XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model

    The ERCC2/XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model Sha Xiao, Su Cui, Xiaobo Lu, Yangyang Guan, Dandan Li, Qiufang Liu, Yuan Cai, Cuihong Jin, Jinghua Yang, Shengwen Wu, Tahar van der Straaten PII: DOI: Reference:

S0887-2333(16)30082-0 doi: 10.1016/j.tiv.2016.04.015 TIV 3773

To appear in: Received date: Revised date: Accepted date:

22 April 2015 1 April 2016 24 April 2016

Please cite this article as: Xiao, Sha, Cui, Su, Lu, Xiaobo, Guan, Yangyang, Li, Dandan, Liu, Qiufang, Cai, Yuan, Jin, Cuihong, Yang, Jinghua, Wu, Shengwen, van der Straaten, Tahar, The ERCC2/XPD Lys751Gln polymorphism affects DNA repair of benzo[a]pyrene induced damage, tested in an in vitro model, (2016), doi: 10.1016/j.tiv.2016.04.015

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ACCEPTED MANUSCRIPT The ERCC2/XPD Lys751Gln polymorphism affects DNA repair

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of Benzo[a]pyrene induced damage, tested in an in vitro model

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Sha Xiao1*, Su Cui2*, Xiaobo Lu1 #, Yangyang Guan1 , Dandan Li 1, Qiufang Liu1, Yuan Cai1,

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Cuihong Jin1, Jinghua Yang1, Shengwen Wu1, and Tahar van der Straaten3

1. Dept. of Toxicology, School of Public Health, China Medical University (Shenyang, P.R. China) 2. Dept. of Thoracic Surgery Ward 2, the first Hospital of China Medical University (Shenyang, P.R. China)

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3. Dept. Clinical Pharmacy and Toxicology, Leiden University Medical Center (Leiden, the Netherlands)

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* The first two authors Sha Xiao and Su Cui contributed to this work equally

#

Correspondence to:

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School of Public Health

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Dept. of Toxicology

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Dr. Xiaobo Lu

China Medical University

No.77 Puhe Road, Shenyang North New Area

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110122, Shenyang

Liaoning Province P. R. China

Tel: +86 024 23256666-5393 E-mail: [email protected]; [email protected]

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ACCEPTED MANUSCRIPT Abstract Nucleotide excision repair (NER) is an important defense mechanism of the body to exogenous carcinogens and mutagens, such as benzo[a]pyrene (B[a]P). Genetic

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polymorphisms in ERCC2/XPD, a critical element in NER, are thought to be

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associated with individual’s cancer susceptibility. Although ERCC2/XPD Lys751Gln (rs13181) is the most studied polymorphism, the impact of this polymorphism on

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DNA repair capacity to carcinogen remains unclear. In the present study, cDNA clones carrying different genotypes of ERCC2/XPD (Lys751Gln) were introduced into an ERCC2/XPD deficient cell line (UV5) in a well-controlled biological system.

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After B[a]P treatment, cell growth inhibition rates and DNA damage levels in all cells were detected respectively. As expected, we found that the DNA repair capacity in

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UV5 cells was restored to levels similar to wildtype parent AA8 cells upon introduction of the cDNA clone of ERCC2/XPD (Lys751). Interestingly, after B[a]P treatment, transfected cells expressing variant ERCC2/XPD (751Gln) showed an

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enhanced cellular sensitivity and a diminished DNA repair capacity. The wildtype

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genotype AA (Lys) was found to be associated with a higher DNA repair capacity as compared to its polymorphic genotype CC (Gln). These data indicate that

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ERCC2/XPD Lys751Gln polymorphism affects DNA repair capacity after exposure to environmental carcinogens such as B[a]P in this well-controlled in vitro system and

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could act as a biomarker to increase the predictive value to develop cancer.

Keywords: ERCC2/XPD Lys751Gln; Nucleotide excision repair (NER); DNA repair capacity; Benzo[a]pyrene (B[a]P); Cancer development

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ACCEPTED MANUSCRIPT 1. Introduction Benzo[a]pyrene (B[a]P), as an ubiquitous environmental pollutant, is a product of

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incomplete combustion of fossil fuels, which can be found in air, tobacco smoke,

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automobile exhaust, and food (Gao et al., 2005). Since B[a]P was isolated and

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identified by Cook in 1933 from coal tar pitch (Cook et al., 1933), thousands of papers have reported its chemical, biochemical, biological and toxicological properties. It is widely accepted that B[a]P is carcinogenic, mutagenic, cytotoxic,

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immunotoxic, and teratogenic in various species and tissues. B[a]P is metabolically activated by CYP1, epoxide hydrolase, and aldo-keto reductases to benzo[a]pyrene-r-

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7, 8-dihydrodiol-9, 10-epoxide (+/-) (anti) (BPDE), which reacts with many biological macromolecules, including DNA, to cause covalent BPDE-DNA adducts and oxidative damage (Mensing et al., 2005; Nesnow et al., 2002; Nguyen et al., 2010).

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The covalent binding of reactive metabolites to DNA is proposed as an important step

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in tumor initiation (Kennedy et al., 2005; Miller, 1978). DNA repair capacity of a cell is vital to maintain the integrity and stability of its genome. The genes involved in

al., 2007).

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DNA repair have been proposed as candidate ones to predict carcinogenesis (Crew et

The biological response to DNA damage is crucial in the maintenance of genomic

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stabilization. Various defenses have evolved to repair DNA damage, such as complex excision repair mechanisms, arrest of the cell cycle and apoptosis (Bhana and Lloyd, 2008). DNA repair capacity in excising DNA adducts induced by carcinogens is important to human. Generally spoken, there are at least 6 main DNA repair pathways known to correct DNA damage: Direct repair (DR), Base excision repair (BER), Nucleotide excision repair (NER), Mismatch repair (MMR), Homologous recombination repair (HR), and Non-homologous end joining (NHEJ) (Bernstein et al., 2002). NER, BER and HR have been shown to be involved in the repair of BPDEDNA adducts (Meschini et al., 2008; Vineis et al., 2009). Among them, NER is the major DNA repair pathway for the removal of a wide variety of bulky DNA lesions induced by environmental genotoxic agents such as B[a]P. There are three steps in 3

ACCEPTED MANUSCRIPT NER: damage recognition; unwinding of DNA and removal of the damaged fragment; DNA synthesis (Conference Report, 2007; Hoeijmakers, 2001; Shell and Zou, 2008). Excision repair cross-complementing complementation group 2 (ERCC2), also known

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as Xeroderma pigmentosum complementation group D (XPD), is located on chromosome 19q13.3. The ERCC2/XPD protein is involved in NER as a critical

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element, it functions as an ATP dependent 5’-3’ helicase joint to the basal transcription factor IIH complex (TFIIH) (Chen and Suter, 2003; Egly and Coin, 2011; Liu et al., 2008).

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Several single nucleotide polymorphisms (SNPs) in ERCC2/XPD have been reported to be related with altered gene expression and an altered phenotype. To date, than

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SNPs

have

been

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more

reported

(http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=2068).

for

ERCC2/XPD Of

those,

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ERCC2/XPD Lys751Gln (rs13181) was found to be associated with cancer

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susceptibility according to some epidemiology studies. ERCC2/XPD Lys751Gln, characterized by an A to C substitution , causes a Lys to Gln amino acid change in the

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C-terminal part of the protein in exon 23 (Wlodarczyk and Nowicka, 2012). This amino acid change is supposed to affect ERCC2/XPD function, which may impact DNA repair capacity, and alter genetic susceptibility to environmental carcinogen

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(Vogel et al., 2000). However, the conclusions in many population studies are not entirely consistent. Furthermore the relationship of ERCC2 Lys751Gln polymorphim and the risk to develop cancer act differently among ethnicity. A meta-analysis suggested that the TT genotype of ERCC2 Lys751Gln polymorphism may decrease the risk of glioma in the Caucasian population (Xin Y et al. 2014). Terry et al. (Terry et al, 2004) reported that carriers of the Gln allele were associated with an increased risk of breast cancer after heavy cigarette smoking, and Zhao et al (Zhao et al., 2008) analyzed the induced BPDE-DNA adducts in cultured lymphocytes from 707 healthy non-Hispanic individuals and found that smoking status was a high risk factor for forming DNA adducts, while other studies (Matullo et al., 2003; Zhao et al., 2008) reported no relationship between ERCC2/XPD Lys751Gln and the level of BPDEDNA adducts. 4

ACCEPTED MANUSCRIPT Due to the lack of knowledge of functional consequences of ERCC2/XPD Lys751Gln, it is difficult to interpret the findings from genetic association studies. Therefore, to clarify whether the wildtype or polymorphic allele of ERCC2/XPD

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codon 751 act differently in DNA repair capacity, we introduced a cDNA clone containing ERCC2/XPD Lys751 or ERCC2/XPD Gln751 into the ERCC2/XPD

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deficient cell line UV5. We investigated the cellular sensitivity and the repair capacity to DNA damage caused by B[a]P. Our findings may contribute to a comprehensive understanding of the involvement of ERCC2/XPD in NER during the repair of DNA

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damage induced by carcinogen B[a]P. In addition, we used the well-controlled in vitro system to further explore the interaction between ERCC2/XPD Lys751Gln

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polymorphism and DNA repair capacity to B[a]P, an environmental carcinogen, and

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may contribute to find a biomarker to increase the predictive value to develop cancer.

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2. Materials and methods

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2.1. Materials

Benzo[a]pyrene (B[a]P), nicotinamide adenine dinucleotide phosphate (NADP), and Glucose-6-phosphate (G-6-P) were purchased from Sigma (Sigma Chemical Co.,

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St. Louis, MO). Rat liver S9, as a metabolic activation system, was afforded from Shenyang Safety Evaluation Center.

2.2. Cell culture and treatment Chinese hamster ovary (CHO) cells: AA8 (wild type; ATCC No.CRL-1859TM) and UV5 (ERCC2/XPD deficient; ATCC No.CRL-1865TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA, 2012). Materials for cell culture were purchased from Gibco (Invitrogen Corporation, USA). The cells were grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented

with

10%

fetal

bovine

serum

(FBS)

and

10

IU/ml

penicillin/streptomycin and maintained at 37°C, 5% CO2 in a humidified incubator. 1.0 mg/ml G418 was added to the medium when required. B[a]P was dissolved in 5

ACCEPTED MANUSCRIPT dimethyl sulfoxide (DMSO, the final concentration =1‰) and cells were treated with 5 or 10 µM concentrations, and 3% (V:V) Rat liver S9-mix. The reduced nicotinamide adenine dinucleotide phosphate regenerating system (NRS) and the S9-mix (S9+NRS)

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were freshly prepared before each experiment. The S9 fraction was added to the mixture immediately before B[a]P treatment [NRS end concentrations: 33 mM

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potassium chloride (KCl), 8 mM magnesium chloride (MgCl2·6H2O), 5 mM G-6-P, 4 mM NADP, 0.1 M Na2HPO4～NaH2PO4 (PH=7.4)].

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2.3. ERCC2/XPD expression in the transfected cells

Bicistronic plasmids expressing the open reading frame of wildtype or mutant

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ERCC2/XPD allele at codon 751, and expressing Green Fluorescent Protein (GFP) for selection of transfected cells, were created by Gateway technology (Invitrogen, Breda,

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The Netherlands). Briefly, the ERCC2 gene was PCR amplified from human cDNA,

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which was genotyped as heterozygous for ERCC2 rs13181, with attB-flanked primers (Forward

5’-

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GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCatgaagctcaacgtggacg-3’, Reverse 5’-GGGGACCACTTTGTACAAGAAAGCTGGGtcagagctgctgagcaatc-3’) The PCR product was recombined in pDONR201. The resulted entry vector was

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sequenced to confirm genotype. Next, the entry vector was recombined with destiny vector pExp-IRES-GFP (Clontech, Oxford, UK) that had been made gateway compatible by insertion of attR-cassette. The resulted plasmids were UV5GFP (only expressing GFP), UV5ERCC2

(AA)

(expressing ERCC2/XPD 751Lys and GFP) and

UV5ERCC2 (CC) (expressing ERCC2 /XPD 751Gln and GFP). Three types of Bicistronic plasmids expressing wild type ERCC2/XPD 751Lys and GFP, polymorphic ERCC2/XPD 751Gln and GFP and only GFP were constructed in Department of Clinical Pharmacy and Toxicology of Leiden University Medical Center (LUMC, the Netherlands). The structure scheme of the plasmid construction was shown in Fig.1. ERCC2/XPD defective cells UV5 were transfected with these three types of plasmids

separately

using

Lipofectamine®2000

reagent

according

to

the

manufacturer’s instructions to get stable transfect cell lines. Plasmid expression was 6

ACCEPTED MANUSCRIPT maintained by the administration of 1.0 mg/ml G418 antibiotic. The transfected cells were designated as UV5GFP (only expressing GFP), UV5ERCC2

(AA)

(expressing

ERCC2/XPD 751Lys and GFP) and UV5ERCC2 (CC) (expressing ERCC2 /XPD 751Gln

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and GFP).

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2.4. Genotyping assay

DNA was extracted according to the instruction of Takara company kit. PCR reactions were run in a 20 µl final volume including Premix Ex TaqTM 10.0 µl, 0.4 µl

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of each probe and primer, 2 µl DNA (l0 ng/µl). Cycling conditions were 95°C for 10 min, and 40 cycles of 95°C for 5 s and 60°C for 34 s. Data analysis for allele

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discrimination was performed using SDS software. ERCC2 751 rs13181:

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Primers: Forward 5'-CAG GAG TCA CCA GGA ACC GT-3'

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Reverse 5'-CTC AGC CTG GAG CAG CTA GAA T-3' Probes: A-allele-5'-FAM-ATC CTC TTC AGC GTC T-MGB NFQ-3'

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C-allele-5'-VIC-TCC TCT GCA GCG TC-MGB NFQ-3'

2.5. Western blotting for detecting ERCC2/XPD protein

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Expression of ERCC2/XPD protein was assessed by Western blotting. Briefly, cells were washed in cold PBS and lysed in 100-200 µl of RIPA buffer containing 10 µl/ml of Protease Inhibitor cocktail at 4°C for 40 min. Samples were collected after centrifuging at 15,000×g for 15 min. Protein concentration was determined using the BCA Protein Assay Kit and 60 µg protein was subjected to electrophoresis on a 5-8% Bis-Tris Gel. Proteins were transferred electrophoretically to a PVDF membrane. The membrane was blocked in 5% (v/w) non-fat milk powder in Tris-buffered saline containing 0.1% Tween20 (TBST) followed by an overnight incubation with a 1:500 dilution of ERCC2/XPD primary antibody (Abcam, ab54676) and 1:800 dilution of βactin primary antibody (ZSGB-BIO) in TBST respectively. After washing with TBST, the membrane was incubated with the secondary antibodies at room temperature for 1 h. Proteins were visualized using the Electrophoresis Gel Imaging Analysis System 7

ACCEPTED MANUSCRIPT (MF-ChemiBIS 312, DNA Bio-Imaging Systems). Quantification of protein expression was performed using Image J (http://rsbweb.nih.gov/ij/) according to the

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developer’s instructions after Western blotting.

2.6. Cell viability by 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium

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bromide (MTT) assay

The MTT assay is a colorimetric assay and a rapid measure of short-term cell viability (Bhana and Lloyd, 2008). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-

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tetrazolium bromide (MTT) was reduced by mitochondrial enzymes in viable cells to form insoluble purple formazan crystals; this conversion was directly proportional to

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the number of viable cells and could be monitored easily by spectrophotometer. Cells were seeded in 96-well plates (5000 cells/well) and after 24 hrs of incubation the

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exponentially growing cells were incubated with various concentrations of B[a]P (0.1,

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0.5, 1, 5, 10, 25, 50, 100 µM) for an additional 24 hrs. Then, after washing thoroughly the cells were cultured for another 24 hrs to allow DNA repair. Later MTT was added

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to each well at a final concentration of 0.05 mg/ml. After 4 hrs of incubation, MTT solution was removed and replaced with 150 µl DMSO. Absorbance of formazan, a MTT metabolite, was detected at 490 nm on a microplate reader (dnm-9602g,

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Perlong).

2.7. Single cell gel electrophoresis (the comet assay) The alkaline comet assay was performed according to the method of Tice et al with some modifications (Tice et al., 2000). Briefly, exponentially growing cells were seeded in 6-well plates (2×105 cells/well). 24 hrs later, cells were treated with 5 or 10 µM B[a]P for 6, 12, 24 hrs and incubated for another 24 hrs to allow DNA repair. Then the cells were harvested for the following procedures: pre-coated microscope slides were prepared by dropping 100 μl 1% NMA (normal melting point agarose dissolved in PBS, PH 7.4) to the glass slide and quickly cover it with a lid slide to make the gel evenly. After at least 10 minutes of incubation at 4oC, the lid slide was carefully removed. The 100 μl gel mixture of cells and 1% low melting point agarose 8

ACCEPTED MANUSCRIPT (LMA ) dissolved in PBS was slowly added as a second layer, and incubated in dark at 4°C. After 10 min, 100 μl 1% LMA was added as the last layer. Gel slides were soaked in cell lysis solution (100 mM disodium EDTA, 2.5 M NaCl, 10 Mm Tris-HCl

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pH 10.5, adding 1% Triton X-100 before using) for 60-90 min and immersed in the electrophoresis solution (1M disodium EDTA, 300 mM NaOH, pH>13) for 30

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minutes to denature DNA. After electrophoresis at 20 V (100 mA) for 20 min, glasses were rinsed three times with 1ml of neutralizing solution (0.4 M Tris-HCl, pH 7.5). Ethidium bromide was used for staining. Finally, DNA damage in a single cell was

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detected using fluorescent microscope. At least 100 tail olives moment (and tail areas)

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in each slide were counted.

2.8. RAD51 Immunofluorescence staining

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For RAD51 foci detection, approximately 2×105 cells were seeded in 6-well plates

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containing sterilized glass cover slips. After 24 hrs, cells were treated with 5 or 10 µM B[a]P for 24 hrs and incubated for an additional 24 hrs to allow DNA repair. After

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treatment, cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100/PBS. The cover slips were blocked with 1% BSA for 30 min and incubated with mouse monoclonal IgG anti-RAD51 (Abcam) primary antibody (1:200)

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overnight at 4°C, followed by TRITC-conjugated AffiniPure Goat Anti-Mouse IgG (ZSGB-BIO) secondary antibody (1:150) for 1 h at room temperature. Next, cells were washed with PBS and incubated with 4, 6-diamidino-2-phenylindole (DAPI) for nuclei staining. Cells on coverslips were visualized using fluorescence microscope. A cell carrying at least 10 foci was denoted as positive and at least 200 cells per coverslip were scored.

2.9. Statistical analysis Data statistical analysis was carried out using the SPSS software. Data are represented as the mean ± SEM. One-way ANOVA and LSD multiple comparison tests were used to estimate the differences among the groups. Cellular sensitivity to B[a]P was expressed as a percentage of survival rate. The difference between the 9

ACCEPTED MANUSCRIPT sensitivity of AA8 and UV5 cells to each dose of B[a]P was expressed as a ‘toxicity ratio’ which was calculated by dividing the mean survival rate of UV5 cells by that of AA8 cells. Each independent experiment performed in triplicate and P<0.05 was

3. Results 3.1. ERCC2/XPD expression in UV5 cells

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considered as statistically significant.

Transfected cells stably expressing ERCC2/XPD with rs13181 either A or C allele, (AA)

and UV5ERCC2

(CC)

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denoted as UV5ERCC2

, were established in our current

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experiment. As shown in Fig. 2, the parental UV5 cells and the control vector GFP transfected cells, did not show ERCC2/XPD protein (87 kD) expression, while the cells transfected with ERCC2/XPD rs13181 containing either A or C allele showed

3.2. Cell viability

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ERCC2/XPD protein expression levels similar to AA8 cells.

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Toxicity ratios were obtained by comparing the toxicity of B[a]P at the same dose treatment between AA8 and UV5 cells (Tables 1 and 2). At each concentrations of B[a]P treatment, UV5 cells showed moderate sensitive to B[a]P as compared to AA8

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after 24 hrs treatment, however, UV5 sensitivity was more pronounced after an additional 24 hrs incubation. Their comparable toxicity ratios ranged from 1.123 to 2.109. For 24 hrs treatment, the differences between the survival rate in AA8 and UV5 cells were statistically significant at each concentration of B[a]P except for 1 µM. After an additional 24 hrs incubation, the differences were found statistically significant at concentrations higher than 5 µM (P<0.05). Differences in survival rate between AA8 and UV5 became more pronounced. For example, at concentration 100 µM the toxicity ratio is 2.109 (Table 2). These results suggested that deficient NER had a deteriorating effect on DNA repair capacity and became a serious challenge to the survival of cells. Cellular sensitivity to B[a]P of UV5 cells were partially restored to wildtype AA8 10

ACCEPTED MANUSCRIPT levels when ERCC2/XPD cDNA is introduced. To determine if the cells transfected with different alleles of ERCC2/XPD codon 751 polymorphism would respond to B[a]P differently, the sensitivity of both the UV5ERCC2 (AA) and UV5ERCC2 (CC) cells to

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B[a]P were detected. There was a significantly increased viability upon B[a]P

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treatment in UV5ERCC2 (AA) and UV5ERCC2 (CC) cells as compared to their parental cell

and UV5ERCC2

(CC)

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UV5 and the vector transfected cell UV5-GFP. The survival rate of the UV5ERCC2 (AA) cells were not affected by 24 hrs B[a]P treatment, but after an

additional 24 hrs of incubation to allow DNA repair, the differences were found

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statistically significant at 5-50 µM B[a]P treatment (P<0.05) (Fig. 3). These results indicate that ERCC2/XPD with an A allele (Lys) in codon 751 had a higher DNA

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repair capacity to B[a]P treatment as compared to C allele (Gln)..

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3.3. ERCC2/XPD protein expression levels in cells

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Fig. 4 and 5 show the dynamic change of ERCC2/XPD protein expression upon B[a]P treatment. The protein expression of ERCC2/XPD in AA8 cells peaked at 6 hrs

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of B[a]P treatment, reaching almost a 3 fold increase with 5 or 10 µM B[a]P treatment respectively (compared to the intact control group, P<0.05), and kept a high level till 12 hrs after treatment (compared to the intact control group, P<0.05). This increasing

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trend stopped at 24 hrs and lasted until a similar level of the control group during another 24 hrs incubation to allow DNA repair (P>0.05). The protein expression of ERCC2/XPD in UV5ERCC2 (AA) and UV5ERCC2 (CC) cell lines peaked at 6 hrs after B[a]P treatment and kept a high level till 12 hrs treatment. Both ERCC2/XPD transfected cells showed an equal ERCC2/XPD protein over-expression.

3.4. DNA damage detected by the comet assay The comet assay was used to evaluate the genotoxic effect of B[a]P. The differences of comet tail moment (OTM, as an index of DNA damage level) between the B[a]P treated groups and the intact control in five cell lines were measured (Tables 3 and 4). Fig. 6 and 7 show the image of the comet assay. For the different time points tested, the OTM increased with B[a]P dose increasing (5, 10µM) in the cell lines as 11

ACCEPTED MANUSCRIPT compared to negative controls (P<0.05). OTM differences were maximal after 6 hrs of B[a]P treatment. Similarly to the report of Hanelt et al. (1997), DNA strand breakage caused by B[a]P raised as a dose dependant way. After 24 hrs of treatment

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followed by an additional 24 hrs incubation, the OTM decreased further for both AA8 and UV5 cells but remained significantly different (P<0.05). In addition, for two

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doses and four time points, the DNA damage level of UV5ERCC2 (CC) cells were all larger than UV5ERCC2 (AA), and showed a significant difference in UV5ERCC2 (AA) and

of 6 hrs treatment with 10 µM B[a]P.

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UV5ERCC2 (CC) cells at 6 hrs and 12 hrs of B[a]P treatment (P<0.05) with the exception

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3.5. Immuno-staining for RAD51 foci formation

BPDE-DNA adducts and DNA double strand breaks (DSBs) induced by B[a]P

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could be repaired by Homologous recombination repair (HR), and could be examined

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by the dynamic redistribution of the key HR protein, RAD51, into DNA damage induced nuclear foci (Godthelp et al., 2002; Wiegant et al., 2006). Here, we analyzed

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the induction of RAD51 foci by immunofluorescence in cells treated with 5 or 10 µM B[a]P. Fig.8 shows impaired formation of nuclear RAD51 foci in the tested cell lines in response to DNA damage. In untreated cell lines, about 1-3% RAD51 foci-positive

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cells could be observed to reflect the background of endogenic damage. After 24 hrs of treatment, the amount of RAD51 foci-positive cells increased to 10-15% for AA8 cells and the two ERCC2/XPD transfected cells. For UV5 and UV5GFP cells 15-25% of RAD51 foci-positive cells were observed (P<0.05). However, after 24 hrs of treatment followed by another 24 hrs of incubation, the amount of foci-positive cells in all cell lines was reduced, and no significant difference in the cell lines could be observed (P>0.05). For UV5ERCC2 (AA) and UV5ERCC2 (CC) cells, no clear difference was found at each dose and each incubation time of B[a]P treatment.

4. Discussion Carcinogenesis is a multistep process which is determined by regulation of cell 12

ACCEPTED MANUSCRIPT proliferation, mutation frequency and also by the efficacy of DNA repair system of cells. One of the most versatile and sophisticated DNA repair pathway is Nucleotide excision repair (NER), which is a fundamental mechanism for protecting the integrity

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of human genome. NER can eliminate a wide variety of different forms of DNA damage and especially deal with bulky DNA damage that can lead to a distortion of

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the DNA helix such as DNA adducts induced by chemical carcinogens PAHs (Vineis et al., 2009). The ERCC2 gene product XPD acts as an ATP-dependent DNA helicase and opens DNA strands around the site of the lesion to make it accessible for

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repairing by other NER proteins (Mathieu et al., 2010). Site-specific ERCC2 germline mutations can lead to a variety of syndromes; XP, XP combined with CS, or TTD,

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and inappropriate function of the ERCC2/XPD gene renders cells more sensitive to DNA damage caused by carcinogens (Wijnhoven et al., 2007; Xing et al., 2012).

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In the present study, we found that ERCC2/XPD has a pivotal role in NER to

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protect cells from gene-toxicity caused by carcinogen B[a]P. Our results have shown that AA8 cells can mostly remove DNA damage after a certain period due to its

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proficient repair system, but for ERCC2/XPD deficient cells (UV5), only a part of DNA damage can be repaired. Therefore these data reminded us that an intact NER system is critical to DNA repair in cells, and if NER is defective, the survival of cells

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will face a big challenge. Usually the alkaline version of the comet assay, a sensitive method offering the possibility of quantifying DNA damage at a single cell level, cannot be chosen to determine the genotoxicity of B[a]P because it was not thought as a suitable method to quantify the DNA adducts directly. But in this study we found that B[a]P can induce DNA strand breaks especially in a NER defective cell line which is consistent with previous studies where genotoxicity of B[a]P was reliably detected with the comet assay(Platt et al., 2008; Speit and Hartmann, 1995). In addition, MTT cell inhibition assay and Rad51 staining assay also reflected that ERCC2/XPD, as a key enzyme in NER system, is required for the repair of DNA damage induced by B[a]P and its metabolites, which is also in line with the previous reports (Liu et al., 2013). Some population studies found that inherited SNPs of DNA repair genes may 13

ACCEPTED MANUSCRIPT contribute to variations in DNA repair capacity and susceptibility to cancer (Hemminki et al., 2001; Schabath, 2005). However, the conclusions seem to be uncertain. Therefore, it is important to understand how inter-individual variations in

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the DNA sequence of specific genes affect the response to environmental genotoxic agents. Takanami et al. (2005) have been established Chinese hamster ovary (CHO)

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EM9 cell lines transfected with XRCC1 (R280H) gene and found the differences of DNA repair ability between the different genotypes of the polymorphism. Gao et al. (2011) also introduced an ERCC1 cDNA clone with either the C or T at the specific

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position into an ERCC1 deficient cell line (UV20) and found that the polymorphism itself is not related to the phenotypic differences in ERCC1 expression or function.

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Recently, another paper from Leiden University Medical Center also tested the effect of ERCC1 codon 118 polymorphism on DNA repair capacity to the damage induced

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by Oxaliplatin in an in vitro transfected cell system (Van Huis-Tanja LH et al, 2014).

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Different to population studies, a well controlled transfected cell line model may provide a unique technical platform to study the effect of different genotypes,

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although care should be taken to translate results obtained from an in vitro system to the in vivo mechanism. In our current study, we introduced plasmids expressing different genotypes of ERCC2/XPD Lys751Gln (rs13181) into an ERCC2/XPD

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deficient cell line (UV5) to examine the the effect of this genetic polymorphism. We found that the protein expression of ERCC2/XPD in UV5ERCC2 (AA) and UV5ERCC2 (CC) cell lines peaked at 6 hrs after B[a]P treatment and kept a high level till 12 hrs after B[a]P treatment. So their dynamic protein expression of ERCC2/XPD showed a similar trend to AA8 cells. In addition, no significant difference in ERCC2/XPD expression of both alleles at each dose and time of B[a]P treatment was found in the transfected model. Since ERCC2/XPD protein joins to the basal transcription factor IIH complex for its protein function, sufficient ERCC2/XPD protein should be produced for optimistic function. As is known, ERCC2/XPD Lys751Gln in exon 23, characterized by an A to C substitution, causes a Lys to Gln amino acid exchange in the C-terminal part of the protein. We hypothesized that the minor polymorphism in ERCC2/XPD codon 751 may change its protein function and then influence DNA 14

ACCEPTED MANUSCRIPT repair capacity to repair the DNA damage caused by carcinogen B[a]P. Interestingly, we found that the two transfected cell lines showed a significant difference of cellular sensitivity and DNA repair capacity to B[a]P according to our data from MTT cell

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inhibition assay and the comet assay. According to our analysis, one of the possible explanations is that the minor polymorphism may change translation kinetics and

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protein folding by altering the structural and functional properties of the gene product. ERCC2/XPD protein functions as an ATP dependent 5’-3’ helicase joint to the TFIIH complex and their potential combination capacity maybe reduced due to the change of

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ERCC2/XPD protein function. Another possible explanation based on the reports from Coin et al. (1998), is that codon 751 affects the protein interactions rather than

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helicase function since codon 751 is in the end of C-terminal domain of ERCC2/XPD, and not in the helicase domain of ERCC2/XPD. It has been shown that mutations in

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the XPD C-terminal, domain, as found in most patients, prevent interaction with p44,

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thus explaining the decrease in XPD helicase activity and the defective function of NER. Furthermore, other studies supported the concept that DNA linked XPB-XPD-

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CAK interactions form a keystone complex that orchestrates DNA damage recognition and verification by TFIIH to coordinate repair with transcription and cell cycle (Chen et al., 2003; Ito et al., 2010; Li et al., 2010; Mui et al., 2011). Similarly,

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the functional difference of ERCC2/XPD protein may alter the modulation of the whole XPB-XPD-CAK chain in cells. Our current study showed that ERCC2/XPD as a DNA repair enzyme is critical to the mammalian cell response to environmental carcinogens such as B[a]P. It is highly unlikely that an additional, as yet uncharacterized, critical DNA adduct exists which relies entirely upon ERCC2/XPD for repair. Different cells expressing different genotypes of ERCC2/XPD Lys751Gln (rs13181) show a significant difference in cellular sensitivity to B[a]P and DNA repair capacity in the well-controlled in vitro system. These data reminded us that this polymorphism may impact the DNA repair capacity due to the alteration of ERCC2/XPD protein and may also act as a biomarker to predict cancer susceptibility. Furthermore, although our conclusion needs to be confirmed in other in vitro and in vivo systems, also needs to be tested using other 15

ACCEPTED MANUSCRIPT DNA damage agents, the present transfected cell line model yields a unique angle of view to study the function of ERCC2/XPD polymorphisms. Therefore our research should be considered part of a battery of complementary assays needed for further

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assessment. We expect that in the near future, the combination of population investigations and functional studies related to gene polymorphisms will provide

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powerful approaches for molecular epidemiological association studies in predicting cancer risk.

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Acknowledgments

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This study was supported by National Natural Science Foundation of China (No. 81273118, 30972506).

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Fig.1. The structure scheme of expression vector construction.

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Fig.2. ERCC2/XPD expression in five cell lines. The parental cell UV5 and the empty vector GFP transfected cells (UV5GFP) did not show detectable ERCC2/XPD protein expression, while cells transfected with ERCC2/XPD cDNA with either genotype of codon 751 (A>C) ploymorphism, designated as UV5ERCC2 (AA) and UV5ERCC2 (CC), showed comparable ERCC2 protein expression levels same to AA8 cells.

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Fig.3. Comparison of cell viability in 5 cell lines after B[a]P treatment The wild type CHO cell AA8, ERCC2/XPD defective parental cell UV5, empty vector transfected cell UV5-GFP, and ERCC2/XPD cDNA with either allele of A or C transfected cell line UV5ERCC2 (AA) and UV5ERCC2 (CC) were treated with B[a]P at 0.1, 0.5, 1, 5, 10, 25, 50, 100 µM doses for 24 h (A) and another 24 h repair after 24 h treatment (B). The cell cytotoxicity was tested by MTT assays. Data obtained from three independent experiments and represent as mean ± SD. *P<0.05 compared between UV5ERCC2 (AA) cells and UV5ERCC2 (CC) cells.

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Fig.4. Kinetics change of ERCC2/XPD expression levels in AA8 following B[a]P treatment. Western blotting for detecting ERCC2/XPD protein: (A) 5 µM B[a]P reatment (B) 10 µM B[a]P treatment. Quantification of ERCC2/XPD expression change following B[a]P treatment: (C) 5 µM B[a]P (D) 10 µM B[a]P. Data obtained from three independent experiments and represent as mean ± SD. The change of ERCC2/XPD protein level is expressed as the fold change compared to the untreated cells. Data were normalized for equal loading. *P<0.05, #P<0.05 compared to the untreated cells.

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Fig.5. ERCC2/XPD expression levels in two transfected cell lines following B[a]P treatment detected by Western blotting: (A) 6 h B[a]P treatment. (B) 12 h B[a]P treatment. 1: UV5ERCC2 (AA) cell. 2: UV5ERCC2 (CC) cell.

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Fig.6. Map of the comet assay of AA8 and UV5 after 5, 10 µM B[a]P treatment.

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Fig.7. Map of the comet assay of UV5ERCC2 (AA) and UV5ERCC2 (CC) after 5, 10 µM B[a]P treatment.

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Fig.8. Impaired formation of nuclear RAD51 foci in 5 cell lines in response to DNA damage. (A) Immunofluorescence and DAPI staining of cells after treatment with B[a]P. Foci were visualized by using rabbit anti-RAD51 antibodies (1 : 200 dilution) and DAPI for nuclear counterstaining. Depicted is the merged picture of the FITC signal used to detect the Rad51 protein and the DAPI signal. (B) 5 µM B[a]P (C) 10 µM B[a]P treatment in five cell lines, and were analysed after 6 h, 12 h, 24 h treatment or 24 h treatment followed by 24 h repair. *P<0.05, AA8 compared to UV5; #P<0.05, AA8 compared to UV5GFP. A cell with more than ten distinct foci in the nucleus was considered to be positive. Depicted is the average and standard deviation of three independent experiments.

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B[a]P(µM)

Table 1 Toxicity ratios of AA8 (wild type) versus UV5 (ERCC2/XPD deficient) cells after 24 h treatment of B[a]P (detected by MTT assay) UV5 Toxicity ratio AA8

P

Cell Viability, ％

OD

Cell Viability, ％

AA8/UV5

0

0.92±0.07

100.00

1.29±0.05

100.00

1.00

-

0.1

0.78±0.04

84.24

0.90±0.09

70.34

1.20

<0.05

0.5

0.74±0.06

79.86

0.86±0.06

66.65

1.20

<0.05

1

0.68±0.08

73.90

0.85±0.06

65.81

1.12

NS

5

0.62±0.03

67.06

0.68±0.03

53.05

1.27

<0.05

10

0.56±0.03

60.69

0.61±0.06

47.30

1.28

<0.05

25

0.52±0.04

56.20

0.59±0.02

45.50

1.24

<0.05

50

0.48±0.04

52.11

0.57±0.03

44.16

1.18

<0.05

100

0.47±0.06

51.12

0.56±0.04

43.56

1.17

<0.05

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Cellular survival between AA8 and UV5 cells was compared at each concentration of B[a]P. NS: not significant.

B[a]P(µM)

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Table 2 Toxicity ratios of AA8 (wild type) versus UV5 (ERCC2/XPD deficient) cells after 24 h treatment followed by 24 h repair of B[a]P (detected by MTT assay) UV5 Toxicity ratio AA8

P

Cell Viability, ％

OD

Cell Viability, ％

AA8/UV5

100

1.25±0.02

100

1

-

1.32±0.08

0.1

1.01±0.05

76.53

0.92±0.09

73.53

1.04

NS

0.5

0.99±0.05

75.19

0.90±0.07

71.93

1.04

NS

1

0.97±0.07

74.08

0.83±0.09

66.48

1.11

NS

5

0.84±0.04

64.23

0.54±0.06

42.78

1.50

<0.05

10

0.70±0.08

53.17

0.33±0.05

26.10

1.95

<0.05

25

0.52±0.03

39.78

0.28±0.02

22.08

1.80

<0.05

50

0.49±0.05

37.41

0.24±0.02

19.53

1.91

<0.05

100

0.48±0.04

36.84

0.22±0.02

17.47

2.11

<0.05

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Cellular survival between AA8 and UV5 cells was compared at each concentration of B[a]P. NS: not significant.

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Table 3 Dynamic changes of DNA damages of AA8 and UV5 after two doses of B[a]P treatment (detected by the comet assay) 5 µM B[a]P 10 µM B[a]P OTM (Olive tail P moment) AA8 UV5 AA8 UV5 control

0.21±0.19

0.22±0.22

-

6h

24.16±8.96

30.20±9.52

<0.05

12 h

9.45±2.98 5.17±2.38

14.40±4.69 9.36±4.44

<0.05

12.44±4.06

<0.05

1.79±0.57

4.79±3.48

<0.05

24 h treatment

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34.04±10.99

<0.05 <0.05

9.36±4.44

19.45±6.79 12.51±4.79

3.29±1.35

9.95±3.67

<0.05

<0.05

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followed by 24 h repair

0.22±0.22

0.21±0.19 28.10±9.24

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OTM between AA8 and UV5 cells was compared at each treatment time of B[a]P.

control

0.20±0.18

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Table 4 Dynamic changes of DNA damages of UV5ERCC2 (AA) and UV5ERCC2 (CC) cells after two doses of B[a]P treatment (detected by the comet assay) 5µM B[a]P 10µM B[a]P OTM (Olive tail P moment) UV5ERCC2 (AA) UV5ERCC2 (CC) UV5ERCC2 (AA) UV5ERCC2 (CC)

12h 24h

by 24h repair

0.21±0.17

-

0.20±0.18

0.21±0.17

-

23.77±11.86 9.17±3.56

27.36±9.50 11.40±4.87

<0.05

30.60±11.12 13.08±6.29

<0.05

<0.05

27.86±11.15 11.24±4.40

5.73±3.67

6.57±4.58

0.11

8.84±4.71

9.58±4.74

0.24

1.70±1.69

0.82

3.40±2.17

3.15±2.05

0.53

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24h treatment followed

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1.76±1.85

OTM between UV5ERCC2 (AA) and UV5ERCC2 (CC) cells was compared at each treatment time of B[a]P.

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ACCEPTED MANUSCRIPT Highlights 1 An in vitro cell model was used to detect the difference of ERCC2/XPD Lys751Gln

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SNP.

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2. Genotype AA of the SNP was found to be associated with a higher DNA repair to

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B[a]P.

3. ERCC2/XPD Lys751Gln could increase the predictive value of cancer susceptibility.

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4. A well controlled in vitro cell model contributed to study the function of a SNP.

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