The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.

A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

Comparison of clastogen-induced gene expression profiles in wild-type and DNA repair-deficient Rad54/Rad54B cells.

BACKGROUND: Previously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC) as compared to wild-type (WT) cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM) and gamma-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts) after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU) and vehicle were taken as controls. RESULTS: Most exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments. CONCLUSION: These data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.

Developmental decline in DNA repair in neural retina cells of chick embryos. Persistent deficiency of repair competence in a cell line derived from late embryos.

Neural retinas of 6-day-old chick embryos synthesize DNA and are able to carry out DNA excision repair. However, in contrast to the situation in human cells, the maximum rate of repair induced by N-acetoxy acetylaminofluorene (AAAF) is no greater than that induced by methyl methanesulfonate (MMS). With advancing differentiation of the retina in the embryo, cell multiplication and DNA synthesis decline and cease, and concurrently the cells lose the ability to carry out DNA excision repair. Thus, in 15-16-day embryos, in which the level of DNA synthesis is very low, DNA repair is barely detectable. If retinas from 14-day embryos are dissociated with trypsin and the cell suspension is plated in growth- promoting medium, DNA synthesis is reinitiated; however, in these cultures there is no detectable repair of MMS-induced damage, and only low levels of repair are observed after treatment with AAAF. A cell line was produced, by repeated passaging of these cultures, in which the cell population reached a steady state of DNA replication. However, the cell population remained deficient in the ability to repair MMS-induced damage. This cell line most likely predominantly comprises cells of retino-glial origin. Possible correlations between deficiency in DNA repair mechanisms in replicating cells and carcinogenesis in neural tissues are discussed.

Involvement of mouse Rev3 in tolerance of endogenous and exogenous DNA damage.

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos. The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions.

UV radiation facilitates methotrexate resistance and amplification of the dihydrofolate reductase gene in cultured 3T6 mouse cells.

Pretreatment of 3T6 murine cells with the carcinogen UV radiation or N-acetoxy-N-acetylaminofluorene increased the number of methotrexate-resistant colonies. This carcinogen-induced enhancement was seen only at low toxicities. The enhancement was transient and was observed at its maximum when cells were subjected to methotrexate selection 12 to 24 h after treatment. The addition of a tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate, during or after carcinogen treatment further enhanced this effect. A large proportion of the resistant colonies had an increase in the dihydrofolate reductase gene copy number and the relative proportions of colonies with amplified genes were similar, regardless of whether selected cells were untreated, treated with carcinogen, or treated with carcinogen plus promoter. We discuss some of the variables which both enhance the generation and improve the detection of methotrexate-resistant colonies, as well as certain implications of our results for the generation and mechanism of gene amplification.

Mitochondrial transcription factor A is the major protein in rodent hepatocytes that recognizes DNA lesions induced by N-acetoxy-acetylaminofluorene.

Extracts from rodent liver cells contain an abundant protein that recognizes DNA adducts induced by the chemical carcinogen N-acetoxy-acetylaminofluorene (AAAF). This protein also has a strong affinity for DNA damaged by cisplatin (DDP), but not by benzo(a)pyrene diolepoxide or UV-radiation, and has been termed AAAF/DDP-DDB. Here we purified this protein from rat tissue and analyzed it by mass spectrometry and identified it as mitochondrial transcription factor A (TFAM). Experiments with bacterially expressed recombinant TFAM confirmed its high affinity for DNA damaged by AAAF. Assuming its abundance and specificity for AAAF induced lesions, TFAM may significantly impede recognition and repair of DNA adducts induced by AAAF and other derivatives of 2-aminofluorene.

Response of human DNA polymerase iota to DNA lesions.

Lesion bypass is an important mechanism to overcome replication blockage by DNA damage. Translesion synthesis requires a DNA polymerase (Pol). Human Pol iota encoded by the RAD30B gene is a recently identified DNA polymerase that shares sequence similarity to Pol eta. To investigate whether human Pol iota plays a role in lesion bypass we examined the response of this polymerase to several types of DNA damage in vitro. Surprisingly, 8-oxoguanine significantly blocked human Pol iota. Nevertheless, translesion DNA synthesis opposite 8-oxoguanine was observed with increasing concentrations of purified human Pol iota, resulting in predominant C and less frequent A incorporation opposite the lesion. Opposite a template abasic site human Pol iota efficiently incorporated a G, less frequently a T and even less frequently an A. Opposite an AAF-adducted guanine, human Pol iota was able to incorporate predominantly a C. In both cases, however, further DNA synthesis was not observed. Purified human Pol iota responded to a template TT (6-4) photoproduct by inserting predominantly an A opposite the 3' T of the lesion before aborting DNA synthesis. In contrast, human Pol iota was largely unresponsive to a template TT cis-syn cyclobutane dimer. These results suggest a role for human Pol iota in DNA lesion bypass.

Molecular characterization of an acidic region deletion mutant of Cockayne syndrome group B protein.

Cockayne syndrome (CS) is a human genetic disorder characterized by post-natal growth failure, neurological abnormalities and premature aging. CS cells exhibit high sensitivity to UV light, delayed RNA synthesis recovery after UV irradiation and defective transcription-coupled repair (TCR). Two genetic complementation groups of CS have been identified, designated CS-A and CS-B. The CSB gene encodes a helicase domain and a highly acidic region N-terminal to the helicase domain. This study describes the genetic characterization of a CSB mutant allele encoding a full deletion of the acidic region. We have tested its ability to complement the sensitivity of UV61, the hamster homolog of human CS-B cells, to UV and the genotoxic agent N-acetoxy-2-acetylaminofluorene (NA-AAF). Deleting 39 consecutive amino acids, of which approximately 60% are negatively charged, did not impact on the ability of the protein to complement the sensitive phenotype of UV61 cells to either UV or NA-AAF. Our data indicate that the highly acidic region of CSB is not essential for the TCR and general genome repair pathways of UV- and NA-AAF-induced DNA lesions.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro.

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.

Lack of susceptibility of F344 rats to mammary tumor induction by topically applied fluorenylacetohydroxamic acids and their acetates.

Carcinogenicity of N-hydroxy-N-3-fluorenylacetamide for the mammary glands of female inbred F344 rats was examined after systemic and topical administration. The compound given ip produced a 60% mammary tumor incidence but was only marginally active after topical application. Female F344 rats did not develop mammary tumors after topical application of N-hydroxy-N-2-fluorenylacetamide, N-acetoxy-N-2-fluorenylacetamide, or N-acetoxy-N-3-fluorenylacetamide. These results contrasted with those reported earlier for female Sprague-Dawley rats and indicated differences in susceptibility of the mammary glands of these rat strains to tumor induction by these carcinogens.

Nonspecific inhibition of DNA repair by promoting and nonpromoting phorbol esters.

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and its nonpromoting structural analogue, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (Me-TPA), on N-acetoxy-2-acetylaminofluorene-elicited DNA repair and replicative DNA synthesis was measured in normal human fibroblasts. Both esters inhibited DNA repair synthesis, and Me-TPA was nearly as effective as TPA. In addition, TPA inhibited replicative DNA synthesis. These findings showed that inhibition of DNA repair synthesis may not be a major factor in the mechanisms of action of tumor promoters.

Reduced capacity for DNA repair synthesis in patients with or genetically predisposed to colorectal cancer.

Peripheral resting mononuclear leukocytes were compared for their capacities to repair DNA lesions induced by a 1-hour exposure to a standardized 10-microM dose of N-acetoxy-N-2-fluorenylacetamide (N-AcO-2-FAA). Leukocytes from the following 3 groups were studied: 39 control subjects, 40 patients after colonic resection because of colorectal cancer (disease-free at the time of this study), and 28 individuals with a hereditary predisposition to colorectal cancer. Although the level of N-AcO-2-FAA that bound to mononuclear leukocyte DNA was the same for the various population groups, the level of N-AcO-2-FAA-induced unscheduled DNA synthesis (UDS) was significantly reduced in the mononuclear leukocytes of individuals who had had colorectal cancer or a genetic predisposition for the disease. These findings indicate that a deficiency in mononuclear leukocyte DNA repair synthesis is associated with the development of colorectal cancer in these populations. Our observation of this nonspecific UDS deficiency (relating to colorectal cancer) was not explained by experimental variations among the sampled groups with regard to individual differences in lymphocyte heterogeneity, age, sex, smoking habits, or blood pressure.

Horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of the carcinogen N-hydroxy-N-acetyl-2-aminofluorene as effected by cyanide and ascorbate.

Horseradish peroxidase and H2O2 mediate N-hydroxy-N-acetyl-2-aminofluorene (N-OH-AAF) conversion into two more potent carcinogens, 2-nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene. Optical studies of this system indicate that horseradish peroxidase is operating as a peroxidase with N-OH-AAF as the electron donor. Our studies confirm the earlier finding that 2-nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene are the products of the type II enzyme-mediated oxidation of N-OH-AAF, but surprisingly, the results with the type VI enzyme indicate that more 2-nitrosofluorene was formed and, in addition, another product absorbing at 245 nm was formed. If ascorbate is present in the N-OH-AAF/horseradish peroxidase/H2O2 system, ascorbate is oxidized preferentially. Cyanide, a known inhibitor of the peroxidase, does not inhibit when N-OH-AAF is the electron donor. The reaction products are the same in the presence or absence of cyanide.

Mutagenesis of Chinese hamster cells in vitro by combination treatments with methyl methanesulfonate and N-acetoxy-2-acetylaminofluorene.

Mutational synergism was examined in Chinese hamster V79 cells exposed to methyl methanesulfonate followed by N-acetoxy-2-acetylaminofluorene (AcAAF) at different time intervals. Treatment with 500 micron methyl methanesulfonate resulted in 95% survival of cloning ability and induced approximately 4 azaguanine-resistant mutants/10(5) survivors. Seven micron AcAAF produced 10 times as many mutants, and the survival was 7%. Lethal synergism was observed for methyl methanesulfonate treatments followed by 7 micron AcAAF, and the resulting lethality was unaffected by increasing the time interval between treatments from 1 to 48 hr. However, no significant changes in the mutant frequency from that induced by AcAAF alone were found for treatment intervals of 1 to 63 hr. This result contrasts with the 6-fold enhancement of the AcAAF-induced transformation of Syrian hamster embryo cells exposed to the same combination with a 48-hr interval between treatments, as previously reported (Chem.-Biol. Interactions, 9: 351-364, 1974). The difference in the response of these two cell types demonstrates the difficulties in attempting to extrapolate the known correlation between individual mutagen and carcinogen treatments to combination treatments, with different cell types for the two cellular responses.

Mammary carcinogenesis in the rat by topical application of fluorenylhydroxamic acids and their acetates.

This work confirms the previous observation that a single application of N-hydroxy-2-fluorenylacetamide or N-hydroxy-3-fluorenylacetamide to the mammary gland of the rat induced a high incidence of tumors, whereas the corresponding arylamides, N-2-fluorenylacetamide (2-FAA) and N-3-fluorenylacetamide, were only weakly active. The results suggested N-hydroxylation of the arylamides as a prerequisite for mammary carcinogenesis. Since N-hydroxylation of 2-FAA by hepatic microsomes is catalyzed by the mixed-function oxidase containing cytochrome P-450 or the 2-methylcholanthrene-inducible cytochrome P1-450, we examined whether these cytochromes are present in mammary microsomes. In contrast to liver, neither cytochrome nor N-hydroxylation of 2-FAA was detected in the mammary gland of normal and 3-methylcholanthrene-treated rats. These experiments indicated that the N-hydroxylation of 2-FAA, although obligatory for induction of mammary neoplasia, is not performed in the mammary gland but may take place in the liver. We also examined the carcinogenicity of N-acetoxy-2-fluorenylacetamide and N-acetoxy-3-fluorenylacetamide for the mammary gland upon topical application. Since both acetates were carcinogenic and since the acetyl group of acetyl coenzyme A is transferred to fluorenylhydroxamic acids at pH 7.4, these esters may be ultimate carciogens in mammary carcinogenesis. Ovariectomized rats did not develop mammary tumors after a single application of the fluorenylhydroxamic acids, and administration of estradiol and fluorenylhydroxamic acids to the ovariectomized rats did not improve the tumor yield. These results indicate that induction of mammary tumors by fluorenylhydroxamic acids is under hormonal control.

DNA excision-repair deficiency of human peripheral blood lymphocytes treated with chemical carcinogens.

Human peripheral blood lymphocytes stimulated with concanavalin A for 72 hr have a 10-fold greater capacity to repair DNA damage induced by N-acetoxy-2-acetylaminofluorene than do unstimulated cells. The increased capacity of concanavalin A-activated cells to repair DNA is not observed after 24 hr in culture, a time at which stimulated cells have not begun to synthesize DNA. The maximum rate of repair synthesis obtained after treatment of stimulated cells with the "large patch"-inducing agent, N-acetoxy-2-acetylaminofluorene, is twice that obtained with methyl methanesulfonate, an agent inducing "small patch" repair. The difference between the maximum rates obtained with N-acetoxy-2-acetylaminofluorene and methyl methanesulfonate is 6-fold in a human lymphoblastoid line. Unstimulated lymphocytes show almost identical rates of repair after treatment with either N-acetoxy-2-acetylaminofluorene or methyl methanesulfonate. There is close correlation between the rate of N-acetoxy-2-acetylaminofluorene-induced repair synthesis and the loss of acetylaminofluorene adducts from DNA. Treatment of lymphocytes with methyl methanesulfonate leads to degradation of cellular DNA with the production of single-stranded regions. Such degradation is not observed with N-acetoxy-2-acetylaminofluroene. We conclude that the rate of excision repair is a function of the capacity of cells for DNA synthesis and that lymphocytes that do not synthesize DNA have a limited repair capacity and cannot be used to distinguish between large and small patch repair.

Clinical symptoms and DNA repair characteristics of xeroderma pigmentosum patients from Germany.

Sixty-one xeroderma pigmentosum (XP) patients living in the Federal Republic of Germany were investigated. Clinical symptoms were correlated with DNA repair parameters measured in fibroblasts grown from skin biopsies. Classification according to the international complementation groups revealed that of the 61 patients 3 belonged to group A, 26 to group C, 16 to group D, 3 to group E, and 2 to group F; 11 were of the XP variant type. A striking clinical aspect was the frequency of histogenetically different skin tumors varying from one XP complementation group to the other: squamous and basal cell carcinomas predominated in XP group C; lentigo maligna melanomas were most frequent in group D; basal cell carcinomas occurred preferentially in group E and XP variants. Three DNA repair parameters were determined for 46 fibroblast strains: colony-forming ability (D0); DNA repair synthesis (G0); and DNA-incising capacity (E0). Dose-response experiments with up to 13 dose levels were performed throughout to achieve sufficient experimental accuracy. DNA-damaging treatments included UV light, the "UV-like" carcinogen N-acetoxy-2-acetylaminofluorene, and the alkylating carcinogens methyl methanesulfonate and N-methyl-N-nitrosourea. Comparison of clinical signs and repair data was made on the basis of D0, G0, and E0 values of both individual cell strains and weighted means of XP complementation groups. Despite considerable clinical and biochemical heterogeneity within complementation groups distinctive features emerged. In general, D0, G0, and E0 values of all XP strains investigated, including XP variants, were found to be reduced upon treatment with UV light or N-acetoxy-2-acetylaminofluorene. After treatment with UV light or N-acetoxy-2-acetylaminofluorene, cell strains in which DNA-incising capacity was reduced also showed a similar reduction in both colony-forming ability and DNA repair synthesis. Consequently, the weighted mean D0, G0, and E0 values of XP complementation groups and XP variants correlated with each other. Furthermore, the onset of both early dermatological symptoms of XP and tumor growth correlated with the extent of DNA repair defects. Of 45 XP fibroblast strains checked for colony-forming ability after treatment with methyl methanesulfonate only 3 cell strains from group D were found to be more sensitive than normal controls, suggesting that overall repair in XP strains was equal to that in controls. Weighted means of DNA repair synthesis of XP complementation groups, however, showed reductions hinting at impaired excision of distinct alkylated bases.(ABSTRACT TRUNCATED AT 400 WORDS)

Excision of N-acetoxy-2-acetylaminofluorene-induced DNA adducts from chromatin fractions of human fibroblasts.

The excision and persistence of covalent DNA adducts formed by N-acetoxy-2-acetylaminofluorene (AAAF) were studied in human skin fibroblasts. The changes in adduct concentration as a function of posttreatment incubation were measured in purified nucleosomal core DNA and in total nuclear DNA, and from these data the changes in adduct concentration in nucleosomal linker DNA were calculated. Immediately after N-acetoxy-2-acetylaminofluorene treatment which introduced 20 to 38 mumol adducts per mol DNA-P, the adduct concentration was 4 to 5 times higher in linker DNA than in core DNA. Adduct removal was rapid during the first 8 hr of incubation and occurred 3.5 to 4 times more efficiently from linker DNA than from core DNA. After 24 hr incubation, adduct removal continued only at a very low rate, leaving a substantial fraction of adducts unexcised. This fraction of persistent adducts had a value of 0.5 and was independent of the initial adduct concentration in the range of 12 to 115 mumol adducts per mol DNA-P. Approximately 65% of the persisting adducts were located in nucleosomal cores. The initial differences in adduct concentration between linker DNA and core DNA diminished during posttreatment incubation. This was entirely due to preferential early adduct excision from linker DNA. No evidence was obtained for repair-induced long-range nucleosomal movement in normal fibroblasts or constitutive movement in the absence of excision repair in xeroderma pigmentosum fibroblasts. Nucleosomal movement would tend to diminish the concentration differences between linker DNA and core DNA.

Role of depurination in mutagenesis by chemical carcinogens.

The effect of modifying phi chi 174 viral DNA by the chemical carcinogens beta-propiolactone, N-acetoxyacetylaminofluorene and anti-benzo[a]pyrene diol-epoxide was investigated by transfecting the modified DNA into Escherichia coli spheroplasts. Modification of the DNA in vitro by each of these agents was mutagenic for the phi chi 174 amber mutants am3 and am86. Mutagenicity depended on the induction of the "SOS" response in the host spheroplasts. Heating beta-propiolactone-treated DNA at neutral pH caused strong inactivation such that the number of lethal hits was increased 4-fold. Sucrose gradient analysis showed the induction of alkali-labile sites in the heated DNA. The "nicked circle assay" with double-stranded phi chi 174 DNA showed greater than 70% of these sites to be apurinic sites. Concomitantly with the production of these new sites, a strong increase in the mutation frequency was observed. This mutagenesis also depended upon the induction of the error-prone SOS response in the spheroplasts, as was previously shown to be the case for mutagenesis at putative apurinic sites induced directly by acid-heat treatment. These results suggest that depurination may be of importance to the mechanism of mutagenesis by beta-propiolactone and other carcinogens.

Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots.

The major mutational hot spots in human cancers occur at CpG sequences in the p53 gene. It is generally presumed that the majority of mutations at these sites result from the endogenous deamination of methylated cytosine. Using a UvrABC incision method, we have found that cytosine methylation greatly enhances guanine alkylation at all CpG sites in the p53 gene by a variety of carcinogens, including benzo(a)pyrene diol epoxide, benzo(g)chrysene diol epoxide, aflatoxin B1 8,9-epoxide, and N-acetoxy-2-acetylaminofluorene. These findings suggest that mutational hot spots at methylated CpG sequences in the p53 gene may be a consequence of preferential carcinogen binding at these sites.