Q106985462
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Here, we compared the effects of inhibitors of three phosphatidylinositol-3-kinase-related kinases, ATM, ATR a DNA-PK, on radiosensitization of cervical carcinoma cells. We demonstrated that DNA-PK inhibitor NU7441 enhanced phosphorylation of Chk1 and Chk2 kinases 2 h after irradiation of HeLa cells at a dose of 8 Gy in contrast to ATM kinase inhibitor KU55933, which completely blocked the Chk2 kinase phosphorylation on threonine 68, and ATR kinase inhibitor VE-821, which blocked the Chk1 kinase phosphorylation on serine 345. Most HeLa cells were accumulated in G2 phase of the cell cycle 24 h after irradiation at a high dose of 15 Gy, which was even potentiated after adding the inhibitors NU7441 and KU55933. Compared to all other irradiated groups, inhibitor VE-821 increased the number of cells in S phase and reduced the number of cells in G2 phase 24 h after irradiation at the high dose of 15 Gy. HeLa cells entered the mitotic cycle with unrepaired DNA, which resulted in cell death and the radiosensitizing effect of VE-821. Short-term application of the inhibitors (2 h before and 30 min after the irradiation by the dose of 8 Gy) significantly decreased the colony-forming ability of HeLa cells. Using real-time monitoring of cell proliferation by the xCELLigence system we demonstrated that while the radiosensitizing effect of VE-821 (ATR inhibitor) is manifested early after the irradiation, the radiosensitizing effect of KU55933 (ATM inhibitor) and NU7441 (DNA-PK inhibitor) is only observed as late as 72 h after the irradiation.
- MeSH
- ATM protein antagonisté a inhibitory metabolismus MeSH
- buněčné klony MeSH
- buněčný cyklus účinky léků MeSH
- HeLa buňky MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- nádory děložního čípku enzymologie patologie MeSH
- počet buněk MeSH
- proliferace buněk účinky léků MeSH
- proteinkinasa aktivovaná DNA antagonisté a inhibitory metabolismus MeSH
- radiosenzibilizující látky farmakologie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie 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
- MeSH
- antigeny CD27 účinky záření MeSH
- B-lymfocyty * účinky záření MeSH
- celková dávka radioterapie MeSH
- dávka záření * MeSH
- fosforylace MeSH
- frakcionace dávky záření MeSH
- histony MeSH
- imunoglobulin M účinky záření MeSH
- imunoglobulinové izotypy účinky záření MeSH
- ionizující záření MeSH
- klinické protokoly normy MeSH
- lidé MeSH
- modely u zvířat MeSH
- prasata krev MeSH
- průtoková cytometrie * MeSH
- radiometrie MeSH
- referenční standardy MeSH
- T-lymfocyty účinky záření MeSH
- tolerance záření MeSH
- ukládání a vyhledávání informací MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Purpose: The purpose of our study is to examine the effect of acetyl-L-carnitine (ALC) pretreatment on radiation pneumonitis and L-arginine-nitric oxide (NO) metabolic pathway after local chest irradiation. Materials and methods: Female Wistar rats were pretreated with either phosphate buffer saline or ALC and locally irradiated by a dose of 15 Gy onto the chest. Samples were collected 7 weeks after irradiation. Exhaled NO was monitored using chemiluminiscence method. In plasma and bronchoalveolar lavage (BAL) fluid, nitrite/nitrate (NOx) and malondialdehyde were measured by high-performance liquid chromatography. Total protein concentration was assayed colorimetrically. In lungs, inducible and endothelial nitric oxide synthase (iNOS, eNOS), arginase (Arg) I and II, cationic amino acid transporter (CAT) 1, 2 and 3 relative mRNA expression were measured by RT-PCR. Expression of Arg I and II was also evaluated by Western blot. Histochemical analysis was used to measure air/tissue ratio and neutrophil infiltration. Results:ALC pretreatment increased survival rate of irradiated rats. It also decreased NOx concentration in BAL, while in lungs, we observed increased air/tissue ratio, reduced neutrophil infiltration, decreased CAT 1 and CAT 3 mRNA expression, and reduced Arg I and Arg II expression. Conclusions:ALC pretreatment improves clinical prognosis and seems to reduce intensity of post-radiation changes on a molecular and microscopic level.
- MeSH
- acylkarnitin * aplikace a dávkování farmakologie MeSH
- arginin * metabolismus účinky záření MeSH
- bronchoalveolární lavážní tekutina chemie MeSH
- dechové testy metody MeSH
- hrudník účinky záření MeSH
- modely nemocí na zvířatech MeSH
- oxid dusnatý metabolismus účinky záření MeSH
- plíce * účinky záření MeSH
- potkani Wistar MeSH
- radiační pneumonitida farmakoterapie MeSH
- radioprotektivní látky * aplikace a dávkování MeSH
- Check Tag
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem 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
