Nucleotide Excision Repair Factor XPC Enhances DNA Damage ... · XPC is a 940-amino acid protein and harbors domains that can bind to damaged DNA and repair factors (10–12). The

Molecular and Cellular Pathobiology

Nucleotide Excision Repair Factor XPC Enhances DNADamage–Induced Apoptosis by Downregulating theAntiapoptotic Short Isoform of Caspase-2

Qi-En Wang1, Chunhua Han1, Bo Zhang1, Kanaga Sabapathy4, and Altaf A. Wani1,2,3

AbstractXPC protein is a critical DNA damage recognition factor in nucleotide excision repair for which genetic

deficiency confers a predisposition to cancer. In this study, we show that XPChas a function that is independent ofits canonical function in DNA repair, potentially altering the interpretation of how XPC deficiency leads toheightened cancer susceptibility. XPC enhances apoptosis induced by DNA damage in a p53 nullizygousbackground, acting downstream of mitochondrial permeabilization and upstream of caspase-9 activation inthe DNA damage–induced apoptosis cascade. We found that deficiency in XPC upregulated production of theshort isoform of caspase-2 (casp-2S). This upregulation occurred at both protein and mRNA levels throughrepression of the caspase-2 promoter by XPC protein. Targeted RNAi-mediated downregulation of casp-2S–enhanced UV-induced apoptosis as well as activation of caspase-9 and caspase-6 in XPC-deficient cells, but not inXPC-proficient cells. In addition, XPC overexpression in various p53-deficient cancer cells resistant to cisplatinimproved their sensitivity to cisplatin-induced apoptosis. Given that casp-2S functions as an antiapoptoticprotein, our findings suggest that XPC enhances DNA damage–induced apoptosis through inhibition of casp-2Stranscription. Together, these findings offer a mechanistic foundation to overcome the resistance of highlyprevalent p53-deficient tumors to cell death induced by DNA-damaging therapeutic agents, by targetingstrategies that inhibit the expression or function of casp-2S. Cancer Res; 72(3); 666–75. �2011 AACR.

Introduction

DNA lesions induced by UV light and the chemotherapeuticagent cisplatin are mainly removed by the nucleotide excisionrepair (NER) consisting of 2 subpathways termed transcrip-tion-coupled repair (TCR) and global genomic repair (GGR;ref. 1). TCR is required for the preferential removal of lesionsfrom the transcribed strand of active genes, whereas GGR isresponsible for the removal of DNA lesions from the bulk of thegenome (1). Cell lines derived from individuals suffering fromthe disorders like xeroderma pigmentosum (XP) and Cockaynesyndrome (CS) have defects in one or both of these NER

subpathways. Specifically, XP-C cells have a defect in GGR butnot TCR. In contrast, CS-A and CS-B cells are defective in TCRbut not GGR (2), whereas XP-A cells have defects in both TCRand GGR (1).

TCR-deficientfibroblasts (e.g., CS-A andXP-A) are extremelysensitive to UV- and cisplatin-induced apoptosis (3–5), where-as XP-C cells with selective defect in GGR but with proficientTCR have normal resistance to cisplatin (6, 7). In addition,murine Xpc�/� keratinocytes are characterized by a completeabsence of apoptosis after UV exposure, in contrast to Xpa�/�

and Csb�/� cells (8). These reports suggest that unrepairedDNA damage in the transcribed strand is an indispensabletrigger for UV/cisplatin-induced apoptosis. However, increas-ing evidence has suggested that unrepaired DNA lesions in thenontranscribed strands or transcriptionally inactive genes cancause replication blockage, which also acts as an apoptosis-inducing signal (9). It has been reported that Xpa�/� kerati-nocytes (TCR and GGR deficiency) showed greater apoptosisthan Csb�/� cells (TCR only deficiency) following UV irradi-ation (8). Given that both cells have deficient TCR, the addi-tional GGR deficiency in Xpa�/� cells must be a contributingfactor in UV-induced apoptosis. Although transcription block-age is absent inXP-C cells due to proficient TCR, the replicationblockage induced by deficient GGR is sufficient to triggerapoptosis in these cells. Therefore, the resistance of XP-C cellsto UV/cisplatin-induced apoptosis could not be simply attrib-uted to the proficient TCR. This prompts us to hypothesize thatan important apoptosis element must be lost in XP-C cells.

Authors' Affiliations: Departments of 1Radiology and 2Molecular andCellular Biochemistry, 3Comprehensive Cancer Center, The Ohio StateUniversity, Columbus, Ohio; and 4Division of Cellular & MolecularResearch, Humphrey Oei Institute of Cancer Research, National CancerCentre, Singapore

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

B. Zhang was a visiting scholar from Department of Urology, TangduHospital, The Fourth Military Medical University, Xi'an, China.

Correspondence Authors: Qi-En Wang, Department of Radiology,The Ohio State University, 460 W. 12th Avenue, Room 718, Colum-bus, OH 43210. Phone: 614-292-9021; Fax: 614-292-9102; E-mail:[email protected]; and Altaf A. Wani, Phone: 614-292-9015;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-2774

�2011 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst December 15, 2011; DOI: 10.1158/0008-5472.CAN-11-2774

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XPC is a 940-amino acid protein and harbors domains thatcan bind to damaged DNA and repair factors (10–12). Themajor function of XPC is to recognize helix-distorting lesionslocated in a transcriptionally inactive genome or a nontran-scribed strand of actively transcribed genes (13). Recent stud-ies have suggested that beyond its role in DNA repair, the XPCprotein is also involved in transcriptional process includingboth transcription activation and repression (14, 15). However,the relationship between XPC- and apoptosis-related genetranscription is yet to be established.Caspases are a family of cysteine aspartate–specific pro-

teases involved in the initiation or execution of apoptosis.Caspase-2 (casp-2) is the most conserved caspase acrossspecies and is one of the initiator caspases activated by variousstimuli (reviewed in ref. 16). The casp-2 gene produces severalalternative splicing isoforms. The inclusion of exon 9 leads tothe inclusion of an in-frame stop codon in casp-2 short isoform(casp-2S) mRNA, thus producing a truncated protein thatinhibits cell death, whereas the exclusion of exon 9 results incasp-2 long isoform (casp-2L) mRNA, whose protein productinduces cell death (17, 18).In this study, we have uncovered a novel function of XPC as a

potent enhancer of apoptosis in the absence of any influence byp53. From a mechanistic standpoint, XPC protein downregu-lates antiapoptotic casp-2S through inhibition of its promoteractivity and thus promotes DNA damage–induced activationof casp-9 and casp-6, which ultimately enhances cellular death.

Materials and Methods

Cell culture and treatmentSV40-transformed XP-C (GM15983) and fully corrected XP-

C (GM16248) cells were purchased from NIGMS HumanGenetic Cell Repository (Coriell Institute for MedicalResearch). HCT116(p53�/�) and HCT116(p53þ/þ) colorectalcarcinoma cells were kindly provided by Dr. B. Vogelstein(Johns Hopkins University). 041-TR cells were provided by Dr.G. Stark (Cleveland Clinic Foundation). A2780-CP70 ovariancancer cell line was provided by Dr. P. Modrich (Duke Uni-versity), SKOV3 ovarian cancer cell line was provided byDr. T. C. Hamilton (Fox Chase Cancer Center), H1299 andA549 cell lines were kindly provided by Dr. W. Duan (The OhioState University). Except for the regular testing for mycoplas-ma contamination using a PCR-based assay, the cell lines werenot specifically authenticated. The cultures were maintainedas described in Supplementary Data, and the cells were treatedwith cisplatin or UV as described before (19).

Plasmids and transfectionpXPC3 plasmid containing XPC cDNAand its corresponding

empty vector pEBS7 were kindly provided by Dr. R. Legerski(The University of Texas MD Anderson Cancer Center; ref. 20).XPC shRNA (short hairpin RNA)-containing plasmids (pLKO.1-puro) were purchased from Sigma. p3XFLAG-CMV-14-casp-2Sconstruct was generated in our laboratory (SupplementaryData). Casp-2S promoter-luciferase construct Del4 has beendescribed (21, 22). The plasmids were transfected into cells byeither FuGENE6 (Roche Applied Science) or Lipofectamine

LTX reagents (Invitrogen) according to manufacturer'sinstructions.

Western blot analysisWhole-cell lysates were prepared by boiling cell pellets for

10 minutes in lysis buffer [2% SDS, 10% glycerol, 62 mmol/LTris-HCl, at pH 6.8, and a complete mini-protease inhibitorcocktail (Roche Applied Science)]. For the detection ofcytochrome c (cyto c) release, cells were harvested by tryp-sinization, and the cytosol was separated as described (23).Equal amounts of proteins were loaded, separated on apolyacrylamide gel, and transferred to a nitrocellulose mem-brane. Protein bands were immunodetected with appropriateantibodies (Supplementary Data).

Apoptosis analysis by Annexin V stainingPhosphatidylserine exposure on the outer leaflet of the

plasma membrane was detected by the Annexin V–GFP apo-ptosis detection Kit II (BD Pharmingen) according to themanufacturer's instructions. Briefly, 1� 106 cells were pelletedfollowing treatment and washed in PBS. Cells were thenresuspended in 500 mL of binding buffer, mixed with AnnexinV–GFP and propidium iodide and incubated at room temper-ature (22�C) for 5 to 10 minutes in the dark. The Annexin V–positive cells were analyzed by flow cytometry.

Mitochondria transmembrane potential detectionXP-C and XP-CþXPC cells growing on the coverslips were

either treated with UV or untreated. The cells were furthercultured for 24 hours and stained with MitoCapture reagents(BioVision) according to the manufacture's instruction. Cellswere observed immediately under a Nikon FluorescenceMicroscope E80i (Nikon) fitted with appropriate filters forFITC and Texas Red. The digital images were then capturedwith a cooled CCD camera and processed with the help of itsSPOT software (Diagnostic Instruments).

Transfection with siRNAssiRNA directed against casp-2S (50-GAAUACUACUG-

GUAAACUAUU-30; 50UTR), and a scramble nontargeting siRNA(50-UUCUCCGAACGUGUCACGUdTdT-30), were synthesizedby Dharmacon Inc. siGENOME SMARTpool XPC siRNA waspurchased from Dharmacon. siRNA (100 nmol/L) was trans-fected into cells with Lipofectamine 2000 transfection reagent(Invitrogen) according to the manufacture's instruction.

Real-time PCRReal-time PCR was conducted as described previously

(19). Special primers were designed to only amplify casp-2S or casp-2L, for example, the forward sequence of casp-2Sprimer covers the exon 9, whereas the forward sequence ofcasp-2L primer covers the junction of exon 8 and 10. Thesequence of the primers is as follows: casp-2L: forward50-GCCTGCCGTGGAGATGAGACTGAT-30; casp-2S: forward50-CACCGCCTCTCTTGCTCTAT-30; casp-2L and casp-2S:reverse 50- TTACCGGCATCACTCTCCTC-30; GAPDH: for-ward 50-GAAGGTGAAGGTCGGAGT -30, reverse 50-GAA-GATGGTGATGGGATTTC-30.

XPC Enhances Apoptosis through Reducing Caspase-2S

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Promoter activity assayCells were transiently transfected with casp-2S promoter-

luciferase constructs (Del4). As an internal control, the pGL4.73plasmid (Promega), which carries a Renilla luciferase gene, wasalso cotransfected into the cells to normalize for transfectionefficiency. Luciferase assays were conducted with Dual-Lucif-erase Reporter Assay System (Promega) according to manu-facturer's instruction.

Chromatin immunoprecipitation assayHCT116(p53�/�) cells were fixed with 1% formaldehyde for

10 minutes at room temperature. The chromatin immunopre-cipitation (ChIP) assay was conducted as described previously(24) using 2 mg of either normal rabbit immunoglobulin G orrabbit anti-XPC antibodies. The casp-2S promoter–specificPCR reactionswere carried outwith 4 different pairs of primers(Supplementary Data).

Results

XPC facilitates DNA damage–induced apoptosisBecause our results (Supplementary Data S1A and B and

S2A–D) and others (6, 7) have shown that XPC-deficient cells

have lower UV and cisplatin sensitivity than other NER-deficient cells, we wanted to determine whether XPC-defi-cient cells are also resistant to DNA damage–induced apo-ptosis. A SV40-transformed human XP-C cell line and itsstably XPC-restored counterpart were used to investigatethe role of XPC protein in various DNA-damaging agent–induced apoptosis. As shown in Fig. 1A–D, following treat-ment with diverse DNA-damaging agents, XPC-restored cellsexhibited enhanced apoptosis than parental XPC-deficientcells, as reflected by the characteristic increase of PARPcleavage in these cells. Enhanced UV-induced apoptosis inXPC-restored cells was also confirmed by Annexin V staining(Fig. 1E) and sub-G1 cell analysis (Supplementary Data S3).Furthermore, we transiently transfected XPC cDNA-contain-ing plasmids into XP-C cells and again found the increasedapoptosis upon UV irradiation compared with the emptyvector–transfected XP-C cells (Supplementary Data S4A andB). To further confirm the independent role of XPC inapoptosis, without the potential influence via its canonicalfunction in NER, we knocked down the expression of XPC inNER-deficient XP-A cells and analyzed their apoptosis uponUV irradiation. Our findings indicated that XPC-deficiencycompromised UV-induced apoptosis in XP-A cells which are

Figure 1. XPC facilitates DNA-damaging agent-inducedapoptosis. A–D, XP-C and XP-CþXPC cells were treated withcisplatin (A), etoposide (B), UVradiation (C), or IR (D), and furthercultured for 24 hours (cisplatin:treated for 1 hour, further culturedin drug-free medium for 24 hours.Etoposide: treated for 24 hours).Whole-cell lysates were preparedand the same amounts of proteinswere loaded for Western blotanalysis of cleaved PARP. Tubulinwas detected as loading control. E,XP-C and XP-CþXPC cells wereirradiated with UV and furthercultured for 24 hours. Apoptoticcells were detected with Annexin Vstaining. The percentage ofAnnexin V–positive cells is anaverage of 3 independent repeats.Statistical significance wasdetermined by 2-sample Student ttest. Bars represent SD; �,P < 0.05;��, P < 0.01, compared with XP-Ccells. F and G, XP-A cells weretransfected with either control orXPC siRNA for 48 hours, UVirradiated, and further cultured for24 hours. F, the expression of XPCand the amount of cleaved PARPwere detected byWestern blotting.G, apoptotic cells were detectedwith Annexin V staining. N ¼ 3,bars: SD; �, P < 0.05; ��, P < 0.01,compared with control siRNA-transfected cells.

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Cancer Res; 72(3) February 1, 2012 Cancer Research668




completely deficient in both GGR and TCR (Fig. 1F and G).These observations showed that XPC protein can facilitateDNA damage–induced apoptosis independent of its knownfunction in DNA repair.

XPC-mediated apoptosis occurs without an influence byp53The above-mentioned data were obtained from SV40-

transformed XP-C and XP-A cells, in which the transcrip-tional properties of p53 are suppressed. To further investi-gate the role of an involvement of p53 in the XPC-mediatedapoptosis, we analyzed the effect of XPC deficiency onUV-induced cellular apoptosis in the presence or absenceof p53. XPC shRNA was stably transfected into 041-TR cellscarrying tetracycline (Tet)-regulated p53 and 2 new cell lineswith downregulated XPC were established (Fig. 2A). Thesecell lines were irradiated with UV either in the presence orabsence of Tet, and the apoptosis was analyzed by PARPcleavage and Annexin V staining. As shown in Fig. 2B–E, XPCknockdown compromised UV-induced apoptosis when p53is eliminated by Tet, while it had no significant influence onapoptosis when p53 was present and responded normally toinduction by DNA damage. This finding was also confirmedin HCT116 cells with differing status of p53 (SupplementaryData S5A–D). These data indicated that XPC-mediatedapoptosis is independent of functional p53, and this noveleffect can be obscured by the regular p53-mediatedapoptosis.

XPC functions downstream of mitochondrial events infacilitating apoptosisFor insights into the mechanism by which XPC protein

enhances apoptosis, we focused on the contribution of XPCto the intrinsic apoptotic pathway. We first analyzed cyto crelease from mitochondria following UV irradiation of XPC-deficient and XPC-proficient cells. As shown in Fig. 3A,despite the higher level of apoptosis in XPC-proficient cellsas reflected by increased PARP cleavage, UV irradiationcaused a comparable release of cyto c into cytosol fractionfrom both cell lines. Furthermore, we analyzed the disrup-tion of the mitochondrial transmembrane potential andfound that both XPC-deficient and XPC-proficient cellsdisplayed the similar diffused green fluorescence upon UVirradiation (Fig. 3B), further supporting that XPC proteindoes not influence DNA damage–induced mitochondrialpermeabilization, and indicated that XPC could be playingits role at steps downstream of cyto c release during apo-ptotic cascade. We then determined the activation of variouscaspases functioning in the downstream of cyto c release inboth XP-C and XP-CþXPC cells following UV irradiation. Asshown in Fig. 3C and D, casp-2L, casp-6, and casp-9 activa-tion prominently coincided with the enhanced PARP cleav-age of XPC-proficient cells, whereas casp-3 and casp-7activation was comparable in XP-C and XP-CþXPC cells.This suggested that XPC-mediated apoptosis might beoccurring through the enhanced activation of casp-2L,casp-9, and casp-6. Furthermore, we also found that pan-caspase inhibitor z-VAD blocked, whereas casp-3 inhibitor

z-DEVD had no influence on UV-induced apoptosis in bothXP-C and XP-CþXPC cells, although casp-3 activity washigher in XP-CþXPC cells than XP-C cells upon UV irradi-ation (Supplementary Data S6A–D). This indicates that thefunctional role of casp-3 in UV-induced apoptosis in XP-Ccells is apparently being substituted by other executorcaspases.

Figure 2. XPC-mediated apoptosis is independent of p53. A, 041-TRcells were stably transfected with empty vector or shXPC, theexpression of XPC was detected by Western blotting in the presenceof Tet (p53�/�). B, 041-TR cells stably transfected with empty vectoror shXPC were cultured in the presence of Tet (p53�/�), UV irradiated,and cultured for 24 hours. Cleaved PARP was detected by Westernblotting. C, Tet was withdrawn from the culture of 041-TR cells16 hours prior to UV irradiation (p53þ/þ) and further cultured in theabsence of Tet for 24 hours. Cleaved PARP was detected.D and E, apoptotic cells in (B and C) were detected with AnnexinV staining. N ¼ 3, bars: SD; �, P < 0.05 compared with emptyvector–transfected cells.






XPC inhibits the expression of casp-2S throughrepressing casp-2S promoter activity

Casp-2 exists as 2 distinct isoforms, proapoptotic casp-2Land antiapoptotic casp-2S (17, 18). So, we determined theeffects of XPC on the expression of both casp-2L and casp-2S in cultured cells. As shown in Fig. 4A, in contrast to casp-2L,casp-2S protein was upregulated in XPC-deficient cells, indi-cating that XPC protein inhibits the expression of casp-2S.Real-time quantitative reverse transcriptase (RT)-PCR ana-lysis further indicated that XPC repressed the transcriptionof casp-2S without an influence on casp-2L transcription(Fig. 4B and C). To further confirm the inhibitory effect ofXPC on the expression of casp-2S, we transiently transfectedvarious amounts of XPC cDNA-containing constructs intoXP-C cells, or down-regulated XPC expression by transfectingXPC siRNA into HCT116(p53�/�) cells, and determined theprotein levels of casp-2S and casp-2L. As expected, withthe increase of XPC expression, casp-2S showed decreasedlevel whereas casp-2L level did not change. Similarly, with thedecrease of XPC expression, casp-2S exhibited increased leveland again casp-2L level kept constant (Fig. 4D and E).

It has been reported that casp-2S can be transcribed froman alternate promoter within intron 1 of the casp-2 gene(21, 22). Thus, we used the casp-2S promoter-luciferasereporter construct, described earlier (21, 22), to investigatethe repressive effect of XPC on the casp-2S promoter activity.Our data showed that XPC expression led to a substantialdecrease in the reporter activity from the construct contain-ing casp-2S promoter (Del4; Fig. 4F), indicating that XPC

may reduce the synthesis of casp-2S pre-mRNA. We furtherdetected the influence of downregulation of XPC on theactivity of casp-2S promoter in HCT116 cells. As shownin Fig. 4G, knockdown of XPC increased the casp-2S pro-moter activity in HCT116 cells. To gain insight into themechanism through which XPC regulates casp-2S promoteractivity, we conducted ChIP assay to investigate whetherXPC protein directly binds to the putative casp-2S promoter(–3,006–1,952) site (21). As shown in Fig. 4H, the strong casp-2S promoter (Seq 1–4), but not casp-2 exon-10 PCR product,in anti-XPC antibody-recruited DNA fragment indicated theenrichment of XPC to casp-2S promoter. Taken together,these data showed that XPC protein specifically regulatesthe expression of antiapoptotic casp-2S by directly bindingto casp-2S promoter region and inhibiting its activity.

Enhanced casp-2S is responsible for the resistance ofXPC-deficient cells to apoptosis

Earlier it has been reported that casp-2 is required for stress-induced apoptosis (25). However, that study could not haveseparated the roles of casp-2L and casp-2S because the siRNAused in their experiments actually targeted both casp-2L andcasp-2S (25). By the siRNA targeting of casp-2L and casp-2S(Supplementary Data S7A and B) and casp-2 inhibition (Sup-plementary Data S7C), we failed to find any influence of casp-2downregulation on UV-induced apoptosis in both XP-C andXP-CþXPC cells observed in this study.

To specifically investigate the role of casp-2S in XPC-mediated apoptosis, we designed an siRNA that only targeted

Figure 3. XPC functionsdownstream of mitochondrialevents in facilitating apoptosis. A,cytosol was isolated from both XP-C and XP-CþXPC cells at 24 hoursafter UV irradiation. The presenceof cyto c in cytosol and the cleavedPARP in whole-cell lysates weredetected by Western blotting. B,XP-C and XP-CþXPC cellsgrowing on the coverslips were UVirradiated at 4 J/m2 and furthercultured for 24 hours.Mitochondrial transmembranepotential was detected byMitoCapture probe underfluorescence microscope. Thepercentages of cells with diffusedgreen fluorescence (apoptoticcells) were calculated. C, XP-C andXP-CþXPC cells were UVirradiated at 4 J/m2 and furthercultured for 24 hours. Whole-celllysates were prepared andsubjected to Western blot analysisfor the detection of cleaved PARPand various caspases cleavage. D,quantification of cleavedPARPandvarious caspases at 24 hours afterUV irradiation. N ¼ 3, bars: SD;��, P < 0.01 compared with XP-Ccells.

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casp-2S (Fig. 5A) and analyzed the UV-induced apoptosis inboth XPC-deficient and XPC-proficient cells before and aftercasp-2S siRNA knockdown. The results indicated that down-regulation of casp-2S significantly enhanced the apoptosis inthe deficient XP-C cells but not the restored XP-CþXPC cells

following UV irradiation (Fig. 5B–D). Overexpression of casp-2S in casp-2S siRNA–transfected XP-C cells reduced UV-induced apoptosis to the original level (Fig. 5E and F). Fur-thermore, downregulation of casp-2S simultaneouslyenhanced the activation of casp-9 and casp-6 in XPC-deficient

Figure 4. XPC inhibits the expression of casp-2S at both protein and mRNA levels. A, the expression of casp-2S and casp-2L in XP-C and XP-CþXPC cellswere detected by Western blotting. B and C, the total RNA was isolated from XP-C and XP-CþXPC cells (B) or HCT116(p53�/�) cells transfected with eithercontrol or XPC siRNA (C), the transcript levels of casp-2S and casp-2Lwere determined by quantitative RT-PCR. N¼ 3, bars: SD; �, P < 0.05 compared withXP-Ccells (B); ��,P<0.01comparedwith control siRNA–transfected cells (C). D, XP-Ccellswere transfectedwith different amounts of XPC-containingpXPC3constructs for 48 hours. Whole-cell lysates were prepared and the expression of XPC, casp-2S, casp-2L, and tubulin were analyzed byWestern blotting. Theintensity of eachbandwasscanned; the relative amountsof casp-2 normalized to tubulinwereplotted. E,HCT116(p53�/�) cellswere transfectedwith differentamounts of XPCsiRNA for 48hours. Casp-2Sandcasp-2Lweredetectedasdescribed in (D). F, casp-2Spromoter-luciferase construct (Del4)was transfectedtogether with pGL4.73 plasmid into XP-C and XP-CþXPC cells for 48 hours. The cultures were lysed and subjected to luciferase assays. N ¼ 3, bars:SD; ��, P < 0.01 compared with XP-C cells. G, HCT116(p53�/�) cells were transfected with control or XPC siRNA for 24 hours. Casp-2S promoter activitywas detected as (F).N¼ 3, bars: SD; ��,P < 0.01 comparedwith cells transfectedwith control siRNA. H, ChIP analysis was conductedwith anti-XPC antibodyand normal rabbit IgG in HCT116(p53�/�) cells. Four promoter sequences on the casp-2S promoter were analyzed by PCR.






cells, but not in XPC-proficient cells (Fig. 5C and D). Notably,wewere unable to observe any influence of casp-2S knockdownon the casp-2L activation. The above results showed that XPCenhances apoptosis through downregulation of antiapoptoticcasp-2 short isoform, which is an inhibitor of casp-9 and casp-6activation.

XPC overexpression enhances cisplatin-inducedapoptosis in various cancer cell lines

To determine whether the level of XPC affects chemo-therapeutic agent–induced apoptosis in human cancercells, we overexpressed XPC in various human cancer celllines of different p53 status and analyzed cisplatin-induced apoptosis. As shown in Fig. 6, XPC overexpressionenhanced the cisplatin-induced apoptosis in p53-deficienthuman ovarian carcinoma cells SKOV3 and human non–small cell lung carcinoma cells H1299, as well as p53heterozygous ovarian carcinoma cells A2780/CP70. Howev-er, XPC overexpression did not exhibit augmented apopto-sis in p53-proficient A549 cells upon cisplatin treatment.These data indicated that elevation of XPC level in p53-deficient cancer cells can overcome their resistance tocisplatin.

Figure 5. Casp-2S is responsible forthe resistance to apoptosis in XP-Ccells. A, XP-C and XP-CþXPCcellswere transfectedwith either controlor casp-2S siRNA for 48 hours, theexpression of casp-2S and casp-2L were detected by Westernblotting. B, XP-C and XP-CþXPCcells were transfected with eithercontrol or casp-2S siRNA for 48hours, UV irradiated at 5 J/m2, andfurther cultured for 24 hours.Apoptotic cells were detected withAnnexin V staining by flowcytometry. N ¼ 3, bars: SD; ��, P <0.01 compared with siControl-transfected cells. C, Whole-celllysates from cells in (B) wereprepared, and the cleavages ofPARP and various caspases weredetected by Western blot. D,quantificationof cleavedPARPandcleaved caspases after UVirradiation in (C).N¼3, bars: SD; ��,P < 0.01 compared with siControl-transfected cells. E, XP-C cellswere transfected with either casp-2S siRNA, or casp-2S siRNAtogether with casp-2S cDNA for 48hours, the expression of casp-2Swas detected by Western blottingwith anti–casp-2S antibody. Thearrow indicates the casp-2S band,the upper band in the third lane is avariant of casp-2S (21). F, cells in(E) were UV irradiated and furthercultured for 24 hours. The amountof cleaved PARP and tubulin wasdetected by Western blotting.

Figure 6. XPC overexpression enhances cisplatin-induced apoptosis invarious cancer cell lines. SKOV3, A2780/CP70, H1299, and A549 cellswere transfected with either empty vector (pEBS7)- or XPC cDNA–containing vector (pXPC3) for 24 hours, treated with cisplatin for 1 hour,and further cultured in drug-freemedium for another 48 hours.Whole-celllysates were prepared and subjected to Western blot analysis for thedetection of XPC and cleaved PARP.

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Discussion

XPC deficiency has been associated with cancer predispo-sition in both human and animal models (26–30). DeficientNER is established as the main contributor to the increasingcancer rates. Here, we have provided evidence showing thatXPC protein can also enhance DNA damage–induced apopto-sis. Thus, the loss of XPC-mediated apoptotic pathway couldalso be a contributory factor in the cancer susceptibility ofXPC-deficient individuals.The resistance of XPC-deficient cells to apoptosis was

initially attributed to the proficient TCR in these cells (6, 7).The intact TCR pathway removes UV- or cisplatin-inducedDNA lesions from the transcribed strand of the active genes,thus eliminates the apoptosis triggers. However, our datashow that XPC-deficient cells are not only resistant toapoptosis induced by UV and cisplatin treatment, whichinduce DNA lesions repaired by NER, but also resistant toapoptosis induced by ionizing radiation (IR) and etoposide,which induce double strand breaks (DSB). Given the puta-tive role of XPC in the repair of DSBs (31), XPC-deficientcells should have more DNA lesions, or in other wordsapoptosis triggers, than XPC-proficient cell. Therefore, thecompromised apoptosis, in response to IR and etoposide inXPC-deficient cells, could not be attributed to diminishedapoptosis signals. This indicates that XPC protein mayparticipate in apoptosis process through a novel functionbeyond its canonical role in DNA repair. Furthermore, ourfinding that downregulation of XPC in XP-A cells compro-mised UV-induced apoptosis further reinforced this notion.Because XP-A cell line is deficient in both GGR and TCR, theknockdown of XPC expression does not further reduceits NER efficiency, and the DNA lesions in the entiregenome including transcribed and nontranscribed strandsare comparable between XPC-knockdown and XPC-proficient XP-A cells. Thus, the reduced apoptosis in XP-A cells transfected with XPC siRNA can only be attributedto the loss of XPC protein and indicates that XPC partici-pates in DNA damage–induced apoptosis independentof NER.It was previously reported that XPC protein plays an

essential role in cisplatin-induced apoptosis, perhapsthrough the activation of p53- and p73-dependent apoptoticpathways (32). Here, we have identified a new pathway inwhich XPC is shown to enhance DNA damage–inducedapoptosis in the absence of functional p53. More interest-ingly, XPC seems to enhance apoptosis only when p53function is deficient, indicating that XPC is redundant incisplatin- or UV-induced apoptosis in the presence of func-tional p53, probably because the dominant p53-driven apo-ptosis obscures the underlying contribution of the XPCprotein. Therefore, the role of this XPC-casp-2S pathway,similar to the role of p73 pathway (33), is not critical toapoptosis in the cells with wild-type p53. However, consid-ering the high p53 mutation prevalence among humantumors, induction of XPC-mediated apoptosis pathwayoffers a unique strategy for sensitizing p53-deficient tumorsto cancer therapy.

Although we were unable to alter UV-induced apoptosisby casp-2 siRNA that targets both casp-2L and casp-2S, weclearly showed that specific knockdown of casp-2S over-comes the resistance of XPC-deficient cells to apoptosis. Asindicated above, casp-2 exists as 2 distinct isoforms withopposing functions: casp-2L induces while casp-2S inhibitscell death upon overexpression (17, 18). Because both iso-forms were knocked out or downregulated simultaneously incasp-2 knockout mice studies and the RNA interference cellculture studies, the biological function of these 2 casp-2isoforms, especially the antiapoptotic function of casp-2Swas not clarified (34). In addition, the results about theantiapoptotic function of casp-2S obtained by overexpres-sion experiments are also controversial (18, 21, 35). By aspecific casp-2S siRNA designed in this study, we haveunambiguously revealed that downregulation of casp-2S canenhance UV-induced apoptosis in XPC-deficient cells, butnot XPC-proficient cells. This indicates that the cellularendogenous XPC level plays a critical role in investigationsof the function of casp-2S by manipulation of cellular casp-2S levels.

The expression of casp-2S can be regulated by alternativepromoter activation (21). It has been reported that p73 isable to specifically transactivate the expression of casp-2Sthrough directly binding to a Sp-1 binding site–containingregion in the promoter of casp-2S. In contrast, our dataclearly showed that the binding of XPC protein to casp-2S

Figure 7. A schematicmodel for XPC function in the enhancement of DNAdamage–induced apoptosis cascade. XPC protein represses thetranscription of antiapoptotic casp-2S through inhibiting casp-2promoter activity. The downregulation of casp-2S attenuates itsantiapoptosis effect, thus enhances DNA damage–triggered casp-9 andcasp-6 activation, and ultimately augments apoptosis.






promoter inhibits casp-2S expression. Given XPC is able toregulate gene transcription in 2 opposite ways with differentmechanisms (14, 15), it is possible that XPC can function as adirect transcriptional repressor of casp-2S.

In summary, XPC protein is proposed to enhance DNAdamage–induced apoptosis through intrinsic apoptotic path-way (Fig. 7). XPC protein downregulates the expression ofantiapoptotic casp-2S through inhibiting its promoter activity.The downregulation of casp-2S would attenuate its inhibitoryeffect on the activation of casp-9 and casp-6, resulting inaugmented apoptosis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors greatly acknowledge Drs. George Stark for 041-TR cell line, BertVogelstein for HCT116 cell line, Paul Modrich for A2780/CP70 cell line, ThomasHamilton for SKOV3 cell line, and Wenrui Duan for H1299 and A549 cell lines.The authors also thank Dr. Randy Legerski for providing pXPC3 plasmids and Yi-Wen Huang for assisting real-time PCR analysis.

Grant Support

This studywas supported by NIH grants CA151248 toQ.-E.Wang and ES2388,ES12991, and CA93413 to A.A. Wani.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 18, 2011; revised December 6, 2011; accepted December 9,2011; published OnlineFirst December 15, 2011.

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−Nucleotide Excision Repair Factor XPC Enhances DNA Damage

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