Abstract
Purpose
Testisin, a serine protease abundantly expressed only in normal testes, is thought to be a tumor suppressor gene silenced by aberrant methylation in testicular germ cell tumors (TGCT). This study aimed at identifying the CpG sites relevant for testisin gene silencing when methylated and evaluating the potential of aberrant methylation as a biomarker in TGCT.
Methods
Bisulfite sequencing and subsequent real-time RT-PCR were carried out in germ cell tumor cell lines and a cervical carcinoma cell line to reveal CpG sites implicated in testisin gene silencing. The normalized index of methylation (NIM) and the relative gene expression (RGE) were calculated in 33 TGCT and 19 normal testicular tissue samples by quantitative methylation-specific PCR (QMSP) and real-time RT-PCR.
Results
CpG sites in the 5′untranslated region proved to be relevant for testisin gene silencing when methylated. Targeting these CpG sites by QMSP, we demonstrated that the median NIM was 8.6 times higher in the TGCT group than in normal testicular samples. This was associated with a median 23.6 times lower RGE in the TGCT probes. Non-seminomas had a significantly higher NIM than seminomas, but RGE was downregulated to comparable levels in both subgroups. QMSP was highly sensitive and distinguished TGCT from normal samples with excellent specificity.
Conclusions
Our results demonstrate for the first time that aberrant methylation of specific CpG sites near the transcription initiation site is an important factor in testisin gene silencing in TGCT.
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References
Baylin SB, Herman JG (2000) DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 16:168–174
Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28:E32
Esteller M (2003) Relevance of DNA methylation in the management of cancer. Lancet Oncol 4:351–358
Esteller M (2005) Aberrant DNA methylation as a cancer-inducing mechanism. Annu Rev Pharmacol Toxicol 45:629–656
Honorio S, Agathanggelou A, Wernert N, Rothe M, Maher ER, Latif F (2003) Frequent epigenetic inactivation of the RASSF1A tumour suppressor gene in testicular tumours and distinct methylation profiles of seminoma and nonseminoma testicular germ cell tumours. Oncogene 22:461–466
Hooper JD, Bowen N, Marshall H, Cullen LM, Sood R, Daniels R, Stuttgen MA, Normyle JF, Higgs DR, Kastner DL, Ogbourne SM, Pera MF, Jazwinska EC, Antalis TM (2000) Localization, expression and genomic structure of the gene encoding the human serine protease testisin. Biochim Biophys Acta 1492:63–71
Hooper JD, Nicol DL, Dickinson JL, Eyre HJ, Scarman AL, Normyle JF, Stuttgen MA, Douglas ML, Loveland KA, Sutherland GR, Antalis TM (1999) Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors. Cancer Res 59:3199–3205
Huyghe E, Matsuda T, Thonneau P (2003) Increasing incidence of testicular cancer worldwide: a review. J Urol 170:5–11
Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415–428
Karge WH III, Schaefer EJ, Ordovas JM (1998) Quantification of mRNA by polymerase chain reaction (PCR) using an internal standard and a nonradioactive detection method. Methods Mol Biol 110:43–61
Kawakami T, Okamoto K, Kataoka A, Koizumi S, Iwaki H, Sugihara H, Reeve AE, Ogawa O, Okada Y (2003) Multipoint methylation analysis indicates a distinctive epigenetic phenotype among testicular germ cell tumors and testicular malignant lymphomas. Genes Chromosomes Cancer 38:97–101
Koul S, Houldsworth J, Mansukhani MM, Donadio A, McKiernan JM, Reuter VE, Bosl GJ, Chaganti RS, Murty VV (2002) Characteristic promoter hypermethylation signatures in male germ cell tumors. Mol Cancer 1:8
Koul S, McKiernan JM, Narayan G, Houldsworth J, Bacik J, Dobrzynski DL, Assaad AM, Mansukhani M, Reuter VE, Bosl GJ, Chaganti RS, Murty VV (2004) Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors. Mol Cancer 3:16
Liu ZJ, Maekawa M (2003) Polymerase chain reaction-based methods of DNA methylation analysis. Anal Biochem 317:259–265
Lo YM, Wong IH, Zhang J, Tein MS, Ng MH, Hjelm NM (1999) Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction. Cancer Res 59:3899–3903
Looijenga LH, Oosterhuis JW (1999) Pathogenesis of testicular germ cell tumours. Rev Reprod 4:90–100
Lutzker SG, Barnard NJ (1998) Testicular germ cell tumors: molecular understanding and clinical implications. Mol Med Today 4:404–411
Manton KJ, Douglas ML, Netzel-Arnett S, Fitzpatrick DR, Nicol DL, Boyd AW, Clements JA, Antalis TM (2005) Hypermethylation of the 5′ CpG island of the gene encoding the serine protease Testisin promotes its loss in testicular tumorigenesis. Br J Cancer 92:760–769
Park JG, Chapman VM (1994) CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene. Mol Cell Biol 14:7975–7983
Smiraglia DJ, Szymanska J, Kraggerud SM, Lothe RA, Peltomaki P, Plass C (2002) Distinct epigenetic phenotypes in seminomatous and nonseminomatous testicular germ cell tumors. Oncogene 21:3909–3916
Smith-Sorensen B, Lind GE, Skotheim RI, Fossa SD, Fodstad O, Stenwig AE, Jakobsen KS, Lothe RA (2002) Frequent promoter hypermethylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene in testicular cancer. Oncogene 21:8878–8884
Summersgill B, Goker H, Weber-Hall S, Huddart R, Horwich A, Shipley J (1998) Molecular cytogenetic analysis of adult testicular germ cell tumours and identification of regions of consensus copy number change. Br J Cancer 77:305–313
Tang T, Kmet M, Corral L, Vartanian S, Tobler A, Papkoff J (2005) Testisin, a glycosyl-phosphatidylinositol-linked serine protease, promotes malignant transformation in vitro and in vivo. Cancer Res 65:868–878
von Eyben FE (2004) Chromosomes, genes, and development of testicular germ cell tumors. Cancer Genet Cytogenet 151:93–138
Acknowledgments
The authors wish to thank Ms. Petra von Kwiatkowski, Ms. Waltraud Jekabsons and Ms. Antonia Maass from the Department of Urology for their excellent support in assessing the samples.
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Kempkensteffen, C., Christoph, F., Weikert, S. et al. Epigenetic silencing of the putative tumor suppressor gene testisin in testicular germ cell tumors. J Cancer Res Clin Oncol 132, 765–770 (2006). https://doi.org/10.1007/s00432-006-0124-6
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DOI: https://doi.org/10.1007/s00432-006-0124-6