Vladusic T et al. / Tumor suppressor genes in testicular germ cell tumors 169 nism, which may be connected with gene activity patterns in different target cells.6 The mechanism involves pRB and its immediate upstream regu- lators, the cyclin dependent kinases (CDK4 and CDK6), their catalytic partners (cyclin D1, cyclin D2 and cyclin D3), and the members of the INK4 family of CDK inhibitors (p16INK4a, p15INK4b, p18INK4c and p19INK4d). This mechanism seems to be a common point for various signaling path- ways, serving as a growth factor dependent cell cy- cle switch. Deregulation of the RB pathway may be an obligatory step in oncogenesis, making tumor cells less dependent on growth stimuli.6,7 The pRB is essential in cell cycle regulation and its function is regulated by phosphorylation. In G0 and the early G1 phase, hypophosphorylated pRB is complexed with the transcription factor E2F.8 In late G1, a significant hyperphosphorylation of the pRB by CDK4 and CDK6 in complex with D cyclins (D1, D2, D3) occurs.9 The CDKN2 locus at chromosomal region 9p21 encodes p16INK4a tumor suppressor protein in- volved in the RB cell cycle control pathway.10 p16INK4a functions as a regulator of G1/S phase transition by inhibiting the activity of CDK4 and CDK6. Thus, by inhibiting pRB phosphorylation, p16INK4a can promote the formation of a pRB-E2F repressive transcriptional complex, which blocks cell cycle progression past G1/S restriction point.11 In diverse types of cancer the RB pathway be- comes deregulated through alterations in one or more of its components. The most common defects of the RB pathway are mutations or deletions of RB1 and inactivating mutations or promoter meth- ylation of the CDKN2A (p16INK4a) tumor suppres- sor gene, as well as the overexpression of the cyclin D2/CDK4 complex.6,12,13 The objective of this study was to evaluate the occurrence of the loss of heterozygosity (LOH) of the CDKN2A and RB1 tumor suppressor genes in TGCTs. Materials and methods Patients and tumor material Fourty TGCT samples (18 seminomas and 22 non- seminomas) were collected from Sisters of Mercy University Hospital and University Hospital Center, Zagreb, Croatia. The samples were for- malin-fixed and paraffin-embedded. Clinical and pathological data for 40 TGCTs according to the WHO 2004 classification are shown in Table 1. DNA extraction For each specimen, 20 µm paraffin-embedded section was prepared for DNA extraction. In ad- dition, 4 µm section was stained with hematoxy- lin-eosin to identify the tumor and normal tissue areas which were removed separately from the microscopic slide, transferred to microtubes and extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). LOH analysis of CDKN2A gene A previously described polymorphic microsatellite marker hMp16α-I1 consisting of a mononucleotide tract of (A)23 located close to intron 1 of the CDKN2A gene was analyzed in this study.14 Primers used for polymerase chain reaction (PCR) amplifications were 5’-CAATTACCACATTCTGCGCTT-3’ and 5’-CAGGCAGAGAGCACTGTGAG-3’, which pro- duced 190-210 bp fragments. PCR amplifications were performed in 25 µl reaction volume with a final concentration 0.2 mM of each dNTP, 3 mM MgCl2, 0.2 µM of each primer (Sigma-Aldrich, St. Louis, MI, USA), 1x Flexi buffer (Promega, Madison, WI, USA) and 0.5 U of GoTaq® Hot Start Polymerase (Promega, Madison, WI, USA). One hundred nanograms of DNA were used in each PCR reaction. PCR amplifications were carried out in a Eppendorf Mastercycler Personal (Hamburg, Germany), with cycling times of 96ºC for 5 min (one cycle), then 45 cycles of 96ºC for 30 s, 57ºC for 45 s, and 72ºC for 30 + 1 s. The final step was incu- bation at 72º C for 10 min. Amplified DNA frag- ments were analyzed on silver-stained 15% poly- acrylamide gels. LOH of CDKN2A was considered to had occured if one out of two alleles (hetero- zygous samples) of a gene marker was missing or significantly reduced in comparison to alleles from adjacent normal tissue. LOH analysis of RB1 gene LOH of RB1 was detected using polymerase chain reaction-restriction fragment length polymor- phism method (PCR-RFLP). Amplification with RB1 primers 5’- TCCCACCTCAGCCTCCTTAG-3’ and 5’-GTAGGCCAAGAGTGGCAGCT-3’ used in our study produced a 190 bp segment of intron 17.15 PCR amplifications were performed under conditions mentioned above. To generate the RFLP pattern for LOH analysis, 10 µl of PCR product were digested with 5 U of XbaI restriction enzyme (Fermentas, Vilnius, Lithuania) in a total volume of Radiol Oncol 2010; 44(3): 168-173.