Vladusic T et al. / Tumor suppressor genes in testicular germ cell tumors 171 Figure 1. Loss of heterozygosity (LOH) of the CDKN2A gene at polymorphic microsatellite marker hMp16α-I1. Silver-stained 15% polyacrylamide gel. Lane 1: 50-bp DNA ladder (Fermentas, Vilnius, Lithuania); lane 2: heterozygous normal testis tissue; lane 3: LOH in the corresponding testicular germ cell tumor (non- seminoma, patient no. 31). Figure 2. Loss of heterozygosity (LOH) of the RB1 gene at intron 17 (XbaI restriction polymorphism). Silver-stained 15% poly- acrylamide gel. Lane 1: 50-bp DNA ladder (Fermentas, Vilnius, Lithuania); lane 2: heterozygous normal testis tissue; lane 3: LOH in the corresponding testicular germ cell tumor (nonseminoma, patient no. 25). 25 µl for 12 h. The restriction digestion resulted in fragments of 75 and 115 bp. DNA fragments were analyzed on silver-stained 15% polyacrylamide gels. LOH was recognized as a partial or complete loss of either the uncleaved (190 bp) or the cleaved (75 + 115 bp) allele. Results In this study 40 TGCTs, 18 seminomas and 22 non- seminomas, were analyzed. First, we searched for LOH of the intragenic polymorphic microsatellite marker hMp16α-I1 in the CDKN2A gene. From 40 TGCTs, 34 (85%) tumors were informative for this polymorphism, 16 (89%) seminomas and 18 (82%) nonseminomas. Our analysis revealed that two (6%) samples showed LOH of hMp16α-I1 marker. The observed changes were assigned to two non- seminomas (11%, patients no. 31 and 34, Table 2). In both tumor cases, one out of two allels of gene marker was missing in comparison to alleles from the adjacent normal tissue (Figure 1). In addition, both LOHs of the CDKN2A were found in nonsem- inomas with a yolk sac tumor component. LOH of the CDKN2A gene was not observed among semi- nomas. The analysis of intragenic polymorphic restric- tion marker of the RB1 gene showed that 34 (85%) of total TGCTs were heterozygous for this poly- morphism; 15 (83%) seminomas and 19 (86%) non- seminomas. LOH was observed in two (6%) sam- ples when looking at the total TGCTs analyzed. Once again the observed allelic losses were as- signed to nonseminomas: two samples (10.5%, pa- tients no. 20 and 25, Table 2) had one of the alleles missing in comparison to bands from the adjacent normal testis tissue. These nonseminoma samples showed loss of the cleaved allele (75- and 115-bp fragments), as the single uncleaved allele (190-bp fragment) appeared on the silver stained 15% poly- acrylamide gel (Figure 2). Furthermore, both LOHs of the RB1 were found in nonseminomas with an embryonal carcinoma component. None of the seminomas demonstrated LOH of the RB1 gene. No statistically relevant correlation between the occurrence of LOH, form of TGCT, histological type of contained components and tumor stage ac- cording to TNM classification could be determined by Fisher’s exact test. Discussion TGCT is associated with characteristic abnormali- ties in the RB pathway including upregulation of cyclin D2, and downregulation of pRB and the CDK inhibitors such as p16INK4a.7 The inactivation of the CDKN2A gene, which encodes an inhibitor of CDK4 and CDK6, is one of the most common molecular events in human neoplasms. The major mechanisms contributing to CDKN2A silencing are promoter methylation, gene mutations and hemizygous or homozygous deletions. When one CDKN2A allele is mutated or methylated, the second allele is often deleted.16 The analysis of the expression of INK4 family has pointed to a down-regulation of CDKN2A in tes- ticular neoplasms.7,12 Honorio et al.17 demonstrated that promoter hypermethylation of that gene is not involved in the decrease of p16INK4a protein ex- pression. In contrast, some studies have found pro- moter mutation, a half of analyzed TGCTs had de novo promoter methylation and approximately half of TGCTs showed hypermethylation of CDKN2A exon 1α. All that correlated with a decreased lev- el of CDKN2A mRNA expression.1,18 However, Chaubert et al.18 have not detected any CDKN2A mutations and observed LOH of the CDKN2A Radiol Oncol 2010; 44(3): 168-173.