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Molecular Cancer Research 3:519-529 (2005)
© 2005 American Association for Cancer Research

DNA Damage and Cellular Stress Responses

Development of New EBV-Based Vectors for Stable Expression of Small Interfering RNA to Mimick Human Syndromes: Application to NER Gene Silencing

Denis S.F. Biard1, Emmanuelle Despras1, Alain Sarasin2 and Jaime F. Angulo1

1 Laboratoire de Génétique de la Radiosensibilité, Commissariat à l'Energie Atomique (CEA), Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, Fontenay-aux-Roses, France and 2 Laboratory of Genetic Instability and Cancer, Centre National de la Recherche Scientifique UPR2169, Institut Gustave Roussy, Villejuif, France

Requests for reprints: Denis S.F. Biard, Laboratoire de Génétique de la Radiosensibilité, Commissariat à l'Energie Atomique, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, BP 6, 92265 Fontenay aux Roses, France. E-mail: denis.biard{at}cea.fr


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPAKD and XPCKD (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced HSAKIN17. Several clones displaying undetectable protein levels of XPA, XPC, or HSAkin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, HSAKIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPAKD and XPCKD HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPCKD HeLa clones were more sensitive to UVC than their XPAKD or KIN17KD counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPAKD cells recovered a normal UVC sensitivity, XPCKD cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, HSAkin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Nucleotide excision repair (NER) is a versatile DNA repair pathway capable of removing a wide variety of helix-distorting base lesions, such as UV-induced pyrimidine dimers as well as chemically induced intrastrand cross-links and bulky adducts. Two distinct subpathways have been identified in the mammalian NER system: transcription-coupled repair, which is specialized to eliminate transcription-blocking lesions on the DNA strand of active genes, and the global genome repair, which acts on DNA damage across the whole genome, including the transcribed strand of active genes at least for some lesions (1, 2). Impaired NER activity affecting either transcription-coupled repair or global genome repair, or both, is associated with several hereditary human disorders, including xeroderma pigmentosum. Xeroderma pigmentosum is a rare, photosensitive, and cancer-prone syndrome with recessive and autosomal inheritance. Two forms of xeroderma pigmentosum exist: a "classic" form and a "variant" form (XP-V). XP-V cells are proficient in NER but are impaired in lesion bypass associated with DNA replication on damaged templates. XP-V cells display a mutated pol {eta} gene encoding a translesional polymerase. Cells from classic xeroderma pigmentosum fall into seven complementation groups (named XP-A to XP-G) where one mutated xeroderma pigmentosum protein hampers one step of the NER process. Noteworthily, mutation of one XP gene induces a variable deficiency in NER. Most XP genes are implicated in both the transcription-coupled repair and global genome repair subpathways, except for XPC which is only deficient in the global genome repair (3).

Transcription-coupled repair and global genome repair differ in the mechanism of DNA damage recognition. transcription-coupled repair is initiated by the blockage of RNA polymerase II by a lesion. In global genome repair, the recognition of the distortion is done by XPC-hHR23B, a global initiator in repair, or the dimer DDB1/DDB2 (XPE group; refs. 4, 5). XPA in conjunction with replication protein A may constitute a repair mediator that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly (6, 7). After damage recognition, which depends on each subpathway, these two DNA repair mechanisms share common steps. The DNA duplex around the lesion is locally unwound by XPB and XPD helicases associated with the transcription factor TFIIH and, at least, replication protein A, XPA, and XPG proteins. The opening of the helix at the damage site is necessary for subsequent dual incision by the two structure-specific NER endonucleases, XPF-ERCC1 and XPG, which causes the release of a stretch of 25 to 30 nucleotides containing the lesion (5).

Because XPA and XPC proteins are key components in the lesion recognition steps of the NER pathway, we sought to determine the implication of these proteins in UVC sensitivity in human syngeneic cells. We have developed a new family of vectors for imposing stable and long-term gene silencing in human cells without using a virus vehicle. In recent years, RNA interference has emerged as a promising technology to knock down the expression of specific genes in human cells. RNA interference is characterized by coordinated cellular pathways that induce the silencing of specific genes by the expression of cognate small double-stranded RNAs. Tuschl et al. (8) described the initial design for the synthesis of a 21-nucleotide small interfering RNA (siRNA) which efficiently suppresses gene expression without inducing the IFN pathway. Transfection of siRNA duplexes subsequently proved effective in the field of reverse genetics (9). However, the transient nature of gene silencing induced by transfection of siRNA duplexes hampered this approach. A major breakthrough was made in a pioneering study describing the pSUPER plasmid (10). pSUPER allowed transient knockdown of both exogenous and endogenous genes. This plasmid carries the H1 RNA polymerase III promoter to drive the transcription of short hairpin RNA (shRNA) giving rise to siRNA-like molecules in vivo. In parallel, long-term expression was attempted using siRNA-based virus systems (retrovirus, lentivirus, or adenovirus). However, safety and ethical concerns about the handling of these viruses could be a major drawback (11). Until now, most of the published articles on gene silencing have described transient transfections of cultured cells with either siRNA duplexes or plasmids. Interestingly, authors always used integrative plasmids carrying (or not) a selective marker. These vectors could lose their siRNA cassette during the selection pressure. Besides, a high copy number of siRNA-based plasmid per cell should saturate the RNA interference machinery (e.g., the RISC complex) triggering unwanted side effects (12). As a consequence, this considerably hampered the establishment of stable cell populations and only a few stable clones were characterized for a reduced period of time in culture (13-15).

To alleviate these shortcomings, we combined the efficiency of both EBV-based vectors with the siRNA approach to establish cell lines expressing a given shRNA for a long time (>300 days). EBV-based vectors have been used to efficiently modify the human cell genotype (16, 17). To show the usefulness of this approach, we used these vectors to mimic the behavior of cells derived from xeroderma pigmentosum patients and to assess the long-term consequences of gene silencing in nearly syngeneic cells. Beside XPA and XPC, we also silenced HSAKIN17 (Homo sapiens KIN17) which encodes a nuclear zinc finger protein involved in DNA replication (18), RNA processing (19), and in the cellular response to DNA damage (20, 21).

We show that HSAKIN17 and XPC gene silencing dramatically impeded cell growth a few weeks after transfection. In contrast, XPA knockdown did not impede cell viability and growth, and numerous clones were easily obtained. Thereafter, these XPAKD, XPCKD, and KIN17KD HeLa clones remained stable even after more than 300 days of culture. The classic features of xeroderma pigmentosum cells were maintained in XPAKD and XPCKD clones (e.g., UVC sensitivity) and presented impaired unscheduled DNA synthesis after UVC irradiation. We show that XPCKD HeLa cells were more sensitive to UVC than their XPAKD or KIN17KD counterparts. As expected, EBV-based siRNA vectors were maintained in HeLa cells in an episomal form even after several months in culture, at about 10 vector copies per cell. Hygromycin B withdrawal rapidly led to the loss of these vectors, triggering the restoration of either XPA or XPC protein levels. Interestingly, we observed the reversion of the XP phenotype in HeLa XPAKD cells but not in HeLa XPCKD cell, which remained sensitive to UVC irradiation. This observation suggested that irreversible defects appeared with time in culture in HeLa XPCKD cells.

Our results indicate that in HeLa cells, (i) HSAkin17 participates indirectly in early events following UVC irradiation and it may be involved in the coordination between different pathways, such as DNA replication when unrepaired (or irreparable) DNA damage is present; and (ii) XPC deficiency strongly affects cell growth and contributes to UVC sensitivity to a greater extent than does XPA. Our data illustrate that the use of EBV-based siRNA vectors is a promising tool in the field of gene silencing and reverse genetics.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Comparison between siRNA Duplexes, Integrative Plasmids, and EBV-Based siRNA Vectors
We used HSAKIN17 gene as a target to determine the experimental conditions for efficient gene silencing. First, we sought to compare gene silencing efficiencies induced by either siRNA duplexes or EBV-based siRNA vectors. Two HSAKIN17 siRNAs (siK180 and siK906) were synthesized as double-stranded RNAs and transfected into HeLa or RKO cells. DNA sequences coding for shRNA were designed and introduced into EBV vectors in dual orientations to check whether the position relative to the other genetic elements modifies their expression. The cloning scheme is summarized in Fig. 1 and the vectors are listed in Table 1.



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  		FIGURE 1. Cloning strategy of EBV-based siRNA vectors. Synthetic double-stranded oligodeoxynucleotides coding for shRNA sequences were synthesized (Eurogentec S.A. Seraing, Belgium) according to a model adapted from Agami and collaborators (10). shRNA sequences contain two identical 19-nucleotide motifs in an inverted orientation, separated by a 9-bp spacer of nonhomologous sequences (TTCAAGAGA). We added six thymidines at the 3' end of the repeat to function as a termination signal for RNA polymerase III and the CCG sequence at the 5' end to determine the initiation site. BglII and HindIII cloning sites were fitted at the 5' and 3' extremities, respectively, of the double-stranded shRNA sequence for subsequent cloning. Sense and antisense oligodeoxynucleotides were annealed to make a double-stranded DNA coding for shRNA with protruding BglII and HindIII ends. They were inserted into the BglII/HindIII sites of the pSUPER plasmid, giving rise to pBD622 (3,230 bp). siRNA cassettes (H1 promoter and shRNA coding sequence) were checked by sequencing, digested with KpnI/BamHI (New England Biolabs Inc., Ipswich, MA), and introduced into an EBV vector (pBD149; "Biard et al. Regulation of the Escherichia coli lac operon expressed in human cells. Biochim Biophys Acta, 1130:68-74, 1992"). For subsequent cloning, annealed shRNA sequences were directly introduced into pBD631 or pBD665 plasmids (empty vectors with a reverse or direct orientation of the siRNA cassette). Integrative plasmids were obtained from EBV vectors after deletion of EBV sequences (HpaI/BstEII digestion, Klenow filling, and self-ligation), giving rise to the pBD642 (4,784 bp). Genetic map of one EBV-based siRNA vector: FR, family of repeats; DS, dyad symmetry element; HygroR, hygromycin B resistance gene; TK pr, herpes simplex virus thymidine kinase promoter; TKpA+, herpes simplex virus-TK polyadenylation signal; pBR ori, pBR322 origin of replication; AMPr, bacterial {alpha}-lactamase gene.

 

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  		Table 1. Replicative pEBV versus Integrative Vectors

 
Three days after transfection with either siRNA duplexes (siK180 and siK906) or EBV vectors carrying these sequences, we observed an 80% to 90% decrease in the HSAkin17 protein level using two different well-characterized monoclonal antibodies, without changes in other nuclear proteins tested (Fig. 2A). This illustrates the efficiency and the specificity of EBV vector–mediated gene silencing. Rapidly during hygromycin B selection, EBV-based vectors triggered an improved gene silencing (Fig. 2B; 10 and 18 days posttransfection). It was noteworthy that the orientation of the siRNA cassette inside EBV vectors slightly influenced the silencing of HSAKIN17 gene. The reverse orientation seemed to be more efficient in imposing gene silencing. Because siRNAs could induce nonspecific effects on protein levels (22), we investigated this possibility by comparing the different knockdown clones themselves and with various controls during the whole culture period. In this way, we constructed a "control" vector carrying an oligonucleotide coding for an shRNA harboring two mismatches in one strand of the hairpin structure (pBD650). We also transfected "empty" EBV vectors carrying the H1 promoter in the two orientations without the shRNA sequence (pBD631 and pBD665). To compare EBV versus integrative vectors, we deleted the EBV sequence from these vectors giving rise to integrative plasmids named pHygro-siRNA (see figure 1).



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  		FIGURE 2. A. Comparison of transient HSAKIN17 gene silencing induced by either siRNA duplexes or EBV-based siRNA vectors. Twenty-four hours after seeding, HeLa cells were transfected with either EBV vectors or siRNA. Three days later, cells were trypsinized, counted, and proteins were analyzed as described. 1, pBD631 (empty vector, reverse); 2, pBD665 (empty vector, direct); 3, pBD650 (control vector, reverse); 4, pBD632 (siK663 vector, reverse); 5, pBD674 (siK180 vector, reverse); 6, pBD676 (siK906 vector, reverse); 7, pBD678 (siK180 vector, direct); 8, pBD680 (siK906 vector, direct); 9, siRNA K180 duplex; 10, siRNA K906 duplex. B. Stability of HSAKIN17 gene silencing 3, 10, and 18 days after transfection of HeLa cells with EBV vectors. 1, pBD631; 2, pBD632; 3, pBD664 (siK663 vector, direct); 4, pBD665; 5, pBD650. C. Enhanced efficiency of gene silencing mediated by EBV-based siRNA vectors in comparison with integrative plasmids. Forty-eight hours after transfection, 150,000 RKO cells were seeded in a 6 cm diameter dish in the presence of hygromycin B in the medium. Immunocytochemical staining of HSAkin17 (immunoglobulin G K36; magnification, x50). Cells were counterstained with 4',6-diamidino-2-phenylindole. 1, pBD641 (empty vector, Hygro); 2, pBD642 (siK663 vector, Hygro); 3, pBD631; 4, pBD632.

 
Thirteen days after transfection, clonogenic cell growth assays revealed a higher number of silenced clones with EBV-based siRNA vectors (pBD632 and pBD634) in comparison with pHygro vectors (pBD642 and pBD657). More precisely, transfection of integrative plasmids (pHygro siRNA) entailed a few growing clones: 194 ± 48 clones with the pBD641 plasmid (no shRNA), 56 ± 48 with pBD642 (KIN17 shRNA), 28 ± 48 with pBD657 (XPC shRNA), and 222 ± 48 with pBD653 (mismatched shRNA). Sixty percent of these clones failed to knock down the gene of interest (for KIN17: Fig. 2C, 2 versus 1). In contrast, numerous clones emerged a few days after transfection with EBV-based siRNA vectors: 44,250 ± 2,136 clones with the pBD631 plasmid (no shRNA), 417 ± 144 with pBD632 (KIN17 shRNA), 250 ± 144 with pBD634 (XPC shRNA), and 50,833 ± 5,700 with pBD650 (mismatched shRNA). Almost all of them down-regulated the gene of interest (Fig. 2C, 4 versus 3).

Strikingly, HSAKIN17 and XPC knockdown dramatically hampered cell survival and the number of growing clones fell more than 100-fold compared with the controls. These results were confirmed in RKO cells (data not shown). The weaker effect of KIN17 shRNA with integrative plasmids was probably due to a lower efficiency of gene silencing. When HeLa clones were picked up for culture propagation, most of them were silenced for the gene of interest (e.g., all isolated XPAKD clones were silenced in Fig. 3D). We concluded that the nuclear retention of EBV vectors in human cells greatly improves the expression of siRNA a few weeks after transfection.



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  		FIGURE 3. Isolation of stable XPAKD and XPCKD HeLa clones. A. Western blot analysis of four XPCKD clones. M, mock-treated cells; C1, control vector (pBD650); C2, empty vector (pBD631). B. Immunocytochemical detection of XPC protein in two XPCKD clones, in XP44RO cells and in the XPC-complemented XP44RO cells. Magnification, x350. C. Immunocytochemical detection of XPA and HSAkin17 in a XPAKD population 13 days after transfection. Magnification, x350. D. Western blot analysis of 12 stable XPAKD clones (48 days in culture) and 2 stable XPCKD clones (113 days in culture).

 
Long-term XPA, XPC, and KIN17 Gene Silencing in Human Cells
We isolated both XPAKD and XPCKD clones after transfection of HeLa cells with the pBD695 and pBD634 vectors. XPA and XPC protein levels both fell a few days after transfection (data not shown) or several months later, and reached undetectable levels in Western blotting or immunocytochemical staining analysis (Fig. 3). In the different XPCKD clones recovered 85 and 113 days after transfection, XPC protein reached a level similar to that observed in classic human XPC cells (Fig. 3B). Similarly, 12 XPAKD clones were picked up at random and grown. Forty-five days after transfection, all isolated clones failed to express detectable XPA protein (Fig. 3C and D). However, residual and undetectable levels of either XPC or XPA proteins might be present in our knockdown HeLa clones.

Interestingly, whereas XPC and HSAkin17 deficiency induced a dramatic decrease in cell growth a few days after transfection, no such decrease was observed with XPA (Fig. 4A). As a consequence, we rapidly obtained XPAKD HeLa clones. In contrast, XPCKD (two vectors) and KIN17KD (four vectors) HeLa cells grew poorly; isolated clones were propagated in culture only after several weeks. These data indicate that XPC and HSAKIN17 gene deficiencies strongly affect cell growth at least in HeLa cells.



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  		FIGURE 4. A. XPCKD and KIN17KD HeLa cells grow poorly 2 weeks after transfection. Forty-eight hours after transfection, cells were plated at the same density and grown in the presence of 250 µg/mL hygromycin B. Fourteen days later, clones were fixed and stained before counting. Columns, mean of three culture dishes; bars, SE. The experiments were replicated more than three times. For each cell line, results are expressed as a percentage of the control cell line. B. Higher UVC sensitivity of XPCKD HeLa cells compared with XPAKD and KIN17KD syngeneic HeLa cells. Clonogenic cell growth assays after UVC irradiation (4 J/m2) were carried out at different times following transfection with control cells (BD650), KIN17KD, XPCKD, or XPAKD HeLa. Cells were seeded at 500 cells per 6 cm diameter dish, irradiated 24 hours later, and analyzed after 14 days of culture. Columns, mean of three culture dishes; bars, SE. Results are expressed as a percentage of the control cell line (BD650) for each UVC irradiation experiment. C. Impaired unscheduled DNA synthesis in XPAKD and XPCKD clones after UVC irradiation. HeLa XPAKD (clones 3 and 6; day 110 after transfection) and XPCKD (clones 21 and 24; day 193) were plated onto coverslips in a growth suppression medium. Cells were irradiated and analyzed as described in Materials and Methods. The level of DNA repair is expressed as number of grains per nucleus as a function of UV dose.

 
We assessed the UVC sensitivity of different knockdown HeLa clones by means of clonogenic survival assays after UVC irradiation (4 J/m2) during several months of culture. In the first months following transfection, we systematically analyzed three XPAKD, five XPCKD, and two KIN17KD HeLa clones, which gave rise to similar results. Afterwards, we followed only one clone per targeted gene. Whereas some fluctuations were observed in cell survival during these months of culture, it seems that about 84% of control cells (BD650) survived after a UVC irradiation at 4 J/m2 (Fig. 4B). In contrast, we clearly showed that XPCKD HeLa cells constantly displayed an elevated sensitivity to UVC, in particular a few months after transfection. Strikingly, XPAKD HeLa cells were less sensitive to UVC than XPCKD HeLa cells. For KIN17KD HeLa clones, we observed a moderate UVC sensitivity (Fig. 4B). Our results showed an exceptionally stable gene silencing over time, even after 269 days of culture (data not shown).

By using the same aforementioned XPCKD and XPAKD clones, we assessed the unscheduled DNA synthesis after UVC irradiation as described elsewhere (23). We analyzed two stable XPCKD HeLa clones (21 and 24) and two XPAKD HeLa clones (3 and 6), which were maintained in continuous culture for 193 and 110 days, respectively. As expected, these clones displayed reduced repair capacity at all doses tested (5 to 20 J/m2) unlike control cells (MRC5-V1 cells or HeLa BD650 control cells; Fig. 4C). These data indicate that XPA and XPC deficiencies, imposed by EBV-based siRNA vectors for a long period of time, are correlated with the expected UV sensitivity characteristic of the XP phenotype.

Lack of Complete Reversion of XPCKD Cells after Suppression of XPC Silencing
We sought to determine whether the long-term deficiency of either XPA or XPC protein could affect cellular integrity. Hygromycin B was removed from the medium for 10 days to cure cells of the episomes and to analyze the long-term consequences of gene knockdown. We observed a rapid (7-15 days) and complete restoration of normal XPA and XPC protein contents in both XPAKD and XPCKD cells as evidenced by Western blotting and immunocytochemical staining (Fig. 5A and B). To check if this restoration of XPA and XPC protein levels was due to the loss of all EBV episomes, genomic DNA was extracted, digested, and analyzed by Southern blotting. Our data clearly indicated that (i) EBV vectors designed for XPA or XPC gene silencing were maintained at about 10 copies per cell in both XPAKD and XPCKD clones; (ii) all of these copies were maintained in an episomal form; and (iii) drug removal was associated with the loss of all EBV copies (Fig. 5C).



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  		FIGURE 5. Recovery of normal XPA and XPC protein levels in XPAKD and XPCKD cells after hygromycin B withdrawal. HeLa clones were grown for 10 days in the presence or absence of 125 µg/mL hygromycin B. A. Cells were irradiated (UVC; 10 J/m2) and harvested 20 hours later. Cells were counted and lysed in Laemmli buffer. The equivalent of 100,000 cells was loaded onto 10% SDS-PAGE. B. Immunocytochemical analysis of XPAKD and XPCKD clones. Magnification, x350. C. Episomal maintenance of EBV-based siRNA vectors and their total loss after hygromycin B removal. Total DNA was extracted, digested with either BamHI or EcoRI, and submitted to Southern blot analysis with a specific probe for EBV-based siRNA vectors. With Hygro, cells were seeded with hygromycin B; w/o Hygro, hygromycin B was removed from the culture for 10 days.

 
Clonogenic survival assays were carried out to assess the sensitivity of reverted knockdown clones. Interestingly, whereas HeLa XPAKD reversed to a nearly wild-type UV sensitivity, HeLa XPCKD cells remained sensitive to UV after hygromycin B withdrawal (Fig. 6A). In parallel, we examined the S-phase arrest after acute UVC irradiation (10 J/m2, cells being harvested 20 hours later) to determine whether remaining DNA damage could be blocking for DNA replication. Whereas control cells exhibited a G2-M blockage, XPAKD and XPCKD cells were severely arrested at the G1-early/S transition (Fig. 6B). A similar result was previously observed for XPV (14). After hygromycin B removal, we observed that reverted XPAKD cells completely restored a normal cell cycle arrest after UVC. Interestingly, reverted XPCKD cells accumulated at the G1-early/S transition maintaining their XP phenotype.



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  		FIGURE 6. Hygromycin B withdrawal failed to reverse the XP phenotype in XPCKD HeLa cells. A. Clonogenic cell growth of XPAKD and XPCKD HeLa cells grown in the presence or absence of 125 µg/mL hygromycin B. Points, mean of three culture dishes; bars, SE. B. Flow cytometry analysis of normal (control cells), XPAKD (clone 6; 95 days after transfection), and XPCKD (clone 21; 177 days after transfection) cells. Twenty-four hours after plating at the same density, cells were irradiated (10 J/m2); adherent cells were harvested 18 hours later and fixed before staining with propidium iodide. With Hygro, cells were seeded with hygromycin B; w/o Hygro, hygromycin B was removed from the culture for 10 days.

 
We sought to determine the early sensitivity of XPAKD, XPCKD, and KIN17KD clones to an elevated irradiation dose (acute UVC irradiation) in the presence or absence of hygromycin B (reversion experiment as described in Fig. 5). Twenty hours after UVC irradiation (15 J/m2), a 5-, 6-, and 15-fold increase in the number of dead cells was observed in KIN17KD, XPAKD, and XPCKD HeLa cells, respectively, in comparison with control cells (as evidenced by 4',6-diamidino-2-phenylindole staining of adherent cells; Fig. 7; Table 2). In particular, 74% of XPCKD cells rapidly died 20 hours after irradiation. This result confirmed the clonogenic survival assays and emphasized the striking sensitivity of XPCKD cells toward UVC irradiation in the HeLa genetic background. After hygromycin B withdrawal, whereas a significant increase in the number of dead cells was always observed for XPCKD (6-fold increase in comparison with control cells), 3- and 2-fold increases were seen with KIN17KD and XPAKD cells, respectively. These observations highlighted that XPCKD HeLa cells were extremely UV sensitive soon after transfection and later.



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  		FIGURE 7. Increased cell death of XPCKD clones in comparison with XPAKD clones soon after acute irradiation. HeLa cells were seeded at the same density (400,000 cells per 6 cm diameter dish), irradiated at 15 J/m2, and analyzed 20 hours later. Cells were stained with HSAkin17, XPC, and XPA antibodies and counterstained with 4',6-diamidino-2-phenylindole. Representative images for each clone. Magnification, x350.

 

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  		Table 2. Percentages of Dead Cells 20 h after Acute UVC Irradiation

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The long-term maintenance of gene silencing in isogeneic cells may open a new area of research, in particular in the field of genetic diseases and cancer in which the loss of function of one gene takes place in a particular genetic background. These defects may be now analyzed over a long period of time. To date, the great majority of approaches have made use of integrative plasmids carrying (or not) a selection marker. Herein, we show that several months after transfection, replicative EBV vectors are stably maintained at about 10 copies per XPAKD and XPCKD cell, as evidenced by Southern blot analysis of genomic DNA. Therefore, replicative EBV vectors overcome these shortcomings and allow the establishment of cell lines presenting a stable down-regulated expression of a target gene.

The main properties of the EBV-based siRNA vectors presented here are that (i) transient gene silencing efficiencies reached a level similar to that observed with siRNA duplexes; (ii) more than 95% of cells were silenced 2 weeks after transfection (in comparison with an integrative plasmid giving rise to <50% of silenced cells); (iii) most isolated clones were silenced (e.g., 100% for XPA); (iv) in the selected clones, the protein of interest is still undetectable more than 300 days after transfection; (v) the phenotype associated with the loss of the targeted protein is maintained over the whole period; and (vi) the silencing is highly reproducible in different cell lines, such as HeLa, RKO, MRC5-V1, and immortalized human fibroblasts (data not shown), and with different vectors.

The striking efficiency of EBV-based siRNA vectors may be explained by their intrinsic features. In the presence of both EBNA-1 protein and the latent replication origin of EBV virus (oriP), EBV plasmids persist in cell nuclei as stable episomes after a long period of culture. Their DNA replication is semiconservative: it initiates at or around the DS sequence (24) and is coordinated with genomic replication (25). Host cellular factors assist the EBV DNA replication. In particular, Orc2 tightly interacted with EBNA-1, which restricted EBV replication to a single round during each cell cycle, ensuring a low vector copy number per cell and a high stability (26, 27). EBNA-1 also tethers EBV episomes to metaphase chromosomes, providing the basis for their nuclear retention and their successful segregation at mitosis (28). Hence, EBV-based siRNA vectors behave like cellular transcription units tightly controlling low levels of siRNA transcription. Our results clearly illustrate that EBV-based siRNA vectors efficiently improve RNA interference by imposing stable and long-term gene silencing in human cells.

The loss of XPA gene expression does not significantly change cell growth and cell viability even several months after transfection. In contrast, XPC and HSAKIN17 gene silencing dramatically decrease cell growth 2 months following transfection. We assume that in this period, cell metabolism compensates the decrease in XPC or HSAKIN17 gene expression. Afterwards, XPCKD and KIN17KD HeLa cells are able to bypass this biological constraint and recover regular growth. In all cases, they maintained their sensitivity to UVC. Established KIN17KD HeLa cells exhibited moderate sensitivity to UVC with about 40% survival after 4 J/m2, in comparison with 84% survival of control cells. These data are consistent with the previously reported implication of HSAkin17 protein in nucleic acid metabolism like replication and RNA processing (18-21).

The system described here can be used to compare XPAKD or XPCKD phenotypes in nearly syngeneic HeLa cells differing only in a small sequence of 40 nucleotides corresponding to the shRNA. XPCKD cells seem to be more UV sensitive than XPAKD cells (21% and 6% survival, respectively, after 4 J/m2). Our data are in apparent contradiction with previous reports on primary cells or established cell lines from XPA patients that are transcription-coupled repair– and global genome repair–deficient. These cells are more sensitive than those from XPC patients which are only global genome repair–deficient (29). However, it must be considered that HeLa cells express the viral E6 protein which impaired p53-dependent apoptosis in the transcription-coupled repair pathway, decreasing the death rate after UV irradiation. At the same time, it should be taken into account that XPAKD cells may synthesize small amounts of XPA protein that are undetectable by Western blotting. It has been previously reported that a low amount of XPA protein could partially restore the UV resistance of an established XPA cell line although it does not lead to a completely normal UV resistance (30). Therefore, we conclude that reduced apoptosis rates and a partial complementation by XPA protein may explain why the UVC sensitivity of the HeLa XPAKD cells, particularly soon after transfection, is lower than the sensitivity of the primary fibroblasts from XPA patients.

Interestingly, when XPCKD cells are cured of the gene silencing imposed by EBV plasmid after hygromycin B withdrawal, the level of XPC protein increases but does not completely reverse all the characteristics of the XP phenotype. On the other hand, when XPAKD cells lost their EBV plasmids, the XP phenotype was fully corrected. These data indicate that XPC deficiency triggers irreversible genetic defects in HeLa cells and lends further support to the idea that XPC protein plays an important role not only in DNA repair and mutagenesis but also in other important biological processes like damage signaling and cell cycle progression. The results of several other groups agree with this idea (31-33). In particular, the molecular characterization of a physical interaction between XPC and centrin-2 further reinforces the idea that a deficiency in XPC protein may decrease the efficiency of several mechanisms in which this protein is involved. We also entertain the idea that the DNA-damage sensor activity of XPC may affect other repair pathways, explaining the extreme difficulty of isolating HeLa XPCKD cells. The inactivation of this sensing activity should also contribute to the enhanced tumor development observed in XPC patients.

Alternatively, a long-term XPC deficiency may lower the concentration of hHR23B protein. A similar effect has been reported in fibroblasts from mHR23A/mHR23B double knockout mice that present reduced amounts of XPC protein as well a specific deficiency in global genome repair, resulting in a UV sensitivity comparable to that of XPC–/– cells (31). Furthermore, hHR23B protein also interferes with the global genome repair reaction through the ubiquitin/proteasome system (34), and a reduced level of hHR23B protein could hamper the recruitment of other repair factors to DNA damage sites. Therefore, a long-term XPC deficiency could trigger a down-regulation (possibly through a hypermethylation process) of the hHR23B protein in HeLa cells, leading to the lack of full recovery on reexpression of XPC. The creation of HeLa cells presenting the silencing of other DNA repair pathways will help to shed some light on the mechanistic bases of the UV sensitivity.

Taken together, our results illustrate the generation of stable cells lines mimicking a human syndrome which should help us to unravel the molecular basis of cancer proneness.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
siRNA Design and Cloning in EBV-Based siRNA Vectors
We designed XPA, XPC, and HSAKIN17 siRNA considering Tuschl's rules with or without the recent thermodynamic recommendations (8, 35-37). siRNA sequences and subsequent vectors are detailed in Table 1. To compare gene silencing efficiencies between siRNA duplexes and EBV vectors, we used two annealed siK180 and siK906 double-stranded RNAs (Ambion, Austin, Texas) stored at –20°C before use. All siRNA sequences were submitted to a BLAST search against the human genome sequence to ensure that only one gene per sequence was targeted.

Synthetic double-stranded oligodeoxynucleotides coding for shRNA sequences were synthesized (Eurogentec S.A., Seraing, Belgium) according to a model adapted from Brummelkamp et al. (10). shRNA sequences contain two identical 19-nucleotide motifs in an inverted orientation, separated by a 9-bp spacer of nonhomologous sequences (TTCAAGAGA). We added six thymidines at the 3' end of the repeat to function as a termination signal for RNA polymerase III, and the CCG sequence at the 5'end to determine the initiation site. BglII and HindIII cloning sites were fitted at the 5' and 3' extremities, respectively, of the double-stranded shRNA sequence for subsequent cloning. The cloning strategy with one oligodeoxynucleotide is summarized in Fig. 1. Two sets of vectors were constructed by introducing siRNA cassettes in a reverse or direct orientation. As control, we used plasmids carrying an H1 promoter without an shRNA sequence (pBD631 and pBD665) or containing an shRNA sequence with two mismatches in one strand of the hairpin structure (pBD650). All plasmid DNAs were purified using an anion-exchange resin and transfected (QIAGEN, GmbH, Hilden).

Cell Culture
HeLa (cervical adenocarcinoma), MRC5-V1 (SV40-transformed normal fetal fibroblasts), RKO (colorectal carcinoma), and XP44RO versus XPC-complemented XP44RO melanoma cells were maintained in DMEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin, under 5% CO2. Culture conditions were optimized for transfection of either siRNA or EBV vectors. HeLa and RKO cells were seeded at 10,000 cells/cm2 and transfected 24 hours later using 3 µL of Lipofectamine 2000 (Invitrogen) with either 2 µg of DNA or siRNA duplexes (60 nmol/L final concentration). Transfection experiments and subsequent analysis of cell populations were replicated more than 10 times.

Transfected cells were propagated in culture in the presence of hygromycin B (Invitrogen) at 250 µg/mL for HeLa and 500 µg/mL for RKO. For clonogenic growth after UVC irradiation, cells were plated at 500 cells per 6 cm diameter dish and irradiated 24 hours later. Growing clones were fixed with 4% paraformaldehyde and stained with methylene blue after 14 days of culture. Clones containing more than 50 cells were counted. Each point represents the mean of three culture dishes. Colony formation was normalized as a percentage of the control. For acute UVC irradiation (15 J/m2, cells being examined 24 hours later), each clone was seeded at 400,000 cells per 6 cm diameter dish 2 days before irradiation.

Western Blot and Immunostaining
Procedures are described elsewhere (38). We used purified immunoglobulin G K36 and immunoglobulin K58 directed against HSAkin17 protein; monoclonal antibody anti-Ku70 (clone N3H10, Lab Vision/Neomarkers, Fremont, CA); anti-Ku80 (clone 111, Vision/Neomarkers), anti-RPA70 (clone RPA70-9, Oncogene Research Products, Calbiochem, Darmstadt, Germany), {alpha}-tubulin (clone B-5-1-2, Sigma Chemical Co., St. Louis, MO), proliferating cell nuclear antigen (clone PC10, Novo Castra, Newcastle, United Kingdom), p34cdc2 (clone sc54, Santa Cruz Biotechnology, Santa Cruz Biotechnology Inc., Santa Cruz, CA), pAb{alpha}XPC (a gift from Wim Vermeulen), and monoclonal antibody XPA (a gift from Yue Zou).

Flow Cytometry
Cells were collected by trypsinization, washed with PBS, and fixed in 75% ethanol at 4°C for at least 24 hours. Cells were washed twice in PBS and nuclear DNA was stained with propidium iodide (4 µg/mL; Sigma) in the presence of RNase (10 µg/mL; Sigma) in PBS for at least 30 minutes. Stained cells were analyzed on a FACScalibur (Becton Dickinson, Franklin Lakes, NJ) using CellQuest software. 10,000 cells gated as single cells using FL2A/FL2W scatter were analyzed.

Unscheduled DNA Synthesis after UVC Irradiation
The ability of cells to repair UV-induced damage was adapted from a method previously described (23). Briefly, cell growth was arrested in a medium containing 1% serum for 48 hours. Hydroxyurea was added to a final concentration of 20 mmol/L 3 hours before UVC irradiation (254 nm) at 0, 5, 10, and 15 J/m2 and labeled for 3 hours in the growth suppression medium supplemented with [3H]thymidine, followed by 1 hour of chase with cold thymidine. Coverslips with cells were mounted onto glass slides dipped in Amersham EM-1 photoemulsion. The mean number of grains per nucleus was obtained by counting 50 non–S-phase nuclei for each UV dose.

Total DNA Extraction and Southern Blotting
Total DNA (genomic and episomal) was extracted using DNA Lock Gel (Eppendorf) and quantified. The equivalent of 8 µg of DNA was digested with either BamHI or EcoRI. Digestions were separated on a 0.6% agarose gel and submitted to Southern blotting on Hybond N+ membranes (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Blots were hybridized with a mix of pBD695 and pBD634, linearized, and [{alpha}-32P]dCTP labeled by random priming (Ready-to-go kit, Amersham).


   Acknowledgements
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank R. Agami (The Netherlands Cancer Institute, Amsterdam, the Netherlands) for kindly providing the plasmid pSUPER. We particularly thank W. Vermeulen and S. Bergink (Centre for Biomedical Genetics, Erasmus Medical Center, Rotterdam, the Netherlands) for having kindly provided XPC antibody and Y. Zou (College of Medicine, East Tennessee State University, Johnson City, Tennessee) for XPA antibody. We are grateful to C. Debacker for her contribution to the unscheduled DNA synthesis assay.

Received 4/13/05; revised 8/ 1/05; accepted 8/ 1/05.


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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