Maria Kapiszewska

Content of iron and copper in the nuclei and induction of pH 9-labile lesions in L5178Y sublines inversely cross-sensitive to H2O2 and x-rays

Cells from the L5178Y murine lymphoma subline LY-R are twice as resistent to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by H2O2, the effect being more pronounced at 37 °C than 0 °C. Initial DNA damage after H2O2 treatment (both temperatures, 5 min) has been estimated by the ‘comet’ assay (single-cell gel electrophoresis) and fluorescent halo technique. According to both methods, the initial damage is significantly higher in LY-R cells, particularly that inflicted at O °C. Differences between DNA unwinding and rewinding abilities at pH 9 and 6.9 (estimated by the fluorescent halo technique) point to a considerable difference in pH-9-labile damage between the sublines, as observed previously for x-irradiated cells (Kapiszewska et al. 1992). In contrast to findings with x-irradiated cells, however, after H2O2 treatment this damage is more extensive in LY-R cells than in LY-S cells. Thus, the initial pH-9-labile damage corresponds to the pattern of sensitivity to H2O2 and x-rays. We suggest that this is caused by different proportions of cuprous and ferric ions found in the nuclei of LY sublines and by the different ability of these ions to react with H2O2 and water radiolysis products. The copper/iron ratio in the nucleus is 1.31 in LY-R cells and 4.84 in LY-S cells.

Prolonged Quercetin Administration Diminishes the Etoposide-Induced DNA Damage in Bone Marrow Cells of Rats

The DNA damage in bone marrow cells induced by etoposide (E) injected intraperitoneally to rats (100 mg/kg b.w.) decreased to the control level when quercetin (Q) was administered subcutaneously for 10 consecutive days (40 mg/kg b.w.per day) before E was injected. The antioxidant power (FRAP assay) increased significantly after Q or E compared with control rats but did not change when Q preceded the E injection. The superoxide dismutase activity significantly increased in Q+E-treated rats compared with quercetin given alone. The study provides evidence that Q protects bone marrow cells against long-lived E-induced DNA damage and alters the redox balance in lung tissue.

Reduced sensitivity to camptothecin of topoisomerase I from a L5178Y mouse lymphoma subline sensitive to X-radiation

Murine L517BY (LY) lymphoma sublines, LY-R (X-radiation resistant) and LY-S (X-radiation sensitive) displayed a difference in susceptibility to camptothecin: susceptibility of LY-S cells to the alkaloid was shifted towards higher concentrations as compared to LY-R cells. A similar difference was observed at the level of genomic DNA when a number of DNA-protein cross-links was determined or single-strand breaks were revealed by the fluorescent nucleoid halo assay. Activities of topoisomerases I and II were the same in both sublines. In turn, a higher resistance to camptothecin was found for the isolated LY-S topoisomerase I in the DNA cleavage test, suggesting that an altered enzyme was responsible for the susceptibility difference observed at the cellular level. In the relaxation test the enzymes from the two sublines showed a different sensitivity to beta-lapachone, an activator of topoisomerase I, but were similarly sensitive to all inhibitors, except camptothecin.

Damage at two levels of DNA folding measured by fluorescent halo technique in X-irradiated L5178Y-R and L5178Y-S cells. II. Repair.

SummaryWe examined, by the fluorescent halo assay, alterations in the nucleoid structure (structure formed from cells under mild lysis conditions: in non-ionic detergent TritonX-100, 0.0005% and 1.5 mol/1 NaCl) of L5178Y (LY) cell sublines which had been untreated, treated with reducing/chelating agents (ß-mercaptoethanol or sodium diethyl dithiocarbamate (DDTC(Na))) or X-irradiated. These sublines differ in radiation sensitivity: LY-R is more resistant (D0 = 1.1 Gy) and LY-S more sensitive (D0 = 0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI)(0.5 to 50 µg/ml) at pH 6.9 or 9. The maximal DNA unwinding in PI was obtained at 7.5 µg/ml PI, at both pH 6.9 and 9 in both sublines; the maximal halo diameter was larger in LY-S than in LY-R cells. In nucleoids from both sublines DNA could be rewound at higher (10–50 µ/ml) PI concentrations both at pH 6.9 and 9. This ability was impaired by mercaptoethanol or DDTC(Na) (at pH 9) or by X-irradiation, indicating damage and/or alteration in the DNA superhelical structure. The susceptibility to reducing/chelating agents was greater in LY-S than in LY-R nucleoids, pointing to differences in chromatin structure between these sublines. The amount of X-ray-inflicted damage was higher, when measured at pH 9 than at pH 6.9 and was about twice larger in LY-S than in LY-R nucleoids, when the cells were irradiated with the same X-ray dose.From analogies between the behaviour of nucleoids under the above-described conditions and nucleoid type I and II sedimentation, as examined by Lebkowski and Laemmli (1982) we conclude that damage at two levels of DNA folding is measured at pH 6.9 and 9.

The effects of reduced temperature and/or starvation conditions on the radiosensitivity and repair of potentially lethal damage and sublethal damage in L5178Y-R and L5178Y-S cells.

Potentially lethal damage (PLD) and sublethal damage (SLD) modification in L5178Y-S(LY-S) and L5178Y-R (LY-R) cells was investigated for postirradiation holding in either plateau phase or log phase at either 25 or 37 degrees C. Incubation in both plateau and log phases increased PLD repair (PLDR) at 25 degrees C but increased PLD fixation (PLDF) at 37 degrees C in LY-S cells, with the opposite result (PLDR at 37 degrees C, PLDF at 25 degrees C) in LY-R cells. Conditioned medium (CM from plateau-phase cells) had only a slight effect on the radiosensitivity of log-phase LY-S cells. CM was highly toxic to log-phase LY-R cells even without radiation. Postirradiation incubation of log-phase cells at 25 degrees C increased PLDF between 5.5 and 18 h in LY-R cells, whereas PLDR was completed by 5.5 h in LY-S cells. Flow cytometric data show that the LY-R vs LY-S plateau-phase PLD results are not caused by a differential cell cycle distribution. Although all cell cycle stages are found in the plateau-phase cells, G1 is enriched and cells in all stages must be slowly cycling or stationary. Split doses decrease survival of LY-S cells at 37 degrees C but maintain survival at the single acute total dose level at 25 degrees C (no SLD repair (SLDR)). SLDR is observed at both 25 and 37 degrees C in LY-R cells. The results for both the split dose and delayed plating experiments are best understood in terms of competition between PLD repair and fixation with their relative contributions depending on cellular metabolism, which can be altered by changes in temperature or medium constituents. The opposite PLD results obtained for LY-S and LY-R cells suggest that radiosensitivity in these cells is determined by two different mechanisms.

Changes in bleb formation following hyperthermia treatment of Chinese hamster ovary cells.

Chinese hamster ovary cells in suspension cultures were heated for various times at 41.5, 43.5, and 45.5 degrees C, and quantitative determinations of microblebbing and macroblebbing of the cell membrane were performed for cells maintained at 4, 25, and 37 degrees C after hyperthermia. The percentage of cells with blebs following heating at 45.5 degrees C was dependent upon the duration of heating with increases from 40% for 5 min to 90% for 30 min. Cells exposed to lower temperatures exhibited less blebbing which was not quantifiable. The changes in bleb formation following 45.5 degrees C were dependent upon the posthyperthermia temperature: a slight decrease of macroblebbing at 25 degrees C, a decrease to 50% by 2 h at 37 degrees C, and a sharp decrease of macroblebbing to less than 10% by 1 h at 4 degrees C. Microblebbing increased slightly at 37 degrees C. When cells were transferred rapidly from the 4 degrees C posthyperthermia incubation to 37 degrees C, the bleb formation percentages returned rapidly to the higher levels which existed before posthyperthermia incubation at the lower temperatures. Gamma irradiation of 20 and 50 Gy produced only a small increase in microblebbing at longer periods (5 to 6 h) but no increase in macroblebbing. The survival of cells heated for 20 min at 45.5 degrees C was decreased 40% for suspension cells maintained at 4 degrees C for 2 to 3 h before incubation at 37 degrees C for colony formation compared to cells immediately incubated at 37 degrees C after heating. The survival of cells maintained at 25 degrees C after heating was not altered in comparison.

Mechanisms of membrane damage for CHO cells heated in suspension

SummaryChinese hamster ovary cells were heated for 20 min at 45.5°C in different conditions, and quantitative determinations of cellular membrane blebbing were performed for cells maintained at 25°C and 37°C after hyperthermia. The percentage of cells with blebs following heating was dependent upon the composition of the medium during heating and the posthyperthermia temperature after heating. The total extent of bleb formation after heating was independent of the calcium-ion concentration in the medium during heating; however, differences in the kinetics of bleb disappearance after heating point to the importance of Ca2+ concentration in the expression of heat damage. Without hyperthermia, blebs were formed on the cell-surface membrane with agents which block sulfhydryl groups or release calcium from cellular stores. The cells were protected from bleb formation when cells were incubated with glutathione before addition of sulfhydryl-blocking agents or heat treatment. Oligomycin did not prevent the formation of blebs, suggesting that this phenomenon is not energy-dependent. Only a small percentage of cells were covered with blebs when they were heated in saline solution. When cells were incubated with dbcAMP before heat, blebs did not appear at 25°C. A possible interpretation for these observations is presented.

The effect of hypothermic treatment on the repair or expression of X-ray damage measured in split dose experiments on L5178Y-S cells

SummaryThe nature of the post-irradiation lesions and processes leading to cellular reproductive death or survival were investigated in mouse lymphoblastic leukemia L5178Y-S (LY-S) cells. Post-(x-)irradiation incubation at 25° C protects LY-S cells against the fixation of biologically expressed damage which takes place at 37° C. An optimal condition for the repair of damage, assayed in split-dose experiments as split-dose recovery (SDR), is 1 h at 37° C followed by 4 h holding at 25° C prior to the second half of a split dose, or 5 h holding at 25° C without a 37° C incubation during the interval between doses. Longer incubations at 37° C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37° C was observed only during the first 30 min; thereafter,3H-dThdR incorporation washigher than in unirradiated controls. Theexcess synthesis effect was removed by shifting irradiated cells to 25° C holding. The inhibition observed at 25° C was reversed by shifting to 37° C. Thus the degree of postirradiation DNA synthesis is inversely related to SDR. DNA filter elution shows complete strand break repair by 20 min at 37° C, and by 3 h at 25° C; DNA double-strand break (DSB) repair plateaus at 80% (37° C) and 60% (25° C) after 90 min. An inverse correlation was found between total strand break repair rate, as assayed by filter elution methods, and cell survival. This work was supported by a grant from The Mathers Charitable Foundation.

Heat-induced inhibition of DNA synthesis in L5178Y-S cells is reversed by caffeine☆

Heating L5178Y cells for 15 min at 43 degrees C caused a decrease in [3H]thymidine incorporation, which could be reversed by post-treatment with 0.75 mM caffeine in an L5178Y-S (radiation-sensitive, heat-resistant) but not in an L5178Y-R (radiation-resistant, heat-sensitive) strain. The reversal was accompanied by a sparing effect of the treatment: survival of L5178Y-S cells increased by a factor of 1.5. The effect of combined (heat + caffeine) treatment of L5178Y-R cells was cumulative.