Cancer Immun 1424-9634Academy of Cancer Immunology Cancer Immunity (1 December 2006) Vol. 6, p. 12 Submitted: 26 September 2006. Accepted: 10 October 2006. Copyright © 2006 by Andrew J. G. Simpson 061012 Article

Physical interaction of two cancer-testis antigens, MAGE-C1 (CT7) and NY-ESO-1 (CT6)

Hearn J. Cho1*,**, Otavia L. Caballero2*, Sacha Gnjatic2, Valéria C. C. Andrade3, Gisele W. Colleoni3, Andre L. Vettore4, Hasina H. Outtz1, Sheila Fortunato2, Nasser Altorki1, Cathy A. Ferrera1, Ramon Chua2, Achim A. Jungbluth2, Yao-Tseng Chen1, Lloyd J. Old2 and Andrew J. G. Simpson2 1Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA 2Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA 3Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, SP, Brazil 4Ludwig Institute for Cancer Research, Sao Paulo Branch, Sao Paulo, SP, Brazil *These authors contributed equally to this work **Present address: NYU Cancer Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA

Contributed by: LJ Old

Cancer/testis (CT) antigens are the products of germ line- encoded on the X (CT-X antigens) and those that associated that are activated in a wide variety of tumors and can are not (non-X CT antigens) (1). elicit autologous cellular and humoral immune responses. CT It is estimated that 10% of the genes on the X-chromosome antigens can be divided between those that are encoded on the X belong to CT-X families (5). CT-X antigens are encoded by chromosome (CT-X antigens) and those that are not (non-X CT genes that have recently undergone rapid evolution and antigens). Among the CT-X antigens, the melanoma antigen amplification (6) and are usually highly expressed in (MAGE) family, defined by a shared MAGE homology domain spermatogonia (7, 8, 9). The CT-X genes are frequently (MHD), is the largest. CT-X genes are frequently expressed in a expressed in a coordinate manner in cancer cells (10) and their coordinate manner in cancer cells, and their expression appears to be expression appears to be modulated by epigenetic mechanisms, modulated by epigenetic mechanisms. The expression of CT-X genes such as promoter hypermethylation and deacetylation is associated with advanced disease and poor outcome in different (11). tumor types. We used the yeast two-hybrid system to identify In several different tumor types, the expression of CT-X genes putative MHD-interacting . The MHD of MAGE-C1 (CT7) is associated with advanced disease and poor outcome. In non- was used as bait to screen a human testis cDNA library. This study small cell lung cancer (NSCLC), cancer/testis , identified NY-ESO-1 (CT6) as a MAGE-C1 binding partner. either cumulatively or individually, showed significant Immunoprecipitation and immunofluorescence staining confirmed associations with advanced tumor type, nodal and pathologic MAGE-C1 interaction with NY-ESO-1, and cytoplasmic co- stages, as well as pleural invasion (10). In the same study, the localization of both proteins in melanoma cells. Co-expression of expression of NY-ESO-1 (CTAG1B) and MAGEA3 was each these two genes was found to occur in cancer cell lines from different found to be a marker for poor prognosis, independent of origins, as well as in primary tumors (multiple myeloma and non- confounding variables. Likewise, expression of MAGEA3 in small cell lung cancer samples). This is the first report of direct pancreatic ductal adenocarcinoma was found to be a prognostic interaction between two CT antigens and may be pertinent in the factor for poor survival (12). In colorectal cancers, NY-ESO-1 light of the frequently coordinated expression of these proteins. gene expression may serve as a marker for local metastasis and advanced disease, while expression of MAGEA4 is significantly associated with vessel emboli (13). Similarly, expression of Keywords: two-hybrid assay, MAGE-C1, NY-ESO-1, human, MAGEA1, MAGEA3, MAGEA4, MAGEC1 (CT-7), and multiple myeloma, NSCLC, RT-PCR NY-ESO-1 in malignant gammopathies correlates with stage and risk status of disease (14). Co-expression of SSX1, 2, 4, and 5 also correlates with adverse prognosis in multiple myeloma patients Introduction (15). Immunohistochemistry (IHC) has demonstrated that 82% Cancer/testis antigens (CT antigens) are present in normal of stage III myeloma specimens expressed CT7 and 70% germ line tissues, such as testis, placenta, and ovary and in a expressed MAGE-A3/6, with CT7 protein expression increasing range of human cancers (1). They constitute a promising class of with advanced stage of disease and higher levels of CT7 and tumor antigens due to their limited expression in somatic tissues MAGE-A3/6 proteins being correlated with elevated plasma-cell and strong immunogenicity (2). Cancer/testis antigens have proliferation (16). been isolated by various methods, including cDNA expression Although these data indicate that CT antigen expression might cloning with tumor-reactive CTLs and patients' sera (SEREX), contribute to tumorigenesis, their biological role in both germ cDNA subtractions, and also by EST or MPSS database mining line and tumors remains poorly understood. Recent studies have (2, 3, 4). More than 44 CT genes and/or gene families have been provided some evidence that CT antigens play a role in human identified to date that can be divided between those that are tumorigenesis. Through a yeast two-hybrid assay, the transcriptional regulator SKIP was identified as a MAGE-A1

www.cancerimmunity.org 1 of 8 Cancer Immunity (1 December 2006) Vol. 6, p. 12 binding partner (17). SKIP connects DNA-binding proteins to most family members remains uncharacterized. The cancer/ other proteins that either activate or repress transcription, and testis antigens of the MAGE family are localized in clusters on participates in a range of signaling pathways, including those the (22): MAGEA genes at Xq28, MAGEB genes involving vitamin D, retinoic acid, estrogens, glucocorticoids, at Xp21, and MAGEC genes at Xp26-27; these are classified as Notch1 and transforming growth factor-β. In the Notch1 Type I MAGE genes (21). Type II MAGE genes (MAGED, pathway, MAGE-A1 was found to disrupt SKIP-mediated MAGEE, MAGEF, MAGEG, MAGEH and necdin) are expressed Notch1 signal transduction by binding to SKIP and recruiting in many normal tissues at various levels (22). The sequence of histone deacetylase, therefore acting as a transcriptional the MAGE homology domain is a common feature of the Type I repressor. Yeast two-hybrid studies using other cancer-related and II MAGE gene families; it can be found in mammalian genes as bait have twice pulled out MAGE proteins: MAGE-A11 species, as well as in Xenopus, Drosophila, and zebrafish (23). and MAGE-A4 (18, 19). MAGE-A11 was found to have a role in The MHD does not contain any regions of significant homology the regulation of androgen receptor function by modulating its with other known proteins, but detailed analysis of a number of internal domain interactions and was found to have a dual type II MAGE proteins shows that this domain is an important amplifying effect on androgen signaling (18). MAGE-A4 was site for protein–protein interactions (24). The promoters and identified in a search for binding partners of the oncoprotein first exons of the MAGEA genes show considerable variability, gankyrin (19). Gankyrin destabilizes the retinoblastoma tumor suggesting that they are subject to different transcriptional suppressor, contributing to unscheduled entry into the controls. and escape from cell-cycle arrest and/or . MAGE-A4 To investigate further the function of members of the CT suppresses the oncogenic activity of gankyrin through the action antigen family, we used the yeast two-hybrid system to identify of a peptide that is naturally cleaved from the carboxyl terminus putative MHD-interacting proteins. The MHD of MAGE-C1 of MAGE-A4 and which induces p53-dependent and p53- (CT7) was used as bait to screen a human testis cDNA library. independent apoptosis. Overexpression of MAGEA4 in a human MAGE-C1 is about 800 amino acids longer than the other embryonic kidney cell line (293 cells) was found to increase MAGE proteins and contains a large number of unique short apoptosis as measured by apoptotic index and caspase-3 activity, repetitive sequences in front of the MAGE homologous while MAGEA4 silencing using a small interfering RNA sequence. As a result of this investigation, we identified another approach resulted in decreased caspase-3 activity in a squamous CT antigen, NY-ESO-1 (CT6) as a MAGE-C1 binding partner. cell lung cancer and in 293/MAGE-A4 cells (20). This is the first report of direct interaction between two CT The MAGE family consists of a large group of proteins that antigens and may be pertinent in the light of the frequently harbor the MAGE homology domain (MHD), a well-conserved coordinated expression of these proteins. domain of about 200 amino acids (21). The functional role of

Table 1 Potential MAGE-C1/CT7 binding partners identified in the yeast two-hybrid screen

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Results cancer cell lines. NY-ESO-1 transcripts were detected in 38 (43%) cancer cell lines. In 74% of the cases, there were Identification of proteins interacting with the MHD of MAGE-C1 concordant results (either negative or positive) between (CT7) expression of MAGEC1 and NY-ESO-1. In 28 cell lines (32%), The MHD of MAGE-C1 (CT7) was cloned into a bait both genes were found to be co-expressed. construct and used to screen a high complexity human testis In general, these preliminary results suggested that NY-ESO-1 cDNA library in yeast two-hybrid assays. Human testis was expression may coincide with MAGEC1 expression in many selected because it is the normal tissue in which type I MAGE cancer cell types. We next decided to examine primary tumors genes are expressed. Eighteen clones selected from a pool of from cohorts of multiple myeloma and NSCLC samples for the positive candidates interacted specifically with the MHD upon co-expression of MAGEC1 and NY-ESO-1. As shown in Table 3, reconstitution analysis. Subsequent sequence analysis showed co-expression of MAGEC1 and NY-ESO-1 also occurs in that most of these clones contained genomic DNA or non- primary tumors. coding regions of cDNAs (Table 1). We selected the clone Thirty-nine multiple myeloma samples were tested by RT-PCR containing a 662 bp insert with 100% identity to the human for both MAGEC1 and NY-ESO-1 expression. Of these, 13 NY-ESO-1 gene (RefSeq Accession No. NM_001327) for further samples were positive for NY-ESO-1 (33%) and 30 positive for analysis. MAGEC1 (77%). A total of 456 NSCLC samples were tested by RT-PCR for expression of both MAGEC1 and NY-ESO-1. There Co-precipitation of MAGE-C1 and NY-ESO-1 from cell lysates were 120 samples positive for NY-ESO-1 (26%) and 101 samples To further analyze protein-protein interactions between positive for MAGEC1 (22%). From these values, assuming that MAGE-C1 (CT7) and NY-ESO-1 (CT6) in human cells, we expression of these antigens is independent, the expected prepared total lysates from the melanoma derived SK-MEL-37 frequency of NSCLC samples positive for both MAGEC1 and cell line that expresses both proteins. Complexes between NY-ESO-1 is 6%, i.e., about 26 samples. The observed frequency endogenous MAGE-C1 and NY-ESO-1 proteins were of NY-ESO-1 positive and MAGEC1 positive samples is 13% (59 precipitated when both anti-NY-ESO-1 antibodies (NY-41 and samples), i.e., more than twice what was expected (P < 0.001). E978) were used, as well as with anti-MAGE-C1 monoclonal antibody (CT7.33). The presence of both proteins in the complex was detected by subsequent Western blotting using Discussion antibodies against MAGE-C1 and NY-ESO-1. As shown in In this study we report the first evidence of specific protein- Figure 1A, MAGE-C1 and NY-ESO-1 proteins co-precipitated protein interaction between two distinct CT-X antigens via the when both antibodies (E978 and NY-41) against NY-ESO-1 MHD of MAGE-C1. This finding suggests the possibility that were used. Similarly, NY-ESO-1 was detected in Western blots cancer/testis antigens exert and/or regulate their activities by anti-CT7 mAb CT7-33, although with a much lower through specific interactions with other CT-X antigens. efficiency. MAGE-C1 and NY-ESO-1 were also co-precipitated However, the positive interaction between MAGE-C1 and when the anti-NY-ESO-1 antibodies were used in two additional NY-ESO-1 could be due to bridging by other proteins and in cells lines: SK-LC-17, a NSCLC cell line (data not shown) and this case MAGE-C1 and NY-ESO-1 may not directly bind to U266, a multiple myeloma cell line (Figure 1B). In contrast, co- each other, but may be part of a multi-protein complex. precipitation reactions with a rabbit polyclonal antibody against In normal testis, the CT-X genes are generally expressed in actin or a monoclonal against influenza hemagglutinin (HA) germ cells, particularly spermatogonia, but their function failed to precipitate MAGE-C1 or NY-ESO-1, indicating that the during male germ cell development is not known. Insights into MAGE-C1/ NY-ESO-1 interaction was specific (Figure 1, panels the function of these genes may provide links between A and B). Additionally, we used SK-MEL-10, another melanoma spermatogenesis and tumor growth and may identify novel cell line that expresses MAGEC1 but does not express therapeutic targets amenable to immunologic or pharmacologic NY-ESO-1. As shown in Figure 1C, CT7.33 could efficiently strategies. immunoprecipitate MAGE-C1, but neither of the NY-ESO-1 The expression of different CT-X antigens in tumors is specific antibodies (E978 and NY-41) could precipitate positively correlated, suggesting that these genes may be MAGE-C1 in the absence of NY-ESO-1, further indicating that activated by a common mechanism. Both promoter the interaction between MAGE-C1 and NY-ESO-1 is specific. demethylation and non-demethylation dependent induction are responsible for the ectopic expression of several CT antigens Subcellular co-localization of NY-ESO-1 and MAGE-C1 (11). CT antigen expression in tumors may be the result of the We next examined whether the association between activation of a coordinated gene-expression program, rather MAGE-C1 and NY-ESO-1 resulted in co-localization of the two than of independent events (1). proteins in vivo using double immunofluorescence staining of The first evidence of the function of MAGE family members SK-MEL-37 cells. As shown in Figure 2, MAGE-C1 and came from recent studies suggesting important roles for type II NY-ESO-1 presented a diffuse distribution throughout the MAGE proteins via transcriptional regulation in cell cycle cytosol in exponentially growing SK-MEL-37 cells. MAGE-C1 control and apoptosis. Clues are also emerging showing that was also distributed in the cell nuclei. MAGE-C1 and NY-ESO-1 type I MAGE genes have roles as transcriptional regulators. appear to co-localize to the cytoplasm of these cells. MAGE-A1 was found to bind SKIP and to recruit histone deacetylases, acting as a potent transcriptional repressor (17). Analysis of MAGEC1 and NY-ESO-1 expression in cultured cells and MAGE-A11 was identified as an androgen receptor (AR) tumors coregulator that increases AR activity by modulating the AR The frequency of expression of MAGEC1 and NY-ESO-1 in 88 interdomain interaction (18). MAGE-A2 was shown to interact human cancer cell lines representing a broad range of solid and and repress p53 activity by recruiting transcription repressors to hematological malignancies was examined by RT-PCR p53 transcription sites, conferring resistance to apoptosis (25). (Table 2). MAGEC1 transcripts were detected in 42 of 88 (48%)

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Figure 1

MAGE-C1 (CT7) specifically interacts with NY-ESO-1. (A) Endogenous CT7-NY-ESO-1 complexes were immunoprecipitated from SK-MEL-37 cell lysates with two anti-NY-ESO-1 antibodies (E-978 and NY-41). Both proteins were detected with anti-NY-ESO-1 and anti-CT7 antibodies. Precipitation with anti-actin antibody served as a negative control. (B) Endogenous CT7-NY-ESO-1 complexes were immunoprecipitated from U266 cell lysates with an anti-NY-ESO-1 antibody (NY-41) and also with anti-CT7 antibody (CT7.33) after longer exposure (lower panel). Both proteins were detected with anti-NY-ESO-1 and anti-CT7 antibodies. Precipitation with anti- influenza hemagglutinin antibody (HA) served as a negative control. (C) CT7 could not be immunoprecipitated from SK-MEL-10 cell lysates using anti-NY-ESO-1 anti- bodies (E-978 and NY-41). The sizes of the proteins detected are indicated on the right. Abbreviations: ACT, actin negative control; WCL, whole cell extract.

Figure 2

MAGE-C1 (CT7) co-localizes with NY-ESO-1 to the cytoplasm of SK-MEL-37 cells. Endogenous MAGE-C1 (CT7) and NY-ESO-1 were detected by immunofluores- cence using (A) anti-CT7 and (B) anti-NY-ESO-1 antibodies and visualized using Alexa Fluor 488 and 633 labeled secondary antibodies, respectively. (C) The images were overlapped (Merge) to reveal co-localization. (D) Nuclei were counterstained with DAPI.

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Table 2 Co-expression of NY-ESO-1 (CT6) and MAGEC1 (CT7) in cancer cell lines

Because in most of these cases regions including the MHD interaction may indicate whether the interaction of these two were involved in protein-protein interactions, we decided to use proteins plays a role in tumorigenesis. the MAGE-C1 MHD as bait in a yeast two hybrid screen. The Finally, CT-X gene products have been extensively investigated physical interaction between the MHD of endogenous as targets for cancer vaccines. Insight into the interactions MAGE-C1 and NY-ESO-1 could be confirmed by co- between these proteins may provide a rational basis for immunoprecipitation in a melanoma cell line, and was also polyvalent vaccine formulations that may target specific tumor- observed in multiple myeloma and NSCLC cells. Both proteins promoting regulatory pathways at multiple levels or that target were shown to co-localize to the cytoplasm. Moreover, analysis multiple pathways. The understanding of the biology of these of expression in a broad range of human tumor cell lines also proteins in both normal and cancer cells is critical to the suggests that coordinate expression of MAGEC1 and NY-ESO-1 development of effective vaccine therapy. It may also lead to may be a common event in a subset of cancers. Together, these other classes of targeted therapy that may be combined with data identify a novel interaction between cancer/testis antigens. vaccines in order to neutralize the tumorigenic effects of CT-X Further studies to identify the functional consequence of this proteins.

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Table 3 Co-expression of MAGEC1 and NY-ESO-1 in cancer cell lines and primary tumors

Abbreviations David R, Davies J, Davis C, Davis J, Delgado O, Deshazo D, Dhami CT, cancer/testis; MHD, MAGE homology domain; NSCLC, P, Ding Y, Dinh H, Dodsworth S, Draper H, Dugan-Rocha S, Dun- non-small cell lung cancer; PBST, PBS with 0.1% Tween 20 ham A, Dunn M, Durbin KJ, Dutta I, Eades T, Ellwood M, Emery- Cohen A, Errington H, Evans KL, Faulkner L, Francis F, Frankland J, Fraser AE, Galgoczy P, Gilbert J, Gill R, Glockner G, Gregory SG, Acknowledgements Gribble S, Griffiths C, Grocock R, Gu Y, Gwilliam R, Hamilton C, HJC was funded by a Clinical Investigation Award from the Hart EA, Hawes A, Heath PD, Heitmann K, Hennig S, Hernandez J, Cancer Research Institute. VCCA is recipient of a fellowship Hinzmann B, Ho S, Hoffs M, Howden PJ, Huckle EJ, Hume J, Hunt from Fundação de Amparo à Pesquisa do Estado de São Paulo, PJ, Hunt AR, Isherwood J, Jacob L, Johnson D, Jones S, de Jong PJ, Brazil. 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(MAGE-C1) antigen expression in normal and neoplastic tissues. 20. Peikert T, Specks U, Farver C, Erzurum SC, Comhair SA. Mela- Int J Cancer 2002; 99: 839-845. (PMID: 12115486) noma antigen A4 is expressed in non-small cell lung cancers and promotes apoptosis. Cancer Res 2006; 66: 4693-4700. (PMID: 9. Takahashi K, Shichijo S, Noguchi M, Hirohata M, Itoh K. Identifica- 16651421) tion of MAGE-1 and MAGE-4 proteins in spermatogonia and pri- mary of testis. Cancer Res 1995; 55: 3478-3482. 21. Barker PA, Salehi A. The MAGE proteins: emerging roles in cell (PMID: 7627949) cycle progression, apoptosis, and neurogenetic disease. J Neurosci Res 2002; 67: 705-712. (PMID: 11891783) 10. Gure AO, Chua R, Williamson B, Gonen M, Ferrera CA, Gnjatic S, Ritter G, Simpson AJ, Chen YT, Old LJ, Altorki NK. Cancer-testis 22. Chomez P, De Backer O, Bertrand M, De Plaen E, Boon T, Lucas S. genes are coordinately expressed and are markers of poor outcome An overview of the MAGE gene family with the identification of all in non-small cell lung cancer. Clin Cancer Res 2005; 11: 8055-8062. human members of the family. Cancer Res 2001; 61: 5544-5551. (PMID: 16299236) (PMID: 11454705)

11. Wischnewski F, Pantel K, Schwarzenbach H. Promoter demethyla- 23. Sasaki A, Hinck L, Watanabe K. RumMAGE-D the members: struc- tion and histone acetylation mediate gene expression of MAGE-A1, ture and function of a new adaptor family of MAGE-D proteins. J -A2, -A3, and -A12 in human cancer cells. Mol Cancer Res 2006; 4: Recept Signal Transduct Res 2005; 25: 181-198. (PMID: 16194933) 339-349. (PMID: 16687489) 24. Taniura H, Kobayashi M, Yoshikawa K. Functional domains of nec- 12. Kim J, Reber HA, Hines OJ, Kazanjian KK, Tran A, Ye X, Amersi FF, din for protein-protein interaction, nuclear matrix targeting, and Martinez SR, Dry SM, Bilchik AJ, Hoon DS. The clinical signifi- cell growth suppression. J Cell Biochem 2005; 94: 804-815. (PMID: cance of MAGEA3 expression in pancreatic cancer. Int J Cancer 15578580) 2006; 118: 2269-2275. (PMID: 16331618) 25. Monte M, Simonatto M, Peche LY, Bublik DR, Gobessi S, Pierotti 13. Li M, Yuan YH, Han Y, Liu YX, Yan L, Wang Y, Gu J. Expression MA, Rodolfo M, Schneider C. MAGE-A tumor antigens target p53 profile of cancer-testis genes in 121 human colorectal cancer tissue transactivation function through histone deacetylase recruitment and adjacent normal tissue. Clin Cancer Res 2005; 11: 1809-1814. and confer resistance to chemotherapeutic agents. Proc Natl Acad (PMID: 15756003) Sci U S A 2006; 103: 11160-11165. (PMID: 16847267)

14. Dhodapkar MV, Osman K, Teruya-Feldstein J, Filippa D, Hedvat 26. NCBI BLAST program. URL: http://www.ncbi.nlm.nih.gov/BLAST/ CV, Iversen K, Kolb D, Geller MD, Hassoun H, Kewalramani T, Comenzo RL, Coplan K, Chen YT, Jungbluth AA. Expression of 27. Vaughan HA, Svobodova S, Macgregor D, Sturrock S, Jungbluth cancer/testis (CT) antigens MAGE-A1, MAGE-A3, MAGE-A4, CT- AA, Browning J, Davis ID, Parente P, Chen YT, Stockert E, St Clair 7, and NY-ESO-1 in malignant gammopathies is heterogeneous and F, Old LJ, Cebon J. Immunohistochemical and molecular analysis of correlates with site, stage and risk status of disease. Cancer Immun human melanomas for expression of the human cancer-testis anti- 2003; 3: 9. (PMID: 12875607) gens NY-ESO-1 and LAGE-1. Clin Cancer Res 2004; 10: 8396-8404. (PMID: 15623618) 15. Taylor BJ, Reiman T, Pittman JA, Keats JJ, de Bruijn DR, Mant MJ, Belch AR, Pilarski LM. SSX cancer testis antigens are expressed in 28. van Baren N, Brasseur F, Godelaine D, Hames G, Ferrant A, Leh- most multiple myeloma patients: co-expression of SSX1, 2, 4, and 5 mann F, Andre M, Ravoet C, Doyen C, Spagnoli GC, Bakkus M, correlates with adverse prognosis and high frequencies of SSX-posi- Thielemans K, Boon T. Genes encoding tumor-specific antigens are tive PCs. J Immunother 2005; 28: 564-575. (PMID: 16224274) expressed in human myeloma cells. Blood 1999; 94: 1156-1164. (PMID: 10438702) 16. Jungbluth AA, Ely S, DiLiberto M, Niesvizky R, Williamson B, Fro- sina D, Chen YT, Bhardwaj N, Chen-Kiang S, Old LJ, Cho HJ. The cancer-testis antigens CT7 (MAGE-C1) and MAGE-A3/6 are com- Materials and methods monly expressed in multiple myeloma and correlate with plasma- cell proliferation. Blood 2005; 106: 167-174. (PMID: 15761016) Yeast two-hybrid screening The NH2-terminal portion of MAGE-C1 (amino acids 775- 17. Laduron S, Deplus R, Zhou S, Kholmanskikh O, Godelaine D, De 1143, nucleotide 2322-3429) encompassing the MAGE Smet C, Hayward SD, Fuks F, Boon T, De Plaen E. MAGE-A1 inter- homology domain (amino acids 902-1029, nucleotides 2704- acts with adaptor SKIP and the deacetylase HDAC1 to repress tran- 3087) of MAGE-C1 (1107 bp) was cloned into the pGBKT7 scription. Nucleic Acids Res 2004; 32: 4340-4350. (PMID: 15316101) vector (Clontech, Palo Alto, CA) to generate a fusion protein downstream of the Gal4 DNA binding domain (BD). This 18. Bai S, He B, Wilson EM. Melanoma antigen gene protein MAGE-11 plasmid was then used as bait to screen a high complexity regulates androgen receptor function by modulating the interdo- human testis cDNA library (Invitrogen, Carlsbad, CA), which main interaction. Mol Cell Biol 2005; 25: 1238-1257. (PMID: was cloned downstream of the Gal4 activation domain (GalAD). 15684378) The yeast strain AH109 was transformed with the bait plasmid, then with the cDNA library. The transformants were plated on 19. Nagao T, Higashitsuji H, Nonoguchi K, Sakurai T, Dawson S, Mayer medium lacking leucine, tryptophan and histidine (L, T, H) in RJ, Itoh K, Fujita J. MAGE-A4 interacts with the liver oncoprotein the presence of 75 mM 3-amino-1,2,4-triazole (3AT) for up to 3 gankyrin and suppresses its tumorigenic activity. J Biol Chem 2003; weeks at 30°C. Colonies were picked and replated on L, T, H agar 278: 10668-10674. (PMID: 12525503) plates. Those that grew in the second plate were plated in medium lacking leucine, tryptophan and adenine (L, T, A). The

www.cancerimmunity.org 7 of 8 Cancer Immunity (1 December 2006) Vol. 6, p. 12 positive colonies were then subjected to several rounds of twice with PBS and fixed with 3% paraformaldehyde in PBS culture in SC without tryptophan (T) to eliminate the bait (138 mM NaCl, 2.7 mM KCl, pH 7.4) for 10 min at room plasmid. Each Trp+ clone was tested again for activation of the temperature, permeabilized with 0.2% Triton X-100 in PBS for reporter genes to eliminate those that transactivate the reporter 2 min and blocked with 5% goat serum in PBST for 30 min at gene in the absence of the bait plasmid. Finally, the plasmid room temperature. The cells were then incubated with the DNA was isolated from the positive yeast clones, amplified in E. primary antibodies (NY-41 and CT7.33) overnight at 4°C and, coli, and analyzed by automated DNA sequencing. All inserts following three washes in PBST, were incubated with a mixture were identified by homology searching with the NCBI BLAST of Alexa Fluor 488 and 633 labeled secondary antibodies program (26). These plasmids were also used to reconstruct the (Molecular Probes, Eugene, OR). After washing, the slides were in vivo interaction by transforming them back into the original incubated with 0.02 µg/ml DAPI (Sigma-Aldrich, St. Louis, yeast strain containing the bait plasmid. MO) to visualize the nuclei. The slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and Cell culture analyzed under an epifluorescence microscope. All cell lines used in this study were obtained from the cell culture bank of the New York Branch of the Ludwig Institute for RT-PCR analysis of tumor cells Cancer Research. They were maintained in RPMI medium Total RNA was prepared from tissues or cell line pellets containing 10% fetal bovine serum (FBS) and non-essential following homogenization by the guanidinium isothiocyanate amino acids. method, followed by CsCl gradient centrifugation. Alternatively, the Ribopure kit (Ambion, Austin, TX) or Trizol reagent Western blot analysis (Invitrogen, Carlsbad, CA) were used according to Cell extracts were prepared in RIPA buffer (25 mM Tris-HCl manufacturer's instructions. Total RNA (2 µg) was reverse- pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, transcribed with 200 units Moloney murine leukemia virus 0.1% SDS and protease inhibitor Complete™ tablet) and reverse transcriptase (Invitrogen, Carlsbad, CA), according to subjected to five cycles of sonication. Fifty microliters of the the manufacturer's instructions, in the presence of 2 µg random protein was mixed with an equal volume of 2x loading buffer hexamers (Applied Biosystems, Foster City, CA), 20 units (125 mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol, 0.006% RNaseOUT (Invitrogen), and 5 mmol/l DTT in a total volume bromophenol blue, 2% β-mercaptoethanol), incubated at 95°C of 20 µl. For individual PCR reactions, 250 ng of cDNA were for 3 min, and loaded onto 10% SDS Bis-Tris gels (Invitrogen, amplified with gene-specific oligonucleotides (2 ng per 25 µl Carlsbad, CA). After electrophoresis, proteins were transferred reaction) in the presence of 1 unit AmpliTaq Gold (Applied to nitrocellulose membranes. The membranes were blocked by Biosystems, Foster City, CA) and 5 µmol/l of each dNTP incubation in PBST (PBS, 0.1% Tween 20) with 3% bovine (Applied Biosystems, Foster City, CA). The MAGEC1, serum albumin (BSA) for 1 h, and then incubated with the NY-ESO-1, MAGEA1 and MAGEA3 specific primers used for primary antibody overnight at 4°C in PBST with 1% BSA. After PCR amplification were the same as those used by Gure et al., washing four times in PBST, the membranes were incubated 2005 (10) and van Baren et al. (28). either with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Jackson Immunoresearch, Bar Harbor, ME) for 1 h at room Patients temperature. Antibody binding was detected using the Western All patients provided written informed consent prior to our Lightening Chemiluminescence Reagent Plus system (Perkin study. Four hundred and fifty six tumor tissue specimens Elmer, Emeryville, CA). The antibodies used were: a rabbit sampled from patients with NSCLC who underwent surgery polyclonal anti-full length NY-ESO-1 (NY-41), a monoclonal between 1991 and 2004 at the Department of Cardio-Thoracic anti-NY-ESO-1 (E978) (27), a monoclonal anti-CT7 (CT7.33) Surgery, Weill Medical College of Cornell University were (8), a rabbit polyclonal anti-actin (20-33, Sigma-Aldrich, St. included in this study. The study was approved by the Louis, MO) and an anti-influenza hemagglutinin (HA-7, Sigma- Institutional Review Board of Weill Medical College of Cornell Aldrich, St. Louis, MO) antibody. University. Thirty nine consecutive, previously untreated multiple myeloma (MM) patients seen in the MM outpatient Co-immunoprecipitations service of the Discipline of Hematology and Hemotherapy, Cells were lysed in IP buffer (50 mM Tris-Cl pH 7.4, 0.15 M UNIFESP/EPM, São Paulo, Brazil, between June 2002 and May NaCl, 2 mM EDTA, 1% NP-40), containing protease inhibitors 2006 were studied. Cells were collected from patients with (Protease Inhibitors Cocktail, Roche, Indianapolis, IN). Five multiple myeloma by bone marrow aspiration. hundred micrograms to one milligram of total lysate were incubated at 4°C for 1 h in a rotator with the polyclonal or monoclonal antibodies, or with normal rabbit serum or mouse Contact IgG1 as negative controls. Immunocomplexes were precipitated Address correspondence to: with 150 µl of 10% protein A/G sepharose beads (Pierce Biotechnology, Rockford, IL) overnight at 4°C. After four Andrew J. G. Simpson washes in IP buffer, beads were boiled in 60 µl of 2x loading E-mail: [email protected] buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% β-mercaptoethanol). Immunoprecipitated proteins were resolved in 12% SDS-PAGE gels, followed by Western blotting.

Immunofluorescence staining Cells were grown on Lab-Tek II 4-well chamber slides (Nalge Nunc, Naperville, IL) until they reached 70% confluence, rinsed

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