p16INK4A-silencing augments DNA damage-induced ... - Nature

Oncogene (2007) 26, 6050–6060

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ORIGINAL ARTICLE

p16INK4A-silencing augments DNA damage-induced apoptosis in cervical cancer cells WM Lau1, TH Ho2,3 and KM Hui1 1 Division of Cellular and Molecular Research, National Cancer Centre, Singapore, Singapore; 2Obstetrics and Gynaecology, Singapore General Hospital, Singapore, Singapore and 3Gynaecological Oncology Unit, KK Women’s and Children’s Hospital, Singapore, Singapore

p16INK4A (p16) has been suggested to be an early biomarker for the detection of cervical cancer. However, its functional role in cervical cancer is not well characterized. In this study, we reported the consistent and significant upregulation of p16 in cervical cancer tissues when compared to both matched non-tumourous tissues of the same patient and normal cervical tissues from non-cancer patients. We have employed p16 small interfering RNA (siRNA) to dissect the role of p16 in cervical carcinogenesis. Although the silencing of p16 was accompanied by the upregulation of p53, p21 and RB in the p16 siRNA-transfected cells, no significant effect on cell cycle progression was observed. When the p16 siRNAsilenced cells were subjected to DNA damage stress including ultraviolet-irradiation and cisplatin treatments, a significantly higher percentage of apoptotic cells could be observed in the p16-siRNA silenced cells compared to control siRNA-treated cells. Moreover, induction of apoptosis was associated with the activation of p53 through phosphorylation, and this process, when studied by gene profiling experiments, involved both the intrinsic and extrinsic apoptotic pathways. The observation that silencing of p16 expression augments DNA damageinduced apoptosis in cervical cancer cells offers alternative strategies for anti-cancer therapies for human cervical cancer. Oncogene (2007) 26, 6050–6060; doi:10.1038/sj.onc.1210405; published online 19 March 2007 Keywords: p16; RNAi; cervical cancer; DNA damage; apoptosis

Introduction Cancer of the cervix is the second most common cancer for women worldwide, with a higher prevalence in Correspondence: Professor KM Hui, Division of Cellular and Molecular Research, National Cancer Centre, Singapore, 11 Hospital Drive, Singapore 169610, Singapore. E-mail: [email protected] Received 8 November 2006; revised 9 February 2007; accepted 9 February 2007; published online 19 March 2007

developing countries (Parkin et al., 2001). Infection with the human papillomaviruses (HPV) subtypes 16 and 18, associated with 99% of all cervical cancers, is a major risk factor in cervical cancer (zur Hausen, 1996; Walboomers et al., 1999). The HPV viral oncoproteins E6 and E7 are crucial for the initiation and maintenance of the malignant phenotype and expression of both E6 and E7 oncoproteins is sufficient to immortalize normal human mammary epithelial cells, fibroblasts and keratinocytes (Yamamoto et al., 2003). Furthermore, E6 and E7 bind and subsequently degrade p53 and Rb respectively, therefore abolishing two major G1-checkpoint controls and consequently dysregulate two important tumour suppressor pathways present in normal cells (Scheffner et al., 1990; Munger et al., 1992). In normal cells, p53 mediates G1 cell-cycle arrest or apoptosis in response to DNA damage, which is often associated with the induction of p21, a cyclin-dependent kinase (CDK) inhibitor and known transcriptional target of p53 (el-Deiry et al., 1993, 1994). Similarly, Rb also exerts G1-checkpoint control by forming a complex with the transcription factor E2F1 and moderating E2F1-dependent transcription of S-phase genes (Nevins, 1992). Formation of the Rb-E2F1 complex depends largely on the phosphorylation status of Rb and when phosphorylated by cyclin D-CDK4 and six complexes, Rb releases E2F1 to enable G1/S progression. The tumour suppressor p16INK4A (p16) functions as a CDK inhibitor of the cyclin D-CDK4 and six complexes, and is therefore important in regulating the phosphorylation of Rb. Hence, a high level of p16 will prevent Rb phosphorylation, which in turn prevents the release of E2F, and leads to G1 arrest. Consequently, p16 is also a potent mediator of growth arrest in response to DNA damage (Shapiro et al., 1998). p16 has been reported to be frequently inactivated in human cancers through the methylation of its promoter, mutations or homozygous deletions, resulting in the reduction or absence of its expression (Kamb et al., 1994; Nobori et al., 1994). Conversely, for cervical cancer cells, it was observed that the inactivation of Rb by E7 resulted in the accumulation and upregulation of p16, possibly owing to a negative-feedback mechanism (Klaes et al., 2001). Clinically, the close association of p16 expression with HPV infection has been reported

p16-silencing augments apoptosis in cervical cancer WM Lau et al

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(Sano et al., 1998; Kanao et al., 2004), and many immunohistochemical studies have employed p16 as a diagnostic biomarker for cervical cancer (Andersson et al., 2006; Ishikawa et al., 2006; Queiroz et al., 2006). In normal cells, high levels of p16 lead to cell-cycle arrest and therefore, the overexpression of p16 in cervical cancer is in stark contradiction to its potential role as a tumour suppressor gene. However, it has been suggested that high levels of p16 in cervical cancer cells would not interfere with cell-cycle progression because of the presence of E7 oncoprotein, and that the upregulation of p16 in cervical cancer is possibly the result of a feedback loop in the p16-Rb pathway (Giarre et al., 2001; Klaes et al., 2001; zur Hausen, 2002). In the present study, we demonstrated the significant upregulation of p16 in cervical cancerous tissues of cervical cancer patients. Furthermore, we showed that the silencing of p16 expression with p16 siRNA-enhanced DNA damage-induced apoptosis in cervical cancer cells.

Figure 1 Overexpression of p16 gene expression in cervical cancer. Gene expression of p16 in normal (N), non-tumourous (NT) or tumour (T) samples as determined by (a) cDNA library screening using spotted cDNA microarrays and (b) real-time PCR analysis. Sample size is indicated by ‘n’. Bars represent median values and (*) indicates significant difference (Po0.05) using one-way analysis of variance with Bonferroni’s post test.

Results Significant upregulation of p16 gene expression in squamous cell cervical carcinoma To identify differentially expressed genes in human cervical cancer, we constructed two reciprocal (forward and reverse) subtracted cDNA libraries from tumourous and non-tumourous cervical tissue taken from a single patient. A total of 1990 clones were obtained from the forward and reverse subtracted libraries. To assess the complexity of the library, we randomly selected 200 clones for sequencing and of these 90% were unique sequences, demonstrating that the majority of the clones were not overlapping (data not shown). These clones were polymerase chain reaction (PCR) amplified along with housekeeping and negative control genes and used to generate spotted cDNA microarrays. Using these microarrays, we screened the subtracted cDNA libraries using 22 tumour samples and 11 paired non-tumourous tissues from cervical cancer patients and seven normal cervical tissues. Amongst the genes overexpressed in cervical cancer, p16 was shown to be significantly upregulated in cervical cancer tissues compared to paired non-tumourous tissues (Po0.01) and to normal cervical tissues (Po0.001; Figure 1a). Quantitative realtime PCR using a further six samples each of tumour, non-tumourous and normal cervical tissue confirmed 20–200-fold p16 upregulation in cervical carcinoma compared to non-tumourous and normal cervical tissue (Po0.05; Figure 1b). p16 siRNA specifically inhibits expression of p16 but not p14ARF, encoded by the same gene locus as p16 In contrast to many human cancers where p16 is inactivated, overexpression of p16 in cervical cancer may have significant impact on the development of cervical cancer. To gain insight into the role of p16 in cervical cancer, SiHa and Cas Ki cervical cancer cells, derived from squamous cell carcinoma of the cervix,

were monitored after transfection with siRNAs that target exon 1a of the p16 gene. We used SiHa and Cas Ki cells specifically because they are derived from squamous cell carcinoma of the cervix, and contain the HPV type 16 viral genome, similar to the tumour samples obtained from cervical cancer patients and employed in our microarray studies. Both cell lines express high levels of p16. A 95% reduction in p16 protein expression was observed after a 24-h transfection of SiHa cells with 20 nM p16 siRNA (Figure 2), and 55% reduction in p16 mRNA was achieved (data not shown). The control siRNA sequence (20 nM) had no effect on p16 mRNA or protein, thus confirming that the reduction in p16 was sequence-specific. More importantly, p14 protein expression encoded by a different reading frame to p16, was not affected, confirming the specificity of p16-targeted siRNA (Figure 2a). Silencing of p16 modulates expression of Rb, E7, p53 and p21 in cervical cell lines As p16 is a key member of the RB tumour suppressor pathway, we monitored expression of Rb in SiHa and Cas Ki cells at 12, 24 and 48 h after p16 siRNA transfection. Although no significant change of Rb mRNA level was detected (Figure 3a), Rb protein expression was increased in the p16-silenced cells compared to control siRNA-treated cells (Figure 3b), suggesting that p16 affects Rb protein expression posttranscriptionally. The observation that p16-silencing downregulated the expression of E7 (Figure 3a), which degrades Rb protein directly in HPV-positive cervical cancer cells, may explain the increase of Rb protein in p16-silenced cells. On the other hand, E6, another HPV viral oncoprotein that binds and degrades p53, did not show altered expression in p16-silenced cells (Figure 3a). Oncogene


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Figure 2 p16 siRNA specifically inhibits expression of p16 but not p14ARF, encoded by the same gene locus as p16. (a) The expression of p16 and p14 were determined by Western blot in SiHa cells at 24 h after transfection with p16 siRNA ( þ ) or control siRNA (). The results shown are representative of two independent experiments. (b) Densitometric analysis of protein bands was performed. After normalization to b-actin, relative protein expression levels were determined in relation to control siRNA, which were given an arbitrary value of 100.

Although E6 remained unchanged, we observed increased levels of p53 gene and protein expression (Figures 3a and b), suggesting that p16-silencing enhanced p53 expression independently of E6. Furthermore, this was accompanied by a sharp increase in protein expression of p21, a p53-downstream transcriptional target (Figure 3b). In our experiments, we did not observe any change in protein levels between control siRNA-treated cells and untreated control cells, confirming that the siRNAs used did not have secondary effects (Figure 3b (i)). Silencing of p16 has no effect on cell-cycle progression in cervical cell lines Induction of p53 and concomitant activation of p21 has been associated with G1-growth arrest. We performed cell-cycle analysis and found that despite high levels of p53 and p21 in p16-silenced cells, G1 and S-phase cell populations remained consistent at approximately 60% and 30%, respectively from 24 to 72 h after p16 siRNA transfection (Figure 4a). BrdU proliferation studies showed that the cells did not stop proliferating (Figure 4b), nor did they undergo apoptosis (data not shown). Hence, upregulation of p53 and p21 in p16silenced cells did not result in cell-cycle arrest, apoptosis or a decrease in cell growth. Silencing of p16 augments UV- and cisplatin-induced apoptosis in cervical cancer cell lines Cervical cancer cells transfected with p16 siRNA or control siRNA were subjected to 100 J/m2 UV irradiation or 20 mM cisplatin treatment to determine the role of p16 in response to DNA damage. A significantly higher percentage of apoptotic cells was observed in p16silenced SiHa cells compared to control siRNA-treated cells (Figure 5a), indicating that p16 may play an antiapoptotic role. At 12 h after UV treatment, the proportion of apoptotic cells in the p16 siRNAtransfected cells was 5% compared to 1.5% in the Oncogene

control-treated cells (Po0.05). At 24 h post UV irradiation, the percentage of apoptotic cells increased to 19% for p16-silenced cells compared to 4% in the control (Po0.001), and at 48 h, induction of apoptosis was 28% for p16-silenced cells compared to 6.5% for the control (Po0.001). Terminal transferase dUTP nick end labeling (TUNEL) assay further confirmed that there were more apoptotic cells in p16 siRNA-transfected cells than controls at 24 h post UV irradiation (Figure 5b). Similarly, after 20 mM cisplatin treatment, apoptotic cell populations were significantly higher in p16 siRNA-transfected SiHa cells compared to controls (Figure 5a). After 12 h of exposure to cisplatin, p16silenced cells showed 32% apoptotic cell population compared to 13% in control cells; at 24 h after cisplatin treatment, the apoptotic population for p16-silenced cells was 41% compared to 15% and at 48 h following cisplatin treatment, p16-silenced cells showed 45% apoptosis compared to 17% in controls. Cas Ki cells with reduced p16 expression were also shown to be more susceptible to apoptosis, compared to control siRNAtreated cells, after UV irradiation and cisplatin treatment (data not shown). Overall, we noticed a higher induction of apoptosis in cisplatin-treated p16-silenced cells compared to UV irradiated p16-silenced cells (Figure 5a), which may suggest that different modes of apoptosis induction are involved in response to UV- and cisplatin-induced DNA damage. Taken together, our data showed that cells, which had reduced p16 expression were more susceptible to apoptosis after UV irradiation and cisplatin treatment. Silencing of p16 in cervical cancer cell lines enhance p53 phosphorylation under UV irradiation and cisplatin treatment Under UV irradiation, increased p53 phosphorylation on serine 15, which is associated with stress-induced apoptosis (Shieh et al., 1997), was observed in p16silenced SiHa cells compared to control siRNA-treated


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Figure 3 Silencing of p16 modulates expression of Rb, p53, E6 and E7 in cervical cell lines. SiHa and CasKi cells were treated with p16 or control siRNA and assayed at 12, 24 and 48 h. (a) mRNA expression levels were determined by real-time PCR analysis in SiHa cells. The relative mRNA levels were determined in relation to control siRNA, which was given an arbitrary value of 100. (*) indicates significant P-value o0.01. (b) Western blot analysis was performed using total cell lysate of SiHa and Cas Ki cells. (i) Endogenous levels of p53 and Rb protein in untreated cells compared to siRNA-transfected cells after 24 h. (ii) The modulation of protein expression after p16 siRNA silencing. ‘ þ ’ represents p16 siRNA treated, ‘’ represents control siRNA treated and ‘C’ represents untreated cells. The data shown are representative of three independent experiments.

Figure 4 Silencing of p16 has no effect on cell-cycle progression in cervical cell lines. The cell proliferation status of SiHa cells after 24, 48 and 72 h transfection of p16 siRNA or control siRNA analysis were determined by (a) Cell-cycle analysis of PI-staining and (b) BrdU proliferation assay. Data were obtained from three independent experiments.

cells (Figure 6). On the other hand, following cisplatin treatment, increased p53 phosphorylation on serine 46 was observed in p16-silenced cells compared to control siRNA-treated cells (Figure 6). This strongly suggests that p53 was activated via different pathways in response to DNA damage by UV irradiation and cisplatin treatment. This is also consistent with our observation that the induction of apoptosis in p16silenced cells after cisplatin treatment was higher compared to UV induced apoptosis in p16-silenced cells (Figure 5a). Our results indicate that the activation of p53 by phosphorylation associated with UV irradiation and cisplatin treatment may regulate apoptosis in p16silenced cells. We also observed a decrease in p53 protein levels after UV irradiated but not cisplatintreated p16-silenced cells, suggesting that activation of p53 on serine residues following UV treatment was concurrent with degradation of total p53 protein. This Oncogene


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Figure 5 Silencing of p16 augments UV- and cisplatin-induced apoptosis in cervical cancer cells. (a) After transfection with p16 siRNA or control siRNA, SiHa cells were then subjected to 100 J/m2 UV irradiation or cisplatin (20 mM). Cells were fixed and stained with propidium iodide (100 mg/ml) for FACS analysis at 12, 24 and 48 h. Percentage of apoptotic cells were determined by sub-G1 populations. (*) P-value o0.01; *** significant P-value o0.001. Data were collected from three independent experiments. (b) TUNEL assay confirms that p16-silencing enhanced UV-induced apoptosis in SiHa cells at 24 h post-irradiation. Cells were counterstained with DAPI. Red bar denotes 10 m .

was further confirmed by the observation that p53 mRNA levels increased in p16-silenced cells after UV irradiation (data not shown). Activation of p53 pathway, intrinsic and extrinsic apoptotic pathways in p16-silenced SiHa cells under UV irradiation To determine apoptotic pathways induced by UV irradiation after p16-silencing, we compared the gene expression profiles of p16 siRNA or control siRNAtreated cells with or without UV irradiation using Affymetrix GeneChip analysis (Santa Clara, CA, USA). We selected genes that were differentially expressed with respect to p16-silencing under UV treatment and focused on genes involved in DNA damage and apoptosis using Spotfire software Gene Oncogene

Ontology (GO) tool (Spotfire, Somerville, MA, USA). Selected genes were further organized into known pathways and networks using Bibliosphere software (Genomatix, GmBH, Germany). These genes included p53 immediate downstream-regulated genes, anti-apoptotic genes, as well as apoptotic genes involved in both the intrinsic mitochondrial apoptosis pathway and the extrinsic death receptor pathway (Table 1). Table 1 summarizes the changes in expression of 60 genes in the p16-silenced cells compared to control siRNA-treated cells at different time points after p16 or control siRNA transfection, with or without UV irradiation. p16silenced cells which were not subjected to UV irradiation did not exhibit changes in the apoptotic genes. At 12 h post-UV irradiation, the majority of genes involved in intrinsic and extrinsic apoptotic pathways remain unchanged. However, 24 and 48 h after UV irradiation,


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Figure 6 Silencing of p16 in cervical cancer cell lines enhance p53 phosphorylation under UV- and cisplatin treatment. After transfection with p16 siRNA ( þ ) or control siRNA (), SiHa cells were then subjected to 100 J/m2 UV irradiation or cisplatin (20 mM). Western blot analysis revealed UV irradiation and cisplatin treatment specifically induced phosphorylation of p53 on serine 15 and 46, respectively. Data shown here are representative of at least three independent experiments.

p53 mRNA levels in p16-silenced cells increased by twoto threefold compared to control siRNA-treated cells at 24 and 48 h after UV irradiation. Of particular interest were the well-known p53 DNA-damage target genes p21, PCNA, and GADD45A, which were also upregulated by 1.5 to twofold in UV irradiated, p16-silenced cells. The anti-apoptotic genes BAG1, TIMP3, AKT1, MCL1 and FAIM were downregulated by 1.5 to threefold, whereas the apoptotic genes belonging to both the mitochondrial and death receptor pathways, such as caspases 8, Bcl family-related genes, APAF1, FAS, FADD and tumor necrosis factor receptor 2family genes were upregulated by two to threefold in UV irradiated, p16-silenced cells (Table 1). These results show that UV-induced apoptosis of p16-silenced cells occurred through well-established mitochondrial apoptosis and death receptor pathways, and was mediated to a certain extent through p53 and its target genes.

Discussion Overexpression of p16 in cervical cancer is well documented. However, its role in cervical carcinogenesis remains largely unknown. Here we report that the silencing of p16 with siRNA in cervical cancer cells promotes apoptosis in cervical cancer cells when subjected to DNA-damaging agents. It has been shown that p53 can directly activate both the intrinsic and extrinsic apoptotic pathways in response to DNA damage (Muller et al., 1998; Mihara et al., 2003). Gene profiling experiments revealed the activation of genes involved in both the intrinsic and extrinsic apoptotic pathways with the upregulation of many p53 target genes following UV irradiation and treatment with cisplatin in p16 siRNA-treated cervical cancer cells (Table 1). Furthermore the activation of p53 was through phosphorylation on serine 15 and 46 in UV irradiated and cisplatin-treated cells respectively. These observations are consistent with reports suggesting that

p53 is phosphorylated at different sites in different cell types under different forms of genotoxic stress (Jimenez et al., 1999; Bode and Dong, 2004). We observed that the induction of apoptosis in p16 siRNA-treated cells after cisplatin treatment was higher compared to UV-induced apoptosis in p16 siRNA-treated cells (Figure 5a), which may suggest that different modes of apoptosis induction are involved in response to UV- and cisplatin-induced DNA damage. This is further supported by reports that the phosphorylation of p53 on serine 46 is associated with more severe forms of DNA damage that triggers apoptosis via pathways different than those associated with phosphorylation of p53 on other residues such as serine 15 (Oda et al., 2000). From our present study, it is apparent that the activation of p53 in p16 siRNA-silenced cells is different in the presence and absence of DNA damaging agents. When p16 was silenced with p16 siRNA, we observed the induction of p53 but not the phosphorylated form of p53. However, when p16-silencing was coupled with DNA-damaging agents, p53 was specifically activated via phosphorylation of its serine residues to promote apoptosis. It is possible that p16-silencing affects pathways, which are also involved in p53-mediated responses to DNA damage, and that p16-silencing may contribute to the DNA damage stress response, directly resulting in activation of p53 by phosphorylation. We also observed that p16-silencing together with UV irradiation resulted in decreased total p53 protein expression despite phosphorylation of p53 on serine 15. This could result from the degradation of p53 in cervical cancer cells after UV irradiation but not cisplatin treatment, possibly mediated by mdm2, E6 or ubiquitin (Freedman and Levine, 1998). This is further confirmed by our observation that p53 mRNA level was increased in p16 siRNA-treated cells compared to control siRNA treated cells under UV treatment (Lau et al., unpublished observation), and suggests that the decrease of p53 protein was not a result of modulation at the transcriptional level. However, induction of phosphorylation of p53 by UV irradiation is still intact. Our observation is similar to that of Lee et al. (2006), who observed the reduction of p53 by acetaminophen, but not etoposide and 50 FU, despite phosphorylation of p53 on serines 15 and 37 . Furthermore, a recent study has also shown that p16 inactivation leads to increased levels of p53 in human mammary epithelial cells, and that the sustained upregulation of p53 may increase the selective pressure to inactivate p53 (Zhang et al., 2006). On the other hand, upregulation of p53 that occurs in p16-silenced cells is abolished under cisplatin treatment, nonetheless, phosphorylation of p53 on serine 46 was observed under cisplatin treatment. It is likely that p16silencing is directly or indirectly involved in p53mediated responses to DNA damage under stress such as UV irradiation and cisplatin treatment, and may result in p53 activation through phosphorylation, without affecting p53 protein levels. We propose that the responses triggered by silencing p16 alone, and in conjunction with DNA damage stress are different, and this may be an interesting avenue for future study in Oncogene


6056 Table 1

Affymetrix GeneChip analysis of expression changes in UV-induced apoptosis related genes in cells treated with p16 siRNA compared to control siRNA

Affymetrix Probe ID

Gene name

Gene symbol

p16 siRNA vs control siRNA UV

202387_at 201150_s_at 207163_s_at 200798_x_at 220643_s_at

Anti-apoptosis BCL2-associated athanogene Tissue inhibitor of metalloproteinase 3 v-Akt murine thymoma viral oncogene homologue 1 Myeloid cell leukemia sequence 1 (BCL2-related) Fas apoptotic inhibitory molecule

211300_s_at 203120_at 204531_s_at 202981_x_at 204947_at 202284_s_at 209112_at 205516_x_at 208878_s_at 33814_at 201202_at 203725_at

Apoptosis - p53 immediate downstream regulated genes Tumor protein p53 (Li–Fraumeni syndrome) Tumor protein p53 binding protein, 2 breast cancer 1, early onset SIAH1 seven in absentia homologue 1 (Drosophila) E2F transcription factor 1 CDK inhibitor 1A (p21, Cip1) CDK inhibitor 1B (p27, Kip1) CDKN1A interacting zinc-finger protein 1 p21 (CDKN1A)-activated kinase 2 p21(CDKN1A)-activated kinase 4 Proliferating cell nuclear antigen GADD45A growth arrest and DNA-damage-inducible, a

209970_x_at 208050_s_at 210026_s_at 205263_at 206665_s_at 222343_at 211725_s_at 202985_s_at 201101_s_at 218732_at 209308_s_at 201848_s_at 208905_at 204859_s_at 219350_s_at

Apoptosis – intrinsic (mitochondrial) pathway Caspase 1, apoptosis-related cysteine protease Caspase 2, apoptosis-related cysteine protease Caspase recruitment domain family, member 10 B-cell CLL/lymphoma 10 BCL2-like 1 BCL2-like 11 (apoptosis facilitator) BH3 interacting domain death agonist BAG5 BCL2-associated athanogene 5 BCL2-associated transcription factor 1 Bcl-2 inhibitor of transcription BCL2/adenovirus E1B 19 kDa interacting protein 2 BCL2/adenovirus E1B 19 kDa interacting protein 3 Cytochrome c, somatic Apoptotic protease activating factor Diablo homologue (Drosophila)

213373_s_at 209939_x_at 215719_x_at 202535_at 208315_x_at 202871_at 204352_at 206907_at 209294_x_at 210654_at 202643_s_at 218368_s_at 201502_s_at

202731_at 203415_at 212422_at 217746_s_at 201715_s_at 204004_at 201095_at 208791_at 218350_s_at 220864_s_at Oncogene

Apoptosis – extrinsic (death receptor) pathway Caspase 8, apoptosis-related cysteine protease CASP8 and FADD-like apoptosis regulator Fas (TNF receptor superfamily, member 6) Fas (TNFRSF6)-associated via death domain TNF receptor-associated factor 3 TNF receptor-associated factor 4 TNF receptor-associated factor 5 TNF (ligand) superfamily, member 9 TNF receptor superfamily, member 10b TNF receptor superfamily, member 10d TNF, alpha-induced protein 3 TNF receptor superfamily, member 12A NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha Apoptosis-related Genes Programmed-cell death 4 (neoplastic transformation inhibitor) Programmed-cell death 6 Programmed-cell death 11 Programmed-cell death 6 interacting protein Apoptotic chromatin condensation inducer 1 PRKC, apoptosis, WT1, regulator Death-associated protein Clusterin Geminin, DNA replication inhibitor Cell death-regulatory protein GRIM19

Without UV

12 h

24 h

48 h

24 h

48 h

72 h

BAG1 TIMP3 AKT1 MCL1 FAIM

NC NC NC NC NC

+ + + + +

+ + + + +

NC NC NC NC NC

NC NC NC NC NC

NC NC NC NC NC

TP53 TP53BP2 BRCA1 SIAH1 E2F1 CDKN1A CDKN1B CIZ1 PAK2 PAK4 PCNA GADD45A

* NC NC NC * NC NC NC NC NC NC NC

* * * * * * * * * * * *

* * * * * * * * * * * *

* NC NC NC NC NC NC NC NC NC NC NC

* NC NC NC NC NC * NC NC NC NC NC

* NC NC * NC NC NC NC NC NC NC NC

CASP1 CASP2 CARD10 BCL10 BCL2L1 BCL2L11 BID BAG5 BCLAF1 BIT1 BNIP2 BNIP3 CYCS APAF1 DIABLO

* * NC * * * NC NC NC NC NC NC * NC NC

* * * * * * * * * * * * * * *

* * * * * * * * * * * * * * *

NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC



CASP8 CFLAR FAS FADD TRAF3 TRAF4 TRAF5 TNFSF9 TNFRSF10B TNFRSF10D TNFAIP3 TNFRSF12A NFKBIA

NC NC NC NC * NC NC NC NC NC NC * *

* * * * * * * * * * * * *

* * * * * * * * * * * * *

NC NC NC NC NC NC NC NC NC NC NC NC NC



PDCD4 PDCD6 PDCD11 PDCD6IP ACIN1 PAWR DAP CLU GMNN GRIM19

NC NC NC NC NC NC NC NC NC NC

* * * * * * * * * *

* * * * * * * * * *





6057 Table 1 (continued ) Affymetrix Probe ID

Gene name

Gene symbol

p16 siRNA vs control siRNA UV

202431_s_at 201710_at 203359_s_at 212099_at 200607_s_at

v-myc myelocytomatosis viral oncogene homologue (avian) v-myb myeloblastosis viral oncogene homologue (avian)-like 2 c-myc binding protein RHOB ras homologue gene family, member B RAD21 RAD21 homologue (S. pombe)

MYC MYBL2 MYCBP RHOB RAD21

Without UV

12 h

24 h

48 h

24 h

48 h

72 h

NC * NC * NC

* * * * *

* * * * *

NC NC NC NC NC

NC NC NC NC NC

NC NC NC NC *

Abbreviations: CDK, cyclin-dependent kinase; TNF, tumour necrosis factor m denotes 1.5 to twofold significant (po0.05) increase, * or + denotes two- to threefold significant (Po0.05) increase or decrease in signal intensity in p16 siRNA compared to control siRNA as computed by Affymetrix Microarray Analysis Suite (v5) change call statistical analysis algorithms.

order to elucidate the relationship between p16 and p53 in cervical cancer cells. Overall, we have shown that p16 is not functionally redundant in cervical cancer cells and under certain circumstances, it could exert an antiapoptotic role. In the absence of DNA damage, we found that p16silenced cells had elevated levels of Rb, p53 and p21 protein, but did not undergo growth arrest or apoptosis that is commonly associated with p53 and p21 upregulation (el-Deiry et al., 1994; Haapajarvi et al., 1999). This could be owing to the effects of E6 and E7, both of which cooperate to bypass p53-induced G1 arrest and subsequently drive cell-cycle progression in cervical cancer cells (Hawley-Nelson et al., 1989; Demers et al., 1994; Jones and Munger, 1997). This statement can be further supported by evidence that when both HPV16 E6 and E7 are repressed, cervical cells are able to undergo senescence or apoptosis in response to DNA damage (Putral et al., 2005). Although we observed a downregulation of E7 mRNA after p16silencing, we did not detect any alteration in E6 mRNA expression. On the basis of observations from this study, we propose that induction of p53 after p16-silencing occurs independently of E6. As SiHa and Cas Ki cells express wild-type p53 (Scheffner et al., 1991), we hypothesize that when p53 levels increase above a ‘basal threshold’ in these cells, such as in the case of p16silencing, p53-mediated pathways are activated. However, it has also been earlier reported that E6 and E7 are expressed from a bicistronic mRNA (Smotkin and Wettstein, 1986). In the light of these data, further study is required to dissect fully the differential effect of p16-silencing on E6 and E7 expression. p16 knockdown in conjunction with DNA-damage triggers the p53 and RB pathways, sensitizing the cells to apoptosis. This confirms that although HPV16 E6 and E7 inactivate p53 and RB respectively in cervical cancer cells, these cells still express basal levels of p53 and Rb proteins, leaving both the RB and p53-tumour suppressor pathways functionally intact and susceptible to re-activation by appropriate stress signals (Butz et al., 1995; Goodwin and DiMaio, 2000). It has also been shown that p53-dependent G1 arrest involves Rb, thus linking these two tumour suppressor pathways in mediating cell-cycle progression and arrest (Slebos

et al., 1994). In addition, the study by Zhang et al. (2006) highlighted that p16 repression resulted in increased p53 levels mediated by the Rb/E2F signalling pathway, providing further evidence that p16 can modulate both p53 and Rb expression. As a result of p16 suppression using siRNA, we observed the upregulation of Rb, p53 and p21, all of which are crucial in tumour suppression. Therefore, the perturbation of the feedback loop that normally maintains Rb at low levels and p16 at high levels in cervical cancer cells is a major factor in driving the cell toward apoptosis. However, further studies are required to conclusively elucidate the underlying molecular mechanism. Nevertheless, the ability of p16 to influence RB and p53 tumour suppressor pathways despite HPV16 infection offers new avenues to develop novel therapeutic strategies. Several studies have described the use of viral- and RNAi-mediated approaches to repress E6 and E7 to induce senescence or apoptosis (Goodwin et al., 2000; Jiang and Milner, 2002; Gu et al., 2006). Our findings suggest that silencing of p16 in conjunction with chemotherapy and radiotherapy could potentially be an effective treatment for cervical cancer.

Materials and methods Cell culture and transfection SiHa and Cas Ki cell lines were obtained from the American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% heat-inactivated foetal bovine serum (Hyclone, Lugan, UT, USA), 0.1 mM non-essential amino acids (Gibco, Invitrogen, Carlsbad, CA, USA) 2 mM L-glutamine and 1 mM sodium pyruvate (Sigma-Aldrich) and maintained in a humidified 371C incubator with 5% CO2. siRNA transfections were performed using Oligofectamine (Invitrogen) according to recommended protocol. siRNA sequences used are as follows. p16 (Oligoengine, Seattle, WA, USA): Forward 50 -CGCACCGAAUAGUUACGGUT T-30 and Reverse 50 -ACCGUAACUAUUCGGUGCGTT-30 . Stealth negative control siRNA (medium GC) was obtained from Invitrogen. Both p16 and control siRNAs were transfected at a concentration of 20 nM. For DNA damage induction, cells were subjected to UV irradiation at 100 J/m2 using a Stratalinker (Stratagene, La Jolla, CA, USA), or Oncogene


6058 treated with 20 mM cisplatin (Mayne Pharma Plc, Warwickshire, UK). Tissue specimens All cervical cancer tissue samples used in our studies were squamous cell carcinoma of the cervix and were obtained from KK Women’s and Children’s Hospital, Singapore. All samples were collected with informed consent from patients. Tissue samples were immediately frozen in liquid nitrogen upon collection. Of the samples collected, 22 tumour samples, 11 paired non-tumourous tissues and seven normal cervical tissues were used for hybridization with cDNA microarrays. Six independent pairs of tumour and paired non-tumourous tissue were used for real-time PCR studies. ‘Paired nontumourous’ tissue indicates that biopsies were taken from histologically normal tissue adjacent to the tumour. ‘Normal’ cervical tissue samples were obtained from non-cancer patients. All tumour samples used contained HPV type 16 viral DNA and were squamous cell carcinoma of the uterine cervix. cDNA library synthesis, microarray fabrication, hybridization and data analysis Using tumourous and non-tumourous normal cervical tissue taken from a single patient, two reciprocal subtracted cDNA libraries were constructed according to the Clontech PCRSelect cDNA subtraction kit protocol and subcloned into pCR2.1-TOPO (Clontech, Mountain View, CA, USA). Cloning vectors sequencing was performed using ABI PRISM Big Dye Terminator v3.1. Both libraries were amplified by PCR and spotted onto glass slides to generate cDNA microarrays. Microarray fabrication, sample hybridization and data analysis were performed according to similar protocols as described previously (Cheng et al., 2002). RNA Isolation and Real-Time PCR RNA was extracted from frozen tissue samples by homogenization (Omni International, Marietta, GA, USA) in 1 ml TRIzol reagent (Invitrogen) according to manufacturer’s protocol. An additional extraction step was performed with 2:1 phenol (pH 5, Invitrogen) and chloroform mixture to eliminate DNA contamination. A total of 5 mg total RNA was reverse-transcribed using both random hexamers and oligo(dT)12-17 primers with Superscript II reverse transcriptase (Invitrogen). Real-time PCR quantification of gene expression was performed on a RotorGene 3000 system (Corbett Research, Sydney, Australia) using QuantiTect SYBR Green PCR kit (Qiagen, GmBH, Hilden, Germany). The primer sequences used are as follows. p16: forward 50 -AGGCTCT GAGAAACCTCGGGAAA-30 and Reverse 50 -AATGGACA TTTACGGTAGTGGGGGAAG-30 ; RB: Forward 50 -CAGA TGCAATTGTTTGGGTG-30 and reverse, 50 -TGAATGGGC AGTCAATCAAA-30 ; p53: forward, 50 -TGGAAGGAAA TTTGCGTGTG-30 and reverse 50 -AAGGCCTCATTCAGCT CTCG-30 ; p21: forward, 50 -CAGGCGCCATGTCAGAAC-30 and reverse, 50 -GGATTAGGGCTTCCTCTTGG-30 ; E6: Forward 50 -AATGTTTCAGGACCCACAGG-30 and Reverse 50 -TCAGGACACAGTGGCTTTTG-30 ; E7: forward, 50 -TG TTAGATTTGCAACCAGAGA CA-30 and Reverse 50 -GAA CAGATGGGGCACACAAT-30 ; 18S rRNA: forward, 50 -CC TGCGGCTTAATTTGACTC-30 and reverse, 50 -CGCTGAG CCAGTCAGTGTAG-30 . All reactions were performed in duplicate. Standard curves for each gene were generated independently by preparing 10 times serial dilutions of template DNA. The relative copy number of each sample was calculated according to their standard curves using Oncogene

Rotor-Gene software (v6.0.23). Normalization for each sample was performed against 18S rRNA relative copy numbers for the same sample. Relative gene expression changes were calculated by adjusting the copy number of control samples to a value of 100. Western blot analysis Cells were lysed for 15 min on ice in lysis buffer containing 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid , 1% Triton X-100, 0.5% NP-40 and 1 Complete protease inhibitor cocktail (Roche, Penzberg, Germany). Lysates were centrifuged for 15 min at 14 000 r.p.m., 41C and protein concentration was determined using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). About 60 mg protein lysate was resolved on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis mini-gels and transferred to nitrocellulose membranes (Amersham, GE Healthcare, Buckinghamshire, England) using a semi-dry transblotting system (Bio-Rad). Blots were developed using Millipore Immobilon western chemiluminescent horse raddish peroxidase substrate and CL-Xposure film (Pierce, Rockford, IL, USA). Antibodies used were p16 (Ab-2 Neomarkers), p14 (Ab-2, Neomarkers), Rb, p53, phospho-p53 serine 15, phospho-p53 serine 46, and p21 (Cell Signaling Technologies, Danvers, MA, USA). Apoptosis assays – fluorescence activated cell sorting analysis and TUNEL assay Cells were harvested with 0.25%. trypsin/phosphate buffered saline (PBS) and washed once in PBS before fixing in 70% icecold ethanol at 4oC overnight. All centrifugation steps were carried out at 500 g for 5 min at 41C. After fixing, cells were washed once in PBS and resuspended in PBS containing a final concentration of 50 mg/ml propidium iodide and 1 mg/ml RNase A. Cell cycle populations were analysed by flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA, USA). TUNEL assay (in situ cell death detection kit, Roche) was used to detect apoptotic cells. Briefly, cells grown on coverslips were fixed in 3.7% formaldehyde for 15 min at room temperature, rinsed once in PBS and permeabilized in 0.1% Triton X-100/0.1% sodium citrate for 2 min on ice. Cells were then incubated in TUNEL reaction mix for 1 h at 371C in a dark humidified chamber. After rinsing twice in PBS, cells were mounted onto glass slides in 40 ,6-diamidino-2-phenylindole, dihydrochloride (DAPI)-containing mounting medium (Vectashield, Vector Laboratories, Burlingame, CA, USA). Slides were then visualized on a LSM-510 laser-scanning confocal microscope (Carl Zeiss, Thornwood, NY, USA). BrdU cell proliferation assay A total of 1x106 cells were labelled with 20(M BrdU (Pharmingen, BD Biosciences) in culture medium at 371C in a CO2 incubator for 2 h. Cells were harvested with 0.25% trypsin/PBS, washed once with PBS and fixed in ice-cold 70% ethanol at 41C overnight. All centrifugation steps were carried out at 500 g for 5 min at 41C. After fixing, cells were washed once in PBS and thoroughly resuspended in 1 ml 2 N/HCl/Triton X-100 for 30 min at room temperature. Cells were then pelleted and the supernatant aspirated before adding 1 ml 0.1 M sodium borate at pH 8.5 as a neutralization step. The cells were pelleted and washed once in 1% bovine serum albumin (BSA)/PBS. About 0.5 mg/ml antiBrdU antibody was then added in a volume of 100(L and incubated at room temperature for 1 h. Cells were washed once in 1% BSA/PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse for 30 min on ice before


6059 washing in 1% BSA/PBS and resuspending in 300(l PBS containing 50 (g/ml propidium iodide and 1 mg/ml RNase A (final concentrations). Cells were analysed on a FACSCalibur (Becton Dickinson). Affymetrix GeneChip analysis We compared the gene expression profiles of p16-silenced and control siRNA-treated cells with and without UV irradiation. A total two independent experiments were performed and all hybridizations were performed at the same time. 5 g total RNA was converted into double-stranded cDNA using a T7-(dT)24 primer containing T7 RNA polymerase promoter and Superscript II reverse transcriptase (Invitrogen). All subsequent cRNA labelling, hybridizations, wash and scan steps were performed according to protocols from Affymetrix Inc. (Santa Clara, CA, USA). After scanning, the average intensity for the genes in total was normalized to 500. Analyses were performed

using Affymetrix Microarray Suite version 5.0 (MAS5) software. Probe sets that were designated ‘absent’ by the detection algorithm were excluded from analysis. Gene annotations were downloaded from Affymetrix NetAffx website and genes of interest were matched to their probe sets. Using Affymetrix MAS software, probe sets representing genes of interest were selected for further analysis. Corresponding increases and decreases in signal intensity for each probe set were generated by MAS5 change call algorithm. Further analysis was performed using Spotfire software (Spotfire Inc., MA, USA) and Bibliosphere software (Genomatix, Germany).

Acknowledgements This work was supported by the National Medical Research Council.

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