Frequent epigenetic inactivation of p53 target genes in seminomatous and nonseminomatous germ cell tumors
Introduction
Testicular germ cell tumors (TGCT) represent the most common male malignancy in the third to fourth decade of life [1]. TGCTs are divided into two major histological groups and it is suggested that all tumors originate from carcinoma in situ and progress, after various cytogenetic and molecular alterations, first into seminoma and subsequently into nonseminoma. Fortunately, testicular cancer is a highly curable disease, even in advanced stages [2].
Several studies of TCGTs have demonstrated the presence of chromosomal alterations including allelic loss or the amplification of chromosome 12p [3], [4]. However, few genes have been identified to contribute to the tumorigenesis of testicular cancer. Recently, epigenetic silencing has emerged as one mechanism thought to impair the activity of specific genes in testicular cancer [5], [6]. Multipoint analysis of testicular neoplasms demonstrates that a minority of genes, typically those affected by promoter methylation such as RASSF1A, MGMT and PRSS21, is also altered in testicular cancer [7], [8], [9], [10]. To date, the findings of all previous studies are limited either by small sample size or the use of a nonquantitative method of methylation-specific PCR (MSP).
Since dysfunctional mutated p53 has been shown to play a role in the resistance of TGCT to chemotherapy, we focused on several downstream target genes of the p53 tumor suppressor gene to assess epigenetic silencing [11], [12]. Although, all the genes selected for investigation in this study are thought to be closely involved in the apoptotic cycle and harbor CpG islands in their promoter regions, and thus serving as potential targets for transcriptional silencing, none has ever been investigated for promoter methylation in testicular cancer.
One of the first p53 target genes found to be methylated and involved in the apoptotic pathway was the death-associated protein kinase (DAPK-1), identified in tumors of epithelial and nonepithelial origin [13], [14]. Another p53 target gene, shown to be methylated in both human leukemic cell lines and metastatic malignant melanoma, the apoptotic protein-activating factor (APAF-1) is thought to play an important role in the induction of apoptosis [15], [16]. One of the executioner caspases, Caspase 8 (CASP-8), plays an antagonistic role with anti-apoptotic survivin, although little is known about whether it is silenced via promoter hypermethylation [17]. Insulin-like growth-factor-binding protein-3 (IGFBP-3) was shown to have reduced mRNA expression in several tumors and also harbors CpG islands. However, no investigation of the IGFBP-3 promoter region has yet been reported [18].
In the current study, we investigated the frequency and percentage of promoter methylation for APAF-1, CASP-8, DAPK-1 and IGFBP-3 in 46 primary TGCTs (26 seminomas and 20 nonseminomas) and 15 normal testicular tissue samples using highly sensitive, quantitative real-time methylation specific PCR (Q-MSP).
Section snippets
Material and methods
Tissue specimens were collected at the Department of Urology, University Charité CBF, Berlin between 2001 and 2005. All patients signed a consent form approved by the Committee on Human Rights in Research of our institution. Normal testicular tissue biopsies (n=15) were obtained from seven patients undergoing subtotal orchiectomy for antihormonal therapy in prostate cancer and from eight patients who underwent a vasovasostomy and requested cryopreservation of a biopsy in case the intervention
Results
TCGT specimens included 26 seminomatous and 20 nonseminomatous testicular tumors. Testicular tissue obtained from 15 patients following testicular biopsy for nonmalignant disease were used as controls. Fifteen (57%) and Eleven (43%) of the seminoma patients were classified as clinical stage (CS) I and ≥II, respectively. In the nonseminomatous cohort, 9 (45%) and 11 (55%) had clinical stage I and ≥II disease, respectively. The median age in the seminoma cohort was 37.5 (±7.4) years as compared
Discussion
In this study, we report the first quantitative analysis of promoter hypermethylation in a subset of p53 target genes in testicular cancer. Promoter hypermethylation of APAF-1, DAPK-1 and IGFBP-3 occurs to varying degrees in testis cancer. For APAF-1, methylation was observed in all neoplastic testicular specimens and in almost two-thirds of the normal testicular tissue. Interestingly, the quantitative MSP analysis revealed that the degree of methylation within the normal tissue samples was
Acknowledgements
We thank Ms Petra von Kwiatkowski for her expert technical assistance and Dr Joanne Weirowski for her linguistic advice.
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