DNA damage response (DDR) is emerging as a physiological anti-cancer barrier in early stages of cancer development, as shown for several types of solid cancers derived from somatic cells. Here we discuss our recently published and unpublished results on the exceptional paucity of such constitutive activation of the DDR machinery in human testicular germ cell tumours (TGCTs), including their common pre-invasive stage of carcinoma in situ (CIS). Our conclusions are supported by immunohistochemical analyses of multiple markers of activated DNA damage signalling, such as the phosphorylated ATM and Chk2 checkpoint kinases and phosphorylated histone H2AX. We propose that the unique lack of DDR activation in TGCTs reflects the biology of their cell of origin, the gonocyte. Furthermore, we propose that the lack of DDR activation avoids the pressure to select for mutations in DDR genes such as p53 or ATM, and the resulting intact DDR machinery may have implications for the exceptional curability of TGCTs by DNA damaging therapies.

• MeSH
• chromozomální aberace MeSH
• germinální a embryonální nádory * genetika   patologie   terapie   MeSH
• invazivní růst nádoru MeSH
• karcinom in situ genetika   MeSH
• lidé MeSH
• mutace MeSH
• poškození DNA * MeSH
• referenční hodnoty MeSH
• testikulární nádory * genetika   patologie   terapie   MeSH
• testis fyziologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH

MDC1 and 53BP1 are critical components of the DNA damage response (DDR) machinery that protects genome integrity and guards against cancer, yet the tissue expression patterns and involvement of these two DDR adaptors/mediators in human tumours remain largely unknown. Here we optimized immunohistochemical analyses of human 53BP1 and MDC1 proteins in situ and identified their virtually ubiquitous expression, both in proliferating and quiescent, differentiated tissues. Focus formation by 53BP1 and/or MDC1 in human spermatogenesis and subsets of breast and lung carcinomas indicated physiological and 'pathological' activation of the DDR, respectively. Furthermore, aberrant reduction or lack of either protein in significant proportions of carcinomas supported the candidacy of 53BP1 and MDC1 for tumour suppressors. Contrary to carcinomas, almost no activation or loss of MDC1 or 53BP1 were found among testicular germ-cell tumours (TGCTs), a tumour type with unique biology and exceptionally low incidence of p53 mutations. Such concomitant presence (in carcinomas) or absence (in TGCTs) of DDR activation and DDR aberrations supports the roles of MDC1 and 53BP1 within the ATM/ATR-regulated checkpoint network which, when activated, provides an early anti-cancer barrier the pressure of which selects for DDR defects such as p53 mutations or loss of 53BP1/MDC1 during cancer progression.

• MeSH
• DNA vazebné proteiny biosyntéza   genetika   MeSH
• germinální a embryonální nádory genetika   metabolismus   patologie   MeSH
• imunohistochemie MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   MeSH
• jaderné proteiny * biosyntéza   genetika   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory plic genetika   metabolismus   patologie   MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• poškození DNA * MeSH
• testikulární nádory genetika   metabolismus   patologie   MeSH
• trans-aktivátory * biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

The ATM kinase is a tumor suppressor and key regulator of biological responses to DNA damage. Cultured cells respond to genotoxic insults that induce DNA double-strand breaks by prompt activation of ATM through its autophosphorylation on serine 1981. However, whether ATM-S1981 becomes phosphorylated in vivo, for example during physiological processes that generate DSBs, is unknown. Here we produced phospho-specific monoclonal antibodies against S1981-phosphorylated ATM (pS-ATM), and applied them to immunohistochemical analyses of a wide range of normal human tissues and testicular tumors. Our data show that regardless of proliferation and differentiation, most human tissues contain only the S1981-nonphosphorylated, inactive form of ATM. In contrast, nuclear staining for pS-ATM was detected in subsets of bone-marrow lymphocytes and primary spermatocytes in the adult testes, cell types in which DSBs are generated during physiological V(D)J recombination and meiotic recombination, respectively. Among testicular germ-cell tumors, an aberrant constitutive pS-ATM was observed especially in embryonal carcinomas, less in seminomas, and only modestly in teratomas and the pre-invasive carcinoma-in-situ stage. Compared with pS-ATM, phosphorylated histone H2AX (gammaH2AX), another DNA damage marker and ATM substrate, was detected in a higher proportion of cancer cells, and also in normal fetal gonocytes, and a wider range of adult spermatocyte differentiation stages. Collectively, our results strongly support the physiological relevance of the recently proposed model of ATM autoactivation, and provide further evidence for constitutive activation of the DNA damage machinery during cancer development. The new tools characterized here should facilitate monitoring of ATM activation in clinical specimens, and help develop future treatment strategies.

• MeSH
• ATM protein MeSH
• cervix uteri cytologie   MeSH
• DNA vazebné proteiny * metabolismus   MeSH
• epitelové buňky účinky záření   MeSH
• ezofágus cytologie   MeSH
• fibroblasty účinky záření   MeSH
• fosforylace MeSH
• germinální a embryonální nádory patologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny * metabolismus   MeSH
• orgánová specificita MeSH
• plod cytologie   MeSH
• poškození DNA genetika   MeSH
• protein-serin-threoninkinasy * metabolismus   MeSH
• proteiny buněčného cyklu * metabolismus   MeSH
• rekombinantní proteiny MeSH
• testikulární nádory * metabolismus   MeSH
• žaludek cytologie   MeSH
• zdraví MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH

AIMS: Spermatocytic seminoma is a rare germ cell derived tumour of the testis that occurs mainly in older men. We analysed the expression of recently discovered markers for germ cell differentiation and the mitosis-meiosis transition in order to define the antigen profile for diagnostic purposes and to clarify the biology and histogenesis of spermatocytic seminoma. METHODS AND RESULTS: Twenty-five spermatocytic seminomas were examined for immunohistochemical expression of germ cell-specific onco-fetal antigens and proteins involved in regulation of germ cell division, DNA repair and differentiation. The panel included Chk2, p19INK4d, p53, MAGE-A4, KIT, TRA-1-60, neurone-specific enolase and placental-like alkaline phosphatase. Four of these proteins/antigens have never before been investigated in spermatocytic seminoma. Proteins highly expressed in gonocytes and spermatogonia, such as Chk2, MAGE-A4 and neurone-specific enolase, were consistently present in spermatocytic seminoma. Antigens expressed in embryonic germ cells but not in the normal adult testis, e.g. TRA-1-60, were undetectable, with the exception of p53 protein, which was demonstrated in 80% of cases. A proto-oncogene p19INK4d, which is involved in the transition from mitotic to meiotic division in germ cells, was not detected in spermatocytic seminoma. CONCLUSIONS: The investigation provided new information concerning the expression of Chk2, MAGE-A4, neurone-specific enolase and p19INK4d in spermatocytic seminoma. The pattern of expression is highly consistent with the origin of spermatocytic seminoma from a premeiotic germ cell, which has lost embryonic traits and has committed to spermatogenic lineage but has not yet passed the meiotic checkpoint, most probably from the spermatogonium of the adult testis.

• MeSH
• antigeny nádorové metabolismus   MeSH
• checkpoint kinasa 2 MeSH
• dospělí MeSH
• fosfopyruváthydratasa metabolismus   MeSH
• imunoenzymatické techniky MeSH
• inhibitor p16 cyklin-dependentní kinasy metabolismus   MeSH
• inhibitor p19 cyklin-dependentní kinasy MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery * metabolismus   MeSH
• nádorové proteiny * metabolismus   MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• protein-serin-threoninkinasy * MeSH
• proteinkinasy metabolismus   MeSH
• proteiny buněčného cyklu * MeSH
• seminom etiologie   metabolismus   patologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• testikulární nádory etiologie   metabolismus   patologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH

Deregulated cell cycle and defective genome-integrity checkpoints are among the hallmarks of cancer. Here we summarize our recent studies of key components of the GI/S machinery in normal human spermatogenesis, and their abnormalities in testicular germ cell tumours (TGCTs), with special emphasis on carcinoma in situ lesions (CIS). Our combined immunohistochemical and immunoblotting analyses of normal human adult and fetal testes, CIS, seminomas, embryonal carcinomas, and teratomas, revealed an 'unorthodox' spectrum of defects within the so-called RB pathway in TGCTs. The early aberrations included lack of expression of the retinoblastoma tumour suppressor (pRB) and the CDK inhibitor pl9ink4d, and overexpression of cyclin D2. Progression from CIS to invasive TGCTswas associated with loss of another two CDK inhibitors and tumour suppressors: pl6ink4a and pl8ink4c. We also found the lack of pRB and pl9ink4d in fetal gonocytes, the candidate target cell for all types of TGCTs. These findings, together with the status of the Chk2-p53 DNA-integrity checkpoint, are considered in relation to the origin, biology and pathogenesis of TGCTs, and potential implications of the GI/S defects for the curability of these tumours.

• MeSH
• buněčný cyklus MeSH
• cyklin D2 MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   MeSH
• cykliny metabolismus   MeSH
• dospělí MeSH
• fosforylace MeSH
• G1 fáze * MeSH
• germinom * etiologie   patologie   MeSH
• inhibitor p16 cyklin-dependentní kinasy metabolismus   MeSH
• inhibitor p18 cyklin-dependentní kinasy MeSH
• inhibitor p19 cyklin-dependentní kinasy MeSH
• inhibitory enzymů metabolismus   MeSH
• karcinom in situ etiologie   patologie   MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• proteiny buněčného cyklu * MeSH
• retinoblastomový protein analýza   metabolismus   MeSH
• S fáze * MeSH
• spermatogeneze MeSH
• testikulární nádory * etiologie   patologie   MeSH
• testis embryologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

Deregulation of the RB pathway is shared by most human malignancies. Components upstream of the retinoblastoma tumour suppressor (pRB), namely the INK4 family of cyclin-dependent kinase (CDK) inhibitors, the D-type cyclins, their partner kinases CDK4/CDK6, and pRB as their critical substrate, are differentially targeted in diverse types of cancer. An 'unorthodox' spectrum of defects within this cascade occurs in testicular germ cell tumours (TGCTs), including silencing of pRB transcription, overexpression of cyclin D2, and loss of p18INK4c. To improve understanding of the role of this pathway in spermatogenesis, and its subversion in TGCTs, we examined immunohistochemical expression patterns of CDK4, p16INK4a, p15INK4b, and pRB, and established an in situ assay for cyclin D-mediated phosphorylation of serine795, a phosphorylation event critical for neutralization of pRB's growth-restraining ability. pRB was expressed throughout adult spermatogenesis and was detectable in teratomas, but was absent or grossly reduced in carcinoma in situ (CIS) and most seminomas and embryonal carcinomas. Unexpectedly, we also found that pRB was absent from fetal human gonocytes, the candidate target cell for all types of TGCTs. Thus, rather than a tumorigenesis-promoting loss of pRB, the lack of pRB in TGCTs likely reflects its developmental control. Widespread expression of p15INK4b, found in normal testes, was preserved in TGCTs. In contrast, p16INK4a was lost or reduced in large subsets of TGCTs. CDK4 was expressed in normal spermatogonia, CIS, and invasive TGCTs, as was serine795-phosphorylated pRB. Our data on expression of pRB support the plausible origin of TGCTs from fetal gonocytes, and the serine795 phosphorylation demonstrates that the cyclin D-dependent kinases are active, and neutralize pRB in spermatogonia and in those TGCTs that express pRB. We hope that this study will inspire further immunohistochemical applications of phosphospecific antibodies in pathology, and examination of the RB pathway defects in relation to curability of TGCTs. Copyright 2003 John Wiley & Sons, Ltd.

• MeSH
• buněčný cyklus MeSH
• dospělí MeSH
• embryonální karcinom metabolismus   MeSH
• fosforylace MeSH
• germinom * patofyziologie   MeSH
• imunoenzymatické techniky MeSH
• karcinom in situ metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• novorozenec MeSH
• retinoblastomový protein * fyziologie   MeSH
• seminom metabolismus   MeSH
• teratom metabolismus   MeSH
• testikulární nádory * patofyziologie   MeSH
• testis metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH

This review summarises the existing knowledge on the phenotype of the carcinoma in situ (CIS) cell. CIS is a common pre-invasive precursor of testicular germ cell tumours of adolescents and young adults. These tumours display a variety of histological forms. Classical seminoma proliferates along the germ cell lineage, whereas embryonal carcinoma retains embryonic features and readily differentiates into teratomas that resemble various somatic cell lineages. A thorough review of the gene expression in CIS cells in comparison to normal testicular germ cells and overt tumours supports the view that CIS is a common precursor for both tumour types. Impaired cell differentiation resulting in a partial retention of the embryonic features, associated with an increasing genomic instability may be responsible for a remarkable phenotypic heterogeneity of CIS cells. Depending on the degree of differentiation and pluripotency, CIS cells found in adult patients seem to be predestined for further malignant progression into one or the other of the two main types of overt tumours. A new concept of phenotypic continuity of differentiation of germ cells along germinal lineage with a gradual loss of embryonic features based on the analysis of gene expression in all types of germ cells during their ontogeny is presented in this review. The data point out that despite the phenotypic continuum of gene expression, there are two periods of rapid changes of gene expression: first at the transition from primordial germ cells to pre-spermatogonia, and later during the pubertal switch from the mitotic to meiotic cell division. The persistent expression of embryonic traits in CIS cells, and the high expression of the cell cycle regulators that are typical of mitotic germ cells support our long-standing hypothesis that CIS cells originate from primordial germ cells or gonocytes and not from germ cells in the adult testis.

• MeSH
• antigeny nádorové genetika   MeSH
• buněčný cyklus genetika   MeSH
• dospělí MeSH
• exprese genu MeSH
• fenotyp * MeSH
• imunohistochemie MeSH
• karcinom in situ * genetika   patologie   MeSH
• kmenové buňky patologie   MeSH
• lidé MeSH
• lidské chromozomy X genetika   MeSH
• lidský chromozom Y genetika   MeSH
• mladiství MeSH
• nádorová transformace buněk genetika   MeSH
• oprava DNA MeSH
• seminom * genetika   patologie   MeSH
• spermie patologie   MeSH
• testikulární nádory * genetika   patologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH

Chk2 is a transducer of DNA damage signals and a tumour suppressor whose germ-line mutations predispose to diverse tumour types. Unlike its downstream targets such as the p53 tumour suppressor, the expression patterns of Chk2 in tissues and tumours remain unknown. As DNA breaks occur commonly during gametogenesis, and p53 is wild-type and overexpressed in testicular cancer, we examined abundance and localisation of the Chk2 protein during normal development of human testes, and at various stages of germ-cell tumour (GCT) pathogenesis. Our results show that Chk2 is abundant in foetal germ cells and adult spermatogonia, yet only weakly expressed or lacking during the meiotic and later stages of spermatogenesis. High levels of Chk2 are detected in the majority of GCTs including all pre-invasive carcinoma-in-situ lesions, contrary to variable expression and even lack of Chk2 in subsets of invasive GCTs and some teratoma structures, respectively. Together with our analyses of cell culture models, these results indicate that downmodulation or lack of Chk2 is not simply attributable to quiescence or differentiation, they suggest a role for Chk2 in mitotic rather than meiotic divisions, support the concept of foetal origin of GCTs, and have implications for protein-based screening for tumour-associated aberrations of Chk2.

• MeSH
• checkpoint kinasa 2 MeSH
• germinom * metabolismus   MeSH
• karcinom in situ * metabolismus   MeSH
• lidé MeSH
• nádorové proteiny * metabolismus   MeSH
• protein-serin-threoninkinasy * MeSH
• proteinkinasy * metabolismus   MeSH
• spermatogeneze * fyziologie   MeSH
• spermie metabolismus   MeSH
• testikulární nádory * metabolismus   MeSH
• testis embryologie   metabolismus   růst a vývoj   MeSH
• tumor supresorové geny MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH

Cell cycle regulators govern cellular proliferation, modulate differentiation and, when defective, contribute to oncogenesis. Here, we examined expression of cyclins A, B1 and E, and cyclin-dependent kinase (CDK) inhibitors p18INK4C (p18), p21WAF1/Cip1 (p21) and p27KiP1 (p27), in normal human adult testis (n = 5), and 53 testicular tumours, including 23 carcinomas in situ (CIS) and 30 germ cell tumours (GCTs). Immunohistochemical analysis revealed a correlation between proliferation and abundance of the cyclin proteins, and abundant p18 and the lack of p21 and p27 in normal spermatogenesis. Expression of p21 and/or p27 was induced in some differentiated structures seen in teratomas, and was recapitulated in cell culture, using human NTera2/D1 teratocarcinoma cells induced to differentiate into neurons. CIS lesions showed abundant p18, low cyclin E, and moderate p27, in contrast with most invasive seminomas and embryonal carcinomas with very low-to-negative p18, often elevated cyclin E, and, unexpectedly, sustained or increased p27. Our results suggest increased abundance of cyclin E, and particularly downmodulation or loss of p18INK4C as the features that correlate with progression from CIS to invasive germ cell tumours of the human testis.

• MeSH
• cyklin-dependentní kinasy * antagonisté a inhibitory   MeSH
• cykliny * metabolismus   MeSH
• dospělí MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• germinom * enzymologie   patofyziologie   patologie   MeSH
• imunoblotting MeSH
• inhibitor p18 cyklin-dependentní kinasy MeSH
• inhibitor p21 cyklin-dependentní kinasy MeSH
• inhibitor p27 cyklin-dependentní kinasy MeSH
• inhibitory enzymů * metabolismus   MeSH
• invazivní růst nádoru MeSH
• karcinom in situ enzymologie   patologie   MeSH
• lidé MeSH
• luminiscenční měření MeSH
• nádorové buňky kultivované MeSH
• nádorové supresorové proteiny * MeSH
• progrese nemoci MeSH
• proteiny asociované s mikrotubuly * metabolismus   MeSH
• proteiny buněčného cyklu * MeSH
• teratokarcinom * enzymologie   patofyziologie   patologie   MeSH
• testikulární nádory * enzymologie   patofyziologie   patologie   MeSH
• transportní proteiny * metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH

p19INK4d, a member of the INK4 family of cyclin-dependent kinase inhibitors, negatively regulates the proto-oncogenic cyclin D/CDK4(6) complexes whose ability to phosphorylate the retinoblastoma tumour suppressor (RB) promotes G1/S transition. In contrast to the related p16INK4a tumour suppressor, expression patterns of 19INK4d in human tissues and tumours remain unknown. As the RB pathway is commonly targeted in cancer, and mouse models suggest a role for p19INK4d in spermatogenesis, we examined the abundance and localization of p19INK4d in the human testis, both during normal development and at various stages of germ-cell tumour pathogenesis. Our data show that the p19INK4d protein is abundant in spermatocytes of normal human adult testes, whereas virtually no p19INK4d is detectable in testicular cancer, including the preinvasive carcinoma in situ stage. Together with the lack of p19INK4d in human foetal germ cells, these results support the concept of foetal origin of the testicular germ-cell tumours, and help better understand the emerging role of the RB pathway in spermatogenesis and tumorigenesis in the human testis. Oncogene (2000) 19, 4146 - 4150

• MeSH
• cyklin D2 MeSH
• cyklin-dependentní kinasa 4 MeSH
• cyklin-dependentní kinasy * antagonisté a inhibitory   genetika   MeSH
• cykliny antagonisté a inhibitory   genetika   MeSH
• geny retinoblastomu MeSH
• germinom * genetika   metabolismus   patologie   MeSH
• imunohistochemie MeSH
• inhibitor p16 cyklin-dependentní kinasy MeSH
• inhibitor p19 cyklin-dependentní kinasy MeSH
• lidé MeSH
• plod MeSH
• proteiny buněčného cyklu * MeSH
• protoonkogenní proteiny * MeSH
• regulace genové exprese u nádorů * MeSH
• spermatogeneze * genetika   MeSH
• testikulární nádory * genetika   metabolismus   patologie   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• tumor supresorové geny MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH