Mutation Research 599 (2006) 116–123

Homologous recombination is involved in repair ofchromium-induced DNA damage in mammalian cells

Helen E. Bryant a, Songmin Ying a,1, Thomas Helleday a,b,∗a The Institute for Cancer Studies, University of Sheffield, Medical School, Beech Hill Road, Sheffield S10 2RX, UK

b Department of Genetics Microbiology and Toxicology, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden

Received 3 August 2005; received in revised form 7 February 2006; accepted 15 February 2006Available online 27 March 2006

Abstract

Chromium is a potent human carcinogen, probably because of its well-documented genotoxic effects. Chromate (Cr[VI]) causesa wide range of DNA lesions, including DNA crosslinks and strand breaks, presumably due to the direct and indirect effects ofDNA oxidation. Homologous recombination repair (HRR) is important for error-free repair of lesions occurring at replication forks.Here, we show that HR deficient cell lines irs1SF and V-C8, deficient in XRCC3 and BRCA2, respectively, are hypersensitive toCr[VI], implicating this repair pathway in repair of Cr[VI] damage. Furthermore, we find that Cr[VI] causes DNA double-strandbreaks and triggers both Rad51 foci formation and induction of HRR. Collectively, these data suggest that HRR is important inrepair of Cr[VI]-induced DNA damage. In addition, we find that ERCC1, XRCC1 and DNA-PKcs defective cells are hypersensitiveto Cr[VI], indicating that several repair pathways cooperate in repairing Cr[VI]-induced DNA damage.© 2006 Elsevier B.V. All rights reserved.

Keywords: Homologous recombination; DNA repair; Mammalian cells; Chromium

1. Introduction

Chromium is a naturally occurring heavy metal whichin the environment exits mainly in trivalent (Cr[III])

Abbreviations: AP site, apurinic/apyrimidinic site; BER, base exci-sion repair; CA, chromosomal aberrations; DSB, DNA double-strandbreak; HAsT, hypoxanthine-l-azaserine-thymidine; HBSS, Hank’sbalanced salt solution; hprt, hypoxanthine-guanine phosphoribosyl-transferase gene; HR, homologous recombination; HRR, homolo-gous recombinational repair; NER, nucleotide excision repair; NHEJ,non-homologous end-joining; PBS, phosphate-buffered saline; PFGE,pulsed-field gel electrophoresis; 6TG, 6-thioguanine

∗ Corresponding author. Tel.: +44 114 271 29 93;fax: +44 114 271 35 15.

E-mail address: t.helleday@sheffield.ac.uk (T. Helleday).1 Present address: Institute for Medical Microbiology, Immunology

and Hygiene, Technical University Munich, D-81675 Munich, Ger-many.

and hexavalent (Cr[VI]) forms. Cr[III] is the most sta-ble form and is unable to enter cells, whereas Cr[VI]is a strong oxidizing agent and more toxic and car-cinogenic. The major routes of exposure to Cr[VI] arefrom industrial productions, such as paints, metal fin-ishes, steel manufacturing, alloy cast irons, chrome andwood treatment. Non-occupational exposure is low butalso occurs through vegetables, meat, urban air andcigarettes. Cr[VI] is known to be a human carcino-gen [1] and increased cancer risks due to exposure toCr[VI] are commonly associated with industrial expo-sure. Inhalation is the most important route for occu-pational exposure to Cr[VI]. The intracellular reductionof Cr[VI] to Cr[III] results in the formation of reactiveintermediates that contribute to the cytotoxicity, geno-toxicity and carcinogenesis of Cr[VI] [2]. Absorptionof chromium may occur through several routes, such asairways, gastrointestinal tract, and sometimes though to

0027-5107/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2006.02.001

H.E. Bryant et al. / Mutation Research 599 (2006) 116–123 117

a lesser extent directly through the skin. The molecularmechanism of chromium-induced carcinogenesis is stillelusive.

Chromium is mutagenic and induces DNA deletionsand base substitutions [3]. These mutations could arisefrom many types of DNA damage as in vitro Cr[VI]treatment causes interstrand crosslinks (ICL) [4–6],DNA–protein crosslinks [7], DNA adducts [8,9], DNAsingle-strand breaks (SSBs) [10] and DNA double-strandbreaks (DSBs) [11,12].

Cr[VI] also activates the ataxia telangiectasia mutated(ATM) pathway [13], which plays a central role in thedamage response pathway in mammalian cells [14]. Thisactivation could explain the observed DNA replicationand S-phase cell cycle arrest seen in Cr[VI] treated cells[15].

Homologous recombination (HR) is an importantpathway involved in repair of ICL and DSBs [16],especially at replication forks [17,18]. We have pre-viously seen that Cr[VI] causes increased rates ofgene rearrangement [19], which could be due to themutagenic effect of Cr[VI] or could suggest thatHR is triggered in order to repair Cr[VI]-inducedDNA lesions. Here, we report that DSBs are pro-duced following Cr[VI] exposure, that the Rad51 pro-tein, involved in HR [20], relocates into nuclear fociand that HR is increased in the hprt gene of SPD8cells [21]. Furthermore, we find that cells deficientin the XRCC3 and BRCA2 proteins, both involvedin HR [22,23], are hypersensitive to Cr[VI]-inducedDpDXswd

2. Methods

2.1. Chemicals and treatment conditions

Sodium chromate (Na2CrO4·6H2O) >98% purity was pur-chased from Sigma-Aldrich (Milwaukee,WI). All treatmentswere performed in Dulbecco’s modified Eagle’s Medium(DMEM) with 10% Foetal bovine serum and penicillin(100 U/ml) and streptomycin sulphate (100 �g/ml) at 37 ◦Cunder an atmosphere containing 5% CO2.

2.2. Cells and cell culture

The AA8, irs1SF [24], V3-3 [25], UV4 [26], EM9 [27]and CXR3 [28] cell lines (Table 1) were provided by LarryThompson (Livermore, CA). Malgorzata Zdzienicka gener-ously provided the VC8 and VC8 + B2 cell lines [23]. Allcell lines (Table 1) in this study were grown in DMEM with10% Foetal bovine serum and penicillin (100 U/ml) and strep-tomycin sulphate (100 �g/ml) at 37 ◦C under an atmospherecontaining 5% CO2.

2.3. Recombination assay

1.5 × 106 cells were inoculated into 100 mm dishes in media4 h prior to a 24-h treatment with Cr[IV]. After treatments,the cells were rinsed three times with PBS and 10 ml mediaadded before allowing the cells to recover for 48 h. After recov-ery, cells were harvest by trypsinisation and counted. HPRT+

revertants were selected by plating 3 × 105 treated cells perdish in the presence of HAsT (50 �M hypoxanthine, 10 �Ml-azaserine, and 5 �M thymidine). To determine cloning effi-ciency, two dishes were plated with 500 cells each. The colonies

TG

C

AV JiC XRCC3E air andU nd crosVS n hprt gVV BRCA2V BRCA2

NA damage. Together these data show that HRlays a role in survival following Cr[VI]-inducedNA lesions. In addition, we find that ERCC1,RCC1 and DNA-PKcs defective cells are hypersen-

itive to Cr[VI], indicating that several repair path-ays cooperate in repairing Cr[VI]-induced DNAamage.

able 1enotype and origin of Chinese hamster cell lines used in this study

ell line Genotype Defect/modification

A8 wt Wild type3-3 DNA-PKcs− DNA-PKcs−, defect in NHE

rs1SF XRCC3− XRCC3−, deficient in HRXR3 XRCC3+ Complemented with humanM9 XRCC1− XRCC1−, defect in SSB repV4 ERCC1− ERCC1−, deficient in NER a79 wt wtPD8 wt 6TGR, carrying duplication i-C8 BRCA2− BRCA2−, deficient in HR-C8 + B2 BRCA2+ Complemented with human-C8#Chr13 BRCA2+ Complemented with human

obtained were stained with methylene blue in methanol (4 g/l),following 7 (in the case of cloning efficiency) or 10 (for rever-sion) days of incubation.

2.4. Immunofluorescence

Cells were plated onto coverslips allowed to settle for 4 hand grown for 24 h in the presence or absence of treatments

Origin Reference

Ovary [24]AA8 [25]AA8 [24]

, functioning HR irs1SF [28]BER AA8 [35,50]slink repair AA8 [26]

Lung [51]ene as HR substrate. V79 [21]

V79 [23], functioning HR V-C8 [23]on chromosome 13, functioning HR V-C8 [23]

118 H.E. Bryant et al. / Mutation Research 599 (2006) 116–123

as indicated. Medium was then removed and coverslips rinsedonce in PBS at 37 ◦C. Cells were fixed in 3% paraformaldehydein PBS containing 0.1% Triton X-100 for 20 min at room tem-perature and coverslips then extensively washed (2 × 15 min inPBS containing 0.1% Triton X-100 and 0.15% bovine serumalbumin, 1 × 10 min in PBS containing 0.3% Triton X-100 and1 × 15 min in PBS containing 0.1% Triton X-100 and 0.15%bovine serum albumin) prior to incubation with rabbit poly-clonal anti Rad51 antibody (H-92, Santa Cruz) at a dilutionof 1:1000 for 16 h at 4 ◦C. The coverslips were subsequentlywashed (as above) followed by 1 h incubation at room tem-perature with Cy-3-conjugated goat anti-rabbit IgG antibody(Zymed) at a concentration of 1:500 and finally washed againas above. Coverslips were washed briefly in PBS, DNA stainedwith 1 �g/ml To Pro (Molecular Probes) and finally mountedin SlowFade Antifade (Molecular Probes).

Images were obtained with a Zeiss LSM 510 inverted confo-cal microscope using planapochromat 63×NA 1.4 oil immer-sion objective and excitation wavelengths 488, 546 and 630 nm.Through focus maximum projection images were acquiredfrom optical sections 0.50 �m apart and with a section thick-ness of 1.0 �m. Images were processed using Adobe Photo-Shop (Abacus Inc.).

The frequencies of cells containing Rad51 foci were deter-mined in three separate experiments. At least 300 nuclei werecounted on each slide. Nuclei containing more than 10 fociwere classified as positive.

2.5. Toxicity assay

Five hundred cells were plated in duplicate onto 100 mmdishes 4 h prior to treatment with increasing doses of chromium

Fig. 1. DNA double-strand breaks induced in AA8 Chinese ham-ster cells by Cr[VI]. (A) DNA double-strand breaks visualised withpulsed-field gel electrophoresis with increasing doses of Cr[VI]. (B)Visualisation of DNA double-strand breaks following a 24-h 25 �MCr[VI] treatment and subsequent repair.

3. Results

3.1. Cr[VI] induces repairable DNA double-strandbreaks

DSBs have previously been visualised followingCr[VI] treatments [11,12]. In agreement with theseresults, we find that Cr[VI] induces DNA fragmenta-tion, visualised by PFGE in SPD8 Chinese hamstercells (Fig. 1A). These arise either during apoptotic frag-mentation or directly from Cr[VI] treatment. The DNAfragments produced during apoptosis are not repaired,while direct induced DSBs are substrate for the DSB-repair machinery, including non-homologous end join-ing (NHEJ) and HR [16].

To further study if Cr[VI]-induced DSBs could berepaired after release from the drug, we carried out arepair assay to detect fragmentations by PFGE at differ-ent time points after 24 h treatment with 25 �M Cr[VI](Fig. 1B). Repair began 2 h after removal of Cr[VI]and was complete after 24 h. These results indicate thatCr[VI]-induced DSBs are substrates for the DSB repairmachinery.

3.2. Cr[VI] triggers Rad51 foci formation andhomologous recombination

To investigate if HRR is triggered following Cr[VI]treatment, we determined Rad51 foci formation. Rad51

as indicated. Ten days later, when colonies could be observed,they were fixed and stained with methylene blue in methanol(4 g/l). Colonies consisting of more than 50 cells were subse-quently counted. Each colony was assumed to represent onecell surviving from the original 500 and surviving fraction foreach dose calculated. Each experiment was repeated betweentwo and five times and the mean and standard deviation calcu-lated.

2.6. Pulse field gel electrophoresis

1.5 × 106 cells were plated onto 100 mm dishes and allowed4 h for attachment. Exposure to chromium at indicated dose wasfor 24 h after which cells were trypsinsied and 106 cells meltedinto each 0.7% agarose insert. These inserts were incubatedin 0.5 M EDTA, 1% N-laurylsarcosyl, proteinase K (1 mg/ml)at 50 ◦C for 48 h then washed four times in TE buffer prior toloading onto a 0.7% agarose (chromosomal grade) gel. Sepa-ration by pulse-field gel electrophoresis was for 24 h (BioRad;120◦ angle, 60–240 s switch time, 4 V/cm). The gel was subse-quently stained with ethidium bromide for analysis. For repairassays agarose inserts were left for increasing lengths of time inmedia prior to transfer to the EDTA/laurylsarcosyl/proteinaseK buffer.

H.E. Bryant et al. / Mutation Research 599 (2006) 116–123 119

Fig. 2. Rad51 foci and homologous recombination induced by Cr[VI].(A) The number of cells containing >10 Rad51 foci following a24 h treatment with Cr[VI] (10 �M). (B) Homologous recombina-tion was determined in SPD8 cells. The reversion frequency from anon-functional to a functional hprt gene, giving resistance to HAsTfollowing a 24 h treatment with Cr[VI] at various concentrations isshown. The average (symbol) and standard deviation (error bars) fromtwo to five experiments are shown.

is involved in the strand transfer reaction in HRR [20],and relocates into stabile nuclear foci during HRR[29,30]. We found that when compared with spontaneouslevels the percentage of cells with >10 Rad51 foci was10-fold increased following a 24 h treatment with 10 �MCr[VI] (Fig. 2A). These results suggest that HR is trig-gered by Cr[VI].

We also wanted to test if Cr[VI] induces HR directly.The system used has been described previously [21].Briefly, partial duplication of exon 7 of the hypoxan-thine guanine phosphoribosyl transferase (hprt) gene,that arose spontaneously in SPD8 cells, leads to expres-sion of non-functional HPRT protein and reversion towild type by homologous recombination can be selectedfor in HAsT media. Colonies formed following selectionare therefore indicative of HR [21]. We found a dosedependent increase in HR in the hprt gene upon treat-

ing SPD8 cells with increased concentrations of Cr[VI](Fig. 2B). There was a 5-fold induction of HR followinga 24 h treatment with 2 �M Cr[VI].

3.3. Homologous recombination deficient cells arehypersensitive to Cr[VI]

We found that HRR is triggered following treatmentwith Cr[VI]. To test if this repair pathway is impor-tant for removal of Cr[VI]-induced DNA damage andsubsequent survival of the cell, we determined survivalfollowing Cr[VI] treatment of wild type and HR deficientcells. We found that the HR deficient cell line irs1SF,deficient in the HRR protein XRCC3 [28,31], is signifi-cantly more sensitive to Cr[VI] than the wild type AA8cell line even at doses as low as 0.3 �M (Fig. 3A). We

(XRCC3 deficient) and V3-3 (DNA-PKcs deficient) cells with increas-ing dose of Cr[VI]. (B) Clonogenic outgrowth of V79 (wild type), V-C8(BRCA2 deficient), V-C8#Chr13 (V-C8 complimented with BRCA2on chromosome 13) and V-C8 + B2 (V-C8 complimented with BRCA2on an expression vector) cells with increasing dose of Cr[VI]. The aver-age (symbol) and standard deviation (error bars) from three to fourexperiments are shown.

Fig. 3. Homologous recombination deficient cell lines are hypersensi-tive to Cr[VI]. (A) Clonogenic outgrowth of AA8 (wild type), irs1SF

120 H.E. Bryant et al. / Mutation Research 599 (2006) 116–123

Fig. 4. Cell deficient in XRCC1 and ERCC1 are hypersensitive toCr[VI]. Clonogenic outgrowth of AA8 (wild type), EM9 (XRCC1 defi-cient), UV4 (ERCC1 deficient) cells with increasing dose of Cr[VI].The average (symbol) and standard error (bars) from two to four exper-iments are depicted.

also found an increased sensitivity to Cr[VI] in the V3-3cell line which has a deficient DNA-PKcs, resulting ina non-functional NHEJ pathway [25], but only at doses>3 �M of Cr[VI] (Fig. 3A). These results are in line withthe overlapping role of HR and NHEJ in repair of DSBs[32].

The BRCA2 protein is involved in HRR of DSBs[23,33,34]. We found that BRCA2 deficient V-C8 cells[23] are hypersensitive to Cr[VI] in agreement with arole of HR in repair of Cr[VI]-induced lesions (Fig. 3B).The sensitivity was specific to the BRCA2 defect in thesecells since complementation with BRCA2 reverted thesensitivity of the V-C8 cells.

3.4. XRCC1 and ERCC1 cells are hypersensitive toCr[VI]

We also investigated the involvement of SSB orbase excision repair (BER) and nucleotide excisionrepair (NER) in repair of Cr[VI]-induced DNA dam-age, using the EM9 and UV4 cell lines, deficientin XRCC1 and ERCC1, respectively [26,35,36]. Wefound that both EM9 and UV4 cells were sensitive toCr[VI] as compared to wild type control cells (Fig. 4),consistent with a role of SSB/BER repair and theXPF/ERCC1 complex in repair of Cr[VI]-induced DNAlesions.

Fig. 5. Model for Cr[VI]-induced DNA repair pathways. Cr[VI] causesDSBs primarily in cells in the S-phase of the cell cycle [12]. The for-mation of such DSBs are dependent on the mismatch repair (MMR)system. These DSBs may be repaired either by HRR or NHEJ. Sponta-neously oxidized bases, such as 8-oxoguanine, are sensitive to furtheroxidation by Cr[VI] to guanidinohydantoin and isomers there of [43].These oxidized bases are excised by the NEIL-1 and NEIL-2 glyco-sylases [45] within BER. The SSB produced following lyasing theAP-site involves repair including XRCC1 and Pol�. It is possible thatCr[VI] DSBs may also arise at replication forks, either during repairwith ERCC1 or when a SSB intermediate during BER is encounteredby the replication fork. DSBs that arise at a collapsed replication forktrigger HRR [17,18]. Cr[VI] may result in interstrand crosslinks, pos-sibly through oxidation [6]. The ERCC1-XPF endonuclease has animportant role in crosslink repair that is likely to involve a mix oftrans-lesion synthesis and NER [40]. Filled lines represent templateDNA and dotted lines nascent DNA.

4. Discussion

The molecular mechanism of Cr[VI]-induced car-cinogenicity is likely to be the result of Cr[VI]-inducedDNA damage. Although the genotoxic effects of Cr[VI]are well documented, the DNA lesions and repair path-ways triggered by Cr[VI] are not thoroughly investi-gated. It has been show that Cr treatment causes ICL[4–6], DNA adducts [8,9], SSBs [10] and DSBs [11,12].More recently, it was shown that the mismatch repairmachinery is required to produce toxic DSBs followingCr[VI] treatment, and that these DSBs are formed afterDNA replication [37]. Here we confirm that DSBs areproduced and can be repaired following Cr[VI] treatmentand show that several DNA repair pathways, includingHR, NHEJ, ICL and BER, cooperate in repair of Cr[VI]-induced DNA damage (Fig. 5).

The nucleotide excision repair pathway is reported tobe involved in removing Cr[VI] DNA adducts [38]. In

H.E. Bryant et al. / Mutation Research 599 (2006) 116–123 121

contrast to this, it was reported that NER deficient UVL-1 (ERCC2 mutant) cells are not hypersensitive to Cr[VI]induced damage itself, but that Cr reduces the NER effi-ciency of other DNA adducts [39]. Here, we find that theNER deficient UV4 (ERCC1 mutated) cell line is hyper-sensitive to Cr[VI]. It is established that ERCC1 deficientcells are especially sensitive to ICL agents, as comparedto cell lines deficient in proteins involved in other partsof the NER pathway. This may reflect a specific roleof the ERCC1/XPF complex in crosslink repair [40,41].Our results, indicating that the ERCC1/XPF complex isinvolved in repair of Cr[VI]-induced lesions, may there-fore relate to the role of this complex in crosslink repairrather than in NER.

Increased toxicity in XRCC1 deficient cells suggeststhat the BER or SSB repair machinery is involved inrepairing Cr[VI] lesions [42]. High valent metals, such asCr[VI] may oxidize 8-oxoG to form guanidinohydantoinand its isomer forms [43], which are highly mutagenicand result in transversions and polymerase arrest [44].The guanidinohydantoin and its isomer forms are recog-nised by the NEIL-1 and NEIL-2 glycosylsases [45].Thus, the reason XRCC1 deficient cells are hypersensi-tive to Cr[VI] is likely to be due to the defect in BER ofoxidized 8-oxoG lesions (Fig. 5).

We confirm that Cr[VI] induces DSBs and find thatCr[VI]-induced DSBs are repaired in mammalian cells.Cr[VI] triggers DSBs primarily in the S-phase of the cellcycle [12], and DSBs formed during S-phase are usu-ally repaired by HR [32,46]. Therefore, as expected, wefChHctlfocnca

coi[

sDo

treatment. Also, it would explain why Cr[VI]-inducedDSBs are repaired and the Cr[VI] sensitivity in HRdeficient cells. As replication fork associated DSBs arerepaired by HR and mainly produce SCE events, whichis the product of HRR [48] this model would explainwhy a positive induction of SCE was found in workersexposed to the Cr[VI] industry [49].

In conclusion, we find that Cr[VI] causes repairableDSBs and that the HRR machinery is activated. HRdeficient cells are highly sensitive to Cr[VI]-inducedlesions suggesting that HR is an important pathway forerror-free repair of Cr[VI]-induced damage. We suggestthat the HRR repair pathway is important in preventingCr[VI]-induced DNA lesions from causing gene rear-rangements or mutations that may inactivate tumour sup-pressor genes or activate proto-oncogenes and therebycause cancer. In addition, our data confirm that otherrepair pathways are also likely to be important to therepair of Cr[VI]-induced DNA damage.

Acknowledgements

We thank Malgorzata Zdzienicka and Larry Thomp-son for materials. The Swedish Cancer Society, TheSwedish Research Foundation, The Swedish Pain ReliefFoundation and Yorkshire Cancer Research supportedthis work financially.

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ound that cells with a deficiency in HR (irs1SF and V-8, deficient in XRCC3 [28] and BRCA2 [23]) wereypersensitive to Cr[VI]-induced damage implicatingR in the repair of these lesions. Interestingly cells defi-

ient in DNA-PK were also sensitive to Cr[VI], implyinghat NHEJ also pays a role in repair of Cr[VI]-inducedesions. NHEJ is mostly associated with repair of DSBsormed outside of S-phase [32] however it can have anverlapping role in repair of DSBs produced in S-phaseells [17,18,32]. Thus, NHEJ may be repairing a smallumber of DSBs occurring in the G1 phase of the cellycle or may work along side HR to repair replicationssociated DSBs.

The lesion caused by Cr[VI] that triggers HRR is notlear. A likely candidate lesion is a DSB formed duringr following replication. DSBs are highly toxic and HRRs a major repair pathway of DSBs at replication forks17,18,32,46,47].

We suggest that Cr[VI]-induced DNA lesions per-ist into the S-phase collapse replication forks, formingSBs (Fig. 5). This model would explain the formationf Rad51 foci and induction of HR following Cr[VI]

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