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Chemico-Biological Interactions 184 (2010) 302–305

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

ole of DNA-PKcs in the biological effect of a benzene metabolite: Phenol toxicity

o human K562 cells in vitro

iao Xiao ∗, Wentao Song, Yongyi Biina

chool of Public Health, Wuhan University, 185 Donghu Rd., Wuhan, Hubei 430071, Ch

. Introduction

Benzene causes leukemia and a range of hematotoxicity char-cterized by peripheral blood cell count reduction in humans [1].xposure of workers to benzene can occur in industries includ-ng shoe making, automobile repair, the oil industry, shipping, andhemical manufacturing. On the other hand, general populationsay be exposed to benzene via cigarette smoking, gasoline vapor,

nd automobile exhaust.The mechanism of benzene hematotoxicity and carcinogene-

is remains unclear. We examined differential gene expressiony microarray analysis of peripheral blood mononucleocytes fromeven workers diagnosed with benzene poisoning and matchedontrols [2]. We found that PRKDC that encodes the DNA-dependentrotein kinase catalytic subunit—DNA-PKcs was consistently ele-ated in benzene-poisoning patients by microarray and realime-PCR [3]. DNA-PKcs is a member of a sub-family of proteininases that contain a phosphoinositol (PI) 3-kinase domain andxhibit serine/threonine protein kinase activity [4]. It is known thatNA-PKcs is required for the non-homologous end joining (NHEJ)athway of DNA double-strand break (DSB) repair, V(D)J recom-ination of immunoglobulin genes and T cell receptor genes, andelomere length maintenance [4]. DNA-PKcs was recently foundo be overexpressed in human cancer. DNA-PKcs expression levelsere also correlated with the development of productive tissues

nd the differentiation and proliferation status of certain cell types5].

The observation that DNA-PKcs was induced in benzene patientsnd the fact that NHEJ is error prone and can lead to aber-ant chromosome formation suggest a potential role of inductionf DNA-PKcs in benzene hematotoxicity and leukemogenesis. Toddress this issue, we analyzed the interaction among benzeneetabolites, DNA-PKcs induction, and benzene toxicity in K562

ells. We found that DNA-PKcs was induced by the benzene

etabolite phenol at both protein and gene levels. Formation of �-2AX foci, which is a marker of NHEJ, was also increased by phenol

n a concentration-dependent manner. Moreover, DNA-PKcs wasound to translocate from the cytoplasm into the nucleus upon

∗ Corresponding author. Tel.: +86 11 86 15926263017.E-mail address: iamfine xx@yahoo.com.cn (X. Xiao).

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.01.023

treatment with phenol. The findings suggest that induction andactivation of DNA-PKcs may contribute to benzene carcinogenesisby increasing NHEJ.

2. Materials and methods

2.1. Cell culture and treatment

K562 cells derived from human chronic myelogenous leukemiawere cultured at 37 ◦C, 5% CO2 in 1640 medium (Hyclone, Logan,Utah) containing 10% fetal bovine serum. Cells were exposed to 0,0.1, 1, and 5 mM phenol (Sigma, New York, NY) for 1–4 days. TotalRNA and protein samples were prepared and analyzed by real timePCR and immunoblotting analyses.

2.2. Real time PCR

Total RNA of each sample was reverse transcribed to cDNA witha reverse transcription kit (TOYOBO, Japan) and real time PCR wascarried out in a reaction mixture containing SYBR Green mix (TOY-OBO, Japan), primers, RNA, and H2O. The amount of the PCR productwas measured as fluorescent signal intensity after standardizing tohuman actin internal control.

2.3. Immunoblotting

The cells were harvested and washed twice in ice-cold phos-phate buffered saline. Cell pellets were treated with lysis buffer(50 mM Tris-HCl, pH 7.5, 1% Noridet P40, 0.5% Sodium deoxy-cholate, 150 mM NaCl, 1 protease inhibitor cocktail tablet in a50 ml solution). Total protein was isolated. Protein of 100 �g wasresolved on SDS/PAGE (6% for DNA-PKcs, 10%for Ku70/80) and thentransferred onto nitrocellulose membranes for immunoblot detec-tion.

2.4. Immunofluorescence

K562 cells were harvested and washed three times in PBS buffer,and then fixed with cold acetone for 15 min followed by perme-abilization with 1% triton for 10 min at RT. Goat Blockinge serum(20%) was used for blocking at 37 ◦C for 30 min. Anti-DNA-PKcs andanti-�-H2AX polyclonal antibodies were from Upstate (New York,

X. Xiao et al. / Chemico-Biological Int

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3.2. Phenol induces �-H2AX foci formation in K562

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ig. 1. Phenol induces DNA-PKcs mRNA expression. K562 cells were treated withhenol as indicated for 1 day and mRNA expression was analyzed by real time PCR.

Y). The blots were incubated with primary antibodies overnight at◦C, and with secondary antibody goat (anti-rabbit IgG-FITC, Pierce,ew York, NY) for 30 min at 37 ◦C. Fluorescence was detected underonfocal microscope.

ig. 2. DNA-PKcs protein was induced by phenol in K562 cells. (A) Cells were treated withing. (B) Quantification of proteins. Quantification was performed by densitometric scaneviation from three independent experiments.

ig. 3. Induction of �-H2AX formation in K562. Cells were treated with phenol as indiells were stained with antibodies against phosphorylated �-H2AX foci and micrographxperimental point, 300–500 nuclei were captured at a constant exposure time and score

eractions 184 (2010) 302–305 303

3. Results

3.1. Phenol induces DNA-PKcs expression at mRNA and proteinlevels

We first examined induction of DNA-Pkcs in K562 cells treatedwith increasing concentrations of phenol. The results showed thatmRNA of DNA-PKcs was slightly elevated by phenol; induction was1.36-fold over control at a concentration of 5 mM phenol (Fig. 1).Next, we further analyzed protein expression of DNA-PKcs, Ku70,and Ku80; the latter two form the dimeric DNA-binding regula-tory subunits of DNA-PK, which are also key factors in NHEJ [6].The results demonstrated that Ku80 and DNA-PKcs proteins wereinduced by phenol in a concentration-dependent manner, whileKu70 expression seemed unchanged with increasing concentrationof phenol (Fig. 2).

�-H2AX is a phosphorylated form of histone H2AX that accumu-lates at the site of DNA double strand breaks. Formation of �-H2AXfoci represents DSB in a 1:1 manner and therefore, is used as a

phenol as indicated for 1 day and protein expression was analyzed by immunoblot-ning of immunoblotting results shown in (A). Data represent means and standard

cated for 1 day and �-H2AX foci were examined by immunofluorescent staining.s were taken under a fluorescence confocal microscope with a FITC filter. For eachd for foci; at least 100 cells/time-point were scored for foci formation.

304 X. Xiao et al. / Chemico-Biological Interactions 184 (2010) 302–305

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ig. 4. DNA-PKcs nuclear translocation. Cells were treated with increasing concentricroscope with an FITC filter.

iomarker of DSB damage [7]. Because benzene is a carcinogen andhenol induces DNA-PKcs that regulates DNA DSB repair, we testedhether phenol induces DSB. The results showed that �-H2AX fociere induced by phenol and induction is concentration-dependent,roviding direct evidence showing that phenol induces DSB (Fig. 3).

.3. Phenol induces DNA-PKcs nuclear translocation

Activated DNA-PKcs translocates from the cytoplasm into theucleus. To confirm the activity of DNA-PKcs, we examined the

ocalization of DNA-PKcs in cells treated with phenol. The resultshowed that DNA-PKcs existed in both the cytoplasm and theucleus in the absence of treatment. Phenol induced nuclear local-

zation of DNA-PKcs in concentration-dependent fashion (Fig. 4).he findings reveal that DNA-PKcs is activated by phenol and acti-ation correlates with �-H2AX foci formation.

. Conclusion

DNA-PK comprises three subunits, the catalytic subunit DNA-Kcs and the Ku heterodimer, which consists of Ku70 and Ku80ubunits abundant in the nucleus. Ku70/80 binds to DNA ter-ini of double-strand breaks (DSBs) with high affinity but without

equence specificity. Ku70/80 recruits DNA-PKcs to the breaknds to activate its kinase function [8]. Localized DNA-PK at thereak sites is critical for double strand break repair through non-omologous end joining.

Benzene metabolites may damage DNA by forming adducts andy causing oxidative stress via redox cycling of quinone deriva-

ive. Given the results of microarray [3], we characterized inductionf DNA-PKcs. Our results show that phenol induces DNA-PKcsnd Ku80 in K562 cells and induction correlates with increasedSBs. Similar results were also obtained in HL60 cells treatedith hydroquinone (data not shown). Since NHEJ is error prone,

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of phenol. DNA-PKcs was stained with antibodies and examined under fluorescent

induction of DNA-PKcs and NHEJ by benzene metabolites maycause DNA mutations and genomic instability and thereby con-tribute to leukemogenesis induced by benzene. In conclusion, ourstudy demonstrated for the first time that benzene and metabolitesinduce DNA-PKcs in benzene poisoning patients and in vitro; induc-tion of the genes may play a role in the pathogenesis of benzenehematotoxicity and serve as biomarkers of benzene exposure.

Conflict of interest

None.

Acknowledgement

The authors thank all members in Professor Y. Bi’s laboratoryin Wuhan University and Dr. Yuanan Lu in Department of Environ-mental Health, University of Hawaii at Manoa for their valuablesuggestions. The project was supported by Chinese National Nat-ural Science Foundation to Y. Bi (Grant No. 30571556). The studywas funded by Grants to YB from National Nature Science Founda-tion, China (30571556) and supported by “Self-research programfor Doctoral Candidates (including Mphil-PhD) of Wuhan Unversityin 2008”.

References

1] ATSDR, Toxicological profile for benzene, Atlanta, GA: Public Health Service,1997.

2] Z. Zhao, X. He, Y. Bi, Y. Xia, N. Tao, L. Li, Q. Ma, Induction of CYP4F3 by ben-zene metabolites in human white blood cells in vivo in human promyelocytic

leukemic cell lines and ex vivo in human blood neutrophils, Drug Metab. Dispos.37 (2009) 282–291.

3] L. Chen, Y.Y. Bi, N. Tao, H. Wang, Y. Xia, Q. Ma, cDNA microarray to identifythe significance of DNA replication and damage repair genes associated withbenzene poisoning, Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 23 (2005)248–251.

cal Int

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X. Xiao et al. / Chemico-Biologi

4] S.P. Jackson, Sensing and repairing DNA double-strand breaks, Carcinogenesis23 (2002) 687–696.

5] J. An, D.-Y. Yang, Q.-Z. Xu, et al., DNA-dependent protein kinase catalytic subunitmodulates the stability of c-Myc oncoprotein, Mol. Cancer 7 (2008) 32.

6] K.K. Khanna, S.P. Jackson, DNA double-strand breaks: signaling, repair, and thecancer connection, Nat. Genet. 27 (2001) 247–254.

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eractions 184 (2010) 302–305 305

7] A. Xu, L.B. Smilenov, P. He, K. Masumura, T. Nohmi, Z. Yu, T.K. Hei, New insight intointrachromosomal deletions induced by chrysotile in the gpt delta transgenicmutation assay, Environ. Health Perspect. 115 (2007) 87–92.

8] L. Stronati, G. Gensabella, C. Lamberti, et al., Expression and DNA bindingactivity of the Ku heterodimer in bladder carcinoma, Cancer 92 (2001) 2484–2492.