DMBA-induced toxic and mutagenic responses vary dramatically between NER-deficient Xpa, Xpc and Csb mice
Susan W.P. Wijnhoven1,
Hanneke J.M. Kool2,
Leon H.F. Mullenders1,3,
Rosalyn Slater2,
Albert A. van Zeeland1,3 and
Harry Vrieling1,3,4
1 Department of Radiation Genetics and Chemical MutagenesisMGC, Leiden University Medical Center,
2 Department of Cell Biology and GeneticsMGC, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam and
3 J.A.Cohen Institute, Inter-University Institute for Radiopathology and Radiation Protection, Leiden, The Netherlands
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Abstract
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Heterogeneity in cancer susceptibility exists between patients with an inherited defect in nucleotide excision repair (NER). While xeroderma pigmentosum (XP) patients have elevated skin cancer rates, Cockayne syndrome (CS) patients do not appear to have increased cancer susceptibility. To investigate whether differences in mutagenesis are the basis for the variability in cancer proneness, we studied mutagenesis at the X-chromosomal Hprt gene and the autosomal Aprt gene in splenic T-lymphocytes after 7,12-dimethyl-1,2-benz[a]anthracene (DMBA) exposure in total NER-deficient Xpa mice, global genome repair (GGR)-deficient Xpc mice and transcription coupled repair (TCR)-deficient Csb mice. Surprisingly, while all intraperitoneally-treated Xpc/ mice survived a dose of 40 mg/kg DMBA, a substantial fraction of the treated Xpa/ and Csb/ mice died a few days after treatment with a 20-fold lower dose. Functional TCR of DMBA adducts in Xpc/ mice thus appears to alleviate DMBA toxicity. However, the mutagenic response in Xpc/ mice was ± 2-fold enhanced at both the Hprt and the Aprt gene compared to heterozygous controls, indicating that GGR at least partially removes DMBA adducts from the genome overall. DMBA-induced SCE frequencies in mouse dermal fibroblasts were significantly enhanced in Xpa- and Csb-, but not in Xpc-deficient background compared to the frequency in normal fibroblasts. These results indicate that both damage-induced cytotoxicity as well as intra-chromosomal recombinational events were not correlated to differences in cancer susceptibility in human NER syndrome patients.
Abbreviations: 6-TG, 6-thioguanine; 8-AA, 8-azaadenine; BER, base excision repair; BrdU, bromodeoxyuridine; CS, Cockayne syndrome; DMBA, 7,12-dimethyl-1,2-benz[a]anthracene; GGR, global genome repair; LOH, loss of heterozygosity; MDF, mouse dermal fibroblasts; NER, nucleotide excision repair; PCR, polymerase chain reaction; SCE, sister chromatid exchange; TCR, transcription coupled repair; TTD, trichothiodystrophy; XP, xeroderma pigmentosum.
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Introduction
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In mammalian cells, a complex network of repair systems has evolved to counteract the deleterious effects of environmental and endogenous genotoxic agents (1). The nucleotide excision repair (NER) pathway is a universal repair pathway mainly dealing with severely helix-distorting DNA injuries including bulky adducts induced by polycyclic aromatic hydrocarbons such as benz[a]anthracene (13). NER consists of two in part overlapping subpathways, i.e. global genome repair (GGR) that eliminates lesions throughout the genome and transcription-coupled repair (TCR) that exclusively removes lesions that interfere with transcription (4,5). In humans, inherited defects in NER are associated with three photosensitive disorders: i.e. xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). Complementation studies with patient cell lines have revealed the existence of seven genes in classical XP (XPA through XPG). Five genes are involved in CS: CSA and CSB, and specific alleles of XPB, XPD and XPG. Within photosensitive TTD, the XPB, XPD and TTDA genes appear to be implicated (6). XP complementation groups AG are all defective in both TCR and GGR, with the exception of XP-C, which is defective in GGR only (7,8). In cells of CS patients, a specific defect is observed in the TCR pathway (9,10). The genetic diversity in photosensitive patients is paralleled by clinical heterogeneity with respect to (skin) cancer susceptibility. XP patients, but not CS and TTD patients, have a >2000-fold increment in all forms of skin cancer, limited to the sun-exposed areas of the skin (2,6). The frequency of internal cancer is also 1020-fold elevated in XP patients (11). CS patients typically suffer from developmental and neurological abnormalities (12).
Since cancer is an in vivo process in which somatic mutations accumulate, we performed mutagenesis studies in mouse models for XP and CS to investigate whether differences in the extent of mutation induction or the kind of induced mutations following exposure to a carcinogen could explain the heterogeneity in cancer susceptibility between XP-A and XP-C patients on the one-hand and CS-B patients on the other. Xpa and Xpc knock-out mice clearly mimic the human XP disorder with respect to skin cancer predisposition as they show a higher susceptibility to UV-induced skin cancer than their heterozygous littermates (1316). Csb/ mice, in contrast to CS patients, appear to be skin cancer prone after dermal exposure to UV. However, compared to Xpa/ or Xpc/ mice, a higher cumulative dose of carcinogen (UV or 7,12-dimethyl-1,2-benz[a]anthracene (DMBA)) and a longer latency time is required in Csb/ mice before they develop skin cancer (17). This discrepancy between mouse and man can be partly explained by the more efficient GGR pathway in human skin fibroblasts compared with rodents, that may compensate the TCR deficiency in CS patients but not in CS mice (18).
In the study presented here, Xpa-, Xpc- and Csb-deficient mice were crossed in an Aprt heterozygous background and intraperitoneally (i.p.) treated with the chemical carcinogen DMBA. The bulky lesions caused by DMBA are substrate for NER and are able to cause both point mutations and loss of heterozygosity (LOH) mutations (19). Surprisingly, we observed an extreme acute toxicity in TCR-deficient mice (Xpa/ and Csb/) after i.p. DMBA treatment, which was not observed in TCR-proficient mice (Xpc/ and wild-type mice). Mutagenesis studies were performed at both the Hprt and Aprt gene in splenic T-lymphocytes of Xpc mice treated with a dose of 40 mg/kg DMBA. The effect of a deficiency in GGR on the spectrum of DMBA-induced mutations was investigated by molecular analysis of the obtained Hprt and Aprt mutants. Furthermore, the induction of sister chromatid exchange (SCE) by DMBA was determined in vivo in bone marrow and spleen cells of Xpc mice and in vitro in dermal fibroblasts of all NER-deficient mouse strains to study the possible involvement of recombinogenic events in this context.
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Materials and methods
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Mice
The generation of NER-deficient mice, i.e. Xpc, Xpa and Csb, has been previously described (13,17,20). All NER-deficient mice as well as the p53+/ were in a C57Bl/6 background (Xpc mice in F5, Xpa and Csb mice were both in F8-F9). NER-deficient (single or double KO) mice were derived from crossing heterozygous (NER+/) and homozygous (NER/) mice. Aprt+/ mice were generated as described earlier (21) and Aprt heterozygous mice as described in these mutational studies were in a C57Bl/6 background (F5 generation).
Genotyping of the different NER-deficient mice was performed via allele-specific PCR analysis of lysates from tail tips. Lysis of tail tips was done overnight at 60°C in a buffer containing 50 mM TrisHCl (pH 8.9) and 12.5 mM MgCl2, in the presence of 0.2 mg proteinase K. After deactivation of proteinase K, 10 min at 95°C, PCR analysis was performed with the different primer sets. XPC primers: XPC10s, 5'-ATTGCGTGCATACCTTGCAC-3'; NEO, 5'-CGCATCGCCTTCTATCGCCT-3'; mXPCin10AS, 5'-TATCTCCTCAAACCCTGCTC-3'. Sequences of the CSB and XPA primers have been described previously (13,17).
DMBA toxicity experiments
Groups of 210 young adult male and female mice of 810 weeks old were used. DMBA (Sigma, Zw
ndrecht, The Netherlands) was dissolved in tricaprilyn (Fluka), and was administered at single (i.p.) doses. Xpa and Csb mice were treated with a dose of 15 mg/kg, Xpa/Xpc and Xpa/p53 double knockout mice with 5 mg/kg DMBA. Xpc mice and wild-type control mice were exposed to a higher dose of DMBA, i.e. 40 mg/kg for the Xpc/ mice and up to 100 mg/kg for the wild-types.
DMBA treatment for induction of Hprt and Aprt mutants
For mutant frequency analysis experiments, young adult male and female mice were used. Hprt-mutant frequencies were determined in Xpc-defective mice as well as heterozygous littermates after treatment with DMBA, at 0, 10, 20 and 40 mg/kg. Aprt (and Hprt) mutant frequencies were determined in Xpc mice crossed in an Aprt +/ background. In this study, mice were treated with 0 and 40 mg/kg DMBA. In all experiments, 310 mice per dose were used. Seven weeks after treatment, mice were killed and spleens were isolated.
Isolation and culturing of splenic T-lymphocytes
Priming and cloning of T-lymphocytes was performed in RPMI culture medium 1640 as described by Tates et al. (22). Details about isolation, freezing and thawing of mouse splenocytes as well as priming of the cells with concavalin A has been previously described (19). Mutant clones were selected by adding either 6-thioguanine (6-TG) or 8-azaadenine (8-AA) to the culture medium for the recovery of Hprt-deficient or Aprt- deficient mutants, respectively (19). Calculation of cloning efficiencies and mutant frequencies was performed as described (22).
Molecular characterization of T-lymphocyte clones
Isolation of Hprt mutant and Aprt mutant clones. 6-TG or 8-AA resistant clones were selected and diluted 1:3 in culture medium containing 2.5 µg/ml 6-TG or 50 µg/ml 8-AA. After 34 days of culturing, cells were collected, centrifuged and cell pellets were washed with phosphate-buffered saline. Hprt mutant clones were stored at 80°C or used directly used for RNA isolation, whereas Aprt mutant clones were processed to give crude cell lysates.
RNA extraction and cDNA synthesis of Hprt mutant clones
RNA isolation from cell pellets using TRIzol (Life Technologies, Breda, The Netherlands) and subsequent chloroform extraction as well as Hprt cDNA synthesis of RNA has previously been described in detail (23).
Amplification of Hprt cDNA by the polymerase chain reactionPCR
Hprt cDNA was amplified by two subsequent PCR reactions with nested primer sets (23). Re-amplified DNA (10 µl) was sequenced using the Thermo Sequenase Fluorescent-labelled Primer Cycle Sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Buckinghamshire, UK) on an automated laser fluorescence (ALF) sequencer (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Allele-specific PCR of Aprt mutant clones
An allele-specific PCR was performed on crude cell lysates of Aprt mutant clones (19) to determine whether mutants had lost the wild-type Aprt allele.
In vivo SCE experiments in DMBA-treated Xpc mice
Xpc homozygous and heterozygous mice of 810 weeks old were treated with DMBA in vivo, and SCE frequencies were determined in bone marrow and spleen cells at different time points. In the first experiment, mice were treated with 0 or 10 mg/kg DMBA and killed at 1, 2, 4 and 7 days after treatment. Both bone marrow and spleen cells were isolated. A second experiment was restricted to splenocytes of mice that had been treated with 0 or 40 mg/kg DMBA and isolated 1 and 7 days later. Per dose and time point, one or two mice were used. The standard culturing medium used was RPMI 1640 (Gibco, Breda, The Netherlands) supplemented with 20% Hybridoma- medium (HL-1, Hycor Brunswig, Amsterdam, The Netherlands), 6% foetal bovine serum (FBS, Hyclone, Logan, Utah, USA) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin sulfate). For culturing of splenocytes 3.39 µg/ml lithium chloride, 22.7 i.e./ml heparin, 50 µM ß-mercaptoethanol, 2.5 µg/ml concavalin A, 5 µg/ml and 5 µM bromodeoxyuridine (BrdU) were added. Bone marrow cells were cultured in standard medium supplemented with 5 ng/ml Granulocyte Macrophage Colony Stimulating Factor and 5 µM BrdU. SCE frequencies were determined in metaphases obtained two cell cycles after addition of bromodeoxyuridine (BrdU) (after 18 h for bone marrow cells and 43 h for splenocytes). Colcemid (0.1 µg/ml) was added 2 h before fixation and slides were stained with acridine orange for SCE determination.
SCE measurement in NER-deficient mouse dermal fibroblasts after DMBA treatment
Mouse dermal fibroblasts were isolated from newborn mice (12 days old) of various NER-deficient mouse strains and cultured in DMEM supplemented with 15% FBS (Hyclone) and antibiotics at 37°C and 10% CO2. At passage 3, the exponentially growing cells were treated with 0, 3, 6, 9 or 12 µg/ml DMBA (dissolved in DMSO) in the presence of a rat microsomal S9 fraction for 3 h. The fibroblasts were washed two times with fresh serum-free medium and cultured for 42.5 h in the presence of 5 µM BrdU. Two hours before harvesting, 0.1 µg/ml colcemid was added. Slides were stained with acridine orange for SCE determination.
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Results
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DMBA-induced toxicity in NER-deficient mice
Young adult NER-deficient mice were exposed to different exposure levels of DMBA, a polycyclic aromatic hydrocarbon that induces bulky lesions that are substrate for NER. DMBA is a potent rodent mutagen and carcinogen (19,2427). Unexpectedly, large differences in toxicity were observed between the various mouse strains. While all treated Xpc/ and wild-type mice survived a dose of 40 mg/kg DMBA without any signs of toxicity, a major fraction of Xpa/ and Csb/ mice suffered from severe abdominal peritonitis and had to be killed within a few days of i.p. treatment with 2030-fold lower doses of DMBA (Table I
). Heterozygous Xpa and Csb mice did not show increased sensitivity to DMBA (data not shown). In addition, Xpa//Xpc/ and Xpa//p53/ double knockout mice were as sensitive to a dose of 5 mg/kg DMBA as Xpa/ mice (Table I
).
DMBA-induced Hprt mutant frequencies in Xpc mice
Mutagenic responses were measured at the Hprt gene in splenic T-lymphocytes of Xpc/ and Xpc+/ mice treated with different subtoxic doses of DMBA (Table II
). In Xpc/ mice, background Hprt mutant frequency levels in untreated mice turned out to be 3-fold higher than in Xpc+/ littermates (Table II
) as reported previously (28). Treatment with high doses of DMBA (10, 20 and 40 mg/kg) led to a dose-dependent increase in mutant frequency in both Xpc/ and Xpc+/ mice. At a dose of 40 mg/kg, however, the Xpc/ mice showed a (±2-fold) hypermutable phenotype (Table II
), indicating that DMBA adducts are partially removed by GGR.
DMBA-induced Aprt and Hprt mutant frequencies in AprtXpc mice
It is conceivable that the observed hypermutability at the Hprt gene in DMBA-treated Xpc/ mice is a reflection of the inability of Xpc/ cells to remove DMBA adducts from the non-transcribed strand of active genes. We have previously shown that DMBA not only causes intragenic mutations, as can be detected at the X-chromosomal Hprt gene, but also has the potency to induce LOH (19). To determine the effect of a GGR deficiency on the LOH-causing potency of DMBA, we crossed Xpc/ and Xpc+/ mice with Aprt heterozygous mice and measured mutant frequencies at both the Aprt and Hprt gene in splenic T-lymphocytes after treatment with 0 and 40 mg/kg DMBA (Table II
). Spontaneous and DMBA-induced Hprt mutant frequencies in Aprt+/ mice of all Xpc-genotypes were in the range of previously obtained data. Therefore, Hprt mutant frequencies of both experiments were pooled. Spontaneous Aprt mutant frequencies were 3-fold higher in Xpc-deficient mice (21.9x106) than in heterozygous Xpc mice (7.3x106; Table II
) and completely repair proficient Aprt+/ mice as reported previously (8.7x106; 21).
After DMBA treatment, the Aprt mutant frequency in Xpc-deficient mice was induced to a level of 91.6x106, while induction in heterozygotes was lower at 45.7x106 (Table II
) and in the range of previous obtained frequencies in Aprt+/ mice (19). The deficiency in GGR of DMBA-exposed Xpc/ mice resulted not only in a hypermutability at the Hprt gene, but also at the Aprt locus after a dose of 40 mg/kg DMBA.
Molecular analysis of DMBA-induced mutants at the Hprt and Aprt locus
Mutational spectrum analysis in Hprt mutants of Xpc mice.
To determine the nature of the DMBA-induced mutants, a DNA sequence analysis of the coding region of the Hprt gene was performed in Hprt mutants. Mutants from the dose-response study and from the AprtXpc experiment were pooled (Table III
). Identical mutations found within one animal were considered to have resulted from clonal expansion. In total, 50 independent Hprt mutants from Xpc+/ mice (four spontaneous and 46 DMBA-induced mutants) and 96 Hprt mutants from Xpc/ mice (28 spontaneous and 68 DMBA-induced mutants) were sequenced. An overview of these results is presented in Table III
.
In Xpc+/ mice, the major classes of DMBA-induced base pair substitutions were GC
TA and AT
TA transversions as found previously (24). These classes of mutations were also the major base substitutions in a Xpc/ background. Most of the mutants that were obtained from untreated Xpc/ mice contained an AT
TA transversion, which is in line with previously obtained data (28). The fraction of mutants originating from a splice mutation was slightly higher in DMBA-treated Xpc/ mice. Possibly, bases at splice acceptor and donor sites in the poorly repaired non-transcribed strand (NTS) in Xpc/ mice, are hotspots for DMBA adduct formation. Indeed, the consensus sequence of splice acceptor sites contains the invariant AG sequence in the NTS, which has been shown to be a preferred target for DMBA-induced lesions (24). Although a ±2-fold increase in Hprt mutant frequency was detected in Xpc/ mice compared to repair proficient mice at the highest dose of DMBA, the mutational spectrum of DMBA-induced base changes was similar. In both genotypes, DMBA-specific AT
TA and GC
TA transversions were the major classes of base pair substitutions. Almost all of the mutated G and A bases were located in the NTS of the Hprt gene (data not shown).
Determination of loss-of-heterozygosity in Aprt mutants of Xpc mice
In repair proficient Aprt+/ mice, 70% of DMBA-induced mutations in the Aprt gene were due to a LOH event (19). We examined whether the deficiency in GGR of DMBA adducts in Xpc/ mice would alter the ratio of point mutations versus LOH mutations. An allele specific PCR was performed to detect the extent of the loss of the wild-type Aprt allele in isolated spontaneous and DMBA-induced Aprt mutants obtained from Xpc/ and Xpc+/ mice, crossed in an Aprt heterozygous background. The results are summarized in Figure 1
. In Xpc heterozygous mice (upper panel), 75% of spontaneous Aprt mutants showed loss of the normal Aprt allele, while this was 67% after DMBA treatment. In Xpc/ mice (lower panel), the percentage LOH in background mutants was 87%, which is substantially higher than the 75% in Xpc+/ mice and 69% previously found under NER-proficient conditions (19). However, after DMBA exposure, the fraction of mutants with loss of the wild-type allele was 67% and similar to the fraction in Xpc+/ mice (Figure 1
). Furthermore, Figure 1
shows that the fraction of the mutant frequency originating from point mutations at Aprt [(total number of Aprt mutants) (mutants with LOH)] is similar to the mutant frequency at Hprt (solid grey bars), in Xpc/ and Xpc+/ mice both spontaneously and after DMBA treatment. This indicates that the target size for base substitutions is similar for Hprt and Aprt.

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Fig. 1. Relative potency of DMBA to induce LOH events at the Aprt locus in Xpc mice. Isolated Aprt mutants from untreated and DMBA-treated Xpc mice of both genotypes were analysed by allele-specific PCR to determine in what fraction of the (DMBA-induced) Aprt mutants the wild-type Aprt allele had been lost. The non-striped part of the Aprt mutant frequency bar represents the fraction of LOH mutations. The percentage (%) next to this bar indicates the exact value. The striped fraction consists of Aprt mutants that contained an intragenic mutation and had retained the wild-type Aprt allele. Upper bars represent the Xpc+/ mice; lower bars the Xpc-deficient mice. The DMBA-induced Aprt frequencies and LOH percentages have been corrected for the background values of untreated mice.
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DMBA-induced SCE frequencies in Xpc mice in vivo
Sister chromatid exchanges are homologous recombinational events between two chromatids of one chromosome. Determination of the number of SCE per cell is considered to be a rapid and sensitive test for the recombinogenic potency of a DNA-damaging agent. Since the predominant mechanism underlying DMBA-induced LOH events at the Aprt locus is most probably mitotic recombination (19) and SCE measurements are considered to be a model for homologous recombinational events between homologous chromosomes, we determined SCE frequencies in Xpc mice after in vivo DMBA treatment. In Table IV
the results are presented on SCE induction in bone marrow and spleen cells at various times points after in vivo treatment of Xpc/ and Xpc+/ mice with different doses of DMBA (0, 10 and 40 mg/kg). DMBA treatment caused slightly enhanced SCE frequencies in both cell types, with induced SCE frequencies being constant for all time points (Table IV
). Interestingly, although the absence of GGR in Xpc/ mice caused a hypermutable effect in splenocytes at both the Hprt and Aprt locus after DMBA exposure, SCE frequencies were not enhanced in Xpc/ compared to Xpc+/ mice.
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Table IV. SCE frequencies in bone marrow and spleen cells of Xpc mice at different time points after in vivo DMBA exposure
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SCE induction after in vitro DMBA treatment of NER-deficient dermal fibroblasts
To compare the mutagenic and recombinogenic response of DMBA in cells with a deficiency in TCR, GGR or both NER subpathways, we determined SCE frequencies in primary cells obtained from the different NER-deficient mouse strains and their heterozygous littermates. Primary mouse dermal fibroblasts (MDF) were in vitro exposed to DMBA. Background SCE levels were similar for fibroblasts of all genotypes indicating no effect of the various NER deficiencies on spontaneous SCE formation (Figure 2
). Since MDF do not have the capacity to metabolically activate DMBA, cells were treated with DMBA in the presence of rat S9 microsomal fraction to allow formation of mutagenic DMBA metabolites. Cell growth of Xpa- and Csb-deficient dermal fibroblasts was completely inhibited at a dose of 12 µg/ml DMBA, whereas Xpc-deficient fibroblasts did not show any sign of cytotoxicity at this dose (unpublished results). This is in line with the observed differences in toxicity between the various NER-deficient mouse strains after in vivo DMBA treatment.
A dose of 6 µg/ml DMBA was used to compare SCE frequencies in fibroblasts obtained from the different NER-deficient mouse strains. At this dose, a clear induction of the SCE frequency over the background level was found in fibroblasts that lacked TCR, i.e. 21.7 in Xpa/ and 26.2 in Csb/. In contrast, the frequency of DMBA-induced SCE levels in Xpc/ cells was 9.2, a level that is only slightly higher than background levels and is comparable with those found for all three heterozygous cell lines (Figure 2
).
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Discussion
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In the present study, NER-deficient mice were i.p. treated with DMBA in order to study differences in mutagenic response in mice that are deficient in TCR, GGR or both of the NER subpathways. Since our recent studies with NER-deficient mouse models showed that both spontaneous and chemical-induced mutagenesis at the Hprt locus, which is a marker for gene mutations, did not correlate well with cancer susceptibility (23,28), the Aprt heterozygous mouse model (19,21,29,30) was used. With this model we could determine the occurrence of both intragenic mutations as well as chromosomal events in splenocytes of DMBA-treated NER-deficient mice and we could try to correlate these responses with cancer susceptibility.
DMBA appeared to be a suitable chemical for this study since in previous experiments a dermal DMBA exposure of NER-deficient mice led to an enhancement of skin cancer levels compared to normal mice, indicating that DMBA adducts are substrate for NER (13,17). However, the data obtained in the present study suggest that proficient TCR plays a crucial role in alleviating the acute toxic effects of a single i.p. dose of DMBA. In line with the much higher resistance to DMBA, Xpc/ mice were found to be also more resistant towards the acute toxic effects of UV-B irradiation on the skin (e.g. erythema and oedema) than Xpa/ and Csb/ mice (31,32). Furthermore, the apoptotic response in UVB-exposed keratinocytes of Xpc/ mice was found to be similar to the response of cells obtained from wild-type animals (33,34), while enhanced levels of apoptosis were found in keratinocytes of UV-exposed Xpa- and Csb-deficient mice (34). These results indicate that survival of cells containing bulky DNA damage is largely dependent on the removal of the small subset of DNA lesions that interfere with transcription. According to this model, impaired TCR of DMBA adducts in Xpa and Csb cells may thus lead to prolonged inhibition of RNA polymerase II driven transcription, triggering induction of (p53-dependent) apoptotic pathways. This hypothesis is supported by reports showing that human XP-A and CS-B fibroblasts accumulate active p53 and go into apoptosis after significantly lower doses of UV (3538) and NA-AAF (van Gijssel, in preparation) than normal or TCR-proficient XP-C cells. In Xpc-deficient mice, as a consequence of the unique combination of functional TCR and defective GGR, damaged cells can survive but with increased mutation rates. In support of this is our recent finding that the spontaneous Hprt mutant frequency in splenic T-lymphocytes of aged Xpc/ mice was dramatically increased, probably as the result of survival of cells with relatively high levels of persistent spontaneous base damage (28). In contrast, mutant frequencies in Xpa/ and Csb/ mice remained at background levels during ageing presumably because of the elimination of heavily damaged cells. However, spontaneous Hprt mutant frequencies did rise in Xpa/ mice that were in a p53-deficient background (28), indicating that in Xpa/ mice at least a fraction of the cells containing spontaneously arisen damage are removed by p53-dependent apoptotic pathways. At 5 mg/kg DMBA no differences in survival rates were observed for double knockout Xpa//Xpc/ and Xpa//p53/ mice compared to single knockout Xpa mice (Table I
). Thus, neither the absence of the XPC protein nor the p53 protein could prevent DMBA toxicity of Xpa/ mice at this dose of DMBA. This result indicates that the XPC damage recognition protein is at least not directly involved in the induction of the observed (cyto-)toxic effects. Furthermore, although DMBA-induced apoptotic pathways may be (partly) p53 dependent, elimination of this route of induction of apoptosis did not protect mice from cell- and animal death.
In DMBA-treated Xpc/ mice, a 2-fold hypermutability at both the endogenous Hprt and Aprt genes was observed compared to Xpc+/ mice after a dose of 40 mg/kg (Table II
). Relative frequencies of intragenic and LOH-causing mutations at Aprt were similar in Xpc+/ and Xpc/ mice, indicating that GGR is equally responsible for the elimination of DMBA adducts causing intragenic mutations at non-transcribed strands as well as of LOH-causing DMBA adducts from the genome overall. GGR of spontaneous arising DNA lesions seemed to be important in reducing the frequency of LOH events, since the background LOH frequency at Aprt was substantially higher in Xpc/ mice than in Xpc+/ mice (Figure 1
) or control mice (21).
Although DMBA-induced Hprt mutant frequencies were ±2-fold enhanced in Xpc/ mice at the highest dose of DMBA, the Hprt mutational spectrum did not undergo a major change in the absence of GGR (Table III
). The purine residues of mutated base pairs, at which the majority of DNA adducts are formed, were in both genotypes almost exclusively located in the NTS of the Hprt gene, probably reflecting TCR of DMBA adducts.
Among both spontaneous and DMBA-induced Aprt mutants isolated from mouse splenic T-lymphocytes, mitotic recombination seems to be the major pathway leading to LOH (19,21,30). Sister chromatid exchanges are homologous recombinational events between two chromatids of one chromosome that are detectable using simple cytogenic techniques and are not mutagenic for the cell. Molecular mechanisms underlying SCE formation are generally believed to be similar to those that cause mitotic recombination, being homologous recombination between two sister chromatids of homologous chromosomes. An important trigger for recombination is thought to be blockage of the replication process as previously described for Escherichia coli, yeast and mammalian cells (3942). DMBA exposure results in the formation of bulky adducts in the DNA that in analogy to other bulky lesions, will probably block the progression of DNA replication forks. Stalling of the replication machinery at DMBA adducts can be overcome either by translesion synthesis using specialized DNA polymerases or by recombinational events with homologous DNA sequences. In Xpc/ cells repair of these bulky lesions is limited to the transcribed strand of active genes. LOH events at Aprt were 23-fold enhanced in the absence of GGR (Figure 1
), suggesting that that unrepaired DMBA adducts in the genome overall trigger additional recombinational events because of their interference with DNA replication.
However, the obtained data on DMBA-induced SCE in the various NER-deficient MDF do not support an important role for replication blocks in the induction of recombinational events between sister chromatids of one chromosome. A defect in GGR of DMBA adducts does not appear to result in an increase in recombinational events between sister chromatids. In contrast, disruption of the TCR pathway seems to play a crucial role in induction of this intra-chromosomal recombination process since cells of both TCR-deficient mouse strains (Xpa and Csb) showed a strong increase of DMBA-induced SCE compared to TCR proficient cells. These results indicate that induction of SCE by DMBA adducts may be triggered by stalled transcription rather than by a blockage of replication. Recently, it has been shown in yeast that transcription is an important factor in modulating the incidence of recombination (43). The mechanisms underlying inter- chromosomal mitotic recombination appear thus to be at least partially different from those that trigger the formation of exchanges between the sister chromatids of a chromosome.
In conclusion, our attempts to relate cancer susceptibility of the various NER-deficient mouse models to in vivo mutagenesis using DMBA as mutagen failed due to the extreme sensitivity of TCR-deficient mice for this compound. We could, however, show that the difference in cancer susceptibility is not related to the (cyto)toxic effects found after induction of adducts, substrate for NER. Furthermore, no distinction could be made between XP on the one hand and CS on the other on the basis of SCE measurements. DMBA-induced stalling of transcription (and subsequent inhibition of DNA synthesis) appears to be a much stronger inducer of SCE than replication blockage. This latter phenomenon could be different for other mutagens and will depend on the relative potency of induced lesions to block replication and transcription, respectively.
The absence of a clear cancer phenotype in CS appears not to be related to its defect in NER. A mutation study similar to the one presented in this paper, but using an agent that selectively induces DNA damage as a substrate for base excision repair (BER), might provide more insight in this matter, since CS cells have been shown to be unable to perform TCR of BER adducts, while this process is fully functional in XP (44,45).
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Notes
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4 To whom correspondence should be addressed at Department of Radiation Genetics and Chemical MutagenesisMGC, Leiden University Medical Center, PO Box 9503, 2300 RA Leiden, The Netherlands Email: H.Vrieling{at}lumc.nl 
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Acknowledgments
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We thank C.van Teijlingen for technical assistance on the isolation and culturing of splenic T-lymphocytes and selection of Aprt and Hprt mutants. Furthermore, we thank Dr T.Jacks, MIT, Dr H.van Steeg, RIVM, Dr G.van der Horst, Erasmus University of Rotterdam and Dr E.Friedberg, University of Texas, for providing us with the p53+/ mice, Xpa, Csb and Xpc mice, respectively. This work was financially supported by the Dutch Cancer Society (Project 96-1321).
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References
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Received January 12, 2001;
revised March 12, 2001;
accepted March 15, 2001.