The HNSCC SQ20B cell line was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The radioresistant SQ20B cells have been reported to express non-funtional p53 protein (10). The hHNSCC UPCI:SCC90 cell line (9) was a kind gift from Professor Susan Gollin (University of Pittsburgh, Pittsburgh, PA, USA). UPC1:SCC90 cells originate from a HPV16-positive oropharyngeal tumor, and bear wild-type TP53 (9). UPCI:SCC90 cells have been shown to be more radiosensitive than SQ20B cells (11). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech GmbH, Dominique Dutscher, Brumath, France) according to standard conditions.

All transfection experiments were carried out using Lipofectamine™ 2000 (Invitrogen, Fisher Scientific, Illkirch, France) according to the manufacturer’s instructions. UPCI:SCC90 cells (5×10⁵) were transfected with 4 μg of the NAE1/APP-BP1 expression vector (12) (kind gift from Dr Dimitris Xirodimas). Mock transfections with 4 μg of the empty pcDNA3 vector (Invitrogen) or the empty pCI-neo vector (Promega, Charbonnières-les-bains, France) were used as the negative controls. p53 was overexpressed by transfection of 5×10⁵ SQ20B cells with 4 μg of pC53-C1N3 plasmid.

The NAE1/APP-BP1 downregulation experiments were performed using the ON-TARGET plus SMARTpool (L-006401-00-0005; Dharmacon, Fisher Scientific, Illkirch, France). This pool is composed by the combination of 4 single siRNAs: siRNA-1, upgrade siRNA NAE1/APP-BP1 J-006401-05; siRNA-2, upgrade siRNA NAE1/APP-BP1 J-006401-06; siRNA-3, upgrade siRNA NAE1/APP-BP1 J-006401-07; and siRNA-4, upgrade siRNA NAE1/APP-BP1 J-006401-08. The ON-TARGET plus non-targeting pool (D-001810-10-05) was used as the negative control. siRNAs (40 pmol) were transfected into 4×10⁴ SQ20B cells using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instructions.

RNA was extracted from 5×10⁵ cells and cDNA synthesis was performed as previously described (3). Cycle threshold (Ct) levels were normalized to the average Ct values of 3 internal controls (housekeeping genes): ubiquitin B (UBB), 18S rRNA and hypoxanthine phospho-ribosyltransferase 1 (HPRT1). Primers specific for the genes of interest were used: HR-HPV16 E6/E7 mRNA forward, 5′-GTTACTGCGACGTGAGGTA TATG-3′ and reverse, 5′-CATTTATCACATACAGCATATGG ATTC-3′ (13); NAE1/APP-BP1 forward, 5′-CTGGAAAGCT GCTCAAGG-3′ and reverse, 5′-TCTTCTCCGCTGACCT GATT-3′; CDKN2A^p16 forward, 5′-GCTGCCCAACGCACCGA ATA-3′ and reverse, 5′-ACCACCAGCGTGTCCAGGAA-3′; WAF1/CIP1^p21 forward, 5′-ATGAAATTCACCCCCTTTCC-3′ and reverse, 5′-CCCTAGCTGTGCTCACTTC-3′; PUMA forward, 5′-ACGACCTCAACGCACAGTACGA-3′ (14) and reverse, 5′-GTAAGGGCAGGAGTCCCATGATGA-3′ (14); MDM2 forward, 5′-GGTGGGAGTGATCAAAAGGA-3′ and reverse, 5′-GTGGCGTTTTCTTTGTCGTT-3′; HPRT1 forward, 5′-TGCTCGAGATGTGATGAAGG-3′ and reverse, 5′-GT CCCCTGTTGACTGGTCATT-3′; UBB forward, 5′-GCTTTG TTGGGTGAGCTTGT-3′ and reverse, 5′-CGAAGATCTGCA TTTTGACCT-3′; 18S rRNA forward, 5′-TGTGGTGTTGA GGAAAGCAG-3′ and reverse, 5′-TCCAGACCATTGGCT AGGAC-3′. Gene expression levels observed in the non-irradiated mock-transfected cells were artificially set to 100, and other expression values were normalized with respect to this level. Differences were considered statistically significant when p<0.05 (ANOVA).

Cells in the exponential growth phase were detached, counted and plated in 6-well plates in 2 ml of growth medium 24 to 48 h prior to irradiation, in order to allow them to attach and undergo transfection. X-ray irradiation was performed with 6 MV photons at the Department for Radiation Therapy of the Paul Strauss Cancer Centre (Strasbourg, France). Clonogenic assays were performed as previously described (15). In brief, cells were collected 24 h after irradiation, diluted, seeded and allowed to grow for up to 3 to 4 weeks after irradiation. Clones were stained with 0.05% crystal violet (Sigma-Aldrich, Lyon, France) in a 5% ethanol solution, and positive colonies (>64 cells) were counted. The SF2 was calculated by dividing the number of positive colonies by the number of cells that were seeded, multiplied by the plating efficiency. The plating efficiency was calculated by dividing the number of positive colonies that grew in the absence of irradiation by the number of cells that were seeded. Differences were considered statistically significant when p<0.05 (ANOVA).

Cells (4×10⁴ to 5×10⁶) were harvested in lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-Cl pH 8.0, 0.5% DOC, 0.1% SDS) and proteins were analyzed by SDS-PAGE according to standard methods. Proteins were detected with the following antibody dilutions: polyclonal mouse AO1 anti-APP-BP1 (1:1000; Abnova, Tebu-Bio, Le-Perray-en-Yvelines, France), monoclonal mouse DO1 anti-p53 (1:1000; Santa Cruz Biotechnologies, CliniSciences, Montrouge, France), polyclonal rabbit anti-p53 FL-393 (1:200; Santa Cruz Biotechnologies), anti-NEDD8 rabbit polyclonal (1:2000; Alexis Biochemicals, Enzo Life Sciences, Villeurbanne, France), mouse monoclonal CM1 anti-β actin (1:1000; Abnova), anti-β tubulin (1:5000; Sigma-Aldrich), enhanced chemiluminescence (ECL) sheep anti-mouse IgG, HRP-conjugated (1:10000; GE Healthcare, Saclay, France), and anti-rabbit IgG, HRP-linked antibodies (1:20000; Cell Signaling, Ozyme, Saint-Quentin-en-Yveline, France). Proteins were revealed with the ECL western blot analysis system (GE Healthcare) according to the manufacturer’s instructions. Quantification of the signals was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) (16,17).

With the use of the Lipofectamine™ 1000 transfection system, 5×10⁵ UPCI:SCC90 cells were co-transfected with 0.75 μg of PG13-Luc plasmid (kind gift from Dr Arnold Levine) or MG15-Luc plasmid, 0.75 μg of RSV-LacZ vector [used as a transfection efficiency control (18)], and increasing amounts (0, 1.0, 1.5, 2.5 μg) of either the NAE1/APP-BP1 expression vector or of the empty vector (pcDNA3). The total amount of transfected plasmid was made up to 4 μg with pUC19 (New England Biolabs). Cells were harvested 36 h after transfection in lysis buffer (25 mM Tris-phosphate, pH 7.8; 2 mM EDTA; 1 mM DTT; 10% glycerol; 1% Triton X-100), and luciferase activity was evaluated by mixing 50 μl of protein extract with luciferase assay buffer (265 μM ATP; 235 μM luciferin; 135 μM coenzyme A; 20 mM tricine; 1.07 mM MgCl₂; 2.70 mM MgSO₄; 0.10 mM EDTA; 33.3 mM DTT) using a MikroWin 2000 microplate reader (Mikrotek Laboratories GmbH, Overath, Germany). The luminescence levels were normalized to the β-galactosidase activity from the RSV-LacZ control plasmid that was measured with an o-nitrophenyl-β-galactoside (ONPG) colorimetric reaction. In brief, β-galatcosidase activity was evaluated by mixing 25 µl of cell extract with 150 µl of ONPG staining solution (0.666 mg/ml ONPG; 2% β-mercaptoethanol; 60 mM Na₂HPO₄; 40 mM NaH₂PO₄; 10 mM KCl; 1 mM MgSO₄), and o-nitrophenol was monitored on a Berthold 900 spectrophotometer using Gen5 Data analysis software. Fluorescence observed in cells transfected with 0 μg of NAE1/APP-BP1 expression vector was set at a value of 100, and other measures were normalized with respect to that point. Differences were considered significant when p<0.05 (Student-Newman-Keuls test for pairwise comparisons).

Cells (1.5×10⁶) were harvested in denaturing lysis buffer (1% SDS, 5 mM EDTA, 10 mM DTT, 15 U/ml DNase, PMSF). A total of 5 μl of each cell lysate was diluted in 45 μl of non-denaturing buffer (20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, PMSF). Diluted cells lysates were pre-incubated with 0.4 μg of anti-p53 antibody (DO1 anti-p53; Santa Cruz Biotechnologies) or with 2 μl of anti-NEDD8 antibody (Alexis Biochemicals). A total of 10 μl of Protein A/G PLUS-Agarose Immunoprecipitation Reagent (sc-2003; Santa Cruz Biotechnologies) was further mixed with extracted protein, pelleted and resuspended in loading buffer and subjected to western blot analysis with an anti-NEDD8 antibody and a polyclonal rabbit anti-p53 FL-393 antibody (Santa Cruz Biotechnologies). Normal mouse IgG-AC (10 μl) (sc-2343; Santa Cruz Biotechnologies) was incubated with protein lysate as the negative control.

UPCI:SCC90 cells were transfected and irradiated as described above. Annexin V staining was monitored by flow cytometry on an Agilent Bioanalyzer 2100 (Agilent Technologies Deutschland, Waldbronn, Germany), according to the manufacturer’s instructions. In brief, cells were incubated first with Annexin V-Biotin (Annexin V-Biotin apoptosis detection kit, Oncogene Research Product, Merck Chemicals, Nottingham, UK), and later with streptavidin-Cy5 (Amersham, GE Healthcare). In order to discriminate early apoptotic cells from dead cells, samples were counterstained with Calcein-AM (Molecular Probes, Invitrogen, Cergy Pontoise, France). Cell samples were loaded on Cell Fluorescence LabChip^® kits, and flow cytrometry data were analyzed with Agilent Cell Fluorescence software. Data were analyzed with one-way ANOVA, together with a Student-Newman-Keuls test of all pairwise possible comparisons, and differences were considered significant when p<0.05.

In order to evaluate the involvement of NAE1/APP-BP1 gene expression levels in HR-HPV-positive oropharyngeal squamous cell carcinoma radiosensitivity, we selected 2 different cell lines: the HPV-negative SQ20B cell line that originates from a laryngeal tumor, harboring a TP53 mutant allele and that is radioresistant (10); and the HR-HPV16-positive UPCI:SCC90 cell line that was established from an oropharyngeal squamous cell carcinoma, that bears a wild-type TP53 locus and is radiosensitive as compared to the SQ20B cell line (11). Using a qRT-PCR approach with 3 independent total RNA extracts from the 2 cell lines, we confirmed that the mRNA that encodes the HPV16 E6 and E7 oncoproteins is expressed in UPCI:SCC90 cells, whereas it was not detectable in RNA extracts from SQ20B cells (Fig. 1A). As expected, the expression of the CDKN2A^p16 gene, which is used as a surrogate marker of HPV infection, was found to be dramatically increased in the UPCI:SCC90 cells as compared to the SQ20B cells (Fig. 1A). These results confirm that HPV16 is biologically active in UPCI:SCC90 cells. We measured the expression of WAF1/CIP1^p21, whose expression is regulated by p53. In agreement with their respective TP53 statuses, the expression of WAF1/CIP1^p21 was found to be higher in the UPCI:SCC90 cells as compared to the SQ20B cells (Fig. 1A). Finally, the expression of the NAE1/APP-BP1 gene was found to be lower in the UPCI:SCC90 cells than in the SQ20B cells. We further evaluated the expression levels of the NEA1/APP-BP1 protein in these 2 cells lines by western blot analysis. Blots were probed with an anti-NEA1/APP-BP1 antibody, and quantification of the signals obtained from total protein extracts confirmed that the NEA1/APP-BP1 protein displays higher expression levels in SQ20B cells than in UPCI:SSC90 cells (Fig. 1B). Thus, these 2 cell lines display NAE1/APP-BP1 expression patterns that are similar to those reported in HR-HPV-positive and HPV-negative HNSCC. They were used to evaluate the influence of the NAE1/APP-BP1 gene on their response to ionizing radiation.

We examined the effect of modulating NAE1/APP-BP1 expression levels in UPCI:SCC90 cells on clonogenic survival. NAE1/APP-BP1 was overexpressed by the transfection of an expression vector. Transfection with the NAE1/APP-BP1 construct resulted in an increase in NAE1/APP-BP1 mRNA levels compared to the mock-transfected cells, as measured by qRT-PCR in 3 independent extracts of cells harvested 24 h after transfection (Fig. 2A). A western blot analysis of protein extracts from the same cells, using an anti-NAE1/APP-BP1 antibody, showed that NAE1/APP-BP1 protein levels also increased (Fig. 2B). In a clonogenic survival assay, the cells were irradiated with a single 2-Gy dose of X-rays, and the number of cells that were able to grow colonies was measured. Non-irradiated cells were used as the control to calculate the fraction of cells that survived the treatment. The SF2 of mock-transfected cells and that of cells that overexpressed NAE1/APP-BP1, calculated from 6 independent measures, was 0.36 and 0.89, respectively (Table I; p<0.001), showing that NAE1/APP-BP1 overexpression augments resistance to ionizing radiation in UPCI:SCC90 cells.

We reasoned that if NAE1/APP-BP1 is involved in cell survival after irradiation, then the response of SQ20B cells to ionizing radiation may be modulated by both restoring functional TP53 and downregulating NAE1/APP-BP1 expression. Consequently, SQ20B cells were co-transfected with a wild-type TP53 expression vector and siRNAs that specifically target NAE1/APP-BP1. A non-related siRNA was used as the negative control. SQ20B cells bear a point mutation in the TP53 gene that results in the production of a non-functional p53 protein, which can be detected by western blot analysis (Fig. 3A, lanes 1 and 3). The transfection of SQ20B cells with an expression vector for wild-type TP53 further increased p53 levels. Non-irradiated and irradiated transfected cells displayed increased amounts of p53 as compared to the mock-transfected cells (Fig. 3A, lanes 2 and 4). The SF2 of mock-transfected SQ20B cells and that of SQ20B cells that overexpressed p53, was 0.46 and 0.35, respectively (Table I; p=0.030), indicating that exogenous p53 function is functional in SQ20B cells. The expression of the NAE1/APP-BP1 protein was downregulated up to 48 and 72 h after transfecting SQ20B cells with an anti-NAE1/APP-BP1 siRNA pool (Fig. 3B, lane 7). This pool is composed of 4 individual siRNAs, which all were found to efficiently downregulate the expression of the NAE1/APP-BP1 gene (Fig. 3A, lanes 3–6). Transfection with a non-related siRNA, used as the negative control, had no effect on NAE1/APP-BP1 protein levels (Fig. 3B, lane 2). Neither the anti-NAE1/APP-BP1 siRNA, nor the control siRNA increased cell sensitivity to ionizing radiation. The SF2 in these conditions was 0.53 and 0.64, respectively (Table I). Strikingly, the irradiation of SQ20B cells that were co-transfected with wild-type TP53 and anti-NAE1/APP-BP1 siRNA resulted in a drop in the SF2 to 0.26, whereas cells that were co-transfected with TP53 and the control siRNA displayed a SF2 of 0.46 (Table I; p=0.045). Taken together, these observations suggest that the inhibition of NAE1/APP-BP1 in SQ20B cells restores cell sensitivity to ionizing radiation in a p53-dependent manner.

In order to examine whether the modulation of NAE1/APP-BP1 expression levels in UPCI:SCC90 cells has an effect on p53 transcriptional activity, we used the pG13-Luc reporter, in which the gene that encodes the luciferase protein is under the control of p53-dependent cis-regulatory elements. UPCI:SCC90 cells were co-transfected with equal amounts of pG13-Luc and increasing amounts of either the NAE1/APP-BP1 expression vector, or the empty control vector. Cells were harvested 36 h after transfection and luciferase activity was measured with 3 independent protein extracts. There was no change in p53 transcriptional activity in the cells that were mock-transfected with various amounts of control vector (Fig. 4A, left histograms). By contrast, p53 transcriptional activity was markedly and significantly repressed in UPCI:SCC90 cells transfected with increasing amounts of the NAE1/APP-BP1 vector, in a dose-dependent manner (Fig. 4A; 1.5 and 2.5 μg of NAE1/APP-BP1 vector; p<0.05). In the control experiment, we used the MG15-Luc reporter plasmid, which contains mutated p53-binding elements upstream of the luciferase gene. The fluorescence that we observed was markedly reduced, and no differences were observed between the cells transfected with either the empty plasmid or the NAE1/APP-BP1 expression construct (Fig. 4A, right histograms). The UPCI:SCC90 mock-transfected cells displayed rapid and significant (p<0.05) induction of p53 target genes, such as WAF1/CIP1^p21, PUMA and MDM2, 30 min after a 2-Gy irradiation (Fig. 4B). This induction was not observed in UPCI:SCC90 cells that overexpressed NAE1-APP-BP1 (Fig. 4B). In order to evaluate whether this modulation of the p53 dependent transcriptional activity depends on a NAE1/APP-BP1-dependent increase in NEDDylation, we performed a western blot analysis to detect NEDDylated p53 in proteins extracted under denaturating conditions. NEDD8-conjugated proteins were immunoprecipitated with a p53 antibody, and the blots were probed with an anti-NEDD8 antibody (Fig. 4C-a). Conversely, NEDD8 was immunoprecipitated from the protein extracts, and the blots were then probed with an anti-p53 antibody (Fig. 4C-b). NEDDylated proteins were present in similar amounts in the extracts from the mock-transfected cells and from the cells transfected with NAE1/APP-BP1 (Fig. 4C-c). In both cases, we observed a band whose size corresponded to NEDDylated p53 (approximately 63 kDa; Fig. 4C-a and -b; asterisk). This signal was stronger in the UPCI:SCC90 cells that overexpressed NAE1/APP-BP1 than in the mock-transfected UPCI:SCC90 cells (Fig. 4C-a and -b). No detectable signal corresponding to a NEDDylated p53 protein was observed when transfected UPCI:SCC90 cell lysates were immunoprecipitated with an unrelated IgG (Fig. 4C-a). These results indicate that the overexpression of NAE1/APP-BP1 in UPCI:SCC90 cells results in an increased NEDDylation of p53. These observations are consistent with the decreased transcriptional activity of p53 (Fig. 4A and B).

We evaluated whether the modulation of radiosensitivity by NAE1/APP-BP1 expression levels and p53 activity is associated with changes in apoptosis in the control and transfected UPCI:SCC90 cells, prior to and 6 h following a 4-Gy irradiation. A flow cytometry approach was used in which living and dead cells were discriminated by staining with a calcein probe, and apoptotic cells by Annexin V. Calcein-positive and -negative cells were cross-gated to cells that displayed high levels of Annexin V (>10²; Fig. 5A), and the proportion of each cell category was counted. The graph represents the statistical analysis of 3 independent experiments (Fig. 5B). The proportion of calcein-negative Annexin V-positive cells was found to be decreased when NAE1/APP-BP1 was overexpressed in irradiated UPCI:SCC90 cells as compared to irradiated mock-transfected cells (p<0.05). Taken together, these observations suggest that NAE1/APP-BP1 regulates cell sensitivity to ionizing radiation by modulating apoptosis.