XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway

Chemico-Biological Interactions 263 (2017) 55e65

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ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway Guopei Zhang a, 1, Yangyang Guan a, 1, Yuejiao Zhao b, 1, Tahar van der Straaten c, Sha Xiao a, Ping Xue a, Guolian Zhu a, Qiufang Liu a, Yuan Cai a, Cuihong Jin a, Jinghua Yang a, Shengwen Wu a, Xiaobo Lu a, * a b c

Dept. of Toxicology, School of Public Health, China Medical University, Shenyang, PR China Cancer Hospital of China Medical University/Liaoning Cancer Hospital & Institute, PR China Dept. Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 August 2016 Received in revised form 8 November 2016 Accepted 22 December 2016 Available online 24 December 2016

Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5ERCC2 (AA) as compared to UV5ERCC2 (CC) after cisplatin treatment. The DNA damage level of UV5ERCC2 (AA) was significant decreased compared to UV5ERCC2 (CC) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5ERCC2 (AA) alleviated the apoptosis compared to UV5ERCC2 (CC), meanwhile P53mRNA levels in UVERCC2 (AA) was also lower when compared UV5ERCC2 (CC). It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: ERCC2/XPDLys751Glnrs13181 Nucleotide excision repair (NER) DNA repair capacity (DRC) Cisplatin resistance Transfected cell model

1. Introduction Platinum analogues are commonly used chemotherapy drugs, treating a wide range of cancer types [1,2]. Making an informed decision on predicting the tumor response to cisplatin allows for tailoring the chemotherapy program based on the biology of the

* Corresponding author. Dept. of Toxicology, School of Public Health, China Medical University, No.77 Puhe Road, Shenyang North New Area, 110122, Shenyang, Liaoning Province, PR China. E-mail address: [email protected] (X. Lu). 1 Guopei Zhang, Yangyang Guan and Yuejiao Zhao contributed to this work equally. http://dx.doi.org/10.1016/j.cbi.2016.12.015 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.

disease and individual genetic background. DNA is a key target for treatment with platinum drugs in tumor cells. Platinum binds to the DNA helix and form Pt-DNA adducts. The intrastrand and interstrand cross-links prevent the cell from replicating its DNA until the damage is repaired. So a valid and efficient DNA repair capacity (DRC) in tumor cells is usually regarded as an explanation for platinum resistance [3]. Generally spoken, there are at least three DNA repair pathways in human cells which are nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). These DNA repair systems are all reported to be associated with platinum resistance. Pt-DNA adducts however, are mainly recognized and repaired by the NER pathway, and NER function is closely

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associated with cellular sensitivity towards platinum analogues [4,5]. NER pathway requires more than 30 proteins and repairs DNA damage through four major steps: recognition of the lesion, unwinding double helix at the lesion site, and removal of the damaged strand and synthesis of a new DNA strand. Excision repair crosscomplementation group 2 (ERCC2), also known as the xeroderma pigmentosum complementary group D (XPD), is one of the key enzymes in NER pathway. The protein ERCC2/XPD is a subunit of the transcription factor II H (TFIIH) complex [6], and works as an ATP dependent 50 -30 helicase after DNA damage recognition [7]. Furthermore, ERCC2/XPD is responsible for performing enzymatic activities to initiate transcription by melting the promoter regions around the transcription origin [8], and it is also involved in P53 mediated apoptosis response [9]. ERCC2/XPD was additionally identified to have a strong correlation between increased expression and cisplatin resistance in NSCLC and glioma cell lines [10]. The ERCC2/XPD gene is located at chromosome 19, and comprises 23 exons. Single nucleotide polymorphisms (SNPs) in ERCC2/ XPD are related to DNA repair deficiency, transcription defects, and hormonal dysfunctions [11]. Several SNPs in this gene have been the subject of intense work aimed at identifying functional consequences and association with cancer susceptibility. Generally, two common single nucleotide polymorphisms (SNPs) in ERCC2/XPD have been identified: Asp312Asn and Lys751Gln, both resulted in decreased DNA repair capacity, and were suggested to be potential biomarkers to predict clinical outcome in osteosarcomas and lung cancers treated with cisplatin [12,13]. The most studied SNP in ERCC2/XPD is rs13181, which is a non-synonymous SNP at codon 751 resulting in an amino acid change from Lysine (Lys) to Glutamine (Gln). This amino acid change caused a change of the electronic configuration of the polypeptide. Given the fact that this change occurs in the domain of interaction between ERCC2/XPD protein and its helicase activator [14], this SNP is predicted to have major impact on DNA repair activity. Two studies have shown that healthy participants with ERCC2/XPD751Gln genotype have a lower DNA repair capacity as compared to its wildtype (751Lys) [15,16]. However other studies on Chinese population can't provide enough evidence to confirm the relationship between ERCC2/XPD Lys751Gln polymorphism and the response to cisplatin chemotherapy [17]. It has shown that this SNP enhances the risk of cancer and could be a potential biomarker for predicting platinum chemotherapy [18]. However, no associations or opposite associations have been reported [19,20]. Most studies on ERCC2/XPD polymorphisms are population based studies, which only investigate the relationship between SNPs and prognosis, leave the mechanisms unknown [21,22]. Questions about the functionality of ERCC2/XPD SNPs should be addressed and the mechanisms involved need to be clarified in order to use this SNP as a valid biomarker or as a target for drug development. In order to explore whether the wildtype and variant genotype of ERCC2/XPD codon 751 act differently in DNA repair capacity to platinum-DNA adducts, we introduced the cDNA clone containing ERCC2/XPD Lys751 or ERCC2/XPD 751 Gln into an ERCC2/XPD deficient cell line (UV5) respectively. We investigated the cellular sensitivity and the DNA repair capacity to platinum-DNA adducts in an in vitro transfected cell model. We also investigated the distribution of cell cycle and the change of apoptosis rates, P53 mRNA expression and phospho-P53 protein level after cisplatin treatment in all transfected cells. Our findings may contribute to a comprehensive understanding of the involvement of ERCC2/XPD in NER during the repair of DNA damage induced by cisplatin and provided some experimental data about the relationship between ERCC2/ XPD polymorphism and resistance of Platinum-based treatment.

2. Materials and methods 2.1. Materials Cisplatin (TCI, Japan), ERCC2/XPD antibody, P53-phosphorylated antibody, Rad51 antibody (Abcam), Cell cycle assay kit and Annexin Ⅴ-PE/7-AAD apoptosis kit (Nanjing KGI bio-Technology Development Co., Ltd.). 2.2. Cell culture and treatment Chinese hamster ovary (CHO) cells: AA8 (wild type; ATCC No. CRL-1859™) and UV5 (ERCC2/XPD deficient; ATCC No. CRL-1865™) 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. Cisplatin was dissolved in PBS (NaCl, KCl, KH2PO4, Na2HPO4) and prepared freshly each time. 2.3. Plasmid construction, transfection and genotyping These methods have been described previously [23]. Briefly, UV5 were transfected with these three types of plasmids separately to get stable transfected cell lines. Plasmid expression was maintained by administration of 1.0 mg/ml G418 antibiotic. The resulted cells were designated as empty plasmid control group cells UV5GFP (only expressing GFP), and ERCC2-overexpression group cells,UV5ERCC2 (AA) (expressing ERCC2/XPD 751Lys and GFP) and UV5ERCC2 (CC) (expressing ERCC2/XPD 751Gln and GFP). DNA extraction and PCR reactions were determined as described previously. 2.4. Protein extraction and western blotting Protein concentration was determined using the BCA Protein Assay Kit (Beyotime, China) and 60 mg protein was subjected to electrophoresis on a 5e8% polyacrylamide gel (Bis-Tris). The proteins were electro-transferred to a polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA) at 100V using a wet membrane-transfer device (CAVOYMini TBC,China). The membrane was blocked in 5% (v/w) non-fat milk powder in Trisbuffered saline followed by an overnight incubation with a 1:500 dilution of ERCC2/XPD primary antibody (Abcam, ab54676) and 1:800 dilution of b-actin primary antibody (ZSGB-BIO) in TBST. 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 (MF-ChemiBIS 312, DNA Bio-Imaging Systems). Quantification of protein expression was performed using Image J (http://rsbweb. nih.gov/ij/) according to the manufaturer's instructions. 2.5. Cell viability using 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium bromide (MTT) assay The MTT assay is a colorimetric assay and a rapid measure of short-term cell viability [24]. Cell lines were seeded in 96-well plates (5000 cells/well), and after 24 h of incubation, exponentially growing cells were incubated with a variety concentrations of cisplatin (0.5, 1, 2, 4, 8, 16, 32, 64 mg/ml) for 24 h. After washing thoroughly, the cells were cultured for another 24 h to allow DNA repair. Next, MTT was added to each well at a final concentration of 0.05 mg/ml. After 4 h of incubation, MTT solution was removed and

G. Zhang et al. / Chemico-Biological Interactions 263 (2017) 55e65

replaced with 150 ml DMSO. Absorbance of formazan was detected at 490 nm on a microplate reader (DNM-9602G, PERLONG).

2.6. Single cell gel electrophoresis (a modified comet assay) Pt-DNA cross-links caused by platinum analogues were studied by Single Cell Gel Electrophoresis (comet assay). The alkaline comet assay was performed according to the method of Tice et al. with some modifications [25]. Briefly, exponentially growing cells were seeded in 6-well plates (2  105 cells/well). After 24 h, cells were treated with 2 or 8 mg/ml cisplatin for 6 h or 24 h, and incubated for another 24 h to allow for DNA repair. Thenthe cells were harvested, ethidium bromide was used for staining after electrophoresis. Finally, DNA damage in a single cell was detected using fluorescent microscope. At least 100 tail olives moment (and tail areas) in each slide were counted. The damage level was obtained with the formula:

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2.9. Apoptosis rate All cells were seeded in 6-well plates for 24hrs attachment, and were treated with 2 or 8 mg/ml cisplatin for 6 h or24 h, followed by an additional 24 h. Next, cells were collected, washed twice with PBS and adjusted to 1e5  106 cells at first. Dye solution was prepared (adding 5 ml 7-AAD dye to 50 ml Binding Buffer) and added to the cells. Cells were kept in the dark at room temperature for 5e15 min. Binding Buffer (450 ml containing 1 ml AnnexinⅤ-PE) was added and incubated for 5e15 min. Apoptosis rate was detected by flow cytometry within 1 h according to the Apoptosis detection method.

2.10. RNA isolation and RT-PCR All cells were collected after cisplatin treatment. Total RNA was isolated using the RNeasy kit (Qiagen) following manufacturers'

ðcontrol group  exposed groupÞ tail olives moment ðor tail areaÞ tail olives moment ðor tail areaÞ of control group

Mean ranks of tail olives moment and tail area ratio were denoted as holistic marking to reflect the integrated evaluation of DNA damage.

2.7. RAD51 immunofluorescence staining RAD51 proteins are concentrated at the damaged DNA strand and can be visualized as foci by immunofluorescence. Approximately 2  105 exponentially growing cells were seeded in 6-well plates containing sterilized glass cover slips. After 24, cells were treated with2 or 8 mg/ml cisplatin for 6 h or 24 h, followed by an additional 24 h to allow DNA repair. After treatment, cells were fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100/PBS. Cover slips were blocked with 1% BSA for 30 min and incubated with mouse monoclonal IgG anti-RAD51 (Abcam) primary antibody (1:200) overnight at 4  C, followed by TRITCconjugated Affini Pure Goat Anti-Mouse IgG (ZSGB-BIO) secondary antibody (1:150) for 1 h at room temperature. Next, cells were washed with PBS andincubated with 4, 6-diamidino-2phenylindole (DAPI) for nuclei staining. Foci on each slide were visualized by fluorescence microscope and cells containing at least 10 foci were denoted as positive. At least 200 cells per slide were scored.

2.8. Cell cycle distribution Single cell suspensions in logarithmic growth phase were seeded in a 6-well plate. After 24 h, the cells were treated with 1 or 2 mg/ml cisplatin for 6 h or 24 h, followed by an additional 24 h to allow DNA repair. The cells were harvested and washed twice with PBS and adjusted to 1e5  106 cells/well. The mix dye liquor (50 ml Binding Buffer containing 7-AAD dye) was added to the cells and kept in the dark at room temperature for 5e15 min. Binding Buffer (450 ml) and 1 ml AnnexinⅤ-PE were added, mixed and incubated for another 5e15 min. Samples were measured within an hour by flow cytometry. Three independent experiments were performed.

instructions, and its concentration, quality and integrity of total RNA were assessed using Nanodrop nucleic acid quantitative analyzer. Two-Step quantitative RTePCR kits (Invitrogen) were used according to manufacturer's instruction. P53 primers (Forward 50 -ATCCTTACCATC ATCACACTGGAA-30 , Reverse 50 -CAGGACAGGCACAAATACGAAC-30 ). Real-time quantifications were performed in ABI 7500 Real-Time PCR instrument. Three parallel samples were performed in each test and b-actin gene was taken as an internal reference. The fluorescence threshold value was calculated by CT value. 2.11. Phospho-P53 protein expression detected by western blot Approximately 2  105 exponentially growing cells were seeded in 6-well plates. After 24hrs, cells were treated with 8 mg/ml cisplatin for 6hrs or24hrs, followed by an additional 24hrs. After cells were collected the total protein was extracted. Western blot was performed as described above (2.4). 2.12. Statistical analysis Statistical data analysis was carried out using the SPSS software. Data are represented as the mean ± SD. One-way ANOVA and LSD multiple comparison tests were used to estimate the differences among the groups. Cellular sensitivity to cisplatin was expressed as a percentage of survival rates. The difference between the sensitivity of transfected cells to each dose of cisplatin was expressed as a ‘toxicity ratio’, which was calculated by dividing the mean survival rate of ERCC2 over-expression groupcells by that of empty plasmid control group cells. Each independent experiment was performed in triplicate and P < 0.05 was considered as statistically significant. 3. Results 3.1. ERCC2/XPD expression in UV5 cells The transfected cells, stably expressing ERCC2/XPD with rs13181 either A or C allele, denoted as UV5ERCC2 (AA) and UV5ERCC2 (CC), and

Fig. 1. A: ERCC2/XPD protein expression in five cell lines. The parental cell line UV5 and the control vector GFP transfected cells (UV5GFP) did not show any detectable ERCC2/XPD protein, while cells transfected with ERCC2, with both genotypes of codon 751 designated as UV5ERCC2 (AA) and UV5ERCC2 (CC), showed ERCC2 protein expression levels similar to AA8 cells. B: Real time PCR plot indicating genotype of ERCC2 of transfected cell lines UV5ERCC2 (AA) and UV5ERCC2 (CC).

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Fig. 2. Comparison of cell viability in 5 cell lines after cisplatin treatment. Wild type CHO cell lines AA8, and ERCC2 defective cell line UV5, empty vector transfected cell UV5GFP, and ERCC2 transfected cell lines UV5ERCC2 (AA) and UV5ERCC2 (CC) were treated with cisplatin at 0.5, 1, 2, 4, 8, 16, 32, 64 mg/ml doses for 24 h (A) and incubated for another 24 h to allow for DNA repair (B). Cell cytotoxicity was tested with MTT assay. Data obtained from three independent experiments and represented as Mean ± SD. *indicates P value < 0.05 between UV5ERCC2 (AA) cells and UV5ERCC2 (CC) cells.

Table 1 Dynamic changes of DNA damages of UV5ERCC2 (AA) and UV5ERCC2

(CC)

ERCC2(AA)

Control 0 h 6h 24 h 24 h treatment followed by 24 h repair OTM between UV5ERCC2

(AA)

cells after two concentrations of cisplatin treatment (detected by the comet assay).

2 mg/ml Cisplatin

OTM (Olive tail moment)

and UV5ERCC2

(CC)

P ERCC2(CC)

8 mg/ml Cisplatin ERCC2(AA)

UV5

UV5

0 2.8707 ± 0.4816 14.6453 ± 0.7114 12.7348 ± 0.7820

0 3.2900 ± 0.4058 15.4317 ± 0.7198 14.2878 ± 1.0377

e 0.312 0.424 0.107

P ERCC2(CC)

UV5

UV5

0 20.2613 ± 1.5822 78.2664 ± 3.5265 44.6561 ± 2.8131

0 21.3364 ± 2.4350 84.4387 ± 2.9500 48.5351 ± 2.9773

e 0.556 0.081 0.423

cells were compared at each time point.

UV5GFPwere successfully established and confirmed by genotyping. As shown in Fig. 1, the parental UV5 cells and the control GFP transfected cell did not show ERCC2/XPD protein (87 kD) expression, while the cells transfected with ERCC2/XPD rs13181 containing either A or C allele showed ERCC2/XPD protein expression levels similar to AA8.

3.2. Cell viability to cisplatin Toxicity ratios were obtained by comparing the survival rate of cisplatin at the same dose treatment of transfectedcells. At each concentration of cisplatin, cellular sensitivity to cisplatinin UV5 cells is partially restored when ERCC2/XPD cDNA is introduced. There was a significantly increased viability in UV5ERCC2 (AA) and

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Fig. 3. DNA damage in a single cell was detected using fluorescent microscope. Quantification of cells with olive tail moment from 100 cells in 30 randomly selected confocal microscopic views. Representive pictures of the modified comet assayof UV5ERCC2 (AA) and UV5ERCC2 (CC) cells were compared at each time point after 2 and 8 mg/ml cisplatin treatment.

Table 2 Dynamic changes of DNA damages of UV5ERCC2 (AA) and UV5ERCC2

cells after two concentrations of cisplatin treatment (detected by the Immunofluorescence assay).

2 mg/ml Cisplatin

Nuclear RAD51 FOCI

Control 0 h 6h 24 h 24 h treatment followed by 24 h repair Nuclear RAD51 foci between UV5ERCC2

(CC)

(AA)

P

UV5ERCC2(AA)

UV5ERCC2(CC)

1.2146 ± 0.0804 4.0699 ± 0.2326 13.7315 ± 0.7303 6.0653 ± 0.3060

1.2322 ± 0.2766 4.3349 ± 0.2814 14.4975 ± 0.4199 6.2281 ± 0.36422

and UV5ERCC2

(CC)

e 0.278 0.19 0.591

8 mg/ml Cisplatin

P

UV5ERCC2(AA)

UV5ERCC2(CC)

1.0906 ± 0.1200 8.8461 ± 0.5505 35.1794 ± 1.2500 16.7755 ± 0.2241

1.0303 ± 0.5051 9.3083 ± 0.4229 37.8803 ± 1.0550 17.4062 ± 0.4785

e 0.314 <0.05 0.108

cells were compared at each time point.

Fig. 4. Comparison of the distribution of cell cycle in transfected cells at each time point after 1 and 2 mg/ml cisplatin treatment. Three independent experiments were carried out.

UV5ERCC2 (CC) cells as compared to the UV5GFP cells after cisplatin treatment. The survival rate of the UV5ERCC2 (AA) and UV5ERCC2 (CC)

cells were not affected by 24 h of cisplatin treatment except for concentrations 8 and 16 mg/ml (Fig. 2 A). However, after additional

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Fig. 5. The dynamic change of apoptosis rate assayed by flow cytometric method. Cells were treated with different concentration of cisplatin 2 mg/ml (A) and 8 mg/ml (B) for 6 h or 24 h, and an additional 24 h. Data obtained from three independent experiments and represented as Mean ± SD. *indicates P value < 0.05 between UV5ERCC2(AA) cells and UV5ERCC2(CC) cells.

Table 3 Apoptosis rates of UV5ERCC2 (AA) and UV5ERCC2

(CC)

cells after two doses of cisplatin treatment (detected by apoptosis rate detection kit).

2 mg/ml Cisplatin

Apoptosis rate, %

ERCC2(AA)

UV5 control 6h 24 h 24 h treatment followed by 24 h repair Apoptosis rate between UV5ERCC2

(AA)

0.9000 0.9667 9.4333 3.5333

and UV5ERCC2

(CC)

± ± ± ±

0.1000 0.1155 0.6658 0.2082

P ERCC2(CC)

UV5

0.9700 ± 0.1153 1.1333 ± 0.1528 10.0000 ± 0.2646 3.8667 ± 0.2517

cells were compared at each time point.

8 mg/ml Cisplatin UV5

e 0.206 0.243 0.152

ERCC2(AA)

0.9233 ± 0.1024 1.2000 ± 0.1000 16.2333 ± 0.3512 6.2333 ± 0.1528

P UV5

ERCC2(CC)

0.9670 ± 0.1003 1.3333 ± 0.1528 17.0667 ± 0.3512 6.6333 ± 0.2517

e 0.275 <0.05 0.078

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Table 4 Comparison of P53 mRNA level between UV5ERCC2

and UV5ERCC2

(CC)

cells after two doses of cisplatin treatment (detected by Real-time PCR assay).

2 mg/ml Cisplatin

P53 mRNA level

control 6h 24 h 24 h treatment followed by 24 h repair P53 mRNA level between UV5ERCC2

(AA)

(AA)

P

UV5ERCC2(AA)

UV5ERCC2(CC)

1 1.0787 ± 0.0601 2.1115 ± 0.2868 1.578 ± 0.1147

1 1.2327 ± 0.1571 2.0072 ± 0.1877 1.8393 ± 0.1987

and UV5ERCC2

(CC)

e 0.188 0.626 0.12

8 mg/ml Cisplatin

P

UV5ERCC2(AA)

UV5ERCC2(CC)

1 1.1703 ± 0.0469 3.1708 ± 0.1358 1.9683 ± 0.1056

1 1.2871 ± 0.0692 5.1945 ± 0.3146 3.7126 ± 0.1145

e 0.108 <0.05 <0.05

cells were compared at each time point.

incubation of 24 h to allow for DNA repair, the differences were found statistically significant for concentrations of 1e32 mg/ml cisplatin (P < 0.05) (Fig. 2 B). These results indicated that A allele in ERCC2Lys751Gln had a higher DNA repair capacity to cisplatin.

incubation, indicating a poor repair capacity. However, we found delayed recovery from S-phase, cell cycle checkpoints in UV5ERCC2 (CC) at the time point of 24 h after cisplatin treatment. It suggested that the UV5ERCC2 (AA) may have a higher DNA repair efficiency compared to UV5ERCC2 (CC).

3.3. DNA damage detected by the modified comet assay 3.6. Apoptosis rate Cisplatin is a DNA cross-linking agent, which can damage DNA and form platinum-DNA adducts. The modified comet assay was used to evaluate the genotoxic effect of cisplatin. After cells exposed to hydrogen peroxide (H2O2) treatment, olive tail moment (OTM, as an index of DNA damage level) was inversed to the degree of DNA damage. After hydrogen peroxide treatment the intact control showed long comet tail. However with the increased concentrations of cisplatin, the comet tails of impaired cells decreased due to existence of platinum-DNA adducts. After 24 h of cisplatin treatment, followed by an additional 24 h of incubation, DNA damage has been restored partially. The differences of OTM between cisplatin treatment and the control in two transfected cells were measured (Table 1). Fig. 3 showed the image of the modified comet assay. For two doses and three time points, the DNA damage level of UV5ERCC2 (AA)was decreased compared with UV5ERCC2 (CC), but no significant difference was found (P > 0.05). 3.4. Immuno-staining for RAD51 RAD51, a key element in homologous recombination pathway, isusually bounded to DNA damage and represents the central of DNA damage [26,27]. Here, we analyzed the induction of RAD51 foci by immunofluorescence in UV5ERCC2 (AA) and UV5ERCC2 (CC) cells treated with 2 or 8 mg/ml cisplatin. With the increase of cisplatin concentration the proportion of damaged cells was gradually increased, and peaked after 24 h of cisplatin treatment. The amount of foci-positive cells of UV5ERCC2 (AA) and UV5ERCC2 (CC) was became significantly different after 24 h of 8 mg/ml cisplatin treatment (P < 0.05). The DNA damage level of UV5ERCC2 (AA) was significant decrease than UV5ERCC2 (CC). However, after 24 hrsof treatment followed by an additional 24 hrsof DNA repair, the amount of focipositive cells in two transfected cells was reduced, and no significantly difference was observed (P > 0.05) (Table 2).

The apoptosis rates of transfected cells after cisplatin treatment were shown in Fig. 5. With the time and concentration of cisplatin treatment, apoptosis rates of all cells increased gradually, and peaked after 24 h of treatment. After an additional 24hrs of DNA repair, the apoptosis rate in the transfected cells was reduced (Table 3). There was a significant difference at24 h of 8 mg/ml cisplatin treatment. Compared to UV5ERCC2 (AA), UV5ERCC2 (CC) showed a slight higher apoptosis rate (P < 0.05) (Fig. 5 B). 3.7. P53 mRNA level P53 mRNA level in UV5ERCC2 (AA) and UV5ERCC2 (CC) was determined at three time points (See Table 4) after 2 and 8 mg/ml of cisplatin treatment. After 24 h, P53 mRNA in these transfected cells increased to maximum (P < 0.05). At two time points of 8 mg/ml cisplatin treatment (24 h and 24 h followed by an additional 24 h repair), P53 mRNA in UV5ERCC2 (CC) was significantly higher compared to UV5ERCC2 (AA) (P < 0.05) (Fig. 6 B). 3.8. Phospho-P53 protein expression In order to analyze the dynamic change of phospho-P53protein expression in two transfected cell lines after 8 mg/ml cisplatin treatment, phospho-P53 expression was detected using western blot (Fig. 7A). We found that phospho-P53 expression level in UV5ERCC2 (AA) and UV5ERCC2 (CC) peaked at 24 h after cisplatin treatment, and kept a high level until an addition 24 h after treatment (compared to the intact control group, P < 0.05). After 24 h of DNA damage repair, phospho-P53 level decreased. However no really significant difference was found in these two transfected cell lines (Fig. 7 B). 4. Discussion

3.5. Cell cycle Cell cycle arrest in three transfected cells was seen after 1 or 2 mg/ml cisplatin treatment at three time points (Fig. 4). With the increase of cisplatin, the more obvious cell cycle arrest and the longer period for DNA damage repair were found. During the whole period of cisplatin treatment, cell cycle arrest happened in S-phase firstly. After 24 h of treatment, followed by an additional 24 h, UV5ERCC2 (AA) and UV5ERCC2 (CC) repaired DNA obviously, and rendered cells into G2 phase, while there was no statistically significant difference between UV5ERCC2 (AA) and UV5ERCC2 (CC). ERCC2/ XPD deficient cell UV5GFP still remained in the S-phase after 24 h of

The individualization of cancer chemotherapy has become a challenge in recent years. Although Platinum-based chemotherapy is the preferred solution in some advanced cancer, the efficiency of platinum-based chemotherapy is only 25%e35% due to drugs resistance [28]. Resistance to platinum-based chemotherapy is attributed to three molecular mechanisms: increased DNA repair, altered cellular accumulation, and increased drug inactivation. Studies have shown that the enhanced capability to repair DNA damage (DNA repair capability, DRC) in tumor cells may be the most important mechanisms to platinum analogue resistance [18,19].

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Fig. 6. Kinetics changes of the level of P53 mRNA between UV5ERCC2 (AA) and UV5ERCC2 (CC) cells. Cells were treated with different concentration of cisplatin 2 mg/ml (A) and 8 mg/ml (B) for 6 h or 24 h, and an additional 24 h. Data obtained from three independent experiments and represented as Mean ± SD. *indicates P value < 0.05 between UV5ERCC2(AA) cells and UV5ERCC2(CC) cells.

Platinum atom binding on DNA chain, which cause distortion and despiralization of DNA chain, disturbs the cellular DNA replication, transcription, and other critical life processes. The platinuminduced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system. [29]: ① identify DNA damage: XPA, XPC genes involved; ② open DNA chain at damaged position: ERCC2/XPD and ERCC3/XPB helicase (two major subunits forming THIIF) open the double-stranded DNA; ③ cut off oligonucleotides

chain: XPG and XPF-ERCC1 complexes break the damaged singlestranded DNA separately from 3 'end and 50 end; ④ PCNA, RPA, RFC complete the resynthesis of single-stranded oligonucleotide on the deficient chain. Numerous studies have confirmed that NER is the main mechanism to repair cisplatin-DNA adducts [30]. ERCC2/XPD as evolutionarily conserved DNA helicases in NER pathway, and also as part of the TFIIH transcription complex participates in transcription. ERCC2/XPD polymorphism has been

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Fig. 7. Quantification of Phospho-p53 expression change after 8 mg/ml cisplatin treatment, detected by Western blot. Data were normalized for equal loading. Data obtained from three independent experiments and represented as Mean ± SD.

found to be closely related to the sensitivity of platinum-based drugs due to their alteration in DNA repair capacity [31]. However, the conclusions in those epidemiology studies were not entirely consistent. It is important to understand how inter-individual variations in the DNA sequence of specific genes affect the response to platinum analogue agents. Our recent publication used the in vitro transfected cell model to analyze the relationship between ERCC2/XPD Lys751Gln polymorphism and DNA repair capacity to BPDE-DNA adducts [23]. Different to population studies, a controlled transfected cell line model may provide a unique technical platform to study the effect of different genotypes, although care should be taken to translate results obtained from an in vitro system to the in vivo mechanism. As expected, our current study showed thatERCC2/XPD is critical to repair DNA damage induced by Platinum in mammalian cells. ERCC2/XPD defective cell line UV5 is much more sensitive to the damage caused by cisplatin and showed a lower repair capacity. As the effect of ERCC2 codon 751 polymorphism on DNA repair capacity to cisplatin was concerning, after additional 24 h of repair, UV5ERCC2 (AA) showed a much higher DNA repair efficiency as compared to UV5ERCC2 (CC). In addition, we also use the modified comet assay and RAD51 staining respectively to further confirm the difference of DNA repair capability among all cells. The results showed that cisplatin-induced DNA damage in all cells peaked at 24 h after cisplatin treatment, while after additional 24 h of recovery, DNA damage has been repaired partly. UV5ERCC2 (AA) showed a much higher DNA repair capability as compared to

UV5ERCC2 (CC). As the main factor of TFIIH complex subunit, ERCC2/XPD has an important significance in P53-mediated apoptosis. P53 pathway plays a central role in signal transduction, interacts with XPC, TFIIH and RPA of NER pathway. As target molecules of tumor resistance, apoptosis-related genes can interact with other pathways to mediate multidrug resistance [32]. In addition, ERCC2/XPD is also required for the association of the cyclin-dependent kinase (CDK)activating kinase (CAK) subcomplex with TFIIH. Since TFIIH is involved in both DNA repair and cell cycle progression, ERCC2 polymorphisms might cause the change of CAK liberation, and alter the distribution of cell cycle [33]. In the present study, we found that the cell cycle distribution was different afterERCC2/XPD transfection. The number of cells in S-phase was increased until it peaked at 24 h in all transfected cells. After additional 24 hrsof DNA repair, both ERCC2/XPD transfected cell lines UV5ERCC2 (AA) and UV5ERCC2 (CC) recovered cell cycle. Cells went into the G2 phase for DNA replication, which indicated that DNA replication continued and the damage can be repaired successfully. ERCC2/XPD deficient cell UV5GFP still remained in the S-phase after 24 h repair, indicating a poor repair capacity. Both transfected cell lines UV5ERCC2 (AA) and UV5ERCC2 (CC) restored the repair capability after the introduction of ERCC2, but they showed different repair efficiency between the two variants. Mutation of ERCC2 rs13181 AA to CC causes a prolonged S-phase. Comparing with UV5ERCC2 (AA), UV5ERCC2 (CC) showed a slight higher apoptosis rate. The apoptosis rate peaked at 24 h of cisplatin

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treatment, and was reduced after an additional 24hrs of DNA repair. It co-incides with a prolonged high expression level of P53 mRNA and phospho-P53 protein which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). In conclusion, the invitro transfected cells showed that ERCC2/ XPD Lys751 has a better repair efficiency to cisplatin treatment when compared to its mutant 751Gln form. Differently from other studies, we explored the possible mechanism by combining cell cycle, apoptosis and phospho-P53 expression. Our current data remind us that ERCC2/XPD Lys751Gln (rs13181) may become a valid biomarker to predict the resistance of cisplatin. Therefore our research should be considered as part of a battery of complementary assays needed for further assessment. We expect that in the near future, the combination of population investigations and functional studies on polymorphisms will provide much more powerful approaches for molecular epidemiological association studies in predicting individual chemotherapy. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments

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

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Transparency document

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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.12.015.

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