The spindle assembly checkpoint (SAC) joins the machinery of chromosome-to-spindle microtubule attachment with that of the cell cycle to prevent missegregation of chromosomes during mitosis. Although a functioning SAC has been verified in a limited number of organisms, it is regarded as an evolutionarily conserved safeguard mechanism. In this report, we focus on the existence of the SAC in a single-celled parasitic eukaryote, Giardia intestinalis. Giardia belongs to Excavata, a large and diverse supergroup of unicellular eukaryotes in which SAC control has been nearly unexplored. We show that Giardia cells with absent or defective mitotic spindles due to the inhibitory effects of microtubule poisons do not arrest in mitosis; instead, they divide without any delay, enter the subsequent cell cycle and even reduplicate DNA before dying. We identified a limited repertoire of kinetochore and SAC components in the Giardia genome, indicating that this parasite is ill equipped to halt mitosis before the onset of anaphase via SAC control of chromosome-spindle microtubule attachment. Finally, based on overexpression, we show that Giardia Mad2, a core SAC protein in other eukaryotes, localizes along intracytoplasmic portions of caudal flagellar axonemes, but never within nuclei, even in mitotic cells with blocked spindles, where the SAC should be active. These findings are consistent with the absence of a conventional SAC, known from yeast and metazoans, in the parasitic protist Giardia.

• Klíčová slova
• Albendazole, Giardia, Kinetochore, Mad2, Single-celled eukaryote, Spindle assembly checkpoint,
• MeSH
• aparát dělícího vřeténka fyziologie   MeSH
• Giardia lamblia genetika   izolace a purifikace   fyziologie   MeSH
• kinetochory fyziologie   MeSH
• kontrolní body M fáze buněčného cyklu fyziologie   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny buněčného cyklu MeSH

Glioblastoma multiforme (GBM) represents approximately 60% of all brain tumors in adults. This malignancy shows a high biological and genetic heterogeneity associated with exceptional aggressiveness, leading to a poor survival of patients. This review provides a summary of the basic biology of GBM cells with emphasis on cell cycle and cytoskeletal apparatus of these cells, in particular microtubules. Their involvement in the important oncosuppressive process called mitotic catastrophe will next be discussed along with select examples of microtubule-targeting agents, which are currently explored in this respect such as benzimidazole carbamate compounds. Select microtubule-targeting agents, in particular benzimidazole carbamates, induce G2/M cell cycle arrest and mitotic catastrophe in tumor cells including GBM, resulting in phenotypically variable cell fates such as mitotic death or mitotic slippage with subsequent cell demise or permanent arrest leading to senescence. Their effect is coupled with low toxicity in normal cells and not developed chemoresistance. Given the lack of efficient cytostatics or modern molecular target-specific compounds in the treatment of GBM, drugs inducing mitotic catastrophe might offer a new, efficient alternative to the existing clinical management of this at present incurable malignancy.

• Klíčová slova
• benzimidazole carbamates, cell death, glioblastoma multiforme, microtubule-targeting agents, mitotic catastrophe,
• MeSH
• glioblastom * metabolismus   mortalita   terapie   MeSH
• kontrolní body fáze G2 buněčného cyklu * MeSH
• kontrolní body M fáze buněčného cyklu * MeSH
• lidé MeSH
• mitóza * MeSH
• nádory mozku * metabolismus   mortalita   patologie   terapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Non-specific chromosomal aberrations (CAs) are microscopically detected in about 1% of lymphocytes drawn from healthy persons. Causes of CAs in general population are not known but they may be related to risk of cancer. In view of the importance of the mitotic checkpoint machinery on maintaining chromosomal integrity we selected 9 variants in main checkpoint related genes (BUB1B, BUB3, MAD2L1, CENPF, ESPL1/separase, NEK2, PTTG1/securin, ZWILCH and ZWINT) for a genotyping study on samples from healthy individuals (N = 330 to 729) whose lymphocytes had an increased number of CAs compared to persons with a low number of CAs. Genetic variation in individual genes played a minor importance, consistent with the high conservation and selection pressure of the checkpoint system. However, gene pairs were significantly associated with CAs: PTTG1-ZWILCH and PTTG1-ZWINT. MAD2L1 and PTTG1 were the most common partners in any of the two-way interactions. The results suggest that interactions at the level of cohesin (PTTG1) and kinetochore function (ZWINT, ZWILCH and MAD2L1) contribute to the frequency of CAs, suggesting that gene variants at different checkpoint functions appeared to be required for the formation of CAs.

• Klíčová slova
• Chromosomal integrity, Cytogenetics, DNA double-stranded break, Spindle checkpoint,
• MeSH
• CDC geny * MeSH
• chromozomální aberace * MeSH
• dvouřetězcové zlomy DNA MeSH
• genetická variace * MeSH
• intracelulární signální peptidy a proteiny genetika   MeSH
• jaderné proteiny genetika   MeSH
• kontrolní body M fáze buněčného cyklu genetika   MeSH
• lidé MeSH
• lymfocyty chemie   patologie   MeSH
• Mad2 protein genetika   MeSH
• modely genetické MeSH
• proteiny buněčného cyklu genetika   MeSH
• sekurin genetika   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• intracelulární signální peptidy a proteiny MeSH
• jaderné proteiny MeSH
• Mad2 protein MeSH
• MAD2L1 protein, human MeSH Prohlížeč
• pituitary tumor-transforming protein 1, human MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• sekurin MeSH
• Zwilch protein, human MeSH Prohlížeč
• ZWINT protein, human MeSH Prohlížeč

The high incidence of chromosomally abnormal human embryos is frequently assumed to be due to a lack of checkpoint controls operating during early embryogenesis. In our study we have analysed when these mechanisms first become functional. Mouse oocytes treated in late metaphase I with either of two different cyclin-dependent kinase inhibitors [butyrolactone 1 (BL1) or 6-dimethylaminopurine (6-DMAP)] form nuclei in the cytoplasm. BL1-treated eggs enter S-phase at 16-18 h post-treatment and, after completion of DNA synthesis, cleave to 2-cell stage embryos. 6-DMAP treatment results in the rapid initiation of DNA synthesis, its completion by 12 h and then arrest in the G2 phase. Thus, two different cell cycle stages can be obtained at the same time point after the initiation of treatment: G1- after BL1 and G2-staged nuclei after 6-DMAP treatment. That this approach greatly facilitates cell cycle studies has been shown by analysing checkpoint function during the first division. Whilst G2-staged eggs enter M phase within 2-3 h when 6-DMAP is washed out, the onset of M phase is delayed after their fusion to G1 (BL1) cells. Here M phase occurs only after the less advanced nucleus completes DNA replication. Our results indicate that checkpoints in mammalian eggs are functional during the first mitotic cycle.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   MeSH
• DNA biosyntéza   MeSH
• G1 fáze MeSH
• G2 fáze * MeSH
• gama-butyrolakton analogy a deriváty   farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• mitóza * MeSH
• myši inbrední C57BL MeSH
• myši inbrední CBA MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• zvířata MeSH
• zygota MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• adenin MeSH
• butyrolactone I MeSH Prohlížeč
• cyklin-dependentní kinasy MeSH
• DNA MeSH
• gama-butyrolakton MeSH
• inhibitory enzymů MeSH
• N(6),N(6)-dimethyladenine MeSH Prohlížeč

As treatment options for patients with incurable metastatic castration-resistant prostate cancer (mCRPC) are considerably limited, novel effective therapeutic options are needed. Checkpoint kinase 1 (CHK1) is a highly conserved protein kinase implicated in the DNA damage response (DDR) pathway that prevents the accumulation of DNA damage and controls regular genome duplication. CHK1 has been associated with prostate cancer (PCa) induction, progression, and lethality; hence, CHK1 inhibitors SCH900776 (also known as MK-8776) and the more effective SCH900776 analog MU380 may have clinical applications in the therapy of PCa. Synergistic induction of DNA damage with CHK1 inhibition represents a promising therapeutic approach that has been tested in many types of malignancies, but not in chemoresistant mCRPC. Here, we report that such therapeutic approach may be exploited using the synergistic action of the antimetabolite gemcitabine (GEM) and CHK1 inhibitors SCH900776 and MU380 in docetaxel-resistant (DR) mCRPC. Given the results, both CHK1 inhibitors significantly potentiated the sensitivity to GEM in a panel of chemo-naïve and matched DR PCa cell lines under 2D conditions. MU380 exhibited a stronger synergistic effect with GEM than clinical candidate SCH900776. MU380 alone or in combination with GEM significantly reduced spheroid size and increased apoptosis in all patient-derived xenograft 3D cultures, with a higher impact in DR models. Combined treatment induced premature mitosis from G1 phase resulting in the mitotic catastrophe as a prestage of apoptosis. Finally, treatment by MU380 alone, or in combination with GEM, significantly inhibited tumor growth of both PC339-DOC and PC346C-DOC xenograft models in mice. Taken together, our data suggest that metabolically robust and selective CHK1 inhibitor MU380 can bypass docetaxel resistance and improve the effectiveness of GEM in DR mCRPC models. This approach might allow for dose reduction of GEM and thereby minimize undesired toxicity and may represent a therapeutic option for patients with incurable DR mCRPC.

• Klíčová slova
• MU380, castration-resistant prostate cancer, checkpoint kinase 1, docetaxel resistance, gemcitabine, mitotic catastrophe,
• MeSH
• buněčná smrt účinky léků   MeSH
• checkpoint kinasa 1 antagonisté a inhibitory   metabolismus   MeSH
• chemorezistence účinky léků   MeSH
• deoxycytidin analogy a deriváty   farmakologie   MeSH
• docetaxel farmakologie   MeSH
• gemcitabin MeSH
• lidé MeSH
• mitóza * účinky léků   MeSH
• myši SCID MeSH
• nádorové buněčné linie MeSH
• nádory prostaty patologie   MeSH
• piperidiny chemie   farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• pyrazoly chemie   farmakologie   MeSH
• pyrimidiny chemie   farmakologie   MeSH
• S fáze účinky léků   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• checkpoint kinasa 1 MeSH
• CHEK1 protein, human MeSH Prohlížeč
• deoxycytidin MeSH
• docetaxel MeSH
• gemcitabin MeSH
• MU380 MeSH Prohlížeč
• piperidiny MeSH
• pyrazoly MeSH
• pyrimidiny MeSH

Genomic instability is a characteristic of a majority of human malignancies. Chromosomal instability is a common form of genomic instability that can be caused by defects in mitotic checkpoint genes. Chromosomal aberrations in peripheral blood are also indicative of genotoxic exposure and potential cancer risk. We evaluated associations between inherited genetic variants in 33 mitotic checkpoint genes and the frequency of chromosomal aberrations (CAs) in the presence and absence of environmental genotoxic exposure. Associations with both chromosome and chromatid type of aberrations were evaluated in two cohorts of healthy individuals, namely an exposed and a reference group consisting of 607 and 866 individuals, respectively. Binary logistic and linear regression analyses were performed for the association studies. Bonferroni-corrected significant p-value was 5 × 10-4 for 99 tests based on the number of analyzed genes and phenotypes. In the reference group the most prominent associations were found with variants in CCNB1, a master regulator of mitosis, and in genes involved in kinetochore function, including CENPH and TEX14, whereas in the exposed group the main association was found with variants in TTK, also an important gene in kinetochore function. How the identified variants may affect the fidelity of mitotic checkpoint remains to be investigated, however, the present study suggests that genetic variation may partly explain interindividual variation in the formation of CAs.

• Klíčová slova
• Chromosomal aberration, Environmental toxicology, GWAS, Genetic Polymorphism, Kinetochore Function, Mitotic checkpoint,
• MeSH
• chromozomální aberace * MeSH
• chromozomální proteiny, nehistonové genetika   MeSH
• cyklin B1 genetika   MeSH
• cyklin-dependentní kinasy genetika   MeSH
• dospělí MeSH
• frekvence genu MeSH
• jednonukleotidový polymorfismus * MeSH
• kinasa aktivující cyklin dependentní kinasy MeSH
• kinetochory metabolismus   MeSH
• kohortové studie MeSH
• kontrolní body M fáze buněčného cyklu genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• lineární modely MeSH
• odds ratio MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CCNB1 protein, human MeSH Prohlížeč
• CENPH protein, human MeSH Prohlížeč
• chromozomální proteiny, nehistonové MeSH
• cyklin B1 MeSH
• cyklin-dependentní kinasy MeSH
• kinasa aktivující cyklin dependentní kinasy MeSH
• TEX14 protein, human MeSH Prohlížeč
• transkripční faktory MeSH

Photodynamic therapy (PDT) is a useful tool against cancer and various other diseases. PDT is capable to induce different cell death mechanisms, due to the PDT evoked reactive oxygen species (ROS) production and is dose dependent. It is known that cytoskeleton is responsible for numerous cell functions, including cell division, maintenance of cell shape, their adhesion ability and movement. PDT initiated redistribution and subsequent disintegration of cytoskeletal components that precedes cell death. Here was present our results in HeLa and G361 cells subjected to sublethal PDT treatments using α,β,χ,δ porphyrin-Tetrakis (1-methylpyridinium-4-yl) p-Toluenesulfonate porphyrin (TMPyP). The photosensitizer (PS) induced transient increasing of mitotic index (MI) observable early after PDT, cell cycle arrest, microtubule (MTs) disorganization of interphase cells, aberrant mitosis and formation of rounded cells with partial loss of adherence. Some cells were partly resistant to PDT induced MTs disorganization. The differences between both cell lines to PDT response were described. This is the first evidence of TMPyP - PDT induced microtubule disorganization and the cell death mechanisms known as mitotic catastrophe and the first detail analysis of microtubule aberrations of mitotic and interphase cells in HeLa and G361 cell lines. New modification of techniques of protein immunolabeling was developed.

• Klíčová slova
• Aberrant mitosis, Cell cycle analysis, Fluorescent immunodetection, Multipolar spindle, PDT resistance,
• MeSH
• cytoskelet účinky léků   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• fotochemoterapie MeSH
• fotosenzibilizující látky farmakologie   terapeutické užití   MeSH
• HeLa buňky MeSH
• histony metabolismus   MeSH
• kontrolní body buněčného cyklu účinky léků   účinky záření   MeSH
• lidé MeSH
• mikrotubuly chemie   metabolismus   MeSH
• mitóza účinky léků   účinky záření   MeSH
• nádorové buněčné linie MeSH
• nádory farmakoterapie   MeSH
• porfyriny farmakologie   terapeutické užití   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• světlo MeSH
• viabilita buněk účinky léků   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fotosenzibilizující látky MeSH
• histony MeSH
• porfyriny MeSH
• reaktivní formy kyslíku MeSH
• tetra(4-N-methylpyridyl)porphine MeSH Prohlížeč

An efficient synthetic route for the synthesis of 2H-pyrazolo[4,3-c]pyridines, primarily varying by the substituents at the 2-, 4- and 6-positions, is described here. A Sonogashira-type cross-coupling reaction was employed to yield 3-alkynyl-1H-pyrazole-4-carbaldehydes, ethanones and propanones from the corresponding 1H-pyrazol-3-yl trifluoromethanesulfonates. Subsequent treatment of the coupling products with dry ammonia afforded a versatile library of 2H-pyrazolo[4,3-c]pyridines, which were then evaluated for their cytotoxicity against K562 and MCF-7 cancer cell lines. The most potent of these compounds displayed low micromolar GI50 values in both cell lines. Active compounds induced dose-dependent cell-cycle arrest in mitosis, as shown by flow cytometric analysis of DNA content and phosphorylation of histone H3 at serine-10. Moreover, biochemical assays revealed increased activities of caspases-3/7 in treated cells, specific fragmentation of PARP-1, and phosphorylation of Bcl-2, collectively confirming apoptosis as the mechanism of cell death.

• Klíčová slova
• Apoptosis, G2/M cell cycle arrest, Pyrazole, Structure-activity relationships,
• MeSH
• antimitotika chemická syntéza   chemie   farmakologie   MeSH
• antitumorózní látky chemická syntéza   chemie   farmakologie   MeSH
• buněčná smrt účinky léků   MeSH
• buňky K562 MeSH
• kontrolní body buněčného cyklu účinky léků   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• mitóza účinky léků   MeSH
• molekulární struktura MeSH
• proliferace buněk účinky léků   MeSH
• pyridiny chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antimitotika MeSH
• antitumorózní látky MeSH
• pyridiny MeSH

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G(1) phase to G(2) and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G(1) cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.

• Klíčová slova
• DNA damage response, Wip1 phosphatase, cell cycle, mitotic progression, γH2AX,
• MeSH
• DNA primery genetika   MeSH
• fluorescenční protilátková technika MeSH
• fosforylace MeSH
• hmotnostní spektrometrie MeSH
• kontrolní body M fáze buněčného cyklu fyziologie   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• mitóza fyziologie   MeSH
• nádorové buněčné linie MeSH
• poškození DNA * MeSH
• proteinfosfatasa 2C MeSH
• proteinfosfatasy metabolismus   MeSH
• regulace genové exprese fyziologie   MeSH
• signální transdukce fyziologie   MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH
• malá interferující RNA MeSH
• PPM1D protein, human MeSH Prohlížeč
• proteinfosfatasa 2C MeSH
• proteinfosfatasy MeSH

In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.

• Klíčová slova
• anaphase promoting complex, cell cycle, meiosis, oocytes, proTAME, spindle assembly checkpoint,
• MeSH
• anafázi podporující komplex metabolismus   MeSH
• embryo savčí účinky léků   metabolismus   MeSH
• embryonální vývoj účinky léků   MeSH
• kontrolní body M fáze buněčného cyklu * MeSH
• myši MeSH
• oocyty účinky léků   růst a vývoj   metabolismus   MeSH
• prekurzory léčiv MeSH
• skot MeSH
• tosylargininmethylester aplikace a dávkování   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• anafázi podporující komplex MeSH
• prekurzory léčiv MeSH
• tosylargininmethylester MeSH