Biological effectiveness of ionizing radiations of various quality in Escherichia coli bacteria (a theoretical analysis). Cell radiosensitivity and DNA repair

On the basis of the ideas reported earlier a study was made of the dependence of radiosensitivity (D0-1) of isogenic mutations of E. coli upon LET. This dependence was shown to be conditioned not only by the physical parameters of radiation but also by the ability of cells to repair definite types of DNA lesions. The influence of the balance in the activity of repair enzymes in E. coli on the shape of D0-1 (LET) curve is discussed.     

Relation of the radiosensitivity of yeast cells to the LET of the radiation. Experiments with diploid cells

A study was made of the dependence of radiosensitivity D0-1 of diploid yeast cells on linear energy transfer L of radiation, and the obtained results were analyzed in terms of the previously developed model concepts. A diploid-specific repair of radiation damages was shown to be equally effective with different L. This mode of repair had no direct effect on D0-1 (L) function. The observed contribution of the diploid-specific repair to cell radioresistance decreased with increasing L. This, however, was due to physical and geometric factors rather than to the decreased efficiency of diploid-specific repair.     

Dependence of the radiosensitivity of yeast cells on radiation LET. A theoretical analysis

A model is proposed for interpreting the radiosensitivity of yeast cells as a function of linear energy transfer (LET) of ionizing radiation. The model takes into account the role of repair processes in sensitivity of yeast cells to ionizing radiation of different LET. Two types of repair are discussed: (1) a nonspecific repair (characteristic of both haploid and diploid cells), and (2) a diploid-specific repair (characteristic of diploid cells only).     

Induction of prophage lambda during the irradiation of E. coli cells exposed to radiations of various LET

Induction of lambda prophage in lysogenic E. coli cells exposed to ionizing radiation of different LET was studied as a function of dose I(D). Activities of pleiotropic RecA protein were shown to contribute to the shape of the I(D) curve. The experimental data were fitted by the function I(D) = alpha D(1-exp(-D0-1.D]exp(-beta D). Inducibility alpha increased with increasing LET which was related to the increased incidence of DNA lesions being a SOS-system call.     

Involvement of DNA polymerase beta in repair of ionizing radiation damage as measured by in vitro plasmid assays

Characteristic of damage introduced in DNA by ionizing radiation is the induction of a wide range of lesions. Single-strand breaks (SSBs) and base damages outnumber double-strand breaks (DSBs). If unrepaired, these lesions can lead to DSBs and increased mutagenesis. XRCC1 and DNA polymerase beta (polbeta) are thought to be critical elements in the repair of these SSBs and base damages. XRCC1-deficient cells display a radiosensitive phenotype, while proliferating polbeta-deficient cells are not more radiosensitive. We have recently shown that cells deficient in polbeta display increased radiosensitivity when confluent. In addition, cells expressing a dominant negative to polbeta have been found to be radiosensitized. Here we show that repair of radiation-induced lesions is inhibited in extracts with altered polbeta or XRCC1 status, as measured by an in vitro repair assay employing irradiated plasmid DNA. Extracts from XRCC1-deficient cells showed a dramatically reduced capacity to repair ionizing radiation-induced DNA damage. Extracts deficient in polbeta or containing a dominant negative to polbeta also showed reduced repair of radiation-induced SSBs. Irradiated repaired plasmid DNA showed increased incorporation of radioactive nucleotides, indicating use of an alternative long-patch repair pathway. These data show a deficiency in repair of ionizing radiation damage in extracts from cells deficient or altered in polbeta activity, implying that increased radiosensitivity resulted from radiation damage repair deficiencies.     

Radiation-sensitive irs mutants rejoin DNA double-strand breaks with efficiency similar to that of parental V79 cells but show altered response to radiation-induced G2 delay.

Induction and repair of DNA double-strand breaks (dsb) was investigated in plateau phase Chinese hamster V79 cells and three radiosensitive mutant cell lines derived from them, irs-1, irs-2 and irs-3, using a pulsed-field gel electrophoresis assay, Asymmetric Field Inversion Gel Electrophoresis (AFIGE). There was no difference in the induction of DNA dsb per Gy and dalton between the radiosensitive mutant cells and wild-type V79 cells despite the wide differences in their radiosensitivity. Also, repair of DNA dsb proceeded in all cell lines with similar kinetics. In contrast to these observations at the DNA level, irradiation of exponentially growing cells showed a prolonged delay in G2 for irs-2 cells and a shortened delay in G2 for irs-1 cells, as compared to wild-type V79 cells. These results confirm previous observations suggesting that a deficiency in the rejoining of DNA dsb is unlikely to be the cause of the increased radiosensitivity of irs cells, and implicate alterations in postirradiation cell cycle progression as a possible cause for this phenomenon, although the mechanism is not known.     

Protection of human tumor cells of differing radiosensitivity by WR-1065

We examined the ability of WR-1065, the biologically active aminothiol form of the clinically used drug amifostine (WR-2721, Ethyol), to protect cultures of two human glioblastoma cell lines of greatly differing radiosensitivity from the cytotoxic effects of gamma radiation. M059J cells are extremely radiosensitive compared to M059K cells (which were derived from the same tumor) and are defective in the DNA-dependent protein kinase (DNAPK)-mediated pathway for the repair of DSBs. In spite of their marked phenotypic differences, the two glioblastoma lines were protected equivalently ( approximately 1.8-fold) after a 30-min preirradiation treatment with 4 mM WR-1065. These findings are in agreement with earlier studies that showed no relationship between the ability of another aminothiol, cysteamine, to protect human tumor cells with differing abilities to repair DSBs and/or radiosensitivity. Thus it appears that differences in intrinsic radiosensitivity and ability to repair DSBs are not important general factors in the modulation of the radiosensitivity of human cells by aminothiols. Because of a previous report that the radiosensitive mutant rodent xrs5 cell line (which, like M059J, is defective in the DNAPK-mediated pathway for repairing DSBs) is unusually refractory to the radioprotective effects of WR-1065, we re-examined the ability of WR-1065 to protect these cells. In contrast to the earlier studies, both the wild-type and mutant rodent lines were protected extensively by WR-1065. This discrepancy might be related to some unknown factor, such as differences in chromatin organization among xrs5 subclones that arise during their karyotypic evolution, possibly leading to altered DNA-drug associations.     

Differential role of DNA-PKcs phosphorylations and kinase activity in radiosensitivity and chromosomal instability

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G(1) cells to (137)Cs γ rays. DNA-PKcs mutant cells defective in phosphorylation at multiple sites within the T2609 cluster or within the PI3K domain displayed extreme radiosensitivity. Cells defective at the S2056 cluster or T2609 single site alone were only mildly radiosensitive, but cells defective at even one site in both the S2056 and T2609 clusters were maximally radiosensitive. Thus a synergism between the capacity for phosphorylation at the S2056 and T2609 clusters was found to be critical for induction of radiosensitivity.     

Base excision repair by hNTH1 and hOGG1: a two edged sword in the processing of DNA damage in gamma-irradiated human cells

Using siRNA technology, we down-regulated in human B-lymphoblastoid TK6 cells the two major oxidative DNA glycosylases/AP lyases that repair free radical-induced base damages, hNTH1 and hOGG1. The down-regulation of hOGG1, the DNA glycosylase whose main substrate is the mutagenic but not cytotoxic 8-oxoguanine, resulted in reduced radiation cytotoxicity and decreased double strand break (DSB) formation post-irradiation. This supports the idea that the oxidative DNA glycosylases/AP lyases convert radiation-induced clustered DNA lesions into lethal DSBs and is in agreement with our previous finding that overexpression of hNTH1 and hOGG1 in TK6 cells increased radiation lethality, mutant frequency at the thymidine kinase locus and the enzymatic production of DSBs post-irradiation [N. Yang, H. Galick, S.S. Wallace, Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks, DNA Repair (Amst) 3 (2004) 1323-1334]. Interestingly, cells deficient in hNTH1, the DNA glycosylase that repairs a major lethal single free radical damage, thymine glycol, were more radiosensitive but at the same time fewer DSBs were formed post-irradiation. These results indicate that hNTH1 plays two roles in the processing of radiation damages: repair of potentially lethal single lesions and generation of lethal DSBs at clustered damage sites. In contrast, in hydrogen peroxide-treated cells where the majority of free radical DNA damages are single lesions, the base excision repair pathway functioned to protect the cells. Here, overexpression of hNTH1 and hOGG1 resulted in reduced cell killing while suppression of glycosylase expression resulted in elevated cell death.     

Effects of hyperthermia on radiation-induced chromosome breakage and loss in excision repair deficient Drosophila melanogaster

Hyperthermia increased radiosensitivity with respect to gamma-ray induced chromosome loss and breakage in all stages of spermatogenesis in the wild type Oregon R strain of Drosophila melanogaster, whereas hyperthermia increased radiosensitivity to a lesser extent in cn mus (2) 201D1, an excision repair mutant with 0 per cent excision capacity and in mus (3) 308D1, a strain with 24 per cent excision capacity. The differences in hyperthermia-induced radiation sensitivity between the excision repair mutants and the wild strain may be due to the hyperthermia affecting the excision repair mechanism, suggesting that one of the possible mechanisms involved in hyperthermia-increased radiosensitivity is an effect on excision repair.     

The effect of He-Ne laser radiation on the survival of HeLa cells subjected to ionizing radiation]

A monolayer of HeLa cells, at the stationary phase of growth, exposed to He-Ne laser radiation (632.8 nm; 100 J/m2) either 5 min or 60 min prior to gamma irradiation (0.1-10 Gy; 6.75 Gy/min), or 5 min after irradiation has been investigated. With a 5-min interval between irradiation sessions (both sequences) the survival curves are virtually the same as those for gamma-irradiated cells only. With He-Ne laser radiation delivered 60 min before gamma irradiation with doses exceeding 5 Gy, a fraction of radioresistant cells is identified whose D0 is almost twice as high as D0 of basic cell mass (3.6 and 1.7 Gy respectively. The survival curve becomes a two-component one. A hypothesis is proposed that He-Ne laser radiation activates, in some cells, the processes that promote the repair of radiation damages.     

DNA repair in cells of the radiosensitive mutant of Drosophila rad(2)201GI after gamma-irradiation]

A radiosensitive mutant of Drosophila melanogaster rad(2)201GI was analysed for the capacity to repair DNA single- and double-strand breaks induced by gamma-rays. Analysis was performed on cell cultures derived from embryos of homozygous mutant stock and wild type strain Oregon R. The viability of irradiated cells was studied. It was shown that the mutant strain cells had increased lethality, just like a whole organism. Single-strand breaks were analysed by alkaline sucrose gradient centrifugation; double-strand breaks were monitored by neutral elution. The similarity of repair kinetics of single- and double-strand breaks in cells of rad(2)201GI and Oregon R was shown. Probable molecular mechanisms of rad(2)201GI mutant radiosensitivity are under discussion.     

Checkpoint controls in Schizosaccharomyces pombe: rad1.

'Checkpoint' controls ensure that the events of the cell cycle are completed in an orderly fashion. For example, such controls delay mitosis until DNA synthesis and repair of radiation-induced DNA damage are complete. The rad series of radiosensitive fission yeast mutants was examined to identify strains deficient for the DNA damage-responsive checkpoint control. Five were identified. A characterization of one (rad1-1) and the wild-type is presented. The rad1-1 mutant does not arrest after irradiation, is sensitive to killing by radiation and is not arrested by hydroxyurea, and thus is also deficient for the DNA synthesis-responsive checkpoint control. The radiosensitivity of the rad1-1 mutant was greatly reduced when irradiated and maintained for 6 h in a non-dividing (density inhibited) state, demonstrating that rad1-1 is repair proficient and radiosensitive only through failure to delay. The checkpoint controls for which rad1 is required appear to regulate G2-M progression through the activity of cdc2, here implicated in this role by the coincidence of the radiation transition point and the cdc2 execution point.     

The effect of low-dose irradiation on unstimulated and PHA-stimulated human lymphocyte subsets

A culture system was used to evaluate the radiosensitivity of CD4+ and CD8+ T cells, Leu 19+ cells, and B cells obtained from normal adult males. Unstimulated CD8+ lymphocytes (D0 = 55 cGy) were twice as radiosensitive as CD4+ cells (D0 = 115 cGy). B cells had an intermediate radiosensitivity (D0 = 100 cGy). Leu 19+ cells were much more radioresistant and expressed a D0 of 550 cGy. When lymphoid cells were irradiated 1 or 4 days before phytohemagglutinin (PHA) stimulation, they were more radiosensitive than if they were first stimulated with PHA and then irradiated. When lymphoid cells were irradiated 1 h after PHA stimulation each lymphocyte subset was characterized by an increase in the D0 to 150 cGy for B cells to 290 cGy for CD4+ cells, and to 240 cGy for CD8+ cells. In contrast, Leu 19+ cells exhibited a decrease in their D0 to 290 cGy after they were stimulated by PHA.     

Lethal action of accelerated heavy ions on mammalian cells as affected by inhibitors of DNA synthesis. A theoretical analysis

Günter-Schulz's model and the authors' own model were used to study the dependence of radiosensitivity (D0(-1)) of Chinese hamster cells on linear energy transfer (LET) upon irradiation in standard conditions and in the presence of DNA synthesis inhibitors, arabinosylcytosine and hydroxyurea. A better agreement of the experimental and theoretical results was obtained using Kozubek-Krasavin's model than the model of Günter and Schulz.     

Variation in the sensitivity of the mouse spermatogonial stem cell population to fission neutron irradiation during the cycle of the seminiferous epithelium

Dose-response studies of the radiosensitivity of spermatogonial stem cells in various epithelial stages after irradiation with graded doses of fission neutrons of 1 MeV mean energy were carried out in the Cpb-N mouse. These studies on the stem cell population in stages IX-XI yielded simple exponential lines characterized by an average D0 value of 0.76 +/- 0.02 Gy. In the subsequent epithelial stages XII-III, a significantly lower D0 value of 0.55 +/- 0.02 Gy was found. In contrast to the curves obtained for stem cells in stages IX-III, the curves obtained in stages IV-VIII indicated the presence of a mixture of radioresistant and radiosensitive stem cells. In stage VII, almost no radioresistant stem cells appeared to be present and a D0 value for the radiosensitive stem cells of 0.22 +/- 0.01 Gy was derived. Previously, data were obtained on the size of colonies (in number of spermatogonia) derived from surviving stem cells. Combining these data with data from the newly obtained dose-response curves yielded the number of stem cells, per stage and with the specific radiosensitivities, present in the control epithelium. In stages IX-XI, there are approximately 6 stem cells per 1000 Sertoli cells with a radiosensitivity characterized by a D0 of 0.76 Gy, which corresponds to one-third of the As population in these stages. (The As spermatogonia are presumed to be the stem cells of spermatogenesis.) IN stages XII-III, there are approximately 12 stem cells per 1000 Sertoli cells with a radiosensitivity characterized by a D0 of 0.55 Gy, which roughly equals the number of A single spermatogonia in these stages. These calculations could not be made for stages IV-VIII since no simple exponential lines were obtained for these stages. In view of the pattern of the proliferative activity of the spermatogonial stem cells during the epithelial cycle, it appears that the stem cell population is most radiosensitive during the period when the majority of these cells are in G0 phase, most resistant when the cells are stimulated again into proliferation, and of intermediate sensitivity during active proliferation.     

Condensed chromatin and cell inactivation by single-hit kinetics

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.     

Repair of gamma-ray-induced DNA base damage in xeroderma pigmentosum cells

The repair of DNA damage produced by 137Cs gamma irradiation was measured with a preparation from Micrococcus luteus containing DNA damage-specific endonucleases in combination with alkaline elution. The frequency of these endonuclease sensitive sites (ESS) was determined after 54 or 110 Gy of oxic irradiation in normal and xeroderma pigmentosum (XP) fibroblasts from complementation groups A, C, D, and G. Repair was rapid in all cell strains with greater than 50% repair after 1.5 h of repair incubation. At later repair times, 12-17 h, more ESS remained in XP than in normal cells. The frequency of excess ESS in XP cells was approximately 0.04 per 10(9) Da of DNA per Gy which was equivalent to 10% of the initial ESS produced. The removal of ESS was comparable in XP cells with normal radiosensitivity and XP3BR cells which have been reported to be moderately radiosensitive.     

Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (⁶⁰Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters β/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

     

Effects of radiation quality on interactions between oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation damages DNA and also induces oxidative stress, which can affect the function of proteins involved in DNA repair, thereby causing repair of DNA damage to become less efficient. We previously developed a mathematical model of this potentially synergistic relationship and applied it to γ-ray exposure data on the radiation-resistant prokaryote Deinococcus radiodurans. Here, we investigate the effects of radiation quality on these processes by applying the model to data on exposures of D. radiodurans to heavy ions with linear energy transfer (LET) of 18.5–11,300 keV/μm. The model adequately describes these data using three parameters combinations: radiogenic DNA damage induction, repair protein inactivation and cellular repair capacity. Although statistical uncertainties around best-fit parameter estimates are substantial, the behaviors of model parameters are consistent with current knowledge of LET effects: inactivation cross-sections for both DNA and proteins increase with increasing LET; DNA damage yield per unit of radiation dose also increases with LET; protein damage per unit dose tends to decrease with LET; DNA and especially protein damage yields are reduced when cells are irradiated in the dry state. These results suggest that synergism between oxidative stress and DNA damage may play an important role not only during γ-ray exposure, but during high-LET radiation exposure as well.