Synthesis of a novel platinum(II) complex with 6,7-dichloro-5,8-quinolinedione and the study of its antitumor mechanism in testicular seminoma.

A new platinum(II) complex, [Pt(ClClQ)(DMSO)Cl] (1), utilizing 6,7-dichloro-5,8-quinolinedione (ClClQ) as a ligand, has been synthesized and fully characterized. Single-crystal X-ray diffraction and other spectroscopic and analytical methods revealed that the coordination geometry of Pt(II) in complex 1 can also be described as a four-coordinated square planar geometry. The aim of the study was to explore the in vitro anticancer properties of complex 1. Our studies showed that complex 1 can regulate the viability of testicular seminoma cells in vitro, including cell proliferation and apoptosis. We further observed negative regulation by complex 1 of the expression levels of the key elements in the phosphoinositide-3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK3ß) pathway, including phosphorylated phosphoinositide-3 kinase (p-PI3K), phosphorylated protein kinase B(p-Akt) and phosphorylated glycogen synthase kinase-3ß (p-GSK3ß). Moreover, the negative effect of complex 1 was reversed by LiCl, a GSK3ß-specific inhibitor of the PI3K signaling pathway. Meanwhile, the levels of Bcl2 associated death promoter (Bad), cytochrome c, active-caspase-3 and active-caspase-9 increased significantly. In conclusion, we observed that complex 1 can regulate the viability of testicular seminoma cells through the PI3K/Akt/GSK3ß signaling pathway and the mitochondria-mediated apoptotic pathway in vitro, and thus, complex 1 may have potential for use as a drug in the treatment of testicular germ cell tumors.

Expression patterns of HENMT1 and PIWIL1 in human testis: implications for transposon expression.

This study aimed to define the expression patterns of HENMT1 and PIWI proteins in human testis and investigate their association with transposon expression, infertility sub-type or development of testicular germ cell tumours (TGCTs). Testis biopsies showing normal spermatogenesis were used to identify normal localisation patterns of HENMT1 and PIWIL1 by immunolocalisation and RT-PCR after laser microdissection. 222 testis biopsies representing normal spermatogenesis, hypospermatogenesis, spermatogenic arrests, Sertoli cell-only (SCO) tumours and TGCTs were analysed by RT-qPCR for expression of HENMT1/PIWIL1/PIWIL2/PIWIL3/PIWIL4 and LINE-1 Additionally, HENMT1-overexpressing TCam2 seminoma cell lines were analysed for the same parameters by RT-qPCR. We found that HENMT1 and PIWIL1 are coexpressed in pachytene spermatocytes and spermatids. Expression of HENMT1, PIWIL1 and PIWIL2 was mainly dependent on germ cell content but low levels of expression were also detected in some SCO samples. Levels of HENMT1, PIWIL1 and PIWIL2 expression were low in TGCT. Samples with HENMT1, PIWIL2 and PIWIL4 expression showed significantly (P < 0.05) lower transposon expression compared to samples without expression in the same histological group. HENMT1-overexpressing TCam2 cells showed lower LINE-1 expression than empty vector-transfected control lines. Our findings support that the transposon-regulating function of the piRNA pathway found in the mouse is conserved in adult human testis. HENMT1 and PIWI proteins are expressed in a germ-cell-specific manner and required for transposon control.

Prognostic impact of LDH levels in patients with relapsed/refractory seminoma.

PURPOSE: To evaluate the impact of age and LDH levels in patients with relapsed seminoma. METHODS: Data on the 204 seminoma from the International Prognostic Factor Study Group (IPFSG) were analyzed. All patients experienced unequivocal relapse/progression after at least three cisplatin-based chemotherapy cycles. Age and LDH at relapse were assessed in addition to previously identified prognostic factors for all germ cell tumor patients from the database (J Clin Oncol 28:4906, 2010). RESULTS: The impact of the IPFSG score remained highly significant in multivariate analysis. In addition, LDH ≥1.5 times the upper limit of normal (ULN) was significant in univariate (HR 1.96; CI 1.06-3.61) and multivariate analysis (HR 1.90; CI 1.00-3.62). Age, however, was not significant. Therefore, LDH was incorporated into a modified new IPFSG seminoma score by moving patients to the next unfavorable group for patients with LDH values ≥1.5 × ULN. Three prognostic groups were thus generated, which better subdivided seminoma patients than the original IPFSG score. Progression-free survival at 2 years: "very low risk" (n = 23) 85.7% (95% CI 62-95), "low risk" (n = 44) 62.7 % (95% CI 46-75) and "intermediate risk" (n = 36) 35.1% (95% CI 20-51). Overall survival at 3 years: "very low risk" 88.8% (95% CI 62-97), "low risk" 71.3% (95% CI 55-83) and "intermediate risk" 51.3% (95% CI 33-67). CONCLUSION: The addition of LDH, but not age, improves the impact of the IPFSG prognostic score in seminoma patients relapsing or progressing after cisplatin-based chemotherapy.

PARP expression in germ cell tumours.

BACKGROUND: Poly(ADP-ribose)polymerase (PARP) inhibitors represent a new class of promising drugs in anticancer therapy. AIMS: To evaluate PARP expression in testicular germ cell tumours (GCTs) and to correlate expression patterns with clinicopathological variables. METHODS: In this translational study, tumour specimens from 124 patients with GCTs (114 patients with testicular primary tumours and 10 with extragonadal GCTs) were identified. PARP expression was detected by immunohistochemistry using monoclonal antibodies, scored by the multiplicative quickscore (QS) method and compared to PARP expression in normal testicular tissue. RESULTS: We observed higher expression of PARP in testicular tumours compared to normal testicular tissue (mean QS=10.04 vs 3.31, p<0.0000001). Mean QS±SD for each histological subtype was as follows: intratubular germ cell neoplasia unclassified (IGCNU)=18.00±0.00, embryonal carcinoma=9.62±5.64, seminoma=9.74±6.51, yolk sac tumour=7.8±7.20, teratoma=5.87±5.34, and choriocarcinoma=4.50±8.33. The PARP overexpression (QS>9) was most often detected in IGCNU (100% of specimen with PARP overexpression), seminona (52.6%), embryonal carcinoma (47.0%), yolk sac tumour (33.3%), teratoma (26.7%) and choriocarcinoma (25.0%), compared to 1.9% of normal testicular tissue specimens. There was no association between PARP expression and clinical variables. CONCLUSIONS: In this pilot study, we showed for the first time, that PARP is overexpressed in testicular germ cell tumours compared to normal testis.

Core 2 N-acetylglucosaminyltransferase-1 expression induces aggressive potential of testicular germ cell tumor.

We studied orchiectomy specimens from 130 patients immuhistochemically with testicular germ cell tumor (TGCT) using anti-core 2 N-acetylglucosaminyltransferase-1 (C2GnT-1) antibody. The incidence of C2GnT-1 positivity in stage I disease (29.5%, 21/71) was significantly lower than that in higher stages (84.7%, 50/59) (P < 0.001, chi(2) test). This significant difference was also found when the cases were divided into seminoma and NSGCT according to histopathological classification. Kaplan-Meier plots and the log rank test showed that in the patients with stage I seminoma, C2GnT-1-positive cases had a higher risk for recurrence (P < 0.001). This was also the case with the patients with stage I NSGCT (P < 0.001). To determine whether C2GnT-1 promotes aggressive behavior of cancer cells, a C2GnT-1-negative human TGCT cell line, JKT-1, was stably transfected with a mammalian expression vector containing C2GnT-1 cDNA. In vitro assays revealed that JKT-1-C2 cells are more invasive than mock transfectants, although there are no differences in proliferation activity. When orthotopically inoculated into athymic nude mice, JKT-1-C2 cells produced larger testicular tumors extending to the retroperitoneum with mesenteric metastasis, while mock transfectants produced small tumors without metastasis (P < 0.01, Mann-Whitney's U-test). When injected via the tail vein, JKT-1-C2 cells produced a number of metastatic lung foci. In contrast, mock transfectants produced a small number of nodules (p < 0.01, Mann-Whitney's U-test). These results strongly suggest that C2GnT-1 enhances the metastatic potential of TGCT and may be a reliable biomarker for aggressive potential of TGCT.

Modified expression of cytoplasmic isocitrate dehydrogenase electrophoretic isoforms in seminal plasma of men with sertoli-cell-only syndrome and seminoma.

Two isoforms of human cytoplasmic isocitrate dehydrogenase (IDPc) of close molecular weights and different isoelectric points were identified in human seminal plasma (SP) by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). These two isoforms were detected in the normospermic men SP and their expressions were markedly altered in patients with testicular seminoma, the most frequent testicular germ cell cancer (TGCC): increase of the more acidic spot and decrease of the more basic one. Since oligospermia has been considered as a high risk pathological condition for developing a testicular cancer, the two IDPc isoforms were analyzed in SP of a group of secretory azoospermic patients. In this group the two spots displayed similar variations of expression to those observed in testicular seminoma. These results propose IDPc as a promising SP biomarker of testicular seminoma. Whether IDPc alteration in secretory azoospermia is predictive of testicular seminoma remains to be elucidated.

Apoptosis in spermatocytic and usual seminomas: a light microscopic and immunohistochemical study.

Despite its alarming appearance, spermatocytic seminoma virtually never metastasizes. We hypothesized that this paradox may at least be partially related to increased apoptosis compared to metastasizing germ cell tumors since high expression of proapoptotic factors correlates with indolent behavior in other tumor systems, notably CD30-positive cutaneous lymphoma, another neoplasm where phenotype and behavior do not match. We therefore compared apoptosis and apoptotic regulators in 17 spermatocytic seminomas (2 with sarcoma) and 18 usual seminomas by light microscopy and using immunostains for caspase-3, p53, bcl-2, bcl-xL, FADD, FAS and survivin. We found significantly greater numbers of apoptotic cells and activated caspase-3-positive cells in spermatocytic seminoma compared to usual seminoma (P<0.01). There was over a 10-fold range in apoptotic cells in usual seminoma but only a 4-fold variation in spermatocytic seminoma. Spermatocytic seminoma had decreased p53 expression compared to usual seminoma, with marked variation in bcl-2 expression and increased FADD. The two sarcomas in spermatocytic seminoma, however, showed decreased apoptosis and caspase-3 reactivity, with upregulation of p53 and bcl-2 and decreased FADD expression. We conclude that apoptosis, caspase-3 and FADD expression are increased in spermatocytic seminoma compared to usual seminoma. Apoptotic parameters are decreased in sarcomatous transformation of spermatocytic seminoma. The increased apoptosis of spermatocytic seminoma, possibly mediated by FAS independent activation of the death receptor pathway, may provide some insight into its excellent prognosis. The variation in apoptosis of usual seminomas merits investigation as a prognostic parameter.

Topoisomerase II alpha expression in testicular germ cell tumors.

OBJECTIVES: Inhibitors of topoisomerase II alpha (TopoIIalpha), an enzyme with a crucial role in DNA maintenance, are included in the chemotherapy protocols for testicular germ cell tumors (GCTs). Despite the success of current chemotherapy regimens, a significant number of patients experience relapse. We analyzed TopoIIalpha expression in primary and metastatic testicular GCTs because this enzyme is a target for some antineoplastic agents. METHODS: Primary GCT specimens from 109 patients, including 57 seminomas and 52 mixed GCTs (41 embryonal carcinomas, 23 yolk sac tumors, 19 seminomas, 8 choriocarcinomas, 17 teratomas with immature elements, and 16 teratomas with mature elements), were obtained from our archives. The metastatic lesions from 11 of the patients with mixed GCTs included seven teratomas with mature components, five embryonal carcinomas, one yolk sac tumor, one choriocarcinoma, and one teratoma with immature components. Representative sections were subjected to immunohistochemistry with monoclonal antibody against TopoIIalpha, and the nuclear staining findings were evaluated. RESULTS: Most embryonal carcinoma (100%), yolk sac tumor (95%), seminoma (88%), and choriocarcinoma (62%) components of the GCTs were TopoIIalpha immunoreactive. None of the teratoma specimens with mature elements expressed TopoIIalpha. CONCLUSIONS: The results of our study have shown that TopoIIalpha is expressed in most seminomas, embryonal carcinomas, yolk sac tumors, and choriocarcinomas, suggesting a possible mechanism of sensitivity of these components to TopoIIalpha inhibitors. Teratomas with mature and immature elements expressed low levels of TopoIIalpha, which might contribute to their chemoresistance. These findings imply that the variable chemoresponsiveness of testicular GCTs could have an underlying molecular basis.

Activation-induced cytidine deaminase (AID) is expressed in normal spermatogenesis but only infrequently in testicular germ cell tumours.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes in antigen-dependent B-cell maturation. SHM is not restricted to immunoglobulin gene loci, raising the possibility of a function for AID in other cell types. In this study, it is shown that AID is expressed in spermatocytes in the human testis. AID was mostly cytoplasmic but nuclear AID was also observed in a proportion of cells, in keeping with the DNA deamination model of AID function. Intratubular germ cell neoplasia unclassified (IGCNU), the precursor lesion of testicular cancers, was AID-negative. Seminomas also lacked AID expression. Nuclear and cytoplasmic AID expression was observed in three of 32 mixed non-seminomatous germ cell tumours. The results provide evidence for a physiological role for AID outside the immune system. AID expression in spermatocytes points to a role in meiosis. It remains uncertain whether AID may also contribute to the genetic aberrations characteristically found in testicular germ cell tumours. The consistent absence of detectable AID expression in atypical spermatogonia of IGCNU and its rare expression in germ cell tumours suggest that continued expression of AID is not involved in the pathogenesis of germ cell tumours.

Comparative proteomic analysis of neoplastic and non-neoplastic germ cell tissue.

A comparative proteomic analysis of neoplastic versus non-neoplastic seminoma identified glutathione S-transferase M3 as a differentially expressed protein. This expression difference could also be observed at the mRNA level, implying neoplasm-associated alterations in transcriptional or post-transcriptional mechanisms.

Lactate dehydrogenase is not a useful marker for relapse in patients on surveillance for stage I germ cell tumours.

As part of surveillance protocols for stage I germ cell tumours, many centres routinely measure human chorionic gonadotrophin (HCG), alpha feto-protein (AFP) as well as lactate dehydrogenase (LDH). In conjunction with regular imaging and clinical examination, does routine measurement of LDH add anything to our relapse/pick up rate? Records of 494 patients at Mount Vernon Hospital who relapsed on surveillance between 1985 and 2005 were examined. Of the 494 patients who relapsed, 125 had raised LDH at the time of relapse. 112 of these had a concurrent rise in either AFP, HCG or both, 11 had their disease detected on CT before the rise in LDH, one had a clinically palpable para-aortic mass and the final patient complained of back pain and his retroperitoneal disease was thus discovered on imaging. Routine measurement of LDH in patients on surveillance for stage I germ cell tumours does not add to the early detection of relapse.

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors.

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.

Localization and expression of TSP50 protein in human and rodent testes.

OBJECTIVES: To present our recent observations obtained from the continuing characterization of the TSP50 gene pertaining to its evolutionary importance and behavior in human testicular germ cell tumors. Previous studies have reported that expression of the human TSP50 gene is testes specific. Its product is similar to many serine proteases but possesses its own unique features. In addition, TSP50 is abnormally activated in most tested patients with breast cancer. METHODS: Testicular tissue from rats, mice, and humans was obtained through biopsy or orchiectomy. The expression of the TSP50 protein was determined using immunohistochemical staining and Western blotting techniques. RESULTS: The Western blot results showed that the polyclonal anti-human TSP50antibody reaction pattern in both rodent testes was the same as that observed in the human testes. In addition, the immunohistochemical staining patterns in the human, mouse, and rat testes were similar. We also discovered that expression of TSP50 was largely downregulated in all testicular germ cell tumors examined by immunohistochemical analysis. CONCLUSIONS: The results of our studies suggest that the TSP50 gene could be of evolutionary importance in mammalian reproduction. Unlike the results generated from patients with breast cancer, in whom upregulation of the TSP50 gene correlates with disease development, the TSP50 gene was downregulated in patients with seminoma. This information indicates that altered expression levels of the TSP50 gene in different microenvironments are associated with different or distinct types of human cancer.

Aurora B expression in normal testis and seminomas.

Aurora/Ipl1-related kinases are a conserved family of proteins that have multiple functions during mitotic progression. High levels of Aurora kinases are characteristic of rapidly dividing cells and tumours. Aurora B encodes a protein that associates with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. In this study the expression and the localisation of Aurora B throughout germinal epithelial progression in normal testis and its neoplastic counterpart were analysed. Immunocytochemistry and RT-PCR analysis of mouse germinal epithelium cells showed the presence of Aurora B in spermatogonia and occasionally in spermatocytes. Western blot analysis revealed the typical Aurora B isoform ( approximately 41 kDa) in the same cellular types. A similar distribution was observed in human testis by immunohistochemistry. Moreover, the distribution and the expression of Aurora B were investigated in neoplasms derived from germ cells. Surgical samples of seminomas were analysed, and a high percentage of Aurora B positive cells (51%) was detected; the expression of Aurora B was significantly related to the MIB-1 proliferation marker (R=0.816). The data presented here demonstrate that Aurora B expression occurs in spermatogonial division. Furthermore, our results indicate that the expression of Aurora B is a consistent feature of human seminomas.

Laboratory markers and germ cell tumors.

The International Germ Cell Consensus Classification (IGCCC) of testicular germ cell tumors (TGCT) in 1997 included three serum tumor markers, serum lactate dehydrogenase catalytic concentration (S-LD), serum alpha fetoprotein concentration (S-AFP), and serum human chorionic gonadotropin concentration (S-hCG). The recommendation should be implemented for all patients with TGCT and is also useful for patients with ovarian and extragonadal germ cell tumors. A fourth serum tumor marker for TGCT, S-LD isoenzyme 1 (S-LD-1), is also relevant for TGCT. Patients with seminoma have a raised S-LD-1 more often than a raised S-AFP and S-hCG, whereas patients with nonseminoma have a raised S-AFP more often than a raised S-LD-1 and S-hCG. A new model combining IGCCC and S-LD-1 predicts survival better than previous staging systems. LD-1 is related to a characteristic chromosomal abnormality in all types of TGCT, a high copy number of chromosome 12p. In contrast, AFP and hCG are found mainly in nonseminomatous germ cell tumors and they related to the histologic differentiation of the tumors. The different biologic background for the serum tumor markers may contribute to the difference in their clinical behavior.

Aneuploidy of human testicular germ cell tumors is associated with amplification of centrosomes.

Testicular germ cell tumors occur in three age groups. Seminomas and nonseminomas of adults, including mature teratomas, and the precursor carcinoma in situ (CIS) are aneuploid. This also holds true for yolk sac tumors of newborn and infants, while the mature teratomas of this age are diploid. In contrast, spermatocytic seminomas occurring in the elderly contain both diploid and polyploid cells. Aneuploidy has been associated with centrosome aberrations, sometimes related to overexpression of STK15. Aneuploidy of non-neoplastic germ cells has been demonstrated in the context of male infertility, a risk factor for the development of seminoma/nonseminoma. We investigated aneuploidy, centrosome aberrations and the role of STK15 in different types of testicular germ cell tumors as well as in normal and disturbed spermatogenesis. The aneuploid seminomas and nonseminomas tumors (including CIS) showed increased numbers of centrosomes, without STK15 amplification or overexpression. Four out of six infantile teratomas had normal centrosomes, the remaining two and an infantile yolk sac tumor showed a heterogeneous pattern of cells with normal or amplified centrosomes. Spermatocytic seminomas had two, four or eight centrosomes. Germ cells in seminiferous tubules with disturbed spermatogenesis shared both aneuploidy and centrosome abnormalities with seminomas/nonseminomas and showed a more intense STK15 staining than those with normal spermatogenesis and CIS. Therefore, aneuploidy of testicular germ cell tumors is associated with amplified centrosomes probably unrelated to STK15.

Changes in activities of enzymes related to energy metabolism in testicular tissues of dogs with seminoma.

Changes in the activities of enzymes related to energy metabolism in the testicular tissues of dogs with seminoma were investigated. The testis was removed surgically from animals anaesthetized with halothane. Cytosolic and mitochondrial fractions were isolated and the total RNA was extracted from testicular homogenates. The activities of enzymes related to energy metabolism were measured and the mRNA of cytosolic malate dehydrogenase (MDH) was investigated by the reverse transcriptase-polymerase chain reaction (RT-PCR). The activities of the glycolytic enzymes glucose-6-phosphate dehydrogenase (G6PD) for the pentose phosphate pathway and malate dehydrogenase (MDH) for the malate-aspartate shuttle, and the expression of the mRNA of cytosolic MDH were significantly increased in the testicular tissues of dogs with seminoma. These enzymatic activities may be useful indicators with which to evaluate changes in the metabolic conditions in testicular tissues of dogs with seminoma.

Serum lactate dehydrogenase isoenzyme 1 in patients with seminoma stage I followed with surveillance.

Serum lactate dehydrogenase isoenzyme I catalytic concentration (S-LD-1) was measured in patients with testicular seminoma clinical stage I followed with surveillance after orchiectomy. The serum samples were obtained before orchiectomy in 110 patients (group A) and soon after orchiectomy in 55 patients (group B). In group A, 60 patients (55%) had elevated S-LD-1 and 10 patients (9%) had elevated serum human chorionic gonadotropin concentrations (S-hCG). In group B, median S-LD-1 was lower than that of group A and decreased with increasing time after orchiectomv (p = 0.001, Jonckheere-Terpstra test, one-sided). After a median follow-up of 5.1 years, 23 patients (21%) in group A had relapses. The patients with elevated S-LD-1 and those with normal S-LD-1 had a similar relapse-free survival (p = 0.79, log-rank test). Thus patients with seminoma stage I had elevated S-LD-1 more often than elevated S-hCG but an elevation in S-LD-1 did not predict a relapse during follow-up with surveillance. Further studies are required to elucidate the value of S-LD-1 in monitoring the surveillance of patients with seminoma stage I.

The combined use of high performance liquid chromatography and immuno-biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins.

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

Germ cell-like telomeric length homeostasis in nonseminomatous testicular germ cell tumors.

Telomere maintenance plays an important role in cell proliferation and tumor survival. Human male germ cells, which carry long telomeres and express telomerase, give rise to a highly heterogeneous group of malignant tumors. We compared telomeric length and telomerase activity between two major histological types of primary testicular germ cell tumors. Fifteen out of 16 seminoma samples revealed telomeric restriction fragment (TRF) length below 13 kb; the remaining seminoma showed a major TRF fraction of 18 kb and a distinct minor fraction of above 23 kb length. In contrast, all 13 samples from nonseminomas showed TRF length >/=23 kb, which is similar to that reported in human sperm. Nine out of 11 seminoma specimens and six out of seven nonseminomas studied showed moderate to high telomerase activity, the only telomerase-negative nonseminoma being pure mature teratoma. These results indicate to a major difference in telomeric length between seminomas and nonseminomas, which is apparently unrelated to the presence of telomerase activity, and suggest a germline-like homeostasis of telomeric length is preserved in human nonseminomas. Oncogene (2000) 19, 4075 - 4078.