Ruthenium oligonucleotides, targeting HPV16 E6 oncogene ... - Nature

Gene Therapy (2013) 20, 435–443 & 2013 Macmillan Publishers Limited All rights reserved 0969-7128/13 www.nature.com/gt

ORIGINAL ARTICLE

Ruthenium oligonucleotides, targeting HPV16 E6 oncogene, inhibit the growth of cervical cancer cells under illumination by a mechanism involving p53 A Reschner1, S Bontems2, S Le Gac3, J Lambermont3, L Marce´lis3, E Defrancq4, P Hubert1, C Moucheron3, A Kirsch-De Mesmaeker3, M Raes5, J Piette2 and P Delvenne1 High-risk Human Papillomaviruses (HPV) has been found to be associated with carcinomas of the cervix, penis, vulva/vagina, anus, mouth and oro-pharynx. As the main tumorigenic effects of the HPV have been attributed to the expression of E6 and E7 genes, different gene therapy approaches have been directed to block their expression such as antisense oligonucleotides (ASO), ribozymes and small interfering RNAs. In order to develop a gene-specific therapy for HPV-related cancers, we investigated a potential therapeutic strategy of gene silencing activated under illumination. Our aim according to this antisense therapy consisted in regulating the HPV16 E6 oncogene by using an E6-ASO derivatized with a polyazaaromatic ruthenium (RuII) complex (E6-Ru-ASO) able, under visible illumination, to crosslink irreversibly the targeted sequence. We examined the effects of E6-Ru-ASO on the expression of E6 and on the cell growth of cervical cancer cells. We demonstrated using HPV16 þ SiHa cervical cancer cells that E6-Ru-ASO induces after illumination, a reactivation of p53, the most important target of E6, as well as the inhibition of cell proliferation with a selective repression of E6 at the protein level. These results suggest that E6-Ru ASOs, activated under illumination and specifically targeting E6, are capable of inhibiting HPV16 þ cervical cancer cell proliferation. Gene Therapy (2013) 20, 435–443; doi:10.1038/gt.2012.54; published online 19 July 2012 Keywords: antisense therapy; phototherapy; ruthenium; photoreactive complex; oligonucleotides; carcinoma

INTRODUCTION Currently, more than 200 different Human Papillomavirus (HPV) types have been described,1,2 with approximately 35 types associated with genital tract infection.3,4 Cancers caused by HPV represent 5.2% of the world cancer burden and include carcinomas of the cervix, penis, vulva/vagina, anus, mouth and oro-pharynx.5 Epidemiological evidence supports the fact that high-risk HPVs have a causal role for all these cancers, while the persistence of infection is associated with 495% of cervical cancers.6–10 The majority of HPV-associated cancer cases are squamous cell carcinomas. Several stages occur before cervical cancer development, designated as cervical intraepithelial neoplasia. Therefore, to be efficient, a therapy should be able to control precursor cervical lesions, as well as cervical cancer. The prognosis for patients diagnosed with HPV-associated carcinomas has not improved dramatically in recent years, with a 73% 5-year survival rate for cervical cancer and 59% for oral cancers, respectively.11 Thus, there is still a clear demand for improving the management of HPV-associated cancers. Surgery is the accepted gold standard for treating squamous cell carcinoma. However it is painful, in many cases cosmetically unacceptable to the patient and does not guarantee complete remission.12 Photodynamic therapy (PDT) represents an alternative to surgery for the treatment of carcinomas associated with HPV, allowing simultaneous treatment of several tumors, good patient tolerance, short healing time and good cosmetic

outcome.13 The technique is based on the application of a photosensitizer drug followed by excitation by light with wavelengths that coincide with the absorption peak of the photosensitizer leading to the generation of reactive oxygen species. Several mechanisms triggered by photosensitization will finally cause the destruction of the lesion. However, for squamous cell carcinoma, PDT with aminolevulinic acid showed poor durability accompanied by high recurrence rates of up to 50%.14,15 Therefore, improved or alternative approaches could be of great interest. Oligonucleotide-based therapeutic strategies for HPV infections have been already investigated with antisense technology and lately RNA interference.16–23 These emerging new approaches of oligonucleotide-based therapy (antisense oligonucleotidehereafter named ASO and small interfering RNA-siRNA) have generated promising results in vitro, but some limitations, such as target selection, specificity and delivery still hold up their clinical development. Therefore, the development of more successful, less toxic oligonucleotides based therapeutic strategies still requires adjustments. In this context, we have shown several years ago that some photo-oxidizing polyazaaromatic ruthenium (RuII) complexes are able to photoreact with guanine (G) bases, leading to photoadducts with the formation of a covalent bond between a G moiety and one of the TAP (1,4,5,8-tetraazaphenanthrene) ligands of the RuII complex (see structure of the photo-adduct in

1 GIGA-Cancer, Laboratory of Experimental Pathology, University of Lie`ge, Lie`ge, Belgium; 2GIGA-Research, Laboratory of Virology and Immunology, University of Lie`ge, Lie`ge, Belgium; 3Organic Chemistry and Photochemistry, Universite´ libre de Bruxelles (ULB), Bruxelles, Belgium; 4De´partement de chimie molećulaire, UMR CNRS 5250, Universite´ Joseph Fourier, Grenoble, France and 5Research Unit of Cell Biology (URBC)-NARILIS, University of Namur (FUNDP), Namur, Belgium. Correspondence: Dr A Reschner, Department of Infectious and Parasitic Disease, Faculty of Veterinary Medicine, University of Lie`ge, Boulevard du Colonster 20, Bat B34b, Lie`ge 4000, Belgium. E-mail: [email protected] Received 10 January 2012; revised 21 May 2012; accepted 12 June 2012; published online 19 July 2012

Photoreactive ruthenium oligos for gene therapy A Reschner et al

436 Figure 1a).24 The formation of such an irreversible link between the G and the Ru complex was thought to be promising in the context of cancer therapy. Indeed, by grafting the photoreactive Ru complex to an ASO (Ru-ASO), the resulting Ru-ASO conjugate could act as a sequence-specific DNA photodamaging agent (antisense strategy). Thus, it was shown that RuII complexes chemically anchored to model oligodeoxyribonucleotides (named Ru-ODNs) are able to photocrosslink with their target sequence provided that G bases, belonging to the target, are located in the vicinity of the tethered complex upon hybridization (Figure 1b, left).25 These Ru-ODN could thus behave as Ru-ASOs that would irreversibly photocrosslink with their target in living cells. The usefulness of such photocrosslinkings was demonstrated with biologically relevant systems. Elongation of a primer in a DNA matrix by two different polymerases (Klenow fragment, exo and polymerase b) was investigated in the presence of a photoreactive 17 mer Ru-ODN conjugate.26 In that case, the elongation was stopped with 100% efficiency at the level of the photocrosslinking. The resistance of the photocrosslinking to the 30 -exonucleolytic activity of the exonuclease III was also demonstrated.27 More recently, the anchoring of Ru complexes to G-containing ODNs (Ru-ODNG) led to a new generation of Ru-ODNs that are able to self-inhibit in the presence of a wrong target and under illumination (this intramolecular photoreaction of the single strand was called ‘seppuku’) (Figure 1b, right).28,29 Such a process is expected to limit undesired off target side reactions. Therefore, using these Ru-ODN probes as Ru-ASO photosensitizers, which are activated by a blue light (less penetrating than the red light4600 nm usually chosen for classical PDT30), represents a promising alternative for gene silencing, with a higher specificity and less off-target effects than in the usual PDT. HPV-associated cancers represent an attractive model for testing gene-specific therapy, because knockdown of HPV oncogenes E6 and/or E7 may result in cancer cell senescence/ apoptosis. E6 and E7 are critical for the ongoing proliferation of HPV-infected cells.31,32 E6 forms complexes with p53 and abrogates its function, resulting in tumor progression.33 The role

of E6 in HPV-mediated carcinogenesis may be to suppress p53-mediated apoptosis.34 This implies that the E6 oncogene is an ideal target for targeted gene-silencing therapy in HPV þ cervical cancer. Therefore, anti-E6 ASOs could be the interesting candidates. In this study, we tested ASO tethered to a photoreactive Ru complex (see structure in Figure 2), targeting E6 in HPV16 þ SiHa cells activated under visible illumination. Our data show that E6-Ru ASOs are able to inhibit efficiently HPV16 þ SiHa cell growth in monolayer and in three-dimensional cultures, while specifically downregulating E6 at the protein level and reactivating p53. RESULTS Evaluation of the efficiency of the different Ru-ASO by gel electrophoresis For the presentation of the results, we will use P for the probe ODN sequence, Ru-P when this sequence is tethered to the photoreactive complex, Scr when the ODN corresponds to a scramble sequence versus the target sequence and the number 6 or 4 when the ODN probe targets a sequence specific of the gene E6 at positions 324 or 187 (which can be found, respectively, in the sixth and fourth lines of the nucleotide sequence, Gene ID 1489078, Gene Bank). Thus, we prepared and examined Ru-P-6, Ru-P-4 and Ru-Scr. Polyacrylamide gel electrophoresis (PAGE) experiments were performed to test the efficiency of these photoreactive probes (Figures 2 and 3). In the absence of the target strand, Ru-P-6 and Ru-P-4, led to ‘seppuku’ adducts (thus to an intramolecular photoreaction with a G base of their own sequences) with yields between 70 and 80% (see lanes 5, 6 and 2 for single strand after 15, 30 or 60 min post illumination Figures 3a and b). In the presence of the complementary sequence (C-6 and C-4), both Ru-P-6 and Ru-P-4, led only to photocrosslinking, with yields in the range of 80% (lanes 4, 7 and 8 for double-strand post illumination, Figures 3a and b). In contrast, the scramble sequence, Ru-Scr, led only to ‘seppuku’ adducts in the presence of the target sequences C-6 and C-4 (Figures 3c and d). These data show that

Figure 1. (a) Structure of the photoreactive [Ru(TAP)2phen]2 þ complex and the resulting photo-adduct with guanosine monophosphate. (b) Left: photocrosslinking between a Ru-ODNG conjugate as the probe sequence and the G-containing complementary strand as a target sequence. Right: intramolecular photoreaction when the Ru-ODN probe is facing the noncomplementary target (seppuku process). The circle around the Ru symbolizes the complex with the 30 end-tethered ODNG probe and, when connected to the G, the adduct formed after illumination, whose structure is shown in Figure 1a. Gene Therapy (2013) 435 – 443

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Figure 2. Structure of the photoreactive [Ru(TAP)2(Phen)]2 þ complex tethered with a linker to the 30 end of the ODN probe (non-modified ODN) and the different sequences used in this study. The circle around the Ru symbolizes the whole-attached complex; Number 6 and 4 corresponds to the sequence for targeting E6 gene at position 324 and 187 E6. C, complementary sequence; P, probe; Scr, scramble sequence.

Figure 3. Photoreactivity of the Ru-ODN from PAGE experiments: (a) Ru-P-6 and Ru-P-6/C-6, (b) Ru-P-4 and Ru-P-4/C-4, (c) Ru-Scr and Ru-Scr/C-6, (d) Ru-Scr and Ru-Scr/C-4. The Ru-ASO were 50 end 32P radiolabelled, the samples (5 mM in 10 mM Tris-HCl buffer pH 7, [NaCl] ¼ 50 mM) were illuminated at 33 1C with a laser source (lex ¼ 442 nm) and the gels were run in denaturing conditions (urea, 7 M). The arrow indicates the direction of migration (from top to bottom). Lanes 1 ¼ non-illuminated single strand Ru-ODN; lanes 2, 5, 6 ¼ illuminated single-strand Ru-ODN during 60, 15, and 30 min respectively. Lanes 3 ¼ non-illuminated Ru-ODN in the presence of target. Lanes 4 ¼ Ru-ODN illuminated in the presence of the target during 60 min, and the same for lanes 7 and 8 but illuminated during 15 and 30 min, respectively.The difference in mobility between the singlestrand and double-strand ODN irreversibly photocrosslinked by a Ru complex for 17 and 21 mer ODNs has been shown in reference Lentzen et al.27 & 2013 Macmillan Publishers Limited

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Figure 5. Ru-P-6 ASO reduces E6 protein expression in SiHa cells in vitro in monolayer cultures. Expression level of E6 protein was determined by western blot in SiHa cells treated with 200 nM of indicated ASO; illuminated under blue light for 2 h 30 min and sampled 24 h post illumination. b-actin expression was used as control of the total protein load. P, probe; Ru, ruthenium; 6 and 4-sequence number; Scr, scramble, Unt, untreated.

Figure 4. Ru-ASO induces growth inhibitory effects in SiHa cells in vitro in monolayer cultures (a) Effects of Ru-P-6 and Ru-P-4 on the growth of SiHa cells; (b) Effects of Ru-P-6 and Ru-P-4 on the growth of HaCat cells. Cells were treated with the indicated ASO (200 nM) and subsequently illuminated for 2 h 30 min. The cell growth was evaluated by measuring the fluorescence in an Alamar Blue assay 24 h post irradiation; black squares, triangles, spheres and diamonds-Non-illuminated conditions; empty squares, triangles, spheres and diamonds-Illuminated conditions. Stars indicate statistical significants *Pp0.01; **Pp0.001; ***Pp0.0001. P, probe; Ru, ruthenium; 6 and 4-sequence number; Scr-scramble, Unt, untreated.

the Ru-probes behave as expected, leading either to photocrosslinking when the specific target (thus the complementary sequence) is present or to ‘sepukku’, that is, intramolecular photoadducts, in the presence of a noncomplementary sequence (thus when Ru-Scr is used) or in the absence of any target. Ru-P-6 ASO exerts inhibitory effects on human cervical cancer SiHa cells growth in vitro Having no fluorescent marker on the Ru-ASO, Ru-free ASO labelled with fluorescein isothiocyanate was used for obtaining an estimation of the ASO uptake. SiHa cells were treated with 200 nM Ru-free ASO labelled with fluorescein isothiocyanate, in the presence of Oligofectamine. FACS analysis performed on treated cells after 24 h showed 490% uptake efficiency (data not shown). To determine the effect of the Ru-ASO on the proliferation status of SiHa cells, a metabolic Alamar Blue assay was used. The two Ru-ASOs tested—Ru-P-4 and Ru-P-6—induced growth inhibition of SiHa cells, but to a different extent, with Ru-P-6 showing higher specificity and efficacy (Figure 4a). The treatment with Ru-P-6 followed by visible illumination resulted in 45–50% cell growth inhibition for the SiHa cells 24 h post illumination, as compared with their counterparts not subjected to illumination (Po0.0001, Figure 4a). The inhibition of growth was higher for the experimental condition corresponding to Ru-P-6 illuminated, when compared with Ru-Scr illuminated (Po0.0001, Figure 4a). In parallel, treatment with Ru-P-4 followed by illumination induced only a 30% growth inhibition when compared with the non-illuminated counterpart (Po0.001, Figure 4a). In contrast, for the Ru-P-4 sequence, the inhibition induced by the specific Gene Therapy (2013) 435 – 443

sequence after illumination was not different from the inhibition induced by the Ru-Scr (Po0.1418, Figure 4a), suggesting a low efficiency for this Ru-P-4 ASO sequence. The antiproliferative effect observed for the Ru-Scr could be due to the cytotoxicity of the Ru complex combined with the illumination, as no such effect was detected for the non-illuminated condition or any of the blank controls (illuminated or not illuminated). When both Ru-ASOs— Ru-P-4 and Ru-P-6—were transfected into HaCat cells, which do not contain the ASO target E6, no growth inhibition was observed after illumination (Figure 4b). The 10–20% inhibition observed for the Ru-sequences in both illuminated and non-illuminated conditions might be due to Oligofectamine cytotoxicity, HaCat nonmalignant keratinocytes being more sensitive than the tumor SiHa cells. Ru-P-6 ASO reduces E6 protein expression in SiHa cells in vitro In order to investigate the specificity of Ru-P-6 ASO treatment, we checked the level of E6 protein expression by western blot. As shown in Figure 5, a remarkable decrease of E6 protein expression (460% when normalized to b-actin) was detected 24 h post illumination. In agreement with the inhibition of proliferation, a limited effect (10–25% when normalized to b-actin) was observed in the cells treated with Ru-Scr ASO, illuminated or not (Figure 5). Ru-P-6 ASO reactivates the expression of the p53 protein in SiHa cells in vitro It is known that E6 protein promotes the degradation of p53, inhibiting its stabilization and leading to apoptosis.35,36 Therefore we verified if the reduction of E6 protein expression observed in our system was able to restore p53 expression. Confocal microscopy performed 3 h after illumination showed increased p53 abundance in all illuminated conditions (Figures 6b,d and f) when compared with the non-illuminated counterparts (Figures 6a,c and e). The most remarkable increase was detected for the Ru-P-6 ASO treated and illuminated cells, suggesting a reactivation of p53 in this specific condition (Figure 6 f1 and f2). Moreover in cells treated with Ru-Scr ASO, no increase in p53 expression was observed (Figures 6c and d). Ru-P-6 ASO induces antiproliferative effects in organotypic cultures Even though many of the oligonucleotide-based therapies proved very efficient in monolayer cultures, this may not be the same in an in vivo microenvironment. Therefore, we tested the efficacy of Ru-ASOs in a three-dimensional culture, namely organotypic culture, using the two E6-Ru-ASO sequences tested in monolayers. Haematoxylin and Eosin staining performed at 2 days post illumination of organotypic cultures did not show any striking difference in terms of morphology and thickness of the culture between illuminated and non-illuminated conditions (data not & 2013 Macmillan Publishers Limited


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Figure 6. Ru-P-6 ASO treatment induces p53 reactivation. SiHa cells were transfected with the indicated complexes and then subjected or not to illumination as indicated above each panel. Three hours after illumination, cells were fixed, permeabilized and stained for p53 using a specific antibody (green). Nuclei were detected with DAPI (blue). Examination was performed using a confocal microscope with the photomultiplier constant. Magnification 40. Panels f1 and f2 are showing two different regions of the same slide. I, illuminated; NI, nonilluminated; P, probe; Ru, ruthenium; 6 and 4-sequence number; Scr, scramble, Unt, untreated.

Figure 7. Ru-P-6 ASO induces growth inhibitory effects in SiHa cells in vitro in organotypic cultures. Ki-67 immunohistochemical staining of organotypic cultures treated with Ru-Scr (a) Ru-P-6 (b) Ru-P-4 (c) magnification 40. Proliferation index –Ki-67 index–in organotypic cultures treated with Ru-ASO (d). I, illuminated; NI, non-illuminated; P, probe; Ru, ruthenium; 6 and 4-sequence number; Scr, scramble, Unt, untreated.

shown). To investigate the proliferation status of the cells stratified in the organotypic cultures we used Ki-67 antigen as marker of proliferation. Ki-67 antigen is present in the S, G2 and M phases of the cell cycle and its cellular expression is indicative of the cell proliferation phase. As shown in Figure 7c and d, the transfection & 2013 Macmillan Publishers Limited

with Ru-P-4 followed by illumination did not result in any inhibition of cell proliferation, supporting the results obtained in monolayer. In contrast, in agreement with the data on monolayers, transfection with Ru-P-6 followed by illumination induced a remarkable decrease (52%) in the proliferation of the cells in the Gene Therapy (2013) 435 – 443


440 organotypic cultures (Figures 7b and d) compared with 12% of growth inhibition observed with the Ru-Scr (Figures 7a and d). DISCUSSION Even though prophylactic Human Papillomavirus (HPV) vaccines were approved in several countries and were shown to be effective in clinical trials, it is estimated that they will reduce cervical cancer incidence by 70% and several decades will be required to determine duration of protection and degree of cross protection from other HPV types.37,38 Therefore, the promising concept presented in this study is nevertheless of interest for cervical cancer and may moreover be applicable to nonmelanoma cancers in the skin as well. Several studies demonstrate high efficiency of topical PDT in non-melanoma skin cancers such as for actinic keratosis (precancerous lesions),39–42 Bowen’s disease43–45 and superficial basal cell carcinomas,46–48 but current evidence does not support the use of topical PDT for invasive squamous cell carcinomas.49–51 PDT can also reduce the number of new lesions in patients at high risk of skin cancers and therefore may have a role as a preventive therapy.52 PDT was shown as being as effective as conventional therapies, but with shorter healing times and the absence of scarring in treatment of vulval intraepithelial neoplasia.53,54 Moreover, the presence of high-grade dysplasia and/or high-risk HPV was associated with a poor response to PDT in vulval intraepithelial neoplasia.55 In spite of some clinical trials performed for different pathologies, for gynaecology, the PDT is still in the experimental stage with not enough clinical evidence in favour of a spread PDT utilization.56 Among metal-based therapeutic agents against cancer that emerged in recent years, ruthenium, a metal from the second generation (post-platinum) agents represents a very promising one.57,58 Ru-ASOs being able to self-inhibit under illumination in the absence of the target (‘seppuku effect’) confers an increased level of selectivity, circumventing the side reactions that can occur in biological systems.28 In the same line the visible illumination needed is the blue light (wavelengths 450–495 nm), which is less penetrant than red light (wavelength 4600 nm), mainly used in oncology PDT.52 Even though the blue light is closely related to the UVA spectrum (320–400 nm; responsible for 20% of carcinogenic effects of sunlight), the short term use of visible blue light in dermatological practice was proven to be safe.59 By tethering Ru to an ASO sequence we aimed at increasing antitumor activity, while keeping nonspecific toxicity at a low level. The presence of the ‘seppuku effect’ reduces the toxicity by eliminating undesired secondary photoeffects such as photoreaction with tryptophan containing proteins.60 Previous antisense based strategies were used for HPV suppression, but the inhibition effects on the gene expression of such approaches were limited and growth inhibition was not sufficient.61,62 The present study demonstrates that a strategy of gene silencing activated under illumination specifically targeting E6 is able to inhibit HPV16 þ cervical cancer cell proliferation, in spite of the fact that HPV16 E6 and E7 target sequences have been shown to be not well suited for antisense inhibitors.63 However effective sequences can be identified when psoralen-conjugated ASOs and UV irradiation were used or a combination of ASOs meant to overcome the potential mutations frequently reported within HPV genome.22,64 This could explain why the Ru-P-4 proved not efficient in the HPV16 þ cells, while it efficiently photocrosslinks in solution in the presence of a synthetic target. The E6-Ru ASOs tested here were generated in an attempt to enhance the effectiveness of the antisense therapy. Our study shows that nonmodified ASO had no antiproliferative effect, while by tethering them to a RuII complex, and after illumination, we succeeded to render them effective against the tumor cells and displaying a low cytotoxicity for normal keratinocytes. Therefore, the Gene Therapy (2013) 435 – 443

photocrosslinking of the Ru-ASO to its target sequence reinforces the regulatory effects of ASOs on gene expression. As shown in this study the Ru-ASO denominated Ru-P-6 proved to be highly efficient in terms of specificity and growth inhibition. A key event in cervical carcinogenesis is the degradation of p53 tumor suppressor pathway by high-risk HPV E6 oncogene.65 Under normal circumstances p53 promotes cell cycle arrest or initiates apoptosis in response to cellular stress. We demonstrated that targeting E6 with Ru-P-6 ASO prevents p53 degradation, restoring its antiproliferative role, which was abrogated by E6. Moreover, its efficacy was of long duration as it inhibited proliferation 3 days after transfection in organotypic cultures. The cellular screening system employed in this study was reliable for selecting effective sequences. The sequence that proved to be efficient in monolayer cultures was also effective in a three-dimensional model (organotypic cultures). This model, which is reminiscent of in vivo situation, may give a better insight regarding the performances of these Ru-ASOs in vivo. A similar combination of PDT and antisense psoralen oligonucleotides was used by Yamayoshi et al.22 and induced apoptosis in HPV-18 þ cervical cancer cells by repressing E6 and stabilising p53. While they used psoralen-conjugated ASO and UV light, we succeeded to obtain similar results in this study by using Ru-ASO and blue light applied on HPV16 þ cells. In addition, when Yamayoshi et al.22 added a low concentration of cisplatin to the treatment they observed a further upregulation of p53, causing massive apoptosis induction. Another study provided evidence that the loss of E6 and E7 results in increased sensitivity to chemotherapeutic agents such as cisplatin, which is commonly used for the treatment of cervical cancer.66 Altogether these data suggest that a strategy of gene silencing activated under illumination photodynamic antisense strategy could represent an interesting approach to complement existing therapy.

MATERIALS AND METHODS Cell culture The HPV16 þ keratinocyte cell line (SiHa-ATCC HTB-35) and normal cervical keratinocyte cell line (HaCat-CLS, Germany) were grown at 37 1C in a humidified atmosphere with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 5% nonessential aminoacids, sodium pyruvate, glutamine and 10% foetal calf serum (Life Technologies, Invitrogen, Paisley, UK). For the organotypic cultures the growth medium was a 1/3 mixture of HAM F12/Dulbecco’s modified Eagle’s medium, supplemented with 10% foetal calf serum, 2 mmol l 1 L-glutamine, 10 mmol l 1 Hepes, 1 mg ml 1 fungizone, 1 mmol l 1 sodium pyruvate, 3000 U ml 1 penicillin streptomycin (Life Technologies), 0.5 mg ml 1 hydrocortisone, 10-10 mol l 1 cholera toxin 5 mg ml 1 insulin, 20 mg ml 1 adenine and 5 mg ml 1 human transferrin (Sigma Chemical Co, St Louis, MO, USA).

Organotypic cultures Organotypic cultures of SiHa cells were prepared by procedures slightly modified from those described previously.67–69 For the preparation of dermal equivalent, the final concentration of collagen (MP Biomedicals, Illkirch, France) was 6.4 mg ml 1 and the final fibroblast density was 5 104 cells ml 1. After gel equilibration with 1 ml of growth medium overnight at 37 1C, 15 104 SiHa cells re-suspended in 100 ml of growth medium were seeded on top of the gel and maintained submerged for 24 to 96 h. The collagen rafts were raised in a 25 mm tissue culture insert (8 mm pore size; Nunc, Roskilde, Denmark) and placed onto stainless-steel grid at the interface between air and liquid culture medium. Epidermal cells were then allowed to stratify over 1 week. After stratification of keratinocytes, Ru-ASO Oligofectamine (Life Technologies) complexes were added on top of the in vitro-formed epithelium. After 24 h at 37 1C and phosphate-buffered saline (PBS) washing, they were illuminated and 2 days post illumination the cultures were embedded in paraffin and sectioned with a microtome for the immunohistochemical analysis. Ki-67 was immunohistochemically detected by the MIB-1 monoclonal antibody (Immunotech, Marseille, France). Ki-67 proliferation index was calculated & 2013 Macmillan Publishers Limited


441 by counting positively and negatively stained cells in 3–5 different microscopic fields on the same slide and expressed as percentage of positively stained cells divided by the total number of cells counted.

Design and choice of the sequence Any sequence of the gene to be silenced can be selected provided that it contains G bases and it satisfies the following conditions. To insure a better stability of the Ru-ASOs towards 30 -exonucleases, the Ru complexes were anchored at the 30 end of the probe strands. Therefore, the target sequences of the E6 gene had to contain G bases at their 50 end in order to obtain a photocrosslinking of the Ru-ASO to the target sequence. Conversely, the presence of cytosine bases at the 50 end of the target sequence should be avoided in order to prevent a self-inhibition of the RuASO in the duplex form. Indeed, if G bases are located on the Ru-ASO strand, close to the Ru attachment site, they can be reached by the tethered Ru complex in the duplex form, and this photoreaction with these G bases in the probe sequence will be in competition with the photocrosslinking process inside the duplex. On the basis of these considerations, two target sequences (21 mer) belonging to the sequence of the E6 oncogene were chosen, namely C-6 corresponding thus to the probe Ru-P-6 (position 324) and C-4 with the probe Ru-P-4 (position 187) (Figure 2). They contain respectively 3 and 4 G bases that are accessible to the attached Ru complex in the duplex form, thus favourable to the photocrosslinking. The closest cytosine bases in these targets, are located 13 ( case of C-6) and 10 (case of C-4 ) bases away from the attachment site of the Ru complex on the probe after hybridization of the two complementary strands, therefore the corresponding G bases on the probe sequence are not reachable in the duplex form (confirmed by PAGE experiments, data not shown). A Ru-ASO scramble was also selected, namely Ru-Scr (Figure 2).

(Life Technologies), to which Oligofectamine was added. The mixtures were incubated at room temperature for 20 min. Transfection complexes were added to wells and incubated overnight at 37 1C. After incubation, the complexes were removed and replaced with complete Dulbecco’s modified Eagle’s medium. An estimation of transfection efficiency was established by transfecting the cells with 200 nM ASO conjugated with fluorescein isothiocyanate under the same experimental conditions as for the Ru-ASO.

Illumination of keratinocytes in monolayer and organotypic cultures Illumination was performed using blue light radiation source 380–480 nm, 4 24 W intense blue bulbs (Actireef-T5-intense-blue 24W, Zolux (Saintes, France)). The distance between the light source and the culture plate was 25 cm. Before illumination the cultures were rinsed with PBS and illuminated in PBS to avoid absorption by coloured culture medium. Plates serving as a dark control were placed on the illumination block but prevented from illumination by covering them with aluminium foil. All the monolayers and organotypic cultures were kept on a heating block set at 33 1C during the 2 h 30 min of illumination. Illuminated and control cultures were immediately returned to the incubator at 37 1C in a humidified environment and cultured in culture media for additional 24 or 72 h.

Growth assay in monolayer Twenty four hours post illumination the cellular proliferation status of SiHa and/or HaCat cells was measured using the Alamar blue assay (AbD Serotec, Oxford, UK) according to the manufacturer’s instructions. Briefly, Alamar Blue was added to the cells in a volume equal to 10% of the well volume and incubated during 2–3 h. Then the media were collected, distributed in triplicate in 96-well plates and the fluorescence was read (excitation at 560 nm and emission at 590 nm) using a spectrofluorimeter (Bio-Rad).

Synthesis of Ru-ASOs The synthesis and purification of [Ru(TAP)2(phen")]2 þ (Figure 2) (TAP, 1,4,5,8-tetraazaphenanthrene; phen, 1,10-phenanthroline) the preparation of the modified oligonucleotides and the procedure for the coupling of the Ru complex to the 30 end of the ODNs were previously reported.70–72 The Ru-ASO conjugates were purified by PAGE and characterized by electrospray mass spectrometry. The complementary sequences (non-modified), namely C-6 and C-4 (Figure 2) were synthesized with a DNA automatic synthesizer and characterized by electro-spray mass spectrometry. The non-modified ASOs P-6 and P-4 (Figure 2) used for in vitro experiments were purchased from Eurogentec, Seraing, Belgium.

Electrophoretic experiments Radiolabelling at the 50 position of the Ru-ASOs were realised with 25 pmol of conjugate by incubation with T4 polynucleotide kinase and [g-32 P] ATP at 37 1C for 20 min. ATP and kinase in excess were removed by exclusion chromatography with Micro Bio-Spin P6 in Tris buffer (Bio-Rad, Hercules, CA, USA). The samples were prepared at a final concentration of 5 mM in an aqueous buffer (Tris-HCl 10 mM, NaCl 50 mM, pH 7). The hybridization was performed by incubating the labelled Ru-ASO with its target strand (1.1 equivalents) at 85 1C for 10 min followed by slow cooling to room temperature. The illuminations for the PAGE experiments were performed with a monochromatic laser (He/Cd, 442 nm, 50 mW, Melles Griot, British Isles, UK). The PAGE experiments were performed through a denaturing (7M urea) 20% polyacrylamide gel (19:1 ratio of acrylamide to bisacrylamide) in TBE buffer (Tris Borate EDTA; 90 mM Tris-borate, pH 8; 2 mM ethylenediaminetetraacetic acid). The ASO bands were visualized by autoradiography with a Storage Phosphor Screen (GE Healthcare, St Gilles, UK) film and were counted with a Phosphor-Imager Storm 860 instrument.

Cell transfection Four groups were included in the experiment: the Ru-P-6/Ru-P-4 ASO complexes as experimental group, Ru-Scr ASO complexes and P-6/P-4 ASO complexes as negative control, and transfection reagent (Oligofectamine) without ASO as blank control. Two different kinds of experiments were carried out for each group using illuminated and non-illuminated cells, respectively. SiHa and/or HaCat cells were transiently transfected with Oligofectamine (Life Technologies) according to the manufacturer’s instructions. Briefly, the day before transfection the cells were plated out at a density of 150 000 cells per well in a six-well plate. ASOs were diluted to concentrations of 200 nM in Opti-MEM I reduced media & 2013 Macmillan Publishers Limited

Immunofluorescence staining and confocal microscopy SiHa cells were seeded at 80 000 cells per well on Lab Teck chamber slides (Nunc), transfected and illuminated or not as described in the experimental conditions. Three hours post illumination medium was removed and cells were fixed 15 min with PBS containing 4% paraformaldehyde. Cells were permeabilized in methanol for 5 min and then blocked in PBS þ 3% bovine serum albumin (Sigma-Aldrich NV/SA, Bornem, Belgium) for 1 h. The primary antibody (mouse anti-p53 DO-7, dilution1/150, Dako, Aachen, Germany) was added in PBS þ 3% bovine serum albumin overnight at 4 1C in a wet chamber. The day after, cells were washed 3 3 min in PBS before adding the secondary antibody (biotinylated goat anti mouse-IgG (H þ L); Biotin XX-Invitrogen, dilution 1/500) in PBS þ 3% bovine serum albumin for 45 min followed by a 30 min incubation with a streptavidin Alexa Fluor488-conjugate (dilution 1/500, Life Technologies) in humid conditions at room temperature. Cells were washed three times with PBS. To visualize the nucleus, cells were incubated 5 min at room temperature in the presence of DAPI (1: 2000 dilutions in PBS þ 3% bovine serum albumin). The slides were finally mounted in Mowiol (Sigma) and observed with a confocal microscope (Olympus, Aartselaar, Belgium) using a constant photomultiplier.

Western blot analysis SiHa cells transfected according to the four experimental groups were lysed in M-PER buffer (Thermo Fisher Scientific, Erembodegem, Belgium) 24 h after transfection. Total protein lysates from HaCat cells were used as negative control. Protein concentrations were determined with Bradford Protein Assay (Bio-Rad). Cell lysates (25 mg) were then resolved on a 12% SDS-PAGE gel, migrated in a Bio-Rad Criterion cuve, blotted on a nitrocellulose membrane, probed with anti HPV16 E6 antibody/ascites fluid (clone 1F1- kind gift from Professor Weiss E, Institute Gilbert Laustrian, Illkirch, France), incubated with rabbit anti-mouse (Dako) IgG-horseradish peroxidase and visualized using Enhanced Chemiluminescence Substrate Signal (ECL-GE Healthcare, Chalfont, UK) detection. The same blot was reprobed for human b-actin used as loading control, using a monoclonal anti-b-actin antibody (Sigma).

Statistical analysis To determine the significant difference of parameters between the samples subjected to different treatments, a paired Student’s test was used (Graph Pad Software, San Diego, CA, USA). Gene Therapy (2013) 435 – 443


442 CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS The research presented here was supported by the Walloon Region (CARCINOM Waleo-2 project). LM thanks the FNRS for a fellowship (‘aspirant’ at the FNRS). The authors are grateful to a NanoBio programme (Grenoble) for the facilities of the Synthesis platform.

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