The role of excision repair in the removal of transient benzo[a]pyrene-induced DNA lesions in Chinese hamster ovary cells

In Chinese hamster ovary (CHO) cells, benzo[a]pyrene induces both persistent and transient lesions that are detected by alkaline sucrose gradient sedimentation analysis (ASG sites). The transient lesions disappear within 15 min while the persistent lesions can be detected for several hours following treatment. Although the persistent ASG sites are believed to be repaired by excision repair, the process responsible for the disappearance of the transient ASG sites is unknown. To determine the contribution of excision repair to the removal of these transient lesions, CHO cells were treated with benzo[a]pyrene (B(a)P) in the presence of the inhibitors of excision repair, araC and novobiocin. The results indicate that: (1) araC inhibits the removal of persistent, but not the transient B(a)P-induced ASG sites; (2) novobiocin, a putative inhibitor of the incision step of DNA excision repair, reduced the number of lesions detected immediately following treatment, indicating that many of these lesions may represent single-strand discontinuities generated during repair; and (3) the lesions detected in the presence of novobiocin disappear rapidly following treatment. Based on these results, we concluded that B(a)P-induced transient ASG sites are repaired by a process other than excision repair.     

Induction of alkaline-labile sites in DNA by benzo [a]pyrene and the repair of those lesions in cultured Chinese hamster cells

Formation of alkaline-labile sites in DNA by S9-activated benzo [a]pyrene (B [a]P) and the repair of those lesions were investigated using the technique of alkaline elution in cultured Chinese hamster V79 cells. When the cells were treated with B [a]P (1-5 micrograms/ml) there was negligible increase in DNA elution at pH 12.1 as compared to untreated controls. However, the elution of DNA increased at pH 12.6 with a concentration dependency, thereby indicating formation of alkaline-labile sites in DNA by B [a]P. After 4 h of repair incubation the elution of DNA at pH 12.6 of B [a]P (5 micrograms/ml) treated cells returned to the control levels. The half-life of alkaline-labile sites formed by B [a]P was approximately 1.5 h. Inhibitors of DNA-repair synthesis, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (ara-C) when added simultaneously with S9-activated B [a]P for 3 h showed an increase in elution of DNA at pH 12.1, indicating that a population of B [a]P-induced DNA lesions could be removed by a rapid DNA-repair process. These results indicate that at least two kinds of DNA lesions, repairable alkaline-labile sites and rapidly repairable DNA single-strand breaks, are detected after B [a]P treatment by the use of the alkaline elution procedure, by changing elution pH.     

Induction of alkaline-labile sites in DNA by benzo[a]pyrene and their repair of the lesions in cultured Chinese hamster cells

Formation of alkaline-labile sites in DNA by S9-activated benzo[a]pyrene (B[a]P) and the repair of those lesions were investigated using the technique of alkaline elution in cultured Chinese hamster V79 cells.When the cells were treated with B[a]P (1–5 μg/ml) there was negligible increase in DNA elution at pH 12.1 as compared to untreated controls. However, the elution of DNA increased at pH 12.6 with a concentration dependency, thereby indicating formation of alkaline-labile sites in DNA by B[a]P. After 4 h of repair incubation the elution of DNA at pH 12.6 of B[a]P (5 μg/ml) treated cells returned to the control lavels. The half-life of alkaline-labile sties formed by B[a]P was approximately 1.5 h. Inhibitors of DNA-repair synthesis, hydroxyrea (HU) and 1-β-arabinofuranosly cytosine (ara-C) when added simultaneously with S9-activated B[a]P for 3 h showed an increase in elution of DNA at pH 12.1 indicating that a population of B[a]P-induced DNA lesions could be removed by a rapid DNA-repair process.These results indicate that at least two kinds of DNA lesions, repairable alkaline-labile sites rapidly repairable DNA single-strand breaks, are detected after B[a]P treatment by the use of the alkaline elution procedure, by changing elution pH.     

Detection of 8-oxodG in Dreissena polymorpha gill cells exposed to model contaminants

Genotoxic end-points are routinely measured in various sentinel organisms in aquatic environments in order to monitor the impact of water pollution on organisms. As a first step towards the evaluation of oxidative DNA damage (8-oxodG) in organisms exposed to chemical water pollution, we have optimized the association between the comet assay and the hOGG1 enzyme for use on zebra mussel (Dreissena polymorpha) gill cells by in vitro exposure to H?O?. Firstly, we observed that in vitro exposure of D. polymorpha gill cells to benzo[a]pyrene (B[a]P, 98.4nM) induced an increase of the Olive Tail Moment (OTM) in both the comet-hOGG1 and comet-Fpg assays, indicating that B[a]P causes oxidative DNA damage. By contrast, methylmethane sulfonate (MMS, 33μM) only induced an increase of the Fpg-sensitive sites, indicating that MMS caused alkylating DNA damage and confirming that hOGG1 does not detect alkylating damage. Thus, the hOGG1 enzyme seems to be more specific towards oxidative DNA damage, such as 8-oxodG than Fpg. Secondly, as was observed in vitro, the in vivo exposure of D. polymorpha to B[a]P (24.6 and 98.4nM) increased oxidative DNA damage in gill cells, whereas only Fpg-sensitive sites were detected in mussels exposed to MMS (240μM). These results show that the comet-hOGG1 assay detects oxidative DNA lesions induced in vitro by H?O? and in vivo with BaP. The comet-hOGG1 assay will be used to detect oxidative DNA lesions (8-oxodG) in mussels exposed in situ.     

Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065.

CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand breaks [Reynolds, R. L., Molineux, I. J., Kaplan, D.J., Swenson, D.H., & Hurley, L.H. (1985) Biochemistry 24, 6228-6237]. We have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, we observe thermally induced breakage of [14C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [3H]thymidine-labeled untreated cells with the [14C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10(6) base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The induction of heat-labile sites in cellular DNA was confirmed by gel electrophoretic analyses of the damage to intracellular simian virus 40 (SV40) DNA in SV40-infected BSC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation, resealing and persistence of DNA breaks produced by 4-demethoxydaunorubicin in P388 leukemia cells

The formation and disappearance of DNA single-strand breaks (SSB) produced by 4-demethoxydaunorubicin (4-dmDR) in P388 murine leukemia cells and in a resistant subline were examined by alkaline elution methods in relation to cellular pharmacokinetics. DNA strand breaks produced by this intercalating agent were essentially DNA lesions mediated by topoisomerase II, even at very high drug concentrations, since they were detected as protein-associated breaks by filter elution. Similarly, the appearance of delayed DNA breaks in cells exposed to high concentrations, following drug removal, showed predominance of protein-associated breaks, thus supporting a similar mechanism of breakage induction. This finding indirectly suggests that, in this experimental model, free radical production makes little (if any) contribution to DNA damage, and also that DNA effects are not the consequence of early cell death. In contrast to a rapid disappearance of protein-associated strand breaks produced by intercalating agents and topoisomerase II inhibitors of different classes, DNA breaks induced by low concentrations of the anthracycline derivative are only partially reversible following drug removal, but they persisted and even increased with high concentrations. Thus, not only the extent of DNA breaks but also their persistence may contribute to the cytotoxic potency of anthracyclines. The importance of DNA lesions to cytotoxic action of the anthracycline is also emphasized by drug effect on the resistant line. A negligible effect on DNA of resistant cells was detected at drug concentrations lethal to sensitive cells. However, exposure to equitoxic drug concentrations resulted in a comparable amount of DNA breaks in sensitive and resistant cells. Although faster DNA rejoining in resistant cells may be in part attributable to increased efflux of drug, no correlation exists between cell drug accumulation and extent of DNA lesions. With equitoxic drug concentrations cellular drug content was higher in resistant cells, suggesting an intrinsic insensitivity of this variant to DNA cleavage effects of the anthracycline.     

Genotoxic effects of a complex mixture adsorbed onto ambient air particles on human cells in vitro; the effects of Vitamins E and C

Genotoxicity of complex mixtures of organic compounds adsorbed onto ambient air particles (extractable organic matter, EOM) collected in Teplice (Czech Republic) as well as genotoxicity of the indirectly acting carcinogens benzo[a]pyrene (B[a]P) and 5,9-dimethyl-7H-dibenzo[c,g]carbazole (5,9-diMeDBC) was studied in human HepG2 and Caco-2 cells cultured in vitro. The level of DNA breaks was detected by conventional single-cell gel electrophoresis (alkaline comet assay). The level of DNA breaks+oxidative DNA lesions was assessed by modified single-cell gel electrophoresis. The indirectly acting chemical carcinogens studied were able to induce DNA breaks as well as oxidative DNA damage in both cell lines, but stronger DNA-damaging effects were observed in HepG2 cells, which contain a higher level of metabolic enzymes. Treatment of cells with the complex mixtures showed a dose-dependent increase of DNA breaks in HepG2 cells as well as in Caco-2 cells, with seasonal differences. Winter samples of EOM from Teplice (TP-W) were more effective in inducing DNA damage than summer samples (TP-S). Both mixtures caused significant oxidative DNA damage in HepG2 cells. The effect was less evident in cells treated with higher concentrations of TP-W, since the comet assay is limited by saturation at a higher level of DNA damage. Possible reduction of B[a]P-, 5,9-diMeDBC- or EOM-induced DNA damage by Vitamins E and C was evaluated in HepG2 cells only. Pre-treatment of these cells with either one of the vitamins considerably reduced the levels of both DNA breaks and oxidative DNA lesions induced by all compounds investigated.     

Metabolism and specific benzopyrene metabolite modification of DNA in early S by human lung epithelial and fibroblast cells leading to the expression of an abnormal phenotype

Examination of the high pressure liquid chromatographic profiles of ethyl acetate extractable benzo [a] pyrene (B(a)P)-metabolites from human lung fibroblast and type II epithelial cells after S phase entry indicated that B(a)P-7,8-diol and 9,10-diol species were produced following the oxygenation of B(a)P. These metabolites were detected intracellularly and in the extracellular growth medium. Both cell types appeared to release extracellularly, elevated amounts of the B(a)P-7,8-diol species. It was interesting to note of the 4 pmol of oxygenated metabolites localized intracellularly, in the fibroblast, that we identified two major metabolites, B(a)P-9,10 and -7,8-diol species. Lung epithelial cells metabolize intracellular B(a)P extensively, greater than or equal to 93% of the parent B(a)P. No tetrols were detected intracellularly or extracellularly in the treated fibroblast cells. The treated epithelial cells produced both tetrols and sulfate conjugates. The extent of observed modification of early S phase nuclear DNA of lung epithelial cells was 7.5 +/- 4.9 adducts per 10(6) bases and 4.2 +/- 2.7 adducts per 10(6) bases in lung fibroblasts. The major adduct formed in both cell types was 7 beta-BPDE-I-dG. Under conditions for transformation, both the B(a)P treated lung epithelial cells and lung fibroblasts treated in early S with either B(a)P or BPDE-I yielded populations that exhibited properties of anchorage independent growth and cellular invasiveness. Metabolism and the presence internally of metabolites did not correlate with the extent of modification of DNA in early S.     

Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells.

Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined. Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL. We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells. 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure. XP-A cells were unable to repair these oxidative DNA base lesions. Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment. Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts. Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.     

DNA lesion-specific co-localization of the Mre11/Rad50/Nbs1 (MRN) complex and replication protein A (RPA) to repair foci

The DNA damage response, triggered by DNA replication stress or DNA damage, involves the activation of DNA repair and cell cycle regulatory proteins including the MRN (Mre11, Rad50, and Nbs1) complex and replication protein A (RPA). The induction of replication stress by hydroxyurea (HU) or DNA damage by camptothecin (CAMPT), etoposide (ETOP), or mitomycin C (MMC) led to the formation of nuclear foci containing phosphorylated Nbs1. HU and CAMPT treatment also led to the formation of RPA foci that co-localized with phospho-Nbs1 foci. After ETOP treatment, phospho-Nbs1 and RPA foci were detected but not within the same cell. MMC treatment resulted in phospho-Nbs1 foci formation in the absence of RPA foci. Consistent with the presence or absence of RPA foci, RPA hyperphosphorylation was present following HU, CAMPT, and ETOP treatment but absent following MMC treatment. The lack of co-localization of phospho-Nbs1 and RPA foci may be due to relatively shorter stretches of single-stranded DNA generated following ETOP and MMC treatment. These data suggest that, even though the MRN complex and RPA can interact, their interaction may be limited to responses to specific types of lesions, particularly those that have longer stretches of single-stranded DNA. In addition, the consistent formation of phospho-Nbs1 foci in all of the treatment groups suggests that the MRN complex may play a more universal role in the recognition and response to DNA lesions of all types, whereas the role of RPA may be limited to certain subsets of lesions.     

DNA damage (comet assay) and 8-oxodGuo (HPLC-EC) in relation to oxidative stress in the freshwater bivalve Unio tumidus.

The relationships between DNA damage and oxidative stress in the digestive gland, gills and haemocytes of the freshwater bivalve Unio tumidus were investigated. Two markers of genotoxicity were measured: DNA breaks by means of the comet assay, and oxidative DNA lesions by means of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) measured using high-performance liquid chromatography (HPLC) coupled to electrochemical detection. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) tissue levels. Effects were studied after exposure of bivalves for 6 days to benzo[a]pyrene (B[a]P) (50 and 100 microg l(-1)) and ferric iron (20 and 40 mg l(-1)), applied alone or in combination. Lipid peroxidation in the digestive gland and gills resulted from exposure to Fe3+ or B[a]P whatever the concentrations tested. DNA oxidatively formed lesions were induced in the two tissues at a higher level after B[a]P exposure than after Fe3+ treatment. No significant dose-response relationship was found with the two compounds and no synergistic effect was observed between Fe3+ and B[a]P. The gills appeared less sensitive than the digestive gland to DNA lesions expressed as 8-oxodGuo and comet results. Good correlations were noted between 8-oxodGuo and comet. MDA and DNA damage did not correlate as well, although it was stronger in the digestive gland than in the gills. Production of mucus by the gills likely served to prevent lesions by reducing the bioavailability of the chemicals tested, which could explain that dose-effect relationships and synergistic effects were not observed.     

DNA strand breaks and repair synthesis in Yoshida sarcoma cells with differential sensitivities to bifunctional alkylating agents and UV light

The induction of DNA-strand breaks and repair synthesis has been examined in cultured Yoshida sarcoma cell lines sensitive (YS) and resistant (YR) to methylene dimethanesulphonate (MDMS). Using an alkaline DNA unwinding-hydroxylapatite technique, we were able to detect breaks in DNA immediately after MDMS treatment and at similar levels in both YS and YR cells. MDMS treatment and post-treatment incubation in the presence of 1-β-D-arabino-furanosylcytosine (araC) lead to a large increase in the numbers of breaks when compared with MDMS treatment alone which indicated that many of the DNA-strand breaks seen after MDMS treatment were intermediates in excision repair. The magnitude of break incidence with the araC treatment was again equal in YS and YR cells indicating that these 2 lines made enzymic incision next to MDMS-induced lesions with equal capacities.During incubation following MDMS treatment, the levels of DNA-strand breaks in YR cells were found to decrease more rapidly than in YS cells. Parallel DNA-repair synthesis estimations, using BND-cellulose chromatography, revealed that the increased rate of decline in breaks in YR cells was accompanied by an increase in repair-synthesis activity compared to YS cells. This was interpreted as indicating that an intermediate step in an excision-repair pathway for MDMS-induced lesions was relatively deficient in YS compared to YR cells.A similar difference in the rates of decline of DNA-strand breaks between YS and YR cells was also observed following treatment with UV light to which MDMS-resistant YR cells also display cross-resistance. However, no such difference was detected following treatment with the monofunctional alkylating agent, methyl methanesulphonate, to which YS and YR cells are equally sensitive. These results suggest that resistance to MDMS in the YR cell line is achieved by an increased efficiency in the gap-sealing component of the excision-repair process.     

Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage

Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system.     

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.     

A novel brain-specific mRNA encoding nuclear protein (necdin) expressed in neurally differentiated embryonal carcinoma cells

A novel DNA sequence has been isolated from a subtraction cDNA library of P19 embryonal carcinoma cells treated with retinoic acid which induces neural differentiation of the stem cells. The cDNA insert (4B) hybridized with a single 1.7 kb mRNA, whose abundance was markedly increased in P19 cells after retinoic acid treatment. The 1.7 kb mRNA was also expressed in the brain, but not in other non-neuronal tissues. A 1.6 kb cDNA insert (4BFL), which was cloned by screening another cDNA library with the 4B probe, encodes a novel protein sequence of 325 amino acids (Mr 36,831). The protein expressed in 4BFL-transfected COS cells was translocated into the nuclei as detected with antibodies against subsequences of the predicted protein. The antibodies stained the nuclei of neurally differentiated P19 cells but not of the undifferentiated stem cells. This novel mRNA encoding the nuclear protein, termed necdin, may represent a useful marker for the differentiation and development of brain cells.     

Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells.

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is essential for both DNA replication and repair. PCNA is required in the resynthesis step of nucleotide excision repair (NER). After UV irradiation, PCNA translocates into an insoluble protein complex, most likely associated with the nuclear matrix. It has not previously been investigated in vivo whether PCNA complex formation also takes place after oxidative stress. In this study, we have examined the involvement of PCNA in the repair of oxidative DNA damage. PCNA complex formation was studied in normal human cells after treatment with hydrogen peroxide, which generates a variety of oxidative DNA lesions. PCNA was detected by two assays, immunofluorescence and western blot analyses. We observed that PCNA redistributes from a soluble to a DNA-bound form during the repair of oxidative DNA damage. PCNA complex formation was analyzed in two human natural mutant cell lines defective in DNA repair: xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). XP-A cells are defective in overall genome NER while CS-B cells are defective only in the preferential repair of active genes. Immunofluorescent detection of PCNA complex formation was similar in normal and XP-A cells, but was reduced in CS-B cells. Consistent with this observation, western blot analysis in CS-B cells showed a reduction in the ratio of PCNA relocated as compared to normal and XP-A cells. The efficient PCNA complex formation observed in XP-A cells following oxidative damage suggests that formation of PCNA-dependent repair foci may not require the XPA gene product. The reduced PCNA complex formation observed in CS-B cells suggests that these cells are defective in the processing of oxidative DNA damage.     

Potassium bromate but not X-rays cause unexpectedly elevated levels of DNA breakage similar to those induced by ultraviolet light in Cockayne syndrome (CS-B) fibroblasts

It has been previously reported that the elevated accumulation of repair incision intermediates in cells from patients with combined characteristics of xeroderma pigmentosum complementation group D (XP-D) and Cockayne syndrome (CS) XP-D/CS fibroblasts following UV irradiation is caused by an "uncontrolled" incision of undamaged genomic DNA induced by UV-DNA-lesions which apparently are not removed. This could be an explanation for the extreme sensitivity of these cells to UV light. In the present study, we confirm the immediate DNA breakage following UV irradiation also for CS group B (CS-B) fibroblasts by DNA migration in the "comet assay" and extend these findings to other lesions such as 8-oxodeoxyguanosine (8-oxodG), selectively induced by KBrO3 treatment. In contrast, X-ray exposure does not induce differential DNA breakage. This indicates that additional lesions other than the UV-induced photoproducts (cyclobutane pyrimidine dimers, CPD, and 6-pyrimidine-4-pyrimidone products, 6-4 PP), such as 8-oxodG, specifically induced by KBrO3, are likely to trigger "uncontrolled" DNA breakage in the undamaged genomic DNA in the CS-B fibroblasts, thus accounting for some of the clinical features of these patients.     

Epstein-Barr Virus Infection in Chronically Inflamed Periapical Granulomas

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/μg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.     

Stability of DNA in mammalian cells irradiated with near-uv light (uv-a)

The induction of lesions in human melanoma DNA by near-uv light (uv-a) has been investigated using a procedure to lyse the cells in dilute alkali. The alkaline treatment denatures the DNA, which results in fragmentation of the DNA in irradiated cells but not in control cells. The solution is neutralized to allow the larger fragments to renature. The smaller, single-stranded DNA fragments (2-10 kb) are then detected using agarose gel electrophoresis. With increasing dose of irradiation there are increasing amounts of the smaller fragments detected. The DNA fragmentation, which can be prevented by treating the cells with the radical scavenger DMSO, is not induced via the incubation medium. Hence uv-a has distinct effects on chromosomal DNA.     

Molecular signals in B cell activation. II. IL-2-mediated signals are required in late G1 for transition to S phase after ionomycin and PMA treatment

We report that sustained increase of intracellular calcium ion concentration and protein kinase C (PKC) activation maintained throughout the G1 phase of cell cycle do not provide sufficient signals to cause S-phase entry in rabbit B cells, and that additional signals transduced by IL-2 and IL-2 receptor interaction are essential for G1 to S transition. We have shown earlier that rabbit B cells can be activated to produce IL-2 and express functional IL-2 receptors after treatment with ionomycin and PMA. Herein we have compared the response of rabbit PBLs, which contain about 50% T cells, with those of purified B cells. After activation with ionomycin or PMA, comparable numbers of PBLs and B cells entered the cell cycle; but DNA synthesis by the PBL cultures was three to four times higher than that of cultures of purified B cells. Interestingly, IL-2 production by the PBL cultures was also three to four times higher than in B cell cultures, suggesting an involvement of IL-2 in inducing DNA synthesis in these cells. The hypothesis that IL-2, which is produced in early G1, acts in late G1 and is required for G1 to S transition in B cells was supported by the following observations: (i) IL-2 production by B cells was detected as early as 6 hr after activation and preceded DNA synthesis by at least 24 hr. (ii) B cell blasts in G1 (produced by treatment of resting B cells with ionomycin and PMA) showed DNA synthesis in response to IL-2, but showed very little DNA synthesis in response to restimulation with ionomycin and PMA. (iii) A polyclonal rabbit anti-human IL-2 antibody caused nearly complete inhibition of DNA synthesis by B cells activated by ionomycin and PMA. (iv) A PKC inhibitor, K252b, inhibited DNA synthesis in ionomycin and PMA-stimulated cells if added at the beginning of culture but was not inhibitory if added 16 hr later. We conclude that increased [Ca2+]i and PKC activation are not sufficient signals for G1 to S transition in B cells; entry into S is signaled by IL-2, and IL-2-mediated signal transduction probably does not involve increased [Ca2+]i or PKC activation.