RBE of densely ionizing radiation for wild-type and radiosensitive mutants of yeast

Survival curves were obtained for haploid and diploid yeasts, Saccharmyces cerevisiae, of wild-type strains and radiosensitive mutants exposed to γ-rays and α-particles. A correlation between the values of the relative biological effectiveness (RBE) of high-LET radiation and cell-repair capacity was found. The difference in radiosensitivities of the wild-type diploid strain and homozygous rad mutants incapable of recovery was significantly higher after low-LET radiation than after high-LET radiation. Possible reasons for the observed radiation responses to low- and high-LET exposure of yeast cells with various genotype are discussed.     

The evaluation of the role of recovery from potentially lethal and sublethal damages in the RBE of densely ionizing radiation

The new data confirming the relations between RBE and recovery of cells are presented. The quantitative evaluation of the contribution of potentially lethal and sublethal damage recovery in radiosensitivity of cells of various origin after exposure to low- and high-LET ionizing radiation was carried out. The conclusion about the greater contribution of potentially lethal damage recovery in the magnitude of RBE in comparison with sublethal damage recovery was made.     

Effects of sparsely and densely ionizing radiation on plants

One of the main purposes leading botanists to investigate the effects of ionizing radiations is to understand plant behaviour in space, where vegetal systems play an important role for nourishment, psychological support and functioning of life support systems. Ground-based experiments have been performed with particles of different charge and energy. Samples exposed to X- or γ-rays are often used as reference to derive the biological efficiency of different radiation qualities. Studies where biological samples are exposed directly to the space radiation environment have also been performed. The comparison of different studies has clarified how the effects observed after exposure are deeply influenced by several factors, some related to plant characteristics (e.g. species, cultivar, stage of development, tissue architecture and genome organization) and some related to radiation features (e.g. quality, dose, duration of exposure). In this review, we report main results from studies on the effect of ionizing radiations, including cosmic rays, on plants, focusing on genetic alterations, modifications of growth and reproduction and changes in biochemical pathways especially photosynthetic behaviour. Most of the data confirm what is known from animal studies: densely ionizing radiations are more efficient in inducing damages at several different levels, in comparison with sparsely ionizing radiation.     

Mutational alterations induced in yeast by ionizing radiation

The cyc1-9 ochre (UAA) mutant and the cyc1-179 amber (UAG) mutant of the yeast Saccharomyces cerevisiae were reverted with X-rays and -particles. The amino acid sequence changes of iso-1-cytochromes c from 36 of the intragenic revertants were determined by amino acid analysis and peptide mapping, aided by partial amino acid sequencing of 4 revertants. In addition, the DNA segments encompassing 3 unusual mutations with complex changes were cloned and sequenced. This study and previous studies of 16 other revertants of cyc1-9 and cyc1-179 revealed that ionizing radiation primarily induces single base-pair substitutions; 47 of the 52 revertants arose by transversions and transitions without any apparent preference. However, the A·T→T·A substitution at the first base pair for the cyc1-179 UAG codon, leading to the normal protein, was not detected, nor was it found previously in 32 revertants of cycl-179 obtained spontaneously or induced with various other mutagens; apparently, there is a prohibition of certain base-pair substitutions at certain sites in DNA. In addition, 5 of the 52 revertants arose by multiple changes within a short region of 11 base pairs. These consisted of the deletion of 6 base pairs, the substitution of 3 base pairs, and 3 different kinds of substitutions of two base pairs. Compared to other mutagens previously tested with the cyc1 system, ionizing radiation produces the most random types of base-pair substitutions.     

Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome

Speculation has long surrounded the question of whether past exposure to ionizing radiation leaves a unique permanent signature in the genome. Intrachromosomal rearrangements or deletions are produced much more efficiently by densely ionizing radiation than by chemical mutagens, x-rays, or endogenous aging processes. Until recently, such stable intrachromosomal aberrations have been very hard to detect, but a new chromosome band painting technique has made their detection practical. We report the detection and quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear-weapons workers who were exposed to plutonium many years ago. Even many years after occupational exposure, more than half the blood cells of the healthy plutonium workers contain large (>6 Mb) intrachromosomal rearrangements. The yield of these aberrations was highly correlated with plutonium dose to the bone marrow. The control groups contained very few such intrachromosomal aberrations. Quantification of this large-scale chromosomal damage in human populations exposed many years earlier will lead to new insights into the mechanisms and risks of cytogenetic damage.     

Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation

In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, we have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells irradiated with either fast neutrons or accelerated argon ions. Individual mutant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loss and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbA1 locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes.     

Protective role of superoxide dismutases against ionizing radiation in yeast

The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Delta), mitochondrial MnSOD (sod2Delta), or both SODs (sod1Deltasod2Delta). Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation. When the oxidation of 2',7'-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells. These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells. Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.     

Photoreactivation of damage induced by ionizing radiation in yeast cells

Summary Experimental data on photoreactivation of damage induced by ionizing radiation in yeast cells are presented. The value of photoreactivation was found to be the highest for the following conditions predicted by us as optimum ones: large volume of irradiated suspension, hypoxia and high energy sparsely ionizing radiation. A comparison of data for yeast and bacterial cells shows that Cerenkov emission from ionizing radiation may produce photoreactivated pyrimidine dimers in both prokaryotic and eukaryotic cell systems.     

Endothelial cell death after ionizing radiation does not impair vascular structure in mouse tumor models

The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato‐labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non‐perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.     

Autophagy induced by ionizing radiation promotes cell death over survival in human colorectal cancer cells

Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.     

Stable intrachromosomal biomarkers of past exposure to densely ionizing radiation in several chromosomes of exposed individuals

A multicolor banding (mBAND) fluorescence in situ hybridization technique was used to investigate the presence inhuman populations of a stable biomarker-intrachromosomal chromosome aberrations-of past exposure to high-LET radiation. Peripheral blood lymphocytes were taken from healthy Russian nuclear workers occupationally exposed from 1949 onward to either plutonium, gamma rays or both. Metaphase spreads were produced and chromosomes 1 and 2 were hybridized with mBAND FISH probes and scored for intra-chromosomal aberrations. A large yield of intrachromosomal aberrations was observed in both chromosomes of the individuals exposed to high doses of plutonium, whereas there was no significant increase over the (low) background control rate in the population who were exposed to high doses of gamma rays.Interchromosome aberration yields were similar in both the high plutonium and the high gamma-ray groups. These results for chromosome 1 and 2 confirm and extend data published previously for chromosome 5. Intrachromosomal aberrations thus represent a potential biomarker for past exposure to high-LET radiations such as alpha particles and neutrons and could possibly be used as a biodosimeter to estimate both the dose and type of radiation exposure in previously exposed populations.     

Gene-dependent cell death in yeast

Caspase-dependent apoptotic cell death has been extensively studied in cultured cells and during embryonic development, but the existence of analogous molecular pathways in single-cell species is uncertain. This has reduced enthusiasm for applying the advanced genetic tools available for yeast to study cell death regulation. However, partial characterization in mammals of additional genetically encoded cell death mechanisms, which lead to a range of dying cell morphologies and necrosis, suggests potential applications for yeast genetics. In this light, we revisited the topic of gene-dependent cell death in yeast to determine the prevalence of yeast genes with the capacity to contribute to cell-autonomous death. We developed a rigorous strategy by allowing sufficient time for gene-dependent events to occur, but insufficient time to evolve new populations, and applied this strategy to the Saccharomyces cerevisiae gene knockout collection. Unlike sudden heat shock, a ramped heat stimulus delivered over several minutes with a thermocycler, coupled with assessment of viability by automated counting of microscopic colonies revealed highly reproducible gene-specific survival phenotypes, which typically persist under alternative conditions. Unexpectedly, we identified over 800 yeast knockout strains that exhibit significantly increased survival following insult, implying that these genes can contribute to cell death. Although these death mechanisms are yet uncharacterized, this study facilitates further exploration.     

Combined action of ultrasound and ionizing radiation on yeast cells

Summary This communication reports the observation of synergistic relationships between ultrasound and gamma-irradiation of stationary phase cultures ofSaccharomyces cerevisiae of different strains. The gamma-ray dose was applied before or after the sound. The extent of synergism depended upon the sequence of application; it was smaller for (US +-ray)-exposure in comparison with (-ray + US)-treatment. The combined action of both modalities had smaller or no synergistic effect for mutant (rad51) yeast cells incapable of recovery. On this basis, it was concluded that possible mechanisms for ultrasound radiosensitization of yeast cells may involve the reduced capacity of cells to recover damages resulted from the combined action and/or the enhanced expression of lethal damage.     

Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation

Abstract. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KGl), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2–4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G₁ were resistant; the sensitivity of G₂ or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G₂ or M, to these inhibitors. The data show that γ-irradiation (0.5–15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G₁ cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G₂ either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G₁, G₂ or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.