Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1β (HP1β) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1β protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture.
- MeSH
- buněčné jadérko účinky léků genetika ultrastruktura MeSH
- buněčné linie MeSH
- Cajalova tělíska účinky léků genetika ultrastruktura MeSH
- chromozomální proteiny, nehistonové genetika metabolismus MeSH
- daktinomycin farmakologie MeSH
- fluorescenční barviva MeSH
- fluorescenční mikroskopie MeSH
- fotochemické procesy MeSH
- heterochromatin účinky léků genetika ultrastruktura MeSH
- histony genetika metabolismus MeSH
- inhibitory histondeacetylas farmakologie MeSH
- inhibitory syntézy proteinů farmakologie MeSH
- interfáze účinky léků genetika MeSH
- kyseliny hydroxamové farmakologie MeSH
- lidé MeSH
- messenger RNA biosyntéza MeSH
- mitóza účinky léků genetika MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Methotrexate (MTX) released from bone cement showed a useful local effect in animal models of bone tumours. However, local toxic reactions such as impaired wound healing were observed in areas surrounding the MTX-loaded implant. Therefore, we hypothesised that MTX released from bone cement would have harmful effects on human mesenchymal stem cells (MSC)-one of the basic components of bone marrow and tissue reparatory processes. Moreover, elution of MTX was calculated from implants prepared either with liquid or powdered MTX. During the 28-day incubation, the cement compounded with liquid MTX showed the highest elution rate of the drug. MTX released from pellets produced a significant decrease in proliferation of MSC as a consequence of a blockade of their cell cycle in the S/G2 phase. These findings indicate impairment of stem cell function in marginal areas surrounding the MTX-loaded cement and may help to explain problems with regeneration of tissues in these locations.
- MeSH
- buňky kostní dřeně patologie účinky léků MeSH
- interfáze účinky léků MeSH
- kostní cementy chemie MeSH
- kultivované buňky MeSH
- lidé MeSH
- methotrexát aplikace a dávkování chemie MeSH
- mezenchymální kmenové buňky patologie účinky léků MeSH
- proliferace buněk účinky léků MeSH
- protinádorové antimetabolity aplikace a dávkování chemie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.
- MeSH
- apoptóza účinky záření MeSH
- buněčné jádro enzymologie genetika metabolismus účinky léků MeSH
- buněčné linie MeSH
- buněčný cyklus účinky léků MeSH
- buňky HT-29 MeSH
- chromatin metabolismus MeSH
- chromozomální proteiny, nehistonové antagonisté a inhibitory metabolismus MeSH
- financování organizované MeSH
- histondeacetylasy metabolismus MeSH
- histony metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- inhibitory histondeacetylas MeSH
- interfáze účinky léků MeSH
- kyselina máselná farmakologie MeSH
- kyseliny hydroxamové farmakologie MeSH
- lidé MeSH
- malobuněčný karcinom enzymologie metabolismus patologie MeSH
- nádorové buněčné linie MeSH
- nádory plic enzymologie metabolismus patologie MeSH
- nádory tračníku enzymologie metabolismus patologie MeSH
- plod MeSH
- Check Tag
- lidé MeSH
Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G(1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.
- MeSH
- anafázi podporující komplex MeSH
- buněčný cyklus * MeSH
- cyklin E metabolismus MeSH
- cyklin-dependentní kinasa 2 MeSH
- cyklin-dependentní kinasy metabolismus MeSH
- DNA vazebné proteiny * MeSH
- fluorescenční protilátková technika MeSH
- interfáze účinky léků MeSH
- kinasy CDC2-CDC28 * MeSH
- komplexy ubikvitinligas * MeSH
- lidé MeSH
- ligasy * metabolismus MeSH
- makromolekulární látky MeSH
- mitóza * MeSH
- nádorové buňky kultivované MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- proteiny buněčného cyklu metabolismus MeSH
- proteiny Cdc20 MeSH
- proteiny Drosophily * MeSH
- protilátky farmakologie MeSH
- průtoková cytometrie MeSH
- replikace DNA * MeSH
- Saccharomyces cerevisiae - proteiny * MeSH
- trans-aktivátory * MeSH
- transkripční faktor DP1 MeSH
- transkripční faktory E2F MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny * MeSH
- ubikvitinligasy MeSH
- vazba proteinů MeSH
- vazebný protein 1 retinoblastomu MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
