (mRNA) in mouse, rat, and human tissues, using a human GSHPx-P cDNA clone as the probe. Unlike the classical cellular glutathione peroxidase (GSHPx-l), GSHPx-P expression appears t o be tissuespecific. In the mouse and rat, kidney expresses an mRNA at a high level detected with the human probe. A signal is also detected in mRNA isolated from mouse and rat heart, rat cardiac myocytes, mouse lung, epididymis, and the mammary gland of midpregnant mice. No signal is detected in mRNA isolated from mouse and rat liver, mouse brain, uterus, and testis. In human tissues, an mRNA hybridizing t o GSHPx-P cDNA is present in liver, as well as kidney, heart, lung, breast, and placenta. We have shown that human kidney expresses a GSHPx-P mRNA, and not a GSHPx-P-like message, by isolating a cDNA clone from a human kidney library in Agtll. From the 412-nucleotide partial sequence of the kidney cDNA, which codes for the 40-170 amino acids of GSHPx-P including the TGA codon for selenocysteine, we found complete sequence identity of the kidney cDNA with GSHPx-P isolated from placenta. The expression of GSHPx-P mRNA in cell lines was also studied. There is some correlation of the expression of GSHPx-P in these cell lines with that in normal tissues. Cell lines that expressed GSHPx-P mRNA or protein included the human hepatocarcinoma HepG2, Hep3B cells, human kidney carcinoma A498 cells, and the human breast cancer SK-BR-3, T47D. MDA-MB-231, and Adr‘MCF-7 cells. Cell lines that did not express GSHPx-P included human choriocarcinoma BeWo cells, human breast cancer MCF-7, ZR-75-1, and Hs578T cells, and mouse hepatoma Hepa-1 cells. o 7992 by The American Society of Hematology. M We and others have previously reported that the human hepatoma cell lines HepG2 and Hep3B secrete GSHPxP.4,10However, Takahashi et all1 failed to detect GSHPx-P mRNA in human liver. Yoshimura et a1 studied GSHPx-P protein expression by Western blotting in 13 rat tissues and found signals in kidney and lung, but not in liver, heart, spleen, thymus, bone marrow, erythrocytes, brain, pituitary and adrenal glands, jejunum, duodenum, or skeletal muscle. These results raised the question of whether there is any correlation between the expression of GSHPx-P in normal tissues and that in cell lines derived from these tissues. Therefore, we have analyzed several normal tissues and cell lines of human, mouse, and rat origin for GSHPx-P expression. We found that human liver, and not mouse or rat liver, expresses GSHPx-P mRNA. Our results are presented in this report. ANY FORMS OF ACTIVE oxygen such as hydrogen peroxide, lipid hydroperoxides, superoxide, hydroperoxy and hydroxyl radicals, and singlet oxygen are implicated in human disease. Evidence exists to support a role for oxidant damage in the pathogenesis of rheumatoid arthritis, reperfusion injury, cardiovascular disease, immune injury, and cancer.1,2Glutathione peroxidase (GSHPx, glutathi0ne:hydrogen-peroxideoxidoreductase, EC 1.11.1.9) has a selenocysteine residue at its active site that catalyzes the reduction of hydrogen peroxide and organic hydroperoxides by oxidizing GSH. Four members of the GSHPx family have been identified in human cells: (1) the classical cellular enzyme, GSHPx-1; (2) the plasma GSHPx, GSHPx-P; (3) the phospholipid hydroperoxide enzyme, PHGPX; and (4) a GSHPx-1-like enzyme, GSHPx-2, expressed in human liver and hepatoma cell^.^-^ Additionally, a mouse 24-Kd epididymal secretory protein regulated by androgen has sequence homology with GSHPx, although it is devoid of ~elenocysteine.~ Based on its epididymisspecific expression, it appears to be distinct from the other members of GSHPx. Thus, it may be the fifth member in the GSHPx family.* Yoshimura et a19 detected a messenger RNA (mRNA) that hybridized to a rat GSHPx-P cDNA, and a polypeptide that reacts with a chicken antirat-GSHPx-P antisera on a Western blot isolated from rat kidney. These results strongly suggested that the kidney is the source of the plasma GSHPx activity. However, there are multiple forms of GSHPx that share significant sequence homology across species: the nucleic acid sequence of GSHPx-P has 57% homology with that of cellular glutathione peroxidase (GSHPx-l), and has 67% homology with that of a 24-Kd mouse epididymal secretory protein. It is possible that the signal detected in various tissues is caused by crosshybridization with yet undetermined members of the GSHPx family. To prove that kidney expresses GSHPx-P we used direct sequencing of a GSHPx-P cDNA isolated from a kidney library. In this study, we showed the sequence identity of the cDNA isolated from a human kidney library to be a GSHPx-P. Blood, Vol79, No 12 (June 15). 1992:pp 3233-3238 MATERIALS AND METHODS Materials. Human cDNA libraries in hgtll from placenta and kidney were purchased from Clontech Lab (San Diego, CA); GSHPx-P-specific oligonucleotides were synthesized by the Core DNA Synthesis Lab of City of Hope Cancer Research Center (Duarte, CA). Phage hgtll forward and reverse primers were purchased from New England Biolabs, Inc (Beverly, MA). Isolation of GSHfi-P cDNA clones. Human stretched placenta and kidney cDNA libraries had 2.3 x 1Olo and 5.0 x 1O’O pfu/mL, From the Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA. Submitted August 28,1991; accepted F e b u l y 12, 1992. Supported by Grant-in-aid No. 901 GI-2 of American Heart Association Greater Los Angeles Affiliate, Inc, and US Public Health Services Grants No. CA31788 and CA33572. Address reprint requests to Fong-Fong Chu, PhD, Dept. of Medical Oncology, City of Hope National Medical Center, 1500 E Duarte Rd, Duarte, CA 91010-0269. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section I734 solely to indicate this fact. 0 1992 by The American Society of Hematology. 0006-497119217912-0003$3.00/0 3233 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CHU ET AL 3234 respectively. Degenerate oligonucleotides were made according to our previously described GSHPx-P peptide fragments." They were used as probes to isolate GSHPx-P cDNA clone from a placenta library. Specific oligonucleotides were made after we sequenced the placenta GSHPx-P clone. These oligonucleotides were used to screen for GSHPx-P clones in a human kidney library. Screening, phage plating, and transfer to nitrocellulose filters (Schleicher & Schuell Inc, Keene, NH) were performed in duplicate as described.I2 Secondary and tertiary screening were performed after isolating the positive plaques to ensure that single clones were obtained in each isolate. DNA sequencing. We completely sequenced our placenta GSHPx-P cDNA clone bidirectionally by subcloning the insert of the Agtll clone into pBluescript (pBS; Stratagene, San Diego, CA). For sequencing reactions, either Sequenase (US Biochemical, Cleveland, OH) or linear polymerase chain reaction (PCR) sequencing13was used. Ig Suite from the Core DNA facility of the Cancer Center, City of Hope (IntelliGenetics, Inc, Mountain View, CA) was used to analyze and manage the sequencing data. The kidney GSHPx-P cDNA clone was sequenced in A g t l l directly. The insert was amplified with Agtll forward and reverse primers by PCR. The amplified fragment was isolated from the unincorporated primers by precipitation with 20% polyethylene glycol in 2 N NaC1.14 It was then sequenced directly by the linear PCR sequencing method.13 Northern analysis. Total RNAs were isolated from human and animal tissues or cell lines. Tissue sources and numbers of successful isolation are summarized as follows. (1) Mouse tissues: liver: 1X from a 0 BaZb\c, 1X from each of a 6 and a 0 Swiss Webster, and 1X from a 0 BM-1 strains; kidney: 3X from 0 Balb\c, 1X from each of a 6 and a P Swiss Webster, and 1X from a 6 Swiss CD-1 strains; heart: 1X from each of a 6 and a 0 Swiss Webster, and 4X from 6 Swiss CD-1 strains; lung: 1X from a 6 Swiss CD-1 strain; mammary gland: 2X from 14 day pregnant P Swiss Webster strain; epididymis: 1X from a 6 BM-1, and 1X from a 6 Swiss Webster strain; uterus: 1X from a 0 BaZb\c strain; brain and testis: 1X from a 6 Swiss Webster strain. (2) Rat tissues were all isolated from 6 Sprague-Dawley rats (Charles River Lab, Wilmington, MA), these including: liver: 2 X kidney cortex and medulla: 2X each; heart: lX, cardiac myocytes: 2x. (3) Human tissues: liver: 3X normal tissue; kidney: 3X normal tissue; heart: 1X normal tissue; lung: 3X normal and 1X tumor tissue; breast: 4X normal tissue. Isolation of cardiac myocytes from 6 Sprague-Dawley rats was performed following a previously described procedure with minor modification^.'^ After the heart was perfused on a LangendorB apparatus with Ca*+-free Tyrode's solution, myocytes were obtained by perfusing the heart with 50 mL of recirculating Tyrode's solution containing 50 mg of collagenase B (Boehringer-Mannheim Biochemicals, Indianapolis, IN), 5 mg protease (type XIV, Sigma) for 45 minutes at 37°C. Total RNA was isolated using the acid guanidinium thiocyanatephenol-chloroform extraction method.I6 Ten micrograms of RNA was resolved by formaldehyde-containing gels. Random primed human 32P-GSHPx-P and chicken 32P-p-actin cDNA were used as probes. The hybridizing and washing conditions were described previously." CeZZ lines and '5Se-ZabeZing. Human cell lines used included hepatoma HepG2 and Hep3B cells, kidney carcinoma A498 cells, breast carcinoma MCF-7, SK-BR-3, T47D, MDA-MB-231, ZR75-1, Hs578T cells, and choriocarcinoma BeWo cells (American Type Culture Collection, Rockville, MD). An adriamycin-resistant variant of MCF-7 cells, AdrIMCF-7, was kindly provided by Kenneth Cowan (National Cancer Institute, Bethesda, MD).ls Mouse hepatoma Hepa-1 cells were kindly provided by Oliver Hankinson (University of California, Los Angeles, CA). A GSHPx-1 transfectant of MCF-7 cells, MCF-7H6, was established as described.17 At least two independent isolations of mRNA were performed on each of these cell lines. BeWo cells were cultured in Kaighn's nutrient mixture F-12 (Irvine Scientific, Santa Ana, CA) supplemented with 15% fetal bovine serum. The other cells were grown in either Dulbecco's modified Eagle's medium (DMEM)\F12 or Richter's improved MEM zinc option medium (GIBCO Laboratories, Grand Island, NY)supplemented with 10% fetal bovine serum. Selenoproteins were labeled by incubating cells in growth media supplemented with 100 nmol/L 75Se-selenious acid for 4 to 6 days before harvesting. Protein analysis and enzyme assays. Immunoprecipitation of 75Se-GSHPx-1 and 75Se-GSHPx-P was performed with rabbit antihuman-RBC-GSHPx-1 antisera and antihuman-GSHPx-P antisera, as previously described." The immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). RESULTS We screened 60,000 plaques and isolated eight GSHPx-P clones from a human placenta cDNA library. We found a cDNA clone that includes the full coding sequence, and extends to the polyadenylation tail. The coding sequence that we have obtained is identical to the sequence published by Takahashi et al," although the 3' noncoding sequence contains a few discrepancies (EMBL accession number: X58295). We have aligned the deduced amino acid sequences of five glutathione peroxidase-like cDNA clones as shown in Fig 1. PxP is human GSHPx-P; Px4 is the porcine monomeric phospholipid hydroperoxide glutathione peroxidase (PHGPX)I9; Px2 is a tetrameric and catalytically active GSHPx expressed only in human liver and hepatoma cells6 (manuscript in preparation); Pxl is the classical human cellular GSHPx-1; and Epi is a 24-Kd mouse epididymal secretory protein regulated by t e s t o ~ t e r o n e . ~ , ~ We have recently determined the nucleotide sequence of 315 bp of the coding region of a human PHGPX cDNA. The deduced amino acid sequence of this portion has 95% homology with porcine PHGPX (unpublished data). First, we studied the expression of GSHPx-P mRNA in mouse tissues and mouse hepatoma Hepa-1 cells using a human GSHPx-P cDNA as the probe. As shown in lanes 1 to 8 of Fig 2A, and lane 9 of Fig 2C, kidney expresses a hybridizing mRNA at a high level, and it is detected in lesser abundance in epididymis, heart, and mammary gland from midpregnant mice. Other mouse tissues, including liver, testis, uterus, and brain, do not have a detectable signal. Similar to mouse liver, Hepa-1, a mouse hepatoma cell line, does not express any mRNA that hybridized with the human GSHPx-P probe. Figure 2B shows the mRNA signals hybridized with the chicken @-actinprobe that are used as a control for the quality and quantity of the mRNA preparation. The weak signal at higher molecular weight is at the same location as the abundant 28s rRNA that often hybridizes with the probes. The 18s rRNA has a molecular weight similar to p-actin. Because actins are composed of a number of isotypes highly conserved in evolution, the p-actin probe appears to hybridize with a-skeletal and From www.bloodjournal.org by guest on June 16, 2017. For personal use only. PLASMA GLUTATHIONE PEROXIDASE (GSHPX-P) PXP 3235 1 &ascllslllaafvsasr&qeksk Px4 Fig 1. Alignment of amino acid sequences from potential members of the glutathione peroxidase family. PxP is human plasma glutathione peroxidase; Pxl is the classic human erythrocyte glutathione peroxidaseza; PxZ is expressed in human liver or hepatoma cellso; Px4 is porcine phospholipid hydroperoxide glutathioneperoxidase'o; and Epi is a 24-Kd, mouse epididymal secretary protein regulated by androgen.' Because the published Px4 and Epi sequences are not full-length, we could not assign numbers for the amino acid positions. The double underlined PxP sequence contains a stretch of hydrophobic amino acids representing the signal sequence for membrane insertion. The boldface letters are the conserved sequences in all five members from three species. SmhEFSAkdID GhmVnLdkYRGyVcivtNVASqCSeGkTevnYTQLvdLhaRyAecG I II I l l II I I I l l I1 I l l II I I I I I I I Ill I Px2 1 mAfIAkSfYdlSAisLD GEkVdFntfRGraVLIENVISLCSeGTTtRDfTQLNELQcRf PRr Pxl 1 mcaarlaAaaAGSvYaFSArpLaGGEpVsLgs1RGKV1LIENVASLCSeGTTvRDYTQmNEIQrRLgPRG PxP 30 I I I I I I I I I l l I l l II II I I I I I II I I I1111II1 I II I l l I l l mdchggIsGtiYEygAltIDGeEyIPFKQYaGKyVLFVNVASYCSBCLTg QYLELNALQEeLAPFG I I IIIIII I IIIIIII I II IIIIIII I Ill Ill sLnGkEhIPFKQYRpKhVLFVNVAtYc GLTi QYpELNAIQEdLkPFG Ep i LrILaFPCNQFGrQEPGsdaEIkef Px4 I I IIIIIII I I II aaGYnvkFdmFsKicVNGddAHPLwkwmK II I I II Ill I Px2 61 LWLGFPCNQFGHQENcqNEEILNS~~RPGGGYqPtFtLvqKCEVNGqNEHPvFAy~dkLPyPyD Pxl 69 LWLGFPCNQFGHQENa~EEIqNSLK~RPGGGFePNFmLFEKCEVNGagAHPLFAFLreaLPaPSD PxP 94 LVILGFPCNQFGKQEPGeNsEILPtLKWRPGGGhrPNFQLFEKGDVNGEkEQKf TFLKnSCPptSE IIIIIIIIIIIIIIII IIII IIIIIlllIII I I I IIIIII Epi I I IIIIIIIII I I I II I I II I I I I I I I I I I I I l l IIII I l l I1 I I I I I I I I I I I I I I I I I I IIII I I I I I I I I I I I I I I I I I I I I I I I l l II I I I IIII Ill I II LVILGFPCNQFGKQEPG~lEILPgLKYVRPGkGFlPNFQLFaKGDVNGENEQKiFTF~rSCPhPSE Px4 VqPKgrgml gNaIkWNFtKFLIdknGcvV kRYgpmeepqVIEkDlpcYL. II I l l iiii I I II II I I IIIII I1 Px2 129 DufsLMTDPKLIiWSPVrRsDVAWNFEKFLIGPeGePf RRYSRTFuTmnIEPDIkrLLkvai. I L I I I I I I I I IIII I IIIIIIIIII II I I IIIII 1 - 1 Pxl 137 DataLMTDPKLItWSPVcRNDVAWNFEKFLVGPDGVPl RRYSRrFqTidIEPDIeALLSqRpsca. I I I I IIIIIIIIIIIII I II I PxP 165 LlgtSdrlfWEPmKVHDIRWNFEKFLVGPDGiPiMRWhHRTtVSnVKm DIlsYmrrqaalgvkrk. Ep i tvVmSKhtsWEPiKVHDIRWNFEKFLVGPDGVPVMRWfHqapVStVKs DImAYLShfkyi. I I l l IIII111111IIIIIIII I Ill I a-cardiac actin mRNAs that are coexpressed at high levels in striated muscles, in addition to two a-smooth muscle actin mRNAs present in sarcomeric muscles.20-22This results in double bands in some of the lanes in the p-actin hybridized blots. To confirm the results obtained with rodent tissues, we also analyzed several human tissues for GSHPx-P expression. Multiple human samples were shown in lanes 5 through 8 of Fig 2C and Fig 3A. With the exception of one human heart mRNA preparation, which was run twice in lane 5 of Fig 2C and lane 5 of Fig 3A, and the normal and tumor lung mRNA in lanes 7 and 8 of Fig 3A, which were isolated from the same individual, all human RNAs of different tissues are isolated from different individuals. These data show that human liver, as well as kidney, heart, lung, and breast, express GSHPx-P mRNA. Because Yoshimura et a19 did not detect any signal from rat heart by Western analysis, we wanted to know if the signal that we detected in mouse heart was specific to mice. As shown in lanes 1 through 4 of Fig 2C, we have detected an mRNA isolated from rat heart and cardiac myocytes, in addition to rat kidney cortex and medulla, that hybridizes with the human GSHPx-P probe. The kidney expresses a message that hybridizes strongly with the human GSHPx-P probe; however, so does epididymis, which expresses a GSHPx-P-like mRNA. To establish that the kidney mRNA codes for GSHPx-P, it was necessary to sequence a kidney cDNA clone that hybridized to GSHPx-P. Thus, we screened 35,000 plaques of a human kidney cDNA library, and found two clones that hybridized to a GSHPx-P-specific oligonucleotide. Despite the fact I1 II II I that both clones are truncated at their 5' ends, we obtained a 412-nucleotide sequence coding for the 40-176 amino acids, including the TGA codon for selenocysteine from one of the kidney clones. It is identical to the GSHPx-P sequence. We have analyzed human cell lines to study the correlation of GSHPx-P expression in vitro versus in vivo. As shown in Fig 2C, lanes 10 through 14, and lanes 3 and 5 of Fig 2E, four of seven human breast cell lines express an mRNA hybridized to GSHPx-P cDNA. SK-BR-3, T47D, MDA-MB-231, and Adr'MCF-7 have a detectable message, whereas ZR-75-1, Hs578T, and MCF-7 do not. Other human cell lines analyzed for GSHPx-P mRNA expression are shown in Fig 2E. These include hepatoma HepG2 (lane l), kidney carcinoma, A498 (lane 2), and choriocarcinoma, BeWo (lane 5), cell lines. HepG2 and A498 cells express GSHPx-P mRNA, whereas BeWo cells do not have a detectable mRNA. We have examined the protein secreted by several human cell lines to determine whether the mRNA expressed by these cells, which hybridized with the GSHPx-P probe, codes for GSHPx-P. As shown in Fig 4, A498 (lanes 1 through 3), Adr'MCF-7 (lanes 4 through 6), MCF-7H6 (lanes 7 through 9 and 13 through 15; a GSHPx-1 transfectant of MCF-7 cells), and HepG2 cells (lanes 10 through 12) were labeled metabolically in media containing 100 nmol/L 75Se-selenious acid for 4 to 6 days and the 75Se-labeled proteins were analyzed by SDS-PAGE. Figure 4 shows that A498, Adr'MCF-7, and HepG2, but not MCF-7H6, secrete From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CHU ET AL 3236 A 1 2 3 4 5 6 7 8 ‘1 2 3 4 5 6 7 8 w-- - 28s rRNA - B-Actin - a - Actin --m= GSHPX-P - 1‘ 2 3 4 5 6 7 8 9 1011 121314 Dl2 3 4 5 6 7 8 9 1011 121314 -28s rRNA -&Actin a-Actin GSHPX-P- E l2 3 4 5 5 , 3 4 5 - 2 8 s rRNA ,a -@Actin GSHPX-P - Fig 2. Northern analysis of GSHh-P mRNA in human and animal tissues, and human cell lines. (A, C and E) show a 1.6-kb mRNA hybridized t o a human GSHPx-P cDNA probe; and (B, D, and F) show a 1.9-kb mRNA hybridized t o an pactin probe. (A and B) show the same set of mouse RNAs isolated from Hepa-1 (lane 1). kidney (lane 2). liver (lane 3). testis (lane 4). epididymis (lane 5). uterus (lane 6). heart (lane 7). and brain (lane 8). (C and D) are the same set of RNAs isolated from rat kidney cortex (lane 1).rat kidney medulla (lane 2). rat heart (lane 3). rat cardiac myocytes (lane 4). human heart (lane 5). human lung (lane 6), human breast (lanes 7 and 8). the mammary glands of a pregnant mouse (lane 9). and five human breast cancer lines (lanes 10 through 14). The breast cell lines are SK-BR-3 (lane 10). T47D (lane 11). MDA-MB-231(lane 12). ZR-75-1 (lane 13). and Hs578T (lane 14). (E and F) are the same set of RNAs isolated from other human cell lines, including HepG2 (lane 1).A498 (lane 2), AdrMCF-7 (lane 3). BeWo (lane 4). and MCF-7 (lane 5). 2 3 4 5 6 7 8 ‘1 2 3 4 5 6 7 0 4-28s rRNA GSHPx-P- +@-Actin Fig 3. Northern analysis of mRNA isolated from human tissue. (A) mRNA hybridized t o GSHPx-P cDNA. (B) mRNA hybridized t o pactin. Duplicate samples were from two individuals. Lanes 1 and 2 are from normal kidney, lanes 3 and 4 are from normal liver, lane 5 is from normal heart, lanes 6 and 7 are from normal lung, and lane 8 is from tumor lung of the same individual as lane 7. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 3237 PLASMA GLUTATHIONE PEROXIDASE (GSHPX-P) Media 1 Cytosol HepG2 MCF-7H6 1011 12 13 14 15 Fig 4. 75Se-labeled proteins obtained from four human cell lines resolved by SDS-PAGE. Lanes 1 through 9,11, and 12 are selenoprotein immunoprecipitatesrecovered in the conditioned media, whereas lanes 13 through 15 are cytosolic selenoproteins that are controls to show the specificity of the antisera. Lanes 1, 4, 7, and 13 are immunoprecipitateswith preimmune sera; lanes 2,5,8.11, and 14 are immunoprecipitateswith anti-RBC-GSHPx-1 antisera; lanes 3, 6, 9, 12, and 15 are immunoprecipitates with anti-GSHPx-P antisera; and lane 10 is the pattern of total cytosolic selenoproteins in HepG2 cells. 22.5 kDa b a 22.5-Kd selenoprotein that is immunoprecipitated with antihuman GSHPx-P antisera. DISCUSSION Yoshimura et a1,9 using immunoblotting and Northern hybridization techniques, identified that rat kidney and lung, but not rat liver or heart, expressed GSHPx-P in 13 tissues analyzed. These data suggest that kidney is the source of plasma glutathione peroxidase. However, as shown in Fig 1, there are apparently five isozymes in the glutathione peroxidase family. To prove that the signal detected in kidney by GSHPx-P probes is that of GSHPx-P per se, a nucleic acid or an amino acid sequence analysis of a kidney GSHPx-P cDNA or protein is necessary. Our partial sequence of a cDNA clone isolated from a human kidney cDNA library has shown its identity. When the deduced amino acid sequence of five potential isozymes in the glutathione peroxidase family are compared, there is higher homology between human GSHPx-P and mouse epididymal 24-Kd protein than between human GSHPx-P and human cellular GSHPx-1. When the nucleic acid sequences at the coding region are compared, human GSHPx-P has 67% homology with mouse epididymal 24-Kd protein and 57% homology with human GSHPx-1. However, the 24-Kd epididymal secretory protein is not likely to be the mouse GSHPx-P, based on the facts that (1) the cDNA of the 24-Kd epididymal protein did not hybridize with any mRNA isolated from kidney and other nonepididymal tissue: (2) the amino acid homology of human and rat GSHPx-P is 90%, whereas that of human GSHPx-P and mouse 24-Kd epididymal protein is 71%,7a9 and (3) the cDNA coding for the 24-Kd protein does not appear to contain a TGA codon.' In addition to the mouse kidney, positive signals were also detected in mRNA isolated from mouse epididymis, heart, lung, and mammary gland. It is likely that the signal detected in epididymis is caused by cross-hybridization of the mRNA of the 24-Kd epididymal secretory protein with a GSHPx-P cDNA probe. However, based on the specificity of the probe, which was shown in the isolation of the human kidney GSHPx-P cDNA, it is more likely that these other tissues are expressing GSHPx-P mRNA. Additionally, the HepG2, A498, and Adr'MCF-7 cell lines that expressed an mRNA that hybridized to GSHPx-P cDNA also secrete a selenoprotein that can be immunoprecipitated by antihuman GSHPx-P antisera. This evidence supports the conclusion that these tissues express a GSHPx-P mRNA. We have detected GSHPx-P mRNA in human liver, kidney, heart, lung, and breast. We are confident in these results because, with the exception of human heart, multiple samples were analyzed. Takahashi et all1 have reported that human liver does not express GSHPx-P mRNA, but no data was shown, and they isolated GSHPx-P clones from a human fetal liver cDNA library.ll However, because GSHPx-P is not expressed in mouse and rat liver, there is a definite species difference in its expression in liver. Additionally, unlike rodent kidncy, human kidney does not have overwhelming levels of GSHPx-P mRNA. Considering the mass of liver, the total GSHPx-P expressed in human liver may be equivalent to that expressed in kidney. We have also detected a signal in mRNA isolated from rat heart, rat cardiac myocytes, mouse heart, and human heart; mouse lung and human lung; mouse breast and human breast. Our result indicates that there may be no species difference in GSHPx-P expression in heart, lung, and breast. However, GSHPx-P is expressed at a very low level, if any, in one human tumorous lung sample. More samples need to be analyzed to determine whether there is a general pattern of reduced GSHPx-P mRNA level in tumorous human tissues. We have also studied the expression of GSHPx-P mRNA or protein in 12 cell lines, and found a fair correlation with From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 3238 CHU ET AL the tissues from which they were derived. HepG2 and Hep3B, human hepatoma cells, are similar to human liver and express GSHPx-P; Hepa-1, a mouse hepatoma cell line similar to mouse liver, does not. A498, a human kidney carcinoma cell line, expresses a GSHPx-P mRNA and protein as does human kidney; human breast cancer cells SK-BR-3, T47D, MDA-MD-231, and Adr'MCF-7 are similar to human breast, and express GSHPx-P. However, other breast cancer cells, ZR-75-1, Hs578T, and MCF-7, and BeWo, a human choriocarcinoma cell line, are unlike their tissues of origin, and do not express GSHPx-P. Although the physiologic role of GSHPx-P has not been established, its expression in heart, lung, and breast tissues, in addition to kidney and human liver, suggest that the antioxidant activity may be important in providing local extracellular protection, in addition to providing a potential oxidant scavenger role in the plasma. This local protection maybe particularly important in humans, because human plasma has several-fold lower levels of GSHPx activity compared with rodent plasma (unpublished data). The differential expression of GSHPx-P in breast cancer cell lines is interesting. Because the normal mouse breast tissues were obtained from pregnant females, and the normal human breast were most likely obtained from postpregnant females, its expression in this tissue may be developmentally regulated. ACKNOWLEDGMENT We thank J.N.A. van Balgooy for his preparations of rat cardiac myocytes. REFERENCES 1. Cerutti P A Prooxidant states and tumor promotion. Science 227:375,1985 2. 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Cleveland DW, Lopata MA, MacDonald RJ, Cowan NJ, Rutter WJ, Kirschner M W Number and evolution conservation of a- and p-tubulin and cytoplasmic p- and y-actin genes using specific cloned cDNA probes. Cell 20:95,1980 23. Mullenbach GT, Tabrizi A, Irvine BD, Bell GI, Hallewell RA: Sequence of a cDNA coding for human glutathione peroxidase confirms TGA encodes active site selenocysteine. Nucleic Acids Res 15:5484,1987 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1992 79: 3233-3238 Expression of plasma glutathione peroxidase in human liver in addition to kidney, heart, lung, and breast in humans and rodents FF Chu, RS Esworthy, JH Doroshow, K Doan and XF Liu Updated information and services can be found at: http://www.bloodjournal.org/content/79/12/3233.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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