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Cancer Research 67, 2526-2534, March 15, 2007. doi: 10.1158/0008-5472.CAN-06-3371
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Long-term XPC Silencing Reduces DNA Double-Strand Break Repair

Emmanuelle Despras1, Petra Pfeiffer2, Bernard Salles3, Patrick Calsou3, Steffi Kuhfittig-Kulle2, Jaime F. Angulo1 and Denis S.F. Biard1

1 Commissariat à l'Energie Atomique, Laboratoire de Génétique de la Radiosensibilité, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, Fontenay-aux-Roses, France; 2 Institute for Genetics, Department of Biology and Geography of the University of Duisburg-Essen, Essen, Germany; and 3 Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, UMR 5089, Toulouse Cedex 4, France

Requests for reprints: Emmanuelle Despras, CEA-DSV-DRR, Laboratoire de Génétique de la Radiosensibilité, BP 6, 92265 Fontenay aux Roses, France. Phone: 33-1-46-54-87-47; Fax: 33-1-46-54-87-34; E-mail: emmanuelle.despras{at}cea.fr.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPAKD (knock down) and XPCKD cells were highly sensitive to UVC. DNA-PKcsKD and XRCC4KD cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPCKD cells also showed intolerance toward acute {gamma}-ray irradiation. We showed that XPCKD cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPCKD and DNA-PKcsKD cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPC-proficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair. [Cancer Res 2007;67(6):2526–34]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Xeroderma pigmentosum (XP) is a human disorder characterized by extreme sunlight sensitivity and markedly increased risk of skin cancer. XP cells exhibit a defect in nucleotide excision repair (NER), the major pathway for the removal of UV-induced and other helix-distorting lesions (1, 2). Seven XP complementation groups have been identified (XP-A to XP-G), with each gene product participating in one particular NER step. UV-induced lesions can also be bypassed by translesion DNA synthesis with polymerase {eta}, the mutations of which lead to a variant form of XP called XP-V (3, 4). It was proposed that in XP-V cells, collapse of replication forks stalled by UV-induced lesions can form DNA double-strand breaks (DSBs) repaired via recombination-based pathways (5). Such a replication-dependent conversion of unrepaired UV-induced lesions into DSBs was also observed in Chinese hamster ovary (CHO) and human cells devoid of NER (5–7). The source of these DNA breaks is likely to be unrepaired cyclobutane pyrimidine dimers (CPD). Their conversion into DNA breaks activates protein networks involved in DSB signaling and repair, as evidenced by functional genomic analysis of CPD-specific photolyase transgenic mice (8). Such a result raises the question of the effect of DSBs in the cytotoxicity of UV light. A defect in DSB repair would not be awaited as a characteristic of XP cells. However, it has been reported that one XP-G (XP3BR) and one XP-C cell line (XP14BR) elicited an impaired response to ionizing radiation (IR; refs. 9, 10). Therefore, it seems interesting to compare the DSB repair capacity of NER-proficient and NER-deficient cells.

Nonhomologous end joining (NHEJ) is the major mechanism of DSB repair in mammalian cells. Contrary to homologous recombination, it requires no homology to rejoin two DNA ends. Biochemical and genetic studies led to the following model for the main NHEJ pathway (for review, see ref. 11): (a) binding of the Ku70/Ku80 (Ku) heterodimer to DNA ends; (b) recruitment and activation of the DNA-PKcs kinase in a complex with Artemis; (c) processing of DNA ends; (d) ligation by DNA ligase IV associated with XRCC4 and presumably Cernunnos-XLF (12, 13). In addition, some studies suggest the existence of secondary pathways that occur in DNA-PK–deficient cells and could involve poly(ADP)ribose polymerase-1, XRCC1, and DNA ligase III (14, 15).

Because the comparison of XP cells derived from patients is delicate due to their different genetic backgrounds, we developed a human syngeneic XP-model based on RNA interference (RNAi). EBV-derived plasmids carrying sequences coding for small interfering RNA (pEBVsiRNA) were used to stably introduce NER defects in HeLa cells (16, 17). We targeted XPA and XPC proteins, the two NER proteins involved in lesion recognition. We characterized different knockdown HeLa clones (XPAKD and XPCKD), which remained stable for >400 days under hygromycin B selection. These clones exhibit undetectable levels of either XPA or XPC protein and present the main features of the XP phenotype (UVC sensitivity and impaired UDS; ref. 16). Here, we silenced the expression of DNA-PKcs and XRCC4 genes (DNA-PKcsKD and XRCC4KD clones) to create a model of NHEJ deficiency in HeLa cells. Both in vitro and in vivo experiments showed that XPCKD cells, unlike XPAKD cells, presented an altered NHEJ activity. XPCKD cells exhibited an enhanced sensitivity to etoposide (VP16) and intolerance to acute doses of {gamma}-rays, although not sensitive to lower doses of IR. Therefore, in a HeLa genetic background, XPC deficiency could hamper DSB repair, certainly in combination with other genetic factors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In accordance with the quality management program of our department, this work is reported in custom laboratory books numbered CEA/DRR/SRBF/LGR 0760, 3090, 3191, 4658, 4659, 6093, and 6094.

siRNA design and cloning in pEBV-based siRNA vectors. Vectors, cloning strategies, and establishment of knockdown HeLa clones were described elsewhere (16, 17). Short hairpin RNA (shRNA) coding sequences were designed with siSearch software,4 including minor change (18) and synthesized by Proligo (Sigma-Aldrich, St. Quentin Fallavier, France). Control cells were obtained by transfection with vector pBD650 (16), which carries a shRNA harboring two mismatches in one strand of the hairpin structure. RNAi sequences for DNA-PKcs (NM_006904; pBD743 plasmid), nucleotides 5,980 to 5,998; XRCC4 (NM_022550; pBD694), nucleotides 674 to 692; XPC (NM_004628; pBD634), nucleotides 267 to 285; XPA (NM_000380; pBD695), nucleotides 587 to 605; KIN17 (NM_012311; pBD674), nucleotides 180 to 198.

Irradiation and chemicals. Cells were irradiated using a 137Cs {gamma}-ray source (IBL 637, CisBio International, Gif-sur-Yvette, France) at a dose rate of 1.9 Gy/min. Neocarzinostatin (kindly provided by Dr. L. Deriano, CEA, France), calicheamicin (kindly provided by Dr. P. Hamann, Wyeth Research, New York, NY), and both VP16 (Vépéside-Sandoz, Sandoz, Levallois-Perret, France) and L-mimosine (Sigma) were diluted in ice-cold 10 mmol/L phosphate buffer (pH 6.6), –20°C ethanol and sterile distilled water, respectively, before cell treatment. Equal volumes of solvents or buffers were added to untreated cells as a control. Wortmannin (Sigma) was diluted in DMSO.

Cell culture. HeLa cells were maintained in DMEM (Invitrogen, Cergy-Pontoise, France) supplemented with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin under 5% CO2. Knockdown clones were grown in presence of 125 µg/mL hygromycin B (Invitrogen). For clonogenic cell growth, cells were irradiated or incubated with neocarzinostatin and VP16 for 1 and 6 h, respectively, 14 days before fixation.

For transient transfection experiments, HeLa cells were trypsinized 72 h after transfection with 2 µg of plasmid and grown under hygromycin B selection for 48 h before irradiation. Gene silencing efficiency was checked by immunocytochemical stainings (data not shown).

Western blot and immunostaining. Procedures were described elsewhere (19). We used monoclonal antibodies directed against Ku80 [clone (cl.) 111], Ku70/80 (cl.162), DNA-PKcs (cl.18.2; Lab Vision/Neomarkers, Newmarket, United Kingdom), {gamma}-H2AX (Upstate/Chemicon Direct, Chandlers Ford, United Kingdom), and human kin17 protein (Ig K36 and Ig K58; ref. 19); rabbit polyclonal antibodies directed against XRCC4 (AbD Serotec, Cergy Saint-Christophe, France); and ligase IV (Abcam, Cambridge, United Kingdom); mouse polyclonal antibody directed against XPC obtained after inoculation of a purified fragment of human XPC protein (CEA-LGR).5

Flow cytometry. The procedure was described elsewhere (16). Briefly, cells were collected by trypsinization and fixed in 75% ethanol. Nuclear DNA was stained with propidium iodide (Sigma; 4 µg/mL) in the presence of RNase (Sigma; 10 µg/mL) in PBS. Ten thousand cells gated as single cells using FL2A/FL2W scatter were analyzed on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) using CellQuest software.

DNA-PKcs activity. Pull-down assay was carried out as already described (20). Protein extracts (25 µg) were mixed with 5 mg of dsDNA cellulose (Amersham Biosciences, GE Healthcare, Courtaboeuf, France). Bound proteins were incubated with 4 nmol of DNA-PK peptide substrate (Promega, Charbonnières-les-Bains, France) in the presence of ATP and [{gamma}-32P]ATP for 10 min at 30°C. The identical reaction devoid of substrate was used as a negative control. As a positive control, we used 50 units of purified DNA-PKcs (Promega) activated by addition of 5 mg dsDNA-cellulose. [{gamma}-32P]ATP incorporation into peptide substrate was measured by liquid scintillation.

In vitro NHEJ assay. Whole-cell extracts and DNA substrates were prepared, and in vitro NHEJ assays were carried out as described elsewhere (21–23). Substrates were generated using following restriction enzymes: PstI (3'-cohesive ends), SmaI (blunt ends), BstXI (3'/3'), AvaI/HincII (5'/blunt), SmaI/PstI (blunt/3'), and BamHI/PstI (5'/3'). Extracts were dialyzed against reaction buffer [50 mmol/L MOPSO-NaOH (pH 7.5), 40 mmol/L KCl, 10 mmol/L MgCl2, 5 mmol/L ß-mercaptoethanol] before NHEJ assay. Standard reactions took place in a total volume of 10 µL containing 8 µL of extracts (40 µg of proteins), 1 µL of substrate (10 ng), and 1 µL of 10x LNB buffer [10 mmol/L Tris-HCl (pH 8), 1.2 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L ß-mercaptoethanol, 10 mmol/L ATP (pH 7), 2 mmol/L deoxynucleotide triphosphates (0.5 mmol/L each), 0.5 mg/mL bovine serum albumin]. For wortmannin inhibition experiments, 7 µL of HeLa cell extracts (48 µg of proteins) were incubated with 1 µL of wortmannin (final concentration 0.1, 1, or 5 µmol/L) for 30 min on ice before addition of 1 µL of substrate and 1 µL of LNB 10x buffer. Samples were incubated at 25°C for 6 h. Reactions were stopped, and an equivalent of 2 ng of DNA substrate was deproteinized, electrophoresed on 1% agarose gels containing 1 µg/mL ethidium bromide, and submitted to Southern blotting. Reaction products were quantified by phosphorimaging (Storm System and ImageQuant software 5.0, Amersham Biosciences). For each NHEJ reaction, the intensity of a band was normalized to the total radioactivity loaded in the lane. NHEJ efficiency was assessed as the conversion of monomeric linear substrate into multimers and circular products, all expressed as a percentage of input substrate.

Biochemical fractionation. Cells were treated with the indicated doses of calicheamicine or neocarzinostatin for 1 h. For VP16 treatment, asynchronous cells were treated for 4 or 24 h or synchronized in S phase by treatment with 400 µmol/L L-mimosine for 24 h followed by drug withdrawal and 6-h incubation before 1-h VP16 treatment. Cell fractionation was carried out as already described (24). Briefly, P2 fraction was obtained after extraction with 0.1% Triton X-100 (15 min on ice) followed by RNase digestion (30 min at 25°C). For whole-cell extracts, cells were resuspended in extraction buffer devoid of Triton X-100. Proteins were denatured by boiling in Laemmli buffer, and volumes equivalent to the same quantity of cells were submitted to Western blotting.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of NHEJ-deficient HeLa cells. We have recently characterized clonal derivatives from HeLa cells in which the expression of XPA or XPC gene has been silenced for a long time in culture (16). HeLa clones stably expressing shRNA sequences directed against either DNA-PKcs or XRCC4 mRNA were termed DNA-PKcsKD and XRCC4KD, respectively. DNA-PKcsKD cells presented a strong reduction of DNA-PKcs protein level as evidenced by immunocytochemical staining and Western blotting (Figs. 1A , 4C, and 5A). We assessed the DNA-PKcs kinase activity in control and DNA-PKcsKD cell extracts using a standard pull-down assay (Fig. 1B). DNA-PKcsKD cells displayed a nearly total loss of the endogenous DNA-PKcs kinase activity, which was close to background levels (Fig. 1B, lane 6). Purified DNA-PKcs was used as a positive control (Fig. 1B, lane 3). Presumably because XRCC4 protein is essential for cell life, all isolated XRCC4KD clones displayed residual levels corresponding to 10% to 15% of the control level (Fig. 1C; data not shown). This remaining XRCC4 level allowed cell survival but dramatically hampered DSB repair as shown below. As expected, both DNA-PKcsKD and XRCC4KD cells elicited a markedly increased sensitivity toward IR. At 2 Gy, 1.4%, 3.1%, 4.6%, and 17.4% survivals were observed for DNA-PKcsKD cl.1, XRCC4KD cl.9, XRCC4KD cl.19, and control cells, respectively (Fig. 1D).


Figure 1
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  		Figure 1. Characterization of DNA-PKcsKD and XRCC4KD clones. A, immunocytochemical staining of control and DNA-PKcsKD cl.1 (day 40) cells with anti–DNA-PKcs antibody. DAPI, 4',6-diamidino-2-phenylindole. B, analysis of DNA-PKcs activity in control and DNA-PKcsKD (D-PKcsKD; day 148) total cell extracts. As a positive control, 50 units of purified DNA-PKcs (D-PKcs) were used. C, immunocytochemical staining of control and XRCC4KD cl.9 (day 40) cells with anti-XRCC4 antibody. D, clonogenic cell growth of DNA-PKcsKD and XRCC4KD clones (day 58) after exposure to IR. Points, mean of three culture dishes; bars, SD.

 

Figure 4
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  		Figure 4. Alteration of NHEJ activity in HeLa XPCKD cell extracts. A, agarose gel separation of NHEJ products formed in (1) control, (2) XPAKD cl.6 (day 97), and (3) XPCKD cl.21 (day 200). P1 and P2 correspond to two independent extract preparations. B, Western blot analysis of total protein content from control cells; XPAKD cl.6 (day 143); XPCKD cl.21 (day 226); and KIN17KD cl.5, cl.6, and cl.12 (day 81). Proliferating cell nuclear antigen (PCNA) was used as a loading control. C, NHEJ protein content of HeLa knockdown clones. Whole-cell extracts were prepared as for biochemical fractionation. The protein equivalent of 600,000 cells was separated on polyacrylamide gels and submitted to Western blotting. 1, control (day 94); 2, DNA-PKcsKD cl.1 (day 94); 3, XRCC4KD cl.9 (day 94); 4, XPCKD cl.4 (day 315); 5, XPCKD cl.21 (day 267); 6, XPCKD cl.24 (day 264); 7, XPAKD cl.6 (day 261). D, agarose gel separation of NHEJ products formed in (1) control, (2) KINKD cl.6, and (3) KINKD cl.12. DNA end configurations are indicated on the top of the blots and NHEJ products are marked on the right side.

 

Figure 5
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  		Figure 5. Recruitment of NHEJ proteins to nuclear structures after DSB induction. Cells were treated at the indicated doses of chemicals. Whole-cell extracts (WCE) and P2 fraction were prepared as described in Materials and Methods. Samples were loaded on 10% SDS-PAGE and submitted to Western blotting. A, control and DNA-PKcsKD cl.1 (day 88); XPCKD cl.24 (day 260); XPAKD cl.6 (day 257) after 1-h calicheamicin (CAL) treatment (700,000 cells per lane). B, control, DNA-PKcsKD cl.1, XRCC4KD cl.9 (day 94), and XPCKD cl.21 (day 267) after 1-h calicheamicin (CAL) treatment (600,000 cells per lane). C, control cells were treated with the indicated doses of neocarzinostatin (NCS) for 1 h (400,000 cells per lane). D, asynchronous or S-phase synchronized control cells were treated with VP16 for the indicated times (400,000 cells per lane).

 
We next analyzed the NHEJ activity of DNA-PKcsKD and XRCC4KD whole-cell extracts as previously reported (22). Extracts from control cells yielded a variety of products comprising linear multimers (intermolecular joining) and circular monomers (intramolecular joining) occurring in the form of open circles and covalently closed circles (Fig. 2A , lanes 1). By contrast, DNA-PKcsKD and XRCC4KD cell extracts presented a 70% to 80% reduction in NHEJ efficiency, depending on DNA end configuration, as shown by phosphorimager quantification (Fig. 2A; Table 1 ). In DNA-PKcsKD extracts, both intermolecular and intramolecular joinings were reduced to 30% of the level in control cell extracts (Table 1). In XRCC4KD extracts, multimer formation was decreased to 30% of the level in control cell extracts, whereas circular products were undetectable. This result indicates that the reduction of XRCC4 protein expression was sufficient to completely abolish recircularization of the substrates. We found that preincubation of HeLa cell extracts with 1 µmol/L wortmannin, an inhibitor of DNA-PKcs kinase activity, abolished NHEJ activity nearly completely (Fig. 2B). This is in agreement with previously reported results in various cell lines (25–27).


Figure 2
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  		Figure 2. Impairment of the NHEJ pathway in DNA-PKcsKD and XRCC4KD cells. A, agarose gel separation of NHEJ products formed in (1) control, (2) XRCC4KD cl.9, and (3) DNA-PKcsKD cl.1 (day 75) extracts followed by Southern blotting. Terminus configurations are indicated on top of the blot (bl., blunt; coh., cohesive). B, HeLa extracts (48 µg of proteins per reaction) were incubated 30 min on ice with increasing doses of wortmannin (0, 0.1, 1, and 5 µmol/L) diluted in DMSO before NHEJ reaction with 3' cohesive DNA end substrate. NHEJ products were separated on 1% agarose gel and submitted to Southern blotting. Reaction products are marked on the right side. M, multimeric products; OC, open circles; L, residual linear substrate; CC, covalently closed circles.

 

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  		Table 1. NHEJ activity in HeLa knockdown cell extracts

 
These compelling data show that the reduction in either DNA-PKcs or XRCC4 protein levels by pEBVsiRNA plasmids led to an increased radiosensitivity in HeLa cells correlated with a significant decrease of NHEJ activity. Moreover, the reduced NHEJ efficiency of DNA-PKcsKD and XRCC4KD cell extracts and NHEJ-proficient cell extracts pretreated with wortmannin confirmed that the DNA end joining observed in this cell-free system is indeed dependent on both DNA-PKcs and XRCC4. In these conditions, long-term RNAi-based gene silencing allowed us to obtain a model of NHEJ deficiency in HeLa cells, in agreement with the biochemical deficiencies reported previously for mutant CHO cells or DNA-PKcs–deficient M059J human cells (28, 29).

Differential sensitivity of NER- and NHEJ-deficient cells to DSB induction. HeLa knockdown cells provided a unique opportunity to compare their sensitivity to various inducers of DSB. NHEJ- and NER-deficient cells were irradiated with {gamma}-rays or treated with two chemicals that induce DSBs either directly (neocarzinostatin; ref. 30) or via a replication-dependent mechanism (VP16; ref. 31).

In addition to their sensitivity to IR (Fig. 1D), DNA-PKcsKD cells exhibited a marked sensitivity to neocarzinostatin (Fig. 3A ). At 1 nmol/L neocarzinostatin, only 1.5 ± 1.3% of DNA-PKcsKD cells survived compared with 21 ± 1.5% of control cells. Both XPAKD and XPCKD cells presented sensitivities similar to that of control cells after exposure to IR or neocarzinostatin (Supplementary Fig. S1).


Figure 3
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  		Figure 3. Sensitivity of NHEJ- and NER-deficient HeLa cells toward DSB induction. A, DNA-PKcsKD cl.1 (day 127) cells were treated with neocarzinostatin (NCS) for 1 h. B, DNA-PKcsKD cl.1 (day 106), XRCC4KD cl.9 (day 106), XPAKD cl.6 (day 255), and XPCKD cl.21 (day 261) and cl.24 (day 218) were treated with VP16 for 6 h. In (A) and (B), cell survival was assessed in clonogenic cell growth assays. Points, mean of three culture dishes; bars, SD. C, effect of acute UVC (10 J/m2) and {gamma}-ray (6 Gy) irradiation after transient XPA and XPC gene silencing. HeLa cells were transfected with 2 µg of plasmid. Three days later, cells were trypsinized and cultivated in the presence of hygromycin B for 48 h before irradiation. D, effect of acute UVC (10 J/m2) and {gamma}-ray (6 Gy) irradiation on stable DNA PKcsKD cl.1 (day 168), XPCKD cl.21 (day 389), and XPAKD cl.6 (day 420). Cells were seeded 1 d before irradiation. In (C) and (D), cells were collected by trypsinization 24 h after irradiation, and fixed in 75% ethanol at 4°C. Cells were stained with propidium iodide (4 µg/mL) in the presence of RNase (10 µg/mL). Stained cells were analyzed on a FACSCalibur (Becton Dickinson) using CellQuest software. Ten thousand cells gated as single cells using FL2A/FL2W scatter were analyzed.

 
DNA-PKcs and XRCC4 gene silencing sensitize HeLa cells to VP16 (Fig. 3B). At 1 µmol/L, no DNA-PKcsKD clones emerged from culture and only 0.5 ± 0.8% survival was observed for XRCC4KD cells, whereas 15 ± 1.7% of control cells survived. Similar survival curves were reported for NHEJ-deficient DT40 chicken cells (32). XPAKD cells behaved like control cells upon VP16 treatment. By contrast, XPCKD cells presented a mild sensitivity to VP16 with 2.5 ± 0.9% and 1.5 ± 0.9% survivals at 1 µmol/L for cl.21 and cl.24, respectively (Fig. 3B).

XPCKD cells showed intolerance to acute {gamma}-ray irradiation. The cell cycle progression of knockdown cells 24 h after acute UVC (10 J/m2, 254 nm) or {gamma}-ray (6 Gy) irradiation was analyzed by flow cytometry. When HeLa cells transiently transfected with pEBVsiRNA plasmids directed against XPA or XPC mRNA were exposed to UVC, they presented a tremendous S-phase blockage compared with cells transfected with control plasmid (Fig. 3C), which revealed that unrepaired UVC-induced lesions blocked DNA replication and cell cycle progression. In HeLa cells, 6-Gy irradiation only triggered a slight G1 arrest and a moderate G2 blockage 24 h after exposure as described elsewhere (33). Strikingly, in the XPCKD cell population, we observed a prominent G2-phase arrest, which was detected at a lower extent in XPAKD cells. These accumulated G2-phase cells might represent damaged ones. As DNA-PKcs gene silencing is very deleterious in the first days of hygromycin B selection, we could not use it as a control in the aforementioned experimental conditions.

The effect of acute irradiation was also determined in stable knockdown clones. IR led to a major G2-phase blockage in DNA-PKcsKD cells (Fig. 3D). On the contrary, in these NER-proficient cells, UVC-induced lesions were removed without hampering progression of DNA replication. In XPAKD and XPCKD cells, we observed the expected S-phase blockage 24 h after 10 J/m2 UVC. Interestingly, we also detected a significant G2-phase blockage in XPCKD after IR. A minor G2-phase arrest was detected in XPAKD cells compared with control cells.

These results show that transient and stable XPC gene silencing triggered intolerance to acute doses of IR. The absence of radiosensitivity as judged by clonogenic cell survival suggests that XPCKD cells may deal with damage produced by lower doses of {gamma}-rays.

In vitro NHEJ was altered in XPCKD cell extracts. To further explore the differences between XPAKD and XPCKD cells toward DSB-inducing agents, we analyzed their NHEJ capacity. Two independent extract preparations (P1 and P2) were made for control, XPAKD cl.6, and XPCKD cl.21. For each cell line, P1 and P2 led to similar repair patterns (Fig. 4A ). XPAKD cl.6 and XPCKD cl.21 extracts presented NHEJ efficiencies comparable with that of control cell extracts as shown by blot quantification (Table 1). Repair profiles from XPAKD and control cell extracts did not show significant differences in the distribution between intermolecular and intramolecular joining. In opposition, XPCKD cl.21 presented a significant 2-fold reduction in circle formation (Fig. 4A, arrows). This decrease was apparently counterbalanced by an increase of multimerization. Extracts prepared from XPCKD cl.4 (independent transfection experiment) and XPCKD cl.24 gave similar results (Table 1; Supplementary Fig. S2). In XPCKD cl.4, circle formation ranged from 55% to 75% of that of control cell extracts, whereas XPCKD cl.24 extracts presented a statistically significant 2-fold reduction. Moreover, NHEJ efficiency dropped to 75% and 60% of that of control cells in XPCKD cl.4 and cl.24, respectively, revealing that the defect in intramolecular joining was not compensated by an enhancement of intermolecular joining.

During this work, knockdown clones were regularly analyzed by immunocytochemical staining and Western blotting. This confirmed that XPAKD and XPCKD clones presented low levels of XPA or XPC protein even after several months of culture (Fig. 4B and C; see also ref. 16). DNA-PKcsKD and XRCC4KD cells also presented reduced levels of DNA-PKcs and XRCC4 proteins, respectively (Fig. 4C). Interestingly, no significant differences in NHEJ key protein amounts were detected in control, XPCKD, and XPAKD cells. Only XRCC4KD cells displayed a reduced ligase IV level (Fig. 4C, lane 3), in agreement with the stabilization of ligase IV by XRCC4 previously observed in CHO cells (34).

To confirm that the impaired NHEJ activity observed in XPCKD clones was not a side effect of pEBVsiRNA plasmids, we prepared extracts from two KIN17KD clones characterized previously (16). kin17 is a nuclear zinc-finger protein involved in DNA replication and in the cellular response to genotoxic stress (35, 36). All isolated KIN17KD clones exhibited a strong but partial reduction of kin17 protein level (Fig. 4B). This residual amount is likely to be required for cell survival (16). NHEJ activity of KIN17KD cell extracts was comparable with that of control cells (Fig. 4D). The fact that control, KIN17KD, and XPAKD cell extracts produced similar NHEJ repair patterns strongly argues that the phenotype observed in XPCKD cells is a consequence of XPC gene silencing and not a bias effect of the RNAi procedure or the NHEJ assay.

Our results indicate that the long-term silencing of XPC gene in HeLa cells led to either a decrease of in vitro NHEJ efficiency or to a repair switch from intramolecular to intermolecular junctions. This alteration was not correlated with any significant modification of the expression of proteins involved in the predominant NHEJ pathway.

Recruitment of XRCC4 and ligase IV to damaged nuclear structures occurred at lower doses of calicheamicin in XPCKD cells. Recently, it was shown that key NHEJ proteins are recruited to detergent-resistant nuclear structures (P2 fraction) after exposure to different DSB inducers (24). We tested whether this recruitment takes place normally in XPCKD clones. Cells were treated with calicheamicin as it is a potent and very specific DSB inducer (30).

Ku80 protein was recruited in a dose-dependent manner without noticeable differences between the tested cell lines. A dose-dependent recruitment of DNA-PKcs was also observed, except in DNA-PKcsKD cells in which DNA-PKcs was, as expected, hardly detected (Fig. 5A and data not shown). We observed that XRCC4 protein was phosphorylated and associated to nuclear structures in a dose-dependent manner, as previously described for neocarzinostatin (24). In control and XPAKD cells, XRCC4 found in the P2 fraction was mostly unphosphorylated after 1 nmol/L calicheamicin, whereas phosphorylation was observed after 4 nmol/L calicheamicin. By contrast, in DNA-PKcsKD cells and in XPCKD cl.21 and cl.24, phosphorylation had already occurred after 1 nmol/L calicheamicin. This was correlated with a recruitment of ligase IV at this lower dose (Fig. 5B). Moreover, in DNA-PKcsKD cells, the band corresponding to phosphorylated XRCC4 migrated further than in other cell lines, revealing a decreased level of phosphorylation (Figs. 5A and B). It is noteworthy that the residual XRCC4 protein in XRCC4KD cells was efficiently recruited and phosphorylated and was sufficient to recruit ligase IV (Fig. 5B).

Our data revealed a similar behavior of XPCKD and DNA-PKcsKD cells upon calicheamicin treatment, ligase IV, and phosphorylated XRCC4 association to nuclear structures occurring at lower doses than in other clones.

DSB induction led to the degradation of XPC protein. XPC protein was found in the P2 fraction of untreated cells, suggesting a tight association of XPC with nuclear structures (Fig. 5). Analysis of whole protein content revealed that XPC was degraded after 1-h incubation with 500 nmol/L neocarzinostatin (Fig. 5C). Both calicheamicin and neocarzinostatin treatments led to a dose-dependent decrease of XPC protein amount in the P2 fraction of control cells (Fig. 5A and C). This reduction was also observed in XPAKD and DNA-PKcsKD cells after treatment with calicheamicin (Fig. 5A). DSB formation was assessed using an antibody directed against phosphorylated histone H2AX after neocarzinostatin treatment of control cells (Fig. 5C). The reduction of XPC level in the P2 fraction was correlated with the increase of H2AX phosphorylation. Similar results were obtained in asynchronous control cells treated with increasing doses of VP16 for 4 and 24 h and in S-phase synchronized control cells treated with VP16 for 1 h (Fig. 5D). As a control, we used kin17 protein, which is tightly associated with chromatin and nuclear matrix (37). These data show that XPC protein is removed from nuclear structures after treatment with chemicals inducing DSB with a high specificity and via replication-dependent or independent mechanisms.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, Arlett et al. (10) described an unusual radiosensitive XP-C patient. They showed that XPC deficiency is not directly involved in the observed IR sensitivity, but impaired XPC pathway could enhance it. We have previously shown that XPC silencing using pEBVsiRNA plasmids dramatically impedes HeLa cell growth during the first few weeks after transfection. In XPCKD cells, hygromycin B withdrawal restores wild-type XPC amount; however, cells remain significantly sensitive to UVC (16). These results suggest that XPC deficiency could trigger unwanted and irreversible genetic changes. This reinforced the idea that XPC could participate to other genetic events than NER. In the present study, we targeted DNA-PKcs and XRCC4, two essential proteins belonging to the NHEJ pathway. XRCC4 gene silencing is particularly interesting because no human cell line lacking the XRCC4 protein is available to date. Thus, we obtained stable HeLa clones harboring specific defects in either NER or NHEJ. We analyzed the relationships between these two allegedly independent DNA repair pathways and compared their respective involvements in the survival of human cells.

We showed that XPC deficiency affected the cellular response to either UVC or IR as evidenced by flow cytometry analysis done 24 h after an acute irradiation. This result was observed 5 days after transfection as well as in stable XPCKD clones. Clonogenic cell survival showed that XPCKD cells are sensitive to UVC (as XPAKD cells; ref. 16) but not to {gamma}-rays at lower doses than those used in acute irradiation experiments. These data suggest that XPCKD cells can tolerate moderate amounts of IR-induced DNA damage but cannot cope with elevated ones.

Interestingly, XPCKD cells also presented a mild sensitivity to VP16, which requires DNA replication for induction of DSBs in contrast to IR or neocarzinostatin. The enhanced sensitivity of DNA-PKcsKD and XRCC4KD cells to VP16 argues for an involvement of DNA-PK–dependent NHEJ in the repair of the VP16-induced DSBs, which is in agreement with previously reported results (38). It is not yet clear how the complexes formed by the VP16 cross-link of topoisomerase II on DNA are processed to become substrates for DSB repair pathways. We assume that these complexes alter the topological structure of the DNA helix, which may activate DNA repair proteins such as XPC. In particular, it has been recently shown in vitro that XPC and XPA proteins can recognize small oligopeptides cross-linked to DNA, which are further excised by the NER machinery (39).

XPCKD cells presented an impaired NHEJ activity as evidenced by in vitro assays, which revealed decreased levels of intramolecular junctions. To date, the biological significance of multimerization versus circularization of DNA substrate is not fully understood. Intramolecular joining was completely abolished in XRCC4KD cells in spite of residual XRCC4 levels and correct mobilization of NHEJ proteins to damaged nuclear structures. This result argues for XRCC4 being a limiting factor in the NHEJ process, at least in vitro, and further supports previously reported results (40). However, radiosensitivity was not strictly correlated with the circularization capacity of cell extracts, because DNA-PKcsKD cells were significantly more sensitive to IR than XRCC4KD cells. XRCC4 phosphorylation and ligase IV recruitment on nuclear structures occurred at lower doses of calicheamicin in both XPCKD and DNA-PKcsKD cells. This similarity strengthens the data from cell-free NHEJ assays. XRCC4 is phosphorylated by DNA-PKcs in vitro; however, to date, there is no direct evidence that this phosphorylation is important for DSB repair in vivo (41, 42), except for the fact that DSB-inducing agents such as X-rays promote XRCC4 phosphorylation (43). In DNA-PKcsKD cells, the fraction of XRCC4 associated with nuclear structures after DSB induction was hypophosphorylated compared with control and XRCC4KD cells. Our results indicate that XRCC4 phosphorylation is essential for DSB repair and cell survival and involves DNA-PKcs in vivo.

XPCKD and XPAKD clones presented very low levels of the target protein, as shown here and elsewhere (16, 17). However, XPA gene silencing did not modify HeLa cell response to DSBs. This suggests that the unexpected behavior toward DSBs observed in XPCKD cells is due to an intrinsic characteristic of XPC rather than being a consequence of NER deficiency. XPC was degraded after treatment with highly specific DSB inducers but the absence of XPC affected NHEJ and sensitized cells to VP16. Taken together, these data suggest that this degradation could be a step of the cellular response to DSBs in HeLa cells. Many authors have postulated that XPC protein could act in broader cellular mechanisms than initiation of global genome-NER alone. First, its association with the centrosomal protein centrin 2 might couple NER to cell division (44). Reduced mRNA level of centrin 2 in a plant mutant is associated with UVC sensitivity, decreased NER efficiency, and increased homologous recombination (45). In cisplatin-treated human cells, XPC defect affects transcription responses of many genes, including DNA DSB repair genes (46). Purified XPC-HR23B complex interacts with thymine DNA glycosylase, an initiator of base excision repair, and stimulates its activity (47). This result physically links XPC to a repair pathway distinct from NER. Compared with wild-type or XPA-deficient mice, XPC-KO mice develop more spontaneous mutations and more tumors upon 2-acetylaminofluorene treatment (48, 49). Furthermore, the absence of XPC leads to the formation of a mutational hotspot at a nondipyrimidine site of the remaining p53 allele of Trp53+/– mice that cannot be detected in the absence of XPA or Cockayne syndrome A (50).

Our results strengthen the notion that the function of XPC protein goes beyond the initial step of NER. Because XPC deficiency could disrupt the cellular response to DSB inducers such as {gamma}-rays (in one XP-C patient–derived cell line; ref. 10) or VP16 (our results in HeLa cells), XPC protein could act at a regulatory level. More broadly, our results emphasize the cross relationships between different DNA repair pathways. At present, we seek to improve this set of silenced clones devoted to study the interwoven pathways of DNA repair.


   Acknowledgments
 
Grant support: Electricité de France contract 8702, fellowships from the Commissariat à l'Energie Atomique and the Ligue Nationale Contre le Cancer (E. Despras), and Wilhelm Sander Stiftung für Krebsforschung grant 2002.108.1 (P. Pfeiffer).

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

We thank E. Feldmann, A. Odersky, and W. Goedecke for their advice on NHEJ assay and S. Britton for his kind help during E. Despras's stay in B. Salles's laboratory.


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

Current address for P. Pfeiffer: Institute for Genetics, University of Cologne, Cologne, Germany. Current address for S. Kuhfittig-Kulle: Pediatric Haematology/Oncology, Children's Hospital of Essen, Essen, Germany.

4 http://sirna.cgb.ki.se/ Back

5 J-B. Charbonnier, E. Renaud, S. Miron, M.H. Le Du, Y. Blouquit, P. Duchambon, P. Christova, A. Shosheva, J.F. Angulo, and C.T. Craescu. Xeroderma pigmentosum C protein recruits human centrin onto ultraviolet-damaged nuclear sites through a direct interaction, submitted for publication. Back

Received 9/12/06. Revised 12/ 5/06. Accepted 1/ 2/07.


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 Introduction
 Materials and Methods
 Results
 Discussion
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