Methylation Status of the Epstein-Barr Virus Major Latent Promoter C in Iatrogenic B Cell Lymphoproliferative Disease
1999; Elsevier BV; Volume: 155; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65157-7
ISSN1525-2191
AutoresQian Tao, Lode J. Swinnen, Jie Yang, Gopesh Srivastava, Keith D. Robertson, Richard F. Ambinder,
Tópico(s)Parvovirus B19 Infection Studies
ResumoThe Epstein-Barr virus (EBV) major latent promoter C drives the expression of viral nuclear proteins important in lymphocyte immortalization and as targets for immune surveillance by cytotoxic T cells. Hypermethylation of the C promoter silences its transcription. This promoter is methylated and silent in Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and nasal lymphoma. However, it is never methylated in the EBV-immortalized lymphoblastoid cell lines that serve as a model for EBV-associated lymphoproliferative disease. We have analyzed C promoter methylation in iatrogenic EBV-associated B-cell lymphoproliferative disease, mainly posttransplant lymphoma, using a sensitive polymerase chain reaction-based C promoter methylation assay. Our results showed heterogeneity in lymphoproliferative disease with methylation of viral DNA in specimens from 3 of 13 patients. In specimens from two of these patients, only methylated viral DNA was detected and viral nuclear antigen expression was correspondingly restricted. Heterogeneity in C promoter methylation and expression of associated transcripts may be an important determinant of the growth properties of lymphoproliferative lesions and may provide an explanation for the failure of some tumors to respond to withdrawal or reduction of immunosuppressive therapy. The Epstein-Barr virus (EBV) major latent promoter C drives the expression of viral nuclear proteins important in lymphocyte immortalization and as targets for immune surveillance by cytotoxic T cells. Hypermethylation of the C promoter silences its transcription. This promoter is methylated and silent in Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and nasal lymphoma. However, it is never methylated in the EBV-immortalized lymphoblastoid cell lines that serve as a model for EBV-associated lymphoproliferative disease. We have analyzed C promoter methylation in iatrogenic EBV-associated B-cell lymphoproliferative disease, mainly posttransplant lymphoma, using a sensitive polymerase chain reaction-based C promoter methylation assay. Our results showed heterogeneity in lymphoproliferative disease with methylation of viral DNA in specimens from 3 of 13 patients. In specimens from two of these patients, only methylated viral DNA was detected and viral nuclear antigen expression was correspondingly restricted. Heterogeneity in C promoter methylation and expression of associated transcripts may be an important determinant of the growth properties of lymphoproliferative lesions and may provide an explanation for the failure of some tumors to respond to withdrawal or reduction of immunosuppressive therapy. The Epstein-Barr virus (EBV) latent C promoter drives expression of six EBV nuclear antigens (EBNAs): 1, 2, 3A, 3B, 3C, and LP.1Kieff E Epstein-Barr virus and its replication.in: Fields BN Knipe DM Howley PM Fields Virology. Lippincott-Raven, Philadelphia1996: 2343-2396Google Scholar, 2Rickinson AB Kieff E Epstein-Barr Virus: Fields Virology.in: Fields BN Knipe DM Howley PM Lippincott-Raven Publishers, Philadelphia1996: 2397-2446Google Scholar The ability of the virus to immortalize B cells is one of the properties that suggest a role for the virus in the pathogenesis of various neoplasia. Among the viral proteins required for lymphocyte immortalization and transformation, four are expressed from the C promoter. All of the immunodominant viral antigens expressed during latency (EBNA3A, 3B, and 3C) are expressed from the C promoter (Figure 1).3Murray RJ Kurilla MG Brooks JM Thomas WA Rowe M Kieff E Rickinson AB Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies.J Exp Med. 1992; 176: 157-168Crossref PubMed Scopus (442) Google Scholar, 4Khanna R Burrows SR Kurilla MG Jacob CA Misko IS Sculley TB Kieff E Moss DJ Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.J Exp Med. 1992; 176: 169-176Crossref PubMed Scopus (330) Google Scholar Thus regulation of the C promoter is likely to be a critical determinant of the growth characteristics of infected cells and of their susceptibility to lysis by cytotoxic T cells. In Burkitt's lymphoma (BL), the C promoter is silent, the growth characteristics of the tumor appear to largely reflect the c-myc translocation rather than viral gene expression, and there is no evidence that this tumor is sensitive to control by virus-specific cytotoxic T cells.5Rowe M Rowe DT Gregory CD Young LS Farrell PJ Rupani H Rickinson AB Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells.EMBO J. 1987; 6: 2743-2751PubMed Google Scholar, 6Rowe M Khanna R Jacob CA Argaet V Kelly A Powis S Belich M Croom-Carter D Lee S Burrows SR Trowsdale J Moss DJ Rickiuson AB Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein-1: coordinate up-regulation of peptide transporters and HLA-class I antigen expression.Eur J Immunol. 1995; 25: 1374-1384Crossref PubMed Scopus (176) Google Scholar, 7Masucci MG Zhang QJ Gavioli R De Campos-Lima PO Murray RJ Brooks J Griffin H Ploegh H Rickinson AB Immune escape by Epstein-Barr virus (EBV) carrying Burkitt's lymphoma: in vitro reconstitution of sensitivity to EBV-specific cytotoxic T cells.Int Immunol. 1992; 4: 1283-1292Crossref PubMed Scopus (22) Google Scholar In contrast, in posttransplant lymphoproliferative disease (PTLD), the C promoter in many cases is active,8Young L Alfieri C Hennessy K Evans H O'Hara C Anderson KC Ritz J Shapiro RS Rickinson A Kieff E Cohen JI Expression of Epstein-Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease.N Engl J Med. 1989; 321: 1080-1085Crossref PubMed Scopus (611) Google Scholar, 9Oudejans JJ Jiwa M Vandenbrule AJC Grasser FA Horstman A Vos W Kluin PM Vandervalk P Walboomers JMM Meijer CJLM Detection of heterogeneous Epstein-Barr virus gene expression patterns within individual post-transplantation lymphoproliferative disorders.Am J Pathol. 1995; 147: 923-933PubMed Google Scholar c-myc translocations are the exception rather than the rule,10Chadburn A Cesarman E Knowles DM Molecular pathology of posttransplantation lymphoproliferative disorders.Semin Diagn Pathol. 1997; 14: 15-26PubMed Google Scholar and viral genes are believed to play a central role in driving cellular proliferation. Similarly, expression of the immunodominant EBNAs from the C promoter in PTLD is presumably responsible for PTLD's exquisite sensitivity to immune manipulations. These include withdrawal of immunosuppressive medicines and adoptive transfer of EBV-specific cytotoxic lymphocytes.11Papadopoulos EB Ladanyi M Emanuel D Mackinnon S Boulad F Carabasi MH Castro-Malaspina H Childs BH Gillio AP Small TN Young JW Kernan NA O'Reilly RJ Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation.N Engl J Med. 1994; 33: 1185-1191Crossref Scopus (953) Google Scholar, 12Rooney CM Smith CA Ng CY Loftin S Li C Krance RA Brenner MK Heslop HE Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation.Lancet. 1995; 345: 9-13Abstract PubMed Google Scholar, 13Lucas KG Small TN Heller G Dupont B O'Reilly RJ The development of cellular immunity to Epstein-Barr virus after allogeneic bone marrow transplantation.Blood. 1996; 87: 2594-2603PubMed Google Scholar, 14Heslop HE Ng CY Li C Smith CA Loftin SK Krance RA Brenner MK Rooney CM Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes.Nat Med. 1996; 2: 551-555Crossref PubMed Scopus (711) Google Scholar However, the patterns of viral antigen expression in PTLD and thus of C promoter usage are not uniform. Several groups of investigators, including our own, have documented heterogeneity of antigen expression in these tumors.9Oudejans JJ Jiwa M Vandenbrule AJC Grasser FA Horstman A Vos W Kluin PM Vandervalk P Walboomers JMM Meijer CJLM Detection of heterogeneous Epstein-Barr virus gene expression patterns within individual post-transplantation lymphoproliferative disorders.Am J Pathol. 1995; 147: 923-933PubMed Google Scholar, 15Murray PG Swinnen LJ Constandinou CM Pyle JM Carr TJ Hardwick JM Ambinder RF Bcl-2 but not the EBV-encoded bcl-2 homologue, BHRF-1, is commonly expressed in post-transplantation lymphoproliferative disorders.Blood. 1996; 87: 706-711PubMed Google Scholar, 16Delecluse HJ Kremmer E Rouault JP Cour C Bornkamm G Berger F The expression of Epstein-Barr virus latent proteins is related to the pathological features of post-transplant lymphoproliferative disorders.Am J Pathol. 1995; 146: 1113-1120PubMed Google Scholar, 17Cen H Williams PA McWilliams HP Breinig MC Ho M McKnight JL Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders.Blood. 1993; 81: 1393-1403PubMed Google Scholar, 18Gratama JW Zutter MM Minarovits J Oosterveer MA Thomas ED Klein G Ernberg I Expression of Epstein-Barr virus-encoded growth-transformation- associated proteins in lymphoproliferations of bone-marrow transplant recipients.Int J Cancer. 1991; 47: 188-192Crossref PubMed Scopus (55) Google Scholar, 19Thomas JA Hotchin NA Allday MJ Amlot P Rose M Yacoub M Crawford DH Immunohistology of Epstein-Barr virus-associated antigens in B cell disorders from immunocompromised individuals.Transplantation. 1990; 49: 944-953Crossref PubMed Scopus (169) Google Scholar One of the mechanisms important in the regulation of the C promoter is CpG methylation. in vitro studies have identified two promoter regions that are especially sensitive to the transcriptional repression of CpG methylation.20Robertson KD Hayward DJ Ling PD Samid D Ambinder RF Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.Mol Cell Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (110) Google Scholar, 21Robertson KD Ambinder RF Mapping promoter regions that are hypersensitive to methylation-mediated inhibition of transcription: application of the methylation cassette assay to the Epstein-Barr virus major latency promoter.J Virol. 1997; 71: 6445-6454PubMed Google Scholar Studies of lymphocytes from healthy EBV seropositive blood donors and in BL, Hodgkin's disease (HD), nasopharyngeal carcinoma (NPC), and nasal lymphoma tumor tissues have shown hypermethylation and transcriptional silence in this promoter.22Robertson KD Manns A Swinnen LJ Zong JC Gulley ML Ambinder RF CpG methylation of the major Epstein-Barr virus latency promoter in Burkitt's lymphoma, and Hodgkin's disease.Blood. 1996; 88: 3129-3136PubMed Google Scholar, 23Robertson KD Ambinder RF Methylation of Epstein-Barr virus genome in normal lymphocytes.Blood. 1997; 90: 4480-4484PubMed Google Scholar, 24Minarovits J Minarovits-Kormuta S Ehlin-Henriksson B Falk K Klein G Ernberg I Host cell phenotype-dependent methylation patterns of Epstein-Barr virus DNA.J Gen Virol. 1991; 72: 1591-1599Crossref PubMed Scopus (51) Google Scholar, 25Ernberg I Falk K Minarovits J Busson P Tursz T Masucci MG Klein G The role of methylation in the phenotype-dependent modulation of Epstein-Barr nuclear antigen 2 and latent membrane protein genes in cells latently infected with Epstein-Barr virus.J Gen Virol. 1989; 70: 2989-3002Crossref PubMed Scopus (100) Google Scholar, 26Minarovits J Hu L-F Imai S Harabuchi Y Kataura A Minarovits-Kormuta S Osato T Klein G Clonality, expression and methylation patterns of the Epstein-Barr virus genomes in lethal midline granulomas classified as peripheral angiocentric T cell lymphomas.J Gen Virol. 1994; 75: 77-84Crossref PubMed Scopus (88) Google Scholar, 27Tao Q Robertson KD Manns A Hildesheim A Ambinder RF Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue.Blood. 1998; 91: 1373-1381PubMed Google Scholar, 28Tao Q Robertson KD Manns A Hildesheim A Ambinder RF The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive.J Virol. 1998; 72: 7075-7083PubMed Google Scholar, 29Chiang AKS Tao Q Srivastava G Ho FCS Nasal NK-, and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma, and Hodgkin's disease.Int J Cancer. 1996; 68: 285-290Crossref PubMed Scopus (154) Google Scholar In Burkitt's cell lines treated with 5-azacytidine, an inhibitor of DNA methyltransferase, the C promoter is activated.20Robertson KD Hayward DJ Ling PD Samid D Ambinder RF Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.Mol Cell Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (110) Google Scholar, 30Masucci MG Contreras-Salazar B Ragnar E Falk K Minarovits J Ernberg I Klein G 5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6, and latent membrane protein in the Burkitt's lymphoma line Rael.J Virol. 1989; 63: 3135-3141PubMed Google Scholar In the investigations reported here, we sought to determine whether methylation of the C promoter occurred in posttransplant and similar iatrogenic EBV-associated lymphoproliferative disease and whether its occurrence correlated with patterns of viral antigen expression. In the past, analysis of C promoter methylation has been performed by Southern blot hybridization with methylation-sensitive and -insensitive restriction enzyme isoschizomers or with bisulfite genomic sequencing. The former approach requires substantial quantities of high quality genomic DNA (10–20 μg) and provides information only about potential methylation sites that happen to coincide with restriction enzyme sites.24Minarovits J Minarovits-Kormuta S Ehlin-Henriksson B Falk K Klein G Ernberg I Host cell phenotype-dependent methylation patterns of Epstein-Barr virus DNA.J Gen Virol. 1991; 72: 1591-1599Crossref PubMed Scopus (51) Google Scholar, 25Ernberg I Falk K Minarovits J Busson P Tursz T Masucci MG Klein G The role of methylation in the phenotype-dependent modulation of Epstein-Barr nuclear antigen 2 and latent membrane protein genes in cells latently infected with Epstein-Barr virus.J Gen Virol. 1989; 70: 2989-3002Crossref PubMed Scopus (100) Google Scholar The latter approach is so laborious as to prohibit its application to large numbers of clinical specimens.20Robertson KD Hayward DJ Ling PD Samid D Ambinder RF Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.Mol Cell Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (110) Google Scholar, 22Robertson KD Manns A Swinnen LJ Zong JC Gulley ML Ambinder RF CpG methylation of the major Epstein-Barr virus latency promoter in Burkitt's lymphoma, and Hodgkin's disease.Blood. 1996; 88: 3129-3136PubMed Google Scholar Therefore, we have developed an alternative rapid polymerase chain reaction (PCR)-based assay for C promoter methylation analysis that requires only small amounts of DNA and specifically targets a region of the C promoter shown to be hypersensitive to the transcriptional inhibitory effects of CpG methylation in vitro. B95-8 is an EBV-immortalized lymphoblastoid cell line. Rael, Akata, Wanyonyi (Wan), Raji, and AG876 are EBV-positive BL cell lines. BJAB is an EBV-negative B-lymphoma cell line. All cell lines were cultivated at 37°C in RPMI 1640 supplemented with 10% fetal bovine serum, 1 mmol/L glutamine, 100 U/ml penicillin and streptomycin. 5-azacytidine was used to treat the Rael cell line (Rael-AzaC).20Robertson KD Hayward DJ Ling PD Samid D Ambinder RF Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.Mol Cell Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (110) Google Scholar The EBV latency types and C promoter methylation status of these cell lines are listed in Table 1.Table 1Latency Types, Methylation Status, and C Promoter Activities in EBV-Positive Cell LinesCell lineLatency typeC promoter methylation statusC promoter activityB95-8IIIuonRaelImoffRael-AzaC*Depending on the dosage and the treatment length of 5-azacytidine to Rael cell line, C promoter can be partially to completely demethylated and active in Rael-AzaC.20,30I + IIIm+ uoff+ onAkataImoffWan†Wan was originally classified as a type I/II cell line.5 However, the Wan cell line we obtained showed a drift to type III in some cells.27II/IIIm+ uoff+ onRaji‡In the Raji cell line, C promoter is methylated and silent and EBNAs are driven by an unknown promoter.IIImoffu, unmethylated; m, methylated.* Depending on the dosage and the treatment length of 5-azacytidine to Rael cell line, C promoter can be partially to completely demethylated and active in Rael-AzaC.20Robertson KD Hayward DJ Ling PD Samid D Ambinder RF Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.Mol Cell Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (110) Google Scholar, 30Masucci MG Contreras-Salazar B Ragnar E Falk K Minarovits J Ernberg I Klein G 5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6, and latent membrane protein in the Burkitt's lymphoma line Rael.J Virol. 1989; 63: 3135-3141PubMed Google Scholar† Wan was originally classified as a type I/II cell line.5Rowe M Rowe DT Gregory CD Young LS Farrell PJ Rupani H Rickinson AB Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells.EMBO J. 1987; 6: 2743-2751PubMed Google Scholar However, the Wan cell line we obtained showed a drift to type III in some cells.27Tao Q Robertson KD Manns A Hildesheim A Ambinder RF Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue.Blood. 1998; 91: 1373-1381PubMed Google Scholar‡ In the Raji cell line, C promoter is methylated and silent and EBNAs are driven by an unknown promoter. Open table in a new tab u, unmethylated; m, methylated. The sources of endemic BL, NPC, HD, and nasal lymphoma specimens have been described previously,22Robertson KD Manns A Swinnen LJ Zong JC Gulley ML Ambinder RF CpG methylation of the major Epstein-Barr virus latency promoter in Burkitt's lymphoma, and Hodgkin's disease.Blood. 1996; 88: 3129-3136PubMed Google Scholar, 27Tao Q Robertson KD Manns A Hildesheim A Ambinder RF Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue.Blood. 1998; 91: 1373-1381PubMed Google Scholar, 28Tao Q Robertson KD Manns A Hildesheim A Ambinder RF The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive.J Virol. 1998; 72: 7075-7083PubMed Google Scholar, 29Chiang AKS Tao Q Srivastava G Ho FCS Nasal NK-, and T-cell lymphomas share the same type of Epstein-Barr virus latency as nasopharyngeal carcinoma, and Hodgkin's disease.Int J Cancer. 1996; 68: 285-290Crossref PubMed Scopus (154) Google Scholar as have 14 lymphoproliferative disease specimens from 12 organ transplant recipients and one lymphoproliferative disease specimen from a patient with aplastic anemia arising after treatment with immunosuppressive therapy (B-LPD).15Murray PG Swinnen LJ Constandinou CM Pyle JM Carr TJ Hardwick JM Ambinder RF Bcl-2 but not the EBV-encoded bcl-2 homologue, BHRF-1, is commonly expressed in post-transplantation lymphoproliferative disorders.Blood. 1996; 87: 706-711PubMed Google Scholar, 22Robertson KD Manns A Swinnen LJ Zong JC Gulley ML Ambinder RF CpG methylation of the major Epstein-Barr virus latency promoter in Burkitt's lymphoma, and Hodgkin's disease.Blood. 1996; 88: 3129-3136PubMed Google Scholar Genomic DNA was extracted from frozen tumor blocks by conventional methods. DNA (2–5 μg) was denatured in 33 μl of 0.3 mol/L NaOH at 37°C for 15 minutes, without using restriction endonuclease. Denatured DNA was mixed directly with 333 μl of bisulfite solution and treated in darkness. The bisulfite solution was prepared as either 2.4 mol/L sodium metabisulfite (pH 5.0–5.2) (Sigma S-1516, St. Louis, MO)/0.5 mmol/L hydroquinone (Sigma H-7148) for a 4-hour treatment31McDonald LE Kay GF Methylation analysis using bisulfite genomic sequencing: application to small numbers of intact cells.BioTechniques. 1997; 22: 272-274Crossref PubMed Scopus (34) Google Scholar or 3.1 mol/L sodium bisulfite (pH 5.0–5.2) (Sigma S-8890)/0.5 mmol/L hydroquinone for a 16-hour treatment.32Frommer M McDonald LE Millar DS Collis CM Watt F Grigg GW Molloy PL Paul CL A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA. 1992; 89: 1827-1831Crossref PubMed Scopus (2542) Google Scholar DNA was desalted and purified using the Wizard DNA Clean-Up system (Promega, Madison, WI). DNA was then treated with 0.3 mol/L NaOH at 37°C for 15 minutes and precipitated with 3 mol/L ammonium acetate and 3 volumes of ethanol. Recovered DNA was dissolved in 20–50 μl of TE buffer (pH 8.0) and stored at −20°C. C promoter methylation status was determined by using methylation-specific PCR33Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5240) Google Scholar (Figure 1). In this method, bisulfite treatment converts unmethylated cytosine to uracil but does not affect the methylated cytosine. Thus, PCR primers can be designed that anneal selectively to methylated or unmethylated DNA after bisulfite conversion.32Frommer M McDonald LE Millar DS Collis CM Watt F Grigg GW Molloy PL Paul CL A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA. 1992; 89: 1827-1831Crossref PubMed Scopus (2542) Google Scholar, 33Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5240) Google Scholar The sequences of the unmethylated DNA and methylated DNA-specific primers are listed in Table 2. The primer pair ub3/ub4 was designed specifically for amplification of the bisulfite-converted unmethylated C promoter, while the mb3/mb4 primer pair was designed specifically for the amplification of the bisulfite-converted methylated C promoter.Table 2Primers for Methylation-Specific PCR Analysis of EBV C PromoterPrimersDNA sequence (5′ → 3′)PCR annealing temperatureCoordinate in B95-8 genomeub3CATCCAAAAACCAAACAACTCA55°C10,702-10,723ub4AGTAAGGTGTAATTAATTTTGTTT11,217-11,194mb3GTCC*GAAAACC*GAAC*GACTC*G58°C10,703-10,723mb4GTAAGGC*GTAATTAATTTC*GTTC*11,216-11,194The cytosine nucleotides in each CpG site that are susceptible to methylation are marked by asterisks. Open table in a new tab The cytosine nucleotides in each CpG site that are susceptible to methylation are marked by asterisks. One microliter of bisulfite-treated DNA (around 40 ng) was PCR-amplified by using 1.875 U of Taq Gold polymerase (Perkin Elmer-Cetus, Norwalk, CT) with 1.5 mmol/L MgCl2 and 0.2 mmol/L dNTP in a 25-μl reaction volume. Primers were used at a final concentration of 0.4 μmol/L each. The PCR involved an initial denaturation at 94°C for 10 minutes, followed by 40 cycles consisting of 94°C for 30 seconds, predetermined optimal annealing temperature for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 3 minutes. Twenty microliters of PCR product were analyzed on a 1.8% agarose gel. Methylation-specific PCR yields a 516-bp product for the unmethylated and a 514-bp product for the methylated C promoter. Primers were designed to amplify a region immediately adjacent to the EBNA2 response element of the C promoter, with the reverse primers (ub4 and mb4) located just within the previously identified methylation hypersensitive region II, which is flanked by two CAAT boxes (Figure 1).21Robertson KD Ambinder RF Mapping promoter regions that are hypersensitive to methylation-mediated inhibition of transcription: application of the methylation cassette assay to the Epstein-Barr virus major latency promoter.J Virol. 1997; 71: 6445-6454PubMed Google Scholar in vitro studies had previously shown that C promoter transcription was hypersensitive to CpG methylation at this locus.20Robertson KD Hayward DJ Ling PD Samid D Ambinder RF Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.Mol Cell Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (110) Google Scholar, 21Robertson KD Ambinder RF Mapping promoter regions that are hypersensitive to methylation-mediated inhibition of transcription: application of the methylation cassette assay to the Epstein-Barr virus major latency promoter.J Virol. 1997; 71: 6445-6454PubMed Google Scholar, 22Robertson KD Manns A Swinnen LJ Zong JC Gulley ML Ambinder RF CpG methylation of the major Epstein-Barr virus latency promoter in Burkitt's lymphoma, and Hodgkin's disease.Blood. 1996; 88: 3129-3136PubMed Google Scholar Differential amplification of methylated and unmethylated sequences by methylation-specific PCR relies on cytosine to uracil conversion after bisulfite treatment but no conversion of cytosine methylated at the 5-position (metC) to uracil after the same treatment.32Frommer M McDonald LE Millar DS Collis CM Watt F Grigg GW Molloy PL Paul CL A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA. 1992; 89: 1827-1831Crossref PubMed Scopus (2542) Google Scholar, 33Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5240) Google Scholar This amplification is strand-specific because bisulfite conversion renders DNA strands noncomplementary. Primer pairs were designed to amplify only bisulfite-reacted DNA sequences of the bottom strand (Table 2). Application of the technique to EBV-positive cell lines whose C promoter methylation status had been previously characterized by bisulfite genomic sequencing verified that the amplification was selective (Tables 1 and 3). There was no amplification of untreated DNAs from B95-8, Akata, Raji, or AG876 cell lines (Figure 2). When these primers were applied to bisulfite-treated DNAs from these cell lines, there was amplification of DNA (Figure 2), indicating that the primer pairs ub3/ub4 and mb3/mb4 were specific for bisulfite-treated DNA. Rael DNA (methylated C promoter)20Robertson KD Hayward DJ Ling PD Samid D Ambinder RF Transcriptional activation of the EBV latency C promoter following 5-azacytidine treatment: evidence that demethylation at a single CpG site is crucial.Mol Cell Biol. 1995; 15: 6150-6159Crossref PubMed Scopus (110) Google Scholar yielded a band with primers (mb3/mb4) specific for bisulfite-treated methylated DNA but not with primers (ub3/ub4) specific for bisulfite-treated unmethylated DNA, whereas B95-8 DNA (unmethylated C promoter) yielded a band with ub3/ub4 primers but not mb3/mb4 primers (Table 3). Other cell lines yielded bands with both sets of primers, reflecting the presence of a mixture of methylated and unmethylated DNA. The C promoter is active in the Wan cell line, which explains the bands obtained with both methylated and unmethylated primers.28Tao Q Robertson KD Manns A Hildesheim A Ambinder RF The Epstein-Barr virus major latent promoter Qp is constitutively active, hypomethylated, and methylation sensitive.J Virol. 1998; 72: 7075-7083PubMed Google Scholar The Raji cell line is a latency III cell line that expresses the immunodominant EBNAs.27Tao Q Robertson KD Manns A Hildesheim A Ambinder RF Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue.Blood. 1998; 91: 1373-1381PubMed Google Scholar It might have been anticipated that the C promoter would be unmethylated. However, bisulfite genomic sequencing studies carried out previously by our group have shown that the C promoter is methylated in Raji, and the immunodominant EBNAs expressed in this cell line derive not from the C promoter but from an alternate uncharacterized promoter (KD Robertson, unpublished observations). The very weak unmethylated PCR band in Raji probably resulted from the presence of unmethylated C promoter or spontaneous lytic activation in a very small proportion of cells. Although Akata is a latency I cell line,5Rowe M Rowe DT Gregory CD Young LS Farrell PJ Rupani H Rickinson AB Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells.EMBO J. 1987; 6: 2743-2751PubMed Google Scholar, 27Tao Q Robertson KD Manns A Hildesheim A Ambinder RF Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue.Blood. 1998; 91: 1373-1381PubMed Google Scholar the spontaneous lytic activation of EBV in a fra
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