Jaroslav Pejchal

Ataxia-telangiectasia Mutated Kinase (ATM) as a Central Regulator of Radiation-induced DNA Damage Response

Ataxia-telangiectasia mutated kinase (ATM) is a DNA damage-inducible protein kinase, which phosphorylates plethora of substrates participating in DNA damage response. ATM significance for the cell faith is undeniable, since it regulates DNA repair, cell-cycle progress, and apoptosis. Here we describe its main signalling targets and discuss its importance in DNA repair as well as novel findings linked to this key regulatory enzyme in the terms of ionizing radiation-induced DNA damage.

Ascorbic Acid: An Old Player with a Broad Impact on Body Physiology Including Oxidative Stress Suppression and Immunomodulation: A Review

Ascorbic acid is a low molecular weight antioxidant well known as anti-scorbut acting vitamin C in humans, primates and guinea pigs. This review summarizes basic data about ascorbic acid in its physiological action point of view. It is divided into biochemistry of ascorbic acid synthesis, mechanism of antioxidant action and participation in anabolism, pharmacokinetics and excretion, exogenous ascorbic acid immunomodulatory effect and participation in infectious diseases, impact on irradiation and intoxication pathogenesis, and supplementary demands. The primary intention was to consider ascorbic acid not only as an antioxidant but also as a chemical compound affecting multiple pathways with a potential beneficial impact in many diseases and processes in human body.

To live or let die: Unclear task of autophagy in the radiosensitization battle.

Radiation-induced autophagy is believed to represent a radioprotective mechanism of cancer cells. Thus, its inhibition should support radiation treatment and increase its efficacy. On the other hand, there is evidence that radiation alone or in combination with various chemical agents can induce autophagy that results into increased cell death, especially within transformed apoptosis-resistant cells. In this paper, besides description of autophagic process and its relation to cancer and radiotherapy, we compared two contradictory radiosensitization approaches that employ inhibition and induction of autophagy. In spite of the classical concept based on cytoprotective model, there is a plethora of recently developed inducers of autophagy, which indicates the future trend in radiosensitization via modulation of autophagy. Because contemporary literature is conflicting and inconsistent in this respect, we reviewed the recent studies focused on enhancement of sensitivity of cancer cells toward radiation in regard to autophagy, revealing some striking discrepancies. The deeper the knowledge, the more complex this situation is. To interpret results of various studies correctly one has to take into account the methodology of autophagy assessment and also the fact that radiosensitization might be mediated by other than intrinsic mechanisms related to autophagy. Notwithstanding, targeting autophagy remains an attractive anti-tumor strategy.

Activation of p38 MAPK and expression of TGF-β1 in rat colon enterocytes after whole body γ-irradiation

Purpose: To examine the p38 mitogen-activated protein kinase (p38) phosphorylation and transforming growth factor beta 1 (TGF-β1) expression in rat colon enterocytes after irradiation and their contribution to pathology of intestinal radiation disease. Materials and methods: Male Wistar rats were irradiated with whole body γ-radiation of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 Gy (60Co, 1.44 Gy.min–1). Samples were taken 4 and 24 h after irradiation, immunohistochemically stained, then p38 phosphorylation and TGF-β1 expression were measured in apical and cryptal enterocytes using computer image analysis. In selected groups, morphometric parameters, mitosis and apoptosis were evaluated. Results: P38 phosphorylation integrated optical density (IOD)-based levels increased 2.4-fold (p ≤ 0.01) and 3.6 to 22.8-fold (p ≤ 0.001) in apical enterocytes 4 h after 0.5 Gy and 24 h after 3–10 Gy, respectively. TGF-β1 IOD-based expression increased 3.3- to 6.9-fold (p ≤ 0.001) and 1.6- to 4.9-fold (p ≤ 0.001) in apical cells 4 h after 0.5–2, 4, 5 Gy and 24 h after 6–10 Gy, respectively. No changes were observed in crypts. Conclusions: We found a chronological and dose-dependent order of p38 activation and TGF-β1 expression in apical enterocytes. Transient up-regulation of p38 and TGF-β1 signalling observed 4 h after low-dose irradiation may participate in molecular mechanisms creating cellular over-expression in apical compartment, while persistent patterns measured 24 h after high-dose irradiation might provide protection of remaining cells in order to maintain tissue integrity.

P-glycoprotein function and expression during obstructive cholestasis in rats.

Objectives The present study was aimed at evaluation of in vivo biliary and renal excretion of rhodamine 123 (Rho123), a P-glycoprotein (P-gp) substrate, in rats during either acute or chronic cholestasis induced by bile duct obstruction (BDO). Methods The Rho123 clearance study was performed either one (BDO1) or seven (BDO7) days after BDO. Bile flow was reconstituted, and bile and urine were collected after steady-state plasma concentration of Rho123 was attained. Tissue expression of P-gp was evaluated by quantitative immunohistochemistry, and immunoblotting. Results Significant up-regulation of the liver P-gp protein was observed in acute and chronic cholestasis. Primary periportal location of P-gp was enlarged also to pericentral areas. In the kidneys, immunohistochemistry showed pancellular increase in P-gp after 1 day of BDO, which subsided after 7 days of BDO. Nevertheless, biliary and renal clearances (CLBile and CLR) of Rho123 did not reflect the induction of P-gp expression. While CLBile was reduced one day after cholestasis and restored on the seventh day, the CLR was preserved in BDO1 group and reduced in BDO7 group without change in glomerular filtration rate. In parallel, biliary and renal clearances of conjugated bilirubin were significantly reduced in both cholestatic groups compared with controls. Conclusion These findings suggest that extrahepatic cholestasis causes time-dependent changes in elimination of Rho123 which do not exactly reflect alteration of P-gp expression in the rat liver and kidney. These data may help to explain impaired elimination of P-gp substrates after short-term cholestasis that may commonly occur in clinical practice.

DNA-dependent protein kinase and its inhibition in support of radiotherapy

Abstract Purpose: Radiotherapy has been used as a treatment of almost 50% of all malignant tumors. The aim of this review is to provide a comprehensive overview of the recent knowledge in the field of molecular mechanisms of radiation-induced double-stranded breaks (DSB) repair. This paper gives particular emphasis to a key DNA repair enzyme, DNA-dependent protein kinase (DNA-PK), which plays a pivotal role in non-homologous end-joining. Furthermore, we discuss possibilities of DNA-PK inhibition and other molecular approaches employed in order to facilitate radiotherapy. Conclusions: We have reviewed the recent studies using novel potent and selective small-molecular DNA-PK inhibitors and we conclude that targeted inhibition of DNA repair proteins like DNA-PK in cancer cells, in combination with ionizing radiation, improves the efficacy of cancer therapy while minimizing side-effects of ionizing radiation. Moreover, the recent discovery of short interfering RNA (siRNA) and signal interfering DNA (siDNA)-based therapeutics, or small peptides and RNA, shows a new opportunity of selective and safe application of biological treatment. All of these approaches are believed to contribute to more personalized anti-cancer therapy.

Specific antibodies protect gamma-irradiated mice against Francisella tularensis infection

The role of antibodies in the course of Francisella tularensis (F. tularensis) infection is still a subject of debate. The understanding of the poorly described role of humoral immunity is more than important for the effort to develop effective prophylactic procedure against the infection with Francisella virulent strains. We utilized the model of gamma-irradiated mice for the studies of the protective role of anti-F. tularensis antibodies in order to partially eliminate cellular responses. The model of gamma-irradiated mice can also demonstrate the responses of immunocompromised host to intracellular bacterial infection. The gamma-irradiation by doses greater than 3 Gy completely impairs the resistance to infection and causes a disbalance of cytokine production in mice. In this study, we demonstrate that passive transfer of immune sera protected irradiated mice against subsequent infection with strains of F. tularensis subsp. holarctica. Naïve mice of BALB/c or C3H/CBi strains were subjected to passive transfer of sera obtained from immunized mice with live vaccine strain (LVS) F. tularensis LVS, F. tularensis subsp. holarctica strain 15, heat-killed F. tularensis LVS, or heat-killed strain 15 two hours before infection with lethal doses of LVS or strain 15. The passive transfer of sera obtained from immunized mice conferred full protection of naïve unirradiated as well as sublethally irradiated mice against low lethal doses of infection with F. tularensis LVS or strain 15, in all variants of the experiments. In addition, the passively protected mice that survived the primary infection with F. tularensis LVS were protected also against further secondary challenge with a highly virulent strain of F. tularensis subsp. tularensis SchuS4. Moreover, the first evidence of combination of successful passive transfer of immunity by specific antisera and subsequent active immunization of immunocompromised animals is demonstrated. In summary, we demonstrate that B cell-mediated effector responses together with the induction of T cell-mediated immunity both play an important role in naïve and also in immunocompromised mice and this fact it would be appropriate to take into the account in the design of new vaccines.

Gamma irradiation of human leukaemic cells HL-60 and MOLT-4 induces decrease in Mcl-1 and Bid, release of cytochrome c, and activation of caspase-8 and caspase-9

Purpose: Apoptosis is significantly controlled by proteins of Bcl-2 (B-cell lymphoma 2) family promoting cell death or maintaining cell survival. We selected two representatives of Bcl-2 family (anti-apoptotic Mcl-1 – myeloid cell line-1 and pro-apoptotic Bid – Bcl-2 homology domain 3 interacting death agonist), cytochrome c (cyt-c), and two initial caspases (-8 and -9) to evaluate their function in ionizing radiation (IR)-induced apoptosis in human leukaemic cell lines diverging in p53 (TP53 tumor suppressor gene) status. Materials and methods: A total of 30 μg of proteins of whole-cell lysates or 10 μg of mitochondrial protein fractions were electrophoretically separated and analyzed by Western-blotting. Results: Here we show that in both HL-60 (p53 null) and MOLT-4 (p53 wild type) leukaemic cells the amount of Mcl-1 initially increased after irradiation by sublethal but not by lethal dose and later (when apoptosis occurred) it decreased in a dose-dependent manner. Caspase-8 was cleaved and afterwards the amount of Bid decreased as it was truncated. We also found cyt-c release from the inner mitochondrial membrane space into cytoplasm to be dose-dependent and it was followed by induction of apoptosis. In the p53-null cells caspase-8 was activated prior caspase-9, whereas the cells harboring p53 exhibited a simultaneous activation of both initial caspases. Conclusion: IR induced a decrease in Mcl-1, activation of Bid, caspase-8, and -9, and release of cyt-c. Presented data indicate that both extrinsic and intrinsic apoptosis signalling pathways were activated in HL-60 and MOLT-4 cells upon exposure to IR regardless to the p53 status.

Lymphocyte subsets and their H2AX phosphorylation in response to in vivo irradiation in rats

Abstract Purpose: The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation. Materials and methods: The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24–72 hours after irradiation in a dose range of 0–5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry. Results: We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation. Conclusion: The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.

Early changes in L-arginine-nitric oxide metabolic pathways in response to the whole-body gamma irradiation of rats

Abstract Purpose: Nitric oxide (NO), a reactive radical, is formed in higher amounts from L-arginine by inducible NO synthase (iNOS) during early response to ionizing radiation presumably as a part of signal transduction pathways. This study investigated the changes in L-arginine-NO metabolic pathways within a 24-hour period after whole-body gamma irradiation of rats with the range of low to supra-lethal doses. Materials and methods: Young adult female Wistar rats received either 0–50 Gy whole-body irradiation or an intraperitoneal injection of bacterial lipopolysaccharide (LPS, 10 mg/kg). Exhaled NO was monitored using chemiluminiscence, nitrite + nitrate (NOx) and L-arginine were assayed by high-performance liquid chromatography, and expression of iNOS was determined using Western blot. Results: Irradiation resulted in a dose-dependent increase of plasma NOx to maximum levels which were 4-fold higher compared to controls (p < 0.001). The NOx levels increased less in the bronchoalveolar lavage fluid (BAL) (1.7-fold, p < 0.001) and liver homogenate (2.5-fold, p < 0.05), respectively, and were dose-independent. Exhaled NO, lung NOx, plasma and BAL L-arginine, and the expression of iNOS in lung and liver tissues of irradiated rats and controls were similar. LPS caused a considerable increase (p < 0.001) in exhaled NO (61-fold), NOx levels (plasma 34-fold, BAL 6-fold, lung 5-fold, liver 4-fold), and in iNOS expression, respectively. Conclusion: In contrast to the LPS treatment of rats, the radiation-induced changes in L-arginine-NO metabolic pathways are modest, particularly in the airways and lungs. Noninvasive measurement of exhaled NO within a 24-h period following the exposure of rats to ionizing radiation has no value for biodosimetry.