Discrepancy between the initial DNA damage and cell survival after camptothecin treatment in two murine lymphoma L5178Y sublines

Two L5178Y (LY) murine lymphoma cell sublines, LY-R, resistant, and LY-S, sensitive, to X-irradiation display inverse cross-sensitivity to camptothecin (CPT): LY-R cells were more susceptible to this specific topoisomerase I inhibitor than LY-S cells. After 1 h incubation with CPT, the doses that inhibited growth by 50 per cent (ID₅₀) after 48 h of incubation were 0·54μM for LY-R cells and 1·25 μM for LY-S cells. Initial numbers of DNA–protein crosslinks (DPCs) measured at this level of growth inhibition were two-fold higher in LY-R (5·6 Gray-equivalents) than in LY-S cells (3·1 Gray-equivalents), which corresponds well with the greater in vitro sensitivity of Topo I from LY-R cells to CPT.^1,2 Conversely, the initial levels of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs) were lower in LY-R cells (4·2 Gray-equivalent SSBs and 5·8 Gray equivalent DSBs) than in LY-S cells (8·0 Gray-equivalent SSBs and 12·0 Gray-equivalent DSBs). Dissimilarity in the replication-dependent DNA damage observed after 1 h of treatment with CPT was not due to a difference in the rate of DNA synthesis between the two cell lines, but may have arisen from a substantially slower repair of DNA breaks in LY-S cells.³ Release from G₂ block by caffeine co-treatment significantly increased cell killing in the LY-S subline, and only slightly inhibited growth of LY-R cells. These results show that after CPT treatment cells arrest in G₂, allowing them time to repair the long-lived DSBs. As LY-S cells are slower in repairing the DSBs, they were more susceptible to CPT in the presence of caffeine.     

Repair of gamma-ray-induced base damage in L5178Y sublines is damage type-dependent and unrelated to radiation sensitivity.

The L5178Y (LY) murine lymphoma sublines LY-R and LY-S are differentially sensitive to ionizing radiation. The high radiation sensitivity of LY-S cells is related to impaired rejoining of DNA double strand breaks. We found previously that the gamma-ray-induced base damage is higher in the more radiosensitive LY-S subline. Here, we examine the role of the repair of ionizing radiation induced base damage in relation to the radiosensitivity difference of these sublines. We used the GS/MS technique to estimate the repair rates of six types of base damage in gamma-irradiated LY cells. All modified DNA bases identified in the course of this study were typical for irradiated chromatin. The total amount of initial base damage was higher in the radiation sensitive LY-S subline than in the radiation resistant LY-R subline. The repair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were similar in both cell lines, the repair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the repair of 5-OHUra was faster in its radioresistant counter, the LY-R. Altogether, the repair rates of the y-ray-induced DNA base damage in LY sublines are related neither to the initial amounts of the damaged bases nor to the differential lethal or mutagenic effects of ionizing radiation in these sublines.     

DNA repair,cell cycle progression and cell death following camptothecin treatment in two murine lymphoma L5178Y sublines

The processes involved in cell response to camptothecin (CPT) were investigated in two sublines of L5178Y (LY) murine lymphoma; LY-R, resistant and LY-S, sensitive to X-irradiation, which are inversely cross-sensitive to the drug. The cells were pulse-treated with 2 μM CPT for 1 h; this resulted in equal numbers of replication-related DNA double-strand breaks (DSBs) in both sublines.¹ After drug removal, at different time points up to 24 h, the levels of DSBs were measured by using field inversion gel electrophoresis (FIGE) and comet assay at neutral pH. Both methods revealed faster DSBs repair in LY-S than in LY-R cells, in contrast with X-ray-induced DSBs. This however, was followed by the appearance of secondary breaks in the former subline. The cell cycle arrest was at S/G₂ phase and comprised equal numbers of cells in LY-S and LY-R populations. In both sublines formation of giant cells took place, as well as delayed apoptosis starting about 20 h post-CPT incubation and proceeding with similar intensity. At the same time, the total number of necrotic cells appearing during post-exposure incubation in the LY-R subline exceeded that in the LY-S subline. We suggest that, beside previously documented higher susceptibility of topoisomerase I (Topo I) from LY-R cells to CPT,^2,3 a higher initial rate of replication-related DSBs repair, but not lower propensity to apoptosis, may contribute to the relative CPT resistance of LY-S versus LY-R cells. Copyright © 1998 John Wiley & Sons, Ltd.     

Caffeine-inhibitable control of the radiation-induced G2 arrest in L5178Y-S cells deficient in non-homologous end-joining

The two L5178Y (LY) sublines bear a heterozygous Tp53 mutation that affects its transactivation function. LY-S (radiation-sensitive) cells are deficient in double strand break (DSB) repair by non-homologous end-joining (NHEJ) and do not express p21^WAF1 (Cdkna1) either constitutively or after x-irradiation, in contrast to their radiation-resistant counterpart LY-R cells, which express p21^WAF1 constitutively. Radiation-induced G2 arrest in LY-S cells is very long (11 h/Gy) but 2 mM caffeine treatment shortens it, decreases the fraction of G2 cells and increases the fraction of apoptotic cells. The treatment also increases the DNA damage that is estimated with the comet assay 18 h after irradiation with 5 Gy (ca. 23% of the initial value for x-rays and ca. 47% for x-rays plus caffeine). This indicates that either the repair has not been completed or the apoptotic DNA fragmentation has been initiated (or both). The same treatment applied to x-irradiated (5 Gy) LY-R cells (G2 arrest, 4 h/Gy) has no radiosensitising effect, induces no apoptosis and does not alter the amount of DNA damage left unrepaired (ca. 28%). The results are compatible with the assumption that inhibition of the Atm-dependent homologous recombination repair by caffeine, brings differential effects in LY sublines because of the defect of the alternative DNA repair system (NHEJ) in LY-S cells. Received: 23 June 2000 / Accepted: 5 January 2001     

Differential induction of apoptosis in x-irradiated L5178Y sublines bearing p53 mutation

We examined apoptosis and expression of p53, E2F-1, bax, bclx_L and bcl2 proteins in two L5178Y (LY) murine lymphoma sublines, LY-R and LY-S, which differ in radiosensitivity and double-strand break (DSB) repair. Both sublines are heterozygous for a p53 mutation in codon 170 that precludes the transactivation function. Accordingly, there is no G1/S arrest after irradiation.We found that there is no change in expression of E2F-1, bax, bclx_L or bcl2 proteins in both LY sublines after x-irradiation. LY-R cells do not constitutively express bcl2, whereas both sublines show high bax content. Radiation induces delayed apoptosis to a greater extent in LY-S than in LY-R cells. The apoptosis can be seen 24 h after irradiation (2 Gy) of LY-S cells, with a maximum at 48 h. LY-R cells need 5 Gy and 72 h post-irradiation incubation to show marked apoptosis (identified by the TUNEL method). The reported observations support the assumption that differential radiosensitivity of LY sublines is associated with the induction of apoptosis that is not related to transactivation by p53 and is primarily related to differential DNA repair ability. Received: 19 August 1999 / Accepted in revised form: 30 November 1999     

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.     

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.     

Novobiocin treatment reverses radiation-induced alterations in higher-order DNA structure in L5178Y nucleoids

We have studied the effect of novobiocin treatment on radiation-induced damage and its repair in higher-order DNA structure in two mouse leukemia cell lines differing in their radiosensitivity, L5178Y-R (LY-R) and L5178Y-S (LY-S). We used the fluorescent halo technique to measure alterations in the superhelical density and the topological constraints of DNA in LY-R and LY-S nucleoids. The results for untreated cells show that both cell lines reached maximal DNA unwinding at the same concentration of propidium iodide (PI), whereas LY-S nucleoids were less efficient in their ability to rewind their DNA. The loop size did not differ significantly between the cell lines. Incubation of LY-R and LY-S cells with novobiocin at a concentration which does not influence survival (0.1 mM for 45 min), but inhibits DNA synthesis in LY-R cells (by 28%) to a greater extent than in LY-S cells (by 10%), also causes more DNA unwinding in LY-R nucleoids than in LY-S nucleoids. However, a decreased superhelical density was observed in nucleoids from both cell lines. Novobiocin applied before, and present during, irradiation prevents radiation-induced alterations in DNA supercoiling more efficiently in LY-R than in LY-S cells. The presence of novobiocin during the repair period increased DNA rewinding to levels not significantly different from control values in nucleoids from both cell lines.     

Recovery from potentially lethal damage of irradiated L5178Y-R and L5178Y-S cells: the effect of temperature and benzamide

Mouse lymphoma L5178 Y-S and Y-R cells differing in radiosensitivity by 1.5 times were treated with benzamide, an inhibitor of poly(ADP-ribosylation), for 24 h before and 18 h after X-irradiation, and incubated after irradiation at 25 degrees C and 37 degrees C. Clonogenic capacity of LY-S cells incubated at 25 degrees C exceeded that of the same cells incubated at 37 degrees C; the clonogenic capacity of LY-R cells did not vary with the postirradiation incubation temperature. Benzamide increased equally the radiosensitivity of LY-R cells incubated at both temperatures, whereas that of LY-S cells was only increased at 37 degrees C. Repair of potentially lethal damages to LY-S cells incubated at 25 degrees C was independent of the effectiveness of poly(ADP-ribosylation).     

The response of L5178Y lymphoma sublines to oxidative stress: antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-kappaB

We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.     

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     

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.     

Labile iron pool correlates with iron content in the nucleus and the formation of oxidative DNA damage in mouse lymphoma L5178Y cell lines

Labile iron pool (LIP) constitutes a crossroad of metabolic pathways of iron-containing compounds and is midway between the cellular need for iron, its uptake and storage. In this study we investigated oxidative DNA damage in relation to the labile iron pool in a pair of mouse lymphoma L5178Y (LY) sublines (LY-R and LY-S) differing in sensitivity to hydrogen peroxide. The LY-R cells, which are hydrogen peroxide-sensitive, contain 3 times more labile iron than the hydrogen peroxide-resistant LY-S cells. Using the comet assay, we compared total DNA breakage in the studied cell lines treated with hydrogen peroxide (25 microM for 30 min at 4 degrees C). More DNA damage was found in LY-R cells than in LY-S cells. We also compared the levels of DNA lesions sensitive to specific DNA repair enzymes in both cell lines treated with H(2)O(2). The levels of endonuclease III-sensitive sites and Fapy-DNA glycosylase-sensitive sites were found to be higher in LY-R cells than in LY-S cells. Our data suggest that the sensitivity of LY-R cells to H(2)O(2) is partially caused by the higher yield of oxidative DNA damage, as compared to that in LY-S cells. The critical factor appears to be the availability of transition metal ions that take part in the OH radical-generating Fenton reaction (very likely in the form of LIP).     

Chelating of iron and copper alters properties of DNA in L5178Y cells, as revealed by the comet assay.

We have previously found different proportions of iron and copper in nuclei of two sublines of murine lymphoma L5178Y (LY) and proposed a model of chromatin organization with these metal ions at the DNA attachment sites. We now examine the effect of chelators, desferal (DFO, iron-specific) and neocupreine (NEO, copper-specific) on DNA of LY-R and LY-S cells, using the comet and micronuclei frequency tests. There is less copper and more iron in LY-R nuclei than in LY-S nuclei. Accordingly, the effect of NEO is more marked in LY-R than in LY-S cells and in both sublines it is expressed as enhanced tail moment (measure of DNA damage in the comet assay) and increased micronuclei frequency. On the contrary, the effect of DFO on the tail moment is less pronounced in LY-R than in LY-S cells. With increasing DFO concentrations, there is a gradual decrease in the tail moment values below the control level in LY-S cells. In LY-R cells the tail moment values initially increase, then gradually decrease, eventually falling below the control level. This points to a dramatic conformational change that masks the effect of DNA discontinuities. The presence of the latter is indicated by the increase in micronuclei frequency. These results support the postulated differential role of iron and copper ions in maintaining the higher order DNA structure in LY sublines.     

PKA controls a level of topoisomerase I mRNA in mouse L5178Y lymphoma cells treated with db-cAMP

The level of topoisomerase I mRNA was measured in cells of two mouse lymphoma (LY) sublines treated with db-cAMP. A transient increase of the level was observed to be of about 60% of the basic level and to have maximum after the 3 h treatment of LY-S cells. The increase in LY-R subline was two-fold lower. The activity of PKA in a cytosol fraction of LY-S cells was 1.75 times higher than that in LY-R cells. The activity of PKA in membranes and nuclear fraction did not differ significantly in both cell types. When the activity of PKA in LY-S cells was inhibited with H8, no increase of the level of topoisomerase I mRNA was observed upon db-cAMP treatment of cells. We suggest that the activity of PKA in the cytosol controls the expression of topoisomerase I gene in LY cells at high concentration of cAMP.Abbreviations db-cAMP dibutyryl-cAMP - H8 N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide - LY mouse L5178Y lymphoma - PKA protein kinase A - topo I topoisomerase I     

Benzamide-inhibitable alterations in spectral properties of chromatin accompany DNA repair in UVC-exposed L5178Y cells

Summary We examined the response of chromatin to increasing NaCl and MgCl₂ concentrations in UVC-irradiated L5178Y (LY) R and S cells, using the spectral index method (Dixon and Burkholder 1985). We have found an alteration in chromatin properties 1 h after UVC-irradiation of repair proficient LY-S cells, but no change in repair deficient LY-R cells. The change was shown as lowered spectral index, indicating that at given Na⁺ and Mg⁺⁺ concentrations (1 or 200 mM NaCl, 0 or 0.5 mM MgCl₂) chromatin from UVC-irradiated LY-S cells was more compact than that from unirradiated ones. Benzamide treatment reversed the effect of UVC-irradiation in LY-S cells and did not change the response pattern of chromatin from LY-R cells or unirradiated LY-S cells.     

Nuclear translocation of the p65 subunit of NF-κB in L5178Y sublines differing in antioxidant defense

We examined the induction of nuclear translocation of the p65 subunit of NF-κB in L5178Y (LY) cells. We used two LY sublines which are inversely cross-sensitive to hydrogen peroxide and x-rays: LY-R cells are radioresistant and oxidant-sensitive, whereas LY-S cells are radiosensitive and oxidant-resistant. Hydrogen peroxide, phorbol ester and x-rays caused a marked translocation of p65-NF-κB in LY-R cells and a weak translocation in LY-S cells. By manipulating the antioxidant defense status, we obtained an alteration in the p65-NF-κB translocation induction in LY-R cells. A similar effect was achieved with lovastatin pretreatment (25 μM, 24 h, 37°C). The response of LY-S cells under all these conditions was considerably weaker. We conclude that differential nuclear translocation of p65-NF-κB in LY sublines is not related to the lethal effect of the activating, damaging agent; rather it is due to the more efficient antioxidant defense in LY-S than in LY-R cells. Received: 10 November 1998 / Accepted in revised form: 2 March 1999     

Hydrogen peroxide induced reproductive and interphase death in two strains of L5178Y murine lymphoma differing in radiation sensitivity

Summary L5178Y-R (LY-R) and L5178Y-S (LY-S) cells, differing in radiation sensitivity and susceptibility to the radiosensitizing effect of benzamide (Bz) were examined for susceptibility to hydrogen peroxide. Survival and chromatid aberration frequency indicated that LY-R cells were considerably more sensitive to H₂O₂ than LY-S cells. So, LY strains were found to be inversely crosssensitive to X/ rays and H₂O₂. The relative resistance to H₂O₂ corresponded with the previously found twofold difference in catalase activity (Jaworska et al. 1987). At higher concentrations H₂O₂ treatment caused interphase death, that was delayed by benzamide (Bz, 2 mM), an inhibitor of po1y(ADP-ribosylation), to a lesser extent in the more resistant cell subline (LY-S). From the examination of the H₂O₂ induced increase in the free Ca²⁺ concentration (with or without 2 mM Bz treatment) with the use of Fura-2 it followed, that the cells responded to the oxidative stress by Ca²⁺ release. The Ca²⁺ concentration increase was neither directly related to the killing effect of H₂O₂ treatment, nor did it correspond with the twofold difference in catalase activity in LY strains.     

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.     

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.