Cisplatin-modification of DNA repair and ionizing radiation lethality in yeast, Saccharomyces cerevisiae

Cis-diamminedichloroplatinum II (cisplatin) is a DNA inter- and intrastrand crosslinking agent which can sensitize prokaryotic and eukaryotic cells to killing by ionizing radiation. The mechanism of radiosensitization is unknown but may involve cisplatin inhibition of repair of DNA damage caused by radiation. Repair proficient wild type and repair deficient (rad52, recombinational repair or rad3, excision repair) strains of the yeast Saccharomyces cerevisiae were used to determine whether defects in DNA repair mechanisms would modify the radiosensitizing effect of cisplatin. We report that cisplatin exposure could sensitize yeast cells with a competent recombinational repair mechanism (wild type or rad3), but could not sensitize cells defective in recombinational repair (rad52), indicating that the radiosensitizing effect of cisplatin was due to inhibition of DNA repair processes involving error free RAD52-dependent recombinational repair. The presence or absence of oxygen during irradiation did not alter this radiosensitization. Consistent with this result, cisplatin did not sensitize cells to mutation that results from lesion processing by an error prone DNA repair system. However, under certain circumstances, cisplatin exposure did not cause radiosensitization to killing by radiation in repair competent wild type cells. Within 2 h after a sublethal cisplatin treatment, wild type yeast cells became both thermally tolerant and radiation resistant. Cisplatin pretreatment also suppressed mutations caused by exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a response previously shown in wild type yeast cells following radiation pretreatment. Like radiation, the cisplatin-induced stress response did not confer radiation resistance or suppress MNNG mutations in a recombinational repair deficient mutant (rad52), although thermal tolerance was still induced. These results support the idea that cisplatin adducts in DNA interfere with RAD52-dependent recombinational repair and thereby sensitize cells to killing by radiation. However, the lesions can subsequently induce a general stress response, part of which is induction of RAD52-dependent error free recombinational repair. This stress response confers radiation resistance, thermal tolerance, and mutation resistance in yeast.     

DNA lesions that signal the induction of radioresistance and DNA repair in yeast

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.     

Exposure to low dose of gamma radiation enhances the excision repair in Saccharomyces cerevisiae

The effect of low doses of ionizing and nonionizing radiation on the radiation response of yeast Saccharomyces cerevisiae toward ionizing and nonionizing radiation was studied. The wild-type strain D273-10B on exposure to 54 Gy gamma radiation (resulting in about 10% cell killing) showed enhanced resistance to subsequent exposure to UV radiation. This induced UV resistance increased with the incubation time between the initial gamma radiation stress and the UV irradiation. Exposure to low doses of UV light on the other hand showed no change in gamma or UV radiation response of this strain. The strains carrying a mutation at rad52 behaved in a way similar to the wild type, but with slightly reduced induced response. In contrast to this, the rad3 mutants, defective in excision repair, showed no induced UV resistance. Removal of UV-induced pyrimidine dimers in wild-type yeast DNA after UV irradiation was examined by analyzing the sites recognized by UV endonuclease from Micrococcus luteus. The samples that were exposed to low doses of gamma radiation before UV irradiation were able to repair the pyrimidine dimers more efficiently than the samples in which low gamma irradiation was omitted. The nature of enhanced repair was studied by scoring the frequency of induced gene conversion and reverse mutation at trp and ilv loci respectively in strain D7, which showed similar enhanced UV resistance induced by low-dose gamma irradiation. The induced repair was found to be essentially error-free. These results suggest that irradiation of strain D273-10B with low doses of gamma radiation enhances its capability for excision repair of UV-induced pyrimidine dimers.     

Relative biological effectiveness of tritiated water to gamma radiation for germ line mutations

The relative biological effectiveness was determined using sex-linked recessive lethals induced in Drosophila spermatozoa as the biological effect. The sex-linked recessive lethal test, a measure of mutations induced in germ cells and transmitted through successive generations, yields a linear dose-response curve in the range used in these experiments. A dose-response curve was determined from three exposures to tritiated water and three exposures to 60Co gamma radiation. The ratio of the slopes of these two response curves is 2.7 +/- 0.3, yielding a relative biological effectiveness that suggests the tritium beta particle is 2.7 times more effective per unit of energy absorbed in inducing gene mutations transmitted to successive generations than 60Co gamma radiation. The increase in relative biological effectiveness with higher linear energy transfer for tritium beta radiation strongly suggests that single-strand breaks are repaired by a nearly error-free repair mechanism. Ion tracks with a high density of ions (high linear energy transfer) are more efficient than tracks with a low ion density (low linear energy transfer) in inducing transmissible mutations, suggesting interaction among products of ionization. Since most transmitted mutations induced by ionizing radiation result from strand breakage, interaction probably occurs at this level with double-strand breaks being repaired by an error-prone mechanism yielding transmissible mutations.     

Intrinsic radiation sensitivity: cellular signaling is the key

The concept that the balance between DNA damage and repair determines intrinsic radiation sensitivity has dominated radiobiology for several decades. There is undeniably a cause- effect relationship between radiation-induced molecular alterations in the genomic DNA and cellular consequences. In the last decade, however, it has become obvious that the chromatin context affects the fate of damaged DNA and that cellular signaling is an important factor in defining intrinsic radiation sensitivity. Damaged DNA is the site of signal generation; however, alternative signaling at the plasma membrane is triggered: Reactive oxygen species (ROS) inactivate phosphatases and consequently cause activation of kinases localized at the plasma membrane; this includes ligand-independent activation of receptor kinases. Cells with an apparently functional DNA repair system may show increased radiation sensitivity due to deficiencies in specific kinases essential for repair activation and checkpoint control. Other signals that determine intrinsic radiosensitivity may affect proneness to apoptosis, the balance between DNA damage fixation and repair, and the translocation of proteins participating in the response to ionizing radiation. Interplay between the various signals decides the extent to which the repair of radiation-inflicted damage is supported or limited; in some cell types, this includes DNA-damage-independent processes guided by plasma membrane-generated signaling. Cellular signaling in the context of specific subcellular structures is the key to understanding how the molecular effects of radiation are expressed as biological consequences in various cell types. A systems approach should bring us closer to this end.     

The effect of riboxine on prophage induction and the survival of bacterial cultures exposed to gamma irradiation

In experiments with lysogenic cultures Pseudomonas aeruginosa and Escherichia coli K-12 proposed as a test for biological indication of relatively low doses of gamma-radiation, it was shown that when estimated by the criterion of prophage induction riboxine had a radioprotective action at nonlethal radiation doses. At the same time, when estimated by the criterion of the survival rate, riboxine produced the radioprotective effect at rather high radiation doses (survival rate of less than 10 per cent). It is suggested that riboxine is involved in the cell repair processes.     

Radioadaptive response revisited

Radiation-induced adaptive response belongs to the group of non-targeted effects that do not require direct exposure of the cell nucleus by radiation. It is described as the reduced damaging effect of a challenging radiation dose when induced by a previous low priming dose. Adaptive responses have been observed in vitro and in vivo using various indicators of cellular damage, such as cell lethality, chromosomal aberrations, mutation induction, radiosensitivity, and DNA repair. Adaptive response can be divided into three successive biological phenomena, the intracellular response, the extracellular signal, and the maintenance. The intracellular response leading to adaptation of a single cell is a complex biological process including induction or suppression of gene groups. An extracellular signal, the nature of which is unknown, may be sent by the affected cell to neighbouring cells causing them to adapt as well. This occurs either by a release of diffusible signalling molecules or by gap-junction intercellular communication. Adaptive response can be maintained for periods ranging from of a few hours to several months. Constantly increased levels of reactive oxygen species (ROS) or nitric oxide (NO) have been observed in adapted cells and both factors may play a role in the maintenance process. Although adaptive response seems to function by an on/off principle, it is a phenomenon showing a high degree of inter- and intraindividual variability. It remains to be seen to what extent adaptive response is functional in humans at relevant dose and dose-rate exposures. A better understanding of adaptive response and other non-targeted effects is needed before they can be confirmed as risk estimate factors for the human population at low levels of ionising radiation.     

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).     

Repair of platinum-DNA lesions in E. coli by a pathway which does not recognize DNA damage caused by MNNG or UV light

The adaptive response is an inducible DNA-repair system which diminishes the mutagenic and toxic effects of alkylating agents. A mutant of E. coli constitutive for adaptative repair, BS21, has been isolated. A spontaneous revertant of this strain, BS23, lacks the adaptive response. When compared to its wild-type parent, mutant BS21 showed an increased resistance to the killing and mutagenic effects of a compound which is not a classical alkylating agent, the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP). However, this resistance to cis-DDP was also found in strain BS23 which lacks the adaptive response. cis-DDP bound to the DNA of all 3 strains with the same efficiency. In addition, we have investigated the effect of UV radiation and we failed to observe a significant difference in the survival and mutagenesis of these strains. This evidence suggests that the resistance of BS21 and BS23 strains to cis-DDP is not a consequence of the adaptive response or increased excision repair.     

Clustered DNA damage induced by heavy ion particles.

Clustered DNA damage (locally multiply damaged site) is thought to be a critical lesion caused by ionizing radiation, and high LET radiation such as heavy ion particles is believed to produce high yields of such damage. Since heavy ion particles are major components of ionizing radiation in a space environment, it is important to clarify the chemical nature and biological consequences of clustered DNA damage and its relationship to the health effects of exposure to high LET particles in humans. The concept of clustered DNA damage emerged around 1980, but only recently has become the subject of experimental studies. In this article, we review methods used to detect clustered DNA damage, and the current status of our understanding of the chemical nature and repair of clustered DNA damage.     

Radioresponsiveness at low doses: hyper-radiosensitivity and increased radioresistance in mammalian cells

The rationale for and importance of research on effects after radiation at "low doses" are outlined. Such basic radiobiological studies on induction of repair enzymes, protective mechanisms, priming, and hypersensitivity are certainly all relevant to treatment of cancer (see Section 1, Studies at low doses - relevance to cancer treatment). Included are examples from many groups, using various endpoints to address the possibility of an induced resistance, which has been compared to the adaptive response [M.C. Joiner, P. Lambin, E.P. Malaise, T. Robson, J.E. Arrand, K.A. Skov, B. Marples, Hypersensitivity to very low single radiation doses: its relationship to the adaptive response and induced radioresistance, Mutat. Res. 358 (1996) 171-183.]. This is not intended to be an exhaustive review--rather a re-introduction of concepts such as priming and a short survey of molecular approaches to understanding induced resistance. New data on the response of HT29 cells after treatment (priming) with co-cultured activated neutrophils are included, with protection against X-rays (S1). Analysis of previously published results in various cells lines in terms of increased radioresistance (IRR)/intrinsic sensitivity are presented which complement a study on human tumour lines [P. Lambin, E.P. Malaise, M.C. Joiner, Might intrinsic radioresistance of human tumour cells be induced by radiation?, Int. Radiat. Biol. 69 (1996) 279-290].It is not feasible to extrapolate to low doses from studies at high doses. The biological responses probably vary with dose, LET, and have variable time frames. The above approaches may lead to new types of treatment, or additional means to assess radioresponsiveness of tumours. Studies in many areas of biology would benefit from considerations of different dose regions, as the biological responses vary with dose. There may also be some implications in the fields of radiation protection and carcinogenesis, and the extensions of concepts of hyper-radiosensitivity (HRS)/IRR extended to radiation exposure are considered in Section 2, Possible relevance of IRR concepts to radiation exposure (space). More knowledge on inducible responses could open new approaches for protection and means to assess genetic predisposition. Many endpoints are used currently--clonogenic survival, mutagenesis, chromosome aberrations and more direct--proteins/genes/functions/repair/signals, as well as different biological systems. Because of scant knowledge of the relevant aspects at low doses, such as inducible/protective mechanisms, threshold, priming, dose-rate effects, LET within one system, it is still too early to draw conclusions in the area of radiation exposure. Technological advances may permit much needed studies at low doses in the areas of both treatment and protection.     

The effects of low doses of low-intensity ionizing radiation on DNA structure and function

Chronic effects of low doses of low-intensity ionizing radiation (IR) on biological objects have gained great social significance. This has given a considerable impetus to research into the biological effects and mechanisms of such exposures, both in Russia and abroad. In this paper, an overview of the physicochemical and molecular basis of IR influence at low doses is provided. Means of cell protection from radiation damage are studied and an analysis of the typical features and differences in the radiation effects at low and high doses is carried out. We considered DNA radiation damage, both in cell cultures and in vivo, as well as the processes and results of their repair. Particular attention is paid to changes in the basic paradigms of biological radiation effects at low doses.     

Mice defective in the mismatch repair gene Msh2 show increased predisposition to UVB radiation-induced skin cancer

Mice defective in the mismatch repair (MMR) gene Msh2 manifest an enhanced predisposition to skin cancer associated with exposure to UVB radiation. This predisposition is further heightened if the mice are additionally defective for the nucleotide excision repair gene Xpc. To test the hypothesis that the predisposition of Msh2 mutant mice to skin cancer reflects a mutator phenotype associated with increased proliferation of skin cells following exposure to UV radiation, Msh2 mutant mice were exposed to the tumor promoter TPA. Such mice showed a robust proliferative response in the skin, but did not manifest evidence of dysplasia or neoplasia. We conclude that the predisposition of Msh2 mice to UVB radiation-induced skin cancer reflects an interaction between the processes of mismatch repair and some other excision repair mode, the exact nature of which remains to be established.     

Effect of low doses of low-intensity ionizing radiation on the DNA structure and functions

Chronic effects of low doses of low intensity ionizing radiation (IR) on biological objects have now become of great social significance. This has given a considerable impetus to research into biological effects and mechanisms of such exposures both in Russia and abroad. This paper provides an overview of physicochemical and molecular bases of the IR influence at small doses and the ways of cell protection from the radiation damage, as well as the analysis of characteristic features and differences in the effects of radiation at small and high doses. We consider the DNA radiation damage both in cell cultures and in vivo, as well as processes and results of their repair. Particular attention is paid to the changes in the basic paradigms of radiation biological effects of small doses.     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

Characterization of a UV-resistant mutant of CHO-K1 with normal repair activity

The biological and repair responses of Mut 8–16, an ultraviolet radiation (UV)-resistant derivative of CHO-K1, were characterized with respect to UV and to the active chemical carcinogen, benzo[a]pyrene-4,5-oxide. In comparison to the parent, the UV-survival response curve of this mutant showed a significantly larger shoulder but little or no difference in the slope of the exponential survival region. In addition, the mutant cell line demonstrated significantly larger mutation frequencies at high survival UV fluences, but smaller mutation frequencies at high survival equitoxic concentrations of the carcinogen benzo[a]pyrene-4,5-epoxide relative to the parent cell. However, these relative differences in mutation frequencies between parent and mutant appeared to decrease as survival decreased. Despite these observations there were no measurable differences in excision-repair, or in post-replication repair although the mutant appeared to show a nominal reduction (not an enhancement) of replication-repair activity following the UV exposure. These data imply there is another lesion recognition system in CHO cells whose effects on survival and mutation are best observed at low doses of carcinogen and/or radiation but which are masked at higher doses where major repair processes dominate. The dissimilar relationship of cytotoxicity to mutation induction frequency observed in UV and carcinogen treated mutant vs. parent cell lines, imply that the probabilities for lethality and mutation are independent of one another in the presence of otherwise unrepaired (residual) damage.     

The role of regulatory networks of the cell response to damages in radiation effects

In view of modern knowledge and concepts about components, function and mechanisms of response of cell molecular structures to damaging effects, response which is generating specialized modules of reactions, it is shown that main components of the mechanism of maintenance of genome constancy at ionizing radiation exposure are checkpoints of cell cycle, DNA repair and apoptosis. They operate under the control of a genetic system at participation of Tp53 gene, corresponding protein and of regulatory networks formed by cascades of mitogen-activated protein kinases (MAPK). At ionizing radiation exposure the MAPK special modules participate in formation of radiation effect: ERK 1/2 (extracellular signal-regulated kinase 1 and 2), JNK/SAPK (c-Jun N-terminal kinase/stress activated protein kinase) and p38 MAPK. Executing physiological functions of maintenance of normal life activity of cells, they do not lose this capacity after exposure to ionizing radiation, participating in formation of radiation effect in a wide range of doses, and are inactivated only by exposure to very high doses. It is concluded that in light of the modern data the main problem is not a problem of mechanisms of biological effect of ionizing radiation but a problem of biological mechanisms of radiation exposure.