E2 Protein Levels Research Articles

Regulator of chromatin condensation 1 abrogates the G1 cell cycle checkpoint via Cdk1 in human papillomavirus E7-expressing epithelium and cervical cancer cells

Regulator of chromatin condensation 1 (RCC1) is a major guanine-nucleotide exchange factor for Ran GTPase and plays key roles in nucleo-cytoplasmic transport, mitosis, and nuclear envelope assembly. RCC1 is known to be a critical cell cycle regulator whose loss causes G1 phase arrest, but the molecular basis for this regulation is poorly understood. Furthermore, little is known about the relationship between RCC1 and carcinomas. Human papillomavirus (HPV) infection is highly associated with the development of cervical cancer. The expression and function of RCC1 in HPV-related cervical cancer and cell cycle regulation have not yet been explored. In this study, we first observed that RCC1 immunostaining was mildly increased in cervical cancer tissues and significantly upregulated in HPV E7-expressing cells; this localization was primarily nuclear. We showed that the transcription factor c-Jun transcriptionally upregulates RCC1 via a direct interaction with the RCC1 promoter. Moreover, siRNA-mediated knockdown of RCC1 inhibited G1/S cell cycle progression and DNA synthesis, while overexpression of RCC1 abrogated the G1 checkpoint. RCC1 knockdown downregulated the protein levels of the transcription factor E2F1, especially nuclear E2F1, by promoting its degradation in HPV E7-expressing cells. Overexpression of E2F1 rescued RCC1 knockdown-mediated inhibition of G1/S progression. Additionally, we showed that cyclin-dependent kinase 1 (Cdk1), a known target of E2F1, is involved in G1 checkpoint regulation, as Cdk1 knockdown hindered G1/S progression, while Cdk1 overexpression rescued RCC1 knockdown-mediated effect on G1 cell cycle progression. Furthermore, RCC1 knockdown reduced HPV E7 protein levels, which may in turn downregulate E2F1. Our study explores the function of RCC1 in G1/S cell cycle progression and suggests that RCC1 may be involved in HPV E7-mediated genomic instability.

Resveratrol and Pterostilbene Exhibit Anticancer Properties Involving the Downregulation of HPV Oncoprotein E6 in Cervical Cancer Cells.

Cervical cancer is one of the most common cancers in women living in developing countries. Due to a lack of affordable effective therapy, research into alternative anticancer compounds with low toxicity such as dietary polyphenols has continued. Our aim is to determine whether two structurally similar plant polyphenols, resveratrol and pterostilbene, exhibit anticancer and anti-HPV (Human papillomavirus) activity against cervical cancer cells. To determine anticancer activity, extensive in vitro analyses were performed. Anti-HPV activity, through measuring E6 protein levels, subsequent downstream p53 effects, and caspase-3 activation, were studied to understand a possible mechanism of action. Both polyphenols are effective agents in targeting cervical cancer cells, having low IC50 values in the µM range. They decrease clonogenic survival, reduce cell migration, arrest cells at the S-phase, and reduce the number of mitotic cells. These findings were significant, with pterostilbene often being more effective than resveratrol. Resveratrol and to a greater extent pterostilbene downregulates the HPV oncoprotein E6, induces caspase-3 activation, and upregulates p53 protein levels. Results point to a mechanism that may involve the downregulation of the HPV E6 oncoprotein, activation of apoptotic pathways, and re-establishment of functional p53 protein, with pterostilbene showing greater efficacy than resveratrol.

Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3–6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

Abstract PR04: HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer

Abstract Human papillomavirus (HPV) 16 plays an etiologic role in a growing subset of head and neck squamous cell carcinoma (HNSCC), where viral expression of the E6 and E7 oncoproteins is necessary for tumor growth and maintenance. Although patients with HPV(+) tumors have a more favorable prognosis, there are currently no HPV-selective therapies. Accumulating evidence indicates that HPV oncoproteins can activate the Phosphoinositol-3-Kinase (PI3K) pathway, which contributes to transformation. Furthermore, PI3K is genomically activated by PIK3CA mutation or amplification in a disproportionately high number of HPV(+) tumors as compared to HPV(-) tumors. Based on this knowledge, we investigated the efficacy of PI3K-targeted therapies in preclinical models of HPV(+) HNSCC. Our results indicate that HPV(+) preclinical models were less sensitive to the PI3K inhibitors BYL719, BKM120, and BEZ235 as compared with HPV(-) models. Sensitivity of HPV(+) cell lines to PI3K inhibitors was increased upon knockdown of the E6 and E7 oncoproteins. Reciprocally, overexpression of E6 and E7 in HPV(-) cells rendered them resistant to PI3K-targeted therapies. Proteomic analyses indicated that treatment of HPV(+) cell lines and patient-derived xenografts (PDXs) with the PI3Kα inhibitor BYL719 induced expression of the receptor tyrosine kinase HER3, as well as E6 and E7. HER3 was found to regulate the abundance of E6 and E7 in the HPV(+) models. Targeting HER3 with siRNAs or the monoclonal antibody, KTN3379, blocked the increase in E6 and E7 protein levels following BYL719 treatment, and enhanced the efficacy of PI3K inhibitors in HPV(+) cell line and PDX models. Taken together, these results suggest that crosstalk between HER3 and HPV16-E6E7 can limit the efficacy of PI3K inhibitors, and that co-targeting HER3 and PI3K may be an effective therapeutic strategy in HPV(+) tumors. This abstract is also being presented as Poster 67. Citation Format: Toni M. Brand, Stefan Hartmann, Neil E. Bhola, Hua Li, Yan Zeng, Rachel O'Keefe, Max V. Ranall, Sourav Bandyopadhyay, Margaret Soucheray, Danielle L. Swaney, Nevan Krogan, Carolyn Kemp, Umamaheswar Duvvuri, Daniel E. Johnson, Michelle A. Ozbun, Julie E. Bauman, Jennifer R. Grandis. HER3 crosstalk with HPV16-E6E7 is a feedback resistance mechanism to PI3K-targeted therapies in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr PR04.

Neurotoxic mechanisms by which the USP14 inhibitor IU1 depletes ubiquitinated proteins and Tau in rat cerebral cortical neurons: Relevance to Alzheimer's disease

In Alzheimer's disease proteasome activity is reportedly downregulated, thus increasing it could be therapeutically beneficial. The proteasome-associated deubiquitinase USP14 disassembles polyubiquitin-chains, potentially delaying proteasome-dependent protein degradation. We assessed the protective efficacy of inhibiting or downregulating USP14 in rat and mouse (Usp14axJ) neuronal cultures treated with prostaglandin J2 (PGJ2). IU1 concentrations (HIU1>25μM) reported by others to inhibit USP14 and be protective in non-neuronal cells, reduced PGJ2-induced Ub-protein accumulation in neurons. However, HIU1 alone or with PGJ2 is neurotoxic, induces calpain-dependent Tau cleavage, and decreases E1~Ub thioester levels and 26S proteasome assembly, which are energy-dependent processes. We attribute the two latter HIU1 effects to ATP-deficits and mitochondrial Complex I inhibition, as shown herein. These HIU1 effects mimic those of mitochondrial inhibitors in general, thus supporting that ATP-depletion is a major mediator of HIU1-actions. In contrast, low IU1 concentrations (LIU1≤25μM) or USP14 knockdown by siRNA in rat cortical cultures or loss of USP14 in cortical cultures from ataxia (Usp14axJ) mice, failed to prevent PGJ2-induced Ub-protein accumulation. PGJ2 alone induces Ub-protein accumulation and decreases E1~Ub thioester levels. This seemingly paradoxical result may be attributed to PGJ2 inhibiting some deubiquitinases (such as UCH-L1 but not USP14), thus triggering Ub-protein stabilization. Overall, IU1-concentrations that reduce PGJ2-induced accumulation of Ub-proteins are neurotoxic, trigger calpain-mediated Tau cleavage, lower ATP, E1~Ub thioester and E1 protein levels, and reduce proteasome activity. In conclusion, pharmacologically inhibiting (with low or high IU1 concentrations) or genetically down-regulating USP14 fail to enhance proteasomal degradation of Ub-proteins or Tau in neurons.

P53-dependent up-regulation of CDKN1A and down-regulation of CCNE2 in response to beryllium.

Beryllium salts (here, beryllium sulphate) can produce a cytostatic effect in some cell types. The basis for this effect may include increased expression of proliferation inhibitors, reduced expression of proliferation promoters, or both. This study sought to determine the role of p53, the tumour-suppressing transcription factor, in mediating beryllium-induced cytostasis. Human A172 glioma cells express wild-type TP53 gene. Activity of p53 was experimentally manipulated using siRNA and related approaches. Key elements of the beryllium-response were compared in normal and p53-knockdown A172 cells using RT-PCR and Western blotting. In A172 cells, 10μm BeSO4 caused 300% increase in CDKN1A (cyclin-dependent kinase inhibitor p21) mRNA and 90% reduction of CCNE2 (cyclin E2) mRNA. The increased p21 mRNA and reduced cyclin E2 mRNA were each dependent on presence of functional p53. For p21, increased mRNA led to commensurately increased protein levels. In contrast, reduction in cyclin E2 mRNA levels did not lead to corresponding reductions in cyclin E2 protein. The proteasomal inhibitor MG-132 caused p53 protein to increase, but it had no effect on cyclin E2 protein levels. Cycloheximide time course studies indicated that the cyclin E2 protein half-life was more than 12hours in these cells. Beryllium elicited p53-dependent changes in mRNA levels of key determinants of cell proliferation such as p21 and cyclin E2. However, cyclin E2 protein appeared to be aberrantly regulated in this cell type, as its turnover was unexpectedly slow.

Antiproliferative and Pro-Apoptotic Effect of Novel Nitro-Substituted Hydroxynaphthanilides on Human Cancer Cell Lines.

Ring-substituted hydroxynaphthanilides are considered as cyclic analogues of salicylanilides, compounds possessing a wide range of pharmacological activities, including promising anticancer properties. The aim of this study was to evaluate the potential anticancer effect of novel nitro-substituted hydroxynaphthanilides with a special focus on structure-activity relationships. The antiproliferative effect was assessed by Water Soluble Tetrazolium Salts-1 (WST-1) assay, and cytotoxicity was evaluated via dye exclusion test. Flow cytometry was used for cell cycle analysis and detection of apoptosis using Annexin V-FITC/PI assay. Protein expression was estimated by Western blotting. Our data indicate that the potential to cause the antiproliferative effect increases with the shift of the nitro substituent from the ortho- to the para-position. The most potent compounds, 3-hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide (2), and 2-hydroxy-N-(4-nitrophenyl)-naphthalene-1-carboxamide (6) showed antiproliferative activity against THP-1 and MCF-7 cancer cells without affecting the proliferation of 3T3-L1 non-tumour cells. Compounds 2 and 6 induced the accumulation of THP-1 and MCF-7 cells in G1 phase associated with the downregulation of cyclin E1 protein levels, while the levels of cyclin B1 were not affected. Moreover, compound 2 was found to exert the pro-apoptotic effect on the THP-1 cells. These results suggest that hydroxynaphthanilides might represent a potential model structure for the development of novel anticancer agents.

A New Sandwich ELISA Test Simultaneously Detecting E7 Proteins of HPV-16, 18 and 45 in Cervical Smears

Persistent infections by high-risk papilloma viruses (HPV) are the main etiological factor for cervical cancer, and E7 oncoproteins were suggested as new markers for tumor progression. The objective of this study was to generate a new Enzyme-Linked Immunosorbent Assay (ELISA)-based detection system to monitor expression of the E7 proteins of the high-risk HPV (hrHPV) types HPV-16, HPV-18, and HPV-45 in cervical smears. Using a combination of rabbit monoclonal antibodies raised against E7 proteins of HPV-16 and HPV-18/HPV-45, respectively, a trivalent E7-ELISA was developed and validated, using recombinant E7 proteins of various HPV types and lysates from E7- positive cervical carcinoma cells. The amount of 0.5 picogram of E7 protein per well was determined as detection limit. The E7-ELISA was used to determine E7 protein levels in cervical smears obtained from a total of 67 women. E7 protein concentration was below the detection limit in all HPV-negative smears, and E7 protein concentrations above background were found in some HPV-positive cervical samples. Together the work described herein provides a new tool for the simultaneous detection of E7 proteins of the three most prevalent in cervical neoplasia hrHPV types.

MiRNA-497 regulates cell growth and invasion by targeting cyclin E1 in breast cancer

BackgroundMicroRNAs are a class of endogenous single strand non-coding RNAs that are involved in many important physiological and pathological processes. The purpose of this study was to examine the expression levels of miR-497 in human breast cancer and its function in MDA-MB-231 breast cancer cells.MethodsQuantitative polymerase chain reaction was used to measure the expression levels of miR-497 in 40 breast cancer specimens and adjacent normal breast tissues. MTT assays, colony formation assays, wound healing assays, transwell assays and cell cycle assays were used to explore the potential function of miR-497 in MDA-MB-231 breast cancer cells. Dual-luciferase reporter assays were performed to analyze the regulation of putative target of miR-497, and western blot assays were used to validate the dual-luciferase results.ResultsThe expression of miR-497 in breast cancer specimens was lower than adjacent normal tissues (P < 0.05). Overexpression of miR-497 inhibited cellular growth, suppressed cellular migration and invasion, and caused a G1 arrest. Dual-luciferase reporter assays showed that miR-497 binds the 3′-untranslated region (3′-UTR) of cyclin E1, suggesting that cyclin E1 is a direct target of miR-497. Western blot assays confirmed that overexpression of miR-497 reduced cyclin E1 protein levels.ConclusionsMiR-497 may act as a tumor suppressor gene in breast cancer. Inhibited cellular growth, suppressed cellular migration and invasion, and G1 cell cycle arrest were observed upon overexpression of miR-497 in cells, possibly by targeting cyclin E1. These results indicate miR-497 could be considered a therapeutic target for the development of treatment for breast cancer.

Immunogenicity of a lettuce-derived vaccine candidate expressing the E2 protein against classical swine fever virus

Classical swine fever (CSF) is a highly infectious hemorrhagic disease that affects domestic and wild swine. Live attenuated vaccines for classical swine fever viruses (CSFV) can provide rapid onset protection against CSF. However, because of serious safety concerns regarding the use of modified-live virus vaccines, the development of novel CSFV vaccines is urgently required. The E2 protein plays a vital role in viral replication and is essential in CSF development; thus represents an ideal vaccine target. Transgenic plants provide cost-effective and scalable systems for the production and delivery of viral protein, and might have potential use as oral vaccines. In this study, we generated a transgenic lettuce expressing the CSFV E2 gene, under the control of the CaMV 35S promoter, using Agrobacterium-mediated transformation. We confirmed the stable integration and transcriptional expression of the E2 gene using genomic DNA PCR amplification and quantitative real-time PCR analysis, respectively. We then confirmed E2 expression using Western blot analysis. Based on ELISA analysis, levels of E2 protein in leaf tissues of different transgenic lines ranged between 0.21 and 0.53 μg mg−1 of the total soluble protein. We detected E2-specific antibodies in mice following their subcutaneous and oral immunization with protein extracts from transgenic lettuce. Following the vaccination of mice with the recombinant E2 protein, we identified upregulated IFN-γ and IL-10 in their splenocytes.

Detection of Human Papillomavirus Type 18 E7 Oncoprotein in Cervical Smears: a Feasibility Study

Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6- to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears.

NRIP, a novel calmodulin binding protein, activates calcineurin to dephosphorylate human papillomavirus E2 protein.

Previously, we found a gene named nuclear receptor interaction protein (NRIP) (or DCAF6 or IQWD1). We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed small interfering RNAs (siRNAs) decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression.

MiRNA Regulation of Liver Growth After 50% Partial Hepatectomy and Small Size Grafts in Rats

The molecular mechanisms underlying the growth of small size grafts and the remaining livers are poorly understood. MicroRNAs (miRNAs) negatively modulate expression of genes that are involved in cellular function and metabolism. The aim of this study is to identify critical miRNA species that modulate the growth of small grafts and the remaining livers after partial hepatectomy (PH). Small size graft liver transplantation was performed in rats. Liver tissue was harvested after transplant or PH for the determination of miRNA expression profile, and the data were confirmed by quantitative reverse-transcriptase polymerase chain reaction. The genes involved in cell cycle and proliferation were analyzed by quantitative reverse-transcriptase polymerase chain reaction and immunohistochemical staining. Compared with control liver, miR_122a, Let_7b, and miR_26a were reduced by more than 90% in 45% volume grafts. In the remaining livers after 50% PH, 30 miRNAs were down-regulated by more than 50%, and among them, miR_22a, miR_26a, miR_30b, Let_7f, and Let_7g were markedly decreased. A negative correlation existed between down-regulated miRNAs and highly up-regulated genes involved in cell cycle and proliferation in the remaining livers. Moreover, overexpression of miR_26a markedly down-regulated cyclin E2 protein levels and significantly decreased proliferation of HepG2 cells. Down-regulated miRNAs play a pivotal role in promoting the growth of small size grafts and the remaining livers. The negative correlation between down-regulated miRNAs and up-regulated genes suggests that these specific miRNAs participate in the modulation of a growth response in both living donors and small size graft recipients.

Suppression of RISC-Independent Decoy and RISC-Mediated mRNA Base-Pairing Activities of MicroRNA-328 Is Required for Differentiation-Arrest and Enhanced Survival of Blast Crisis CML Progenitors.

Abstract 855MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind mRNA in a sequence-specific manner. We have reported that a) hnRNP-E2 suppresses CEBPA mRNA translation and inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BCCD34+; Perrotti et al, Nat Genet 2002); and b) miR-328 expression is lost in myeloid CML-BCCD34+ progenitors (n=6) and its restored expression at physiological levels rescues granulocytic differentiation and impairs clonogenic potential of primary BCR/ABL+ blasts (Eiring et al, ASH 2007). Here we show by Northern blot, real-time PCR, and microarray analyses that miR-328 levels increase during granulocytic differentiation of normal human CD34+ and mouse Lin− BM progenitors, but not during differentiation towards erythroid, megakaryocytic or monocytic lineages. BCR/ABL uses the same MAPKERK1/2-hnRNP-E2 signaling pathway to suppress both C/EBPα and miR-328, as pharmacologic or shRNA-mediated inhibition of these molecules restored miR-328 expression in BCR/ABL+ cells. In fact, two functional C/EBPα binding sites are present in the miR-328 promoter region and C/EBPα interacts in vivo with these regulatory elements to enhance miR-328 transcription.Importantly, we also show that restored maturation of BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the C-rich regions of miR-328. Indeed, RNA-immunoprecipitation (RIP) assays demonstrated that miR-328 directly binds to hnRNP-E2 independent of the RNA-induced silencing complex (RISC). Furthermore, ectopic miR-328, but not miR-181b, resulted in decreased in vivo binding of hnRNP-E2 to the uORF/spacer region of CEBPA mRNA, thereby releasing CEBPA from hnRNP-E2 translation inhibition and rescuing C/EBPa-driven neutrophil maturation (decoy activity). Differentiation of miR-328-expressing CML-BCCD34+ blasts (88.8±2.4% post-mitotic cells) correlated with induction of C/EBPa protein expression, whereas CEBPA mRNA and hnRNP E2 protein levels remained unchanged. The existence of a direct miR-328/hnRNP-E2/CEBPA interplay was formally demonstrated in vitro using RRL-directed translation assays and in vivo using the 6.15 clone of 32D-BCR/ABL cells that do not express endogenous CEBPA mRNA and require ectopic C/EBPα (wt-uORF-CEBPA) for differentiation. Addition of miR-328, but not miR-330, to hnRNP-E2-containing RRL reactions increased newly synthesized 35S-C/EBPa levels by >100%. Likewise, forced miR-328 expression in vivo resulted in decreased hnRNP-E2 binding to CEBPA mRNA, induction of C/EBPa protein but not mRNA and rescued granulocytic differentiation of 6.15-wt-uORF-CEBPA but not vector-transduced 6.15 cells.While hnRNP-E2 was not found in complex with basic RISC components (Dicer, TRBP2 and Ago2), RIP assays detected miR-328 associated to Dicer and Ago2 in miR-328-expressing cells, suggesting that it also acts through canonical RISC-dependent base-pairing with mRNA targets. Indeed, we identified the BCR/ABL-regulated PIM1 serine-threonine kinase as a bona fide miR-328 target in BCR/ABL+ cells. Ectopic miR-328 suppressed PIM1 protein but not mRNA levels, and this effect required integrity of the miR-328 binding site present in the PIM1 3'UTR. Forced expression of a wild-type but not kinase-deficient PIM1 lacking the 3'UTR into miR-328-expressing cells fully rescued BCR/ABL clonogenicity, suggesting that miR-328-induced PIM1 suppression accounts for reduced survival of miR-328-infected BCR/ABL+ blasts. To show that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 seed sequence (miR-328-Mut) while retaining its C-rich character. Similar to wild-type miR-328, miR-328-Mut efficiently interacted with hnRNP-E2, restored C/EBPa protein expression and rescued granulocytic differentiation, but was unable to silence PIM1 in 32D-BCR/ABL cells, indicating that the C-rich character of miR-328 is essential for its decoy activity, while its seed sequence integrity is necessary for RISC-dependent pairing to mRNA targets.Thus, the discovery of dual activities for miR-328 not only adds a new layer of complexity to the mechanisms regulating CML disease progression, but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA-binding proteins. Disclosures:Cortes:Novartis: Research Funding.

Estrogen Regulation of Cyclin E2 Requires Cyclin D1 but Not c-Myc

During estrogen-induced proliferation, c-Myc and cyclin D1 initiate independent pathways that activate cyclin E1-Cdk2 by sequestration and/or downregulation of the CDK inhibitor p21(Waf1/Cip1), without significant increases in cyclin E1 protein levels. In contrast, cyclin E2 undergoes a marked increase in expression, which occurs within 9 to 12 h of estrogen treatment of antiestrogen-pretreated MCF-7 breast cancer cells. Both E cyclins are important to estrogen action, as small interfering RNA (siRNA)-mediated knockdown of either cyclin E1 or cyclin E2 attenuated estrogen-mediated proliferation. Inducible expression of cyclin D1 upregulated cyclin E2, while siRNA-mediated knockdown of cyclin D1 attenuated estrogen effects on cyclin E2. However, manipulation of c-Myc levels did not profoundly affect cyclin E2. Cyclin E2 induction by estrogen was accompanied by recruitment of E2F1 to the cyclin E1 and E2 promoters, and cyclin D1 induction was sufficient for E2F1 recruitment. siRNA-mediated knockdown of the chromatin remodelling factor CHD8 prevented cyclin E2 upregulation. Together, these data indicate that cyclin E2-Cdk2 activation by estrogen occurs via E2F- and CHD8-mediated transcription of cyclin E2 downstream of cyclin D1. This contrasts with the predominant regulation of cyclin E1-Cdk2 activity via CDK inhibitor association downstream of both c-Myc and cyclin D1 and indicates that cyclins E1 and E2 are not always coordinately regulated.

The stability of the human papillomavirus E6 oncoprotein is E6AP dependent

Human papillomavirus (HPV) E6 oncoproteins target numerous cellular proteins for ubiquitin-mediated degradation. In the case of p53 this is mediated by the E6AP ubiquitin ligase. However, there are conflicting reports concerning how central E6AP is to the global function of the HPV-16 and HPV-18 E6 oncoproteins. To investigate this further we have analysed the effects of E6AP removal upon the stability of endogenously expressed E6 protein. We show that when E6AP is silenced in HPV-positive cells, E6 protein levels are dramatically decreased in a proteasome-dependent manner. Further, we show that when E6AP is depleted in HeLa cells, E6 has a greatly decreased half-life. In addition, overexpression of E6AP stabilises ectopically expressed HPV-16 and HPV-18 E6 in a manner that is independent of its ubiquitin ligase activity. These results demonstrate that the stability of HPV E6 is critically dependent upon the presence of E6AP.

The Ubiquitin-Specific Peptidase USP15 Regulates Human Papillomavirus Type 16 E6 Protein Stability

Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.

Paclitaxel combined with siRNA targeting HPV16 oncogenes improves cytotoxicity for cervical carcinoma

Cervical cancer is attributable to continuous expression of the E6 and E7 oncoproteins of the high-risk human papillomaviruses. These proteins target p53 and members of the retinoblastoma cellular regulatory protein family respectively for degradation, disrupting cellular control over apoptosis, senescence and the cell cycle. Delivery of short interfering RNAs (siRNAs) targeting mRNA from the HPV16 E6/E7 open reading frame to HPV16-positive cell lines, led to an 80% reduction in full-length transcripts and 60% reduction in total (full-length and spliced) transcripts. Downregulation of E6 mRNA led to increased levels of p53 detectable by western blot and resulted in an eightfold increase in luciferase expression from a p53-responsive reporter plasmid. Downregulation of E7 mRNA reduced the levels of E7 protein and increased the levels of hypophosphorylated pRb. Cellular proliferation was reduced after siRNA delivery; the effect being greater in SiHa cells than CaSki cells and when full-length transcripts encoding E6 and spliced transcripts encoding E7 were both targeted. There was no loss of proliferation in human papillomavirus-negative cell lines. Elevation of p53 in the absence of changes to Rb led to 35% of CaSki cells undergoing apoptosis, whereas in SiHa cells restoration of both p53 and hypophosphorylated Rb had the greatest effect with 25% of cells undergoing apoptosis. The combined use of oncogene-targeting siRNA with either carboplatin, irinotecan, leptomycin B or doxorubicin led to additive toxicity, whereas, with cisplatin, led to sub-additive toxicity. In contrast, siRNA combined with paclitaxel treatment resulted in synergistic toxicity, with intronic siRNA (which mainly targets E6) more effective than exonic siRNA (which targets both E6 and E7). The growth of SiHa xenograft tumors was reduced using paclitaxel combined with intronic and exonic siRNA, compared with exonic siRNA alone, confirming the synergistic relationship between p53 restoration and paclitaxel.

Cidofovir induces an increase in levels of low‐risk and high‐risk HPV E6

Cidofovir is a nucleoside analogue that is used off-license to treat recurrent respiratory papillomatosis (RRP) caused by HPV6/11. However, the effect of this drug upon low-risk HPV 6/11 gene expression is unknown. The expression of E6 was evaluated by RT-PCR in HPV-ve C33A cervical carcinoma cells stably transfected with both low- and high-risk HPV E6 cDNA's and in SiHa (HPV16+ve) cervical carcinoma cells after treatment with 2 doses and durations of exposure to cidofovir. Compared to the vector only transcript, E6 RNA levels showed an 8-fold increase in low-risk and 20-fold increase in high-risk E6-expressing cells. High-risk E6 protein levels were also detected by Western blot in cidofovir-treated C33A Type16 E6-transfected cells. These data may indicate a potential rationale for increased risk of genetic instability and thus transformation due to drug-induced increase in the level of E6.

Host cell sumoylation level influences papillomavirus E2 protein stability

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329–338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125–136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.