cyclin-d1 and Germinoma

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.

Topics: Adolescent; Adult; beta Catenin; Cell Cycle Proteins; Cell Transformation, Neoplastic; Child; Child, Preschool; CpG Islands; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cytoskeletal Proteins; DNA, Neoplasm; Female; Genes, p16; Germinoma; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Methylation; Mutation; Neoplasm Proteins; Ovarian Neoplasms; Polymerase Chain Reaction; Promoter Regions, Genetic; Trans-Activators

Testicular germ cell tumors (GCT) characteristically display two chromosome 12 abnormalities: the isochromosome i(12p) and concomitant deletions of the long arm. Some genes important in the control of the G(1)-S cell cycle checkpoint G(1)-S, i.e., cyclin-dependent kinases 2 and 4, cyclin D2 are located on this chromosomal region. Therefore, testicular GCTs were analyzed as to the expression of CDK2, CDK4, CDK6, and the expression of their catalytic partners cyclins D1, D2 and E by semiquantitative reverse transcription-PCR. Cyclin D2, located on 12p, was overexpressed in 69% (31 of 45) of the tumors by a mean factor of 8, including all histological subtypes. In addition, the cyclin D2 partner CDK4 was increased in 41% (21 of 51) of all tumors by a factor of 6, most strongly in embryonal carcinomas. Sixty-four percent of the seminomas and 23% of the non-seminomas had decreased expression of CDK6 by a mean factor of 5 (P = 0.009). Statistical analysis using configural frequency analysis and regression analysis revealed that cyclin D2 and CDK4 expression were strongly correlated (r(2) = 0.682; P = 0.000052), whereas expression of CDK6 did not correlate with either of them (r(2) = 0.382; P = 0.00085). CDK2 and its catalytic partner cyclin E were down-regulated in 40% (19 of 47) and 42% (19 of 45) of the tumors, respectively, by a factor of 7 each. Western blots and immunohistochemical experiments confirmed cyclin D2 and CDK4 overrepresentation and reduced expression of cyclin E and CDK2 tumors in the few tumors under protein study. Despite its localization on 12q13, a hot spot for loss of heterozygosity in testicular GCTs (>40%), Southern blotting revealed no gross DNA alteration of the CDK2 gene. Because up-regulation of the cyclin D2/CDK4 complex and down-regulation of cyclin E/CDK2 complex were found in seminomas as well as in non-seminomas and in all tumor stages, these findings seem to be early events during tumorigenesis of testicular GCTS: Together with previous findings that retinoblastoma mRNA and protein expression is strongly decreased in these tumors, these data suggest an unusual deregulated G(1)-S checkpoint as a decisive event for germ cell tumors.

Topics: CDC2-CDC28 Kinases; Cyclin D1; Cyclin D2; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; G1 Phase; Gene Expression Regulation, Neoplastic; Germinoma; Humans; Immunohistochemistry; Male; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S Phase; Testicular Neoplasms; Up-Regulation