Modulation of the effect of camptothecin in x-irradiated L5178Y-R and L5178Y-S cells by benzamide

The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 μM CPT enhanced the radiosensitivity of LY-S cells (D₀ decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly(ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D₀ decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e. g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases α and δ) applied concomitantly with CPT in cells not pre-treated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication. Received: 28 November 1995 / Accepted in revised form: 20 April 1996     

Effect of UVC and araC on L5178Y-R and L5178Y-S cells. Nucleoid sedimentation

The effects of UVC radiation (lambda = 254 nm, 85 J/m2) and/or 1-beta-D-arabino-furanosylcytosine (araC, 2 x 10(-3) M, 2 h) on two mouse lymphoma cell lines, UVC-sensitive and X-ray resistant L5178Y-R and UVC-resistant and X-ray sensitive L5178Y-S, were investigated. AraC treatment inhibited the semiconservative DNA replication to 1.4% and 3.8% in L5178Y-R and L5178Y-S cells, respectively, and decreased the sedimentation distance of nucleoids from the cells of both lines. The shortening of sedimentation distances induced by UVC and araC treatment was 8.1 mm for L5178Y-R cells and 11.8 mm for L5178Y-S, and indicated a higher number of DNA breaks in L5178Y-S cells. Assuming that such breaks are the result of the inhibition of DNA repair replication by araC, we conclude that L5178Y-S cells have a greater number of repaired sites than L5178Y-R cells.     

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.     

Ion content and the response of L5178Y-R and L5178Y-S cells to X rays and ionophore A23178

Summary We examined the response of L5178Y-S (radiosensitive, LY-S) and L517SY-R (radioresistant, LY-R) lymphoblasts to X-irradiation with concomittant treatment with divalent cation ionophore, A23187 (3 h or 5 h, 5 µg/ml). Cells treated with A23187 alone progressed through the cell cycle more slowly than the untreated cells and their cloning efficiency was reduced. In both cell strains the ionophore prolonged duration of the postirradiation mitotic delay. Radiation-induced inhibition of DNA synthesis was reversed by A23187 in LY-S but not in LY-R cells.Cells subjected to the ionophore treatment survived X-irradiation in almost the same way as untreated cells, as if the effect of A23187 treatment were reversed by irradiation. There was also a reversion in the ion content: A23187 caused a marked increase in Na⁺ content and a decrease in K⁺ content, irradiation itself did not change the ion content, whereas in the A23187-treated cells it restored almost the same pattern as that found in the control cells. We found less Mg²⁺ ions in LY-S cells after treatment with A23187 and A23187 + X than in LY-R cells, in relation to untreated (control) cells. These observations point to the possible importance of ion transport for recovery from radiation damage.     

DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells

DNA supercoiling ability was assayed following irradiation in two cell lines of differing radiosensitivity, L5178Y-S (LY-S) and L5178Y-R (LY-R). Cells treated with NaCl and Triton X-100 were exposed to increasing concentrations of the fluorescent, DNA-intercalating dye, propidium iodide (PI), and the diameter of the resulting fluorescent halo of DNA was measured. As the PI concentration was increased from 0.5 to 5 micrograms/ml, halo diameter increased from 20-25 to 45-55 microns due to the unwinding of the DNA supercoils. This process was similar for both cell lines under all conditions studied. As the PI concentration was increased to 50 micrograms/ml, the halo rewound to a diameter of 25-30 microns in unirradiated cells from both lines. However, following exposure to 3-12 Gy of 137Cs gamma rays, the ability of the DNA to be rewound was inhibited in a dose-dependent manner. Rewinding inhibition was greater in LY-S cells than in LY-R cells. Since the induction of DNA damage (e.g., single-strand DNA breaks) appears to be the same for both cell lines, this result implies that a similar extent of damage results in a greater loss of topological constraints on the DNA loops in LY-S. Such a change might be related to the protein composition of the nucleoid cores. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that nucleoids from LY-S cells were missing a 55-kDa protein present in LY-R.     

Damage at two levels of DNA folding measured by fluorescent halo technique inX-irradiated L5178Y-R and L5178Y-S cells

Summary We 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 (D ₀ = 1.1 Gy) and LY-S more sensitive (D ₀ = 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.     

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

In the preceding paper we described the properties of nucleoids analyzed with the fluorescent halo assay at pH 6.9 and 9, as well as in the presence of reducing and chelating agents and after X-irradiation. We found analogies between the properties of type I and II nucleoids, as examined by Lebkowski and Laemmli (1982), and nucleoids analyzed with the fluorescent halo assay. We concluded that radiation-inflicted damage at two levels of DNA folding is measured at pH 6.9 and 9. In this paper we examined repair of damage to the nucleoid structure as assayed by the fluorescent halo method in X-irradiated L5178Y (LY) sublines; R (radiation resistant,D ₀=1.4 Gy) and S (radiation sensitive,D ₀=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 and 9. The ability of DNA to be rewound at 10–50 µg/ml of PI was impaired by X-irradiation and partly restored during 90-min post-irradiation incubation, indicating damage to the superhelical structure and its partial restoration. The exponential time constants for repair were 10.1 min (LY-S, 6 Gy), 11.2 min (LY-R, 12 Gy), and 20.3 min (LY-s, 12 Gy) when measured at pH 9. In X-irradiated (12 Gy) LY-S cells, slower restoration of DNA supercoiling was observed at pH 9 than at pH 6.9. The presence of labile lesions at pH 9 did not prevent restoration of the higher-order DNA structure, as estimated from DNA rewinding at pH 6.9 in LY-S cells.Work performed at SUNY-Health Science Center at Brooklyn     

The repair of potentially lethal damage and sublethal damage in strains of mouse L5178Y lymphoma cells differing in radiation sensitivity

Mouse lymphoma strains L5178Y-R (LY-R) and L5178Y-S (LY-S), which are differentially sensitive to the cytotoxic effects of ionizing radiation, were found to differ in their abilities to repair potentially lethal damage (PLD) and sublethal damage (SLD). The results showed that strain LY-R was more proficient than strain LY-S in the repair of SLD. The split dose recovery observed in strain LY-S could be accounted for by its recovery during postirradiation incubation. In contrast, SLD repair occurred in the absence of PLD repair in strain LY-R. The possibility that the repair of PLD might be completed prior to the postirradiation incubation in strain LY-R was suggested by the decreased survival observed when the cells were irradiated in a hypotonic solution. The repair of PLD and SLD in strain LY-S was temperature sensitive, occurring during postirradiation incubations between 15 and 34 degrees C, but not at 37 or 40 degrees C. This temperature sensitivity is very similar to the temperature sensitivity of the repair of pH 9.6-labile lesions in DNA in strain LY-S, as reported previously. Thus postirradiation cellular recovery processes in strain LY-S may involve the repair of pH 9.6-labile lesions in DNA. Temperature-dependent changes in the postirradiation distribution of cells throughout the cell cycle were observed which could contribute to the temperature sensitivity of the postirradiation recovery of strain LY-S.     

Recovery from potentially lethal damage and recruitment time of noncycling clonogenic cells in 9L confluent monolayers and spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.     

Recovery from sublethal and potentially lethal damage in an X-ray-sensitive CHO cell

It has been suggested that DNA strand breaks are the molecular lesions responsible for radiation-induced lethality and that their repair is the basis for the recovery of irradiated cells from sublethal and potentially lethal damage. EM9 is a Chinese hamster ovary cell line that is hypersensitive to killing by X rays and has been reported to have a defect in the rate of rejoining of DNA single-strand breaks. To establish the importance of DNA strand-break repair in cellular recovery from sublethal and potentially lethal X-ray damage, those two parameters, recovery from sublethal and potentially lethal damage, were studied in EM9 cells as well as in EM9's parental repair-proficient strain, AA8. As previously reported, EM9 is the more radiosensitive cell line, having a D0 of 0.98 Gy compared to a D0 of 1.56 Gy for AA8 cells. DNA alkaline elution studies suggest that EM9 cells repair DNA single-strand breaks at a slower rate than AA8 cells. Neutral elution analysis suggests that EM9 cells also repair DNA double-strand breaks more slowly than AA8 cells. All of these data are consistent with the hypothesis that DNA strand-break ligation is defective in EM9 cells and that this defect accounts for increased radiosensitivity. The kinetics and magnitude of recovery from sublethal and potentially lethal damage, however, were similar for both EM9 and AA8 cells. Six-hour recovery ratios for sublethal damage repair were found to be 2.47 for AA8 cells and 1.31 for EM9 cells. Twenty-four-hour recovery ratios for potentially lethal damage repair were 3.2 for AA8 and 3.3 for EM9 cells. Both measurements were made at approximately equitoxic doses. Thus, the defect in EM9 cells that confers radiosensitivity and affects DNA strand-break rejoining does not affect sublethal damage repair or potentially lethal damage repair.     

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

Summary The 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.A preliminary report of this work was presented at the 35th Annual Meeting of the Radiation Research Society, Atlanta, GA 1987, USA     

Aphidicolin promotes repair of potentially lethal damage in irradiated mammalian cells synchronized in S-phase

Aphidicolin, an inhibitor of the α-polymerase in mammalian cells, at a concentration of 0.5 μg/ml, is shown to enable cells which are growing exponentially and synchronized in the S-phase of the cell cycle, to repair potentially lethal damage caused by exposure to either x-rays or UV-light. The drug holds cells up in the S-phase which may serve to allow time for repair and could prevent fixation of damage which may occur when the cells progress through the cell cycle. The possible involvement of α- and β-polymerase in repair of potentially lethal damage is discussed.     

The action of caffeine on X-irradiated HeLa cells. VIII. Recovery from potentially lethal damage

Recovery from potentially lethal radiation damage in HeLa S3 cells has been studied by irradiating synchronous cultures with 4 Gy at selected ages in the cell cycle, initiating treatment with 4 mM caffeine, which prevents recovery, at progressively later times up to 24-30 h after irradiation, and determining the plateau level of survival after incubation with the caffeine until 36-40 h after mitotic collection. Cell recovery appears to begin immediately after irradiation at any time during interphase: an accelerating increase in survival gives way after several hours to a linear increase which lasts for an additional several hours. The median recovery time is approximately 13 h after irradiation at any time during G1, but is markedly shorter (5-7 h) after irradiation in S or G2. The rate of recovery is slightly depressed if DNA replication is inhibited with aphidicolin after irradiation and slightly enhanced if protein synthesis is inhibited with cycloheximide. Both the rate and the extent of recovery are dependent on the location of the cells in the cycle at the time of irradiation--both functions increasing with cell age from the beginning of S, but having different age dependencies in G1. Blocking cell progression with a DNA-synthesis inhibitor before irradiation halts the age-dependent changes.     

Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts

The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells.     

Evidence for the repair of potentially lethal damage in irradiated bone marrow

Summary The effects on cell survival of maintaining bone marrow cells (CFU-S) in situ following irradiation and before assay by transplantation was investigated. When the CFU-S cells are maintained in situ following irradiation survival drops and plateaus at about 9 h post-irradiation. Evidence is presented that this decrease in survival may be due to potentially lethal damage repair (PLD) inhibition caused by post-irradiation in situ holding. This effect on PLD repair is different than that usually found in cells in vitro and in vivo tumors in that it mainly alters the shoulder rather than the slope of the survival curve of CFU-S cells. It is different than PLDR found in vivo for normal mammary and thyroid gland epithelial cells because in situ holding decreases rather than increases the survival of CFU-S cells. Evidence is also presented that the radiation survival curve for in situ bone marrow cells (CFU-S) may not have a shoulder.Supported in part by NIH, NCI grants P01 CA 19298 and P30 CA 14520Supported in part by an American Cancer Society Clinical Fellowship     

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.     

Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells

The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.     

The effect of pH on potentially lethal damage recovery in A549 cells

The radiation sensitivity and potentially lethal damage recovery (PLDR) capacity of A549 human lung carcinoma cells have been studied. For unfed monolayer cultures, radiation sensitivity was greater in plateau phase than in log phase of growth. PLDR was observed when plateau-phase cells were held in their own spent medium postirradiation, such that the dose-response curve with 24 h holding was similar to that for log-phase cells plated immediately after irradiation. The high PLDR capacity of A549 plateau-phase cells (recovery factor between 40 and 70 for 24 h holding after 10 Gy) was reduced 10-fold or more by alkalinizing the pH of the spent medium immediately after irradiation from a value of 6.5 +/- 0.1 to a value of 7.6. Medium alkalinization resulted in an increase in the rate of glycolysis, with subsequent reacidification to a pH of 7.3 within 2 h of the pH adjustment. No change in cell cycle distribution was observed in the plateau-phase cultures up to 32 h after change of medium pH, and no increase in cell density was found after 48 h. A slight increase in the rate of incorporation of radiolabeled thymidine into acid-precipitable material was observed at 4 and 24 h after alkalinization of the medium. While it is not possible at present to define a mechanism for this pH effect, our results demonstrate that, at least for this cell line, variables such as medium pH and glucose concentration can profoundly influence the observation of PLDR.