Skip to main content

Advertisement

Springer Nature Link
Log in

Treatment of ovarian cancer cell lines with 5-aza-2′-deoxycytidine upregulates the expression of cancer-testis antigens and class I major histocompatibility complex-encoded molecules

  • Original Article
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Purpose

To test the hypothesis that decrease in DNA methylation will increase the expression of cancer-testis antigens (CTA) and class I major histocompatibility complex (MHC)-encoded molecules by ovarian cancer cells, and thus increase the ability of these cells to be recognized by antigen-reactive CD8+ T cells.

Methods

Human ovarian cancer cell lines were cultured in the presence or absence of varying concentrations of the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC) for 3–7 days. The expression levels of 12 CTA genes were measured using the polymerase chain reaction. The protein expression levels of class I MHC molecules and MAGE-A1 were measured by flow cytometry. T cell reactivity was determined using interferon-γ ELISpot analysis.

Results

DAC treatment of ovarian cancer cell lines increased the expression of 11 of 12 CTA genes tested including MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, NY-ESO-1, TAG-1, TAG-2a, TAG-2b, and TAG-2c. In contrast, DAC treatment decreased the already low expression of the MAGE-A2 gene by ovarian cancer cells, a finding not previously observed in cancers of any histological type. DAC treatment increases the expression of class I MHC molecules by the cells. These effects were time-dependent over a 7-day interval, and were dose-dependent up to 1–3 μM for CTA and up to 10 μM for class I MHC molecules. Each cell line tested had a unique pattern of gene upregulation after exposure to DAC. The enhanced expression levels increased the recognition of 2 of 3 antigens recognized by antigen-reactive CD8+ T cells.

Conclusions

These results demonstrate the potential utility of combining DAC therapy with vaccine therapy in an attempt to induce the expression of antigens targeted by the vaccine, but they also demonstrate that care must be taken to target inducible antigens.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

CTA:

Cancer-testis antigen

CTL:

Cytotoxic T lymphocyte

DAC:

5-Aza-2′-deoxycytidine

DNMT:

DNA methyltransferase

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

mAb:

Monoclonal antibody

MHC:

Major histocompatibility complex

PCR:

Polymerase chain reaction

References

  1. Berger AE, Davis JE, Cresswell P (1982) Monoclonal antibody to HLA-A3. Hybridoma 1:87–90

    PubMed  CAS  Google Scholar 

  2. Brasseur F, Marchand M, Vanwijck R, Herin M, Lethe B, Chomez P, Boon T (1992) Human gene MAGE-1, which codes for a tumor-rejection antigen, is expressed by some breast tumors. Int J Cancer 52:839–841

    Article  PubMed  CAS  Google Scholar 

  3. Buick RN, Pullano R, Trent JM (1985) Comparative properties of five human ovarian adenocarcinoma cell lines. Cancer Res 45:3668–3676

    PubMed  CAS  Google Scholar 

  4. Carr TM, Adair SJ, Fink MJ, Hogan KT (2008) Immunological profiling of a panel of ovarian cancer cell lines. Cancer Immunol Immunother 57:31–42

    Article  PubMed  CAS  Google Scholar 

  5. Chen YT, Old LJ (1999) Cancer-testis antigens: targets for cancer immunotherapy. Cancer J Sci Am 5:16–17

    Article  PubMed  CAS  Google Scholar 

  6. Chianese-Bullock KA, Pressley J, Garbee C, Hibbitts S, Murphy C, Yamshchikov G, Petroni GR, Bissonette EA, Neese PY, Grosh WW, Merrill P, Fink R, Woodson EM, Wiernasz CJ, Patterson JW, Slingluff CL Jr (2005) MAGE-A1-, MAGE-A10-, and gp100-derived peptides are immunogenic when combined with granulocyte-macrophage colony-stimulating factor and Montanide ISA-51 adjuvant and administered as part of a multipeptide vaccine for melanoma. J Immunol 174:3080–3086

    PubMed  CAS  Google Scholar 

  7. Cho B, Lee H, Jeong S, Bang YJ, Lee HJ, Hwang KS, Kim HY, Lee YS, Kang GH, Jeoung DI (2003) Promoter hypomethylation of a novel cancer/testis antigen gene cage is correlated with its aberrant expression and is seen in premalignant stage of gastric carcinoma. Biochem Biophys Res Commun 307:52–63

    Article  PubMed  CAS  Google Scholar 

  8. Coral S, Sigalotti L, Altomonte M, Engelsberg A, Colizzi F, Cattarossi I, Maraskovsky E, Jager E, Seliger B, Maio M (2002) 5-aza-2′-deoxycytidine-induced expression of functional cancer testis antigens in human renal cell carcinoma: Immunotherapeutic implications. Clin Cancer Res 8:2690–2695

    PubMed  CAS  Google Scholar 

  9. Coral S, Sigalotti L, Colizzi F, Spessotto A, Nardi G, Cortini E, Pezzani L, Fratta E, Fonsatti E, Di Giacomo AM, Nicotra MR, Natali PG, Altomonte M, Maio M (2006) Phenotypic and functional changes of human melanoma xenografts induced by DNA hypomethylation: Immunotherapeutic implications. J Cell Physiol 207:58–66

    Article  PubMed  CAS  Google Scholar 

  10. Coral S, Sigalotti L, Gasparollo A, Cattarossi I, Visintin A, Cattelan A, Altomonte M, Maio M (1999) Prolonged upregulation of the expression of HLA class I antigens and costimulatory molecules on melanoma cells treated with 5-aza-2′-deoxycytidine (5-aza-cdr). J Immunother 22:16–24

    Article  PubMed  CAS  Google Scholar 

  11. De Plaen E, Arden K, Traversari C, Gaforio JJ, Szikora JP, De Smet C, Brasseur F, van der Bruggen P, Lethe B, Lurquin C, Chomez P, De Backer O, Boon T, Arden K, Cavenee W, Brasseur R (1994) Structure, chromosomal localization, and expression of 12 genes of the MAGE family. Immunogenetics 40:360–369

    Article  PubMed  CAS  Google Scholar 

  12. De Smet C, De Backer O, Faraoni I, Lurquin C, Brasseur F, Boon T (1996) The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc Natl Acad Sci U S A 93:7149–7153

    Article  PubMed  Google Scholar 

  13. De Smet C, Lurquin C, Lethe B, Martelange V, Boon T (1999) DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol Cell Biol 19:7327–7335

    PubMed  Google Scholar 

  14. De Smet C, Lurquin C, Van der Bruggen P, De Plaen E, Brasseur F, Boon T (1994) Sequence and expression pattern of the human MAGE2 gene. Immunogenetics 39:121–129

    Article  PubMed  Google Scholar 

  15. dos Santos NR, Torensma R, De Vries TJ, Schreurs MWJ, de Bruijn DRH, Kater-Baats E, Ruiter DJ, Adema GJ, van Muijen GNP, van Kessel AG (2000) Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines. Cancer Res 60:1654–1662

    PubMed  CAS  Google Scholar 

  16. Ellis SA, Taylor C, McMichael A (1982) Recognition of HLA-B27 and related antigen by a monoclonal antibody. Hum Immunol 5:49–59

    Article  PubMed  CAS  Google Scholar 

  17. Fogh J, Tremple G (1975) New human tumor cell lines. In: Fogh J (ed) Human tumor cell lines in vitro. Plenum Press, New York, pp 115–141

    Google Scholar 

  18. Fogh J, Wright WC, Loveless JD (1977) Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J Natl Cancer Inst 58:209–214

    PubMed  CAS  Google Scholar 

  19. Fonsatti E, Nicolay HJM, Sigalotti L, Calabro L, Pezzani L, Colizzi F, Altomonte M, Guidoboni M, Marincola FM, Maio M (2007) Functional up-regulation of human leukocyte antigen class I antigens expression by 5-aza-2′-deoxycytidine in cutaneous melanoma: Immunotherapeutic implications. Clin Cancer Res 13:3333–3338

    Article  PubMed  CAS  Google Scholar 

  20. Fonsatti E, Sigalotti L, Coral S, Colizzi F, Altomonte M, Maio M (2003) Methylation-regulated expression of HLA class I antigens in melanoma. Int J Cancer 105:430–431

    Article  PubMed  CAS  Google Scholar 

  21. Fujie T, Mori M, Ueo H, Sugimachi K, Akiyoshi T (1997) Expression of MAGE and BAGE genes in Japanese breast cancers. Ann Oncol 8:369–372

    Article  PubMed  CAS  Google Scholar 

  22. Guo ZS, Hong JA, Irvine KR, Chen GA, Spiess PJ, Liu Y, Zeng G, Wunderlich JR, Nguyen DM, Restifo NP, Schrump DS (2006) De novo induction of a cancer/testis antigen by 5-aza-2′-deoxycytidine augments adoptive immunotherapy in a murine tumor model. Cancer Res 66:1105–1113

    Article  PubMed  CAS  Google Scholar 

  23. Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, Whang-Peng J, Rogan AM, Green WR, Ozols RF (1983) Characterization of a human ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors. Cancer Res 43:5379–5389

    PubMed  CAS  Google Scholar 

  24. Hogan KT, Coppola MA, Gatlin CL, Thompson LW, Shabanowitz J, Hunt DF, Engelhard VH, Ross MM, Slingluff CL (2004) Identification of novel and widely expressed cancer/testis gene isoforms that elicit spontaneous cytotoxic T lymphocyte reactivity to melanoma. Cancer Res 64:1157–1163

    Article  PubMed  CAS  Google Scholar 

  25. Jager D, Jager E, Knuth A (2001) Immune responses to tumour antigens: Implications for antigen specific immunotherapy of cancer. J Clin Pathol 54:669–674

    PubMed  CAS  Google Scholar 

  26. Jungbluth AA, Chen YT, Stockert E, Busam KJ, Kolb D, Iversen K, Coplan K, Williamson B, Altorki N, Old LJ (2001) Immunohistochemical analysis of NY-ESO-1 antigen expression in normal and malignant human tissues.[erratum appears in Int J Cancer 2002 February 20;97(6):878]. Int J Cancer 92:856–60

    Google Scholar 

  27. Jungbluth AA, Stockert E, Chen YT, Kolb D, Iversen K, Coplan K, Williamson B, Altorki N, Busam KJ, Old LJ (2000) Monoclonal antibody MA454 reveals a heterogeneous expression pattern of MAGE-1 antigen in formalin-fixed paraffin embedded lung tumours. Br J Cancer 83:493–497

    Article  PubMed  CAS  Google Scholar 

  28. Juretic A, Spagnoli GC, Schultz-Thater E, Sarcevic B (2003) Cancer/testis tumour-associated antigens: immunohistochemical detection with monoclonal antibodies. Lancet Oncol 4:104–109

    Article  PubMed  CAS  Google Scholar 

  29. Karlan BY, Amin W, Band V, Zurawski VR, Littlefield BA (1988) Plasminogen activator secretion by established lines of human ovarian carcinoma cells in vitro. Gynecol Oncol 31:103–112

    Article  PubMed  CAS  Google Scholar 

  30. Karpf AR, Lasek AW, Ririe TO, Hanks AN, Grossman D, Jones DA (2004) Limited gene activation in tumor and normal epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine. Mol Pharmacol 65:18–27

    Article  PubMed  CAS  Google Scholar 

  31. Kirkin AF, Dzhandzhugazyan KN, Zeuthen J (2002) Cancer/testis antigens: structural and immunobiological properties. Cancer Invest 20:222–236

    Article  PubMed  CAS  Google Scholar 

  32. Kuppen PJK, Schuitemaker H, van’t Veer LJ, de Bruijn EA, van Oosterom AT, Schrier PI (1988) Cis-diamminedichloroplatinum(II)-resistant sublines derived from two human ovarian tumor cell lines. Cancer Res 48:3355–3359

    PubMed  CAS  Google Scholar 

  33. Lau DHM, Lewis AD, Ehsan MN, Sikic BI (1991) Multifactorial mechanisms associated with broad cross-resistance of ovarian carcinoma cells selected by cyanomorpholino doxorubicin. Cancer Res 51:5181–5187

    PubMed  CAS  Google Scholar 

  34. Li J, Yang Y, Fujie F, Baba K, Ueo H, Mori M, Akiyoshi T (1996) Expression of BAGE, GAGE, and MAGE genes in human gastric carcinoma. Clin Cancer Res 2:1619–1625

    PubMed  CAS  Google Scholar 

  35. Lurquin C, De Smet C, Brasseur F, Muscatelli F, Martelange V, De Plaen E, Brasseur R, Monaco AP, Boon T (1997) Two members of the human mageb gene family located in xp21.3 are expressed in tumors of various histological origins. Genomics 46:397–408

    Article  PubMed  CAS  Google Scholar 

  36. Maier JA, Voulalas P, Roeder D, Maciag T (1990) Extension of the life-span of human endothelial cells by an interleukin-1 alpha antisense oligomer. Science 249:1570–1574

    Article  PubMed  CAS  Google Scholar 

  37. Menendez L, Walker D, Matyunina LV, Dickerson EB, Bowen NJ, Polavarapu N, Benigno BB, McDonald JF (2007) Identification of candidate methylation-responsive genes in ovarian cancer. Mol Cancer 6:10

    Article  PubMed  Google Scholar 

  38. Menendez L, Walker LD, Matyunina LV, Totten KA, Benigno BB, McDonald JF (2008) Epigenetic changes within the promoter region of the HLA-G gene in ovarian tumors. Mol Cancer 7:43

    Article  PubMed  Google Scholar 

  39. Nie Y, Yang G, Song Y, Zhao X, So C, Liao J, Wang LD, Yang CS (2001) DNA hypermethylation is a mechanism for loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas. Carcinogenesis 22:1615–1623

    Article  PubMed  CAS  Google Scholar 

  40. Parham P, Barnstable CJ, Bodmer WF (1979) Use of a monoclonal antibody (W6/32) in structural studies of HLA-A, b, c antigens. J Immunol 123:342–349

    PubMed  CAS  Google Scholar 

  41. Parmiani G, Castelli C, Dalerba P, Mortarini R, Rivoltini L, Marincola FM, Anichini A (2002) Cancer immunotherapy with peptide-based vaccines: what have we achieved? Where are we going? J Natl Cancer Inst 94:805–818

    PubMed  CAS  Google Scholar 

  42. Platsoucas CD, Fincke JE, Pappas J, Jung WJ, Heckel M, Schwarting R, Magira E, Monos D, Freedman RS (2003) Immune responses to human tumors: development of tumor vaccines. Anticancer Res 23:1969–1996

    PubMed  CAS  Google Scholar 

  43. Provencher DM, Lounis H, Champoux L, Tetrault M, Manderson EN, Wang JC, Eydoux P, Savoie R, Tonin PN, Mes-Masson AM (2000) Characterization of four novel epithelial ovarian cancer cell lines. In Vitro Cell Dev Biol Anim 36:357–361

    Article  PubMed  CAS  Google Scholar 

  44. Ries LAG, Melbert D, Krapcho M, Marriotto A, Miller BA, Feuer EJ, Clegg L, Horner MJ, Howlader N, Eisner MP, Reichman M, Edwards BK (2007) Seer cancer statistics review, 1975–2004. Natl Cancer Inst, Bethesda

    Google Scholar 

  45. Russo C, Ng AK, Pellegrino MA, Ferrone S (1983) The monoclonal antibody CR11-351 discriminates HLA-A2 variants identified by T cells. Immunogenetics 18:23–35

    Article  PubMed  CAS  Google Scholar 

  46. Scanlan MJ, Simpson AJ, Old LJ (2004) The cancer/testis genes: review, standardization, and commentary. Cancer Immun 4:1

    PubMed  Google Scholar 

  47. Schrump DS, Fischette MR, Nguyen DM, Zhao M, Li X, Kunst TF, Hancox A, Hong JA, Chen GA, Pishchik V, Figg WD, Murgo AJ, Steinberg SM (2006) Phase I study of decitabine-mediated gene expression in patients with cancers involving the lungs, esophagus, or pleura. Clin Cancer Res 12:5777–5785

    Article  PubMed  CAS  Google Scholar 

  48. Seliger B, Cabrera T, Garrido F, Ferrone S (2002) HLA class I antigen abnormalities and immune escape by malignant cells. Semin Cancer Biol 12:3–13

    Article  PubMed  CAS  Google Scholar 

  49. Serrano A, Tanzarella S, Lionello I, Mendez R, Traversari C, Ruiz-Cabello F, Garrido F (2001) Expression of HLA class I antigens and restoration of antigen-specific CTL response in melanoma cells following 5-aza-2′-deoxycytidine treatment. Int J Cancer 94:243–251

    Article  PubMed  CAS  Google Scholar 

  50. Sigalotti L, Altomonte M, Colizzi F, Degan M, Rupolo M, Zagonel V, Pinto A, Gattei V, Maio M, Lubbert M, Wijermans PW, Jones PA, Hellstrom-Lindberg E (2003) 5-aza-2′-deoxycytidine (decitabine) treatment of hematopoietic malignancies: A multimechanism therapeutic approach? Blood 101:4644–4646

    Article  PubMed  CAS  Google Scholar 

  51. Sigalotti L, Coral S, Altomonte M, Natali L, Gaudino G, Cacciotti P, Libener R, Colizzi F, Vianale G, Martini F, Tognon M, Jungbluth A, Cebon J, Maraskovsky E, Mutti L, Maio M (2002) Cancer testis antigens expression in mesothelioma: role of DNA methylation and bioimmunotherapeutic implications. Br J Cancer 86:979–982

    Article  PubMed  CAS  Google Scholar 

  52. Sigalotti L, Fratta E, Coral S, Tanzarella S, Danielli R, Colizzi F, Fonsatti E, Traversari C, Altomonte M, Maio M (2004) Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2′-deoxycytidine. Cancer Res 64:9167–9171

    Article  PubMed  CAS  Google Scholar 

  53. Slingluff CL Jr, Petroni GR, Chianese-Bullock KA, Smolkin ME, Hibbitts S, Murphy C, Johansen N, Grosh WW, Yamshchikov GV, Neese PY, Patterson JW, Fink R, Rehm PK (2007) Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting. Clin Cancer Res 13:6386–6395

    Article  PubMed  CAS  Google Scholar 

  54. Suyama T, Ohashi H, Nagai H, Hatano S, Asano H, Murate T, Saito H, Kinoshita T (2002) The MAGE-A2 gene expression is not determined solely by methylation status of the promoter region in hematological malignancies. Leuk Res 26:1113–1118

    Article  PubMed  CAS  Google Scholar 

  55. Wang RF, Johnston SL, Zeng G, Topalian SL, Schwartzentruber DJ, Rosenberg SA (1998) A breast and melanoma-shared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames. J Immunol 161:3598–3606

    PubMed  CAS  Google Scholar 

  56. Weber J, Salgaller M, Samid D, Johnson B, Herlyn M, Lassam N, Treisman J, Rosenberg SA (1994) Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2′-deoxycytidine. Cancer Res 54:1766–1771

    PubMed  CAS  Google Scholar 

  57. Weiser TS, Guo ZS, Ohnmacht GA, Parkhurst ML, Tong-On P, Marincola FM, Fischette MR, Yu X, Chen GA, Hong JA, Stewart JH, Nguyen DM, Rosenberg SA, Schrump DS (2001) Sequential 5-aza-2′ deoxycytidine-depsipeptide FR901228 treatment induces apoptosis preferentially in cancer cells and facilitates their recognition by cytolytic T lymphocytes specific for NY-ESO-1. J Immunother 24:151–161

    Article  CAS  Google Scholar 

  58. Weiser TS, Ohnmacht GA, Guo ZS, Fischette MR, Chen GA, Hong JA, Nguyen DM, Schrump DS (2001) Induction of MAGE-3 expression in lung and esophageal cancer cells. Ann Thorac Surg 71:295–302

    Article  PubMed  CAS  Google Scholar 

  59. Wischnewski F, Pantel K, Schwarzenbach H (2006) Promoter demethylation and histone acetylation mediate gene expression of MAGE-A1, -A2, -A3, and -A12 in human cancer cells. Mol Cancer Res 4:339–349

    Article  PubMed  CAS  Google Scholar 

  60. Yamshchikov GV, Barnd DL, Eastham S, Galavotti H, Patterson JW, Deacon DH, Teates D, Neese P, Grosh WW, Petroni G, Engelhard VH, Slingluff CL Jr (2001) Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients. Int J Cancer 92:703–711

    Article  PubMed  CAS  Google Scholar 

  61. Zendman AJ, Ruiter DJ, Van Muijen GN (2003) Cancer/testis-associated genes: identification, expression profile, and putative function. J Cell Physiol 194:272–288

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grant W81XWH-05-1-0012 from the United States Department of Defense to K. T. Hogan. The authors wish to thank Dr. Craig L. Slingluff Jr. for reviewing the manuscript and the members of the UVA Human Immune Therapy Center core laboratory for performing the ELISpot analyses.

Author information

Authors and Affiliations

  1. Department of Surgery and the Human Immune Therapy Center, University of Virginia, Box 801359, Charlottesville, VA, 22908, USA

    Sara J. Adair & Kevin T. Hogan

Authors
  1. Sara J. Adair
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Kevin T. Hogan
    View author publications

    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence to Kevin T. Hogan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figures (DOC 584 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Adair, S.J., Hogan, K.T. Treatment of ovarian cancer cell lines with 5-aza-2′-deoxycytidine upregulates the expression of cancer-testis antigens and class I major histocompatibility complex-encoded molecules. Cancer Immunol Immunother 58, 589–601 (2009). https://doi.org/10.1007/s00262-008-0582-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-008-0582-6

Keywords