Effects of inhibitors of DNA synthesis and protein synthesis on the rate of DNA synthesis after exposure of mammalian cells to ultraviolet light.

Chinese hamster V-79 cells were treated with metabolic inhibitors o DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J m-2. After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [14C]thymidine into acid-insoluble material. A 2.5 or 5.0 h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involvement of an inducible DNA repair process.

Effects of growth media on cell cycle progression in CHO cells exposed to the radioprotector WR-1065.

WR-1065 (2-[(aminopropyl)amino]ethanethiol) reduces cytotoxic and mutagenic effects caused by exposure of cells to radiation and chemotherapeutic drugs, but the mechanisms involved are not fully known. We have observed an accumulation of cells in G2 in WR-1065 treated Chinese hamster ovary cells grown in alpha-minimal essential medium, while others have found no cell cycle effects in WR-1065 treated Chinese hamster ovary cells grown in McCoy's 5A medium. To determine if the two types of media had an effect on cells treated with WR-1065, we examined survival and cell cycle progression. Population doubling times of 12 h were observed for cells grown in both media. Incubation of AA8 cells grown in McCoy's 5A medium with 4 mM WR-1065 30 min prior to and during irradiation with 137Cs gamma-rays resulted in a protection factor of 2.2, in close agreement with the value of 2.0 we previously obtained for AA8 cells grown in alpha-minimal essential medium. Treatment with WR-1065 caused an alteration in the cell cycles of cells grown in both media. An increase in the G2 population and a decrease in the G1 population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065, with a redistribution of the cells throughout the cell cycle occurring following removal of the drug. These data suggest that exposure of cells to WR-1065 is the cause of perturbations in cell cycle progression, and is not affected by the type of medium the cells are grown in.

Reversion of radiosensitivity in azacytidine-treated XRS5 cells does not result in full radioprotection by WR-1065.

A series of cell lines were previously generated from the radiation sensitive Chinese hamster ovary line xrs5 after treatment with azacytidine. Six of these lines have been examined for their resistance to killing by 0 to 20 Gray of 60Co gamma rays and the amount of radioprotection afforded by treatment with the drug 2-[(aminopropyl)amino]ethanethiol (WR-1065). As xrs5 cells have lost the ability to be protected by WR-1065, studies were performed to determine whether reversion to radio-resistance correlated with recovery of aminothiol radioprotection. Treatment of azacytidine-treated, radiation sensitive and resistant cells with four millimolar WR-1065 30 minutes prior to irradiation enhanced survival after exposure to gamma radiation, although the enhancement in survival was less than for wild type Chinese hamster ovary K1 cells. The data suggest that there is not an absolute linkage between recovery of gamma ray radiation resistance and protection by WR-1065 and other factors, such as chromatin organization, must play a role.

Delayed inhibition of subchromosomal DNA synthesis in V-79 Chinese hamster cells after gamma irradiation.

Existence of a substantial fraction of replicon initiation events refractory to the effects of X irradiation in Chinese hamster cells has been reported by several laboratories. The work reported here examined whether this apparently refractive fraction resulted from a delayed inhibition of initiation events. Data obtained from velocity sedimentation studies indicated that the extent of inhibition increased over the first hour after irradiation from 35% inhibition immediately following exposure to 3 kR to 75% inhibition of initiation 1 hr after irradiation. Analysis of subsequent recovery of initiation radiosensitivity was performed using DNA fiber autoradiograms prepared from cells incubated up to 4 hr between 2-kR exposures. The data from these experiments indicated that some recovery occurs within 1 hr of irradiation and thus separation of the inhibition and recovery processes in V-79 cells may not be feasible.

Accumulation of CHO cells in G2 phase following exposure to WR-1065.

The radioprotector WR-1065 (2-[(aminopropyl)amino]ethanethiol) is known to protect mammalian cells from the cytotoxic and mutagenic effects of radio- and chemotherapeutic agents, but the exact mechanisms involved in this protection are not fully known. To help determine the effects of WR-1065 alone on cells, we examined its effect on a variety of cellular processes. Incubation of AA8 cells in 4 mM WR-1065 did not significantly affect the rate of DNA synthesis. Autoradiographic analysis of heavily labeled (S-phase population) nuclei of AA8 cells showed no significant difference in the S-phase population of WR-1065-treated versus control cells for up to 3 h. An examination of the effect of WR-1065 on repair synthesis, as measured by unscheduled DNA synthesis (UDS) in cells exposed to 15 Gy, showed no difference between treated and sham-treated cells for up to 2 h exposure. A significant reduction in the amount of UDS was seen in cells treated with the protector for 2.5 and 3 h. Incubation of cells in WR-1065 did alter the cell cycle distributions. An increase in the G2-phase population with a corresponding decrease in the G1-phase population was observed in cells incubated up to 3 h in the presence of 4 mM WR-1065. After the removal of WR-1065 at 3 h, a redistribution of the cells throughout the cell cycle occurred as has been observed in cells treated with other synchronization agents. These data suggest that perturbations in cell cycle progression, rather than direct effects on the rate of DNA synthesis, could play a role in the increased survival and reduced mutation frequencies observed in the presence of WR-1065.

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation.

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.

Association of WR-1065 with CHO AA8 cells, nuclei, and nucleoids.

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.

Varying levels of radioprotection from the effects of JANUS neutrons in repair-deficient xrs-5 hamster cells treated with azacytidine.

A series of cell lines have been generated from the radiation-sensitive Chinese hamster ovary line xrs-5 by treatment with azacytidine. Several of these lines have been shown to be resistant to gamma radiation. Survival curves have been generated for several of these lines and the parental lines after exposure to 0 to 5 Gy of JANUS neutrons in the presence or absence of a 30-min pretreatment with the aminothiol radioprotector WR-1065. These studies were performed to determine whether the parental xrs-5 cell line was radioresistant to exposure to JANUS neutrons and whether reversion to a neutron-resistant phenotype correlated with recovery of aminothiol radioprotection. Exposure to 4 mM WR-1065 enhanced survival after exposure to neutron radiation for most "revertant" lines, although the increase in survival varied. The xrs-5 cell line was sensitive to JANUS neutrons and showed no protection by WR-1065. These data indicate that xrs-5 cells are also sensitive to neutron radiation, that azacytidine-induced revertants for gamma-ray survival demonstrate the wild-type phenotype for survival after neutron exposure, and that the gene product that is defective is responsible for repairing only a small portion of neutron-induced damage.

Effects of ultraviolet light on thymidine incorporation and DNA chain elongation in photoreactivable insect cells.

An insect cell line, IAL-PID2, was exposed to UV and analyzed for its ability to incorporate [3H]-thymidine and to elongate replicon-sized DNA fragments. After exposure to 5 or 10 J/m2 UV, the cells exhibited a rapid and prolonged depression in the rate of thymidine incorporation. Photoreactivation reduced this depression but did not entirely reverse it. For exposures of 5 J/m2 or above, full recovery did not occur until 18 h after exposure. The blockage of fork progression after UV exposure was fluence-dependent, with replication segments after exposure to 20 J/m2 being shorter than those observed after exposure to 10 J/m2. Immediately after exposure to either 10 or 20 J/m2, photoreactivation reversed blockage of fork progression, indicating that the (5-6) cyclobutyl pyrimidine dimer is responsible for blockage. This also indicates that blockage of fork progression may not be the only factor responsible for the prolonged depression seen in thymidine incorporation. Three hours after exposure to either 10 or 20 J/m2, replication segments were still significantly shorter than control segments. Photoreactivation completely reversed blockage after exposure to 10 J/m2, but did not completely reverse blockage after exposure to 20 J/m2, indicating that at such fluences, other lesions may play a role in UV-induced blockage of fork progression.

Evaluation of homology between cloned Escherichia coli and yeast DNA photolyase genes and higher eukaryotic genomes.

Repair of ultraviolet-induced pyrimidine dimers by photoreactivation is catalyzed by a single enzyme, DNA photolyase. However, the process of photoreactivation is difficult to detect reproducibly in cultured mammalian cells. We have used clones containing yeast and Escherichia coli DNA photolyase genes to determine whether their sequences are conserved and whether there is homology between either cloned sequence and chick or human genomic DNA and mRNA sequences. The cloned sequences failed to hybridize to each other even under nonstringent conditions, indicating little conservation of sequence between the yeast and E. coli genes. Furthermore, only weak hybridization under nonstringent conditions was found between the cloned photoreactivating genes and human or chick genomic DNA or mRNA. This indicates that there is negligible homology between the cloned probes and mammalian DNA, but we are unable to conclude whether this indicates sequence divergence for prokaryotic and eukaryotic photoreactivation genes or the absence of such genes from the mammalian genome.

Color quenching in "environmentally friendly" cocktails.

Fluorescent and colored compounds used in molecular biology labs may generate color quench in wipe test samples taken from such labs. The ability of conventional chemical quench curves to correct for color quench caused by fluorescent and colored compounds in "environmentally friendly" cocktails has not been reported. Series of colored samples spiked with 3H or 14C were generated for three fluorescent compounds: ethidium bromide; Hoechst 33342; and fluorescein, and three colored compounds: thiazol yellow G; tartrazine; and a mixture of bromphenol blue and xylene cyanol. Three different "environmentally friendly" cocktails were used: one with a linear alkylbenzene solvent; one with a diisopropylnapthalene solvent; and a third with a phenylxylylethane solvent. The data, generated from two different liquid scintillation spectrometers that use different quench indicating parameters, suggest that the quench from the fluorochromes and colored dyes with moderate amounts of quench can be corrected by chemical quench curves. The data also suggest that the quench curve of diisopropylnapthalene-based cocktails is significantly different from the other "environmentally friendly" cocktails, when examined using tSIE as the quench indicating parameter.

Adding water to liquid scintillation cocktail for laboratory wipe tests.

As part of an ongoing laboratory survey process improvement program, we evaluated the addition of water to liquid scintillation cocktail to improve wipe test counting efficiency. Both polar and non-polar 3H and 14C-labeled compounds were used as model contaminants. Our results support the recommendations in the literature regarding the addition of water to scintillation cocktail We found an increase in the counting efficiency of the water-soluble material as a function of water content of the cocktail, but also observed a decrease in the efficiency of detection of the non-polar compound. The offsetting effects are believed to be the result of increased solubility of the polar compounds in water and increased quench of the already solubilized non-polar compound. The finding that adding water to the cocktail brought counting efficiencies of both polar and non-polar molecules to roughly the same value is novel and allows the use of a single quench curve for each radionuclide, regardless of chemical form.

Conformational changes in chromatin structure induced by the radioprotective aminothiol, WR 1065.

WR 1065, 2-[(minopropyl) amino] ethanethiol is an effective scavenger of free radicals. When present during irradiation it reduces cellular DNA damage as analysed by alkaline elution from filters. The same technique indicates that without irradiation, WR 1065 has no effect of DNA integrity. Using nucleoid analysis, where DNA damage is detected at the level of replicon clusters, WR 1065 distorts replicon supercoiling without breaking the DNA molecule. This confirmational change in nucleoid structure occurs with no detectable change in nucleoid protein content. It is proposed that perturbation of replicon supercoiling affects the process of normal DNA synthesis and strand break rejoining, allowing a longer time for the accurate repair of DNA damage.