Loss of Expression of the Metastasis Suppressor Gene Kiss1 During Melanoma Progression and Its Association with LOH of Chromosome 6Q16.3-Q231

[CANCER RESEARCH 61, 7422–7425, October 15, 2001] Advances in Brief

Loss of Expression of the Metastasis Suppressor Gene KiSS1 during Melanoma Progression and Its Association with LOH of Chromosome 6q16.3-q231

Fumiaki Shirasaki, Minoru Takata,2 Naohito Hatta, and Kazuhiko Takehara Department of Dermatology, Graduate School of Medical Science, School of Medicine, Kanazawa University, Kanazawa 920-8641, Japan

Abstract Although the function of the protein encoded by KiSS1 is unknown, the KiSS1 gene product has recently been shown to repress 92-kDa KiSS1 is a putative melanoma metastasis suppressor gene, the expression type IV collagenase (MMP-9) expression by effecting reduced NF-␬B of which may be regulated by another gene(s) mapping to chromosome binding to the promoter (14). Furthermore, very recent investigations 6q16.3-q23. To additionally elucidate the role of KiSS1 in the progression of human melanoma in vivo, we examined KiSS1 mRNA expression in 51 have shown that KiSS1 encodes a COOH-terminally amidated peptide melanocytic tumors with various stages of progression by in situ hybridiza- with 54 amino acid residues, which is a ligand of a novel human tion. We also examined a correlation between loss of KiSS1 mRNA expression G-protein-coupled receptor (named AXOR12 and hOT7T175, respec- and loss of heterozygosity (LOH) of 6q16.3-q23 in 27 melanoma metastases. tively; Refs. 15 and 16). The peptide ligand named as “metastin” by All of the four nevocellular nevi and eight primary melanomas <4mmin one of the investigators inhibits chemotaxis and invasion of thickness showed KiSS1 mRNA expression, whereas only 50% (6 of 12) of hOT7T175-transfected Chinese hamster ovary cells in vitro and at- primary melanomas >4 mm in thickness expressed KiSS1. Loss of KiSS1 tenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 mRNA was equally frequent in metastases; 44% (12 of 27) of tumors lost melanomas in vivo. (16). These findings show functional mechanisms KiSS1 expression. LOH of 6q16.3-q23 was observed in 52% (14 of 27) of metastases. There was a strong association between LOH and loss of KiSS1 by which KiSS1 may act as a metastasis suppressor gene. nine metastases with LOH of 6q16.3-q23 lost KiSS1 To additionally elucidate the role of KiSS1 in the progression of ;(0.03 ؍ expression (P expression, whereas 10 tumors with no LOH showed positive KiSS1 mRNA human melanocytic tumors in vivo, we examined KiSS1 mRNA ex- expression. The findings in this study show, for the first time, KiSS1 down- pression in various stages of progression by in situ hybridization. regulation during the progression of melanoma in vivo and strongly suggest Furthermore, to test the hypothesis that expression of KiSS1 is regu- that inactivation of a tumor suppressor gene(s) mapping to 6q16.3-q23 by lated by a gene(s) mapping to 6q16.3-q23, we also examined a deletion or mutation coupled with LOH may lead to the down-regulation of correlation between loss of KiSS1 mRNA expression and LOH of KiSS1. 6q16.3-q23 in melanoma metastases. Introduction Materials and Methods Cytogenetic analysis and LOH3 studies in melanoma metastases have demonstrated that chromosomes 1, 6, and 10 are the most frequent sites Tissue Samples. Twenty primary tumors and 27 metastases were obtained of chromosome alterations in advanced stage disease (1, 2). In particular, from 41 surgical patients with melanoma (25 males and 16 females with ages the long arm of chromosome 6 is preferentially lost during the progres- ranging from 31 to 84 years). In 5 patients, both primary and metastatic tumors were available. The types of melanoma included 24 acral lentiginous melano- sion of the majority of melanomas, suggesting the existence of a metas- mas, nine nodular melanomas, four superficial spreading melanomas, two tasis suppressor gene(s) important for melanoma metastasis on 6q (3–6). lentigo maligna melanomas, one mucosal melanoma, and one melanoma of Direct experimental evidence for a metastasis suppressor gene(s) on 6q unknown subtype. Eight of the 20 primary tumors were Ͻ4 mm in thickness has been provided by showing suppression of metastasis after introduc- (1.3–3 mm; median, 1.8 mm), and the remaining 12 primary tumors were Ͼ4 tion of a normal copy of chromosome 6 into highly metastatic human mm in thickness (4.2–23 mm; median, 5.5 mm). Metastatic tumors consisted melanoma cell lines (7, 8). By the use of differential display and sub- of 10 lymph node metastases, 12 in-transit metastases, and five distant skin tractive hybridization, a novel cDNA designated as KiSS1 was isolated metastases. In 1 patient, both lymph node and skin metastases were examined. from melanoma cells that had been suppressed for metastatic potential by Four lesions of nevocellular nevus were also included. the introduction of human chromosome 6 (9). Transfection and consti- Partial tissue samples were fixed in 10% neutral buffered formaldehyde and tutive expression of the full-length KiSS1 cDNA suppressed metastasis of processed for routine pathology. The remaining tissue was embedded in human melanoma and breast carcinoma cells, strongly suggesting that Tissue-Tek OCT compound (Miles, IN), snap frozen in liquid nitrogen, and Ϫ ␮ KiSS1 is a novel metastasis suppressor (9–11). Surprisingly, however, stored at 70°C until use. Ten serial 6- m sections were cut from each frozen KiSS1 mapped to 1q32-q41 by fluorescence in situ hybridization, the tissue embedded in OCT compound. The sections were placed on silan-coated glass slides (Dako Japan, Kyoto, Japan). One section was stained with H&E, region not implicated in melanoma progression (12). Evidence from and the remaining sections were subjected to in situ hybridization and DNA subsequent experiments suggests that the expression of KiSS1 is very extraction for PCR-microsatellite LOH analysis. likely to be regulated by a gene(s) localized in the 40-cM region between Probes Preparation. Probes of cDNA for the human KiSS1 were synthe- 6q16.3-q23 (13). sized by PCR based on GenBank nucleotide sequence (accession no. U43527). Amplified cDNA of 282 bp (nucleotide number 124–405) was cloned into Received 7/16/01; accepted 8/28/01. pBluescript vector (Stratagene, La Jolla, CA) and linearized with XbaI for the The costs of publication of this article were defrayed in part by the payment of page sense probe and HindIII for the antisense probe. We confirmed that the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sequence for this clone was identical to the human KiSS1 sequence. DIG- 1 Supported by a Grant-in Aid for Scientific Research (C2_12670812) from the Japan labeled cRNA probes were made using the DIG RNA Labeling Kit (Boeh- Society for the Promotion of Science. ringer Mannheim, Mannheim, Germany) following the protocol provided by 2 To whom requests for reprints should be addressed, at Department of Dermatology, the manufacturer. The labeling efficiency was tested by dot blots of the probes Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8640, Japan. Phone: 81-76-265-2341; Fax: 81-76-234-4270; E-mail: [email protected]. at increasing dilutions on nitrocellulose membranes and detection by anti-DIG 3 The abbreviations used are: LOH, loss of heterozygosity; DIG, Digoxigenin. antibody and substrates, as described below. 7422

In Situ Hybridization. Cryostat sections (6 ␮m) were washed with diethyl Table 1 Expression of KiSS1 mRNA in melanocytic lesions of different pyrocarbonate-treated PBS and fixed in freshly prepared 4% paraformaldehyde progression stages (Wako, Tokyo, Japan) in 0.1 M PBS for 5 min. After washing in three changes of No. of cases with KiSS1 PBS, the slides were treated with 0.1 M triethanolamine ϩ 0.25% acetic anhydride Tumor expression/No. of cases examined (volume for volume; both from Sigma Chemical Co., Poole, United Kingdom) for Nevocellular nevus 4 of 4 (100%) 10 min. The slides were prehybridized for1hat42°C with 60 ␮l of hybridization Primary melanoma Ͻ4 mm in thickness 8 of 8 (100%) Ͼ buffer. The hybridization buffer comprised 50% formamide, 4 ϫ SSC, 1 ϫ Den- Primary melanoma 4 mm in thickness 6 of 12 (50%) Metastasis 15 of 27 (56%) hardt’s solution, 125 ␮g/ml tRNA, and heat-denatured 100 ␮g/ml salmon sperm DNA (Sigma Chemical Co.). The mixture was drained from the slide and 30 ␮lof the hybridization buffer containing 1:100 diluted DIG-labeled cRNA probe for 20hat42°C. The sections were washed once with 2 ϫ SSC at 65°C, once with plastic epidermis overlying nevocellular nevus and primary mela- 2 ϫ SSC at 50°C, twice with 0.1 ϫ SSC at 50°C, and treated with 100 ng/ml noma, KiSS1 mRNA was frequently detected. Although KiSS1 ex- RNase A for 30 min at 37°C. Then, immunological detection was performed using pression was reported in cultured melanocytes using reverse the DIG Nucleic Acid Detection Kit (Boehringer Mannheim) following the man- transcription-PCR (9), KiSS1 transcripts were not detected in normal ufacturer’s instructions. Briefly, the sections were washed for 5 min in buffer 1 melanocytes by in situ hybridization. [0.1 M Tris-HCl and 0.15 M NaCl (pH 7.5)] followed by 60 min in buffer 1 KiSS1 mRNA expression was detected in all four lesions of the containing 1% blocking reagent. Anti-DIG antibody diluted to 1:500 in buffer 1 nevocellular nevus (Fig. 1A). The intensity of labeling was strong in the was added to the sections and incubated for 30 min at room temperature. Chro- cytoplasm of nevus cells located in the reticular dermis. In contrast, nevus mogenic reactions were carried out in the presence of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, and the slides were counter- cells within the epidermis showed negative or weak hybridization signals. stained with methyl-green. The stained slides were evaluated by two independent In primary melanomas with variable thickness, 70% (14 of 20) of tumors observers and scored as diffusely positive (ϩϩ) when the percentage of stained showed KiSS1 mRNA expression. Although the expression was hetero- melanoma cells in the entire lesion was Ͼ75%, heterogeneously positive (ϩ) when geneous with only 25–75% melanoma cells positive in 2 tumors, the the percentage was between 25 and 75%, and negative (Ϫ) when the percentage remaining 12 tumors showed diffuse KiSS1 mRNA expression with was Ͻ25%. The sections stained with the sense probe were used as controls for Ͼ75% of tumor cells positive. All eight primary melanomas Ͻ4mmin nonspecific hybridization. thickness (ranged from 1.3 to 3 mm; median, 1.8 mm) showed KiSS1 Microdissection and DNA Extraction. Microdissection and DNA extrac- mRNA expression. In these tumors, nests of proliferating atypical mela- tion were carried out using the Pinpoint Slide DNA Isolation System (Zymo nocytes within the epidermis were intensely labeled (Fig. 1B). In contrast, Research, Orange, CA). The cryostat sections mounted on glass slides were placed loss of KiSS1 mRNA expression was frequently observed in thicker on an inverted microscope. Adhesive solution was then applied to a tumor area with the aide of matching H&E-stained section. The tumor tissue embedded within lesions (Fig. 1C). Only 50% (6 of 12) of primary melanomas with a Ͼ the dried solution was then lifted with a scalpel blade, transferred into a microfuge tumor thickness 4 mm (thickness ranged from 4.2 to 23 mm; median, tube, and digested for4hat55°C in buffer containing proteinase K. The lysate was 5.5 mm) showed KiSS1 mRNA expression. Loss of KiSS1 mRNA ex- heated at 95°C for 10 min to inactivate proteinase K. Aliquots of 1 ␮l of this lysate pression was equally frequent in metastases; 56% (15 of 27) of the lesions were used directly for PCR. Control DNA was obtained from either peripheral showed a diffuse expression with Ͼ75% of labeled cells, whereas the blood lymphocytes using the DNA Extraction Kit (Stratagene) or normal tissue expression was lost in the remaining 44% (12 of 27) of metastases. (either epithelial or stromal) from the same tissue section. LOH of 6q16.3-q23 in Melanoma Metastases (Table 2). Previ- Microsatellite-PCR LOH Analysis. A total of 13 microsatellite polymor- ous analyses in cell lines as well as in vivo tumors have provided phisms that are mapped to chromosome 6q16.3–23.3 (Table 2) was used for compelling evidence that at least one metastasis suppressor gene impor- analysis. All of the primers for PCR amplification were obtained from Research tant for melanoma progression resides on chromosome 6 (7, 8, 17). Genetics (Huntsville, AL). One of each primer pair was end-labeled with ␥32P- Recently, this putative melanoma metastasis suppressor locus has been ATP. PCR amplification was performed in reaction buffer containing 0.1 ␮M each ϳ oligonucleotide primer, 0.2 mM each deoxynucleotide-triphosphate, 1.5 mM mapped to 40-cM region between D6S468 and D6S314 in 6q16.3-q23 by microcell-mediated chromosomal transfer experiments of a deletion MgCl2, and 0.5 unit of Taq DNA polymerase (Promega, Madison, WI) in a final volume of 10 ␮l. PCR cycles consisted of 1 cycle at 94°C for 3 min followed by variant of human chromosome 6 to highly metastatic human melanoma 29 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. PCR products were cell line C8161 (13). To examine whether the melanoma metastases in separated through 6% denaturing polyacrylamide gels. All samples showing two vivo actually show LOH of this particular chromosome region, and to distinct allelic bands in normal DNA were considered to be informative. The signal additionally refine this metastasis suppressor locus, we carried out PCR- intensities for all informative samples were examined visually by two independent based LOH analysis in 27 metastases by using seven polymorphic loci Ͼ observers. LOH was scored as positive when a clear reduction ( 75%) in signal (D6S268, D6S266, D6S267, D6S287, D6S262, D6S457, and D6S292) intensity was detected in one of the alleles in the tumor DNA compared with the mapped within this region, as well as an additional six flanking loci same allele in the paired normal DNA. Statistics. Two-by-two tables were compared using the ␹2 test. (D6S275, D6S300, D6S468, D6S314, D6S311, and D6S473; Table 2). Overall, 52% (14 of 27) of metastases showed LOH of the 40-cM region Results and the telomeric, as well as centromeric, flanking regions in 6q16.3-q23 (Fig. 1D), confirming that this region is the frequent site of somatic allelic KiSS1 mRNA Expression in Normal Skin, Nevocellular Nevus, loss in melanoma metastases. Additional refinement of this metastasis Primary Melanoma, and Metastasis (Table 1). Analysis of frozen suppressor locus was not possible, however, because all of the 14 me- tissue sections by in situ hybridization showed KiSS1 mRNA expres- tastases except 1 tumor (case 24) showed LOH spanning the entire 40-cM sion in the cytoplasm of cells of nevocellular nevus and melanoma. region between D6S468 and D6S314. A skin metastasis from case 24 The detection of KiSS1 mRNA was carried out using the DIG-labeled showed retention of heterozygosity at D6S268 and D6S262 flanked by antisense RNA probe. The corresponding sense RNA probe was used LOH at the adjacent loci. However, this tumor retained KiSS1 expression. as a control of specificity, and all of the control hybridization was Relationship between the Loss of KiSS1 Expression and LOH of negative. Because of the clear hybridization signals, the scoring of 6q16.3-q23 in Melanoma Metastases (Table 3). In 27 melanoma sections by two independent observers was identical. metastases, 9 tumors that showed LOH of 6q16.3-q23 lost KiSS1 In normal skin, hybridization signals for KiSS1 mRNA was de- expression, whereas 10 tumors with no LOH showed positive KiSS1 tected in pilosebaceous units and eccrine sweat glands. Keratinocytes mRNA expression. Despite the retention of 6q16.3-q23, 3 tumors in normal skin were not labeled with KiSS1. However, in the hyper- were negative for KiSS1. Expression of KiSS1 was detected in 5 7423

Fig. 1. KiSS1 mRNA expression and LOH of 6q16.3-q23 in nevocellular nevus and melanoma. In situ hybridization on cryostat sections with a DIG-labeled antisense cRNA probe for KiSS1 (A–C) and microsatellite-PCR LOH analysis (D). A, a nevocellular nevus showing intense labeling for KiSS1 mRNA in nevus cells in the dermis. Keratinocytes in the overlying epidermis also show positive labeling for KiSS1. Scale bar, 200 ␮m. B, an early primary melanoma with 1.5 mm in thickness showing expression of KiSS1 mRNA in atypical melanocytes proliferating within the epidermis (arrowheads). Scale bar, 100 ␮m. C, an invasive primary melanoma with 5 mm in thickness showing loss of expression of KiSS1 mRNA in contrast to the labeling in the overlying epidermis. Scale bar, 400 ␮m. D, representative autoradiographs of LOH analysis in a lymph node metastasis. Micro- satellite markers D6S267 (left) and D6S457 (right) were amplified from normal (N) and tumor (T) DNA. Arrows, respective alleles. The smaller-sized allele is lost at both loci.

tumors that showed LOH of 6q16.3-q23. The association between that deeply invasive vertical growth phase melanoma cells already LOH and loss of KiSS1 expression was statistically significant harbor numerous cytogenetic abnormalities and are metastasis com- (P ϭ 0.03). petent (19) and that no major additional genetic changes may be required for additional progression to metastatic dissemination (20). Discussion There is compelling evidence that the long arm of chromosome 6 Since the cloning of KiSS1, a number of experiments using cell contains at least four tumor suppressor genes important for melanoma, lines have provided functional evidence showing KiSS1 as a metas- including SOD2 and AIM1 (reviewed by Welch and Goldberg, Ref. tasis suppressor of melanoma and breast carcinoma (9–11, 16). How- 21). Recently, a novel candidate gene was mapped to a 40-cM region ever, the actual expression or loss of KiSS1 in in vivo tumors was between 6q16.3-q23 by microcell-mediated chromosome transfer of a unknown. In the study described here, we show for the first time that deletion variant of neomycin-tagged human chromosome 6 into met- the KiSS1 expression is lost during the progression of melanocytic astatic C8161.9 melanoma subline (13). Because this subline did not tumors in vivo; i.e., all of the lesions of benign nevocellular nevus and express KiSS1 (while it was expressed in the same cell transferred early stage primary melanoma express KiSS1 mRNA, but the expres- with an intact chromosome 6), it is suggested that a tumor suppressor sion is frequently lost in deeply invasive primary melanomas and gene mapping to this region may be a critical regulator of KiSS1 (13). metastases. Marked decrease of KiSS1 expression in primary mela- The finding in the present study showing significant associations nomas Ͼ4 mm in thickness is striking and may in part account for the between loss of KiSS1 mRNA expression and LOH of 6q16.3-q23 in dramatic fall of survival in patients presented with such fairly thick melanoma metastases supports this hypothesis and strongly suggests primary tumors (although these patients are rather rare and constitute that inactivation of the tumor suppressor gene mapping to 6q16.3-q23 Ͻ10% of all melanoma patients; Ref. 18). By contrast, the frequency by deletion or mutation coupled with LOH may lead to the down- of KiSS1 loss between deeply invasive primary melanomas and me- regulation of KiSS1. A tumor suppressor gene may also be inactivated tastases is nearly the same (ϳ50%). This is keeping with the notion by homozygous deletion, promoter methylation, or protein inactiva-

Table 2 LOH of 6q16.3–23.3 in melanoma metastasesa Case no. Cytogenetic Mapping distance Locusb localizationc (cM)c 10 12 14 21 23 24 26 28 29 32 33 34 38 EF ءFEEFFFF ء D6S275 6q16.3–21 102.81 F ءFFFF E ءFFFءء D6S300 6q16.3–21 103.45 FEF ءFFFءءءD6S468 6q16.3–21 107.88 FF Fءء D6S268 6q16.3–21 114.93 FFF EF F ءء FFFءءء D6S266 6q21–23 120.31 FFF FFFFFFءءءFFF ء D6S267 6q21–23 121.97 ءFFءء F ءFF F ء D6S287 6q21–23 121.97 ء F ء FءءءFFFE ء D6S262 6q22.3–23.1 121.97 F FFFF ءFFFFF F ء D6S457 6q21–23.2 130.00 FF ءFF ءD6S292 6q16.3–23.2 136.97 FFFF FF ءء F ءFEءءءءD6S314 6q16.3–27 143.40 FF FEFFءءD6S311 6q21–23.3 147.13 FEFF FF FEFF EF ءFF ءD6S473 6q21–23.3 155.17 FF .uninformative; blank, not examined ,ء ;a F, LOH; E, retention of heterozygosity b Microsatellite markers deleted in a deletion variant of neomycin-tagged human chromosome 6 (neo6qdel; Ref. 13) are indicated in bold letters. c Cytogenetic localization and mapping distance of individual markers is according to information obtained at www.marshfieldclinic.org/research/genetics/. 7424

Table 3 Relationship between KiSS1 mRNA expression and LOH of 6q16.3–q23 in alterations and clonal evolution during progression. J. Investig. Dermatol., 111: melanoma metastasesa 919–924, 1998. 6. Takata, M., Morita, R., and Takehara, K. Clonal heterogeneity in sporadic melanomas LOH of 6q16.3–q23 as revealed by loss-of- heterozygosity analysis. Int. J. Cancer, 85: 492–497, 2000. KiSS1 mRNA expression Positive Negative 7. Trent, J. M., Stanbridge, E. J., McBride, H. L., Meese, E. U., Casey, G., Araujo, D. E., Witkowski, C. M., and Nagle, R. B. Tumorigenicity in human melanoma cell lines Positive 5 10 controlled by introduction of human chromosome 6. Science (Wash. DC), 247: Negative 9 3 568–571, 1990. a P ϭ 0.03. 8. Welch, D. R., Chen, P., Miele, M. E., McGary, C. T., Bower, J. M., Stanbridge, E. J., and Weissman, B. E. Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorige- tion that would not involve LOH (22). This may explain why three nicity. Oncogene, 9: 255–262, 1994. 9. Lee, J. H., Miele, M. E., Hicks, D. J., Phillips, K. K., Trent, J. M., Weissman, B. E., metastases that did not show LOH of 6q16.3-q23 lost KiSS1 expres- and Welch, D. R. KiSS-1, a novel human malignant melanoma metastasis-suppressor sion. An alternative possibility explaining loss of KiSS1 expression in gene [Published erratum appears in J. Natl. Cancer Inst. (Bethesda), 89: 1549, 1997]. these tumors might be direct chromosomal alterations involving chro- J. Natl. Cancer Inst. (Bethesda), 88: 1731–1737, 1996. 10. Lee, J. H., and Welch, D. R. Suppression of metastasis in human breast carcinoma mosome 1q32-q41 where KiSS1 resides. MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1. The findings in this study confirm the importance of KiSS1 down- Cancer Res., 57: 2384–2387, 1997. 11. Lee, J. H., and Welch, D. R. Identification of highly expressed genes in metastasis- regulation in the progression of melanoma in vivo. Recent investiga- suppressed chromosome 6/human malignant melanoma hybrid cells using subtractive tion shows that the KiSS1 gene product is subjected to proteolytic hybridization and differential display. Int. J. Cancer, 71: 1035–1044, 1997. cleavage followed by amide transfer generating a 54 amino acid 12. West, A., Vojta, P. J., Welch, D. R., and Weissman, B. E. Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene (KISS1). Genomics, peptide “metastin,” which is a ligand of an orphan G-protein coupled 54: 145–148, 1998. receptor hOT7T157 (16). Although metastin could be a potential new 13. Miele, M. E., Jewett, M. D., Goldberg, S. F., Hyatt, D. L., Morelli, C., Gualandi, F., therapeutic agent for advanced stage of melanoma (16), perhaps we Rimessi, P., Hicks, D. J., Weissman, B. E., Barbanti-Brodano, G., and Welch, D. R. A human melanoma metastasis-suppressor locus maps to 6q16.3-q23. Int. J. Cancer, need more information; e.g., whether KiSS1 gene product is properly 86: 524–528, 2000. processed in melanoma cells and whether the receptor hOT7T157 is 14. Yan, C., Wang, H., and Boyd, D. D. KiSS-1 represses 92-kDa type IV collagenase expressed in melanomas in vivo. The investigation is now underway expression by down-regulating NF-␬ B binding to the promoter as a consequence of I␬ B␣-induced block of p65/p50 nuclear translocation. J. Biol. Chem., 276: 1164– in our laboratory. Another important issue is the identification of a 1172, 2001. putative KiSS1 regulator gene mapping to 6q16.3-q23. Our attempt to 15. Muir, A. I., Chamberlain, L., Elshourbagy, N. A., Michalovich, D., Moore, D. J., additionally refine the region by constructing deletion maps of met- Calamari, A., Szekeres, P. G., Sarau, H. M., Chambers, J. K., Murdock, P., Steplewski, K., Shabon, U., Miller, J. E., Middleton, S. E., Darker, J. G., Larminie, astatic tumor samples failed because of the preferential large deletions C. G., Wilson, S., Bergsma, D. J., Emson, P., Faull, R., Philpott, K. L., and Harrison, in this chromosome area (23). More sophisticated approaches, such as D. C. AXOR12: A novel human G protein-coupled receptor, activated by the peptide comparison of mRNA expression profiling between a nonmetastatic KiSS-1. J. Biol. Chem., 31: 31, 2001. 16. Ohtaki, T., Shintani, Y., Honda, S., Matsumoto, H., Hori, A., Kanehashi, K., Terao, neo6 cell line and a metastatic neo6qdel cell line (13) by using DNA Y., Kumano, S., Takatsu, Y., Masuda, Y., Ishibashi, Y., Watanabe, T., Asada, M., microarray, might be necessary. 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Fumiaki Shirasaki, Minoru Takata, Naohito Hatta, et al.

Cancer Res 2001;61:7422-7425.

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