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     

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).     

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.     

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.     

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     

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.     

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     

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.     

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.     

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     

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.     

Differential poly(ADP-ribose) metabolism in repair-proficient and repair-deficient murine lymphoma cells

The UV254nm-sensitive, tumorigenic murine lymphoma cell line LY-R undergoes spontaneous conversion into a UV254nm-resistant, non-tumorigenic LY-S subline after prolonged culture in vitro. Here we describe that this conversion leads to distinct changes in several features of intranuclear ADP-ribose polymer metabolism, which may contribute to the altered processing of DNA damage in these cells. The UV254nm-resistant LY-S cells contain 3-fold higher levels of ADP-ribose polymers than LY-R cells. The initial catabolic rate of degradation of these polymers is more than 6-fold higher in LY-S cells. UV254nm irradiation raises the catabolic rates of ADP-ribose polymers in both cell lines. As a consequence, the polymer half-lives decrease from 15 min to 4 min in LY-S cells, and from 96 min to 19 min in LY-R cells. In addition, the rapidly turning over fraction of polymers is much larger in the UV254nm-resistant LY-S cells. These data suggest that the catabolism of poly(ADP-ribose) may be an important factor in the biological expression of DNA damage.     

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     

Does the cellular labile iron pool participate in the oxidation of 2',7'-dichlorodihydrofluorescein?

The fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF-DA) is widely used for the estimation of oxidative stress in cells. It is known that 2',7'-dichlorodihydrofluorescein (H₂DCF), product of intracellular hydrolysis of H₂DCF-DA, is oxidized to the fluorescent compound, DCF, mainly by hydrogen peroxide (H₂O₂) in the presence of catalysts. The present study was aimed at answering the question whether the labile iron pool (LIP) may contribute to the oxidation of H₂DCF in cellular systems. The membrane-permeable lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) was found to inhibit oxidation of the probe by H₂O₂ dependent on ferrous ions but not by peroxidase or superoxide dismutase in defined in vitro systems. When applied to cells, the probe inhibited considerably oxidation of H₂DCF in V79 Chinese hamster fibroblasts and two murine lymphoma L5178Y(LY) sublines (LY-R, LY-S) differing in LIP level, the extent of inhibition being greater in the LY-R line of higher LIP level. These results demonstrate that LIP is a significant factor determining the rate of intracellular H₂DCF oxidation.     

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.     

Does ionizing radiation induce an adaptive response in mouse L5178Y lymphoma cells?]

Growth kinetics of LY-S and LY-R cells (radiosensitive and radioresistant sublines of murine lymphoma L5178Y) has been investigated after beta-irradiation at cumulative doses of 1.5 to 20 cGy and dose rates of 0.8-10 mGy/h. It has been found that after 48 h culture in a complete medium the number of cells differed 5 times, whereas after X- and gamma-irradiation, Do values differed 1.62 times. Using the growth rate as the end point in evaluating the combined effect of beta-irradiation (10 cGy) and subsequent X-irradiation with lethal doses, we observed an increased relative cell number, in comparison to that after X-irradiation alone (an "adaptive response", using this criterion), in LY-S cells irradiated with a dose of 2 Gy. In contrast, when reproductive death of LY-S and LY-R cells the end-point analyzed, the lethal effect of consecutive beta- and X-irradiation in LY-S cells was higher than that expected for X-radiation alone (the synergistic effect).     

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.     

Relationship between topoisomerase II and radiosensitivity in mouse L5178Y lymphoma strains

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radiation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and ellipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors. m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.     

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.     

Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells

In previous studies, the two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. Thus, in comparison to strain LY-R, strain LY-S has been found to be more sensitive to the lethal effects of ionizing radiation, more resistant to the lethal effects of UV radiation, but less mutable at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus by both UV and X-radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na+/K+-ATPase locus as well as the HGPRT locus was determined. As previously reported, we found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, we found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na+/K+-ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na+/K+-ATPase locus. We have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells.