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Up-Regulation of Cyclin-Dependent Kinase 4/Cyclin D2 Expression but Down- Regulation of Cyclin-Dependent Kinase 2/Cyclin E in Testicular Germ Cell Tumors1

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Up-Regulation of Cyclin-Dependent Kinase 4/Cyclin D2 Expression but Down- Regulation of Cyclin-Dependent Kinase 2/Cyclin E in Testicular Germ Cell Tumors1 [CANCER RESEARCH 61, 4214–4221, May 15, 2001] Up-Regulation of Cyclin-dependent Kinase 4/Cyclin D2 Expression but Down- Regulation of Cyclin-dependent Kinase 2/Cyclin E in Testicular Germ Cell Tumors1 Bettina A. Schmidt,2 Achim Rose, Christine Steinhoff, T. Strohmeyer, Michael Hartmann, and Rolf Ackermann Department of Urology, Heinrich-Heine-University of Duesseldorf, 40225 Duesseldorf, Germany [B. A. S., A. R., C. S., R. A.], and Department of Urology, Hospital of the Armed Forces, 22099 Hamburg, Germany [M. H.] ABSTRACT causes death in about 20–30% of patients with highly advanced stages. The present state of research provides only marginal insights Testicular germ cell tumors (GCT) characteristically display two chro- into the molecular pathogenesis of these tumors and the mechanisms mosome 12 abnormalities: the isochromosome i(12p) and concomitant underlying the effectiveness of chemotherapy. deletions of the long arm. Some genes important in the control of the G -S 1 The most frequent cytogenetic observation indicative of testicular cell cycle checkpoint G1-S, i.e., cyclin-dependent kinases 2 and 4, cyclin D2 are located on this chromosomal region. Therefore, testicular GCTs were GCT is the presence of an isochromosome of the short arm of analyzed as to the expression of CDK2, CDK4, CDK6, and the expression chromosome 12, i12(p), in up to 80% of all tumors. Although this of their catalytic partners cyclins D1, D2 and E by semiquantitative finding has been confirmed by various investigators (6–8), its diag- reverse transcription-PCR. Cyclin D2, located on 12p, was overexpressed nostic and prognostic relevance is still under discussion (9, 10). in 69% (31 of 45) of the tumors by a mean factor of 8, including all Additional abnormalities involve deletions of the long arm of chro- histological subtypes. In addition, the cyclin D2 partner CDK4 was in- mosome 12, particularly the 12q13 and 12q22 regions, in 40% of all creased in 41% (21 of 51) of all tumors by a factor of 6, most strongly in histological subtypes (11). These findings suggest the localization of embryonal carcinomas. Sixty-four percent of the seminomas and 23% of the non-seminomas had decreased expression of CDK6 by a mean factor one or more tumor suppressor genes on 12q and the localization of Statistical analysis using configural frequency analysis relevant proto-oncogenes on 12p (12). The acquired data supports the .(0.009 ؍ of5(P and regression analysis revealed that cyclin D2 and CDK4 expression hypothesis of a possible role for chromosome 12 in the development .whereas expression and progression of testicular GCTs ,(0.000052 ؍ P ;0.682 ؍ were strongly correlated (r2 An important molecular change in testicular GCTs involves the loss .(0.00085 ؍ P ;0.382 ؍ of CDK6 did not correlate with either of them (r2 CDK2 and its catalytic partner cyclin E were down-regulated in 40% (19 of mRNA and protein expression of the RB tumor-suppressor gene. of 47) and 42% (19 of 45) of the tumors, respectively, by a factor of 7 each. We found a highly significant suppression or lack of expression of the Western blots and immunohistochemical experiments confirmed cyclin RB gene at both mRNA and protein levels in 95% and 71%, respec- D2 and CDK4 overrepresentation and reduced expression of cyclin E and CDK2 tumors in the few tumors under protein study. Despite its local- tively, of seminomas and non-seminomas (13). The RB protein was ization on 12q13, a hot spot for loss of heterozygosity in testicular GCTs not detected by immunohistochemistry in undifferentiated tumors (>40%), Southern blotting revealed no gross DNA alteration of the CDK2 (seminomas, embryonal carcinoma and chorion carcinomas). Only gene. Because up-regulation of the cyclin D2/CDK4 complex and down- differentiated cells of teratocarcinomas and mixed tumors showed regulation of cyclin E/CDK2 complex were found in seminomas as well as some protein expression. However, using Southern blotting, no alter- in non-seminomas and in all tumor stages, these findings seem to be early ations were found at the DNA level (13). events during tumorigenesis of testicular GCTs. Together with previous The nuclear protein encoded by the RB gene is consistently ex- findings that retinoblastoma mRNA and protein expression is strongly pressed by all normal mammalian cells and tissues. However, a decreased in these tumors, these data suggest an unusual deregulated G -S 1 number of different tumor types (RB, bronchial carcinoma, osteosar- checkpoint as a decisive event for germ cell tumors. coma, glioma, and carcinoma of the prostate and bladder) are asso- ciated with inactivation of the RB gene by deletion, mutation and/or INTRODUCTION lack of down-regulation (14–16). The RB protein is an essential protein in cell cycle regulation, and its function is regulated by With an incidence of 6–7 per 100,000 men, GCTs3 are the most phosphorylation. In G0 and the early G1 phase, hypophosphorylated frequent solid malignant tumors in men 20–40 years of age and RB is complexed with the cellular transcription factor E2F (17, 18). In represent the most frequent cause of death from solid tumors in this late G1, a significant hyperphosphorylation of the RB protein by age group (1, 2). The rapid progress of GCTs causes early lymph node cyclin-dependent kinases (CDK2, CDK4, and CDK6) in complex metastases and/or distant metastases. At the time of diagnosis, up to with their catalytic partners (cyclins D1, D2, D3, and E) occurs 50% of the patients suffer from metastatic disease (3). Because of (19–22). As a consequence, E2F is set free and binds to promoters of highly effective combinations of cytostatic drugs, cure rates of 85– genes that are involved in the early steps of the subsequent S phase, 90% can be reached (4, 5). Despite this success, the disease still i.e., DNA polymerase ␣ and ␦, dihydrofolate reductase, thymidine kinase, cyclin E, CDK 2, c-myc, and E2F-1 itself. During the subse- Received 3/6/00; accepted 2/28/01. quent S, G , and mitosis phases, the RB protein remains hyperphos- The costs of publication of this article were defrayed in part by the payment of page 2 charges. This article must therefore be hereby marked advertisement in accordance with phorylated. 18 U.S.C. Section 1734 solely to indicate this fact. Several regulators of RB are located on the regions of chromosome 1 The work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ac 29/1-2). 12 frequently affected by aberrations in GCTs (23–25). While human 2 To whom requests for reprints should be addressed Bettina Schmidt, Heinrich-Heine- cyclin D2 gene (CCND2) is located on 12p13, the human CDK2 gene University of Duesseldorf, Department of Urology; Moorenstr. 5, 40225 Duesseldorf, (CDK2) and CDK4 gene (CDK4) are localized on 12q13. These Germany Phone: ϩ49–211-8118110; Fax: ϩ 49–211-8117804 e-mail: bschmidt@uni- duesseldorf.de. findings, combined with the above-mentioned observations on RB 3 The abbreviations used are: GCT, germ cell tumor; RB, retinoblastoma; RT-PCR, expression in GCTs, give rise to the hypothesis that the cell cycle reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CFA, configural frequency analysis; ISA, interacting structure analysis; FISH, fluorescence in restriction point G1-S may be entirely disrupted in testicular GCTs. situ hybridization. The aim of this study was to test this hypothesis in primary testicular 4214 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2001 American Association for Cancer Research. CYCLINS AND CDKS IN TESTICULAR TUMORS GCTs by measuring the mRNA expression of those proteins which facturer’s protocol. Chemiluminescence was detected on enhanced chemilu- directly regulate pRB function: the cyclin-dependent kinases CDK2, minescence-Hyperfilm (Amersham). Signals were quantified by video scan- CDK4, and CDK6 and their catalytic partners (cyclin D1, cyclin D2, ning densitometry (One D-SCAN; Scanalytics, Billerica, Madison, WI) and and cyclin E). standardized for ␤-actin expression in each matched normal testicular tissue. One set of experiments was performed with GAPDH as the internal control. However, because of the localization of the GAPDH gene on chromosome 12p, MATERIALS AND METHODS GAPDH expression was additionally standardized to ␤-actin expression in each tumor/normal pair. Overexpression in tumor was defined as expression Tumor and adjacent normal testicular tissue was obtained by surgery, shock Ͼ2-fold of the corresponding normal tissue; normal as between 0.5-fold and frozen in liquid nitrogen, and stored at Ϫ70°C. The tissue cohort included 58 2.0- fold; and decreased expression as Ͻ0.5-fold, after standardization to the GCTs and the adjacent nonmalignant testicular tissues. Histological tumor ␤-actin expression ratios of tumor and normal tissue. subtyping and staging was performed according to the WHO International Southern Blot Analysis. Genomic DNA (10 ␮g) was digested with 8 units Histological Classification of Tumors (26) and the American Joint Committee of TaqI restriction enzyme (Life Technologies, Inc., Karlsruhe, Germany), on Cancer (27). resolved on an 0.8% agarose gel, and transferred to a nylon membrane Isolation of Total RNA. Total cellular RNA was extracted from frozen (Hybond; Amersham, Buckinghamshire, UK) according to standard protocols tissues using guanidinium thiocyanate, phenol-chloroform, and CsCl density (28). Prehybridization and hybridization were performed in Dig-Hybridization gradient centrifugation as described (28). Purified RNA was quantified by UV buffer (Roche Diagnostics, Mannheim, Germany) at 42°C. Fifty ng of the spectroscopy scans from 340 nm to 210 nm (UviKon 900; Kontron Instru- 778-bp CDK2 probe (pCDK2), representing nucleotide numbers 166–943 ments, Neufarn, Germany). Integrity of RNA was confirmed by denaturing PAGE. (kindly provided by Guido Reifenberger, Department of Neuropathology, Primers. Primers specific for cyclin E, cyclin D1, cyclin D2, cyclin D2 University of Duesseldorf, Duesseldorf, Germany) was digoxigenine-labeled pseudogene, CDK2, CDK4, and CDK6 cDNA were designed using sequences by oligonucleotide random priming (38) and used for hybridization.
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