Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells.

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

Intrinsic radiation sensitivity: cellular signaling is the key.

The concept that the balance between DNA damage and repair determines intrinsic radiation sensitivity has dominated radiobiology for several decades. There is undeniably a cause- effect relationship between radiation-induced molecular alterations in the genomic DNA and cellular consequences. In the last decade, however, it has become obvious that the chromatin context affects the fate of damaged DNA and that cellular signaling is an important factor in defining intrinsic radiation sensitivity. Damaged DNA is the site of signal generation; however, alternative signaling at the plasma membrane is triggered: Reactive oxygen species (ROS) inactivate phosphatases and consequently cause activation of kinases localized at the plasma membrane; this includes ligand-independent activation of receptor kinases. Cells with an apparently functional DNA repair system may show increased radiation sensitivity due to deficiencies in specific kinases essential for repair activation and checkpoint control. Other signals that determine intrinsic radiosensitivity may affect proneness to apoptosis, the balance between DNA damage fixation and repair, and the translocation of proteins participating in the response to ionizing radiation. Interplay between the various signals decides the extent to which the repair of radiation-inflicted damage is supported or limited; in some cell types, this includes DNA-damage-independent processes guided by plasma membrane-generated signaling. Cellular signaling in the context of specific subcellular structures is the key to understanding how the molecular effects of radiation are expressed as biological consequences in various cell types. A systems approach should bring us closer to this end.

Loss of tumorigenicity with simultaneous changes in radiosensitivity and photosensitivity during in vitro growth of L5178Y murine lymphoma cells.

Murine leukemic lymphoblasts L5178Y-R (LY-R) undergo conversion into their L5178Y-S (LY-S) variant as a result of prolonged (5 months to 4 years) cultivation in vitro. LY-R cells are highly tumorigenic in DBA/2 mice; resistant to X-rays [D0 (mean lethal dose [reciprocal of the slope of the linear portion of dose-survival curve] ) = 0.91 grays]; and sensitive to ultraviolet radiation (D0 = 0.7 J/sq m), short (up to 60 min) heat (43 degrees) treatment, and certain potential cancer drugs. LY-S cells are practically nontumorigenic in DBA/2 mice; sensitive to X-rays (D0 = 0.56 grays); and resistant to ultraviolet radiation (D0 = 5.5 J/sq m), short heat treatment, and the drugs. The differences in sensitivity of these two cell strains to physical and chemical agents are paralleled by differences in DNA repair efficiency. Although both strains can be cloned in soft agar, LY-S cells invariably show higher plating efficiencies. In vitro mean doubling times are 12 to 15 hr and approximately 10 hr for LY-R and LY-S cells, respectively. The actual loss of tumorigenicity and changes in radio- and photosensitivity associated with conversion of LY-R cells into LY-S cells occur within a short time. This indicates that these changes (and probably other phenotypic changes mentioned above) result from a single event or from several events occurring within a relatively short time elicited by in vitro culture conditions.

Telomere length abnormalities in mammalian radiosensitive cells.

Telomere lengths in radiosensitive murine lymphoma cells L5178Y-S and parental radioresistant L5178Y cells were measured by quantitative fluorescence in situ hybridization. Results revealed a 7-fold reduction in telomere length in radiosensitive cells (7 kb) in comparison with radioresistant cells (48 kb). Therefore, it was reasoned that telomere length might be used as a marker for chromosomal radiosensitivity. In agreement with this hypothesis, a significant inverse correlation between telomere length and chromosomal radiosensitivity was observed in lymphocytes from 24 breast cancer patients and 5 normal individuals. In contrast, no chromosomal radiosensitivity was observed in mouse cell lines that showed shortened telomeres, possibly reflecting differences in radiation responses between primary cells and established cell lines. Telomere length abnormalities observed in radiosensitive cells suggest that these two phenotypes may be linked.

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.

Topoisomerase I is differently phosphorylated in two sublines of L5178Y mouse lymphoma cells.

Two sublines of LY murine lymphoma, differing in sensitivity to CPT, served as source of topoisomerase I in order to compare the enzyme's properties. The activity of topoisomerase I isolated from LY-S cells of reduced sensitivity to CPT increased about 2-times more upon phosphorylation with casein kinase but was inhibited to a lesser extent upon dephosphorylation with alkaline phosphatase than the enzyme from the CPT-sensitive LY-R cells. The in vitro phosphorylation of LY-S enzyme restored its sensitivity to CPT. The in vitro incorporation of 32P into topoisomerase protein was about 1.7-times higher in LY-S than in LY-R enzyme. A reversed incorporation ratio was observed upon metabolic labelling. The level of topoisomerase I protein, determined by Western blot analysis using scleroderma anti-topoisomerase I antibodies, was about 1.5-times higher in LY-S than in LY-R cells. The level of topoisomerase I mRNA was similar in both sublines. These results indicate that the reduced sensitivity of LY-S cells to CPT is based on the lowered phosphorylation of topoisomerase I protein but does not depend on the expression of topoisomerase I gene.

L5178Y sublines: a look back from 40 years. Part 1: general characteristics.

The aim was to review and summarize the results of studies done over the last 40 years concerning the general characteristics and response to ultraviolet C (UV-C) radiation and hydrogen peroxide of the pair of L5178Y (LY) sublines, LY-R and LY-S, that differ in their sensitivity to various DNA damaging agents. (The response of the sublines to ionizing radiation is described in the second part of the paper.) Comparison of subline karyotypes shows a number of differences in their banding patterns. The sublines differ in their ion transport, the ganglioside pattern of plasma membranes, and in the content and turnover rate of poly(adenosine diphosphoribose) polymers. Nuclear matrix proteins show a differential affinity to these polymers. A unique property of the pair of LY sublines is an inverse cross-sensitivity to X-rays and hydrogen peroxide, with cross-sensitivities to hydrogen peroxide and UV-C, as well as to UV-C and a platinum (Pt) complex (cisplatin analogue). Initial DNA damage and repair and various aspects of the cellular response of the sublines were determined in cells damaged with these agents. The higher sensitivity of LY-R cells to hydrogen peroxide, as compared with LY-S cells, is causally related to the higher content of iron ions in these cells and a less efficient anti-oxidant defence system (including a lower catalase activity). Sensitivity of LY-R cells to UV-C radiation and Pt complexes is explained by impaired excision repair (the incision step is missing).

L5178Y sublines: a look back from 40 years. Part 2: response to ionizing radiation.

The aim was to review and summarize the results of 40 years of study concerning the response to ionizing radiation of the pair of L5178Y (LY) sublines, LY-R and LY-S, that differ in sensitivity to various DNA-damaging agents, among them X- and gamma-rays. The reviewed data indicate the key importance of DNA damage repair and fixation for the ultimate fate of the irradiated LY cell. The cause of slow double-strand break (DSB) repair in LY-S cells is not identified, but a defect in non-homologous end-joining (NHEJ) would explain most features of the cellular response of LY-S cells to irradiation, as compared with repair-competent LY-R cells. The most prominent features are the very high radiosensitivity of G1 cells, extensive poly(ADP-ribose)-dependent damage fixation, long G2 arrest, considerable chromosomal damage seen as premature chromatin condensation (PCC) fragments and aberrations in metaphase cells. The main cause of radiosensitivity difference between LY sublines is in DNA repair/damage fixation ability. At the level of damage corresponding to a comparable lethal effect, the type of death differs between LY sublines; LY-S cells die in considerably greater proportion by apoptosis than LY-R cells, whereas the latter die in greater proportion by necrosis. This observation is consistent with differential expression of proteins that are pro- or anti-apoptotic. The prominent role of poly(ADP-ribosylation) in the response of LY-S cells apparently is connected with damage fixation, but is in contrast to other cell lines hypersensitive to X- or gamma-radiation with DSB repair defects.

Adaptive response: stimulated DNA repair or decreased damage fixation?

The aim was to review the present state of knowledge on the adaptive response and attempt to redefine the acknowledged model in the framework of the transcription-based model of damage fixation of Radford (2002). Data are reviewed that suggest that the priming stimulus is the source of signalling that eventually leads to expression of the adaptive response. For a certain time, the 'primed' cell can then respond to the challenge dose by an increased recovery, as compared with the control one. An essential part of the adaptive response is generation or receipt and transmission of a signal that is the direct cause of initiation of a cellular response that diminishes the effects of DNA damage. The often accepted view that DNA repair is stimulated in the 'primed' and challenged cell is not supported by all the available data. Taking into account the abrogation of radio-adaptation by poly(ADP-ribosylation) inhibitors applied simultaneously with the challenge dose and the fact that adaptation is revealed as a decrease in chromosomal aberration frequency, one can apply to the adaptive response the same arguments as those that support the fixation model of Radford. Adaptive response (at least in part) is due to diminished fixation of double-strand breaks in the transcription factories by the mechanism proposed by Radford.

Antioxidant defense system in differentially hydrogen peroxide sensitive L5178Y sublines.

Two sublines of L5178Y (LY) murine lymphoma, differing in sensitivity to hydrogen peroxide, served as a cellular model for examination of the antioxidant defense system. The contribution of catalase, glutathione peroxidase (G-Px) and glutathione were evaluated. Sensitivity to 3-amino-1,2,4-triazole (AMT), inhibitor of catalase, was higher in LY-R (hydrogen peroxide sensitive) than in LY-S (hydrogen peroxide resistant) cells. Accordingly, activity of catalase was twofold lower in LY-R than in LY-S cells. G-Px activity was about two times higher in LY-R than in LY-S cells. After induction with selenium it increased 15.6 times in LY-R cells and 50.3 times in LY-S cells. Reduced glutathione (GSH) content (and possibly other monobromobimane-reactive thiols) were determined fluorimetrically with monobromobimane and fluorescence found 54% higher in LY-S than in LY-R cells. Inhibition of catalase caused GSH decrease in LY-S cells; this decrease was abrogated by inducing G-Px by selenium treatment. On the contrary, in LY-R cells inhibition of catalase decreased GSH content only slightly and selenium treatment did not further change the GSH level. DNA damage (estimated by "comet" assay) was the same in hydrogen peroxide-treated cells in the presence or absence of AMT; however, after induction of G-Px by selenium, DNA damage was considerably lowered. This sparing effect of selenium was accompanied by decreased growth inhibition in selenium pretreated, hydrogen peroxide-treated cell cultures.

Monitoring and signaling of radiation-induced damage in mammalian cells.

This paper reviews the functions of and connections between the presumed DNA damage sensors: poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase (DNA-PK), the protein product of the ataxia telangiectasia mutated (ATM) gene, and the tumor suppressor, p53. Recognition of DNA damage is associated with the generation of alarm signals. The possible alarm signals include synthesis of poly(ADP-ribose) polymers and initiation of phosphorylation cascades by kinases complexed with the DNA damage sensors, DNA-PK and ATM; the role of other factors is discussed, among them BRCA1 and 2, IRF-1 and RB (retinoblastoma). Alarm signal molecules generated in the cytoplasm or plasma membrane are reactive oxygen species and ceramide. Some of the signal pathways are discussed. The p53 protein, which is poised in the central junction of the postirradiation signaling, as well as p53-independent signaling pathways form an intricate network that executes concerted and partly overlapping functions in the cellular response to ionizing radiation. These functions comprise activation of specific groups of genes, control of progression through the cell cycle checkpoints, inhibition of replication and transcription, induction of apoptosis, or an adaptive response; these features of the cellular response to radiation are discussed. They affect the fate of the irradiated mammalian cell as markedly as the DNA repair efficiency. This is shown in examples of the effect of inhibition of signaling on the adaptive response of human lymphocytes and on survival of tumor cells.

Arachidonic acid metabolism in murine lymphoma cell sublines differing in radiation sensitivity.

14C arachidonic acid incorporation and 14C radioactivity release as well as prostaglandin (PG) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis were measured in the pair of murine lymphoma L5178Y (LY) cell sublines differing in radiation sensitivity. Both LY sublines, LY-R (resistant) and LY-S (sensitive), incorporated exogenous arachidonic acid and released it from membrane phospholipids. Ca2+ ionophores (ionomycin and A23187) but not PMA stimulated the liberation of 14C arachidonic acid in LY cells. PMA did not potentiate the 14C arachidonic acid release both in the presence or in the absence of A23187; this observation suggests that protein kinase C activation is not essential for the regulation of arachidonic acid release by LY-R and LY-S cells. X-irradiation (5 Gy) did not change the uptake of 14C arachidonic acid into LY-R and LY-S cells and did not potentiate the release of its total radioactivity from the cells. PG synthesis was stimulated in irradiated LY-R cells but not in LY-S cells. The susceptibility of eicosanoid metabolism to A23187 and H2O2 was altered in irradiated LY-R cells. A23187 stimulated only PG and 5-HETE synthesis in irradiated LY-R cells. H2O2 did not stimulate the synthesis of PG from exogenous arachidonic acid in irradiated LY-R and LY-S cells and 5-HETE synthesis in LY-R cells. An implication of the increased PG synthesis in LY-R cells in the protection against radiation is discussed.

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.

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.

Ionizing radiation-induced cell death.

Selected aspects of radiation-induced cell death, connected with signal transduction pathways are reviewed. Cell death is defined as insufficiency of the cellular signal transducing system to maintain the cell's physiological functions. The insufficiency may be due to impaired signal reception and/or transduction, lack or erroneous transcription activation, and eventual cellular 'misexpression' of the signal. The molecular basis of this insufficiency would be damage to genomic (but also other cellular) structures and closing of specific signalling pathways or opening of others (like those leading to apoptosis). I describe experimental data that suggest an important role of RAS/NF1 and p53/p105 Rb proteins in cell cycle control-coupled responses to DNA damage.

Effect of signal transduction inhibition in adapted lymphocytes: micronuclei frequency and DNA repair.

Irradiation of human lymphocytes (1 cGy X rays, 37 degrees C) or their treatment with 10 microM hydrogen peroxide (30 min at 37 degrees C) evoked a ca 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). The response was reflected in a lower micronuclei frequency, but no change in DNA repair rate was observed as measured by the comet assay, directly after the challenge dose. Treatment of lymphocytes with staurosporine, an inhibitor of protein kinases, or with TMB-8, a calcium antagonist, carried out in parallel with the adaptive dose prevented the development of the adaptive response measured as micronuclei frequency. In lymphocytes that were staurosporine- or TMB-8-treated and irradiated under adaptive conditions showed that the rate of DNA repair was not changed. We conclude that treatment with agents that interfere with the transduction of the signal triggered by the low dose prevents the development of the adaptive response induced by X rays or hydrogen peroxide. Lower chromosome damage revealed by the cytokinesis block-micronuclei test in the adapted lymphocytes is unrelated to DNA repair rate as measured by comet assay.

Ca2+ mobilization is related to the lethal effect of X-irradiation in L5178Y cells.

Radiation-resistant L5178Y-R (LY-R) and radiation-sensitive L5178Y-S (LY-S) murine lymphoma cells were X-irradiated and their free Ca2+ concentration was examined with Fura-2 in Ca2(+)-free salt solution. The release of free Ca2+ from intracellular stores was linear between 10 and 60 min after irradiation (1-5 Gy X-rays) and was higher in LY-S than in LY-R cells. Pre-treatment with 2 mM benzamide (Bz) further increased the concentration of free Ca2+ in LY-S cells but not in LY-R cells. In contrast with LY-R cells, LY-S cells had previously been found to be radiosensitized by continuous 2 mM Bz treatment (Szumiel et al. 1984b). Thus, there was a parallel effect of Bz on survival and on the increase in free Ca2+ concentration in LY cells. Moreover, the rates of Ca2+ release after irradiation with 1-5 Gy of X-rays with or without Bz treatment were inversely related to the respective surviving fractions of LY cells.

Effects of topoisomerase I-targeted drugs on radiation response of L5178Y sublines differentially radiation and drug sensitive.

The murine L5178Y (LY) lymphoma sublines, LY-R (radiation resistant) and LY-S (radiation sensitive) display a difference in susceptibility to camptothecin (CPT): LY-S cells are less sensitive to killing by this inhibitor of topoisomerase I than LY-R cells. Post-treatment (CPT present until 3 h after irradiation) sensitizes only LY-S cells. In agreement with this, only in LY-S cells is the relative number of DNA-protein cross-links formed after treatment with CPT + X higher than expected for additivity of X-ray and CPT-induced damage. The pattern of changes in the labelling indices and cell cycle distribution in cells that underwent combined treatment is essentially like that seen for single-agent treatment: for LY-S cells like that for radiation, for LY-R cells like that for CPT. We found no direct relation between the patterns of cell cycle distributions and the enhancement of the lethal effect of X-irradiation by CPT post-treatment. The sublines are not markedly differentially sensitive to beta-lapachone, which modifies topoisomerase I activity, and not sensitized to X-rays by post-irradiation treatment with the drug.

Calcium antagonist, TMB-8, prevents the induction of adaptive response by hydrogen peroxide or X-rays in human lymphocytes.

Treatment of human lymphocytes with hydrogen peroxide (10 microM, 30 min, 37 degrees C in PBS) or with 1 cGy X-rays evoked about a 30% decrease in the frequency of micronuclei upon subsequent X-irradiation (1.5 Gy). In addition to a lower micronuclei frequency, we also found an increase in the sedimentation distance of the nucleoids, when measured 90 min (duration of the isolation procedure carried out at 4 degrees C) after the adaptive dose (hydrogen peroxide or X-rays) and preceding the challenge dose. To test whether Ca2+ is involved in the induction of the adaptive response pathway, we treated cells with the calcium chelator, EGTA. When EGTA was given at the same time as the adaptive dose, it prevented the development of the adaptive response. In addition, the calcium antagonist, TMB-8, also prevented the development of the adaptive response as it prevented the reduction of both micronuclei and increased nucleoid sedimentation. Cellular treatment with TMB-8 increased the free [Ca2+] by 40%, when given together with hydrogen peroxide. The faster sedimenting nucleoids from adapted cells were also examined by ethidium bromide titration; there was no indication of any change in supercoil density or loop size. Psi-tectorigenin, an inhibitor of phosphatidylinositol turnover, did not modify the adaptive response, indicating that inositol (1,4,5)-trisphosphate is not involved in the induction of the adaptive response, but free Ca2+ ions are.

Differential DNA double strand break fixation dependence on poly(ADP-ribosylation) in L5178Y and CHO cells.

PURPOSE: To investigate the role of poly(ADP-ribosylation) in DNA double-strand break repair and fixation in murine lymphoma L5178Y (LY) sublines, LY-R and LY-S, and a pair of Chinese hamster ovary lines: wild-type and mutant xrs6 cells, that have differences in repair competence and degree of radiosensitization with poly(ADP-ribosylation) inhibitors. MATERIALS AND METHODS: Cells (asynchronous, logarithmic phase) were pre-incubated with 2 mM aminobenzamide at 37 or 25 degrees C, X-irradiated with 10 Gy and allowed to repair DNA breaks for 15, 60 and 120 min at 37 or 25 degrees C. The remaining double-strand break were estimated by the neutral comet assay. RESULTS: At 37 degrees C, no effect of AB treatment on the repair kinetics was observed either in xrs6 or Chinese hamster ovary (wild-type) cells. In contrast, aminobenzamide decreased the repair of double-strand break in the LY-S line but not the LY-R line, in agreement with the previously observed radiosensitization of LY cells by poly(ADP-ribosylation) inhibition. However, double-strand break rejoining in the repair competent cell lines, Chinese hamster ovary and LY-R, also was affected by aminobenzamide when the post-irradiation incubation was carried out at 25 degrees C. Analysis of these results together with earlier data on LY-S cells have been interpreted in terms of Radford's model of radiation damage fixation. CONCLUSION: The reported results indicate that poly(ADP-ribosylation) can be an important modulator of the conversion of DNA damage to lethal events.