Effect of ADP-Ribosyl Transferase Inhibitors on the Survival of Human Lymphocytes after Exposure to Different DNA-Damaging Agents (original) (raw)
Related papers
Physics of Particles and Nuclei Letters, 2011
The influence that inhibitors of repair and replicative DNA synthesis, 1 β D arabinofuranosyl cytosine and hydroxyurea, have on the formation and repair kinetics of double strand breaks (DSBs) in peripheral human blood lymphocytes under the influence of radiation with a different linear energy transfer (LET) (gamma quanta and accelerated heavy ions) is studied. It is demonstrated that lithium and boron ions with LETs of 20 and 40 keV/μm, respectively, possess higher biological effectiveness with respect to the DNA DSB induction criterion. The value of the relative biological effectiveness of accelerated lithium and boron ions is 1.5 ± 0.1 and 1.6 ± 0.1, respectively. It is found that, upon cell irradiation by gamma quanta in the absence of inhibitors, efficient DNA DSB repair is observed during incubation. Under the conditions of cell incubation and in the presence of inhibitors, some growth in the number of DNA DSBs, rather than a reduc tion, is observed after 5 h incubation. In the case of the action of accelerated boron ions (as well as gamma quanta), under normal conditions, the efficient repair of induced DNA lesions takes place. Unlike the action of gamma quanta, in the case of cell incubation in the presence of radiomodifiers, the number of induced DNA DSBs falls. These results may testify to the fact that the repair of double strand DNS breaks takes place under the action of ionizing radiation with a different LET on mammalian cells in the presence of DNA syn thesis inhibitors Ara C and HU. It is concluded that, for cells subject to gamma irradiation, no DNA DSB repair is observed due to the large contribution of single strand incision DNA breaks formed in the postradi ation period in the course of excision nucleotide repair.
International Journal of Radiation Biology, 2005
The purpose was to compare the radiation-induced apoptosis in human lymphocytes with DNA-loop relaxation and DNA damage as a function of radiation dose and time after exposure. Morphological changes were analysed by staining with fluorescent dyes and apoptotic fragmentation of DNA with conventional agarose gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and alkaline comet assay. Viability was estimated by trypan blue assay. The levels of protein p53 (TP53) were determined with Western blot. Relaxation of DNA-loops was analysed by the method of anomalous viscosity time dependence (AVTD) and neutral comet assay. Induction and repair of double-strand breaks (DSB) was studied by PFGE and by immunostaining of the TP53 binding protein 1 (53BP1). At various time points of apoptosis, there was a linear dose dependence for all apoptotic end-points up to 1 -2 Gy followed by a plateau at higher doses. Immediately after irradiation, relaxation of DNA-loops due to strand breaks was observed. This relaxation had a similar dose -response with saturation at 2 -3 Gy. This dose induced approximately one single-strand break (SSB) per 2 Mb of DNA, a value close to the average size of DNA-loops in resting lymphocytes. Similar saturations in dose -responses for apoptosis and DNA-loop relaxation were also observed if cells were treated by camptothecin (CPT) or etoposide VP-16, drugs that relax DNA-loops by induction of SSB and DSB, respectively. The PFGE data showed that the vast majority of DSB were repaired within few hours after irradiation. However, approximately 1.4 foci/Gy/cell, that corresponded to around 3.5% of initial DSB, remained in cells even 24 h after irradiation as measured with immunostaining. The probability to produce one or more than one residual foci per cell was calculated. Radiation at 2 -3 Gy induced at least one residual 53BP1 focus per cell. The doseresponses for DNA-loop relaxation, induction of at least one residual 53BP1 foci per cell and apoptosis saturated at 2 -3 Gy. The correlation between dose -responses obtained suggested that the DSB in residual foci and relaxation of DNA-loops may be linked to induction of radiation-induced apoptosis in lymphocytes.
European Journal of Biochemistry, 1997
Poly(ADP-ribosy1)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADPribose) polymerase (PARP), an enzyme which uses NAD' as substrate. Binding of PARP to DNA singlestrand or double-strand breaks leads to enzyme activation. Inhibition of poly(ADP-ribose) formation impairs the cellular recovery from DNA damage. Here we describe stable transfectants of the Chinese hamster cell line C060 that constitutively overexpress human PARP (COCF clones). Immunofluorescence analysis of y-irradiation-stimulated poly(ADP-ribose) synthesis revealed consistently larger fractions of cells positive for this polymer in the COCF clones than in control clones, which failed to express human PARP. HPLC-based quantitative determination of in vivo levels of poly(ADP-ribose) confirmed this result and revealed that the basal polymer levels of undamaged cells were significantly higher in the COCF clones. The COCF clones were sensitized to the cytotoxic effects of y irradiation compared with control transfectants and parental cells. This effect could not be explained by depletion of cellular NAD' or ATP pools. Together with the well-known cellular sensitization by inhibition of poly(ADP-ribosyl)ation, our data lead us to hypothesize that an optimal level of cellular poly(ADP-ribose) accumulation exists for the cellular recovery from DNA damage.
Dna Repair, 2005
Poly(ADP-ribosyl)ation is one of the first responses to DNA damage in mammals. Although it is involved in base excision repair, its exact role has not been ascertained yet. Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 mediate most of the poly(ADP-ribosyl)ation response in mammals and are well conserved in evolution. Their respective homologues PME-1 and PME-2 are found in the nematode Caenorhabditis elegans, a well-known genetically tractable model currently used in DNA damage response research. Here we report the functional analysis of PME-1 and PME-2 in presence of DNA damage. Worms irradiated with high doses of ionizing radiations displayed a sharp drop in their NAD + content immediately after treatment, and a biphasic increase in poly(ADP-ribose). The physiological importance of the poly(ADP-ribosyl)ation response was highlighted when worms were preincubated with mammalian PARP inhibitors (3AB, DHQ, PJ34) and irradiated. The embryonic survival rate of the progeny was significantly decreased in a dose-dependent manner. The inhibitor 3AB had a weak effect on embryonic survival, followed closely by DHQ. However, PJ34, a member of the phenantridinone family, was very effective even when used at low concentration (100 nM). In vitro PARP assay using recombinant PME-1 and PME-2 showed a similar pattern of inhibition where 3AB and DHQ were weak inhibitors, and PJ34 a stronger one. Inhibitors affect mostly the poly(ADP-ribose) polymers elongation at high concentrations. These results suggest that poly(ADP-ribosyl)ation in response to DNA damage is an ancient and very important biochemical process protecting DNA from deleterious modification.
Physics of Particles and Nuclei Letters
With the use of the DNA comet assay and immunocytochemical staining, regularities have been studied in the induction and repair of DNA double-strand breaks(DSBs) in human cells after exposure to 60 Co γ-rays and accelerated heavy ions with different linear energy transfer (LET) in the presence of the DNA repair inhibitors cytosine arabinoside and hydroxyurea. It is shown that for heavy ions the agents' modifying effect decreases with increasing particles' LET. The approach involving DNA synthesis inhibitors used in this study allows an estimation of the proportion of enzymatic DNA DSBs in total DSB yield after exposure to ionizing radiations of different quality.
Modification of radiation-induced apoptosis in radiation- or hyperthermia-adapted human lymphocytes
Biochemistry and Cell Biology, 1994
We have investigated the influence of the cellular adaptive response to ionizing radiation on radiation-induced apoptosis in human cells. The adaptive response is believed to be a protective mechanism that confers resistance to the detrimental effects of ionizing radiation and that can be induced by different agents, including hyperthermia and radiation. We have used fluorescence analysis of DNA unwinding (FADU) to assay the induction of apoptosis in human peripheral blood lymphocytes by ionizing radiation. Using the FADU assay, we have observed the initial radiation-induced DNA damage, its subsequent disappearance due to enzymatic repair, and its time- and dose-dependent reappearance. We believe this reappearance of DNA damage to be indicative of the DNA fragmentation event associated with apoptosis. This interpretation has been supported at the individual cell level using an in situ terminal deoxynucleotidyl transferase (TDT) assay (Apoptag, Oncor Inc.), which detects the 3′-hydro...
DNA repair inhibitors sensitize cells differently to high and low LET radiation
Scientific Reports
The aim of this study was to investigate effects of high LET α-radiation in combination with inhibitors of DDR (DNA-PK and ATM) and to compare the effect with the radiosensitizing effect of low LET X-ray radiation. The various cell lines were irradiated with α-radiation and with X-ray. Clonogenic survival, the formation of micronuclei and cell cycle distribution were studied after combining of radiation with DDR inhibitors. The inhibitors sensitized different cancer cell lines to radiation. DNA-PKi affected survival rates in combination with α-radiation in selected cell lines. The sensitization enhancement ratios were in the range of 1.6–1.85 in cancer cells. ATMi sensitized H460 cells and significantly increased the micronucleus frequency for both radiation qualities. ATMi in combination with α-radiation reduced survival of HEK293. A significantly elicited cell cycle arrest in G2/M phase after co-treatment of ATMi with α-radiation and X-ray. The most prominent treatment effect was ...
British journal of cancer, 1986
The radioprotector 2-[(aminopropyl)amino] ethanethiol (WR1065) was investigated with respect to its ability to affect radiation-induced DNA damage and repair in V79 cells. Studies were performed to evaluate the protector under conditions in which it is known to be effective in reducing the cytotoxic and mutagenic effects of gamma-irradiation. At a concentration of 4 mM, WR1065 protected against the formation of single strand breaks (SSB), as determined by the method of alkaline elution, when it was present during irradiation. The protector appeared, however, to inhibit the subsequent postirradiation repair or rejoining of SSB. While repair was complete within 24 h, the protector reduced the rate of repair by a factor of 3. This inhibitory effect on the rate of repair did not correlate with either measured differences in cell survival or mutagenesis. The radioprotector was also investigated with respect to its ability to affect cell cycle progression. WR1065 present in the growth med...