E7 genes in HeLa cervical cancer cells

Gene 553 (2014) 98–104

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SAHA inhibits the transcription initiation of HPV18 E6/E7 genes in HeLa cervical cancer cells Hongpeng He a, Xuena Liu a, Dandan Wang a, Yijie Wang a, Lei Liu a, Hao Zhou a, Xuegang Luo a, Nan Wang a, Bingyan Ji b, Yan Luo b,⁎, Tongcun Zhang a,c,⁎⁎ a b c

Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China School of Basic Medical Sciences, Zhejiang University College of Medicine, #388, YuHangTang Road, Hangzhou, Zhejiang 310058, PR China College of Life Sciences, Wuhan University of Science and Technology, Wuhan 430081, PR China

a r t i c l e

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Article history: Received 14 July 2014 Received in revised form 16 September 2014 Accepted 3 October 2014 Available online 7 October 2014 Keywords: Human papillomavirus E6 and E7 Transcription SAHA Histone acetylation Cervical cancer

a b s t r a c t High risk human papillomavirus (HPV) is a well recognized causative agent of cervical cancer. Suberoylanilide hydroxamic acid (SAHA) is a potential anti-cervical cancer drug; however, its effect on the expression of HPV E6 and E7 genes remains unclear. Here, we show that, in SAHA treated HeLa cells, HPV18 E6 and E7 mRNA and protein levels were reduced, HPV18 promoter activity was decreased, and the association of RNP II with HPV18 promoter was diminished, suggesting that SAHA inhibited the transcription initiation of HPV18 E6 and E7 genes. In SAHA-treated HeLa, although the level of lysine 9-acetylated histone H3 in the whole cell extracts increased obviously, its enrichment on HPV18 promoter was significantly reduced which is correlated with the down-regulation of HPV E6 and E7. © 2014 Published by Elsevier B.V.

1. Introduction Cervical cancer is the second common female malignant tumor. Globally, there were more than half million new cases of cervical cancer diagnosed in 2010 (de Sanjose et al., 2010). Carcinogenesis of cervical cancer is highly correlated with human papillomavirus (HPV) infection, which is different from other cancers or malignant tumors. There are more than 100 types of HPVs isolated so far and 13 of them are defined to be high risk HPVs which have a causative relationship with cervical cancer. Among them, HPV 16 and HPV 18 are the two types being most frequently detected in cervical cancer specimens (de Villiers et al., 2004; zur Hausen, 2009).

Abbreviations: SAHA, suberoylanilide hydroxamic acid; HPV, human papillomavirus; RT-PCR, reverse transcription-PCR; RNPII, RNA polymerase II; ChIP, chromatin immunoprecipitation; FACS, fluorescence activated cell sorting; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor. ⁎ Correspondence to: Y. Luo, Research Building Room A709, School of Basic Medical Sciences, Zhejiang University College of Medicine, YuHangTang Road #388, Hangzhou 310058, Zhejiang, China. ⁎⁎ Correspondence to: T. Zhang, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th. Avenue, Tianjin Economic and Technological Development Area (TEDA), Tianjin 300457, China. E-mail addresses: [email protected] (Y. Luo), [email protected] (T. Zhang).

http://dx.doi.org/10.1016/j.gene.2014.10.007 0378-1119/© 2014 Published by Elsevier B.V.

Genome of HPV is a circular double strand DNA encoding early and late virus genes. The tandemly linked HPV E6 and E7 genes are usually integrated into host cellular genome and are constitutively expressed in HPV-positive cervical cancer cells (zur Hausen, 2009). It has been demonstrated that high risk HPV E6 and E7 proteins contribute to oncogenesis by facilitating cell immortalization, migration, altering cell cycle and apoptosis control, evading host immuno-surveillance, etc. (Ghittoni et al., 2010; Au Yeung et al., 2011; Liu et al., 2009). Introduction of HPV E6 and E7 into normal epithelial cells will immortalize the cells. On the contrary, downregulation of HPV E6 and E7 genes by antisense RNA, siRNA or ectopically expressed HPV E2 which is a repressor of HPV E6/ E7 genes resulted in growth arrest and apoptosis of cervical cancer cells (Gu et al., 2011; Hong et al., 2009; Morrison et al., 2011; Sima et al., 2007). Therefore, HPV E6/E7 genes are specific targets for cervical cancer therapy (Shillitoe, 2006; Stern et al., 2012). In the last 30 years, knowledge about histone post-translational modifications has accumulated quickly and certain chemicals affecting histone modifications are taken as potential anti-cancer drugs. The most successfully developed chemicals are inhibitors of histone deacetylase (HDACi) which are supposed to induce apoptosis and differentiation and inhibit proliferation of cancer cells (Grant and Dai, 2012; Glass and Viale, 2013; Marchion and Munster, 2007). The therapeutic potential of HDACi on HPV-positive cervical cancer was as well investigated in a few of laboratories (Finzer et al., 2001, 2002; Darvas et al., 2010;

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Borutinskaite et al., 2012; Shao et al., 2004; Lin et al., 2009; Jin et al., 2010). However, the effects of HDACi on the expression of HPV E6 and E7 oncogenes were less studied (Finzer et al., 2001, 2002; Darvas et al., 2010; Luczak and Jagodzinski, 2008). So far, chemists had developed four classes of HDACis, namely small molecule HDACi, depsipeptide HDACi, cyclictetrapeptide HDACi and nonpeptide macrocyclic HDACi (Gryder et al., 2012). Among these HDACis, SAHA (suberoylanilide hydroxamic acid, also named vorinostat, Zolinza™) and FK228 (romidepsin, Istodax™), have been approved by FDA USA for treating refractory C cutaneous T-cell lymphoma (CTCL) in 2006 and 2009, respectively (Gryder et al., 2012; Duvic and Vu, 2007). However, there are still limitations and side effects, for instance, cardiac toxicity and trabecular bone loss, observed during the applications of SAHA and FK228 in cancer patients (Gryder et al., 2012; McGee-Lawrence et al., 2011). Hence, bioinformatics scientists and organic chemists are engaged in designing and synthesizing novel HDACis with side effects (Tambunan and Parikesit, 2012; Spencer et al., 2011). JAHA (Jay Amin hydroxamic acid) was recently synthesized as a SAHA analogue with selective HDACi activity (Spencer et al., 2011). Including JAHA, new compounds with HDACi activity are potential alternatives of SAHA in cancer treatment, which should be further verified in wet laboratory experiments and clinical trials. In this study, we used HeLa cell which carries HPV18 DNA in cellular genome as a model to investigate whether SAHA, the first HDACi approved by FDA USA for cancer therapy, would affect the transcription of HPV18 E6 and E7 genes, and further, underlying mechanism was explored.

2. Materials and methods 2.1. Cell culture and treatments HeLa cells were cultured in Dulbecco's modified Eagle's medium with L-Glutamine (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou China) and 1% Antibiotic/Antimycotic (Solarbio, China). SAHA was purchased from Sigma and dissolved in DMSO.

2.2. MTT assay HeLa cells seeded in 96-well culture plates (1.2 × 104 per well) were treated with SAHA at different concentrations. Twenty-four hours after SAHA treatment, 20 μl of MTT (5 mg/ml, Solarbio, China) was added to each well and incubated for 4 h, then the medium was removed and formazan crystals were dissolved with 100 μl of DMSO. Cell viability was analyzed by measuring the absorbance at 590 nm using an ELISA reader (BioTek, USA).

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2.4. Fluorescence activated cell sorting (FACS) HeLa cells treated with 5 μM of SAHA for 24 h were harvested, washed with PBS, fixed with 80% cold ethanol and left at 4 °C for N30 min. Afterwards, cells were washed twice with PBS and incubated in 200 μl of propidium iodide (50 μg/ml, Sigma) containing RNase A (20 μg/ml, Sigma) for 30 min in the dark, before being analyzed by flow cytometry (BD company). 2.5. Extraction of total RNA and RT-PCR Total RNA was extracted with Trizol reagent (Invitrogen). Reverse transcription was performed with M-MLV reverse transcriptase (Promega). Complementary DNA was quantified by Semi-quantitative PCR using EasyTaq DNA Polymerase kit (TransGen Biotech). PCR primers were designed with NCBI online software Primer-BLAST and synthesized by Sigma-Aldrich. Sequences of the primers were: 18s rRNA-forward: 5′CACGGGAAACCTCACCCGGC-3′; 18s rRNA-reverse: 5′-CGGGTGGCTGAA CGCCACTT-3′; GAPDH-forward: 5′-GGGCTCCGGGTCTTTGCAGTCGTA-3′; and GAPDH-reverse: 5′-GGGACCTCCTGTTTCTGGGGACTA-3′. Primers for HPV18 E6, E7 and p21 were as described (He and Luo, 2012). PCR products were visualized by electrophoresis using 2% agarose gels. For realtime qPCR, cDNA was quantified by with Biosystems StepOne™ RealTime PCR and Fast SYBR Green Master Mix (Applied Biosystems). 2.6. Cell transfection and promoter activity assay HeLa cells cultured in 12-well plates at about 80% confluency were co-transfected with 1 μg of pGL4.20 HPV18-LCR-luciferase plasmids (Schweiger et al., 2007) (Addgene, Plasmid #22859) and 1 μg of pSV40renilla luciferase (lab stocks) using Lipofectamine 2000 (Invitrogen). Twenty-four hours post-transfection, cells were treated with SAHA for another 24 h, and then luciferase activities were measured with DualGlo Luciferase Assay System (Promega E2940) in a Veritas Microplate Luminometer. 2.7. Chromatin immunoprecipitation (ChIP) HeLa cells were treated with 5 μM of SAHA for 1 h before being harvested. For ChIP assays with anti-AcH3 (Upstate, #06-599), anti-AcH4 (Upstate, #06-866), anti-RNA polymerase II (Covance, #MMS-126R), anti-H3K4me3 (Abcam, ab8580), AcH3K9 (Millipore, ABE18) or antiH3 (Abcam, ab1790) antibodies, HeLa cells were cross-linked with 1% of formaldehyde (Sigma) for 15 min at room temperature with gentle shaking. For ChIP assays with anti-p300 (Santa Cruz, sc-585) or antiBrg1 (Abcam) antibodies, double cross-linking was performed as previously described (He and Luo, 2012). Procedures for ChIP assays and primers for HPV18 promoter were performed as described (He and Luo, 2012). ChIP products were measured by real-time qPCR using KAPA SYBR FAST qPCR MasterMix kit (Kapa Biosystems) and the realtime PCR system (Applied Biosystems).

2.3. Western-blot 2.8. Statistical analyses HeLa cells were treated with SAHA at different times and whole cell extracts were loaded for SDS-PAGE (15% gel). Proteins were transferred to nitrocellulose membrane (Hybond-C Extra, Amersham Bioscience). Primary antibodies included HPV18 E6 (Santa Cruz, sc-1586), HPV18 E7 (Santa Cruz, sc-365035), AcH3 (Upstate, #06-599), AcH3K9 (Millipore, ABE18), GAPDH (house raised), caspase 3 (Santa Cruz, sc-271759) and beta-actin (Santa Cruz). For western-blot showed in Fig. 1, secondary antibody was HRP-conjugated goat anti-rabbit IgG (Amersham Bioscience) and membranes were visualized with ECL plus kit (GE Healthcare). In Figs. 3 and 4, secondary antibodies were IRDye-conjugated donkey anti-mouse or anti-goat IgG (Licor Biosciences) and membranes were visualized with Odyssey Infrared Imaging System (Gene Company Limited).

Data were analyzed with unpaired student t test. Two tailed p value b0.05 or b0.01 was indicated by * or **, respectively. Data were presented as Mean ± SD. 3. Results 3.1. SAHA inhibits the expression of HPV18 E6/E7 in HeLa cells To select an optimal dosage and to confirm the HDACi activity of SAHA, HeLa cells were treated with serial diluted SAHA for 24 h as indicated in Fig. 1A. The acetylation level of histone H3 was measured by Western-blot using whole cell extracts. As shown in Fig. 1A, the global

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Fig. 1. The expression of HPV18 E6 and E7 decreased after SAHA treatment. (A) The global level of acetylated histone H3 in SAHA treated HeLa cells. Cells treated with SAHA of different concentrations as indicated. Whole cell lysates were loaded for Western-blot. GAPDH was used as a loading control. (B) SAHA downregulated HPV18 E6 and E7 mRNA levels in a dosagedependent manner. (C) Quantitative analysis of HPV18 E6 E7 mRNA with real-time PCR. Data represents mean ± SD, n = 3. (D) SAHA downregulated HPV18 E6 and E7 protein levels in a dosage-dependent manner. In (A–D), HeLa cells were treated for 24 h. (E) SAHA treatment decreased HPV18 E6 and E7 transcript levels constantly. (F) Stable downregulation of HPV18 E6 and E7 protein levels in SAHA treated HeLa. In (E) and (F), HeLa cells were treated with 5 μM of SAHA for 1 to 4 days as indicated. GAPDH and 18s rRNA were loading controls in RT-PCR assay. β-Actin was used as a loading control in Western-blot.

level of acetylated histone H3 increased obviously when SAHA concentration reached 2.5 μM, suggesting that SAHA efficiently inhibited HDAC activity in HeLa cells at the concentrations above 2.5 μM. Therefore, 5 μM of SAHA was applied in following experiments unless otherwise described. HeLa cell constitutively expresses HPV18 E6/E7 genes. To test whether SAHA inhibits HPV18 E6/E7 gene transcription, total RNA was isolated and RT-PCR was carried out. As shown in Fig. 1B, the mRNA levels of HPV18 E6 and E7 were down-regulated gradually following the increase of SAHA concentration. The effects were significant once SAHA concentration was above 2.5 μM. The results of quantitative real-time PCR showed that HPV18 E6 and E7 mRNA levels decreased by nearly 80% with 2.5 μM of SAHA treatment (Fig. 1C). In parallel with the RT-PCR analysis, Western-blot was performed to examine the protein levels of HPV18 E6 and E7. According to the reduced mRNA levels, E6 and E7 protein levels decreased in SAHAtreated HeLa cells in a dosage-dependent manner as well (Fig. 1D). These results suggest that SAHA treatment diminishes the expression of HPV18 E6/E7 in HeLa cells. A previous study reported that sodium butyrate, a different type of HDACi, downregulated HPV18 E7 expression transiently during the

period of 3–12 h of drug treatment however HPV18 E7 protein levels recovered to the control levels for prolonged treatment up to 20 h (Finzer et al., 2002). To test whether the effect of SAHA on HPV18 E6/E7 expression is also transient, HeLa cells were treated with 5 μM of SAHA for different periods. The results of RT-PCR showed that HPV E6 and E7 mRNA levels were constantly downregulated without any recovery up to 96 h of treatment (Fig. 1E). In agreement, the results of Western-blot showed permanent downregulation of HPV E6 and E7 protein levels (Fig. 1F). Therefore, the inhibitory effect of SAHA on HPV18 E6/E7 genes in HeLa cells is stable and is different from that of sodium butyrate.

3.2. SAHA inhibits HPV18 E6/E7 genes at transcription level Apicidin, another type of HDACi, promote a decay of E6/E7 mRNA in SiHa cells, by which the expression of HPV E6 and E7 genes was downregulated (Luczak and Jagodzinski, 2008). To explore the mechanism for the inhibition of HPV18 E6/E7 expression by SAHA, HPV18 promoter activity was analyzed with luciferase assay and the results were shown in Fig. 2A. HPV18 promoter activity was inhibited by SAHA in a dosage-dependent manner and the effect was dramatic when

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Fig. 2. HPV18 E6 and E7 genes were inhibited by SAHA at transcription level. (A) HPV18 promoter activity in HeLa cells treated with SAHA. HeLa cells were co-transfected with pGL4.20 HPV18-LCR-firefly luciferase reporter plasmids and pSV40-renilla luciferase plasmids. Data represents mean ± SD, n = 6. (B) HPV18 E6 and E7 mRNA levels in HeLa cells treated with 5 μM of SAHA for 1 and 6 h, respectively. Real-time PCR was performed and 18s rRNA was the normalizer. Data represents mean ± SD, n = 3. (C) The association of RNA polymerase II and the enrichment of histone H3 trimethylated at lysine 4 on HPV18 promoter in HeLa cells treated with SAHA. Data represents mean ± SD, n = 3.

SAHA concentration was 5 μM. These results suggest that SAHA may inhibit the expression of HPV18 E6/E7 at transcription level. In Fig. 2A, cells were treated for 24 h. To examine whether HPV18 genes respond to SAHA more quickly, HeLa cells were treated with 5 μM of SAHA for 1 h and 6 h, respectively. The results showed that HPV18 E6 and E7 mRNA levels slightly decreased 1 h later and the inhibition became significant 6 h after SAHA treatment (Fig. 2B). These results suggest that SAHA quickly diminished HPV18 E6 and E7 gene transcription. To confirm that SAHA down-regulates HPV18 E6/E7 genes at the transcription level, ChIP assays were carried out with antibodies against RNA polymerase II. As shown in Fig. 2C, after 1 h of SAHA treatment, the association of RNA polymerase II on HPV18 promoter region already decreased by about 60%. The significantly reduced RNA polymerase II association with the HPV18 E6/E7 promoter suggests that the transcription initiation of HPV18 E6/E7 genes was impaired by SAHA. On the same region, the enrichment of tri-methylated histone H3K4 which is positively correlated with transcription activation was measured with ChIP assays. As shown in Fig. 2C, the level of trimethylated histone H3K4 dramatically decreased supporting that the downregulation of HPV18 E6/E7 genes resulted from the defect in transcription initiation. 3.3. The effects of SAHA on HPV18 E6/E7 promoter modification The global level of acetylated histone H3 increased in SAHA treated samples. To measure the acetylation of histones on HPV18 promoter, ChIP was carried out with antibodies against histone H3, acetylated histone H3 or acetylated histone H4. Results showed that the density of histone H3 was unchanged but the acetylated histones H3 and H4 increased by 5 and 3 fold, respectively (Fig. 3A). Histone acetylation was supposed to affect gene transcription either by reducing the static electric interaction between histones and DNA or

by recruiting transcription factors through the physical binding of bromo-domain proteins with the acetylated histone lysine residues (Filippakopoulos and Knapp, 2012; Jenuwein and Allis, 2001). Our previous work showed that the bromodomain-transcription factors p300 and Brg1 which associate with HPV18 promoter are important for the transcription of HPV18 genes (He and Luo, 2012). To test whether the recruitments of p300 and Brg1 were inhibited by SAHA, the association of p300 and Brg1 with HPV18 promoter was examined. The results of ChIP showed that the enrichment of p300 and Brg1 on HPV18 promoter was coincidently upregulated for about 2-folds following SAHA treatment (Fig. 3B), suggesting that the recruitment of transcriptional coactivators to HPV18 promoter was enforced by SAHA treatment. Usually the hyperacetylation of gene promoter and enriched association of transcriptional co-activators should dictate activated transcription. One thing we kept in mind was that the antibodies against acetylated histones H3 and H4 used in ChIP assays target multiple acetylated lysine residues instead of a specific lysine. It is possible that the majority but not all lysine residues of histone were affected by SAHA. Thus we performed ChIP with antibody against acetylated histone H3K9, and the results showed that the enrichment of acetylated H3K9 on HPV18 decreased (Fig. 3C, top row). In contrast to HPV18, the enrichment of H3K9ac on GAPDH and C-myc promoters was not altered (Fig. 3C, middle rows). Myocardin which is a tissue specific gene being silent in HeLa cells was used as a negative control and there was no band detected for this chromatin locus (Fig. 3C, bottom row) indicating the specificity of ChIP assay. ChIP produced DNA was also analyzed with real-time PCR and the results showed that, on HPV18 promoter, the abundance of H3K9ac decreased by about 40% after 1-hour SAHA treatment and further decreased by about 70% after 6-hour treatment (Fig. 3D), which is correlated with the repression of HPV18 E6/E7 transcription. Therefore, the inhibiting effects of SAHA on HPV18 E6/E7 might be mediated by the defect in histone H3K9 acetylation.

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Fig. 3. The modification of HPV18 promoter changed in HeLa cells treated with SAHA. (A) The enrichment of acetylated histones H3 and H4 on HPV18 promoter region. ChIP was performed with antibodies against poly-acetylated H3 or H4. (B) The association of transcription cofactors Brg1 and p300 on HPV18 promoter region. (C) The enrichment of K9-acetylated histone H3 on HPV18. ChIP assays with antibodies specific to acetylated H3K9. GAPDH and c-myc genes serve as positive controls and myocardin gene as a negative control. In (A–C), HeLa cells were treated with 5 μM of SAHA for 1 h. (D) Quantitative analysis of H3K9ac on HPV18 promoter following 5 μM of SAHA treatment for 1 h and 6 h, respectively. Data represents mean ± SD, n = 3. (E) The global levels of acetylated histone H3K9 in whole cell extracts. HeLa cells were treated with the same method as described in (D).

We then examined the global level of histone H3K9 acetylation in whole cell extracts. The results of Western-blot showed that acetylated H3K9 accumulated obviously after 1- or 6-hour of SAHA treatment (Fig. 3E), suggesting that the decrease of H3K9 acetylation on HPV18 promoter is specific to the genomic locus. There are at least 15 HATs identified in mammalian cells (Sterner and Berger, 2000). p300 and Tip60 HAT activities are known to be recruited to HPV18 promoter (Bouallaga et al., 2003; Jha et al., 2010), while the involvement of additional HATs in HPV18 gene regulation cannot be excluded. Some HATs themselves are acetylated via autoacetylation and their conformation is thereby changed which affects their HAT activities and binding affinity with the target DNA sequence (Arif et al., 2007; Stiehl et al., 2007; Yang et al., 2012a, 2012b; Albaugh et al., 2011). It is very possible that HATs modifying H3K9 on HPV18 promoter are hyperacetylated following SAHA treatment. Thus, one possibility for the reduced H3K9 acetylation on HPV18 promoter is that the HAT activity responsible for H3K9 acetylation is indirectly inhibited by SAHA. Another possible reason is that the association of specific HAT(s) with HPV18 promoter is reduced, which leads to the decrease of H3K9 acetylation specifically. For the whole genome, the reaction rate of H3K9 acetylation might be unaffected due to the functional

redundancy of different HATs, then the inhibition of HDACs resulted in the accumulation of H3K9 acetylation in the whole genome. 3.4. SAHA inhibits the proliferation of HeLa cells Constitutive expression of HPV E6 and E7 are critical causative factors of cervical cancer. Since SAHA efficiently inhibited the expression of HPV18 E6/E7 in HeLa cells, the effects of SAHA on cell growth were further investigated. HeLa cells were treated with SAHA for 24 h and the morphological changes were examined under a microscope. As shown in Fig. 4A, HeLa cells treated with SAHA became larger in size, spine-like in shape and lower in cell density. The cell viability was measured by MTT method and the results showed that the amount of viable cells decreased gradually following the increase of drug concentration (Fig. 4B) which suggests that SAHA inhibits HeLa cell growth in a dosage-dependent manner. To examine the cell division cycle of SAHA treated HeLa cells, FACS analysis was performed. As shown in Fig. 4C, the population of cells at G1 phase increased by about 20%, whereas cells at S and G2/M phases were much less in SAHA treated samples than those in control samples, indicating that SAHA induced cell cycle arrest by blocking cell cycle progression from G1 to S phase.

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Fig. 4. SAHA inhibited the proliferation of HeLa cells but did not induce apoptosis. HeLa cells were treated with 5 μM of SAHA for 24 h. (A) The morphological change of HeLa cells following treatment. Magnification, 40×. (B) The proliferation of HeLa cells treated with SAHA. The viable cells were measured with the MTT method. Data represents mean ± SD, n = 6. (C) FACS analysis of HeLa cells treated with SAHA. (D) The examination of pro-caspase 3 and caspase 3 in SAHA treated HeLa cells with Western-blot. β-Actin was used as a loading control.

It was previously reported that HDACi may induce apoptotic cell death (Finzer et al., 2001; Shao et al., 2004), whereas we did not observe apoptosis induced by SAHA as HeLa cells attached firmly to the bottom of culture plates and there were no cells in sub-G1 phase in FACS analysis. The results of Western-blot showed that there was only procaspase 3 but no activated caspase 3, the cleaved form, in SAHA treated HeLa cells (Fig. 4D), supporting that SAHA alone does not induce apoptosis. Taken together, the above results demonstrate that SAHA inhibits cervical cancer cell proliferation by blocking the cell cycle at G1/S transition and has little effect on inducing apoptosis. 4. Discussion HDACis were usually utilized to activate epigenetically repressed genes, such as tumor suppressor genes in malignant tumor cells, by boosting the histone acetylation which is well known as a positive marker for gene activation. However, profiling of gene expression in HDACi treated cells reveals that, actually, some genes are upregulated and others are downregulated by HDACi although the underlying mechanism is unclear (Chambers et al., 2003; Joseph et al., 2004). Here, with the promoter activity assays and ChIP assays we demonstrated that SAHA inhibits the transcription initiation of HPV18 E6/E7 genes

(Fig. 2). Alteration of chromatin structure is important for transcription regulation. With polyclonal antibodies against multiple acetylated histones H3 and H4, the results of ChIP assays showed that histones H3 and H4 were hyperacetylated on HPV18 promoter (Fig. 3A). In addition, the recruitments of positive transcriptional factors p300 and Brg1 were increased as well (Fig. 3B). To dissect the effects of SAHA on lysinespecific acetylation, ChIP assays with specific antibodies against acH3K9 showed that the acetylation of histone H3K9 was reduced on HPV18 promoter by SAHA (Fig. 3C and D). Taken together, the results suggest that not all lysine residues of histone are hyperacetylated by SAHA on HPV18 promoter and the acetylation of histone H3K9 is more crucial for HPV18 gene transcription. In addition to histones, some transcription factors, such as p53 and p300 (Thompson et al., 2004; Black et al., 2008; Brooks and Gu, 2011), are also modified by histone acetylases and HDACs resulting in the regulation of their transcription activities. Thus, it is also possible that the activities of transcriptional factors critical for HPV18 E6/E7 transcription are affected by SAHA treatment, which in turn leads to the defect in HPV18 E6/E7 transcription initiation. The detailed mechanism by which SAHA inhibits the transcription initiation of HPV18 E6 and E7 is worth of further study. For the treatment of cervical cancer, various HDACis were investigated in different laboratories and one common observation is that HDACis

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decrease the proliferation of cancer cells, whereas, different results were obtained regarding the expression of HPV E6/E7 genes (Finzer et al., 2001, 2002; Darvas et al., 2010; Luczak and Jagodzinski, 2008). One possible reason for the discrepancy might be that different types of HDACi function differently. In this study, we treated HeLa cells with SAHA and we found that both mRNA and protein levels of HPV18 E6 and E7 were reduced by SAHA treatment. Given the importance of viral oncoproteins E6 and E7 to the maintenance of cancer phenotype, the downregulation of HPV18 E6/E7 by certain HDACis should, in some way, contribute to the anti-cervical cancer effects. Although being efficient for tumor treatment, SAHA has constitutive, gastrointestinal and hematologic side effects. Therefore, a few of new compounds, such as JAHA and Largazole, have been developed as SAHA replacements (Gryder et al., 2012; Spencer et al., 2011). These SAHA alternatives are potential anti-cervical cancer drugs which are worth of further study in laboratory and clinic in the future. Conflict of interest These authors claim no actual or potential conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (31301073) and Applied Basic Science and Frontier Technology Program of Tianjin (13JCYBJC38000). Zhejiang University provided starting fund to Y Luo through the National 985 Platform; Y Luo has also been supported in part by the China National 973 project (2014CB542003), China Natural Sciences Foundation project (81372179), Zhejiang Provincial Natural Sciences Foundation project (LY13C070001), and the Fundamental Research Funds for the Central Universities, National Ministry of Education, China. References Albaugh, B.N., et al., 2011. Autoacetylation of the histone acetyltransferase Rtt109. J. Biol. Chem. 286 (28), 24694–24701. Arif, M., et al., 2007. Autoacetylation induced specific structural changes in histone acetyltransferase domain of p300: probed by surface enhanced Raman spectroscopy. J. Phys. Chem. B 111 (41), 11877–11879. Au Yeung, C.L., et al., 2011. Human papillomavirus type 16 E6 induces cervical cancer cell migration through the p53/microRNA-23b/urokinase-type plasminogen activator pathway. Oncogene 30 (21), 2401–2410. Black, J.C., et al., 2008. The SIRT2 deacetylase regulates autoacetylation of p300. Mol. Cell 32 (3), 449–455. Borutinskaite, V.V., Magnusson, K.E., Navakauskiene, R., 2012. Histone deacetylase inhibitor BML-210 induces growth inhibition and apoptosis and regulates HDAC and DAPC complex expression levels in cervical cancer cells. Mol. Biol. Rep. 39 (12), 10179–10186. Bouallaga, I., et al., 2003. HMG-I(Y) and the CBP/p300 coactivator are essential for human papillomavirus type 18 enhanceosome transcriptional activity. Mol. Cell. Biol. 23 (7), 2329–2340. Brooks, C.L., Gu, W., 2011. The impact of acetylation and deacetylation on the p53 pathway. Protein Cell 2 (6), 456–462. Chambers, A.E., et al., 2003. Histone acetylation-mediated regulation of genes in leukaemic cells. Eur. J. Cancer 39 (8), 1165–1175. Darvas, K., et al., 2010. Histone deacetylase inhibitor-induced sensitization to TNFalpha/ TRAIL-mediated apoptosis in cervical carcinoma cells is dependent on HPV oncogene expression. Int. J. Cancer 127 (6), 1384–1392. de Sanjose, S., et al., 2010. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol. 11 (11), 1048–1056. de Villiers, E.M., et al., 2004. Classification of papillomaviruses. Virology 324 (1), 17–27. Duvic, M., Vu, J., 2007. Vorinostat: a new oral histone deacetylase inhibitor approved for cutaneous T-cell lymphoma. Expert Opin. Investig. Drugs 16 (7), 1111–1120.

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