Sprouty proteins are modulators of the MAPK/ERK pathway. Amongst these, Sprouty2 (SPRY2) has been investigated as a possible factor that takes part in the initial phases of osteogenesis. However, the in vivo context has not yet been investigated and the underlying mechanisms taking place in vitro remain unknown. Therefore, in this study, the impact of Spry2 deficiency was examined in the developing tibias of Spry2 deficient (-/-) mouse. The investigation was performed when the osteogenic zone became clearly visible and when all three basic bone cells types were present. The main markers of osteoblasts, osteocytes and osteoclasts were evaluated by immunohistochemistry and RT-PCR. RT-PCR showed that the expression of Sost was 3.5 times higher in Spry2-/- than in the wild-type bone, which pointed to a still unknown mechanism of action of SPRY2 on the differentiation of osteocytes. The up-regulation of Sost was independent of Hif-1α expression and could not be related to its positive regulator, Runx2, since none of these factors showed an increased expression in the bone of Spry2-/- mice. Regarding the RANK/RANKL/OPG pathway, the Spry2-/- showed an increased expression of Rank, but no significant change in the expression of Rankl and Opg. Thanks to these results, the impact of Spry2 deletion is shown for the first time in the developing bone as a complex organ including, particularly, an effect on osteoblasts (Runx2) and osteocytes (Sost). This might explain the previously reported decrease in bone formation in postnatal Spry2-/- mice.

• MeSH
• buněčná diferenciace MeSH
• cytoplazma metabolismus   MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus   MeSH
• ligand RANK metabolismus   MeSH
• membránové proteiny genetika   fyziologie   MeSH
• myši inbrední ICR MeSH
• myši knockoutované MeSH
• myši MeSH
• osteoblasty cytologie   metabolismus   MeSH
• osteocyty cytologie   metabolismus   MeSH
• osteogeneze * MeSH
• osteoklasty cytologie   metabolismus   MeSH
• osteoprotegerin metabolismus   MeSH
• proliferace buněk MeSH
• protein-serin-threoninkinasy genetika   fyziologie   MeSH
• vývoj kostí MeSH
• vývojová regulace genové exprese * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Jednotlivé proteiny z rodiny polo-like kináz (Plk) plní rozdílné, avšak kritické funkce při regulaci buněčného cyklu a koordinují buněčnou odpověď na poškození DNA. Nejvíce prostudovaným z pěti členů této rodiny je protein Plk1. Jedná se o serin/treonin kinázu, která hraje klíčovou roli v mnoha fázích mitózy, a s její deregulací se setkáváme u různých typů nádorů, kde je její zvýšená hladina většinou asociována s horší prognózou. Z pohledu léčby je také zajímavý vztah Plk1 a proteinu p53. Nejen z těchto důvodů se Plk1 stala jedním z atraktivních cílů pro vývoj protinádorových léčiv. Nejnadějněji se nyní jeví inhibitor ATP vazebné oblasti Plk1 volasertib (BI 6727), který v doposud provedených klinických studiích prodloužil přežití pa­cientů s akutní myeloidní leukemií a nyní je testován v klinické studii fáze III. Ve fázi preklinického testování se nachází také několik inhibitorů polo‑box domény (druhého možného místa inhibice polo‑like kináz), které by měly zajistit větší specifitu vůči Plk1.

Individual proteins from polo-like kinase (Plk) family fulfil different but critical functions in regulating cell cycle and coordinate cell response to DNA damage. The most studied one from this five member family is Plk1. It is a serine/threonine kinase that plays a pivotal role in many aspects of mitosis and its deregulation is common in various tumor types where the elevated level is mostly associated with worse prognosis. From the therapeutical point of view, intertwin­ed relationship between Plk1 and p53 protein is very interesting and will be discussed. Not only for these reasons, Plk1 has become an attractive target for anti‑tumor drug development. The most promising seems to be ATP binding site inhibitor Volasertib (BI 6727) which provided a survival benefit for patients with acute myeloid leukemia and is now tested in phase III clinical trial. A new generation of Plk1 inhibitors that target the second druggable domain of Plk1, the polo-box domain, is currently being tested preclinically and are believed to improve Plk1 specificity. Key words: polo like kinase 1 – tumor suppressor protein p53 – ATP competitive inhibitors – polo-box domain – drug evaluation studies This study was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101), MEYS – NPS I – LO1413, MH CZ – DRO (MMCI, 00209805) and BBMRI_CZ (LM2010004). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 9. 4. 2015 Accepted: 19. 6. 2015

• Klíčová slova
• polo like kináza 1, polo box doména, ATP kompetitivní inhibitory, volasertib,
• MeSH
• adenosintrifosfát chemie   MeSH
• antitumorózní látky * farmakologie   chemie   MeSH
• buněčný cyklus fyziologie   MeSH
• lidé MeSH
• nádorová transformace buněk MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• nádory farmakoterapie   metabolismus   patofyziologie   MeSH
• objevování léků MeSH
• proliferace buněk účinky léků   MeSH
• protein-serin-threoninkinasy * antagonisté a inhibitory   fyziologie   genetika   metabolismus   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   fyziologie   genetika   metabolismus   MeSH
• protoonkogenní proteiny antagonisté a inhibitory   fyziologie   genetika   metabolismus   MeSH
• pteridiny farmakologie   terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.

• MeSH
• DNA metabolismus   MeSH
• endonukleasy chemie   genetika   metabolismus   MeSH
• enzymy opravy DNA chemie   genetika   metabolismus   MeSH
• intracelulární signální peptidy a proteiny fyziologie   MeSH
• lysin metabolismus   MeSH
• mutace MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• protein-serin-threoninkinasy fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   fyziologie   MeSH
• sumoylace * MeSH
• ubikvitinligasy fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.

• MeSH
• aktivace enzymů účinky léků   MeSH
• choriogonadotropin farmakologie   MeSH
• HeLa buňky MeSH
• kultivované buňky MeSH
• lidé MeSH
• meióza účinky léků   genetika   fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• oocyty enzymologie   metabolismus   fyziologie   MeSH
• oogeneze účinky léků   genetika   fyziologie   MeSH
• organizační centrum mikrotubulů účinky léků   metabolismus   MeSH
• protein-serin-threoninkinasy genetika   metabolismus   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

This review describes a drug target for cancer therapy, family of phosphatidylinositol-3 kinase related kinases (PIKKs), and it gives a comprehensive review of recent information. Besides general information about phosphatidylinositol-3 kinase superfamily, it characterizes a DNA-damage response pathway since it is monitored by PIKKs.

• MeSH
• buněčný cyklus MeSH
• DNA vazebné proteiny MeSH
• fosfatidylinositol-3-kinasy třídy I MeSH
• fosfatidylinositol-3-kinasy třídy II MeSH
• fosfatidylinositol-3-kinasy třídy III MeSH
• fosfatidylinositol-3-kinasy * fyziologie   genetika   MeSH
• fosfatidylinositol-4-fosfát-3-kinasa MeSH
• ionizující záření * MeSH
• lidé MeSH
• nádorové supresorové proteiny fyziologie   genetika   MeSH
• nádorový supresorový protein p53 fyziologie   genetika   MeSH
• nádory genetika   radioterapie   MeSH
• oprava DNA genetika   MeSH
• poškození DNA * účinky záření   MeSH
• protein-serin-threoninkinasy fyziologie   genetika   MeSH
• proteiny buněčného cyklu fyziologie   genetika   MeSH
• radioterapie škodlivé účinky   MeSH
• teleangiektatická ataxie genetika   MeSH
• TOR serin-threoninkinasy fyziologie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: DeltaNp63alpha is an epithelial progenitor cell marker that maintains epidermal stem cell self-renewal capacity. Previous studies revealed that UV-damage induced p53 phosphorylation is confined to DeltaNp63alpha-positive cells in the basal layer of human epithelium. RESULTS: We now report that phosphorylation of the p53 tumour suppressor is positively regulated by DeltaNp63alpha in immortalised human keratinocytes. DeltaNp63alpha depletion by RNAi reduces steady-state ATM mRNA and protein levels, and attenuates p53 Serine-15 phosphorylation. Conversely, ectopic expression of DeltaNp63alpha in p63-null tumour cells stimulates ATM transcription and p53 Serine-15 phosphorylation. We show that ATM is a direct DeltaNp63alpha transcriptional target and that the DeltaNp63alpha response element localizes to the ATM promoter CCAAT sequence. Structure-function analysis revealed that the DeltaNp63-specific TA2 transactivation domain mediates ATM transcription in coordination with the DNA binding and SAM domains. CONCLUSIONS: Germline p63 point mutations are associated with a range of ectodermal developmental disorders, and targeted p63 deletion in the skin causes premature ageing. The DeltaNp63alpha-ATM-p53 damage-response pathway may therefore function in epithelial development, carcinogenesis and the ageing processes.

• MeSH
• ATM protein MeSH
• DNA vazebné proteiny * genetika   fyziologie   MeSH
• fosforylace MeSH
• genetická transkripce * fyziologie   MeSH
• lidé MeSH
• nádorové supresorové proteiny * chemie   genetika   fyziologie   MeSH
• promotorové oblasti (genetika) MeSH
• protein-serin-threoninkinasy * genetika   fyziologie   MeSH
• proteiny buněčného cyklu * genetika   fyziologie   MeSH
• serin * metabolismus   MeSH
• trans-aktivátory * chemie   fyziologie   MeSH
• transformované buněčné linie MeSH
• transkripční faktory MeSH
• Check Tag
• lidé MeSH

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G(2) and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G(2) to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6-treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Overexpression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.

• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• blastodisk metabolismus   MeSH
• buněčný cyklus fyziologie   genetika   MeSH
• buňky NIH 3T3 MeSH
• cyklin-dependentní kinasy fyziologie   metabolismus   MeSH
• financování organizované MeSH
• HeLa buňky MeSH
• lidé MeSH
• meióza fyziologie   genetika   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty enzymologie   fyziologie   MeSH
• organizační centrum mikrotubulů metabolismus   MeSH
• protein-serin-threoninkinasy fyziologie   genetika   metabolismus   MeSH
• protoonkogenní proteiny c-akt fyziologie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

Complete or partial inability to sense and repair DNA damage increases the risk of developing cancer. The ataxia telangiectasia mutated (ATM) protein kinase has a crucial role in response to DNA double-strand breaks. Hereditary mutations in the ATM gene are the cause of a rare genomic instability syndrome ataxia telangiectasia (AT) characterized, among others, by elevated cancer risk. Although clear in homozygotes, numerous studies have failed to find a link between heterozygotes and cancer. However, there is increasing evidence that ATM heterozygotes have an increased risk of developing breast cancer. First, epidemiological studies conferred an increased risk of breast cancer among AT relatives. Second, in vitro studies of heterozygous cells provide strong evidence of hyperradiosensitivity. Third, some clinical studies found an increased frequency of ATM mutations among high-risk breast cancer families.

• MeSH
• DNA vazebné proteiny fyziologie   MeSH
• financování organizované MeSH
• lidé MeSH
• mutace MeSH
• nádorové supresorové proteiny fyziologie   MeSH
• nádory prsu etiologie   MeSH
• protein-serin-threoninkinasy fyziologie   MeSH
• proteiny buněčného cyklu fyziologie   MeSH
• teleangiektatická ataxie genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.

• MeSH
• ATM protein MeSH
• buněčné jádro metabolismus   MeSH
• buněčný cyklus * MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• DNA vazebné proteiny fyziologie   chemie   MeSH
• fosforylace MeSH
• HeLa buňky MeSH
• jaderné proteiny chemie   metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové supresorové proteiny fyziologie   chemie   MeSH
• poškození DNA * MeSH
• protein-serin-threoninkinasy fyziologie   chemie   metabolismus   MeSH
• proteinkinasy metabolismus   MeSH
• proteiny buněčného cyklu fyziologie   chemie   metabolismus   MeSH
• replikační protein A chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH

Checkpoint adaptation was originally defined in yeast as the ability to divide despite the presence of damaged DNA. An important unanswered question is whether checkpoint adaptation also occurs in human cells. Here, we show that following the ionizing radiation-induced G(2) checkpoint, human osteosarcoma cells entered mitosis with gamma-H2AX foci, a marker for unrepaired DNA double-strand breaks. Exit from the G(2) checkpoint was accelerated by inhibiting the checkpoint kinase 1 (Chk1) and delayed by overexpressing wild-type Chk1 or depleting the Polo-like kinase 1 (Plk1). Chk1 and Plk1 controlled this process, at least partly, via independent signaling pathways. Our results suggest that human cells are able to exit the checkpoint arrest and divide before the damage has been fully repaired. Such cell division in the presence of damaged DNA may be detrimental for genetic stability and could potentially contribute to cancer development.

• MeSH
• fyziologická adaptace MeSH
• G2 fáze * účinky záření   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• poškození DNA MeSH
• protein-serin-threoninkinasy * fyziologie   MeSH
• proteinkinasy * fyziologie   MeSH
• proteiny buněčného cyklu * fyziologie   MeSH
• protoonkogenní proteiny * fyziologie   MeSH
• Check Tag
• lidé MeSH