Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

The induction of DNA-protein crosslinks in hypoxic cells and their possible contribution to cell lethality

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.     

Radiosensitization in multifraction schedules. II. Greater sensitization by 2-nitroimidazoles than by oxygen

The objective of this study was to characterize the extent of and mechanisms involved in radiosensitization by 2-nitroimidazoles in multifraction schedules using low doses per fraction. For this purpose, contact-inhibited monolayers of C3H 10T1/2 cells were given 1.7 Gy every 12 h and plated 12 h after the last dose received to allow full repair of potentially lethal damage (PLD). Severe hypoxia was obtained by a 1-h gassing procedure at room temperature immediately before each irradiation. No toxicity occurred as a consequence of multiple exposures to 5 mM misonidazole (MISO) or SR 2508 (2508) during the deoxygenation procedure. Experimental conditions during the pregassing and irradiation (presence of drug and gas mixture) were appropriately manipulated to test for the different mechanisms of radiosensitization demonstrated by nitroimidazoles. A very low oxygen enhancement ratio (OER) results under these conditions (1.34). Exposure to 5 mM MISO or 2508 during the deoxygenation and irradiation of hypoxic cells resulted in greater radiosensitization than could be accounted for by oxygen-mimetic sensitization alone (MISO and 2508 enhancement ratios were greater than the OER). Pregassing cells with N2 in the presence of 5 mM drug sensitized cells which were subsequently irradiated under aerobic conditions (drug free), indicating the occurrence of the "preincubation effect" (which does not require hypoxia or the drug's presence during the irradiation). Thus, for the hypoxic irradiations, the preincubation effect could account for the greater sensitization by nitroimidazoles than by oxygen. The presence of 5 mM drug only during the irradiation of aerobic cells produced radiosensitization in both multifraction and single-dose experiments with delayed plating. This sensitization has been previously shown to involve reduced PLD repair. Finally, maximum radiosensitization occurred in the multifraction schedule when a transient period of hypoxia with drug preceded an aerobic irradiation with drug present, thus combining the benefits of both the preincubation effect and PLD repair inhibition. This work demonstrates the possibility that effects other than oxygen-mimetic radiosensitization could be largely responsible for the sensitization seen in multifraction schedules, particularly when the OER is already low and only transient periods of hypoxia occur.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

Contribution of a photoreactivable component to the oxygen effect observed in certain rad mutants of Saccharomyces cerevisiae after exposure to high energy electrons

It is shown that a fraction of damage induced by high energy electrons (25 MeV) in certain rad mutants of the yeast Saccharomyces cerevisiae can be photoreactivated. The photoreactivable damage contributes to the lethal effect of this type of irradiation and modifies the oxygen effect. Using photoreactivating light or nigrosin, the amount of photoreactivable damage is reduced and the oxygen enhancement ratio (OER) for yeast mutants increases approximately to the OER found in wild-type cells.     

Radiosensitization in multifraction schedules. I. Evidence for an extremely low oxygen enhancement ratio

Stable monolayers of contact-inhibited C3H 10T1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.     

Mutant cells defective in DNA repair pathways provide a sensitive high-throughput assay for genotoxicity

Chemicals used industrially and commercially are required by law to be assessed for their genotoxic potential. However, all currently used assays have major limitations and despite intense effort, there is no universal agreement on which tests should be employed, or how to interpret results. We have developed a new assay system using the chicken DT40 B cell line that offers a number of significant advantages over current methodologies. Our assay could provide enhanced sensitivity using genetically defined and phenotypically characterized mutants defective in DNA repair pathways. Furthermore, analysis of the mutants, using DNA repair proficient wild-type cells as a negative control, minimizes false negative outcomes. Assessing the different responses of a panel of mutants representative of all repair pathways, mechanistic detail of genotoxicity can be determined. This unique feature, as well as reducing the false positive rate, strengthens positive identifications and is useful when extrapolating results to the human context. Our panel of mutants is likely to be useful in screening large compound libraries for an emerging class of chemotherapeutic drugs, which includes inhibitors of DNA repair enzymes such as PARP and DNA polymerases.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and alpha-polymerase

The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)     

uvrC Gene Function in Excision Repair in Toluene-Treated Escherichia coli

We have examined the role of the uvrC gene in UV excision repair by studying incision, excision, repair synthesis, and DNA strand reformation in Escherichia coli mutants made permeable to nucleoside triphosphates by toluene treatment. After irradiation, incisions occur normally in uvrC cells in the presence of nicotinamide mononucleotide (NMN), a ligase-blocking agent, but cannot be detected otherwise. We conclude that repair incisions are followed by a ligation event in uvrC mutants, masking incision. However, a uvrC polA12 mutant accumulates incisions only slightly less efficiently than a polA12 strain without NMN. Excision of pyrimidine dimers is defective in uvrC mutants (polA(+) or polA12) irrespective of the presence or absence of NMN. DNA polymerase I-dependent, NMN-stimulated repair synthesis, which is demonstrable in wild-type cells, is absent in uvrC polA(+) cells, but the uvrC polA12 mutant exhibits a UV-specific, ATP-dependent repair synthesis like parental polA12 strains. A DNA polymerase I-mediated reformation of high-molecular-weight DNA takes place efficiently in uvrC polA(+) mutants after incision accumulation, and the uvrC polA12 mutant shows more reformation than the polA12 strain after incision. These results indicate that normal incision occurs in uvrC mutants, but there appears to be a defect in the excision of pyrimidine dimers, allowing resealing via ligation at the site of the incision. The lack of NMN-stimulated repair synthesis in uvrC polA(+) cells indicates that incision is not the only requirement for repair synthesis.     

Protection against radiation-induced mutagenesis in V79 cells by 2-[(aminopropyl)amino] ethanethiol under conditions of acute hypoxia

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.     

Hyperthermia responses in cell lines with normal and deficient DNA repairs systems

Earlier data showed that cultured cells are more hyperthermia sensitive in S phase of the cell cycle. In part this sensitivity may be related to the disruption of DNA repair/processing activity in S phase. It is well known that many components of DNA repair systems are in fact involved in DNA replication/processing. For this study we set out to evaluate a wide range of DNA repair mutants to determine their thermal responses compared to the wild-type cells. Mutants of nucleotide excision repair (NER), homologous recombination repair (HR) and nonhomologous endjoining (NHEJ) were studied. In all cases the mutants were more thermally sensitive than the wild-type counterparts. In addition, studies on thermal tolerance (TT) showed that mutants did not have a smaller TT response than the wild type thus ruling out TT as a cause in the increased sensitivity. Cell cycle analysis showed no significant difference amongst mutant and wild-type cell line pairs and thus effects of differing cell cycle distribution was also ruled out. It is speculated that it may be the involvement of the mutated pathways in DNA replication/progressing that make mutated cells more sensitive than the wild types.     

The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway

Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.     

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.     

The role of thiols in cellular response to radiation and drugs

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.     

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.     

Cytostatic, cytotoxic and mutagenic effects of voacristine, an indole alkaloid in wild-type and repair-deficient yeasts

Voacristine, an indole alkaloid isolated from the leaves of Ervatamia coronaria (Stapf.) (Apocynaceae) has dose-dependent cytostatic and cytotoxic effects on cultures of Saccharomyces cerevisiae. These inhibitory effects take place only in growing cells. Among the different repair-deficient mutants examined, a mutant defective in excision-resynthesis repair pathway (rad3-e5) was found to be the most sensitive to such a toxic effect. The mutant rad52-1 blocked in the DNA strand break repair pathway showed an intermediary sensitivity to the lethal effect induced by this indole alkaloid, whereas the mutant defective in the mutagenic repair pathway (rad6-1) demonstrated practically the same sensitivity as the wild-type strain. The nuclear reversion mutation for the locus lysl-1 was induced by voacristine, whereas the mitochondrial "petite" mutation was not induced by this alkaloid. These results indicate that the lesions induced by voacristine in vivo are likely to be of the adducts type; such damage is repairable in the wild-type; the DNA strand break repair pathway plays a minor role in the repair of voacristine-induced lesions.     

Role of gene 59 of bacteriophage T4 in repair of UV-irradiated and alkylated DNA in vivo.

Nonsense mutants in gene 59 (amC5, amHL628) were used to study the role of this gene in the repair of UV-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to UV irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after UV irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to UV irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that UV repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B (su minus), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of UV lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules.     

Compromised cellular responses to DNA damage accelerate chronological aging by incurring cell wall fragility in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Elevated levels of reactive oxygen species (ROS) can attack almost all cell components including genomic DNA to induce many types of DNA damage. In this study, we used Saccharomyces cerevisiae with various mutations in a biological network supposed to prevent deleterious effects of endogenous ROS to test the effect of such a network on yeast chronological aging. Our results showed that cells with defects in cellular antioxidation, DNA repair and DNA damage checkpoints displayed a mutation rate higher than that of wild-type strain. Moreover, the chronological life span of most mutants as determined by colony formation was found to be shorter than that of wild-type cells, especially for the mutants defective in DNA replication and DNA damage checkpoints, although the observed cell number was almost the same for wild-type and mutant strains. The mutants were finally found to be more sensitive to SDS and lysing enzyme treatment, and that the degree of sensitivity was correlated with their chronological life span.     

Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here.     

Hypoxic cell radiosensitization by moderate hyperthermia and glucose deprivation

Cell culture studies were carried out to determine whether moderate hyperthermia reduces the oxygen enhancement ratio of cells under well-defined cultural conditions. Using asynchronously growing HeLa cells, the OER of cells with and without glucose was determined following exposure of cells to moderate hyperthermia, 40.5 degrees C for 1 hr, immediately after X irradiation. The OER of cells with 5 mM glucose was 3.2, whereas the OER of glucose-deprived cells was reduced to 2.0. The pH of the cell culture medium was kept at 7.4 throughout the experiments. The present finding may provide a clue toward further enhancing the radiosensitization of hypoxic cells by heat.