BACKGROUND: Increased expression of PCNA gene was detected in chronic myeloid leukemia (CML) patients in our laboratory. The gene may participate in the disease development. The aim of the study was to develop appropriate conditions for PCNA-siRNA transfection into K562 and MOLM-7 cell lines (which both have the up-regulated PCNA gene) and to silence the increased expression. METHODS AND RESULTS: Key parameters of successful siRNA delivery into the cells are type and quantity of transfection reagent, cells and siRNA concentration or cultivation time before an expression analysis. Transfection reagents ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) and siPORT Amine (Ambion) were tested. Transfection efficiency was monitored by fluorescence microscopy of fluorescein labeled siRNA. Gene silencing was determined at mRNA level by real-time PCR and at protein level by western blots. As the most suitable reagent was chosen Oligofectamine, which achieved 70% decrease of PCNA mRNA level. Further, 50 nM siRNA concentration, 1 x 10(6) cells/ml and amount of Oligofectamine 4 microl per 1 ml of transfected cells were selected. The best cultivation time after siRNA delivery was 48 h. CONCLUSIONS: Based on the results of this study, transfection method for siRNA delivery into the K562 and MOLM-7 cell lines was proposed. The procedure can be transferred also on further selected genes potentially involved in CML and afterwards it will be possible to monitor the impact of siRNA-inhibition on expression profile. In the future siRNAs against some over-expressed genes would be used for gene therapy of CML.

• MeSH
• buňky K562 MeSH
• lidé MeSH
• malá interferující RNA * MeSH
• nádorové buněčné linie MeSH
• proliferační antigen buněčného jádra genetika   MeSH
• regulace genové exprese u leukemie * MeSH
• technika přenosu genů * MeSH
• transfekce * MeSH
• umlčování genů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá interferující RNA * MeSH
• proliferační antigen buněčného jádra MeSH

DNA repair is a complex process that prevents genomic instability. Many proteins play fundamental roles in regulating the optimal repair of DNA lesions. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after injury. Here, in very early S-phase, we show that the fluorescence intensity of mCherry-tagged PCNA after local micro-irradiation was less than the fluorescence intensity of non-irradiated mCherry-PCNA-positive replication foci. However, PCNA protein accumulated at locally irradiated chromatin in very late S-phase of the cell cycle, and this effect was more pronounced in the following G2 phase. In comparison to the dispersed form of PCNA, a reduced mobile fraction appeared in PCNA-positive replication foci during S-phase, and we observed similar recovery time after photobleaching at locally induced DNA lesions. This diffusion of mCherry-PCNA in micro-irradiated regions was not affected by cell cycle phases. We also studied the link between function of PCNA and A-type lamins in late S-phase. We found that the accumulation of PCNA at micro-irradiated chromatin is identical in wild-type and A-type lamin-deficient cells. Only micro-irradiation of the nuclear interior, and thus the irradiation of internal A-type lamins, caused the fluorescence intensity of mCherry-tagged PCNA to increase. In summary, we showed that PCNA begins to play a role in DNA repair in late S-phase and that PCNA function in repair is maintained during the G2 phase of the cell cycle. However, PCNA mobility is reduced after local micro-irradiation regardless of the cell cycle phase.

• Klíčová slova
• DNA repair, Lamins, Micro-irradiation, PCNA, S/G2 phases, rDNA,
• MeSH
• buněčné dělení genetika   fyziologie   MeSH
• buněčné jádro metabolismus   MeSH
• buněčný cyklus genetika   fyziologie   MeSH
• chromatin genetika   metabolismus   MeSH
• G2 fáze genetika   fyziologie   MeSH
• oprava DNA genetika   fyziologie   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• S fáze genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• proliferační antigen buněčného jádra MeSH

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.

• MeSH
• buněčné jádro metabolismus   MeSH
• folikulární buňky cytologie   metabolismus   MeSH
• imunohistochemie MeSH
• oocyty cytologie   metabolismus   MeSH
• prasata MeSH
• proliferace buněk * MeSH
• proliferační antigen buněčného jádra biosyntéza   MeSH
• regulace genové exprese fyziologie   MeSH
• thekální buňky cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proliferační antigen buněčného jádra MeSH

The expression of PCNA was proved in some organs of human fetuses aged from 10 to 25 weeks of the intrauterine development. The distinct expression of PCNA was demonstrated in the nuclei of the myoblasts of the ventricles and atria of the fetal heart. The high expression of PCNA was found in the nuclei of the epithelial cells of the fetal bronchial tree, surrounding mesenchyme and in the nuclei of the endothelial cells of the primitive vessels. The highest expression of PCNA was demonstrated in the nuclei of the cells of the "neogene zone" of the primitive kidney. The distinct expression of PCNA exhibited the nuclei of the cells of the surface zone of the primitive adrenal cortex and the cells of the primitive islets of the adrenal medulla. The expression of PCNA was demonstrated in the primitive hepatocytes, endothelial cells of the liver sinusoids and epithelial cells of the bile ducts and in the epithelial cells of the intestinal anlage.

• MeSH
• imunoenzymatické techniky MeSH
• lidé MeSH
• plod imunologie   MeSH
• proliferační antigen buněčného jádra analýza   MeSH
• techniky in vitro MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proliferační antigen buněčného jádra MeSH

The immunolocalization of cyclin/PCNA in synchronized 3T3 cells was performed with human autoantibodies using an immunogold technique performed on thin cryosections. Previous immunofluorescent studies demonstrated that the DNA replication sites correspond to the localization of bound cyclin. We have found that in the early periods of S phase, the DNA replication sites (or sites potentially ready for the replication during the hydroxyurea DNA synthesis block) are situated in the perichromatin region and correspond to clustered gold particles present frequently over a morphologically distinct small nuclear area. Heavily labeled chromocenters, including perinucleolar condensed chromatin, exhibiting several such distinct areas were found in later periods of S phase.

• MeSH
• buněčné jádro metabolismus   ultrastruktura   MeSH
• buněčné linie MeSH
• hydroxymočovina farmakologie   MeSH
• imunohistochemie MeSH
• jaderné proteiny metabolismus   MeSH
• kompartmentace buňky MeSH
• myši MeSH
• proliferační antigen buněčného jádra MeSH
• replikace DNA * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydroxymočovina MeSH
• jaderné proteiny MeSH
• proliferační antigen buněčného jádra MeSH

Analysis of expression of the proliferating cell nuclear antigen (PCNA) was used to determine the presumed hyperplastic character of morphological changes in the rat thyroid evoked by bromide administration. Male rats fed by a standard diet with determined iodine and bromine content were given potassium bromide. Control animals received no bromide. Experimental animals were given 10, 50 or 100 mg Br- per 11 drinking water for 16 and 66 days, or 100, 200, 400 mg Br-/l drinking water for 133 days. The thyroids of treated animals showed activation of growth of the epithelial follicular component as well as diffuse and focal microfollicular rearrangement of the parenchyma with higher follicular cells accompanied by a decrease of the amount of colloid even at low bromine concentrations (10-100 mg Br-/l drinking water). Using the PCNA-LI index (PCNA-positive nuclei.100/total number of follicular cell nuclei in the section), immunohistochemical analysis of PCNA in the nuclei of the follicular cells was carried out in parrafin sections. The index was significantly higher in bromide exposed animals (P < 0.01) and correlated well with the histological changes, with bromide concentration and with a increased mitotic activity of the follicular cells. PCNA analysis showed that morphological changes resembling a parenchymatic goitre reflect a microfollicular rearrangement of the thyroid of rats exposed to bromide and have the character of hyperplasia owing to the increased mitotic activity of the follicular epithelium.

• MeSH
• bromidy toxicita   MeSH
• epitel účinky léků   patologie   MeSH
• hyperplazie MeSH
• imunohistochemie MeSH
• krysa rodu Rattus MeSH
• mitóza MeSH
• proliferační antigen buněčného jádra biosyntéza   MeSH
• štítná žláza účinky léků   patologie   MeSH
• struma chemicky indukované   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bromidy MeSH
• proliferační antigen buněčného jádra MeSH

Srs2 plays many roles in DNA repair, the proper regulation and coordination of which is essential. Post-translational modification by small ubiquitin-like modifier (SUMO) is one such possible mechanism. Here, we investigate the role of SUMO in Srs2 regulation and show that the SUMO-interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation of SIM in asrs2ΔPIMstrain leads to a decrease in recombination, indicating a pro-recombination role of SUMO. Thus SIM has an ambivalent function in Srs2 regulation; it not only mediates interaction with SUMO-PCNA to promote the anti-recombination function but it also plays a PCNA-independent pro-recombination role, probably by stimulating the formation of recombination complexes. The fact that deletion of PIM suppresses the phenotypes of Srs2 lacking SIM suggests that proper balance between the anti-recombination PCNA-bound and pro-recombination pools of Srs2 is crucial. Notably, sumoylation of Srs2 itself specifically stimulates recombination at the rDNA locus.

• Klíčová slova
• DNA repair, homologous recombination, proliferating cell nuclear antigen (PCNA), protein-protein interaction, small ubiquitin-like modifier (SUMO),
• MeSH
• aminokyselinové motivy MeSH
• DNA fungální genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• oprava DNA fyziologie   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• protein SUMO-1 genetika   metabolismus   MeSH
• rekombinace genetická fyziologie   MeSH
• ribozomální DNA genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• sumoylace fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA fungální MeSH
• DNA-helikasy MeSH
• proliferační antigen buněčného jádra MeSH
• protein SUMO-1 MeSH
• ribozomální DNA MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.

• MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA-polymerasa II genetika   metabolismus   MeSH
• DNA-polymerasa III genetika   metabolismus   MeSH
• homologní rekombinace * MeSH
• mutace genetika   MeSH
• nestabilita genomu MeSH
• oprava DNA genetika   účinky záření   MeSH
• poškození DNA genetika   účinky záření   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• protein SUMO-1 genetika   metabolismus   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• replikace DNA genetika   účinky záření   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sumoylace MeSH
• ultrafialové záření škodlivé účinky   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• DNA-polymerasa II MeSH
• DNA-polymerasa III MeSH
• proliferační antigen buněčného jádra MeSH
• protein SUMO-1 MeSH
• RAD51 protein, S cerevisiae MeSH Prohlížeč
• rekombinasa Rad51 MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SRS2 protein, S cerevisiae MeSH Prohlížeč

Cell proliferation in developing tooth germs has been studied particularly using bromodeoxyuridine (BrdU) incorporation into growing tooth primordia and by counting and three-dimensional (3D) reconstruction of mitoses in serial sections of developing teeth. PCNA has been proposed as an alternative marker of proliferation activity. The aim of our study was to detect immunohistochemically locations of PCNA-positive cells in developing tooth germs of Microtus agrestis (Rodentia). PCNA expression could be distinguished in oral epithelium and mesenchyme before first signs of dental lamina elevation. During bud, cap, and bell stages, positive immunostaining could be observed at defined sites in enamel organ, tooth papilla, and dental follicle. Rudimental tooth germs of the upper diastema, enamel knots, and inner enamel epithelium at day of ontogeny 18 and 19 showed negative reaction. PCNA marks cycling and early G0 cells and can be used successfully as a proliferation marker even in collection material.

• MeSH
• Arvicolinae embryologie   metabolismus   MeSH
• dentice MeSH
• embryo savčí metabolismus   MeSH
• imunohistochemie MeSH
• odontogeneze fyziologie   MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• zubní zárodek embryologie   MeSH
• zuby embryologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proliferační antigen buněčného jádra MeSH

BACKGROUND: Proper DNA replication is essential for faithful transmission of the genome. However, replication stress has serious impact on the integrity of the cell, leading to stalling or collapse of replication forks, and has been determined as a driving force of carcinogenesis. Mus81-Mms4 complex is a structure-specific endonuclease previously shown to be involved in processing of aberrant replication intermediates and promotes POLD3-dependent DNA synthesis via break-induced replication. However, how replication components might be involved in this process is not known. RESULTS: Herein, we show the interaction and robust stimulation of Mus81-Mms4 nuclease activity by heteropentameric replication factor C (RFC) complex, the processivity factor of replicative DNA polymerases that is responsible for loading of proliferating cell nuclear antigen (PCNA) during DNA replication and repair. This stimulation is enhanced by RFC-dependent ATP hydrolysis and by PCNA loading on the DNA. Moreover, this stimulation is not specific to Rfc1, the largest of subunit of this complex, thus indicating that alternative clamp loaders may also play a role in the stimulation. We also observed a targeting of Mus81 by RFC to the nick-containing DNA substrate and we provide further evidence that indicates cooperation between Mus81 and the RFC complex in the repair of DNA lesions generated by various DNA-damaging agents. CONCLUSIONS: Identification of new interacting partners and modulators of Mus81-Mms4 nuclease, RFC, and PCNA imply the cooperation of these factors in resolution of stalled replication forks and branched DNA structures emanating from the restarted replication forks under conditions of replication stress.

• Klíčová slova
• Mus81 complex, Proliferating cell nuclear antigen, Recombination, Replication, Replication factor C,
• MeSH
• "flap" endonukleasy genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• rekombinace genetická MeSH
• replikace DNA MeSH
• replikační protein C genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DNA vazebné proteiny MeSH
• endonukleasy MeSH
• MMS4 protein, S cerevisiae MeSH Prohlížeč
• MUS81 protein, S cerevisiae MeSH Prohlížeč
• POL30 protein, S cerevisiae MeSH Prohlížeč
• proliferační antigen buněčného jádra MeSH
• replikační protein C MeSH
• Saccharomyces cerevisiae - proteiny MeSH