Unscheduled DNA synthesis induced by 4-nitroquinoline-1-oxide in xeroderma pigmentosum cells and their complementing heterodikaryons

Unscheduled DNA synthesis (UDS) induced by 4-nitroquinoline-1-oxide (4NQO) in the complementation groups A to E and variant xeroderma pigmentosum (XP) cells, and various pairs of complementing XP heterokaryons were investigated. The pattern of UDS induced by various concentrations of 4NQO in normal cells was quite different from those in groups A to D XP cells. The patterns of UDS in group E and variant XP cells were indistinguishable from those of normal cells under our experimental conditions. The levels of UDS induced by 5×10^–6 M 4NQO were 13 % of normal in group A, 9% in group B, 17% in group C, 25 % in group D. The heterokaryons obtained by pair-wise fusions between different complementation groups of XP strains showed restored UDS induced by 5×10^–6 M 4NQO, while the dikaryons obtained from fusion between the same groups did not.     

Persistence of 4-nitroquinoline-1-oxide induced lesions in human lymphocytes

Liquid holding (LH) recovery was matched with three-way differential staining (TWD) to assess the reduction of damage induced in DNA following treatment with 4-nitroquinoline-1-oxide (4NQO) in resting (G0) lymphocytes. Human peripheral lymphocytes (HPL) from three donors were used to evaluate lesion persistence and individual repair capacity. Our data are in contrast to those for diepoxybutane (Ponzanelli et al., 1995) and suggest that LH recovery is completely inefficient in removing 4NQO induced lesions, which are only partially repaired after one cell cycle.     

Newly synthesised DNA in ageing human cells in culture treated with 4-nitroquinoline-1-oxide

Two lines of normal human embryonic lung fibroblasts, MRC-5 and F2002, were serially subcultured until senescence was attained. When cells were exposed to varying concentrations of the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO), the rate of DNA synthesis (as measured by thymidine incorporation) was reduced in a dosedependent fashion in cells from both early and late passages. While the overall amount of incorporation was considerably lower in old cells, the extent of inhibition caused by 4-NQO treatment (relative to appropriate controls) was not related to culture age.Alkaline sucrose density gradient analysis of newly synthesised DNA from cells pre-treated with 4-NQO failed to detect any significant variation in the size of labelled DNA from cells examined immediately after incubation with radioactive thymidine. The shift of this labelled material to high molecular weight in 4-NQO-treated cells also showed no age-related difference.     

The molecular nature of mutations induced by adriamycin at the hprt locus of V79 cells

Adriamycin (AM), a widely used chemotherapeutic drug, induceda broad spectrum of gene mutations at the hprt locus of V79cells. The frequency and distribution of AM-induced deletionswas analyzed with multiplex polymerase chain reaction in twoV79 cell lines, which differed considerably in their spontaneousdeletion frequency. Among AM-induced mutants, deletions predominatedin both cell lines. Apart from total deletions of the hprt gene,partial deletions were found which were distributed all overthe hprt gene with breakpoints in nearly all introns. Underthe same experimental conditions, chromosome aberrations wereinduced by AM which mainly represented chromatid-type aberrations.Neither the induction of gene mutations nor the induction ofchromosome aberrations was enhanced by the repair inhibitor3-aminobenzamide. These results are discussed in the contextwith our earlier findings on bleomycin-induced mutations andit is suggested that at least two mechanisms lead to the formationof gene deletions. One of them seems to be associated with amisrepair process of frank DNA double-strand breaks and relatedto chromosome aberrations while the other is not. ¹To whom correspondence should be addressed     

The repair of 4-nitroquinoline-1-oxide induced DNA adducts in hypersensitive Chinese hamster mutants: lack of repair of UV induced (6-4) photoproduct correlates with reduced repair of adducts at the N2 of guanosine.

UV sensitive Chinese hamster mutants belonging to ERCC groups 1, 2 and 6 together with one cross-link- and one X-ray-sensitive mutant have been examined for sensitivity to 4-nitroquinoline-1-oxide (4NQO) and the ability to repair 4NQO adducts at the N2 and C8 of guanosine. Despite the fact that all of the mutants examined were hyper-sensitive to 4NQO there was little difference between the mutants V-H1, V-H4, V-C4 and UV61 and the parental cell lines as regards the ability to remove these lesions from bulk DNA. The UV5 and UV20 mutants were both defective in the ability to remove N2 guanosine adducts yet repaired the C8 guanine adduct as normal. The fact that the mutants V-H1, V-H4, V-C4 and UV61 are 4NQO sensitive but repair the above adducts suggests that either some other lesion(s) is responsible for increased toxicity in these mutants, or that some regions of the genome may not be repaired as effectively as bulk DNA in these mutants, or that the quality of the repair is less than in the parental cells. Clearly the inability to remove UV induced pyrimidine dimers and the (6-4) photoproduct associated with the UV5 and UV20 mutants correlates with the inability to repair 4NQO-N2 guanosine adducts. However, mutants capable of (6-4) photoproduct repair but not dimer repair (VH-1 and UV61) can repair this lesion. Hence it is possible that the same domains in these repair proteins are required for the recognition of (6-4) photoproduct repair and 4NQO-N2 guanosine adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in chinese hamster ovary cells

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3–8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene. © 1995 Wiley-Liss, Inc.     

Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents

The role of the nucleotide excision repair (NER) pathway inremoval of DNA ethylation damage was investigated by means ofhprt mutational spectra analysis in the NER-deficient Chinesehamster ovary cell line UV5, which lacks ERCC2/XPD, and in itsparental cell line AA8. Both cell lines were exposed to ethylmethanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). EMS gavea similar dose-dependent increase in hprt mutants in UV5 comparedwith AA8. In both cell lines EMS-induced mutations in the hprtcoding region consisted almost exclusively of GC     

Mutational spectrum of ICR-191 at the hprt locus in human lymphoblastoid cells

Human TK6 lymphoblasts were treated with the acridine derivative ICR-191, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshift mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remaining +1 frameshifts occurred at six different GGG sequences (14 mutants) and a single GGGG sequence (2 mutants) in other hprt exons. The repetitive guanine sequences that underwent frameshift mutagenesis were located in both the transcribed and nontranscribed strands of hprt. No single base deletions (-1 frameshift mutations) were observed. Base substitutions were observed in 13% (5/39) of the clones analyzed and occurred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven consecutive guanines (produced from a +1 frameshift in a GGGGGG sequence) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10^?7 and contained the wild-type sequence (GGGGGG) in all clones analyzed. The observed frequency of ICR-191-induced -1 frameshift reversion in the GGGGGGG sequence was ～500-fold lower than the estimated frequency of +1 frameshifts observed in the wild-type GGGGGG sequence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt locus in human cells. © 1994 Wiley-Liss, Inc.     

Genotoxicity of tamoxifen citrate and 4-nitroquinoline-1-oxide in the wing spot test of Drosophila melanogaster

Tamoxifen (TAM) is an anti-oestrogen used for treatment and prevention of human breast cancer, but it is also related to human endometrial and uterine cancer. The wing spot test in Drosophila melanogaster was employed to determine the genotoxic effects of TAM and 4-nitroquinoline-1-oxide (4-NQO), a carcinogen that produces adducts similar to TAM-DNA adducts detected in rodent liver and human liver microsomes. As Drosophila spp. have no oestrogen receptor, no effects can result in binding of TAM to a receptor. Chronic treatments with TAM citrate were performed with 3-day-old larvae of the standard (ST) and high bioactivation (HB) crosses of the wing spot test at concentrations of 0.66, 1.66 and 3.33 mM. In addition, the carcinogen 4-NQO was administered at 2.5 and 5.0 mM. Somatic spots on normal wings from marker-heterozygous flies and on serrate wings from balancer-heterozygous flies were scored to determine mutation and recombination events in somatic cells for each compound. The results showed genotoxic effects of TAM at 1.66 and 3.33 mM in the ST cross only and without a clear dose-response effect. This suggests a weak genotoxicity of this anti-oestrogen. The negative results obtained with TAM in the HB cross may indicate efficient detoxification of the compound by the increased xenobiotic metabolism present in this cross. As reported before, 4-NQO showed genotoxic effects in the ST cross with a clear dose-response effect. For the first time, we report enhanced effects of this compound in the HB cross. It is concluded that the genotoxicity of TAM in the Drosophila wing spot test is different from that of 4-NQO.     

DNA damage and repair induced by bleomycin in mammalian and insect cells.

We assessed the response of mosquito (ATC-15) and mammalian (CHO) cells to bleomycin (BLM). Comparison of the data obtained in both cell lines indicates that DNA in the chromatin of mosquito cells is 10 to 20 times more resistant to BLM, and that the DNA damage induced by this antibiotic is better repaired in mosquito than in mammalian cells. Permeability of the cell membrane for BLM was found to be the same for both cell lines. Moreover, the time-kinetics of BLM damage to nuclear DNA was similar for ATC-15 and CHO cells. The low sensitivity of mosquito cells to BLM is reflected in better growth efficiency. These cells exhibit a satisfactory growth at BLM doses that produce a permanent arrest of growth in CHO cells. It is proposed that variations in the chromatin structure and in the intracellular free amino acid pool may play an important role in the differential response of insect and mammalian cells to BLM.     

Analysis of mutations at the neurofibromatosis 1 (NF1) locus.

A panel of 200 unrelated NF1 individuals has been screened for mutations using a panel of specific clones for the entire gene. DNA analysis on conventional Southern blots indicated that (20) 10% of NF1 patients showed aberrant bands. Small lesions involving nucleotide alterations were detected in a further 10 patients; 5 of these alterations have been fully characterised and are the novel mutations in the NF1 gene. A number of mutations were identified in exon 2. Identical mutations in this exon in two unrelated individuals involved an insertion of cytosine into codon 5662 and resulted in an inappropriate stop codon. This mutation also created a new MnlI site. Another novel mutation in exon 2 resulted from the insertion of thymidine at nucleotide 5678, which also created an inappropriate stop codon. We have so far completed the screen of exons 1-9 of the NF1 gene for the identification of mutations and have found no evidence of clustering of such mutations in the gene.     

Analysis of DNA alkylation damage and repair in mammalian cells by the comet assay

The single cell gel electrophoresis (SCGE) or comet assay, whichmeasures DNA strand breaks in individual cells, was used toanalyse DNA damage and repair induced by the SN₁-type alkylatingcarcinogens N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosoureain CHO cells. The comet assay was comparable in sensitivityto the alkaline elution assay. The alkyl-adducts detected asDNA single-strand breaks (ssb) by this technique were completelyrepaired within 24 h after treatment. These data indicate thatlong-lived lesions, such as alkylphosphotriesters, are not convertedinto ssb under the standard SCGE alkaline conditions (pH 13.5).The lesions revealed by the comet assay are mainly apurinic/apyrimidinic(AP) sites and breaks formed as intermediates in the base excisionrepair process of N-alkylpurines. When SCGE was performed atpH 12.5 instead of pH 13.5 a lower level of ssb was detectedand these breaks were completely resealed within 2 h after treatmentThese data suggest that different subsets of lesions are detectedunder different pH conditions. The SCGE combined with inclusionwithin the cells of endo-nuclease III revealed that a high portionof AP sites induced by alkylation damage were not convertedinto ssb by alkali. The level of endonuclease III-sensitivesites decreased as a function of the repair time and by 24 hafter treatment no sites were left on the DNA. The use of thismodified SCGE assay allows the estimation of the total amountof unrepaired AP sites present on DNA. Alkylation-induced ssbas detected by the comet assay should be regarded as an indicatorof repair rate and balance more than a measure of actual DNAdamage. ³To whom correspondence should be addressed     

The effect of non-ionic surfactants on the SOS-inducing potency of 4-nitroquinoline-1-oxide in Escherichia coli PQ37

Various non-ionic surfactants affect the SOS-inducing potency (SOSIP) of the model genotoxin, 4-nitroquinoline-1-oxide, in Escherichia coli PQ37 to varying degrees, as measured by an automated version of the SOS chromotest. While there is little effect on the SOSIP value and other test parameters from Tween 20 and 80 and, with some reservation, Triton X305 and Tyloxapol, over the critical micelle concentration range, the SOSIP value increases in the presence of comparable concentrations of Triton X15, 45 and 100. A possible dependence of the tester strain's beta-galactosidase production and its growth inhibition on the HLB of the non-ionic surfactants added is discussed.     

Analysis of mutations at the fragile X locus using the DNA probe Ox1.9.

In this study, 40 families segregating for fragile X [fra (X)] syndrome were examined for the presence of a mutation within the FMR-1 gene. Using the DNA probe Ox1.9, both carriers and affected individuals were found to contain an insertion/amplification-type of mutation with somatic instability. Variability in the size of the mutation, which ranged from less than 0.2 kb to approximately 13 kb, was observed both between individuals (even from the same family) and within individuals, who showed a smear rather than a discrete band(s) on Southern blot analysis. Transmission of the mutation by males resulted in little change of its size, while transmission by females usually resulted in an increase in size. Correlations were observed between the size of inserted/amplified DNA and the level of chromosome fragility and the presence or absence of mental impairment. Overall, a mutation was detected in 66 of 67 (99%) clinically affected males, in 12 of 13 (92%) transmitting males and in 95 of 112 (85%) carrier females. Equivocal results were obtained in 12 (11%) of the carrier females. No mutation was detected in 58 females and 33 males predicted to be normal by linkage, or in one female and 36 normal control males. These results strongly suggest that the mutation detected by Ox1.9 is closely associated with the cytogenetic and clinical expression of fra (X) syndrome. Additionally, the use of this probe along with other probe/enzyme combinations should provide a sensitive clinical assay for the detection of carriers of fra (X) syndrome.     

Molecular spectrum of background mutation at the hprt locus in human T-lymphocytes

The molecular basis of somatic mutation at the hypoxanthineguaninephosphoribosyl-transerase (hprt) locus in human 6-thioguanineresistant T-cell clones from 17 individuals has been studiedby Southern blot analysis, multiplex PCR (polymerase chain reaction)and direct sequencing of PCR amplified hprt cDNAs or genomicDNA. Twenty-three novel mutations were detected, which in additionto previously described mutations provide a background mutationalspectrum based on a total of 45 hprt mutations in human T-cells.Twenty T-cell mutants had base substitutions in the coding regionleading to 15 missense and five nonsense mutations. In additionto five frameshift mutations caused by four small deletionsand one duplication, seven splice mutations, three of them withskipping of exon 8, were detected. Thirteen genomic structuralalterations have also been identified; one of these had a genomicexon 1 deletion with a GGCCGG-hexamer in both breakpoints.     

DNA sequence analysis of mutations induced by melphalan in the CHO aprt locus.

In previous work, we established that treatment with melphalan (L-phenylalanine mustard) produced a predominance of A.T-->T.A transversions in the Simian virus 40 (SV40)-based shuttle vector pZ189 during replication in human 293 cells. Mutations were induced with varying doses (4-12 microM) melphalan in the aprt gene of the hemizygous Chinese hamster ovary (CHO) cell line D422 to determine whether a similar mutation spectrum would be observed in an endogenous gene. DNA sequence alterations were determined for 39 spontaneous and 41 melphalan-induced independent mutant clones. Other than a predominance of transversions in both systems, the spectrum of melphalan-induced aprt mutations bears little resemblance to the spectrum observed in the supF gene of the shuttle plasmid pZ189. In aprt, mutations at G.C base pairs (bp) predominated (29 of 41 base substitutions). Significantly enhanced mutagenesis was observed at 5' G-G-C 3' and 5' G-G-C-C 3' sites in the aprt gene. Almost half of the melphalan-induced base substitutions occurred at 5' G-N-C 3' sequences, which are believed to be potential interstrand crosslink sites.     

DNA sequence of mutations induced in cells by herpes simplex virus type-1

The shuttle vector plasmid pZ189 was used to find the kinds of mutations that are induced in cells by herpes simplex virus type-1 (HSV-1). A significant increase in mutation frequency was detected as early as 2 hr after infection, and reached a peak of two- to sevenfold over background at 4 hr after infection. Several differences were detected between spontaneous mutants and those induced by HSV-1 when they were analyzed by gel electrophoresis and DNA sequencing. Point mutations accounted for 63% of spontaneous mutants but for only 44% of HSV-1-induced mutants (P less than 0.05). In each case the predominant type of point mutation was the G:C to A:T transition, which comprised 51% of point mutations induced by HSV-1, and 32% of spontaneous point mutations. Deletions of DNA were seen in HSV-1-induced mutants at a frequency of 44%, compared with only 29% in spontaneous mutants. HSV-1-induced deletions were less than half the length of spontaneous deletions, and 3 contained short filler sequences. An increase in size was seen in 13% of HSV-1-induced mutants and was due either to duplication of plasmid DNA, or, in 8 instances, to insertion of sequences derived from cellular DNA. Among spontaneous mutants, only 8% were increased in size and none of them had inserted cellular DNA. The proportion of complex mutants increased as infection by the virus progressed and they accounted for 79% of mutants at 24 hr after infection. The observed mutations have implications for understanding the "hit and run" mechanism of malignant transformation of cells by HSV-1.     

Evidence for preferential repair of 3-carbethoxypsoralen plus UVA induced DNA lesions in the active MAT{alpha} locus in Saccharomyces cerevisiae using the UvrABC assay

The occurrence of preferential repair in Saccharomyces cerevisiaeof the active MAT locus compared with the inactive HML locuswas confirmed after 254 nm UV irradiation. Experiments carriedout using the UvrABC excinuclease assay with the monofunctionalfurocoumarin 3-carbethoxypsoralen (3-CPs) plus UVA radiationwhich induce mainly monoadducts in DNA demonstrated preferentialrepair of the active MAT locus compared with the inactive HMLlocus in a SIR⁺ strain. However, as after 254 nm UV irradiation,no difference in the rate of removal of 3-CPs plus UVA inducedlesions was observed between the two loci in the sir-3 mutantin which both loci are active. Thus, it appears that 3-CPs plusUVA induced monoadducts as well as pyrimidine dinners a e subjectto preferential repair. ³To whom correspondence should be addressed     

XRCC3 promotes homology-directed repair of DNA damage in mammalian cells

Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.