Clinical Study DNA Damage Sensor -H2AX Is Increased in …downloads.hindawi.com/journals/tswj/2013/597095.pdf · 2019. 7. 31. · e Scientic World Journal -H2AX DAPI Case 2 Case 7

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 597095 7 pageshttpdxdoiorg1011552013597095

Clinical StudyDNA Damage Sensor 120574-H2AX Is Increased in PreneoplasticLesions of Hepatocellular Carcinoma

Yasunobu Matsuda1 Toshifumi Wakai2 Masayuki Kubota3 Mami Osawa3 MasaakiTakamura4 Satoshi Yamagiwa4 Yutaka Aoyagi4 Ayumi Sanpei1 and Shun Fujimaki1

1 Department of Medical Technology Niigata University Graduate School of Health Sciences2-746 Asahimachi-dori Niigata 951-8518 Japan

2Division of Digestive and General Surgery Niigata University Graduate School of Medical and Dental Sciences2-746 Asahimachi-dori Niigata 951-8518 Japan

3Division of Pediatric Surgery Niigata University Graduate School of Medical and Dental Sciences2-746 Asahimachi-dori Niigata 951-8518 Japan

4Division of Gastroenterology and Hepatology Niigata University Graduate School of Medical and Dental Sciences2-746 Asahimachi-dori Niigata 951-8518 Japan

Correspondence should be addressed to Yasunobu Matsuda yasunobumedniigata-uacjp

Received 6 December 2012 Accepted 5 February 2013

Academic Editors M Ladekarl P-D Line and P Stal

Copyright copy 2013 Yasunobu Matsuda et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background Phosphorylated histone H2AX (120574-H2AX) is a potential regulator of DNA repair and is a useful tool for detecting DNAdamage To evaluate the clinical usefulness of 120574-H2AX in hepatocellular carcinoma (HCC) we measured the level of 120574-H2AX inHCC dysplastic nodule and nontumorous liver diseasesMethods The level of 120574-H2AX was measured by immunohistochemistryin fifty-eight HCC 18 chronic hepatitis 22 liver cirrhosis and 19 dysplastic nodules Appropriate cases were also examined by fluo-rescence analysis and western blotting Results All cases with chronic liver disease showed increased levels of 120574-H2AX expressionIn 40 (699) of 58 cases with HCC the labeling index (LI) of 120574-H2AX was above 50 and was inversely correlated with thehistological grade Mean 120574-H2AX LI was the highest in dysplastic nodule (741 plusmn 221) which was significantly higher thanHCC (119875 lt 0005) Moreover 120574-H2AX was significantly increased in nontumorous tissues of HCC as compared with liver cirrhosiswithout HCC (625plusmn247 from 51 to 960 119875 lt 0005) Conclusions 120574-H2AXwas increased in the preneoplastic lesions of HCCand might be a useful biomarker for predicting the risk of HCC

1 Introduction

Hepatocellular carcinoma (HCC) is one of themost commonmalignancies in developing and industrial countries and isincreasing worldwide [1ndash4] HCC is unique as it frequentlyreoccurs after treatment irrespective of the different etiolog-ical factors including hepatitis virus B (HBV) and C (HCV)alcohol abuse and nonalcoholic steatohepatitis [2 4 5] Onepossible reason for the frequent recurrence of HCC mightbe due to many patients being affected with hepatitis virus-associated chronic liver inflammation [1ndash3]

To date many reports have described a possible relation-ship between hepatitis virus and DNA damage For exampleHBV has been reported to directly regulate the DNA damage

response in host cells [6] HBV stimulates ATM- and Rad3-related protein kinase (ATR)and checkpoint kinase 1 (Chk1)pathways [7] leading to the acquisition of strengthened sur-vival against DNA damage Moreover HBV X gene product(HBX) widely recognized as a possible viral carcinogen [8 9]plays a critical role in the phosphorylation and inactivationof Rb via activating p38 mitogen-activated protein kinase[10] HBX also binds and inhibits the functional efficiency ofp53 [11 12] leading to DNA damage accumulation in HBV-infected cells HCV has been also reported to be involved inthe deregulation of the DNA repair system HCV nonstruc-tural proteins NS3 and NS4A inhibit Ataxia-telangiectasia-mutated (ATM) kinase in response to DNA damage [13]HCV core protein inhibits the functional formation of

2 The Scientific World Journal

the Mre11NBS1Rad50 complex which causes the ATM-mediated DNA repair system to be markedly impaired inHCV-infected cells [14] Together these lines of evidencestrongly suggest that DNA damage response machinery issignificantly involved in hepatocarcinogenesis and might beused as biomarkers for predicting the risk of HCC develop-ment

Recently several studies reported that the level of oxida-tive DNA damage is a good biomarker For example hepatic8-oxo-21015840-deoxyguanosine (8-OHdG) an oxidized derivativeof deoxyguanosine which reflects oxidative stress was closelycorrelated with the risk of HCC recurrence after surgery [1516] To search for more sensitive and reliable biomarkers ofDNA damage we investigated the levels of 120574-H2AX in HCCtissues which mark the site of DNA double-strand breaksand evoke theDNA repair system [17 18] To address whether120574-H2AX might be a good indicator for the risk of HCCdevelopment we also examined and compared the level of120574-H2AX in nontumorous chronic liver diseases

2 Materials and Methods

21 Patients The pathological diagnoses and analyses of dys-plastic nodule and HCC were made according to the GeneralRules for the Clinical and Pathological Study of PrimaryLiver Cancer [19] HCC tissue samples were obtained from58 patients (7 cases with hepatitis B virus-positive 35 withhepatitis C virus-positive and 16 with unknown etiology 9females 49 males mean age 62 plusmn 9) that underwent hepaticresection at Niigata University Medical and Dental HospitalTissue samples of dysplastic nodules were obtained from 19patients (2 cases with hepatitis B virus-positive 12 with hep-atitis C virus-positive and 5 with unknown etiology 13malesand 6 females mean age 63plusmn8) by ultrasound-guided biopsy(Table 1) Tissue samples of 18 cases with chronic hepatitis (6with hepatitis B virus-positive and 10 with hepatitis C virus-positive 14 males and 4 females) and 22 with liver cirrhosis(4 with hepatitis B virus-positive and 18 with hepatitis Cvirus-positive 19 males and 3 females) were obtained byultrasound-guided or laparoscopic biopsy All tissue sampleswere fixed in formalin and the tissue sections were subjectedto hematoxylin and eosin staining for histopathologicalevaluation by two pathologists Freshly frozen tissues wereobtained from 12 cases with HCC and 8 with liver cirrhosisand were used for western blot analysis Normal liver tissuesamples were surgically obtained from 5 individuals withoutliver disease Informed consent was obtained from all thehuman subjects included in the study under an InstitutionalReview Board-approved protocol and the study protocolconformed to the ethical guidelines of the 1975 Declarationof Helsinki as reflected in a priori approval by the institutionrsquoshuman research committee

22 Immunohistochemical Analysis Tissue sections weredeparaffinized in xylene rehydrated in alcohol and quenchedin 3hydrogen peroxide withmethanol to block endogenousperoxidase activity Slides were heated in a microwave in

Table 1 Clinical characteristics of the patient groups of DN andHCC

Clinical variables DN(119899 = 19)

HCC(119899 = 58) 119875 valuelowast

Mean age (year) 63 plusmn 8 62 plusmn 9 0792Gender

Male 13 49female 6 9

0117

HBs Ag+ 2 7minus 17 51

0610

Anti-HCV+ 12 35minus 7 23

0525

DN dysplastic nodule HCC hepatocellular carcinomalowast119875 value of independent Studentrsquos t-test for continuous data and Chi2 test forcategorical data

10mmsodiumcitrate (pH65) for antigen retrieval Immuno-histochemical reactions were performed by immersing tissuesections in 5 normal goat serum for 60 minutes andincubating them at 4∘C overnight with mouse anti-phospho-histone H2AX monoclonal antibody (Ser139) clone JBW301(Upstate Biotech Charlottesville VA) at a dilution of 1 500in blocking buffer As a negative control control mouseimmunoglobulin G (Dako Cytomation Glostrup Denmark)was used instead of the primary antibody After the sectionswere rinsed a secondary antibody from the VectastainElite ABC Kit (Vector Laboratories Burlingame CA) wasapplied and color development was performed using 33-diaminobenzidine (Sigma Chemical Co St Louis MO)Counterstain was provided by staining with hematoxylinLabeling indices (LIs) for 120574-H2AX were determined as thenumber of positive nuclei in 100 hepatocytes or tumor cellsin 3 randomly selected fields In HCC cases the patients weredivided into two groups according to the levels of 120574-H2AXLIas low expressors (LI lt50) and high expressors (LI gt50)

23 Immunofluorescence Staining For immunofluorescenceanalysis appropriate tissue slides were incubated in 100mmglycine for 15 minutes three times to reduce fluorescentbackground Slides were reacted with the same primaryantibody as used for immunohistochemistry and washedin tris-buffered saline containing 005 Tween-20 3 timesfor 5 minutes to reduce background They were incubatedfor 30 minutes with Alexa Fluor 488 goat anti-mouse IgG(H + L) (Molecular Probes Eugene OR) in the dark andmounted with 02120583gmL 41015840-6-diamidino-2-phenylindole(DAPI) Immunofluorescence imageswere visualized by fluo-rescence microscope (BZ-9000 Keyence Osaka Japan)

24Western Blotting Liver tissueswere homogenized using aTissueRuptor (Qiagen Valencia CA USA) with a buffer con-taining 20mmTris-HCl (pH74) 150mmNaCl 2mmEGTA5mm 120573-glycerophosphate 1mm MgCl

2 1 Triton X-100

The Scientific World Journal 3

120574-H2AX

DAPI

Case 2 Case 7

(a)

120574-H2AX

Case 2 Case 7

120573-actin

H1 H2 T NT N

(b)

Case 2

Case 7Case 1

Case 11

(c)

Figure 1 120574-H2AX is expressed at different levels in HCC (a) Immunofluorescence staining shows that phosphorylated histone H2AX(120574-H2AX) is located in the nuclei of HCC cells (green) (original magnification times100) Case 2 HCC with increased 120574-H2AX expressionCase 7 HCC with sparse expression of 120574-H2AX DAPI (blue) nucleus counterstain (b) Representative data of western blotting for 120574-H2AX in liver tissues H1 and H2 healthy livers Cases 2 and 7 HCC cases T tumor tissues N adjacent nontumorous liver tissues (c)Immunohistochemical staining of 120574-H2AX Cases 1 and 7 HCC cases with negative to low expression of 120574-H2AX Cases 2 and 11 HCCs withhigh expression of 120574-H2AX (original magnification times40) Arrows indicate positive staining in the nuclei

1mm sodium orthovanadate 10 120583gmL protease inhibitors1 120583gmL aprotinin 1120583gmL leupeptin and 1 120583gmL pepstatinLysates were cleared by centrifugation and the supernatantscontaining 20 120583g of protein were electrophoresed on 5ndash20SDS-polyacrylamide gels After samples were blotted ontoHybond-P membranes (GE Healthcare Milwaukee WI)membranes were incubated with rabbit anti-120574-H2AX poly-clonal antibody (Bethyl Lab Inc Montgomery TX) Proteinblots were visualized using an enhanced ECL Western blot-ting detection system (GEHealthcare) and equal amounts ofthe protein loading were confirmed by reprobing with anti-120573-actin antibody (Sigma Chemical Co)

25 Statistical Analysis Data were analyzed using SPSSsoftware (Statistical Product and Service Solutions 115 for

Windows SPSS Inc Chicago IL) Chi-square test was usedfor examining the association between the status of 120574-H2AXand clinic-pathologic features in HCC When appropriatea Mann-Whitney U-test or independent Studentrsquos t-test wasused to test for statistical differences between the groups

3 Results

31 120574-H2AX Expression Is Increased in HCC Immunoflu-orescence analysis demonstrated that 120574-H2AX appearedas diffuse and discrete foci in the nuclei of HCC cells(Figure 1(a)) The results of western blotting correlated withthe immunofluorescence staining (Figure 1(b)) confirm-ing the potential reliability for detecting 120574-H2AX in theHCC samples Immunohistochemical analysis showed that


CH LC DN

(a)

120574-H

2AX

labe

ling

inde

x (

)

Normal

119886lowastlowast119886lowastlowast

119886lowastlowast

119886lowastlowast

119887lowastlowast 119887lowastlowast

(119899 = 5) (119899 = 18) (119899 = 22) (119899 = 19) (119899 = 58)CH LC DN HCC

100

80

60

40

20

0

(b)

Figure 2 120574-H2AX is significantly increased in preneoplastic lesions in the liver (a) Representative images of immunostaining for 120574-H2AXin non-HCC tissues CH chronic hepatitis LC liver cirrhosis DN dysplastic nodule (original magnification times40) (b) Dot plots showing the120574-H2AX labeling index in normal livers (119899 = 5) chronic hepatitis (CH 119899 = 18) liver cirrhosis (LC 119899 = 22) dysplastic nodule (DN 119899 = 19)and HCC (119899 = 58) Horizontal bars depict the mean value and vertical bars indicate the standard deviation 119886lowastlowast 119875 value of lt001 versusnormal livers 119887lowastlowast 119875 value of lt001 versus dysplastic nodule

the mean value of LI for 120574-H2AX in HCC was 562 plusmn 314(range from 30 to 951) which was significantly increasedcompared with normal livers LI 10 plusmn 06 Fifty-eight HCCpatients were categorized into two groups 40 (690) charac-terized by significantly increased levels of 120574-H2AX expression(high expressors LI gt50) and 18 (319) with very low tonegative expression (low expressors LI lt50) (Figure 1(c))120574-H2AX expression levels showed no positive correlationwith clinical features in HCC patients (Table 2) howeverthere was an inverse relationship between the histologicalgrade of the tumors (119875 = 0011) (Table 2)

32 120574-H2AX Is Increased in Chronic Liver Diseases and Dys-plastic Nodules Immunohistochemical analysis showed thatthe LI of 120574-H2AX in chronic hepatitis and liver cirrhosis wasincreased compared with normal livers (chronic hepatitis275 plusmn 158 range from 50 to 593 119875 lt 0005 liver cir-rhosis 562 plusmn 314 range from 72 to 630 119875 lt 0005resp) Intriguingly dysplastic nodules showed a significantlyincreased 120574-H2AX expression (741 plusmn 221 range from 201

to 940) which was significantly increased compared withthose in liver cirrhosis (119875 lt 0005) and HCC (119875 lt 0005)(Figures 2(a) and 2(b))

33 120574-H2AX Is Increased in Adjacent Nontumorous LiverTissues from HCC Patients To investigate the clinical sig-nificance of 120574-H2AX we determined whether fundamentaldifferences existed between the nontumorous liver tissueswith and without the coexistence of HCC Western blottingdetected 120574-H2AX in 3 (375) of 8 liver cirrhosis patientswith no evident HCC occurrence while 9 (75) of 12 HCCcases showed a detectable protein band for 120574-H2AX in theadjacent nontumorous liver tissues (Figure 3(a)) Immuno-histochemical analysis showed that the mean LI of 120574-H2AXin nontumorous liver tissues obtained from HCC patientswith chronic hepatitis was relatively but not statisticallyincreased as compared with that obtained from individualswith chronic hepatitis withoutHCCoccurrence (357plusmn172range from 80 to 655 119875 = 011) Moreover the mean LIof 120574-H2AX in nontumorous tissues from HCC patients with


HCC

Cases L3 L5 L12 H3 H5 H12

minus +

120574-H2AX

120573-actin

(a)

HCC minus +

(b)

minus minus ++

120574-H

2AX

labe

ling

inde

x (

)

NS

lowastlowast

100

80

60

40

20

0

(119899 = 18) (119899 = 28) (119899 = 22) (119899 = 30)LCCH

HCC

(c)

Figure 3 120574-H2AX is increased in the adjacent nontumorous liver tissues of HCC patients (a) Representative data of western blotting for120574-H2AX L3 5 and 12 cases with liver cirrhosis without the coexistence of HCC H3 5 and 12 adjacent nontumorous liver tissues obtainedfromHCC patients (b) Representative images of 120574-H2AX immunostaining in liver tissues with and without the coexistence of HCC (originalmagnification times40) (c) Dot plots showing the 120574-H2AX labeling index CH chronic hepatitis without the coexistence of HCC (119899 = 18) andwith HCC (119899 = 28) LC liver cirrhosis without HCC (119899 = 22) and with HCC (119899 = 30) NS not significant lowastlowast119875 lt 001

liver cirrhosis was significantly increased when comparedwith the cases with liver cirrhosis withoutHCC (625plusmn247range from 51 to 960 119875 lt 0005) (Figure 3(c))

4 Discussion

Over the last few decades alpha-fetoprotein (AFP) normallyproduced by the fetal liver and yolk sac in pregnant indi-viduals has commonly been used as a clinically availablebiomarker for HCC Unfortunately serum levels of AFP donot correlate well with the risk of the development of HCCand reliable biomarkers have been long awaited for improvingthe poor prognosis of HCC As DNA double-strand breaksaccumulate during long periods of chronic inflammationinvestigating the molecules involved in the DNA repairsystem for use as biomarkers for the risk ofHCCdevelopmentwould be of value

During the period of chronic inflammation intracellularreactive oxygen species (ROS) are increased and have a strongcapability to induce oxidative DNA damage [20] In this

setting the highly conserved phosphatidylinositol-3-relatedkinases ATM and ATR play critical roles in regulating thecell cycle checkpoints and DNA repair [21] Of note both ofthese kinases have a strong capability to increase the level ofphosphorylated histone H2AX (120574-H2AX) which immedi-ately traffics to the damaged sites ofDNA 120574-H2AXplays a keyrole in theDNA repair process because it recruitsmany othermolecules involved in the DNA repair [17 22] Intriguingly120574-H2AX is now regarded as a useful biomarker for assessingradio sensitivity of cancer cells after treatment [23] andmorerecently it was reported that this unique molecule might beincreased in preneoplastic lesions such as colon adenoma [24]and dysplastic lesions in the bronchial epithelium [25]

There have been few reports of 120574-H2AX during hepato-carcinogenesis Kim et al [26] reported that 120574-H2AX fociwere significantly increased in HBV-related liver cirrhosisand HBV-related HCC compared with normal hepatocytesbut there have been no following reports and no such studiesto investigate the clinical significance of 120574-H2AX in individ-uals with a risk of HCC In this study we found that 120574-H2AX


Table 2 Associations of 120574-H2AX with clinicopathological featuresin HCC

Clinicopathologicalvariables

120574-H2AX immunoreactivityLow

(119899 = 18)High

(119899 = 40) 119875 valuelowast

Age (year)lt50 2 4ge50 16 36 0613

Gendermale 15 34female 3 6 0576

Tumor size (cm)lt3 5 11ge3 13 29 0609

Intrahepatic metastasisminus 14 30+ 4 10 0550

Venous invasionminus 16 37+ 2 3 0497

Histological gradedagger

III 10 35IIIIV 8 5 0011lowast

daggerHistological grade was assessed according to the Edmondson-Steiner gradelowast119875 value of Chi2 test

was sequentially increased from normal to chronic hepatitisand liver cirrhosis Importantly dysplastic nodule showeda significantly high level of 120574-H2AX LI which was increasedcompared with HCC (741 plusmn 221 versus 562 plusmn 314 119875 lt0005) Together with our data suggesting that histologicalgrades of HCC were inversely correlated with the level ofLI 120574-H2AX might play a critical role in the developmentof HCC especially during the early stages of carcinogenesisFurthermore the levels of LI increased in CC-coexistingtissues with liver cirrhosis than those without tumors (625 plusmn247 versus 562 plusmn 314 119875 lt 0005) Together 120574-H2AXmay be a useful biomarker for predicting individuals with ahigh risk of HCC

Until recently there have been no useful biomarkers forpredicting the potential of HCC development Because 120574-H2AX foci can be representatively detected using standardpathological techniques this may be a promising and stan-dard biomarker for HCC surveillance To confirm the clinicalusefulness of this DNAdamage sensor repeated liver biopsiesand a long followup study of individuals with chronic liverdiseases would be required

Conflict of Interests

None of the authors has any financial or other interest withregard to the submitted paper that might be construed as aconflict of interests

Acknowledgment

This work was supported by a Grant-in-Aid for ScientificResearch (no 24590962) from the Ministry of EducationCulture Sports Science and Technology of Japan

References

[1] J M Llovet S Ricci V Mazzaferro et al ldquoSorafenib inadvanced hepatocellular carcinomardquo The New England Journalof Medicine vol 359 pp 378ndash390 2008

[2] P A Farazi and R A DePinho ldquoHepatocellular carcinomapathogenesis from genes to environmentrdquo Nature ReviewsCancer vol 6 no 9 pp 674ndash687 2006

[3] N Ince and J R Wands ldquoThe increasing incidence of hepato-cellular carcinomardquo The New England Journal of Medicine vol340 no 10 pp 798ndash799 1999

[4] Y Matsuda T Ichida and M Fukumoto ldquoHepatocellular car-cinoma and liver transplantation clinical perspective onmolec-ular targeted strategiesrdquoMedicalMolecularMorphology vol 44pp 117ndash124 2011

[5] C Della Corte and M Colombo ldquoSurveillance for hepatocellu-lar carcinomardquo Seminars in Oncology vol 39 pp 384ndash398 2012

[6] I J Groisman R Koshy F Henkler J D Groopman and MA Alaoui-Jamali ldquoDownregulation of DNA excision repair bythe hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cellsrdquo Carcinogenesis vol 20 no 3 pp 479ndash483 1999

[7] F Zhao N B Hou X L Yang et al ldquoAtaxia telangiectasia-mutated-Rad3-relatedDNAdamage checkpoint signaling path-way triggered by hepatitis B virus infectionrdquo World Journal ofGastroenterology vol 14 no 40 pp 6163ndash6170 2008

[8] C M Kim K Koike I Saito T Miyamura and G Jay ldquoHBxgene of hepatitis B virus induces liver cancer in transgenicmicerdquo Nature vol 351 no 6324 pp 317ndash320 1991

[9] Y Matsuda and T Ichida ldquoImpact of hepatitis B virus X proteinon the DNA damage response during hepatocarcinogenesisrdquoMedical Molecular Morphology vol 42 no 3 pp 138ndash142 2009

[10] W H Wang R L Hullinger and O M Andrisani ldquoHepatitis Bvirus X protein via the p38MAPK pathway induces E2F1 releaseand ATR kinase activation mediating p53 apoptosisrdquo Journal ofBiological Chemistry vol 283 no 37 pp 25455ndash25467 2008

[11] S Prost J M Ford C Taylor J Doig and D J HarrisonldquoHepatitis B x protein inhibits p53-dependent DNA repair inprimary mouse hepatocytesrdquo Journal of Biological Chemistryvol 273 no 50 pp 33327ndash33332 1998

[12] L Jia XWWang and C C Harris ldquoHepatitis B virus x proteininhibits nucleotide excision repairrdquo International Journal ofCancer vol 80 no 6 pp 875ndash879 1999

[13] C K Lai K S Jeng K Machida Y S Cheng andM M C LaildquoHepatitis C virus NS34A protein interacts with ATM impairsDNA repair and enhances sensitivity to ionizing radiationrdquoVirology vol 370 no 2 pp 295ndash309 2008

[14] K Machida G Mcnamara K T H Cheng et al ldquoHepatitisC virus inhibits DNA damage repair through reactive oxygenand nitrogen species and by interfering with the ATM-NBS1Mre11Rad50 DNA repair pathway in monocytes and hepato-cytesrdquo Journal of Immunology vol 185 no 11 pp 6985ndash69982010

[15] K Matsumoto Y Satoh H Sugo et al ldquoImmunohistochemicalstudy of the relationship between 8-hydroxy-21015840-deoxyguano-sine levels in noncancerous region and postoperative recur-rence of hepatocellular carcinoma in remnant liverrdquoHepatologyResearch vol 25 no 4 pp 435ndash441 2003


[16] H TanakaN Fujita R Sugimoto et al ldquoHepatic oxidativeDNAdamage is associated with increased risk for hepatocellularcarcinoma in chronic hepatitis Crdquo British Journal of Cancer vol98 no 3 pp 580ndash586 2008

[17] L J Kuo and L X Yang ldquo120574-H2AX- a novel biomaker for DNAdouble-strand breaksrdquo In Vivo vol 22 no 3 pp 305ndash310 2008

[18] N F Lowndes and G W L Toh ldquoDNA repair the importanceof phosphorylating histone H2AXrdquo Current Biology vol 15 no3 pp R99ndashR102 2005

[19] Liver Cancer Study Group of Japan General Rules for the Clin-ical and Pathological Study of Primary Liver Cancer KaneharaTokyo Japan 5th edition 2008

[20] C Bertram and R Hass ldquoCellular responses to reactive oxygenspecies-induced DNA damage and agingrdquo Biological Chemistryvol 389 no 3 pp 211ndash220 2008

[21] A Poehlmann and A Roessner ldquoImportance of DNA damagecheckpoints in the pathogenesis of human cancersrdquo PathologyResearch and Practice vol 206 no 9 pp 591ndash601 2010

[22] J Kobayashi ldquoMolecular mechanism of the recruitmentof NBS1hMRE11hRAD50 complex to DNA double-strandbreaks NBS1 binds to 120574-H2AX through FHABRCT domainrdquoJournal of Radiation Research vol 45 no 4 pp 473ndash478 2004

[23] D Klokov S M MacPhail J P Banath J P Byrne and P LOlive ldquoPhosphorylated histoneH2AX in relation to cell survivalin tumor cells and xenografts exposed to single and fractionateddoses of X-raysrdquo Radiotherapy and Oncology vol 80 no 2 pp223ndash229 2006

[24] J Bartkova ZHorejsı K Koed et al ldquoDNAdamage response asa candidate anti-cancer barrier in early human tumorigenesisrdquoNature vol 434 no 7035 pp 864ndash870 2005

[25] V G Gorgoulis L V F Vassiliou P Karakaidos et al ldquoActi-vation of the DNA damage checkpoint and genomic instabilityin human precancerous lesionsrdquo Nature vol 434 no 7035 pp907ndash913 2005

[26] H Kim B K Oh M Roncalli et al ldquoLarge liver cell change inhepatitis B virus-related liver cirrhosisrdquoHepatology vol 50 no3 pp 752ndash762 2009

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the Mre11NBS1Rad50 complex which causes the ATM-mediated DNA repair system to be markedly impaired inHCV-infected cells [14] Together these lines of evidencestrongly suggest that DNA damage response machinery issignificantly involved in hepatocarcinogenesis and might beused as biomarkers for predicting the risk of HCC develop-ment

Recently several studies reported that the level of oxida-tive DNA damage is a good biomarker For example hepatic8-oxo-21015840-deoxyguanosine (8-OHdG) an oxidized derivativeof deoxyguanosine which reflects oxidative stress was closelycorrelated with the risk of HCC recurrence after surgery [1516] To search for more sensitive and reliable biomarkers ofDNA damage we investigated the levels of 120574-H2AX in HCCtissues which mark the site of DNA double-strand breaksand evoke theDNA repair system [17 18] To address whether120574-H2AX might be a good indicator for the risk of HCCdevelopment we also examined and compared the level of120574-H2AX in nontumorous chronic liver diseases

2 Materials and Methods

21 Patients The pathological diagnoses and analyses of dys-plastic nodule and HCC were made according to the GeneralRules for the Clinical and Pathological Study of PrimaryLiver Cancer [19] HCC tissue samples were obtained from58 patients (7 cases with hepatitis B virus-positive 35 withhepatitis C virus-positive and 16 with unknown etiology 9females 49 males mean age 62 plusmn 9) that underwent hepaticresection at Niigata University Medical and Dental HospitalTissue samples of dysplastic nodules were obtained from 19patients (2 cases with hepatitis B virus-positive 12 with hep-atitis C virus-positive and 5 with unknown etiology 13malesand 6 females mean age 63plusmn8) by ultrasound-guided biopsy(Table 1) Tissue samples of 18 cases with chronic hepatitis (6with hepatitis B virus-positive and 10 with hepatitis C virus-positive 14 males and 4 females) and 22 with liver cirrhosis(4 with hepatitis B virus-positive and 18 with hepatitis Cvirus-positive 19 males and 3 females) were obtained byultrasound-guided or laparoscopic biopsy All tissue sampleswere fixed in formalin and the tissue sections were subjectedto hematoxylin and eosin staining for histopathologicalevaluation by two pathologists Freshly frozen tissues wereobtained from 12 cases with HCC and 8 with liver cirrhosisand were used for western blot analysis Normal liver tissuesamples were surgically obtained from 5 individuals withoutliver disease Informed consent was obtained from all thehuman subjects included in the study under an InstitutionalReview Board-approved protocol and the study protocolconformed to the ethical guidelines of the 1975 Declarationof Helsinki as reflected in a priori approval by the institutionrsquoshuman research committee

22 Immunohistochemical Analysis Tissue sections weredeparaffinized in xylene rehydrated in alcohol and quenchedin 3hydrogen peroxide withmethanol to block endogenousperoxidase activity Slides were heated in a microwave in

Table 1 Clinical characteristics of the patient groups of DN andHCC

Clinical variables DN(119899 = 19)

HCC(119899 = 58) 119875 valuelowast

Mean age (year) 63 plusmn 8 62 plusmn 9 0792Gender

Male 13 49female 6 9

0117

HBs Ag+ 2 7minus 17 51

0610

Anti-HCV+ 12 35minus 7 23

0525

DN dysplastic nodule HCC hepatocellular carcinomalowast119875 value of independent Studentrsquos t-test for continuous data and Chi2 test forcategorical data

10mmsodiumcitrate (pH65) for antigen retrieval Immuno-histochemical reactions were performed by immersing tissuesections in 5 normal goat serum for 60 minutes andincubating them at 4∘C overnight with mouse anti-phospho-histone H2AX monoclonal antibody (Ser139) clone JBW301(Upstate Biotech Charlottesville VA) at a dilution of 1 500in blocking buffer As a negative control control mouseimmunoglobulin G (Dako Cytomation Glostrup Denmark)was used instead of the primary antibody After the sectionswere rinsed a secondary antibody from the VectastainElite ABC Kit (Vector Laboratories Burlingame CA) wasapplied and color development was performed using 33-diaminobenzidine (Sigma Chemical Co St Louis MO)Counterstain was provided by staining with hematoxylinLabeling indices (LIs) for 120574-H2AX were determined as thenumber of positive nuclei in 100 hepatocytes or tumor cellsin 3 randomly selected fields In HCC cases the patients weredivided into two groups according to the levels of 120574-H2AXLIas low expressors (LI lt50) and high expressors (LI gt50)

23 Immunofluorescence Staining For immunofluorescenceanalysis appropriate tissue slides were incubated in 100mmglycine for 15 minutes three times to reduce fluorescentbackground Slides were reacted with the same primaryantibody as used for immunohistochemistry and washedin tris-buffered saline containing 005 Tween-20 3 timesfor 5 minutes to reduce background They were incubatedfor 30 minutes with Alexa Fluor 488 goat anti-mouse IgG(H + L) (Molecular Probes Eugene OR) in the dark andmounted with 02120583gmL 41015840-6-diamidino-2-phenylindole(DAPI) Immunofluorescence imageswere visualized by fluo-rescence microscope (BZ-9000 Keyence Osaka Japan)

24Western Blotting Liver tissueswere homogenized using aTissueRuptor (Qiagen Valencia CA USA) with a buffer con-taining 20mmTris-HCl (pH74) 150mmNaCl 2mmEGTA5mm 120573-glycerophosphate 1mm MgCl

2 1 Triton X-100


120574-H2AX

DAPI

Case 2 Case 7

(a)

120574-H2AX

Case 2 Case 7

120573-actin

H1 H2 T NT N

(b)

Case 2

Case 7Case 1

Case 11

(c)





3 Results



CH LC DN

(a)

120574-H

2AX

labe

ling

inde

x (

)

Normal


119886lowastlowast

119886lowastlowast


(119899 = 5) (119899 = 18) (119899 = 22) (119899 = 19) (119899 = 58)CH LC DN HCC

100

80

60

40

20

0

(b)







HCC


minus +

120574-H2AX

120573-actin

(a)

HCC minus +

(b)

minus minus ++

120574-H

2AX

labe

ling

inde

x (

)

NS

lowastlowast

100

80

60

40

20

0

(119899 = 18) (119899 = 28) (119899 = 22) (119899 = 30)LCCH

HCC

(c)



4 Discussion









(119899 = 18)High

(119899 = 40) 119875 valuelowast

Age (year)lt50 2 4ge50 16 36 0613












Acknowledgment


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120574-H2AX

DAPI

Case 2 Case 7

(a)

120574-H2AX

Case 2 Case 7

120573-actin

H1 H2 T NT N

(b)

Case 2

Case 7Case 1

Case 11

(c)





3 Results



CH LC DN

(a)

120574-H

2AX

labe

ling

inde

x (

)

Normal


119886lowastlowast

119886lowastlowast


(119899 = 5) (119899 = 18) (119899 = 22) (119899 = 19) (119899 = 58)CH LC DN HCC

100

80

60

40

20

0

(b)







HCC


minus +

120574-H2AX

120573-actin

(a)

HCC minus +

(b)

minus minus ++

120574-H

2AX

labe

ling

inde

x (

)

NS

lowastlowast

100

80

60

40

20

0

(119899 = 18) (119899 = 28) (119899 = 22) (119899 = 30)LCCH

HCC

(c)



4 Discussion









(119899 = 18)High

(119899 = 40) 119875 valuelowast

Age (year)lt50 2 4ge50 16 36 0613












Acknowledgment


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CH LC DN

(a)

120574-H

2AX

labe

ling

inde

x (

)

Normal


119886lowastlowast

119886lowastlowast


(119899 = 5) (119899 = 18) (119899 = 22) (119899 = 19) (119899 = 58)CH LC DN HCC

100

80

60

40

20

0

(b)







HCC


minus +

120574-H2AX

120573-actin

(a)

HCC minus +

(b)

minus minus ++

120574-H

2AX

labe

ling

inde

x (

)

NS

lowastlowast

100

80

60

40

20

0

(119899 = 18) (119899 = 28) (119899 = 22) (119899 = 30)LCCH

HCC

(c)



4 Discussion









(119899 = 18)High

(119899 = 40) 119875 valuelowast

Age (year)lt50 2 4ge50 16 36 0613












Acknowledgment


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HCC


minus +

120574-H2AX

120573-actin

(a)

HCC minus +

(b)

minus minus ++

120574-H

2AX

labe

ling

inde

x (

)

NS

lowastlowast

100

80

60

40

20

0

(119899 = 18) (119899 = 28) (119899 = 22) (119899 = 30)LCCH

HCC

(c)



4 Discussion









(119899 = 18)High

(119899 = 40) 119875 valuelowast

Age (year)lt50 2 4ge50 16 36 0613












Acknowledgment


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















(119899 = 18)High

(119899 = 40) 119875 valuelowast

Age (year)lt50 2 4ge50 16 36 0613












Acknowledgment


References

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Clinical Study DNA Damage Sensor -H2AX Is Increased in …downloads.hindawi.com/journals/tswj/2013/597095.pdf · 2019. 7. 31. · e Scientic World Journal -H2AX DAPI Case 2 Case 7