Molecular analysis of P16(Ink4)/CDKN2 and P15(INK4B)/MTS2 genes in primary human testicular germ cell tumors.

PURPOSE

p16 (MTS 1) and p15 (MTS 2) are negative regulators of cell cycle progression at the G1 cell cycle checkpoint and function as tumor suppressor genes (TSG). Both p15 and p16 are located on chromosome 9p21 and alterations have been demonstrated in a variety of human malignancies and human cancer cell lines. In testicular germ cell tumors (TGCT) loss of heterozygosity of 9p21 to 23 has been observed in 41% to 72% of informative cases. The aim of our study was to examine TGCT and testicular cancer cell lines for deletions and mutations of the p15 and p16 genes.

MATERIALS AND METHODS

24 testicular germ cell tumors, 4 testicular cancer cell lines (TERA I, TERA II, HTE, HTH), 8 benign testicular tumors and 9 samples of normal testicular parenchyma were examined. Examinations for loss of heterozygosity (LOH) on 9p21 were performed for IFNA, D9S171, D9S126, D9S161, D9S1748 and PKY9 locus using Southern Blot analysis. Southern Blot analysis of the p16 gene to examine gross alterations was done using random primer labeled p16 cDNA. In addition all samples were examined for mutations of exons 1 to 3 of p16 and exons 1 to 2 of p15 by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis on MDE-gels. All observed band shifts on SSCP were further evaluated by direct DNA sequencing techniques.

RESULTS

No major deletions were detected for p16 and p15. 42% of all TGCT demonstrated LOH of the IFNA locus, 16% of the D9S161 locus and 5.5% of the D9S171 locus. No LOH was seen for the D9S126 locus. 5/24 TGCT (21%) and 1 testicular cancer cell line (TERA II) demonstrated band shifts for exon 2, 8/24 TGCT (33%) and 1 testicular cancer cell line (TERA I) showed band shifts for exon 3; 4 TGCT had an allelic loss for exon 3 on SSCP. DNA sequencing analysis for exon 2 demonstrated a single base substitution (G-->A) in codon 140 resulting in an amino acid exchange (ala-->thre) in 4 cases and a single base deletion with frame shift mutation in codon 142 in 2 cases. DNA sequencing analysis for exon 3 demonstrated a polymorphism in intron 2 in all cases. For p15 no band shifts were observed for exons 1 to 2 in TGCT or testicular cancer cell lines; none of benign testicular tumors or normal control tissues demonstrated any band shifts for p15 or p16.

DISCUSSION

We demonstrated a high frequency of mutations for exon 2 in specimens of primary testicular germ cell tumors, but not in benign tumors or normal controls. We therefore believe that p16 might be involved in the pathogenesis of human TGCT. The polymorphisms described for exon 3 might be utilized for further LOH studies. The absence of mutations in p15 gene in TGCT specimens suggests that p15 might not play an important role in the pathogenesis of testicular germ cell tumors.