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.     

Chromosomal instability and delayed apoptosis in long-term T-lymphocyte cultures irradiated with carbon ions and x rays

In this study, we examined genomic instability induced by 250 kV X rays and 100 MeV/nucleon carbon ions in long-term lymphocyte cultures from two healthy donors. Two biological end points, delayed apoptosis and chromosomal instability, were studied in descendants of cells irradiated with three different doses of the particular radiation up to 22 population doublings. The delayed apoptosis showed no clear dependence on radiation dose, culture time or radiation quality. A persistent significant increase in the rate of apoptosis up to 36 days after X irradiation was observed for a dose of 4 Gy in donor 1 only. For both donors and radiations, de novo aberration yields were significantly increased in comparison to control values up to day 36. For both radiations, chromosome-type aberrations were seen more frequently than chromatid-type aberrations in both donors up to 22 days postirradiation. In both donors, carbon ions were more effective than X rays with respect to the induction of chromosome instability. A dose of 0.25 Gy of carbon ions corresponding to 1.4 ion traversals per cell nucleus was effective in the induction of instability in our cell system.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Serial analysis of chromosomal aberrations and proliferation kinetics of mouse spermatogonia after single or fractionated X-ray exposures

Dose-fractionation studies on translocation induction in stem-cell spermatogonia of mice, as measured by spermatocyte analysis many cell generations after irradiation, revealed that a small conditioning dose of X-rays sensitizes the stem cells to the induction of translocations by a second dose 24 h later (Van Buul and Léonard, 1974, 1980). To find out whether such sensitization effects also occur at other spermatogonial stages, a comparison was made of the effects of single (50, 100 and 150 rad) and fractionated (100 + 50 rad, with 24 h in between) doses of X-rays on the induction of chromosomal aberrations in spermatogonia by analysing spermatogonial metaphases shortly after irradiation at multiple sampling times (0–48 h; every 4 h). In addition, the kinetics of spermatogonial proliferation was studied by using, in vivo, a BrdU chromosome-labelling procedure. The recorded frequencies of chromosomal aberrations did not indicate any sensitization effect of dose fractionation. It is concluded that the sensitization effects, as observed for chromosomal aberrations in male premeiotic germ cells, are characteristic for the stem-cell spermatogonia and do not occur in the more differentiated spermatogonia.     

Effect of confluent holding on potentially lethal damage repair, cell cycle progression, and chromosomal aberrations in human normal and ataxia-telangiectasia fibroblasts

The effects of confluent holding recovery on survival, chromosomal aberrations, and progression through the life cycle after subculture of human diploid fibroblasts X-irradiated during density inhibition of growth have been examined. The responses of three normal strains were determined and compared with those of four ataxia-telangiectasia (AT), an AT heterozygote, and two hereditary retinoblastoma strains. The capacity for potentially lethal damage repair (PLDR) was slightly reduced in retinoblastoma cells and almost absent in AT cells, but normal in an AT heterozygote. The decline in chromosomal aberrations seen in normal cells during confluent holding was absent in AT cells, consistent with the lack of PLDR. Following subculture, all irradiated AT fibroblasts progressed through the cell cycle to the first mitosis with no delay. AT heterozygotic and retinoblastoma cells showed both an enhanced delay in the initiation of DNA synthesis and a large fraction of cells irreversibly blocked in G1 as compared with normal cells. Both the delayed entry into S and the G1 block were reduced by confluent holding. These results indicate that AT homozygotic and heterozygotic cells respond quite differently to X irradiation.     

Effect of cisplatin on Allium cepa root meristem cells

Allium cepa root growth was retarded by cisplatin treatment in a dose-dependent manner. A decrease in the mitotic index (MI) and an increase in the number of interphase cells was seen in cisplatin treated root tips. An increase in the frequency of abnormal mitoses and chromosomal aberrations was also observed in cisplatin treated groups which indicates its genotoxic effect on plant cells. The endogenous glutathione (GSH) level in the root tips decreased significantly after cisplatin treatment which may favour its increased interaction with cellular DNA thereby developing enhanced chromosomal aberrations and affecting cell divisions and root growth. It is suggested that the decrease in endogenous GSH may be related to the development of cisplatin-mediated genotoxic effects in plants.     

Effect of nitric oxide donor (NaNO2) on the stability of non-transformed and malignant cells to ultraviolet and gamma-radiation

A decreased sensitivity of the Chinese hamster cells (line V-79) to gamma-radiation under the influence of nitric oxide induction was shown elsewhere. This effect is connected hypothetically with post-radiation reparation of DNA. The investigation of the nitric oxide donor effect on sensitivity of these to UV-radiation is of interest, because this radiation is an important ecological factor of the environment. The question of retention of nitric oxide positive effect on UV and gamma-radiation sensitivity in malignant HeLa cells is no less actual, because these cells significantly differ from normal cells of line V-79. We demonstrated that the donor of nitric oxide enhances stability of the Chinese hamster cells (line V-79) to UV-radiation, as well as to gamma-radiation independently of the time of cell incubation with sodium oxide donor before or after irradiation. The inefficiency of nitric oxide as a factor increasing UV-stability of cells was shown for malignant HeLa cells. A 1 h long incubation of these cells with NO-donor before gamma-irradiation decreased the number of chromosome aberrations, and conversely, the addition of this agent to the HeLa cell culture after gamma-irradiation did not change the radiostability. It may be inferred that distinctions in behaviour of nitric oxide in cultures of V-79 and HeLa cells using UV-radiation may be explained by transformation of the latter special features of their damage, and by the following reparation.     

Accumulation in the regenerating liver of hepatocytes with pathological nuclei as affected by daunorubicin

The action of the antitumor antibiotic rubomycin on dividing cells in the regenerating liver was studied. The antibiotic was administered 2 hours before partial hepatectomy in single doses of 1 to 8 mg/kg. It was shown that any of these doses provided equal suppression of the cell division. Mitosis always started on the 5th or 6th day. Various forms of mitosis pathology were observed. On the 7th day after the partial hepatectomy there was detected a large number of pathologically changed nuclei in the liver. With an increase in the rubomycin dose their number increased. With an increase in the dose there was also observed a large number of affections associated with impairment of the mitotic apparatus. After some time the morphologically visible nuclear disorders in the population disappeared. In six months there were practically no pathological nuclei in the liver. Three aspects of the antibiotic action i.e. toxic action, cytostatic action and induction of chromosomal aberrations are discussed. The toxic action and induction of chromosomal aberrations increased with increasing the drug dose while the cytotoxic action did not change.     

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).     

Effect of treatment schedule on the interaction of Cisplatin and radiation in human lung cancer cells

This study was designed to determine the effects of the treatment schedule on the interaction between cisplatin and radiation. Cells of a human squamous cell lung cancer cell line were treated with cisplatin and radiation using three treatment protocols: 1-h exposure to cisplatin immediately followed by irradiation (A), 4-day continuous exposure to cisplatin immediately followed by irradiation (B), and 1-h exposure to cisplatin followed by irradiation after a 4-day interval (C). The interactions were assessed by isobologram, cell cycle distribution and apoptosis. The combination resulted in a additive effect in every protocol. Cell cycle accumulation at G(2)/M phase before irradiation was observed in Protocols B and C, whereas no cell cycle shift in the limited time course was noted in Protocol A. Although a 4-day continuous exposure to cisplatin and a 1-h exposure to cisplatin followed by a 4-day interval before irradiation caused significantly increased apoptosis, an additional increase in apoptosis after irradiation was not observed in Protocols B and C, whereas Protocol A showed an additional increase. Despite a cell cycle shift favoring radiation sensitivity, the drug-radiation interactions in Protocols B and C were additive, possibly because of negative effects including induction of a durable G(2)/M-phase arrest and suppression of apoptosis by cisplatin.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Streptonigrin induces delayed chromosomal instability involving interstitial telomeric sequences in Chinese hamster ovary cells

We analyzed the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells exposed to the radiomimetic compound streptonigrin (SN), in order to determine whether interstitial telomeric sequences (ITSs) are involved in the long-term clastogenic effect of this antibiotic. CHO cells were treated with a single concentration of SN (100ng/ml), and the frequency of unstable chromosomal aberrations was determined at three times after treatment (18h, and 6 and 15 days) by using PNA-FISH with a pan-telomeric probe. Cytogenetic analysis revealed a higher frequency of aberrations at 18h and 6 days after treatment in SN-exposed cultures vs. untreated cultures. The percentage of damaged cells and the yield of SN-induced aberrations at 6 days after treatment increased on average twofold compared with the ones at 18h after treatment. Moreover, a significant decrease in the frequency of aberrations was observed in SN-exposed cells at 15 days after treatment, resulting in a frequency of aberrations significantly lower than the frequency of aberrations observed in the corresponding control cultures. These data indicate that SN induces delayed chromosomal instability in CHO cells, and that the in vitro clastogenic effect of this compound persists for at least 6 days but less than 15 days after treatment. In addition, we found that SN induces delayed ITSs instability, cytogenetically detectable as additional FISH signals and centromeric breaks involving dissociation of the telomeric signal 6 days after treatment. We propose that the delayed effect of SN on ITSs results from breakage of heterochromatic centromeric ITSs blocks and further insertion of these sequences at the sites of mono- or isochromatid breaks occurring at G2 or G1-S phases of the cell cycle, respectively, since most of the additional FISH signals were present as single or double dots, and located at interstitial sites of the involved chromosomes.     

The radioenhancement of two human head and neck squamous cell carcinomas by 2'-2' difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs

PURPOSE: The present study aimed at investigating if 2'-2' difluorodeoxycytidine (dFdC) radioenhancement was mediated by an effect on induction and/or repair of radiation-induced DNA DSBs and chromosome aberrations in cells with different intrinsic radiosensitivity. METHODS: Confluent human head and neck squamous cell carcinoma cell lines designated SCC61 and SQD9 were treated with 5 microM dFdC for 3 or 24 h prior to irradiation. DNA DSBs induction and repair were analyzed by PFGE. Radiation-induced chromosome aberrations were examined with a FISH technique. RESULTS: In both cell lines, dFdC did not modify radiation-induced DNA DSBs in a dose range between 0 and 40 Gy. After a single dose of 40 Gy, dFdC affected neither the kinetic of repair nor the residual amount of DNA DSBs up to 4 h after irradiation. Whereas dFdC did not increase the induction of chromosome aberrations, after a single dose of 5 Gy, the percentage of aberrant cells and the number of aberrations per aberrant cells were significantly higher in combination with dFdC. CONCLUSION: Our data suggest that under experimental conditions yielding substantial radioenhancement, dFdC decreases the repair of genomic lesions inducing secondary chromosome breaks but has no effect on DNA DSBs repair as measured by PFGE.     

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation

BACKGROUND: In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. MATERIAL AND METHODS: DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. RESULTS: Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G(2) phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 microg/l TRAIL for 16 h reduced D(0) value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. CONCLUSION: TRAIL in concentration of 6 microg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction.     

Effect of ionizing radiation on Fc-receptors of mouse peritoneal mononuclear cells

The influence of different gamma-radiation doses on the number of mouse peritoneal mononuclears bearing Fc-receptors was studied. At doses of 7 and 9.5 Gy, phase changes were observed in the number of cells bearing Fc-receptors: a short-term rise registered 24 h to 2 days after irradiation was followed by depression. In addition, the total number of peritoneal mononuclears decreased. At a dose of 100 Gy, the number of cells with Fc-receptors was reduced as early as 1 h after irradiation, and the pronounced cell lysis occurred.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Increase in the frequency of gamma-ray induced chromosomal aberrations in mammalian cells by post-treatment with novobiocin

The effect of novobiocin (an inhibitor of DNA topoisomerase and polymerase) on the frequency of chromosomal aberrations was examined in Chinese hamster V79 cells irradiated with gamma-rays in the plateau phase of growth and subcultured in the presence of novobiocin until the first mitosis after irradiation. Novobiocin alone affected cell survival, DNA synthesis and the mitotic frequency of unirradiated cells in a dose-dependent manner, without causing any significant increase in the frequency of chromosome- or chromatid-type aberrations. The frequency of chromosome-type aberrations induced by gamma-radiation was not influenced by novobiocin at 200 microM, but the frequency of chromosome deletions (but not rings and dicentrics) showed a two-fold increase when 300 microM novobiocin was present. Irradiation produced a low level of chromatid-type aberrations and post-treatment with novobiocin at concentrations greater than 100 microM significantly increased the frequency of chromatid gaps and breaks. The results support the idea that different radiation-induced lesions lead to chromosome- as opposed to chromatid-type aberrations.     

Cytogenetic effect of the anticancer drug epirubicin on Chinese hamster cell line in vitro

The present study demonstrated the cytogenetic effect of the anticancer drug epirubicin on cultures of Chinese hamster cell line in vitro. The cultures were exposed to the drug for 24 h at three final concentrations; 10, 20 and 40 microg/ml. All treatments were carried out in the absence of any exogenous metabolic activation system.The different types of structural chromosomal aberrations, including gaps, breaks, deletions and fragments were increased in epirubicin-treated cultures. This increase was dose dependent where there was a positive correlation between increased drug concentration and induction of structural chromosomal aberrations. Also, the numerical chromosomal aberrations, including hypodiploidy and hyperdiploidy, were increased significantly in epirubicin-treated cultures. Like structural aberrations, the increase of numerical chromosomal aberrations was also dose-dependent.The frequency of sister chromatid exchanges in cultures treated with epirubicin increased significantly and this increase was dose-dependent. On the other hand, the epirubicin significantly decreased the mitotic index in treated cultures of Chinese hamster cell line.     

Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system.

In this study, we investigated the importance of redox and Bcl-2 status on cytochrome c-mediated apoptosis. Two mouse lymphoma cell lines, LYas and LYar that express Bcl-2 protein at different levels, were used to reconstitute a cell-free system. Cytoplasmic extracts made from apoptosis-sensitive LYas cells 2.5 h after exposure to 5 Gy gamma-radiation were able to induce apoptosis in isolated nuclei, whereas extracts made from LYas cells at time points earlier than 2. 5 h, or from Bcl-2-overexpressing, apoptosis-resistant LYar cells at all time points after irradiation were inactive. Apoptotic activity was restored to inactive extracts by the addition of oxidized but not reduced cytochrome c. Cytochrome c reductase was able to inhibit apoptosis in extracts made from LYas cells 2.5 h after irradiation and LYar extracts activated by addition of oxidized cytochrome c. Antioxidants, but not oxidant defensive enzymes, blocked apoptosis implying that antioxidants might alter the redox state of factors important in mediating apoptosis. These findings confirm the importance of cellular redox state during apoptosis and are consistent with a role for Bcl-2 in regulating this redox state.