Radiomimetic Drug Bleomycin Research Articles

Nono induces Gadd45b to mediate DNA repair.

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP (KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b.

GSE4 peptide suppresses oxidative and telomere deficiencies in ataxia telangiectasia patient cells.

Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.

Microgravity Impairs DNA Damage Repair in Human Hematopoietic Stem/Progenitor Cells and Inhibits Their Differentiation into Dendritic Cells.

Astronauts on missions beyond low-Earth orbit are exposed to a hostile environment in which they are continually bombarded with unique high-energy species of radiation, while in conditions of microgravity (μG), which can alter radiation response and immunity. In the present studies, we examined the impact exposing human hematopoietic stem/progenitor cells (HSC) to μG had upon their capacity to repair DNA damage and their ability to generate immune cells critical for mounting an effective antitumor response. To this end, we first treated a human HSC-like cell line with an acute dose of the radiomimetic drug bleomycin, cultured them in normal gravity (1G) or simulated μG, and quantitated double-strand breaks through γ-H2AX foci. Calculating the median fluorescence intensity ratio at 1-to-4 h post-bleomycin revealed a 26% decrease in 1G, but a 20% increase in μG, suggesting that μG compromised HSC DNA damage repair and thus has the potential to enhance the genotoxic effects of space radiation. We next examined whether μG negatively affected the development of dendritic cells (DC), critical regulators of both the innate and acquired arms of the immune system. Primary human HSC were cytokine induced in 1G or μG and analyzed for generation of plasmacytoid (CD123+) and myeloid (CD11c+) DC. HSC cultured in 1G gave rise to significantly higher numbers of both myeloid and plasmacytoid DC than those cultured in μG, suggesting μG impairs production of these critical antigen-presenting cells. Our studies thus indicate that conditions of μG present during spaceflight perturb multiple pathways that could potentially enhance astronaut risk from exposure to space radiation.

Evolutionarily Distant Streptophyta Respond Differently to Genotoxic Stress.

Research in algae usually focuses on the description and characterization of morpho—and phenotype as a result of adaptation to a particular habitat and its conditions. To better understand the evolution of lineages we characterized responses of filamentous streptophyte green algae of the genera Klebsormidium and Zygnema, and of a land plant—the moss Physcomitrella patens—to genotoxic stress that might be relevant to their environment. We studied the induction and repair of DNA double strand breaks (DSBs) elicited by the radiomimetic drug bleomycin, DNA single strand breaks (SSB) as consequence of base modification by the alkylation agent methyl methanesulfonate (MMS) and of ultra violet (UV)-induced photo-dimers, because the mode of action of these three genotoxic agents is well understood. We show that the Klebsormidium and Physcomitrella are similarly sensitive to introduced DNA lesions and have similar rates of DSBs repair. In contrast, less DNA damage and higher repair rate of DSBs was detected in Zygnema, suggesting different mechanisms of maintaining genome integrity in response to genotoxic stress. Nevertheless, contrary to fewer detected lesions is Zygnema more sensitive to genotoxic treatment than Klebsormidium and Physcomitrella

BET-bromodomain inhibitors modulate epigenetic patterns at the diacylglycerol kinase alpha enhancer associated with radiation-induced fibrosis

Background and purposeFibrosis is a frequent adverse effect of radiotherapy and no effective treatments are currently available to prevent or reverse fibrotic disease. We have previously identified altered epigenetic patterns at a gene enhancer of the diacylglycerol kinase alpha (DGKA) locus in normal skin fibroblasts derived from fibrosis patients. An open chromatin pattern related to radiation-inducibility of DGKA is associated with onset of radiation-induced fibrosis. Here, we explore epigenetic modulation of DGKA as a way to mitigate predisposition to fibrosis. Material and methodsWe studied the effect of the BET-bromodomain inhibitors (JQ1, PFI-1) on DGKA inducibility in primary fibroblasts. Hence, DGKA transcription was additionally induced by the radiomimetic drug bleomycin, and DGKA mRNA expression, histone H3K27 acetylation and downstream markers of profibrotic fibroblast activation after BET-bromodomain inhibition were determined. ResultsBET-bromodomain inhibition suppressed induction of DGKA in bleomycin-treated fibroblasts, reduced H3K27ac at the DGKA enhancer and repressed collagen marker gene expression. Alterations in fibroblast morphology and reduction of collagen deposition were observed. ConclusionFor the DGKA enhancer, we show that BET-bromodomain inhibitors can alter the epigenetic landscape of fibroblasts, thus counteracting profibrotic transcriptional events. Interference with epigenetic patterns of fibrosis predisposition may provide novel preventive therapies that improve radiotherapy.

DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts.

BackgroundStudies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after several passages in standard conditions of cultivation due to what is known as «culture stress». Using proliferating young and non-dividing presenescent cells in primary cultures of Syrian hamster fibroblasts, we defined their response to the action of radiomimetic drug bleomycin (BL) that induces DNA double-strand breaks (DSBs).ResultsThe effect of the drug was estimated by immunoblotting and immunofluorescence microscopy using the antibody to phosphorylated histone H2AX (gH2AX), which is generally accepted as a DSB marker. At all stages of the cell cycle, both presenescent and young cells demonstrated variability of the number of gH2AX foci per nucleus. gH2AX focus induction was found to be independent from BL-hydrolase expression. Some differences in DSB repair process between BL-treated young and presenescent Syrian hamster cells were observed: (1) the kinetics of gH2AX focus loss in G0 fibroblasts of young culture was faster than in cells that prematurely stopped dividing; (2) presenescent cells were characterized by a slower recruitment of DSB repair proteins 53BP1, phospho-DNA-PK and phospho-ATM to gH2AX focal sites, while the rate of phosphorylated ATM/ATR substrate accumulation was the same as that in young cells.ConclusionsOur results demonstrate an impairment of DSB repair in prematurely aged Syrian hamster fibroblasts in comparison with young fibroblasts, suggesting age-related differences in response to BL therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0046-4) contains supplementary material, which is available to authorized users.

The effect of growth architecture on the induction and decay of bleomycin and X-ray-induced bystander response and genomic instability in lung adenocarcinoma cells and blood lymphocytes

Purpose: Cancer patients treated with radiomimetic drug bleomycin (BLM) have shown incidence of 7% second malignancy. Studies regarding BLM-induced genomic instability in bystander cells are scarce, and experiments with cells grown on three-dimensional (3D) cultures to mimic the in-vivo condition have never been attempted.Materials and methods: A549 and NCI-H23 (human lung adenocarcinoma) cells were grown as 3D cultures using Cytomatrix™, exposed to BLM or X-radiation and co-cultured with their respective unexposed cells. The DNA damage in direct and bystander cells were assessed by the induction of micronuclei (MN) or phosphorylated serine-15 residue in protein 53 (p53ser-15), a reflection of DNA damage, and by up-regulation of protein 21 (p21Waf1). The persistence of DNA damage was measured using MN assay and fluorescence in situ hybridization (FISH) in cancer cells and human peripheral blood lymphocytes (PBL) respectively.Results: BLM or X-irradiation induced DNA damage in both A549 and NCI-H23 cells and their respective bystander cells grown in 2D or 3D cultures. Further persistence of these damages in bystander PBL at delayed times indicated genomic instability in these cells.Conclusion: BLM-induced genomic instability in the progeny of bystander cells and their significance in therapy-induced second malignancy may not be eliminated completely.

Genotoxicity testing of combined treatment with cisplatin, bleomycin, and 5-fluorouracil in somatic cells of Drosophila melanogaster

The simultaneous treatment with the cross-linking agent cisplatin, the radiomimetic antitumoral drug bleomycin, and the anti-metabolite drug 5-fluorouracil has been used as a regimen to treat patients with squamous cell carcinoma of the head and neck. Considering that these drugs interact directly with DNA, one of the important late-occurring complications from treatment of primary malignancies is the therapy-related secondary cancers as a result of the genotoxic activity of the drugs on normal cells. In this sense, the genotoxicity of this combination was evaluated using the wing somatic mutation and recombination test in Drosophila melanogaster. The mutant spots observed in marker-heterozygous and balancer-heterozygous flies were compared in order to quantitatively and qualitatively estimate the genotoxic effect of these drugs. Cisplatin (0.003 and 0.006mM), bleomycin (0.005 and 0.01mM), and both combinations preferentially induced recombinational events, while mutation is the major event regarding the genetic toxicity of 5-fluorouracil (0.025 and 0.05mM). The combination of these drugs produced synergistic and antagonistic genotoxic effects, depending on the concentrations used, which could impose a higher risk of secondary effects associated with their genotoxic effects, emphasizing the importance of long-term monitoring in patients being treated with these drugs.

Gene expression profiling of human dermal fibroblasts exposed to bleomycin sulphate does not differentiate between radiation sensitive and control patients

BackgroundGene expression profiling of the transcriptional response of human dermal fibroblasts to in vitro radiation has shown promise as a predictive test of radiosensitivity. This study tested if treatment with the radiomimetic drug bleomycin sulphate could be used to differentiate radiation sensitive patients and controls in patients who had previously received radiotherapy for early breast cancer.FindingsEight patients who developed marked late radiation change assessed by photographic breast appearance and 8 matched patients without any change were selected from women entered in a prospective randomised trial of breast radiotherapy fractionation. Gene expression profiling of primary skin fibroblasts exposed in vitro to bleomycin sulphate and mock treated fibroblast controls was performed. 973 genes were up-regulated and 923 down-reguated in bleomycin sulphate treated compared to mock treated control fibroblasts. Gene ontology analysis revealed enriched groups were cellular localisation, apoptosis, cell cycle and DNA damage response for the deregulated genes. No transcriptional differences were identified between fibroblasts from radiation sensitive cases and control patients; subgroup analysis using cases exhibiting severe radiation sensitivity or with high risk alleles present in TGF β1 also showed no difference.ConclusionsThe transcriptional response of human dermal fibroblasts to bleomycin sulphate has been characterised. No differences between clinically radiation sensitive and control patients were detected using this approach.

Delineation of the Xrcc4-interacting Region in the Globular Head Domain of Cernunnos/XLF

In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor. Ten of 27 screened mutants were nonfunctional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg(64), Leu(65), and Leu(115)) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.

A direct view by immunofluorescent comet assay (IFCA) of DNA damage induced by nicking and cutting enzymes, ionizing 137Cs radiation, UV-A laser microbeam irradiation and the radiomimetic drug bleomycin

In DNA repair research, DNA damage is induced by different agents, depending on the technical facilities of the investigating researchers. A quantitative comparison of different investigations is therefore often difficult. By using a modified variant of the neutral comet assay, where the histone H1 is detected by immunofluorescence [immunofluorescent comet assay (IFCA)], we achieve previously unprecedented resolution in the detection of fragmented chromatin and show that trillions of ultraviolet A photons (of a few eV), billions of bleomycin (BLM) molecules and thousands of gamma quanta (of 662 keV) generate, in first order, similar damage in the chromatin of HeLa cells. A somewhat more detailed inspection shows that the damage caused by 20 Gy ionizing radiation and by a single laser pulse of 10 microJ are comparable, while the damage caused by 12 microg/ml BLM depends highly on the individual cell. Taken together, this work provides a detailed view of DNA fragmentation induced by different treatments and allows comparing them to some extent, especially with respect to the neutral comet assay.

Modifications of Bleomycin Induced Cytogenetic Damages by 2-Deoxy-D-Glucose on Normal and Tumor Cells

The radiomimetic drug bleomycin induced chromosomal aberrations (CA) and micronuclei (MN) were studied in peripheral blood lymphocytes (PBL), as reference to normal cells and Glioma (BMG -1) as reference to tumor cells, with and without exposure to 2-deoxy D- glucose (2-DG, an analogue of glucose). Treatment with bleomycin increased both CA and MN frequency in a dose dependent manner in both cell types. The frequencies of CA are 2 fold higher than MN for a given concentration of bleomycin. Exposure to bleomycin predominantly induced exchange type chromosome aberrations. While in the presence of 2-DG the aberrations induced by bleomycin reduced significantly in PBL, the same was increased significantly in BMG cells (P<0.001) showing a protective effect and sensitizing effect on normal and tumor cells respectively. The dose modulatory factor (protection) for different concentration of bleomycin exposure varied between 0.38 and 0.72 for CA and 0.1 and 0.84 for MN in PBL. In the case of BMG-1 cells, the modulatory factor (sensitization) varied between 1.42 and 2.59 for CA, 1.25 and 1.66 for MN at different concentration of bleomycin exposure. The modulatory effect of 2-DG was also evidenced from the coefficients obtained for the dose-response curves of the aberrations studied. The paper discusses the types of aberrations induced by bleomycin and the mechanism involved for differential modifications of cytogenetic damage by 2-DG in normal (PBL) and tumor (Glioma) cells.

Protective Effect of 2-Deoxy-D-Glucose on Chemotherapeutic Drugs Induced Damages on Peripheral Blood Lymphocytes Exposed in-Vitro

KEYWORDS Bleomycin; Mitomycin-C; chromosomal aberrations; sister chromatid exchanges ABSTRACT The effect of 2-deoxy-D-glucose (2-DG), an antimetabolite of glucose was studied in peripheral blood lymphocytes (PBL) exposed to radiomimetic drug bleomycin and an alkylating agent mitomycin-C. The PBL were exposed to 2-DG (5 mM), 30 minutes pretreatment and with Bleomycin (10 to 80 µg/ml) and Mitomycin-C (2 to12 µg/ml) for three hours. The drug as well as 2-DG was removed by washing the cells with HBSS buffer. Then the cells were cultured for 48 hours to study chromosomal aberrations (CA), Translocations (TL) and 72 hours for micronuclei (MN) and Sister Chromatid Exchanges (SCE). Exposures of PBL to Bleomycin and Mitomycin-C showed, a concentration dependent increase in the aberration frequencies, both in the presence and absence of 2-DG. While, the regression analysis showed, that the presence of 2-DG reduced bleomycin induced TL, CA frequencies and Mitomycin- C induced CA and MN frequencies significantly (P<0.001) when compared to PBL treated with the drugs alone, Bleomycin induced MN frequencies and Mitomycin-C induced SCE's reduction were not significant. The difference could be attributed to the mechanism of the action of drugs on the cells. Furthermore the alteration in the cell cycle kinetics, suggest that the presence of 2-DG during drug exposure, alter the cellular environment and delay the cell proliferation and provide sufficient time to repair the damages, could resulted in the reduced aberration frequencies.

The glycolytic inhibitor 2-deoxy-D-glucose enhances the efficacy of etoposide in ehrlich ascites tumor bearing mice

Earlier studies have shown that 2-deoxy-D-glucose (2-DG), a glucose analogue and inhibitor of glycolytic ATP production significantly enhances the cytotoxic effects of anticancer agents like topoisomerase inhibitors (etoposide and camptothecin) and a radiomimetic drug (bleomycin) in established human tumor cell lines. Therefore, combination of 2-DG and DNA damage causing cytotoxic agents could be very useful in enhancing local tumor control. The purpose of the present studies was to investigate the therapeutic effects of etoposide and 2-DG in Ehrlich ascites tumor (EAT) bearing mice, grown as solid tumor as well as in the ascites form. Cell growth, cell cycle perturbations (flow cytometry), cytogenetic damage (micronuclei assay) and apoptosis (DNA content, morphological changes) were studied as parameters of cellular response, while delay in tumor growth and cure rate (tumor free survival) were evaluated as parameters of systemic response. Body weight and general condition as well as the damage to bone marrow and spleen was monitored to evaluate normal tissue toxicity. Intraperitoneal administration of etoposide (30 mg/Kg b. wt.) resulted in significant tumor growth delay and cure (~11 %) only in subcutaneous tumors leading to local tumor control. When etoposide was combined with 2-DG (2 g/Kg b. wt.; i.v./i.p.; 4 h after etoposide injection), these effects were further enhanced resulting in a cure rate of ~22 % in case of subcutaneous tumors and 20% in ascites bearing mice. Analysis of cells obtained from ascitic fluid as well as solid tumors during follow up clearly showed that etoposide induced cell death was mainly due to apoptosis, which was enhanced further by 2-DG. Although, there was a significant level of toxicity revealed by reduced animal survival, decrease in body weight and damage to sensitive organ status like spleen and bone marrow at 60 mg/Kg b. wt. of etoposide, it was not significant at 30 mg/Kg b.wt. 2-DG, however, did not enhance the etoposide toxicity at both the doses. These results indicate that the administration of 2-DG can improve the local control of tumors without increasing normal tissue toxicity; thereby enhancing the therapeutic efficacy of topoisomerase inhibitor based anticancer drugs like etoposide.

Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks.

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.

Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells.

In this study, we examined DNA repair synthesis in human cells treated with the radiomimetic drug bleomycin, which efficiently induces double-strand breaks (DSBs). Using tyramide-biotin to amplify fluorescent signals, discrete nuclear foci from the incorporation of 5-iododeoxyuridine (IdU) were detected in proliferating human cells treated with bleomycin. We believe this comes from the repair of DSBs. An increase in the number of foci (>5 per nucleus) was detected in a major fraction (75%) of non-S-phase cells labeled for 30 min with IdU 1 h after the end of bleomycin treatment. The fraction of cells with multiple IdU-containing foci was found to decrease 18 h after treatment. The average number of foci per nucleus detected 1 h after bleomycin treatment was found to decrease twofold between 1 and 3.5 h, indicating that the foci may be associated with the slow component of DSB repair. The presence of DSBs in bleomycin-treated cells was confirmed using antibodies against phosphorylated histone H2AX (gamma-H2AX), which is strictly associated with this type of DNA damage. After treatment with bleomycin, non-S-phase cells also displayed heterogeneous nuclear foci containing tightly bound proliferating cell nuclear antigen (PCNA), suggesting an ongoing process of unscheduled DNA synthesis. PCNA is known to be involved in base excision repair, but a fraction of the PCNA foci may also be associated with DNA synthesis occurring during the repair of DSBs.

In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts.

We describe a new assay for in vitro repair of oxidatively induced DNA double-strand breaks (DSBs) by HeLa cell nuclear extracts. The assay employs linear plasmid DNA containing DNA DSBs produced by the radiomimetic drug bleomycin. The bleomycin-induced DSB possesses a complex structure similar to that produced by oxidative processes and ionizing radiation. Bleomycin DSBs are composed of blunt ends or ends containing a single 5'-base overhang. Regardless of the 5'-end structure, all bleomycin-induced DSBs possess 3'-ends blocked by phosphoglycolate. Cellular extraction and initial end joining conditions for our assay were optimized with restriction enzyme-cleaved DNA to maximize ligation activity. Parameters affecting ligation such as temperature, time, ionic strength, ATP utilization and extract protein concentration were examined. Similar reactions were performed with the bleomycin-linearized substrate. In all cases, end-joined molecules ranging from dimers to higher molecular weight forms were produced and observed directly in agarose gels stained with Vistra Green and imaged with a FluorImager 595. This detection method is at least 50-fold more sensitive than ethidium bromide and permits detection of </=0.25 ng double-stranded DNA per band in post-electrophoretically stained agarose gels. Consequently, our end-joining reaction requires </=100 ng substrate DNA and >/=50% conversion of substrate to product is achieved with simple substrates such as restriction enzyme-cleaved DNA. Using our assay we have observed a 6-fold lower repair rate and a lag in reaction initiation for bleomycin-induced DSBs as compared to blunt-ended DNA. Also, end joining reaction conditions are DSB end group dependent. In particular, bleomycin-induced DSB repair is considerably more sensitive to inhibition by increased ionic strength than repair of blunt-ended DNA.

Chromosome instability in the murine melanoma cell line K-1735 is due to drug-specific mechanisms

The purpose of the present study was to investigate chromosomal instability and DNA repair by exposing clones from the murine melanoma cell line K-1735 to the radiomimetic drug bleomycin and to the DNA polymerase a inhibitor aphidicolin. Results from previous experiments conducted with human lymphocytes have suggested synergistic chromosomal damage after simultaneous exposure to these two agents. However, in the murine cell line studied here, there was no direct correlation between the effects of these two agents. Indeed, the extensive variation in the responses to aphidicolin and bleomycin suggested different mechanisms for the repair of bleomycin-induced DNA damage by the clones. Evaluation of the unexplained propensity of some bleomycin-treated metaphase cells to disintegrate suggested that this phenomenon was most likely the result of a direct action of bleomycin, rather than a potential manifestation of tumor cell instability.

Bleomycin-induced DNA damage and DNA repair in chicken embryo cells as compared to X-irradiation.

Following in vitro- and in ovo-exposure of chicken embryo cells, the level of bleomycin (BM)-induced damage was evaluated by using DNA synthesis, nucleoid sedimentation (SED), and viscometry of alkaline cell lysates (VISC). This damage was compared to X-irradiation, using 5.9-378 nM BM in vitro, 1.5-116 micrograms BM/egg in ovo, and 2-32 Gy, respectively, in vitro as well as in ovo. With respect to BM, the most notable result is the increase in DNA synthesis and VISC at the lowest concentrations of the drug. A decrease in both parameters was observed at high BM concentrations and following exposure to X-rays, concomitantly with an increase in SED. Regarding the radiomimetic drug BM and X-rays, different modes of DNA damage and DNA repair are suggested by previous investigations and the present results. Therefore, further evidence is presented, that the chicken embryo can act as a simple, rapid and inexpensive test system to characterize the biological effects of many nucleo- and/or cytotoxic agents.

Fluorescent labelling of closely-spaced aldehydes induced in DNA by bleomycin-Fe(III)

Purpose: To test the ability of FARPhc and FARP, two novel fluorescent reagents, to label aldehyde-containing sites (principally abasic sites) generated in DNA by the radiomimetic drug bleomycin, and to use fluorescent energy transfer from FARPhc (donor) to FARP (acceptor) to quantitate such closely-spaced sites. Materials and methods: FARPhc, 7-hydroxycoumarin-3-carboxylic acid (((((amino-oxymethyl) carbonyl) hydrazino) carbonylethyl) amide) was synthesized with a protocol similar to the one recently reported for FARP (a fluorescein-based probe). Results: Both FARPhc and FARP form stable oxime bonds with the open-chain aldehydes generated upon acidic depurination of DNA. Plasmid DNA exposed to bleomycin-Fe(III)-ascorbate undergoes extensive strand breakage, and upon subsequent reaction with FARPhc and/or FARP it becomes fluorescently labelled, indicating the generation of aldehyde-containing sites. The binding of the probes to calf thymus or plasmid DNA results in significant fluorescent energy transfer among closely-spaced fluorophores, as revealed by the fluorescence increase following digestion of fluorescently labelled samples with nuclease P1. The fluorescent quenching is most evident when both FARPhc and FARP are used simultaneously to trap aldehyde sites. When single-stranded oligonucleotides engineered to contain either one or two closely spaced bleomycin binding sites are exposed to bleomycin and then fluorescently labelled, the oligonucleotides demonstrate significantly increased fluorescent energy transfer with two binding sites indicating a dependence of aldehyde site generation and clustering on the local sequence of a single strand. Conclusions: A new detection method for DNA damage induced by bleomycin following fluorescent labelling of aldehyde groupcontaining sites (FLAGS) and their clustering via fluorescent energy transfer is demonstrated. The method is applicable to any form of DNA. This work may lead to a general approach for the quantification of multiply damaged sites in DNA, a subset of DNA lesions that may have major biological significance.