The aim of this study was to compare the effects of DNA repair inhibitors in the context of radio-sensitization of human lung cells. The radio-sensitizing effects of NU7441 (1 mM), an inhibitor of DNA-dependent protein kinase (DNA-PK); KU55933 (10 μM), an inhibitor of ataxia-telangiectasia mutated kinase (ATM); and VE-821 (10 μM), an inhibitor of ATM-related kinase (ATR) were tested by the xCELLigence system for monitoring proliferation, fluorescence microscopy for DNA damage detection, flow-cytometry for cell cycle and apoptosis analysis and western blotting and ELISA for determination of DNA repair proteins. We employed normal human lung fibroblasts (NHLF, p53-wild-type) and non-small cell lung cancer cells (H1299, p53-negative). DNA-PK inhibition (by NU7441) in combination with ionizing radiation (IR) increased the number of double strand breaks (DSB), which persisted 72 h after irradiation in both cell lines. Additionally, NU7441 and KU55933 in combination with IR caused G2-arrest. ATR inhibitor (VE-821) together with IR markedly inhibited proliferation and induced G2/M arrest accompanied by apoptosis in H1299, but not in NHLF cells, and thus diminished DNA-repair of tumour cells but not normal lung fibroblasts. Our findings indicate that ATR inhibition could be a promising therapeutic strategy in p53-deficient lung tumours.

• MeSH
• enzymy opravy DNA antagonisté a inhibitory   genetika   MeSH
• fibroblasty účinky záření   MeSH
• kultivované buňky účinky záření   MeSH
• nádorové buňky kultivované účinky léků   účinky záření   MeSH
• nemalobuněčný karcinom plic genetika   radioterapie   MeSH
• oprava DNA účinky léků   MeSH
• proliferace buněk účinky záření   MeSH
• radiosenzibilizující látky farmakologie   izolace a purifikace   terapeutické užití   MeSH
• techniky in vitro MeSH
• Publikační typ
• práce podpořená grantem MeSH

BACKGROUND: DNA repair pathways play a major role in tumour resistance towards chemo- and radiotherapy. Therefore, inhibitors of specific DNA repair pathways might be advantageous when used in combination with DNA-damaging agents, such as ionizing radiation. This review put particular emphasis on the key DNA repair enzymes: DNA-dependent protein kinase (DNA-PK), ataxia-telangiectasia mutated kinase (ATM) and ATM-Rad3-related kinase (ATR) and their specific inhibitors in the context of radio-sensitization. RESULTS: We reviewed recent studies on novel and potent inhibitors and found evidence that inhibitors of DNA repair pathways such as small molecule inhibitors could be efficient and selective in tumour cells. Interpretation of recent literature results accompanied with implications for practice and further research are presented. CONCLUSIONS: The prospects of targeting DNA repair enzymes to treat cancer are optimistic, but future work will show if this approach has a significant in vivo efficacy, since we are still waiting for the inhibitor which would pass all phases in clinical trials. In spite of the fact that a number of drugs possess interesting synergy of radiotherapy in vitro, the future use will depend on developing compounds with improved solubility and the serum half-life. Normal tissue toxicity leading to a significant increase of radiotherapy efficiency remains a key question that might be answered only by clinical trials.

• MeSH
• ATM protein antagonisté a inhibitory   MeSH
• dvouřetězcové zlomy DNA MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• nádory terapie   MeSH
• oprava DNA fyziologie   MeSH
• proteinkinasa aktivovaná DNA antagonisté a inhibitory   MeSH
• proteinkinasa CDC2 antagonisté a inhibitory   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

We evaluated the impact of ataxia-telangiectasia mutated kinase inhibitor KU55933, DNA-dependent protein kinase inhibitor NU7441 and ataxia telangiectasia and rad3-related kinase inhibitor VE821 in human peripheral lymphocytes in vitro. The lymphocytes were divided into 5 groups: non-irradiated control, irradiated group (2 Gy) and 3 groups pretreated with inhibitors 30 min before irradiation. We used flow cytometry to evaluate phosphorylated H2AX (γ-H2AX) and cytotoxicity (Apoptest). Micronucleus assay was used to assess genotoxicity. After irradiation, γ-H2AX, incidence of micronuclei (MN), nucleoplasmatic bridges (NPBs) and nuclear buds in binuclear cells, MN in mononuclear cells and apoptosis were increased. KU55933 decreased γ-H2AX and inhibited ionizing radiation-induced cytotoxicity. NU7441 showed no effect on γ-H2AX but it significantly increased MN and NPBs in binuclear cells and apoptosis. VE821 decreased γ-H2AX, whereas genotoxicity and cytotoxicity were not affected. In conclusion, KU55933 protected lymphocytes, which might be employed to preserve the immune system during anticancer therapy. NU7441 radiosensitized lymphocytes, thus, undesirable side effects toward immune system could be expected. VE821 showed decrease of γ-H2AX with no radiosensitizing effects in our model likely due to p53 positive status, which underlies the concept of its application in p53 negative environment.

• Klíčová slova
• Účinky ionizujícího záření způsobené inhibitory ATM (KU55933), DNA-PK (NU7441) a ATR (VE821) na lymfocyty periferní krve,
• MeSH
• ATM protein účinky záření   MeSH
• biomedicínský výzkum MeSH
• financování organizované MeSH
• fosfatidylinositol-3-kinasy účinky záření   MeSH
• fosforylace imunologie   účinky záření   MeSH
• histony chemie   účinky záření   MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• ionizující záření * MeSH
• krevní a imunitní systémy cytologie   imunologie   účinky záření   MeSH
• lidé MeSH
• lymfocyty * cytologie   imunologie   účinky záření   MeSH
• statistika jako téma MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH

In this paper we describe the influence of NU7026, a specific inhibitor of DNA -dependent protein kinase, phosphoinositide 3-kinase, and AT M-kinase on molecular and cellular mechanisms triggered by ionising irradiation in human T-lymphocyte leukaemic MOLT -4 cells. We studied the effect of this inhibitor (10 μM) combined with gammaradiation (1 Gy) leading to DNA damage response and induction of apoptosis. We used methods for apoptosis assessment (cell viability count and flow-cytometric analysis) and cell cycle analysis (DNA content measurement) and we detected expression and post-translational modifications (Western blotting) of proteins involved in DNA repair signalling pathways. Pre-treatment with NU7026 resulted into decreased activation of checkpoint kinase-2 (Thr68), p53 (Ser15 and Ser392), and histone H2A.X (Ser139) 2 hours after irradiation. Subsequently, combination of radiation and inhibitor led to decreased amount of cells in G2-phase arrest and into increased apoptosis after 72 hours. Our results indicate that in leukaemic cells the pre-incubation with inhibitor NU7026 followed by low doses of ionising radiation results in radio-sensitising of MOLT -4 cells via diminished DNA repair and delayed but pronounced apoptosis. This novel approach might offer new strategies in combined treatment of leukaemia diseases.

• MeSH
• apoptóza účinky záření   MeSH
• buněčný cyklus účinky záření   MeSH
• chromony farmakologie   MeSH
• leukemie T-buněčná radioterapie   MeSH
• lidé MeSH
• morfoliny farmakologie   MeSH
• nádorové buněčné linie účinky záření   MeSH
• oprava DNA účinky záření   MeSH
• poškození DNA účinky záření   MeSH
• proliferace buněk účinky záření   MeSH
• proteinkinasa aktivovaná DNA antagonisté a inhibitory   MeSH
• radiosenzibilizující látky farmakologie   MeSH
• tolerance záření účinky léků   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem 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