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Complicated Mechanisms of Class II Transactivator Transcription Deficiency in Small Cell Lung Cancer and Neuroblastoma

2002; Elsevier BV; Volume: 161; Issue: 1 Linguagem: Inglês

10.1016/s0002-9440(10)64181-8

1525-2191

Takuya Yazawa, Takaaki Ito, Hiroshi Kamma, Takehisa Suzuki, Koji Okudela, Hiroyuki Hayashi, Hisashi Horiguchi, Takesaburo Ogata, Hideaki Mitsui, Masaichi Ikeda, Hitoshi Kitamura,

NF-κB Signaling Pathways

Small cell lung cancer (SCLC) and neuroblastoma (NB), the most aggressive adult and infant neuroendocrine cancers, respectively, are immunologically characterized by a severe reduction in major histocompatibility complex (MHC) that is indispensable for anti-tumor immunity. We had reported that the severe reduction of MHC in SCLC was caused by a deficient interferon (IFN)-γ-inducible expression of class II transactivator (CIITA) that is known as a very important transcription factor for IFN-γ-inducible class II and class I MHC expression (Yazawa T, Kamma H, Fujiwara M, Matsui M, Horiguchi H, Satoh H, Fujimoto M, Yokohama K, Ogata T: Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer. J Pathol 1999, 187:191–199). Here, we demonstrate that the reduction of MHC in NB was also caused by a deficient IFN-γ-inducible expression of CIITA and that the deficiency in SCLC and NB was caused by similar mechanisms. Human achaete-scute complex homologue (HASH)-1, L-myc, and N-myc, which are specifically overexpressed in SCLC and NB, bound to the E-box in CIITA promoter IV and reduced the transcriptional activity. Anti-sense oligonucleotide experiments revealed that overexpressed L-myc and N-myc lie upstream in the regulatory pathway of HASH-1 expression. The expression of HASH-1 was also up-regulated by IFN-γ. Our results suggest that SCLC and NB have complicated mechanisms of IFN-γ-inducible CIITA transcription deficiency through the overexpressed HASH-1, L-myc, and N-myc. These complicated mechanisms may play an important role in the escape from anti-tumor immunity. Small cell lung cancer (SCLC) and neuroblastoma (NB), the most aggressive adult and infant neuroendocrine cancers, respectively, are immunologically characterized by a severe reduction in major histocompatibility complex (MHC) that is indispensable for anti-tumor immunity. We had reported that the severe reduction of MHC in SCLC was caused by a deficient interferon (IFN)-γ-inducible expression of class II transactivator (CIITA) that is known as a very important transcription factor for IFN-γ-inducible class II and class I MHC expression (Yazawa T, Kamma H, Fujiwara M, Matsui M, Horiguchi H, Satoh H, Fujimoto M, Yokohama K, Ogata T: Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer. J Pathol 1999, 187:191–199). Here, we demonstrate that the reduction of MHC in NB was also caused by a deficient IFN-γ-inducible expression of CIITA and that the deficiency in SCLC and NB was caused by similar mechanisms. Human achaete-scute complex homologue (HASH)-1, L-myc, and N-myc, which are specifically overexpressed in SCLC and NB, bound to the E-box in CIITA promoter IV and reduced the transcriptional activity. Anti-sense oligonucleotide experiments revealed that overexpressed L-myc and N-myc lie upstream in the regulatory pathway of HASH-1 expression. The expression of HASH-1 was also up-regulated by IFN-γ. Our results suggest that SCLC and NB have complicated mechanisms of IFN-γ-inducible CIITA transcription deficiency through the overexpressed HASH-1, L-myc, and N-myc. These complicated mechanisms may play an important role in the escape from anti-tumor immunity. Small cell lung cancer (SCLC) and neuroblastoma (NB) are the most aggressive adult and infant neuroendocrine neoplasms, respectively.1Travis WD Colby TV Corrin B Shimosato Y Brambilla E World Health Organization: Histological Typing of Lung and Pleural Tumours. ed 3. Springer-Verlag, Heidelberg1999: 1-156Google Scholar, 2Schwab M Shimada H Joshi V Brodeur GM Neuroblastic tumours of adrenal gland and sympathetic nervous system.in: Kleihues P Cabenee WK Pathology and Genetics of Tumours of the Nervous System. International Agency for Research on Cancer, Lyon2000: 153-161Google Scholar Both are less associated with tumor-infiltrating lymphocytes, which are morphological findings of anti-tumor immunity, and show a more severe reduction in major histocompatibility complex (MHC) than nonneuroendocrine cancers.3Doyle A Martin WJ Funa K Gazdar A Carney D Martin SE Linnoila I Cuttitta F Mulshine J Bunn P Minna J Markedly decreased expression of class I histocompatibility antigens, protein, and mRNA in human small-cell lung cancer.J Exp Med. 1985; 161: 1135-1151Crossref PubMed Scopus (302) Google Scholar, 4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar, 5van't Veer LJ Beijersbergen RL Bernards R N-myc suppress major histocompatibility complex class I gene expression through down-regulation of the p50 subunit of NF-κB.EMBO J. 1993; 12: 195-200Crossref PubMed Scopus (65) Google Scholar The MHC expressed on the cell surface is indispensable for contact with T lymphocytes. Class I MHC (MHC-I) expressed on cancer cells plays an important role in the killer T lymphocyte-mediated immune response. Class II MHC (MHC-II) expressed on cancer cells could present antigenic peptides to helper T lymphocytes and could contribute to anti-tumor immunity as well as nonprofessional antigen-presenting cells such as endothelial cells.6Yun S Rose ML Fabre JW The induction of major histocompatibility complex class II expression is sufficient for the direct activation of human CD4+ T cells by porcine vascular endothelial cells.Transplantation. 2000; 69: 940-944Crossref PubMed Scopus (17) Google Scholar, 7Ostrand-Rosenberg S Tumor immunotherapy: the tumor cell as an antigen-presenting cell.Curr Opin Immunol. 1994; 6: 722-726Crossref PubMed Scopus (130) Google Scholar, 8Hock RA Reynolds BD Tucker-McClung CL Kwok WW Human class II major histocompatibility complex gene transfer into murine neuroblastoma leads to loss of tumorigenicity, immunity against subsequent tumor challenge, and elimination of microscopic preestablished tumors.J Immunother Emphasis Tumor Immunol. 1995; 17: 12-18Crossref PubMed Scopus (28) Google Scholar, 9Heuer JG Tucker-McClung C Gonin R Hock RA Retrovirus-mediated gene transfer of B7–1 and MHC class II coverts a poorly immunogenic neuroblastoma into a highly immunogenic one.Hum Gene Ther. 1996; 7: 2059-2068Crossref PubMed Scopus (26) Google Scholar The immune systems of neuroendocrine cancer-laden hosts are therefore not considered to function well. The expression mechanisms of MHC have recently been clarified. Nucleated cells constitutively express varying amounts of MHC-I. Although nonimmune competent cells hardly express MHC-II constitutively, several cytokines induce MHC-II in nonimmune competent cells. Interferon-gamma (IFN-γ), the most powerful MHC inducer, evokes both MHC-I and MHC-II expression.10Kamma H Yazawa T Ogata T Horiguchi H Iijima T Expression of MHC class II antigens in human lung cancer cells.Virchows Arch B Cell Pathol. 1991; 60: 407-412Crossref Scopus (32) Google Scholar Therefore, IFN-γ is now widely used for immunotherapy against cancers such as melanomas.11Fujii S Huang S Fong TC Ando D Burrows F Jolly DJ Nemunaitis J Hoon DS Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-gamma retroviral vector in melanoma patients.Cancer Gene Ther. 2000; 7: 1220-1230Crossref PubMed Scopus (39) Google Scholar It has been clarified that RFX5, RFXAP, RFXB, CREB1, NF-Y, and class II transactivator (CIITA) are responsible for gene activation of MHC-II.12DeSandro A Nagarajan UM Boss JM The bare lymphocyte syndrome: molecular clues to the transcriptional regulation of major histocompatibility complex class II genes.Am J Hum Genet. 1999; 65: 279-286Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar CIITA, one of the bare lymphocyte syndrome genes,12DeSandro A Nagarajan UM Boss JM The bare lymphocyte syndrome: molecular clues to the transcriptional regulation of major histocompatibility complex class II genes.Am J Hum Genet. 1999; 65: 279-286Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar is not only a master transcription factor for the IFN-γ-inducible MHC-II expression but also very important for MHC-I expression.13Steimle V Siegrist CA Mottet A Lisowska-Grospierre B Mach B Regulation of MHC class II expression by interferon-γ mediated by the transactivator gene CIITA.Science. 1994; 265: 106-109Crossref PubMed Scopus (690) Google Scholar, 14Martin BK Chin KC Olsen JC Skinner CA Dey A Ozato K Ting JP Induction of MHC class I expression by the MHC class II transactivator CIITA.Immunity. 1997; 6: 591-600Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar Different cellular compartments are controlled by the differential usage of the CIITA promoters, and the epithelial cells use promoter IV for the IFN-γ-inducible expression of CIITA.15Muhlethaler-Mottet A Otten LA Steimle V Mach B Expression of MHC class II molecules in different cellular and functional compartments is controlled by different usage of multiple promoters of the transactivator CIITA.EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (432) Google Scholar CIITA promoter IV has three specific transcription factor-binding sites, the IFN-γ-activating site (GAS), the E-box (CACGTG sequence), and the IFN regulatory factor (IRF)-binding site.15Muhlethaler-Mottet A Otten LA Steimle V Mach B Expression of MHC class II molecules in different cellular and functional compartments is controlled by different usage of multiple promoters of the transactivator CIITA.EMBO J. 1997; 16: 2851-2860Crossref PubMed Scopus (432) Google Scholar, 16Muhlethaler-Mottet A Di Berardino W Otten LA Mach B Activation of the MHC class II transactivator CIITA by interferon-γ requires cooperative interaction between Stat1 and USF-1.Immunity. 1998; 8: 157-166Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar The binding of signal transducer and activator of transcription (STAT)-1α, upstream stimulatory factor (USF)-1, and IRF-1 to the respective binding site is crucial for transcription of CIITA.16Muhlethaler-Mottet A Di Berardino W Otten LA Mach B Activation of the MHC class II transactivator CIITA by interferon-γ requires cooperative interaction between Stat1 and USF-1.Immunity. 1998; 8: 157-166Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar Immature neuronal and neuroendocrine cells express unique transcription factors with a basic helix-loop-helix (bHLH) structure such as human achaete-scute complex homologue (HASH)-1, L-myc, and N-myc. The expression of HASH-1 is physiologically restricted to neuronal and neuroendocrine cells in the early stages of development, and therefore HASH-1 is expected to be associated with the determination of cell fate.17Ball DW Azzoli CG Baylin SB Chi D Dou S Donis-Keller H Cumaraswamy A Borges M Nelkin BD Identification of a human achaete-scute homolog highly expressed in neuroendocrine tumors.Proc Natl Acad Sci USA. 1993; 90: 5648-5652Crossref PubMed Scopus (154) Google Scholar, 18Ito T Udaka N Yazawa T Okudela K Hayashi H Sudo T Guillemot F Kageyama R Kitamura H Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium.Development. 2000; 127: 3913-3921Crossref PubMed Google Scholar, 19Kageyama R Nakanishi S Helix-loop-helix factors in growth and differentiation of the vertebrate nervous system.Curr Opin Genet Dev. 1997; 7: 659-665Crossref PubMed Scopus (328) Google Scholar Fetal nervous and neuroendocrine tissues express much L-myc and N-myc, which are considered to be involved in regulating cell proliferation and differentiation.20Stanton BR Perkins AS Tessarollo L Sassoon DA Parada LF Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop.Genes Dev. 1992; 6: 2235-2247Crossref PubMed Scopus (306) Google Scholar, 21Zimmerman KA Yancopoulos GD Collum RG Smith RK Kohl NE Denis KA Nau MM Witte ON Toran-Allerand D Gee CE Minna JD Frederick WA Differential expression of myc family genes during murine development.Nature. 1986; 319: 780-783Crossref PubMed Scopus (367) Google Scholar Neuroendocrine cancers specifically overexpress HASH-1.17Ball DW Azzoli CG Baylin SB Chi D Dou S Donis-Keller H Cumaraswamy A Borges M Nelkin BD Identification of a human achaete-scute homolog highly expressed in neuroendocrine tumors.Proc Natl Acad Sci USA. 1993; 90: 5648-5652Crossref PubMed Scopus (154) Google Scholar SCLC and NB are known to involve gene amplification and/or overexpression of L-myc and N-myc, respectively.22Littlewood TD Evan GI Sheterline P Helix-Loop-Helix Transcription Factors. ed 3. Oxford University Press, New York1998: 1-151Google Scholar, 23Nesbit CE Tersak JM Prochownik EV MYC oncogenes and human neoplastic disease.Oncogene. 1999; 18: 3004-3016Crossref PubMed Scopus (959) Google Scholar The amplification of N-myc has been used clinically as a prognostic factor for a poor outcome in patients with NB.2Schwab M Shimada H Joshi V Brodeur GM Neuroblastic tumours of adrenal gland and sympathetic nervous system.in: Kleihues P Cabenee WK Pathology and Genetics of Tumours of the Nervous System. International Agency for Research on Cancer, Lyon2000: 153-161Google Scholar However, the precise relation between the overexpression of these bHLH transcription factors and disease initiation/progression has been controversial.23Nesbit CE Tersak JM Prochownik EV MYC oncogenes and human neoplastic disease.Oncogene. 1999; 18: 3004-3016Crossref PubMed Scopus (959) Google Scholar We recently clarified that SCLC, among various histological types of lung cancer, showed a severe reduction of MHC caused by a deficiency of CIITA expression.4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar Because a reduction of MHC has also been reported in NB7Ostrand-Rosenberg S Tumor immunotherapy: the tumor cell as an antigen-presenting cell.Curr Opin Immunol. 1994; 6: 722-726Crossref PubMed Scopus (130) Google Scholar and because the E-box, the consensus binding motif of HASH-1, L-myc, and N-myc,22Littlewood TD Evan GI Sheterline P Helix-Loop-Helix Transcription Factors. ed 3. Oxford University Press, New York1998: 1-151Google Scholar is located in CIITA promoter IV, it has been proposed that NB also has a deficiency of CIITA and the bHLH transcription factors overexpressed in SCLC and NB modulate the IFN-γ-inducible CIITA expression. This study demonstrates that the overexpressed HASH-1, L-myc, and N-myc in SCLC and NB competitively bind to the E-box in CIITA promoter IV and repress the transcriptional activity, that IFN-γ up-regulates HASH-1 expression, and that L-myc and N-myc are involved in HASH-1 expression. Therefore, it is considered that SCLC and NB fatefully have complicated molecular mechanisms that negatively affect CIITA expression. The human SCLC cell lines (TKB15, TKB16, and TKB17),4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar and the human NB cell lines (LA-N-1, LA-N-2, and LA-N-5) (purchased from Riken Cell Bank, Tsukuba, Japan), NH12 (provided by Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan), and SK-N-DZ (kindly provided by Dr. M. Kaneko, Department of Pediatric Surgery, University of Tsukuba, Tsukuba, Japan), were grown in RPMI 1640 medium. The human nonneuroendocrine cancer cell lines, TKB5 (large cell lung cancer)4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar and HeLa (purchased from Riken Cell Bank), were grown in Dulbecco's modified Eagle's medium. Both media were supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin, and cells were incubated at 37°C in 5% CO2. Recombinant IFN-γ (Genzyme, Cambridge, MA) was added at 100 IU/ml for the CIITA and MHC induction experiments.4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar RT-PCR using a RNA-PCR kit (Takara, Shiga, Japan) was conducted with 1 μg of total RNA. Specific primers for CIITA, NFY-A, NFY-B, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as described previously.4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar The upper and lower primers for RFX5, RFXAP, RFXB, CREB1, L-myc, N-myc, and HASH-1, which, respectively, amplify 787-bp, 858-bp, 507-bp, 527-bp, 239-bp, 381-bp, and 374-bp cDNA fragments, were as follows: RFX5 upper primer 5′-AACAGAGAGGTAGGCATAGGT-3′ and lower primer 5′-GAAAAGGTCAGAGGCAGCAAC-3′, RFXAP upper primer 5′-AAGAAACACGCAACAAGAT-G-3′ and lower primer 5′-AACACAAAATAGCCATCAA-CA-3′, RFXB upper primer 5′-CTCATCCAGACCCAGCAGACC-3′ and lower primer 5′-CTCCAGCAGCAGCCC-CACAAT-3′, CREB1 upper primer 5′-CCCCAGCACTTCCTACACAGC-3′ and lower primer 5′-TTTCCTCATTTC-TCCCATCAA-3′, L-myc upper primer 5′-TATGACTGC-GGGGAGGATTTC-3′ and lower primer 5′-CGGCGTATGA-TGGAGGCGTAG-3′, N-myc upper primer 5′-GACGC-TTCTCAAAACTGGACA-3′ and lower primer 5′-TCA-AATGGCAAACCCCTTATG-3′, HASH-1 upper primer 5′-CAGCGCCCAAGCAAGTCAAGC-3′ and lower primer 5′-GGCCATGGAGTTCAAGTCGTT-3′. Northern blot analyses using 15 μg of total RNA were performed as described previously.4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar Probes of digoxigenin-labeled HLA-DRα and GAPDH cDNA, which was used for the internal control, were as described.4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar The 402-bp human MHC-I cDNA fragments produced by RT-PCR using specific primers (upper primer 5′-CGACGGCAAGGATTACAT-3′ and lower primer 5′-CCAGAAGGCACCACCACA-3′), which amplify nonpolymorphic regions of MHC-I, were labeled with digoxigenin (Roche Diagnostics, Basel, Switzerland) and used as the probe. RT-PCR products of HASH-1, L-myc, and N-myc, and the c-myc exon 2 fragments (purchased from Takara) were also digoxigenin-labeled and used as probes. Semiquantitative analysis of mRNA levels was performed by NIH image version 1.62. The inserts of HASH-1, L-myc, and N-myc expression vectors were constructed using RNA of TKB15 and SK-N-DZ and primer sets specific for the 5′- and 3′-untranslated regions of HASH-1 (upper primer 5′-GATTCCGCGACTCCTTGG-3′ and lower primer 5′-CCTGA-CCAGGCCGAGCCCCTCAGA-3′), L-myc (upper primer 5′-CTGGAGCGAGGGAGCGGACAT-3′ and lower primer 5′-ACTAAAGGGGAGAGGGAGGTT-3′), and N-myc (upper primer 5′-GCTGCTCCACGTCCACCATG-3′ and lower primer 5′-CATGGAGGTGAGGTGGAGGAG-3′). The products were cloned with pT7Blue cloning vector (Novagen, Madison, WI) and sequenced. Each purified HASH-1, L-myc, or N-myc cDNA was inserted into a pZeoSV2 expression vector (Invitrogen, Carlsbad, CA). HASH-1 was also inserted into a pIRES2 expression vector (Clontech, Palo Alto, CA). We used an expression vector for human CIITA (pZeoSV2-CIITA) that was previously constructed.4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar The recombinant proteins for HASH-1, L-Myc, and N-Myc were produced using a T7 promoter-dependent in vitro transcription and translation system (Novagen). The reactions were conducted according to the manufacturer's instructions. Western blot analyses for HASH-1, L-Myc, N-Myc, Max, E12/E47, USF-1, 701Tyr-phosphorylated STAT-1α, and IRF-1 were conducted using 50 μg of whole cell lysate from cultured cancer cells and anti-mammalian achaete-scute complex homologue (MASH)-1 (BD Pharmingen, San Diego, CA), anti-L-Myc (Sigma-Genosys, Cambridge, UK), anti-N-Myc (Sigma-Genosys), anti-Max (Santa Cruz, Santa Cruz, CA), anti-E12/E47 (Santa Cruz), anti-USF-1 (Santa Cruz), anti-701Tyr-phosphorylated STAT-1 (New England Biolab, Beverly, MA), and anti-IRF-1 (Santa Cruz) antibodies. The cross-reactivity of anti-MASH-1 antibody with HASH-1 was confirmed by immunoprecipitation assay using recombinant HASH-1 protein as mentioned below. The electrophoresed samples were electroblotted on a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and detected with the ECL system (Amersham Pharmacia, Buckinghamshire, UK). Anti-β-actin antibody (Sigma, St. Louis, MO) was used for the internal control. TKB15 (a SCLC cell line), SK-N-DZ (a NB cell line), and TKB5 (a non-SCLC cell line) cells were stimulated with IFN-γ (100 IU/ml) for 30 minutes before the preparation of nuclear extract. The following steps were performed at 4°C. The IFN-γ-treated cells were collected and washed three times with phosphate-buffered saline (154 mmol/L NaCl and 10 mmol/L sodium phosphate buffer, pH 7.6) containing 2 mmol/L of MgCl2, and then suspended in isolation buffer [20 mmol/L HEPES, pH 7.6, 10 mmol/L NaCl, 0.5 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.5 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 0.5 μg/ml pepstatin A, 2 μg/ml leupeptin, 1 mmol/L dithiothreitol, and 0.25% Triton X-100]. Nuclei were collected by centrifugation, washed twice with isolation buffer without Triton X-100, and then suspended in extraction buffer (20 mmol/L HEPES, pH 7.6, 20 mmol/L NaCl, 0.5 mmol/L EDTA, 10% glycerol, 0.5 mmol/L PMSF, 1 μg/ml aprotinin, 0.5 μg/ml pepstatin A, 2 μg/ml leupeptin, and 1 mmol/L dithiothreitol). Because a highly concentrated salt buffer is necessary for effective extraction of Myc and Max proteins,24Nozaki N Naoe T Okazaki T Immunoaffinity purification and characterization of CACGTG sequence-binding proteins from cultured mammalian cells using an anti-c-Myc monoclonal antibody recognizing the DNA-binding domain.J Biochem. 1997; 121: 550-559Crossref PubMed Scopus (7) Google Scholar NaCl was added at up to 1 mol/L in the nuclear suspensions. After gentle agitation for 1 hour at 4°C, the suspensions were layered on the sucrose cushion buffer (20 mmol/L HEPES, pH 7.6, 1 mol/L NaCl, 0.5 mmol/L EDTA, 10% glycerol, 0.5 mmol/L PMSF, 1 μg/ml aprotinin, 0.5 μg/ml pepstatin A, 2 μg/ml leupeptin, 1 mmol/L dithiothreitol, and 30% sucrose) and centrifuged at 15,000 × g for 1 hour. The top layer was desalted and concentrated with microcon-10 (Amicon, Beverly, MA) and ddH2O containing 0.5 mmol/L PMSF, 1 μg/ml aprotinin, 0.5 μg/ml pepstatin A, 2 μg/ml leupeptin, and 1 mmol/L dithiothreitol. To analyze the availability of anti-MASH-1 (BD Pharmingen), anti-L-Myc (Sigma-Genosys), and anti-N-Myc (Sigma-Genosys) antibodies for chromatin immunoprecipitation assay, immunoprecipitation and immunoblot studies using respective antibody-conjugated protein G-Sepharose beads (Pharmacia, Uppsala, Sweden), 50 μl of in vitro transcription translation reactant containing recombinant HASH-1, L-Myc, and N-Myc, and 25 μg of nuclear lysate from TKB15 or SK-N-DZ containing the intrinsic forms were performed. Immunoprecipitation experiments using anti-STAT-1α, anti-USF-1, anti-c-Myc, anti-Max, and anti-E12/E47 (all from Santa Cruz) antibodies, which were used in chromatin immunoprecipitation studies mentioned below, were also performed to examine the ability to specifically immunoprecipitate the respective cognate antigen. Chromatin immunoprecipitation assays were essentially performed as described by Moreno and colleagues.25Moreno CS Beresford GW Louis-Plence P Morris AC Boss JM CREB regulates MHC class II expression in a CIITA-dependent manner.Immunity. 1999; 10: 143-151Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar TKB15 or SK-N-DZ cells (1 × 107) were exposed to formaldehyde (final concentration, 1%) added directly to the tissue culture medium for 10 minutes. Cells were pelleted and washed in phosphate-buffered saline three times, lysed in lysis buffer [5 mmol/L PIPES (pH 8.0), 85 mmol/L KCl, and 0.5% Nonidet P-40 containing protease inhibitors (1 mmol/L PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin)], and incubated on ice for 5 minutes. Nuclei were pelleted and lysed in 500 μl of nuclei lysis buffer [50 mmol/L Tris-HCl, pH 8.1, 10 mmol/L EDTA, and 1% sodium dodecyl sulfate (SDS)] containing protease inhibitors. Lysed nuclei were sonicated using a microtip until the average DNA fragment was ∼600 bp. The samples were centrifuged at 16,000 × g for 5 minutes and the supernatants (50 μl) were diluted 1:10 with immunoprecipitation dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mmol/L EDTA, 16.7 mmol/L Tris-HCl, pH 8.1, and 167 mmol/L NaCl) containing protease inhibitors, 50 μg/ml of yeast tRNA, and 20 μg/ml of sonicated salmon sperm DNA. Immunoprecipitations were performed at 4°C for 2 hours using 550 μl of diluted supernatant and protein G-Sepharose beads (Pharmacia) conjugated with antibody against STAT-1α (Santa Cruz), USF-1 (Santa Cruz), L-Myc (Sigma-Genosys), N-Myc (Sigma-Genosys), Max (Santa Cruz), HASH-1 (BD Pharmingen), or anti-E12/E47 (Santa Cruz). Protein G-Sepharose beads without antibody were used as the negative control. Protein G-Sepharose beads conjugated with immune complexes were washed three times for 10 minutes each with the following buffers: immunoprecipitation dilution buffer, washing buffer A (0.1% SDS, 1% Triton X-100, 2 mmol/L EDTA, 20 mmol/L Tris-HCl, pH 8.1, and 500 mmol/L NaCl), and washing buffer B (0.1% SDS, 1% Nonidet P-40, 100 mmol/L Tris-HCl, pH 8.1, and 500 mmol/L NaCl), followed by a final wash with TE (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA). Immune complexes were disrupted with elution buffer (50 mmol/L NaHCO3 and 1% SDS) and the covalent links were reversed by addition of NaCl to a final concentration of 300 mmol/L and heating to 65°C for 6 hours. DNA was ethanol-precipitated and further purified by proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation. Purified DNA fragments sonicated under the same conditions were used as the positive control. PCR was conducted using Ex Taq polymerase (Takara). The upper and lower primers that amplify CIITA promoter IV including GAS, E-box, and the IRF-binding site, or the 3′ untranslated region of the CIITA gene, which, respectively, amplifies 143-bp and 181-bp DNA fragments, were as follows: CIITA promoter IV upper primer 5′-CAGGGACCTCTTGGATG-3′ and lower primer 5′-CCCCGCAGTTCTTTTTC-3′, 3′ untranslated region upper primer 5′-AGGCCAGAGGGAGTG-ACA-3′ and lower primer 5′-GCTGAAGCAGAAGAATCG-3′. For the binding reaction with the CIITA promoter IV sequence, 5 μg of nuclear extract was mixed with 5 pmol of digoxigenin-labeled double-stranded CIITA promoter IV-specific DNA probe containing the GAS and the E-box (ProIV) or mutated E-box (ProIVm) sequence in a final volume of 20 μl containing 20 mmol/L Tris-HCl, pH 7.9, 50 mmol/L NaCl, 1 mmol/L EDTA, 5% glycerol, 0.75 μg poly dIdC, and 5 mmol/L dithiothreitol. The mixture was incubated 30 minutes at 20°C. The sense sequence of the ProIV probe was 5′-GCCCCAGGCAGTTGGGATGCCACTTCTGATAAAGCACGTGGTGGCCACAG-3′ and the sense sequence of the ProIVm probe was 5′-GCCCCAGGCAGTTGGGATGCCACTTCTGATAAAGGAATTCG-TGGCCACAG-3′. Mutations are underlined. For supershift experiments, antibodies against USF-1 (Santa Cruz), L-Myc (Sigma-Genosys), N-Myc (Sigma-Genosys), HASH-1 (BD Pharmingen), and c-Myc (Santa Cruz) were added to the mixture and left 20 minutes at 4°C before gel electrophoresis in 5% acrylamide/bisacrylamide (29:1) gels with 0.25× TBE buffer for 2 hours at 200 V and 4°C with recirculating buffer. The protein-probe complexes were contact-blotted and fixed with UV for 5 minutes on positively charged nylon membranes (NEN), and the digoxigenin-labeled probes were detected with a chemiluminescence detection kit (Roche Diagnostics). The expression vectors were transfected into TKB5, using the lipofection method (Qiagen, Hilden, Germany) as previously described,4Yazawa T Kamma H Fujiwara M Matsui M Horiguchi H Satoh H Fujimoto M Yokohama K Ogata T Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.J Pathol. 1999; 187: 191-199Crossref PubMed Scopus (70) Google Scholar and stable transfectants were obtained after 3 weeks selection. To check the expression of transfected genes, RT-PCR was performed using primers specific for each. A 416-bp fragment of cloned CIITA promoter IV sequence (−335 to +81)16Muhlethaler-Mottet A Di Berardino W Otten LA Mach B Activation of the MHC class II transactivator CIITA by interferon-γ requires cooperative interaction between Stat1 and USF-1.Immunity. 1998; 8: 157-166Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar obtaine