Differential Expression of FEZ1/LZTS1 Gene in Lung Cancers and Their Cell Cultures1

2292 Vol. 8, 2292–2297, July 2002 Clinical Cancer Research

Shinichi Toyooka, Yasuro Fukuyama, NSCLC cell lines, it was strongly correlated to D8S261 Ignacio I. Wistuba, Melvyn S. Tockman, and LPL loci in SCLC cell lines. No mutation was found John D. Minna, and Adi F. Gazdar2 within cording region of FEZ1 by PCR-single-strand conformational polymorphism. Hamon Center for Therapeutic Oncology Research [S. T., Y. F., Conclusions: We found differential FEZ1 expression in J. D. M., A. F. G.], and Departments of Pathology [A. F. G.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], University of NSCLC and SCLC cell lines, and the absent expression in 3 Texas Southwestern Medical Center, Dallas, Texas 75390-8593; of 6 short-term cultures of NSCLC tumors. FEZ1 may be Department of Pathology, Pontificia Universidad Catolica de Chile, related to tumorigenesis of lung cancer. Santiago, Chile [I. I. W.]; and Molecular Screening Laboratory, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612-9497 [M .S. T.] INTRODUCTION Lung cancer is the most common cause of cancer deaths in the United States (1) and on clinicopathological grounds is ABSTRACT divided into two major types, NSCLCs and SCLCs. The mo- Purpose: The FEZ1/LZTS1 (FEZ1) gene, located on lecular genetic changes in these two types of lung cancer are chromosome 8p22 (8p22), was identified recently as a can- very different, including specific patterns of allelic loss (2–5). didate tumor suppressor gene. Because loss of heterozygos- Frequent allelic loss at the short arm of chromosome 8p21–23 ity at 8p21–22 is a frequent event in lung cancers, we studied (8p21–23) region has been reported in lung cancers (2, 3, 6–9). FEZ1 alteration in short-term cultures of resected lung In microdissected lung cancers, we found LOH3 in 86% of cancer tumors and cell lines. SCLCs, 100% of squamous cell carcinomas, and 81% of ade- Experimental Design: We examined FEZ1 expression in nocarcinomas (7). Furthermore, deletions commence early dur- 17 non-small cell lung cancer (NSCLC), 19 small cell lung ing the multistage development at the hyperplasia/metaplasia cancer (SCLC) cell lines, and 6 pairs of short-term cultures stage in NSCLC tumor patients and in smokers without cancer of resected NSCLCs and accompanying nonmalignant bron- (7). The deletions may persist for several decades after smoking chial cells (NBECs) by reverse transcription-PCR and West- cessation (7). ern blotting. To investigate the mechanism for silencing, Allelic losses on the 8p have also been reported as a ,cells were cultured with 5-aza-2؅-deoxycytidine or tricho- frequent event in several kinds of cancer including prostate statin A. We screened for genomic mutations by PCR-single- colon, breast, head and neck, urinary bladder, hepatocellular, strand conformational polymorphism. and cholangiocarcinomas (10–21). These findings suggest the Results: Thirteen of 17 NSCLC (76%) and 3 of 19 presence of TSGs in this region. Functional evidence for this hypothesis was provided by chromosome transfer and microcell ؍ SCLC (16%) of cell lines showed absent expression (P 0.001). Of the paired NSCLC-NBEC cultures, 3 of 6 fusion experiments (22–25). showed loss of expression in tumor cell cultures. In the At least two major candidate TSGs have been identified on cell lines retaining expression, the amplicon products in 8p (26, 27). One of these, the FEZ1/LZTS1 (FEZ1) gene located

SCLCs were more intense than those of NSCLCs and on 8p22 encodes a Mr 67,000 leucine-zipper protein (27). NBECs. Expression of FEZ1 was not restored by 5- Genomic mutation and loss of expression of FEZ1 was reported aza-2؅-deoxycytidine and trichostatin A. Although FEZ1 in some solid tumors including esophagus, prostate, and gastric expression was moderately correlated with loss of hetero- cancers (27, 28). In this study, we examined the expression and zygosity of specific microsatellite makers at 8p21–22 in mutation status of FEZ1 gene in NSCLC and SCLC cell lines, and also primary cultures of NSCLC tumors and corresponding NBECs.

Received 2/18/02; revised 4/15/02; accepted 4/15/02. MATERIALS AND METHODS The costs of publication of this article were defrayed in part by the Lung Cancer Cell Lines. Thirty six lung cancer cell lines payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to (17 NSCLC and 19 SCLC cell lines) and corresponding B lym- indicate this fact. phoblastoid lines (n ϭ 30) were established by us (29). The 17 1 Supported by grants from the University of Texas Specialized Program of Research Excellence in Lung Cancer (P50CA70907) and the Early Detection Research Network (5U01CA8497102), National Cancer In- stitute, Bethesda, MD. 2 To whom requests for reprints should be addressed, at Hamon Center 3 The abbreviations used are: LOH, loss of heterozygosity; SCLC, small for Therapeutic Oncology Research, University of Texas Southwestern cell lung cancer; NSCLC, non-small cell lung cancer; TSG, tumor Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390- suppressor gene; RT-PCR, reverse transcription-PCR; NBEC, nonma- 8593.Phone:(214) 648-4921;Fax:(214) 648-4940;E-mail:Adi.gazdar@ lignant bronchial cell; 5-Aza-CdR, 5-aza-2Ј-deoxycytidine; TSA, tricho- UTsouthwestern.edu. statin A; SSCP, single-strand conformational polymorphism.

Table 1 Primer sequences for PCR-SSCP Exon Forward primer Reverse primer Size (bp)a 15Ј-TGCTATGACCTCAGTCCCCTC-3Ј 5Ј-CCATTTTGGAGCTGGACTTGCC-3Ј 280 15Ј-GACGGGCTGCTGAGGTTTGG-3Ј 5Ј-ACTTACCCTTGCCAGCGACC-3Ј 218 25Ј-GCATGAGTCACCGCGGTCCTT-3Ј 5Ј-GGGTGTGACCAGCGGGTCCA-3Ј 284 25Ј-ACACAGCACCAGCAGCAGCTA-3Ј 5Ј-CTGCGCTGCAGCTTCTGGAGG-3Ј 298 25Ј-AGAAGCTGTTGGAGAGGGAGG-3Ј 5Ј-CAAACCCATGAGCCCTGTGTG-3Ј 375 35Ј-ACTCACCTCTTGGCACTCTGTC-3Ј 5Ј-CTTCTTGCGCTGCAGCTCATTC-3Ј 266 35Ј-CCTGCGCACCAAGGGCCTG-3Ј 5Ј-ACATGGCCACGTAGCTCTGCT-3Ј 344 35Ј-GGTGATTCAGTACCAGAAACAGC-3Ј 5Ј-AGAGGGGTCTGAATTGCTGAGC-3Ј 269 a Size, PCR product size in bp.

NSCLC cell lines consisted of 13 adenocarcinomas, 2 squamous 5-Aza-CdR and TSA Treatment. Six lung cancer cell cell carcinomas, and 2 large cell carcinomas. Most NSCLC cell lines (4 cases of NSCLC and 2 cases of SCLC) with loss of lines were derived from primary tumors, and most SCLC cell lines FEZ1 expression were incubated in culture medium with 5-Aza- were from metastases. Cells cultures were grown in RPMI 1640 CdR and TSA each (30). Drug treatment was accomplished by (Life Technologies, Inc., Rockville, MD) supplemented with 5% adding reagents to the culture medium to final concentrations as fetal bovine serum and incubated in 5% CO2 at 37°C. follows: (a) 5-Aza-CdR, 2 ␮g/ml; and (b) TSA, 150 and 300 nM. Primary Culture of Resected Lung Cancer and NBECs. Cells were treated with 5-Aza-CdR for 5 days and TSA for Six NSCLC primary cells (2 cases each of adenocarcinomas, 2 24–48 h. Medium were changed every 48 h for 5-Aza-CdR and squamous cell carcinomas, and 2 large carcinomas) and its every 24 h for TSA. corresponding NBECs were cultured. Primary cultures of re- Western Blot Analysis. A sample of protein (30 ␮g) sected nonmalignant lung tissue were selected for culture and from the cell lysates were separated by SDS-PAGE in 10% placed in trypsin in the cold room for 22–24 h. The next day, the polyacrylamide gels and transferred to nitrocellulose mem- specimen was removed from the cold room and 1 ml of medium branes. The membranes were incubated first with primary anti- was added. The medium was MCDB1532ϩ, consisting of body to Fez1 protein kindly given by Drs. Carlo Croce and MCDB 153 basal medium (Sigma Chemical Company, St. Hideshi Ishii (Kimmel Cancer Center, Jefferson Medical Col- Louis, MO) supplemented with growth factors in a collagen- lege of Thomas Jefferson University, Philadelphia, PA) and then coated dish. Every day, fresh medium was added to the dish. For with rabbit polyclonal antibody as secondary antibody coupled primary lung tumor cell culture, small tumor pieces were enzy- to horseradish peroxidase, after which the membranes were matically disassociated and cultured in MCDB153ϩ medium. developed by SuperSignal West Pico Chemiluminescent Sub- All of the culture pairs have been immunophenotyped with strate (PIERCE, Rockford, IL). vimentin, cytokeratin, and thyroid transcription factor. Early DNA Extraction and PCR-SSCP Assay. Genomic passages from all of the lines were preserved in liquid nitrogen DNA was isolated from all of the cultured cells by SDS/ until tested. proteinase K (Life Technologies, Inc.) digestion, phenol-chlo- RT-PCR Assay. The RT-PCR assay was used to exam- roform extraction, and ethanol precipitation (31). PCR-SSCP ine FEZ1 mRNA expression. Total RNA was extracted from the assay was performed for the cording region of FEZ1 gene. The samples (6 NSCLC primary cells and its corresponding NBECs, primer information used for PCR-SSCP is summarized in 17 NSCLC, and 19 SCLC cell lines) with Trizol (Life Technol- Table 1. PCR amplification was carried out 12 min for 95°C for ogies, Inc.) following the manufacturer’s instructions. Reverse initial denaturation, followed by 35 cycles of 94°C for 20 s, transcription reaction was performed on 2 ␮g of total RNA with 60–64°C for 40 s, and 72°C for 40 s. The PCR products were the SuperScript II First-Strand Synthesis using oligodeoxythy- then denatured, loaded on 6% polyacrylamide gels with and midylic acid primer System (Life Technologies, Inc.), and ali- without 5% (vol/vol) glycerol, electrophoresed, and exposed to quots of the reaction mixture were used for the subsequent PCR X-ray film. amplification. The forward PCR amplification primer was Data Analysis. The frequencies of FEZ1 expression in 5Ј-GAGCCTCATGAAGGAGCAGG-3Ј targeted the end of NSCLCs and SCLCs were compared using ␹2 tests. Probability exon 2, and the reverse primer 5Ј-CAGGTCCTGGGTCCT- values of P Ͻ 0.05 were regarded as statistically significant. The CAGGT-3Ј the beginning of exon 3 of FEZ1 gene, and con- statistical test was two-sided. firmed that genomic DNA was not amplified with these primers. PCR amplification was carried out in a reaction volume of 25 ␮l for 12 min at 95°C for initial denaturation, followed by RESULTS 33 cycles of 94°C for 20 s, 60°C for 30 s, and 72°C for 30 s. We analyzed FEZ1 expression of mRNA in 17 NSCLC and The housekeeping gene GAPDH was used as an internal 19 SCLC cell lines by RT-PCR (Table 2). Thirteen of 17 (76%) control to confirm the success of the reverse transcription NSCLC cell lines lacked FEZ1 expression. By contrast, only 3 reaction. The primers for GAPDH amplification were followed: of 19 (16%) SCLC cell lines lacked FEZ1 expression. The forward primer, 5Ј-ACAGTCCATGCCATCACTGCC-3Ј and re- difference of EFZ1 expression status between NSCLCs and verse primer, 5Ј-GCCTGCTTCACCACCTTCTTG-3Ј. PCR prod- SCLCs was significant (P ϭ 0.001). There was no difference in ucts were analyzed on 2% agarose gels. expression status between adenocarcinoma, squamous cell car-

Table 2 FEZ1 mRNA expression in NSCLC and SCLC cell lines A. NSCLCs NSCLC cell mRNA Protein Any line expression expression D8S261 D8S254 LPL marker HCC366 (AD)a ϩϩNI LOH LOH LOH HCC827 (AD) ϪϪNI LOH LOH LOH H2126 (LC) ϪϪNI RE NI RE H2009 (AD) Ϫ ND LOH LOH LOH LOH H2087 (AD) ϪϪLOH LOH LOH LOH HCC44 (AD) ϪϪLOH LOH NI LOH H1395 (AD) ϪϪNI LOH NI LOH H1437 (AD) ϩϩNI NI NI NI HCC1171 (LC) Ϫ ND RE LOH NI LOH HCC515 (AD) ϪϪNI LOH RE LOH HCC95 (SQ) Ϫ ND LOH RE LOH LOH HCC15 (SQ) Ϫ ND RE RE NI RE H2347 (AD) ϩϩNI RE NI RE HCC193 (AD) Ϫ ND NI RE NI RE HCC78 (AD) Ϫ ND NI NI NI NI H1819 (AD) ϩ ND RE NI RE RE H2122 (AD) Ϫ ND RE NI NI RE Total (%) 4/17 (24) 3/9 (33) 4/8 (50) 8/13 (62) 5/7 (71) 9/15 (60) B. SCLCs mRNA Protein Any SCLC cell line expressiona expression D8S261 D8S254 LPL marker H2171 ϪϪLOH NI LOH LOH HCC33 Ϫ ND LOH RE NI LOH H2107 ϩϩ ϩϩ NI NI LOH LOH H1607 ϩϩ ϩϩ RE NI RE RE H1339 ϩϩ ϩϩ NI LOH NI LOH H209 ϩϩ ϩϩ RE RE NI RE H1184 ϩϩ ND RE RE RE RE H1672 ϩϩ ϩϩ RE RE RE RE H1963 ϩϩ ϩϩ NI NI RE RE H1450 ϩϩ ϩϩ NI RE RE RE H195 ϩϩ ND RE NI RE RE H2028 ϩϩ ND RE NI RE RE H2141 ϩϩ ϩϩ RE NI RE RE H69 ϩϩ ND ND ND ND ND H82 ϩϩ ND ND ND ND ND H146 ϩϩ ND ND ND ND ND H182 ϩϩ ND ND ND ND ND H211 Ϫ ND ND ND ND ND H220 ϩϩ ND ND ND ND ND Total (%) 16/19 (84) 8/9 (89) 2/9 (22) 1/6 (17) 2/10 (20) 4/13 (31) a Expression, FEZ1 expression; AD, adenocarcinoma; SQ, squamous cell carcinoma; LC, large cell carcinoma; ϩϩ, strong positive expression; ϩ, positive expression; Ϫ, negative expression; RE, retained allele; NI, non informative; ND, not done.

cinoma, and large cell carcinoma cell lines. A total of 18 cell pressed stromal cell proliferation, and they consisted entirely of lines (9 NSCLCs and 9 SCLCs) were tested for protein expres- epithelial cells. All 6 of the NBEC cultures and 3 (50%) of the sion by Western blot, and the concordance with the RT-PCR NSCLC cultures demonstrated FEZ1 expression by RT-PCR expression was 100% (Table 2). Representative samples are (Fig. 3; Table 3). The number of cultured cells available was shown in Figs. 1 and 2. Of interest, in positive cell lines, insufficient to perform Western blot study. expression of FEZ1 by both RT-PCR and Western blot was We compared the presence or absence of LOH at 8p21–22 stronger in SCLC cell lines than in NSCLC cell lines (Figs. 1 using three polymorphic markers with FEZ1 expression in 17 and 2). Expression of FEZ1 was not restored by neither 5-Aza- NSCLC and 13 SCLC cell lines (Table 2). Except for 2 NSCLC CdR nor TSA treatment in 6 cell lines of which FEZ1 expres- cell lines, all were informative for at least one marker. LOH at sion was negative. one or more markers was present in 9 of 15 (60%) informative Because of stromal cell contamination, we could not test NSCLC cases and in 5 of 13 (31%) informative SCLC cases. tumors directly for loss of FEZ1 expression. Thus, we chose to The concordances between LOH and FEZ1 expression status test short-term cultures of NSCLC tumors and corresponding were summarized in Table 4. The FEZ1 expression was mod- NBECs. These cells were cultured under conditions that sup- erately correlated with LOH of tested microsatellite makers at

Fig. 3 RT-PCR assay for FEZ1 and GAPDH in 6 NSCLCs and their corresponding NBEC primary cultures. Expression of the housekeeping Fig. 1 Representative examples of RT-PCR assay for FEZ1 RNA gene GAPDH was used as a control for RNA integrity. PC, positive expression in NSCLC and SCLC cell lines. Expression of the house- control; NC, negative control (genomic DNA); T, NSCLC cell culture keeping gene GAPDH was used as a control for RNA integrity. PC, sample; N, corresponding nonmalignant epithelial cell culture sample. positive control; NC, negative control (genomic DNA); [NS], NSCLC; [S], SCLC. Table 3 FEZ1 mRNA expression in primary NSCLC and corresponding bronchial epithelial cell cultures RT-PCR Pair no. Sample no. Histology expressiona 1 Tumor LC Ϫ NBEC ϩ 2 Tumor LC ϩ NBEC ϩ 3 Tumor SQ ϩ NBEC ϩ 4 Tumor AD ϩ Fig. 2 Representative figure of Western bolt analysis for Fez1 in NBEC ϩ NSCLC and SCLC cell lines. Expression of the housekeeping gene 5 Tumor SQ Ϫ Actin was used as a control for protein loading. [NS], NSCLC; [S], NBEC ϩ SCLC. 6 Tumor AD Ϫ NBEC ϩ a Expression, FEZ1 mRNA expression; ϩ, positive expression; Ϫ, negative expression; AD, adenocarcinoma; SQ, squamous cell carci- 8p21–22 in NSCLC cell lines, it was strongly correlated to noma; LC, large cell carcinoma. D8S261 and LPL loci in SCLC cell lines (both concordances were 82%). We examined the genomic mutation by PCR-SSCP using Expression in SCLC cell lines was relatively strong compared eight sets of primer covering cording region of FEZ1 gene. with NSCLC cell lines, tumors, and nonmalignant bronchial However, no evidence of genomic mutation was detected by this epithelium. Whereas the origin of SCLC is controversial, one technique. possibility is that they arise from bronchial cells programmed to become neuroendocrine cells and that FEZ1 expression in neu- DISCUSSION roendocrine cells is greater than in the other epithelial cells. Of FEZ1 was identified recently as a candidate TSG at 8p22, interest, FEZ1 is strongly expressed in brain tissue (27). and absence or reduction of FEZ1 expression and occasional The mechanism of down-regulation of FEZ1 expression in genomic mutation were reported in several kinds of cancers human cancers has not been demonstrated convincingly. Muta- (27). Introduction of FEZ1 into Fez1-negative cells resulted in tions are relatively rare, and we could find no evidence of inhibition of cell growth, suggesting that FEZ1 is a candidate mutations in expression negative lung cancer cell lines. Recently TSG. Fez1 is associated with microtubule components and aberrant methylation of CpG islands in the promoter region has interacts with p34 (cdc2) at late S-G2/M stage in vivo (32). become established as an important epigenetic mechanism for Our data demonstrate a highly significant difference (P ϭ gene silencing (33). We reported that the profile of aberrant 0.001) between the rates of FEZ1 inactivation in NSCLC (76%) methylation was different between NSCLCs and SCLCs (34). and SCLC (16%) cell lines. There was complete concordance Vecchione, et al. (28) reported that hypermethylation of 5Ј between RNA expression (as detected by RT-PCR) and protein region of FEZ1 (1 kb including exon 2) was present in gastric expression (as detected by Western blot). These findings are in cancer cell lines and also peripheral leukocyte DNA; however, accord with previous reports of more frequent allelic loss at the the status of methylation of the 5Ј region of FEZ1 was not gene location (8p22) in NSCLC carcinomas and cell lines com- correlated with the level of expression. Because the expression pared with SCLC cell lines (7). Expression data correlated with of FEZ1 was not restored by the 5-Aza-CdR treatment in our allelic loss, particularly with LOH at marker D8S261. Exami- study, aberrant methylation may not be related to silencing of nation of a limited number of short-term cultures of paired FEZ1 expression in both NSCLCs and SCLCs. Like other NSCLC tumors and their corresponding bronchial epithelial mechanisms for controlling gene expression, histone deacetyla- cultures demonstrated expression in all of the NBEC cultures tion causes silencing of gene expression (35). However, failure but loss of expression in 50% of the paired tumors. This illus- of restoration of FEZ1 expression by treatment with the histone trates that the loss of FEZ1 was likely sustained during neoplas- deacetylation inhibitor TSA indicated that histone deacetylation tic transformation and is not an artifact of long-term cell culture. was not the cause of silencing of FEZ1 expression. Thus, the

Table 4 Concordances between FEZ1 expression and 8p21–22 short arm of chromosome 8 in non-small cell lung carcinoma. Genes polymorphic markers in NSCLC and SCLC cell lines Chromosomes Cancer, 7: 85–88, 1993. Data of all the informative cell lines are presented. Loss of FEZ1 10. Kagan, J., Stein, J., Babaian, R. J., Joe, Y. S., Pisters, L. L., expression was correlated with allelic loss at three informative markers Glassman, A. B., von Eschenbach, A. C., and Troncoso, P. Homozygous near the FEZ1 gene at 8p21–22. deletions at 8p22 and 8p21 in prostate cancer implicate these regions as the sites for candidate tumor suppressor genes. Oncogene, 11: 2121– Concordance (%) 2126, 1995. Type D8S261 D8S254 LPL Any marker 11. Bova, G. S., Carter, B. S., Bussemakers, M. J., Emi, M., Fujiwara, NSCLC 5/8 (63) 8/13 (62) 5 /7 (71) 10/15 (67) Y., Kyprianou, N., Jacobs, S. C., Robinson, J. C., Epstein, J. I., Walsh, SCLC 9/9 (100) 4/6 (67) 9 /10 (90) 11/15 (85) P. C., and et al. Homozygous deletion and frequent allelic loss of Total 14/17 (82) 12/19 (63) 14 /17 (82) 23/29 (79) chromosome 8p22 loci in human prostate cancer. Cancer Res., 53: 3869–3873, 1993. 12. Kerangueven, F., Essioux, L., Dib, A., Noguchi, T., Allione, F., Geneix, J., Longy, M., Lidereau, R., Eisinger, F., Pebusque, M. J., and et al. Loss of heterozygosity and linkage analysis in breast carcinoma: mechanism of FEZ1 gene silencing in lung and other tumors indication for a putative third susceptibility gene on the short arm of remains unknown. chromosome 8. Oncogene, 10: 1023–1026, 1995. Our findings indicate that FEZ1 is inactivated frequently in 13. Yaremko, M. L., Wasylyshyn, M. L., Paulus, K. L., Michelassi, F., NSCLC tumors and cell lines, and that inactivation is associated and Westbrook, C. A. Deletion mapping reveals two regions of chro- with frequent allelic loss at 8p21–22. These findings support a mosome 8 allele loss in colorectal carcinomas. Genes Chromosomes role for FEZ1 as a TSG in NSCLCs. Of interest, other onco- Cancer, 10: 1–6, 1994. genes and TSGs demonstrate striking differences between the 14. Yaremko, M. L., Recant, W. M., and Westbrook, C. A. Loss of heterozygosity from the short arm of chromosome 8 is an early event in two major forms of lung cancer, including RAS, MYC ampli- breast cancers. Genes Chromosomes Cancer, 13: 186–191, 1995. fication, and inactivation of the p16 and caspase 8 genes (36– 15. Emi, M., Fujiwara, Y., Nakajima, T., Tsuchiya, E., Tsuda, H., 39). These studies support the concept that these forms of lung Hirohashi, S., Maeda, Y., Tsuruta, K., Miyaki, M., and Nakamura, Y. cancer may arise via different pathogenetic pathways. Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer. Cancer Res., 52: ACKNOWLEDGMENTS 5368–5372, 1992. 16. Fujiwara, Y., Emi, M., Ohata, H., Kato, Y., Nakajima, T., Mori, T., We thank Drs. 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Shinichi Toyooka, Yasuro Fukuyama, Ignacio I. Wistuba, et al.

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