Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance

Differences in the global methylation pattern, ie hyper‐ as well as hypo‐methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5‐$^{\rm m}$cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam‐2 before and after demethylation using 5‐azacytidine. Exposure to 5‐azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell‐specific marker VASA, showed increased expression. Following treatment with 5‐azacytidine, TCam‐2 cells were analysed using a high‐throughput methylation screen for changes in the methylation sites of 14 000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

[1]  A. Horwich,et al.  Second-line chemotherapy in patients with relapsed extragonadal nonseminomatous germ cell tumors: results of an international multicenter analysis. , 2001, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[2]  R. Spang,et al.  New insights into the biology and origin of mature aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling. , 2009, Blood.

[3]  K. Okamoto,et al.  Epigenetic profile of testicular germ cell tumours. , 2007, International journal of andrology.

[4]  K. Biermann,et al.  Epigenetics in male germ cells. , 2007, Journal of andrology.

[5]  S. Fosså,et al.  European consensus conference on diagnosis and treatment of germ cell cancer: a report of the second meeting of the European Germ Cell Cancer Consensus Group (EGCCCG): part II. , 2008, European urology.

[6]  S. Fosså,et al.  Prognostic variables for response and outcome in patients with extragonadal germ-cell tumors. , 2002, Annals of oncology : official journal of the European Society for Medical Oncology.

[7]  A. Multani,et al.  N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA , 2004, Molecular Cancer.

[8]  Yi Guo,et al.  Drug sensitivity prediction by CpG island methylation profile in the NCI-60 cancer cell line panel. , 2007, Cancer research.

[9]  L. Looijenga,et al.  Testicular germ-cell tumours in a broader perspective , 2005, Nature Reviews Cancer.

[10]  E. Dmitrovsky,et al.  High DNA methyltransferase 3B expression mediates 5-aza-deoxycytidine hypersensitivity in testicular germ cell tumors. , 2009, Cancer research.

[11]  Marina Bibikova,et al.  A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms , 2009, PloS one.

[12]  C. Bokemeyer,et al.  Comparative proteome, transcriptome, and genome analysis of a gonadal and an extragonadal germ cell tumor cell line. , 2008, Journal of proteome research.

[13]  C. Bokemeyer,et al.  Pharmacotherapy of relapsed metastatic testicular cancer. , 2008, Expert opinion on pharmacotherapy.

[14]  W L Allen,et al.  c-FLIP inhibits chemotherapy-induced colorectal cancer cell death , 2006, Oncogene.

[15]  C. Bokemeyer,et al.  Towards an understanding of the biological basis of response to cisplatin-based chemotherapy in germ-cell tumors. , 2003, Annals of oncology : official journal of the European Society for Medical Oncology.

[16]  Li-Xuan Qin,et al.  Identification and validation of a gene expression signature that predicts outcome in adult men with germ cell tumors. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[17]  J. Hicks,et al.  Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors , 2008, Modern Pathology.

[18]  V. Reuter.  Origins and molecular biology of testicular germ cell tumors , 2005, Modern Pathology.

[19]  Hongtao Yu,et al.  Short hairpin RNA targeting c-FLIP sensitizes human cervical adenocarcinoma Hela cells to chemotherapy and radiotherapy. , 2008, Cancer letters.

[20]  L. Einhorn.  Curing metastatic testicular cancer , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R. Pfundt,et al.  Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene. , 2006, Cancer research.

[22]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[23]  M. Esteller.  Cancer epigenomics: DNA methylomes and histone-modification maps , 2007, Nature Reviews Genetics.

[24]  R. Siebert,et al.  Array-based DNA methylation profiling of primary lymphomas of the central nervous system , 2009, BMC Cancer.

[25]  C. Bokemeyer,et al.  Efficacy of cytotoxic agents used in the treatment of testicular germ cell tumours under normoxic and hypoxic conditions in vitro , 2003, British Journal of Cancer.

[26]  S. Kitazawa,et al.  Further characterization of the first seminoma cell line TCam‐2 , 2008, Genes, chromosomes & cancer.

[27]  R. Siebert,et al.  Hunting for the 5th base: Techniques for analyzing DNA methylation. , 2009, Biochimica et biophysica acta.

[28]  W. Reik,et al.  Epigenetic Reprogramming in Mammalian Development , 2001, Science.

[29]  J. Bacik,et al.  Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors , 2004, Molecular Cancer.

[30]  A. Grothey,et al.  Failure of activation of caspase-9 induces a higher threshold for apoptosis and cisplatin resistance in testicular cancer. , 2003, Cancer research.

[31]  E. Steyerberg,et al.  Microsatellite instability, mismatch repair deficiency, and BRAF mutation in treatment-resistant germ cell tumors. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[32]  D. Heimburger,et al.  Immunohistochemical Evaluation of Global DNA Methylation: Comparison with in Vitro Radiolabeled Methyl Incorporation Assay , 2000, Biotechnic & histochemistry : official publication of the Biological Stain Commission.

[33]  C. Plass,et al.  Distinct epigenetic phenotypes in seminomatous and nonseminomatous testicular germ cell tumors , 2002, Oncogene.

[34]  S. Fosså,et al.  European consensus conference on diagnosis and treatment of germ cell cancer: a report of the second meeting of the European Germ Cell Cancer Consensus group (EGCCCG): part I. , 2008, European urology.

[35]  R. Siebert,et al.  The Human Retinoblastoma Gene Is Imprinted , 2009, PLoS genetics.

[36]  R. Lothe,et al.  The epigenome of testicular germ cell tumors , 2007, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.