CHK2
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Checkpoint kinase 2 (Chk2) is emerging as a key mediator of diverse cellular responses to genotoxic stress, guarding the integrity of the genome throughout eukaryotic evolution. Recent studies show the fundamental role of Chk2 in the network of genome-surveillance pathways that coordinate cell-cycle progression with DNA repair and cell survival or death. Defects in Chk2 contribute to the development of both hereditary and sporadic human cancers, and earmark this kinase as a candidate tumour suppressor and an attractive target for drug discovery.
- MeSH
- aktivace enzymů MeSH
- biologická evoluce MeSH
- biologické modely MeSH
- buněčný cyklus MeSH
- checkpoint kinasa 2 MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- mutace MeSH
- nádory genetika MeSH
- protein-serin-threoninkinasy * MeSH
- proteinkinasy fyziologie genetika chemie MeSH
- racionální návrh léčiv MeSH
- signální transdukce MeSH
- tumor supresorové geny MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Checkpoint kinase 2 (Chk2) is a tumour suppressor and signal transducer in genome integrity checkpoints that coordinate cell-cycle progression with DNA repair or cell death in response to DNA damage. Defects of Chk2 occur in subsets of diverse sporadic malignancies and predispose to several types of hereditary carcinomas. However, the status of Chk2 in tumours of the urinary bladder remains unknown. Here, we report that among 58 advanced (grade T2-T4) human bladder carcinomas, immunohistochemical analysis revealed tumour-specific reduction or lack of Chk2 protein in 6 (10.3%) cases. Genetic analysis of the latter subset showed that a Chk2-negative carcinoma #668 harboured a truncating mutation 1100delC, in one Chk2 allele and loss of the corresponding second allele. The 1100delC mutation was also found in the germ line of this patient. Sequencing of TP53 in tumour #668 identified two missense mutations. Furthermore, the vast majority of the tumours showed 'unscheduled' activatory phosphorylation on Thr68 of Chk2 in the absence of any DNA-damaging treatment. Our results indicate that the otherwise dormant DNA damage signal transducer Chk2 is aberrantly and constitutively activated in invasive urinary bladder carcinomas, and that such likely proapoptotic checkpoint signalling can be disabled by inactivation of Chk2 and/or p53 tumour suppressors in subsets of these tumours.
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
The Chk2 kinase is a tumor suppressor and key transducer of DNA-damage checkpoints. We show that the human Chk2 protein is relatively stable, nuclear, and responding to gamma-radiation throughout the cell cycle. Contrary to the retinoblastoma protein-regulated, labile Chk1 kinase restricted to S-G(2) phases, Chk2 remains activatable even in quiescent and differentiating cells. In human tissues, Chk2 is homogeneously expressed in renewing cell populations such as epidermis or intestine, heterogeneous in conditionally renewing tissues, and absent or cytoplasmic in static tissues such as muscle or brain. These data highlight striking differences between Chk2 and Chk1 and show unexpected correlation of Chk2 expression with tissue biology.
- MeSH
- aktivace enzymů MeSH
- buněčná diferenciace fyziologie MeSH
- buněčné dělení fyziologie MeSH
- buněčné linie MeSH
- buněčný cyklus fyziologie MeSH
- checkpoint kinasa 2 MeSH
- fibroblasty cytologie enzymologie MeSH
- G1 fáze fyziologie MeSH
- lidé MeSH
- monoklonální protilátky MeSH
- nádorové buňky kultivované MeSH
- osteosarkom enzymologie patologie MeSH
- poškození DNA * fyziologie MeSH
- protein-serin-threoninkinasy * MeSH
- proteinkinasy fyziologie imunologie metabolismus MeSH
- S fáze fyziologie MeSH
- Check Tag
- lidé MeSH
Recent evidence identified a genetic and functional link between Chk2 kinase and p53 as a candidate genome integrity checkpoint and a tumour suppressor pathway. Here we report that in human cells, Chk2 and p53 form protein-protein complexes whose abundance increased upon DNA damage, and whose formation was abrogated through cancer associated mutations in the FHA domain of Chk2, or mutations in the tetramerization domain of p53. Whereas among Li-Fraumeni syndrome families mutations of Chk2 or p53 occur in a mutually exclusive manner, we document that the colon cancer cell line HCT-15 concomitantly lacks functions of both Chk2 and p53, the latter demonstrated by a non-invasive reporter assay monitoring p53-dependent transactivation in live cells. Despite the preserved ability of common cancer-derived mutant p53 proteins to bind and potentially 'titrate' activated Chk2, the integrity of the S phase checkpoint response to ionizing radiation remained largely intact and dependent on Chk2 in cells with wild-type, mutant, or no p53. These results provide new mechanistic insights into the Chk2-p53 interplay, suggest how mutations in Chk2 may abrogate its tumour suppressor function, and indicate that compared with individual defects in either Chk2 or p53, concomitant mutations in both of these cell cycle checkpoint regulators may provide some additional selective advantage to tumour cells.
- MeSH
- aktivace transkripce MeSH
- checkpoint kinasa 2 MeSH
- DNA biosyntéza MeSH
- geny p53 * MeSH
- ionizující záření MeSH
- lidé MeSH
- mutace MeSH
- nádorové buňky kultivované MeSH
- nádorový supresorový protein p53 * fyziologie MeSH
- nádory * etiologie genetika metabolismus MeSH
- poškození DNA * MeSH
- protein-serin-threoninkinasy * MeSH
- proteinkinasy * fyziologie genetika MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
Accumulation of mutations and chromosomal aberrations is one of the hallmarks of cancer cells. This enhanced genetic instability is fueled by defects in the genome maintenance mechanisms including DNA repair and cell cycle checkpoint pathways. Here, we discuss the emerging roles of the mammalian Chk1 and Chk2 kinases as key signal transducers within the complex network of genome integrity checkpoints, as candidate tumor suppressors disrupted in sporadic as well as some hereditary malignancies and as potential targets of new anticancer therapies.
- MeSH
- biologické modely MeSH
- buněčný cyklus MeSH
- checkpoint kinasa 2 MeSH
- lidé MeSH
- mutace * MeSH
- myši knockoutované MeSH
- myši MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- nádory prsu enzymologie metabolismus MeSH
- nádory tračníku enzymologie MeSH
- nádory * genetika MeSH
- oprava DNA MeSH
- protein-serin-threoninkinasy fyziologie genetika MeSH
- proteinkinasy fyziologie genetika MeSH
- signální transdukce MeSH
- terciární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
When exposed to ionizing radiation (IR), eukaryotic cells activate checkpoint pathways to delay the progression of the cell cycle. Defects in the IR-induced S-phase checkpoint cause 'radioresistant DNA synthesis', a phenomenon that has been identified in cancer-prone patients suffering from ataxia-telangiectasia, a disease caused by mutations in the ATM gene. The Cdc25A phosphatase activates the cyclin-dependent kinase 2 (Cdk2) needed for DNA synthesis, but becomes degraded in response to DNA damage or stalled replication. Here we report a functional link between ATM, the checkpoint signalling kinase Chk2/Cds1 (Chk2) and Cdc25A, and implicate this mechanism in controlling the S-phase checkpoint. We show that IR-induced destruction of Cdc25A requires both ATM and the Chk2-mediated phosphorylation of Cdc25A on serine 123. An IR-induced loss of Cdc25A protein prevents dephosphorylation of Cdk2 and leads to a transient blockade of DNA replication. We also show that tumour-associated Chk2 alleles cannot bind or phosphorylate Cdc25A, and that cells expressing these Chk2 alleles, elevated Cdc25A or a Cdk2 mutant unable to undergo inhibitory phosphorylation (Cdk2AF) fail to inhibit DNA synthesis when irradiated. These results support Chk2 as a candidate tumour suppressor, and identify the ATM-Chk2-Cdc25A-Cdk2 pathway as a genomic integrity checkpoint that prevents radioresistant DNA synthesis.
- MeSH
- alely MeSH
- ATM protein MeSH
- buněčné linie MeSH
- buněčný cyklus * genetika účinky záření MeSH
- checkpoint kinasa 2 MeSH
- DNA vazebné proteiny MeSH
- fosfatasy cdc25 * fyziologie účinky záření MeSH
- fosforylace MeSH
- ionizující záření MeSH
- lidé MeSH
- myši MeSH
- nádorové supresorové proteiny MeSH
- protein-serin-threoninkinasy * fyziologie MeSH
- proteinkinasy fyziologie genetika MeSH
- proteiny buněčného cyklu MeSH
- replikace DNA * účinky záření MeSH
- S fáze účinky záření MeSH
- serin metabolismus MeSH
- signální transdukce MeSH
- tolerance záření MeSH
- transfekce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
BACKGROUND: Haemanthamine (HA) and sodium butyrate (NaB) are promising candidates for chemotherapy as a treatment for cancer. PURPOSE: We aimed to determine the anticancer potential of HA and NaB, alone and in combination, in A2780 ovarian cancer cells and concurrently investigated anticancer potential in contrast to non-cancer human MRC-5 fibroblasts. METHODS: Antiproliferative effects were determined by WST-1 assay and by Trypan blue exclusion staining. Cell cycle distributions were studied by flow cytometry and protein levels were determined by Western blotting. RESULTS: The combination of HA and NaB caused a significant decrease in the proliferation of A2780 cells compared to the stand-alone treatment of cells by HA or NaB. This effect was less pronounced in non-cancer MRC-5 fibroblasts. In the later intervals, the number of A2780 living cells was strongly decreased by treatment using a combination of NaB and HA. This simultaneous application had no considerable effect in MRC-5 fibroblasts. The combination of NaB and HA led to the suppression of cells in the G1 phase and caused an accumulation of cells in the S and G2 phase in comparison to those treated with NaB and HA alone. Treatment of cells with NaB alone led to the activation of proteins regulating the cell cycle. Notably, p21WAF1/Cip1 was upregulated in both A2780 and MRC-5 cells, while checkpoint kinases 1 and 2 were activated via phosphorylation only in A2780 cells. Unexpectedly, NaB in combination with HA suppressed the phosphorylation of Chk2 on threonine 68 and Chk1 on serine 345 in A2780 cells and downregulated p21WAF1/Cip1 in both tested cell lines. The sensitization of cells to HA and NaB treatment seems to be accompanied by increased histone acetylation. NaB-induced acetylation of histone H3 and H4 and histone acetylation increased markedly when a combination of NaB and HA was applied. Whereas the most prominent hyperacetylation after HA and NaB treatment was observed in A2780 cells, the acetylation of histones occurred in both cell lines. CONCLUSION: In summary, we have demonstrated the enhanced activity of HA and NaB against A2780 cancer cells, while eliciting no such effect in non-cancer MRC-5 cells.
- MeSH
- acetylace MeSH
- aktivace transkripce účinky léků MeSH
- alkaloidy amarylkovitých farmakologie MeSH
- buněčné dělení účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- checkpoint kinasa 1 metabolismus MeSH
- checkpoint kinasa 2 metabolismus MeSH
- fenantridiny farmakologie MeSH
- fosforylace MeSH
- histony metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy metabolismus MeSH
- kyselina máselná farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků patologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články 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
