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1

Oncoprotein E7 from Beta Human Papillomavirus 38 Induces Formation of an Inhibitory Complex for a Subset of p53-Regulated Promoters  

PubMed Central

Our previous studies on cutaneous beta human papillomavirus 38 (HPV38) E6 and E7 oncoproteins highlighted a novel activity of I?B kinase beta (IKK?) in the nucleus of human keratinocytes, where it phosphorylates and stabilizes ?Np73?, an antagonist of p53/p73 functions. Here, we further characterize the role of the IKK? nuclear form. We show that IKK? nuclear translocation and ?Np73? accumulation are mediated mainly by HPV38 E7 oncoprotein. Chromatin immunoprecipitation (ChIP)/Re-ChIP experiments showed that ?Np73? and IKK? are part, together with two epigenetic enzymes DNA methyltransferase 1 (DNMT1) and the enhancer of zeste homolog 2 (EZH2), of a transcriptional regulatory complex that inhibits the expression of some p53-regulated genes, such as PIG3. Recruitment to the PIG3 promoter of EZH2 and DNMT1 resulted in trimethylation of histone 3 on lysine 27 and in DNA methylation, respectively, both events associated with gene expression silencing. Decreases in the intracellular levels of HPV38 E7 or ?Np73? strongly affected the recruitment of the inhibitory transcriptional complex to the PIG3 promoter, with consequent restoration of p53-regulated gene expression. Finally, the ?Np73?/IKK?/DNMT1/EZH2 complex appears to bind a subset of p53-regulated promoters. In fact, the complex is efficiently recruited to several promoters of genes encoding proteins involved in DNA repair and apoptosis, whereas it does not influence the expression of the prosurvival factor Survivin. In summary, our data show that HPV38 via E7 protein promotes the formation of a multiprotein complex that negatively regulates the expression of several p53-regulated genes.

Saidj, Djamel; Cros, Marie-Pierre; Hernandez-Vargas, Hector; Guarino, Francesca; Sylla, Bakary S.; Tommasino, Massimo

2013-01-01

2

Hypoxia inducible factor-1 alpha expression is increased in infected positive HPV16 DNA oral squamous cell carcinoma and positively associated with HPV16 E7 oncoprotein  

PubMed Central

Background There is increasing evidence for the role of High Risk (HR) Human PapillomaVirus (HPV) in the pathogenesis of Oral Squamous Cell Carcinoma (OSCC). The E6 and E7 oncogenes from HR HPVs are responsible for the deregulation of p53 and pRB proteins involved in cell cycle and apoptotic pathways. In cell lines experiments, the HPV E7 protein seems to be able to enhance Hypoxia Inducible Factor-1 alpha (HIF-1?) activity, normally involved in the response to hypoxia and able to enhance angiogenesis. Results We studied tumor specimens from 62 OSCC; a higher prevalence of tumors in TNM stage II and also in pT2 class between OSCC infected positive HPV16 DNA than non-infected ones was observed. HIF-1? positivity was detected throughout the analysed fields, not associated with areas of necrosis and also observed in cells immediately adjacent to blood vessels. A significant increase in mean values of the HIF-1? labeling indexes was observed for pT1-T2, as well for stage I-II, in the infected positive HPV16 DNA tumors than non-infected ones. HIF-1? and HPV16 E7 labeling indexes showed a significantly positive correlation which suggested a positive association between HPV16 E7 and HIF-1? expression. Conclusions In our specimens HIF-1? immunoreactivity hints for an O2-independent regulatory mechanism in infected positive HPV16 DNA tumors, especially for pT1-T2 and stage I-II tumors, suggesting a very early involvement in the development of HPV-induced OSCC. HIF-1? and HPV16 E7 labeling indexes suggest also a positive association between the two proteins in infected positive HPV16 DNA OSCC.

2011-01-01

3

Expression of the HPV E7 Oncoprotein Mimics but Does Not Evoke a p53Dependent Cellular DNA Damage Response Pathway  

Microsoft Academic Search

Acute expression of the human papillomavirus E7 oncoprotein in preimmortal human fibroblasts induces changes in the abundances of multiple cellular regulatory proteins. These alterations include a destabilization of the retinoblastoma tumor suppressor protein pRB, stabilization of the tumor suppressor protein p53, and increases in the level of the cyclin-dependent kinase inhibitor p21cip1. Since the HPV E7 oncoproteins can interfere with

D. Leanne Jones; David A. Thompson; Elizabeth Suh-Bürgmann; Miranda Grace; Karl Münger

1999-01-01

4

A postulated role of p130 in telomere maintenance by human papillomavirus oncoprotein E7.  

PubMed

High-risk human papillomaviruses (HR-HPVs) infections is highly associated with the development of cervical cancer. It is now recognized that telomere length maintenance or extension is indispensable for carcinogenesis. The early oncoproteins E6 and E7 are the main malignant transformation factors of HR-HPVs and they maintain telomeres by different mechanisms, of which E6 protein activating telomerase is well documented. Reports showed that E7 protein utilized an alternative lengthen of telomere (ALT) mechanism to restore telomere length, yet the underlying molecular basis remains largely unknown. We propose that degradation of tumor suppressor pRb family member p130 plays an essential role in E7-regulated telomere extension by ALT. ALT is a mechanism based on homologous recombination (HR) between telomere sister chromatids, and a number of proteins involved in the HR pathway, such as MRN [MRE11 (meiotic recombination 11)-Rad50-NBS1 (Nijmegen breakage syndrome 1)] complex are required for the ALT pathway. Rb family member p130 could inhibit ALT by interacting with Rad50, while HPV E7 could activate ALT by degrading p130. We will make E7 mutants which are defective in p130 degradation to test whether these cells have a limited life span. Besides, immunofluorescence assay will show an ALT-related promyelocytic leukemia (PML) body (APBs) in E7-expressing cells. Although cervical cancer usually has high telomerase activities since the expressing of HPV E6, the anti-telomerase therapy will be unavailable for cervical cancer since it may activate E7-induced ALT. Our hypothesis not only enrich the knowledge of the regulation of ALT, but also indicate that p130 may serve as a potential suppressor of ALT, and gene therapy of p130 may be used in cervical cancers. PMID:22595804

Zhang, WeiFang; Tian, YongHao; Chen, Jason J; Zhao, WeiMing; Yu, XiuPing

2012-08-01

5

Identification of the nuclear localization and export signals of high risk HPV16 E7 oncoprotein  

SciTech Connect

The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence ({sub 76}IRTLEDLLM{sub 84}) changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments.

Knapp, Alixandra A.; McManus, Patrick M.; Bockstall, Katy [Biology Department, Boston College, Higgins Hall, Room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States); Moroianu, Junona [Biology Department, Boston College, Higgins Hall, Room 578, 140 Commonwealth Avenue, Chestnut Hill, MA 02467 (United States)], E-mail: moroianu@bc.edu

2009-01-05

6

Indoleamine 2,3-dioxygenase Activity Contributes to Local Immune Suppression in the Skin Expressing Human Papillomavirus Oncoprotein E7  

PubMed Central

Chronic infection of anogenital epithelium with human papillomavirus (HPV) promotes development of cancer. Many pathogens evoke immunosuppressive mechanisms to enable persistent infection. We have previously shown that grafted skin expressing HPV16 E7 oncoprotein from a keratin-14 promoter (K14E7) is not rejected by a syngeneic, immunocompetent host. In this study we show that indoleamine 2, 3-dioxygenase (IDO) 1, an IFN-? inducible immunoregulatory molecule, is more highly expressed by langerin?ve dermal dendritic cells from K14E7 skin than nontransgenic control skin. Furthermore, inhibiting IDO activity using 1-D/L-methyl tryptophan promotes K14E7 skin graft rejection. Increased IDO1 expression and activity in K14E7 skin requires IFN-? and iNKT cells, both of which have been shown to negatively regulate T-cell effector function and suppress K14E7 graft rejection. Further, dendritic cells from K14E7 skin express higher level of IFN-? receptor (IFN-?R) than dendritic cells from control skin. K14E7 transgenic skin recruits significantly higher number of dendritic cells, independent of IFN-? and IFN-?R expression. Consistent with these observations in a murine model, we found higher expression of IDO1 and IFN-? but not IDO2 in the cervical epithelium of patients with HPV-associated cervical intraepithelial neoplasia (CIN) 2/3. Our data support a hypothesis that induction of IDO1 in HPV infected skin contributes to evasion of host immunity.

Mittal, D; Kassianos, AJ; Tran, LS; Bergot, AS; Gosmann, C; Hofmann, J; Blumenthal, A; Leggatt, GR; Frazer, IH

2013-01-01

7

Expression of the human papillomavirus type 16 E7 oncoprotein induces an autophagy-related process and sensitizes normal human keratinocytes to cell death in response to growth factor deprivation  

SciTech Connect

Expression of oncogenes, such as the human papillomavirus type 16 (HPV16) E7 oncoprotein, promotes aberrant cell proliferation. In the absence of concurrent mitogenic stimuli, this triggers a cell-intrinsic defense mechanism, the 'trophic sentinel response', which eliminates such aberrant cells. The molecular pathways that elicit this response, however, remain obscure. We set up an experimental system to investigate the trophic sentinel pathway triggered by HPV16 E7 expression in normal human keratinocytes, the natural host cells of HPVs. Keratinocytes expressing HPV16 E7 cultured in E-medium undergo cell death and show increased sub-G1 DNA content when grown to confluence or under conditions of serum deprivation. Moreover, HPV16 E7 expressing human keratinocytes express higher levels of the autophagy marker, LC3-II, which can be abrogated by 3-methyladenine, an autophagy inhibitor. These findings indicate that even under normal culture conditions, HPV16 E7 expression triggers metabolic stress that may result in autophagy, a pathway implicated in carcinogenesis.

Zhou Xiaobo [Infectious Diseases Division, Channing Laboratories, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School (United States); Muenger, Karl [Infectious Diseases Division, Channing Laboratories, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School (United States)], E-mail: kmunger@rics.bwh.harvard.edu

2009-03-01

8

The human papillomavirus-16 E7 oncoprotein exerts antiapoptotic effects via its physical interaction with the actin-binding protein gelsolin.  

PubMed

The oncoprotein E7 from human papillomavirus-16 (HPV-16 E7) plays a pivotal role in HPV postinfective carcinogenesis, and its physical interaction with host cell targets is essential to its activity. We identified a novel cellular partner for the viral oncoprotein: the actin-binding protein gelsolin (GSN), a key regulator of actin filament assembly and disassembly. In fact, biochemical analyses, generation of a 3D molecular interaction model and the use of specific HPV-16 E7 mutants provided clear cut evidence supporting the crucial role of HPV-16 E7 in affecting GSN integrity and function in human immortalized keratinocytes. Accordingly, functional analyses clearly suggested that stable HPV-16 E7 expression induced an imbalance between polymeric and monomeric actin in favor of the former. These events also lead to changes of cell cycle (increased S phase), to the inhibition of apoptosis and to the increase of cell survival. These results provide support to the hypotheses generated from the 3D molecular interaction model and encourage the design of small molecules hindering HPV-induced host cell reprogramming by specifically targeting HPV-16 E7-expressing cells. PMID:23729654

Mileo, Anna M; Abbruzzese, Claudia; Vico, Carmen; Bellacchio, Emanuele; Matarrese, Paola; Ascione, Barbara; Federico, Antonio; Della Bianca, Stefano; Mattarocci, Stefano; Malorni, Walter; Paggi, Marco G

2013-10-01

9

Eradication of Established Tumors by Vaccination With Recombinant Bordetella pertussis Adenylate Cyclase Carrying the Human Papillomavirus 16 E7 Oncoprotein  

Microsoft Academic Search

High-risk human papillomaviruses (HPV) such as HPV16 are associated with the development of cervical cancer. The HPV16-E6 and HPV16-E7 oncoproteins are expressed through- out the replicative cycle of the virus and are necessary for the onset and maintenance of malignant transformation. Both these tumor-specific antigens are considered as potential targets for specific CTL-mediated immunotherapy. The adeny- late cyclase (CyaA) of

Xavier Preville; Daniel Ladant; Benedikt Timmerman; Claude Leclerc

2005-01-01

10

Novel action modality of the diterpenoid anisomelic acid causes depletion of E6 and E7 viral oncoproteins in HPV-transformed cervical carcinoma cells.  

PubMed

Cervical cancer, the second most common malignancy among women, is mainly caused by human papilloma virus (HPV) infection. In HPV-positive cervical cancer cells, the activity of p53 and the induction of p21 are inhibited by the HPV oncoproteins E6 and E7. Therefore, blocking the activity of E6 and E7 would serve as an important therapeutic target in these cancer cells. In this study, anisomelic acid (AA), a natural compound belonging to the same diterpenoid family of bioactive compounds as taxol, was found to deplete the E6 and E7 proteins in HPV-positive cervical cancer cells. Consequently, p53 and the p53-responsive gene, p21, were dramatically induced, leading to G2/M-phase cell cycle arrest. AA-mediated cell cycle arrest and p21 expression were canceled when p53 was down-regulated by p53-shRNA. AA also induced p53-independent intrinsic apoptosis by depletion of the cellular inhibitor of apoptosis protein 2 (cIAP2) whose proteosomal degradation is inhibited by E6. The in ovo chick embryo chorioallantoic membrane (CAM) assay showed that anisomelic acid inhibited the tumor growth of the cervical cancer SiHa cells. AA is revealed to hold a novel action modality based on specific targeting of the HPV oncoproteins, which restores p53-mediated growth arrest and induces apoptosis by terminating E6-mediated cIAP2 stabilization. PMID:24565908

Paul, Preethy; Rajendran, Senthil Kumar; Peuhu, Emilia; Alshatwi, Ali A; Akbarsha, Mohammad A; Hietanen, Sakari; Eriksson, John E

2014-05-15

11

Human Papillomavirus Type 16 E7 Oncoprotein Associates with E2F6?  

PubMed Central

The papillomavirus life cycle is intimately coupled to the differentiation state of the infected epithelium. Since papillomaviruses lack most of the rate-limiting enzymes required for genome synthesis, they need to uncouple keratinocyte differentiation from cell cycle arrest and maintain or reestablish a replication-competent state within terminally differentiated keratinocytes. The human papillomavirus (HPV) E7 protein appears to be a major determinant for this activity and induces aberrant S-phase entry through the inactivation of the retinoblastoma tumor suppressor and related pocket proteins. In addition, E7 can abrogate p21 and p27. Together, this leads to the activation of E2F1 to E2F5, enhanced expression of E2F-responsive genes, and increased cdk2 activity. E2F6 is a pRB-independent, noncanonical member of the E2F transcription factor family that acts as a transcriptional repressor. E2F6 expression is activated in S phase through an E2F-dependent mechanism and thus may provide a negative-feedback mechanism that slows down S-phase progression and/or exit in response to the activation of the other E2F transcription factors. Here, we show that low- and high-risk HPV E7 proteins, as well as simian virus 40 T antigen and adenovirus E1A, can associate with and inactivate the transcriptional repression activity of E2F6, thereby subverting a critical cellular defense mechanism. This may result in the extended S-phase competence of HPV-infected cells. E2F6 is a component of polycomb group complexes, which bind to silenced chromatin and are critical for the maintenance of cell fate. We show that E7-expressing cells show decreased staining for E2F6/polycomb complexes and that this is at least in part dependent on the association with E2F6.

McLaughlin-Drubin, Margaret E.; Huh, Kyung-Won; Munger, Karl

2008-01-01

12

Human Papillomavirus 16 Oncoprotein E7 Stimulates UBF1-Mediated rDNA Gene Transcription, Inhibiting a p53-Independent Activity of p14ARF  

PubMed Central

High-risk human papillomavirus oncoproteins E6 and E7 play a major role in HPV-related cancers. One of the main functions of E7 is the degradation of pRb, while E6 promotes the degradation of p53, inactivating the p14ARF-p53 pathway. pRb and p14ARF can repress ribosomal DNA (rDNA) transcription in part by targeting the Upstream Binding Factor 1 (UBF1), a key factor in the activation of RNA polymerase I machinery. We showed, through ectopic expression and siRNA silencing of p14ARF and/or E7, that E7 stimulates UBF1-mediated rDNA gene transcription, partly because of increased levels of phosphorylated UBF1, preventing the inhibitory function of p14ARF. Unexpectedly, activation of rDNA gene transcription was higher in cells co-expressing p14ARF and E7, compared to cells expressing E7 alone. We did not find a difference in P-UBF1 levels that could explain this data. However, p14ARF expression induced E7 to accumulate into the nucleolus, where rDNA transcription takes place, providing an opportunity for E7 to interact with nucleolar proteins involved in this process. GST-pull down and co-immunoprecipitation assays showed interactions between p14ARF, UBF1 and E7, although p14ARF and E7 are not able to directly interact. Co-expression of a pRb-binding-deficient mutant (E7C24G) and p14ARF resulted in EC24G nucleolar accumulation, but not in a significant higher activation of rDNA transcription, suggesting that the inactivation of pRb is involved in this phenomenon. Thus, p14ARF fails to prevent E7-mediated UBF1 phosphorylation, but could facilitate nucleolar pRb inactivation by targeting E7 to the nucleolus. While others have reported that p19ARF, the mouse homologue of p14ARF, inhibits some functions of E7, we showed that E7 inhibits a p53-independent function of p14ARF. These results point to a mutually functional interaction between p14ARF and E7 that might partly explain why the sustained p14ARF expression observed in most cervical pre-malignant lesions and malignancies may be ineffective.

Dichamp, Isabelle; Seite, Paule; Agius, Gerard; Barbarin, Alice; Beby-Defaux, Agnes

2014-01-01

13

Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600  

PubMed Central

The human papillomavirus type 16 (HPV-16) E7 gene encodes a multifunctional oncoprotein that can subvert multiple cellular regulatory pathways. The best-known cellular targets of the HPV-16 E7 oncoprotein are the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. However, there is ample evidence that E7 has additional cellular targets that contribute to its transforming potential. We isolated HPV-16 E7 associated cellular protein complexes by tandem affinity purification and mass spectrometry and identified the 600-kDa retinoblastoma protein associated factor, p600, as a cellular target of E7. Association of E7 with p600 is independent of the pocket proteins and is mediated through the N terminal E7 domain, which is related to conserved region 1 of the adenovirus E1A protein and importantly contributes to cellular transformation independent of pRB binding. Depletion of p600 protein levels by RNA interference substantially decreased anchorage-independent growth in HPV-positive and -negative human cancer cells. Therefore, p600 is a cellular target of E7 that regulates cellular pathways that contribute to anchorage-independent growth and cellular transformation.

Huh, Kyung-Won; DeMasi, Joseph; Ogawa, Hidesato; Nakatani, Yoshihiro; Howley, Peter M.; Munger, Karl

2005-01-01

14

Correlation between serological immune response analyzed by a new ELISA for HPV16\\/18 E7 oncoprotein and clinical characteristics of cervical cancer patients  

Microsoft Academic Search

Summary.  Human papillomaviruses (HPVs), particularly HPV-16\\/18, are linked to cervical cancer development. Full-length, recombinant\\u000a HPV-16\\/18 E7 oncoproteins were used in a new streptavidin-biotin capture ELISA method to investigate anti-HPV E7 antibody\\u000a prevalence in serum. Sera from 99 healthy women, 70 cervical cancer patients, and 30 patients with cervical pre-invasive neoplasia\\u000a were analyzed. Anti-HPV-16\\/18 E7 positivity was found in 53% of cervical

A. Ravaggi; C. Romani; B. Pasinetti; R. A. Tassi; E. Bignotti; E. Bandiera; F. E. Odicino; M. Ragnoli; C. Donzelli; M. Falchetti; S. Calza; A. D. Santin; S. Pecorelli

2006-01-01

15

Human Papillomavirus Type 16 E6 and E7 Oncogenes Abrogate Radiation-Induced DNA Damage Responses in vivo through p53Dependent and p53Independent Pathways  

Microsoft Academic Search

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis

Shiyu Song; Gene A. Gulliver; Paul F. Lambert

1998-01-01

16

The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints  

SciTech Connect

HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.

Zhang Weifang [Department of Medicine, University of Massachusetts Medical School, Worcester, MA (United States); Department of Microbiology, School of Medicine, Shandong University, Jinan, Shandong (China); Li Jing [Department of Microbiology, School of Medicine, Shandong University, Jinan, Shandong (China); Kanginakudru, Sriramana [Department of Medicine, University of Massachusetts Medical School, Worcester, MA (United States); Zhao Weiming [Department of Microbiology, School of Medicine, Shandong University, Jinan, Shandong (China); Yu Xiuping, E-mail: yuxp@sdu.edu.c [Department of Microbiology, School of Medicine, Shandong University, Jinan, Shandong (China); Chen, Jason J., E-mail: Jason.chen@umassmed.ed [Department of Medicine, University of Massachusetts Medical School, Worcester, MA (United States)

2010-02-05

17

High level HPV-16 E7 oncoprotein expression correlates with reduced pRb-levels in cervical biopsies.  

PubMed

High-risk human papillomaviruses (HPVs) are major etiological agents of cervical cancer. Despite excellent epidemiological evidence for a direct role of HPV-16 in cervical carcinogenesis, molecular pathways underlying carcinogenesis in vivo remain obscure. The E7 gene is required for immortalization and maintenance of the transformed phenotype in vitro; however, little is known about its role for tumorigenesis in vivo. The E7 gene codes for an unstable protein the abundance of which in cervical biopsies is unknown. We show here that E7 protein levels strongly increase during cervical carcinogenesis, underlining its fundamental role in cervical cancer. The E7 protein was found predominantly in the nucleus and to a minor extent in the cytoplasm in the cervical cancer cell line Ca Ski in vitro and in invasive cervical carcinoma in situ, suggesting that nuclear resident E7 plays a major role in cervical carcinogenesis in humans. The retinoblastoma protein (pRb) is a major E7-target in vitro. We show here that pRb expression is initially upregulated in LSIL and disappears in later stages concomitant with increased E7 levels, suggesting that E7-driven degradation of pRb is involved in cervical tumorigenesis in humans. PMID:15155561

Fiedler, Marc; Müller-Holzner, Elisabeth; Viertler, Hans-Peter; Widschwendter, Andreas; Laich, Andreas; Pfister, Gerald; Spoden, Gilles A; Jansen-Dürr, Pidder; Zwerschke, Werner

2004-07-01

18

An RNA Aptamer Provides a Novel Approach for the Induction of Apoptosis by Targeting the HPV16 E7 Oncoprotein  

PubMed Central

Background Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions. Methodology/Principal Findings This study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting. Conclusions/Significance This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future.

Nicol, Clare; Cesur, Ozlem; Forrest, Sophie; Belyaeva, Tamara A.; Bunka, David H. J.; Blair, G. Eric; Stonehouse, Nicola J.

2013-01-01

19

Effects of the human papilloma virus HPV-16 E7 oncoprotein on glycolysis and glutaminolysis: role of pyruvate kinase type M2 and the glycolytic-enzyme complex.  

PubMed Central

Proliferating and tumour cells express the glycolytic isoenzyme, pyruvate kinase type M2 (M2-PK), which occurs in a highly active tetrameric form and in a dimeric form with low affinity for phosphoenolpyruvate. The switch between the two forms regulates glycolytic phosphometabolite pools and the interaction between glycolysis and glutaminolysis. In the present study, we show the effects of oncoprotein E7 of the human papilloma virus (HPV)-16 (E7)-transformation on two NIH 3T3 cell strains with different metabolic characteristics. E7-transformation of the high glycolytic NIH 3T3 cell strain led to a shift of M2-PK to the dimeric form and, in consequence, to a decrease in the cellular pyruvate kinase mass-action ratio, the glycolytic flux rate and the (ATP+GTP)/(UTP+CTP) ratio, as well as to an increase in fructose 1,6-bisphosphate (FBP) levels, glutamine consumption and cell proliferation. The low glycolytic NIH 3T3 cell strain is characterized by high pyruvate and glutamine consumption rates and by an intrinsically large amount of the dimeric form of M2-PK, which is correlated with high FBP levels, a low (ATP+GTP)/(CTP+UTP) ratio and a high proliferation rate. E7-transformation of this cell strain led to an alteration in the glycolytic-enzyme complex that correlates with an increase in pyruvate and glutamine consumption and a slight increase in the flow of glucose to lactate. The association of phosphoglyceromutase within the glycolytic-enzyme complex led to an increase of glucose and serine consumption and a disruption of the linkage between glucose consumption and glutaminolysis. In both NIH 3T3 cell lines, transformation increased glutaminolysis and the positive correlation between alanine and lactate production.

Mazurek, S; Zwerschke, W; Jansen-Durr, P; Eigenbrodt, E

2001-01-01

20

Human papillomavirus type 38 E6 and E7 act as tumour promoters during chemically induced skin carcinogenesis.  

PubMed

Many findings support a possible involvement of a subgroup of human papillomaviruses (HPVs), called cutaneous beta HPV types, in the development of non-melanoma skin cancer. The skin of transgenic (Tg) mice expressing viral oncoproteins E6 and E7 from different cutaneous beta HPV types, including HPV38, showed an increased susceptibility to UV-induced and/or chemically induced skin carcinogenesis compared with wild-type animals. In this study, we show that beta HPV38 E6 and E7 oncoproteins act as promoter and progression factors in multi-stage skin carcinogenesis, strongly cooperating with the initiator and DNA damage agent 7,12-dimethylbenz[a]anthracene. In contrast, exposure of HPV38 E6/E7 Tg mice to the promoter 12-O-tetradecanoylphorbol-13-acetate did not significantly result in the development of skin lesions. These findings further support the role of beta HPV types in skin carcinogenesis, providing additional insight into their precise contribution to the multi-step process. PMID:23223623

Viarisio, Daniele; Decker, Karin Müller; Aengeneyndt, Birgit; Flechtenmacher, Christa; Gissmann, Lutz; Tommasino, Massimo

2013-04-01

21

Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7  

Microsoft Academic Search

The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does

SONIA L. GONZALEZ; MATT STREMLAU; XI HE; JOHN R. BASILE; KARL MUNGER

2001-01-01

22

Overexpression of c-erbB-2 and p21 oncoproteins in human radiation-induced skin ulcers  

SciTech Connect

We studied the overexpression of c-erbB-2 and p21 oncoproteins in human radiation-induced skin ulcers using immunohistochemistry. We found that the positive rate of overexpression of c-erbB-2 and p21 oncoproteins was 92.0 and 92.9%, respectively. The overexpression of c-erbB-2 oncoprotein was observed mainly in the cell membrane of squamous epithelial cells and in the cytoplasm of fibroblasts, endothelial cells, leiomyocytes in the media, and in fibrocytes of the adventitia of mesenchymal arterioles. The location of the p21 oncoprotein overexpression was mostly similar to that of c-erbB-2 with stronger staining in the cytoplasm of squamous epithelial cells and weaker staining in mesenchymal arteriolar walls. The overexpression of c-erbB-2 and p21 oncoproteins may be corresponding to the cancer transformation and poor healing of radiation-induced skin ulcers. 6 refs., 2 figs.

Zhao Po, Yang Zhixiang, Wang De-wen [Inst. of Radiation Medicine, Beijing (China)] [and others

1995-12-31

23

Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways  

PubMed Central

E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7’s transforming function, were also required for E7 to modulate DNA damage responses in vivo. Thus pRb and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.

Song, Shiyu; Gulliver, Gene A.; Lambert, Paul F.

1998-01-01

24

Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers.  

PubMed

Human papillomavirus (HPV) is involved in the development of anogenital tumors and also in the development of oropharyngeal head and neck carcinomas, where HPV-16, expressing the E6 and E7 oncoproteins, is the most frequent serotype. Although vaccines encoding L1 and L2 capsid HPV proteins are efficient for the prevention of HPV infection, they are inadequate for treating established tumors. Hence, development of innovative vaccine therapies targeting E6/E7 is important for controlling HPV-induced cancers. We have engineered a nononcogenic mutated E7-specific plasmo-retroVLP vaccine (pVLP-E7), consisting of plasmid DNA, that is able to form recombinant retrovirus-based virus-like particles (VLPs) that display E7 antigen into murine leukemia virus Gag proteins pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G). pVLP-E7 vaccinations were studied for their ability to generate specific immune responses and for induction of protective immunity against tumor cell challenge in preventive and therapeutic models. The produced VLPs induce the maturation of human dendritic cells in vitro and mount specific E7 T cell responses. Intradermic vaccinations of mice with pVLP-E7 show their efficacy to generate antigen-specific T cell responses, to prevent and protect animals from early TC-1 tumor development compared with standard DNA or VLP immunizations. The vaccine efficacy was also evaluated for advanced tumors in mice vaccinated at various time after the injection of TC-1 cells. Data show that pVLP-E7 vaccination can cure mice with already established tumors only when combined with Toll-like receptor-7 (TLR7) and TLR9 agonists. Our findings provide evidence that pVLPs, combining the advantages of DNA and VLP vaccines, appear to be a promising strategy for the treatment of HPV-induced cancers. PMID:23521528

Lescaille, Geraldine; Pitoiset, Fabien; Macedo, Rodney; Baillou, Claude; Huret, Christophe; Klatzmann, David; Tartour, Eric; Lemoine, François M; Bellier, Bertrand

2013-05-01

25

Roles of PI3K/Akt and c-Jun Signaling Pathways in Human Papillomavirus Type 16 Oncoprotein-Induced HIF-1?, VEGF, and IL-8 Expression and In Vitro Angiogenesis in Non-Small Cell Lung Cancer Cells  

PubMed Central

Background and Objectives Human papillomavirus (HPV)-16 infection may be related to non-smoking associated lung cancer. Our previous studies have found that HPV-16 oncoproteins promoted angiogenesis via enhancing hypoxia-inducible factor-1? (HIF-1?), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) expression in non-small cell lung cancer (NSCLC) cells. In this study, we further investigated the roles of PI3K/Akt and c-Jun signaling pathways in it. Methods Human NSCLC cell lines, A549 and NCI-H460, were stably transfected with pEGFP-16 E6 or E7 plasmids. Western blotting was performed to analyze the expression of HIF-1?, p-Akt, p-P70S6K, p-P85S6K, p-mTOR, p-JNK, and p-c-Jun proteins. VEGF and IL-8 protein secretion and mRNA levels were determined by ELISA and Real-time PCR, respectively. The in vitro angiogenesis was observed by human umbilical vein endothelial cells (HUVECs) tube formation assay. Co-immunoprecipitation was performed to analyze the interaction between c-Jun and HIF-1?. Results HPV-16 E6 and E7 oncoproteins promoted the activation of Akt, P70S6K, P85S6K, mTOR, JNK, and c-Jun. LY294002, a PI3K inhibitor, inhibited HPV-16 oncoprotein-induced activation of Akt, P70S6K, and P85S6K, expression of HIF-1?, VEGF, and IL-8, and in vitro angiogenesis. c-Jun knockdown by specific siRNA abolished HPV-16 oncoprotein-induced HIF-1?, VEGF, and IL-8 expression and in vitro angiogenesis. Additionally, HPV-16 oncoproteins promoted HIF-1? protein stability via blocking proteasome degradation pathway, but c-Jun knockdown abrogated this effect. Furthermore, HPV-16 oncoproteins increased the quantity of c-Jun binding to HIF-1?. Conclusions PI3K/Akt signaling pathway and c-Jun are involved in HPV-16 oncoprotein-induced HIF-1?, VEGF, and IL-8 expression and in vitro angiogenesis. Moreover, HPV-16 oncoproteins promoted HIF-1? protein stability possibly through enhancing the interaction between c-Jun and HIF-1?, thus making a contribution to angiogenesis in NSCLC cells.

Liu, Fei; Zhang, Peihua; Liang, Jie; Tang, Xudong

2014-01-01

26

Mdm2 associates with Ras effector NORE1 to induce the degradation of oncoprotein HIPK1  

PubMed Central

The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2–NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.

Lee, Deresa; Park, Sang-Joon; Sung, Ki Sa; Park, Jikyoung; Lee, Sean Bong; Park, Sang-Yoon; Lee, Hyo Jeong; Ahn, Jang-Won; Choi, So Jung; Lee, Seok-Geun; Kim, Sung-Hoon; Kim, Duk-Hwan; Kim, Jhingook; Kim, Yongsok; Choi, Cheol Yong

2012-01-01

27

Intranasal immunization with recombinant Lactococci carrying human papillomavirus E7 protein and mouse interleukin-12 DNA induces E7-specific antitumor effects in C57BL/6 mice  

PubMed Central

The use of Lactococcus lactis for the co-delivery of antigens and cytokines has been shown to successfully induce a special immune response. However, it is unknown whether the same results may be triggered through immunization of animals with L. lactis simultaneously carrying protein antigen and cytokine DNA. The present study evaluated the protective effects of intranasally administered live L. lactis strains carrying human papillomavirus 16 E7 protein and murine interleukin-12 (IL-12) DNA (LL-E7P-IL-12D) in a TC-1 tumor animal model. C57BL/6 mice were intranasally immunized with recombinant lactococci, and assays for cytotoxicity measurement and tumor protection were carried out to assess the immunological effects of the vaccine candidates. IL-12 and interferon-? serum levels were measured and immunization with LL-E7P-IL-12D was shown to induce an E7-specific immune response and to confer protection against TC-1-induced tumors in vivo. Mice in the LL-E7P-IL-12D group showed an 80% survival rate when the control mice had died. Therapeutic immunization with recombinant L. lactis strains 7 days after TC-1 injection led to a reduction in the number of palpable tumors in treated mice. The antitumor effects of the vaccination occurred through an E7-specific cytotoxic T-lymphocyte response. In the present study, the use of a single L. lactis strain, to co-administer protein antigen and adjuvant DNA, successfully induced an antigen-specific immune response. These observations demonstrate a new strategy for the use of L. lactis as a delivery vector of therapeutic molecules and antigens.

LI, YIJIE; LI, XINPING; LIU, HUANHUAN; ZHUANG, SHUZHEN; YANG, JIANHUA; ZHANG, FUCHUN

2014-01-01

28

Tumor necrosis factor-induced microtubule stabilization mediated by hyperphosphorylated oncoprotein 18 promotes cell death.  

PubMed

Tumor necrosis factor (TNF)-induced cell death in the fibrosarcoma cell line L929 occurs independently of caspase activation and cytochrome c release. However, it is dependent on mitochondria and is characterized by increased production of reactive oxygen intermediates that are essential to the death process. To identify signaling molecules involved in this TNF-induced, reactive oxygen intermediate-dependent cell death pathway, we performed a comparative study by two-dimensional gel electrophoresis of phosphoproteins from a mitochondria-enriched fraction derived from TNF-treated and control cells. TNF induced rapid and persistent phosphorylation of the phosphorylation-responsive regulator of the microtubule (MT) dynamics, oncoprotein 18 (Op18). By using induced overexpression of wild type Op18 and phosphorylation site-deficient mutants S25A/S38A and S16A/S63A in L929 cells, we show that TNF-induced phosphorylation on each of the four Ser residues of Op18 promotes cell death and that Ser(16) and Ser(63) are the primary sites. This hyperphosphorylation of Op18 is known to completely turn off its MT-destabilizing activity. As a result, TNF treatment of L929 cells induced elongated and extremely tangled microtubules. These TNF-induced changes to the MT network were also observed in cells overexpressing wild type Op18 and, to a lesser extent, in cells overexpressing the S25A/S38A mutant. No changes in the MT network were observed upon TNF treatment of cells overexpressing the S16A/S63A mutant, and these cells were desensitized to TNF-induced cell death. These findings indicate that TNF-induced MT stabilization is mediated by hyperphosphorylation of Op18 and that this promotes cell death. The data suggest that Op18 and the MT network play a functional role in transduction of the cell death signal to the mitochondria. PMID:10913145

Vancompernolle, K; Boonefaes, T; Mann, M; Fiers, W; Grooten, J

2000-10-27

29

Human Papillomavirus16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival  

Microsoft Academic Search

BackgroundHuman Papillomavirus (HPV)-16 is a paradigm for “high-risk” HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.Methodology\\/Principal FindingsBy protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a

Anna M. Mileo; Claudia Abbruzzese; Stefano Mattarocci; Emanuele Bellacchio; Paola Pisano; Antonio Federico; Vittoria Maresca; Mauro Picardo; Alessandra Giorgi; Bruno Maras; M. Eugenia Schininà; Marco G. Paggi; Dong-Yan Jin

2009-01-01

30

Oncoprotein Akt/PKB induces trophic effects in murine models of Parkinson's disease  

PubMed Central

Despite promising preclinical studies, neurotrophic factors have not yet achieved an established role in the treatment of human neurodegenerative diseases. One impediment has been the difficulty in providing these macromolecules in sufficient quantity and duration at affected sites. An alternative approach is to directly activate, by viral vector transduction, intracellular signaling pathways that mediate neurotrophic effects. We have evaluated this approach in dopamine neurons of the substantia nigra, neurons affected in Parkinson's disease, by adeno-associated virus 1 transduction with a gene encoding a myristoylated, constitutively active form of the oncoprotein Akt/PKB. Adeno-associated virus Myr-Akt has pronounced trophic effects on dopamine neurons of adult and aged mice, including increases in neuron size, phenotypic markers, and sprouting. Transduction confers almost complete protection against apoptotic cell death in a highly destructive neurotoxin model. Activation of intracellular neurotrophic signaling pathways by vector transfer is a feasible approach to neuroprotection and restorative treatment of neurodegenerative disease.

Ries, Vincent; Henchcliffe, Claire; Kareva, Tatyana; Rzhetskaya, Margarita; Bland, Ross; During, Matthew J.; Kholodilov, Nikolai; Burke, Robert E.

2006-01-01

31

MUC1-C oncoprotein suppresses reactive oxygen species-induced terminal differentiation of acute myelogenous leukemia cells  

PubMed Central

Acute myeloid leukemia (AML) cells are characterized by unlimited self-renewal and an impaired capacity to undergo terminal differentiation. The MUC1 oncoprotein is aberrantly expressed in AML cells; however, there has been no evidence for involvement of MUC1 in myeloid leukemogenesis. Cell-penetrating peptide inhibitors of the MUC1-C subunit block its oligomerization and thereby oncogenic function. The present results demonstrate that treatment of human MOLM-14 and MV4-11 AML cells with these inhibitors is associated with arrest of growth, induction of late apoptosis/necrosis, and loss of self-renewal capacity. Similar results were obtained with primary blasts from patients with AML. Inhibition of MUC1-C was associated with increases in reactive oxygen species (ROS) and depletion of glutathione. Increases in ROS have been linked to induction of hematopoietic cell differentiation along the myeloid lineage. In this regard, inhibition of MUC1-C was associated with induction of a terminally differentiated myeloid phenotype in AML cell lines and primary blasts by an ROS-dependent mechanism. These findings indicate that MUC1-C function is of importance to AML cell self-renewal and that inhibition of MUC1-C represents a potential therapeutic approach to induce terminal differentiation of AML cells.

Yin, Li; Wu, Zekui; Avigan, David; Rosenblatt, Jacalyn; Stone, Richard; Kharbanda, Surender

2011-01-01

32

DNA vaccine encoding HPV-16 E7 with mutation in L-Y-C-Y-E pRb-binding motif induces potent anti-tumor responses in mice.  

PubMed

Cervical cancer is the second most common cancer among women worldwide and remains a clinical problem despite improvements in early detection and therapy. The human papillomavirus (HPV) type 16 (HPV16) E7 oncoprotein expressed in cervical carcinoma cells are considered as attractive tumor-specific antigen targets for immunotherapy. Since the transformation potential of the oncogenes, vaccination based of these oncogenes is not safe. In present study, DNA vaccine expressing the modified variant with mutation in pRb-binding motif of the HPV-16 E7 oncoprotein was generated. A novel modified E7 gene with mutation in LYCYE motif was designed and constructed and the immunogenicity and antitumor effect of therapeutic DNA vaccines encoding the mutant and wild type of E7 gene were investigated. The L-Y-C-Y-E pRb-binding motif of E7 proteins has been involved in the immortalization and transformation of the host cell. The results showed that the mutant and wild type HPV-16 E7 vectors expressed the desired protein. Furthermore, the immunological mechanism behind mutant E7 DNA vaccine can be attributed at least partially to increased cytotoxic T lymphocyte, accompanied by the up-regulation of Th1-cytokine IFN-? and TNF-? and down-regulation of Th3-cytokine TGF-?. Immunized mice with mutant plasmid demonstrated signi?cantly stronger cell immune responses and higher levels of tumor protection than wild-type E7 DNA vaccine. The results exhibit that modified E7 DNA vaccine may be a promising candidate for development of therapeutic vaccine against HPV-16 cancers. PMID:24880067

Bahrami, Armina Alagheband; Ghaemi, Amir; Tabarraei, Alijan; Sajadian, Azadeh; Gorji, Ali; Soleimanjahi, Hoorieh

2014-09-01

33

p53 oncoprotein overexpression correlates with mutagen-induced chromosome fragility in head and neck cancer patients with multiple malignancies.  

PubMed Central

In this study, we analysed immunocytochemically p53 expression in first primary and second primary cancers from 25 head and neck cancer patients (HNCPs) with multiple malignancies in comparison with oncoprotein expression in tumour tissues from 25 historical HNCP controls with single cancer in a match-paired analysis. Moreover, we investigated bleomycin-induced chromosome fragility in both groups of HNCPs and in 21 additional healthy controls. Thirty-nine out of 75 tumour specimens analysed (52%) showed positive p53 immunostaining. Eleven out of 25 (44%) from single cancer patients and 28 out of 50 (56%) tumours from HNCPs with multiple malignancies were p53 positive. In the group of multiple primary cancers, nine patients (36%) showed positive staining of both first and second primaries, whereas six (24%) had positive labelling of first primary cancer but not of the subsequent second primary, four (16%) patient showed p53 expression only in the second primary cancer and six (24%) patients showed no p53 immunoreactivity in both tumours. Chromosomal analysis demonstrated a higher sensitivity to clastogens of HNCPs with multiple tumours than of HNCPs with a single cancer (P < 0.01), and a significant correlation between chromosome fragility and p53 overexpression (P < 0.01) only in HNCPs with multiple malignancies more than in those with single head and neck cancer (P = 0.11). Moreover, we found that patients with p53-positive staining of both first and second primaries showed a statistically significant higher mutagen sensitivity than those with a single p53 immunoreactive tumour or those in whom both cancers were p53 negative (P < 0.01). Our data suggest that subjects with increased susceptibility to carcingogens after exposure to tobacco or alcohol are at higher risk for multiple cancers in which one of the most common genetic events is aberrant p53 expression. Images Figure 1

Gallo, O.; Bianchi, S.; Giovannucci-Uzzielli, M. L.; Santoro, R.; Lenzi, S.; Salimbeni, C.; Abbruzzese, M.; Alajmo, E.

1995-01-01

34

Expression of LIGHT/TNFSF14 Combined with Vaccination against Human Papillomavirus Type 16 E7 Induces Significant Tumor Regression  

PubMed Central

LIGHT, a ligand for the lymphotoxin-beta receptor, establishes lymphoid-like tissues inside tumor sites and recruits naïve T-cells into the tumor. However, whether these infiltrating T-cells are specific for tumor antigens is not known. We hypothesized that therapy with LIGHT can expand functional tumor-specific CD8+ T-cells that can be boosted using HPV16E6E7-Venezuelan Equine Encephalitis Virus Replicon Particles (HPV16-VRP) and that this combined therapy can eradicate HPV16-induced tumors. Our data show that forced expression of LIGHT in tumors results in an increase in expression of interferon gamma (IFNg) and chemottractant cytokines such as IL-1a, MIG and MIP-2 within the tumor and that this tumor microenvironment correlates with an increase in frequency of tumor-infiltrating CD8+ T-cells. Forced expression of LIGHT also results in the expansion of functional T-cells that recognize multiple tumor-antigens, including HPV16 E7, and these T-cells prevent the outgrowth of tumors upon secondary challenge. Subsequent boosting of E7-specific T-cells by vaccination with HPV16-VRP significantly increases their frequency in both the periphery and the tumor, and leads to the eradication of large well-established tumors, for which either treatment alone is not successful. These data establish the safety of Ad-LIGHT as a therapeutic intervention in pre-clinical studies and suggest that patients with HPV16+ tumors may benefit from combined immunotherapy with LIGHT and antigen-specific vaccination.

Kanodia, Shreya; Da Silva, Diane M.; Karamanukyan, Tigran; Bogaert, Lies; Fu, Yang-Xin; Kast, W. Martin

2010-01-01

35

MUC1-C ONCOPROTEIN INDUCES TAMOXIFEN RESISTANCE IN HUMAN BREAST CANCER CELLS  

PubMed Central

Resistance of estrogen receptor positive (ER+) breast cancer cells to tamoxifen has been linked in part to activation of (i) certain receptor tyrosine kinases, such as HER2, and (ii) the PI3K?AKT pathway. Mucin 1 (MUC1) is aberrantly overexpressed in about 90% of human breast cancers and the oncogenic MUC1-C subunit associates with ER?. The present studies using HER2 overexpressing BT-474 breast cancer cells, which are constitutively resistant to tamoxifen, demonstrate that silencing MUC1-C is associated with (i) downregulation of p-HER2 levels, and (ii) sensitivity to tamoxifen-induced growth inhibition and loss of clonogenic survival. The results also demonstate that overexpression of MUC1-C in tamoxifen-sensitive MCF-7 breast cancer cells results in upregulation of p-AKT and tamoxifen resistance. We show that MUC1-C forms complexes with ER? on the estrogen-responsive promoter of the Rab31 gene and that MUC1-C blocks tamoxifen-induced decreases in ER? occupancy. MUC1-C also attenuated tamoxifen-induced decreases in (i) recruitment of the coactivator CREB binding protein, (ii) Rab31 promoter activation, and (ii) Rab31 mRNA and protein levels. The importance of MUC1-C is further supported by the demonstration that targeting MUC1-C with the cell-penetrating peptide inhibitor, GO-203, sensitizes tamoxifen-resistant cells to tamoxifen treatment. Moreover, we show that targeting MUC1-C in combination with tamoxifen is highly synergistic in the treatment of tamoxifen-resistant breast cancer cells. These findings indicate that MUC1-C contributes to tamoxifen resistance and provide support for the investigation of MUC1-C inhibitors in the setting of tamoxifen refractory disease.

Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Raina, Deepak; Kufe, Donald

2013-01-01

36

E6 and E7 proteins from different beta-papillomaviruses types do not interfere in UVB-induced apoptosis of HaCaT keratinocytes.  

PubMed

Beta-papillomaviruses (beta-HPV) have been linked to the development of skin cancer in humans. Because both E6 and E7 proteins from beta-HPV have been involved in the potential carcinogenicity of these viruses, we investigated their role on UVB-induced apoptosis in HaCaT cell line. HaCaT cells have been transduced with both E6/E7 using a retroviral system and treated with PRIMA-1. Apoptosis was assessed by flow cytometry to measure mitochondrial membrane potential and DNA fragmentation. HaCat keratinocytes transduced with both E6 and E7 genes of seven beta-HPV types (HPV5, HPV8, HPV14, HPV24, HPV36, HPV38 and HPV49) did not demonstrate any inhibition of UVB-induced apoptosis, even after p53 reactivation through PRIMA-1. Our data suggest that the expression of E6 and E7 exert different modulatory effects on UVB-induced apoptosis according to beta-HPV types and to the cellular genetic context. PMID:21158941

Guerrini, Jean-Sébastien; Bouvard, Véronique; Oswald, Evelyne; Alonso, Angel; Prétet, Jean-Luc; Tommasino, Massimo; Mougin, Christiane; Aubin, François

2011-01-01

37

Viral oncoprotein-induced mislocalization of select PDZ proteins disrupts tight junctions and causes polarity defects in epithelial cells  

PubMed Central

Summary The development of human cancers is frequently associated with a failure of epithelial cells to form tight junctions and to establish proper apicobasal polarity. Interestingly, the oncogenic potential of the adenovirus E4-ORF1 protein correlates with its binding to the cellular PDZ proteins MUPP1, MAGI-1, ZO-2 and SAP97, the first three of which assemble protein complexes at tight junctions. Given that E4-ORF1 sequesters these three PDZ proteins in the cytoplasm of fibroblasts, we postulated that E4-ORF1 would inhibit tight junction formation in epithelial cells. Providing further support for this idea, we identified MUPP1-related PATJ, a key component of the tight junction-associated CRB3-PALS1-PATJ polarity complex, as a new PDZ-protein target for both the E4-ORF1 and high-risk human papillomavirus type 18 E6 oncoproteins. Moreover, in epithelial cells, E4-ORF1 blocked the tight junction localization of PATJ and ZO-2, as well as their interacting partners, and disrupted both the tight junction barrier and apicobasal polarity. These significant findings expose a direct link between the tumorigenic potential of E4-ORF1 and inactivation of cellular PDZ proteins involved in tight junction assembly and polarity establishment.

Latorre, Isabel J.; Roh, Michael H.; Frese, Kristopher K.; Weiss, Robert S.; Margolis, Ben; Javier, Ronald T.

2012-01-01

38

Rabbits immunised with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L2E7E2 genes induces regression of established papillomas.  

PubMed

We previously demonstrated in a cottontail rabbit papillomavirus (CRPV) challenge model that recombinant Bacille Calmette-Guerin (rBCG) could potentially be used as a prophylactic vaccine vehicle to deliver papillomavirus proteins. In this study we investigated whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with rBCG expressing CRPVL2, CRPVE2, CRPVE7 or CRPVL2E7E2 proteins. Rabbits immunised with rBCG/CRPVL2E7E2 had papillomas that were largely suppressed and were significantly smaller compared to the rBCG negative control group (PE7E2 had papillomas that completely regressed 1.5 weeks post third immunisation. Rabbits immunised with rBCG/CRPVL2, rBCG/CRPVE7, or rBCG/CRPVE2 had papillomas that were significantly smaller than the negative control rabbits (P

Govan, V A; Williamson, A-L

2007-07-01

39

Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system.  

PubMed

Certain types of human papillomaviruses have been etiologically associated with malignant lesions, most notably with cervical cancer. The major oncoproteins of these cancer-associated viruses are encoded by the viral E6 and E7 genes. Thorough characterization of these oncoproteins and their interaction with cellular proteins has shown that both E6 and E7 exploit the ubiquitin-proteasome system to degrade and, thus, to functionally inactivate negative cell-regulatory proteins including members of the p110(RB) family and p53. This act of piracy is assumed to contribute to both the efficient propagation of HPVs and HPV-induced carcinogenesis. PMID:12507557

Scheffner, Martin; Whitaker, Noel J

2003-02-01

40

Long-lasting immunoprotective and therapeutic effects of a hyperstable E7 oligomer based vaccine in a murine human papillomavirus tumor model.  

PubMed

Cervical cancer and many other anogenital and oropharyngeal carcinomas are strongly associated with high-risk human papillomavirus (HPV) persistent infections. HPV E7 oncoprotein is the major viral transforming factor, emerging as a natural candidate for immunotherapy, since it is constitutively expressed in HPV-induced cancer cells. We have previously shown that E7 can self-assemble into soluble and homogeneous spherical oligomers, named E7 soluble oligomers (E7SOs). These are highly resistant to thermal denaturation, providing an additional advantage given the demand for highly stable vaccine formulations. Here, we present a new chemically stabilized form of the E7SOs (E7SOx) and analyzed its effect in a murine HPV-tumor model. Vaccination of female mice with low doses of E7SOx combined with a CpG-rich oligonucleotide (ODN) as adjuvant elicits a strong long-lasting protection against E7-expressing tumor cells, preventing tumor outgrowth after rechallenge 90-days later. Therapeutic experiments showed that E7SOx/ODN vaccination significantly delays tumor growth and extends the time of survival of the treated mice in a dose-dependent manner. These proof-of-principle preclinical experiments denote the potential applicability of our E7SOx-based vaccine to the treatment of cervical cancer and other mucosal HPV-related neoplastic lesions. In addition to thermal, chemical and proteolysis stability, the combined recombinant and chemical modification nature of the E7SOx vaccine candidate, results in low-cost, of particular interest in developing countries, where most of the cervical cancer cases occur and the most affected population is at reproductive age. PMID:21780110

Cerutti, María L; Alonso, Leonardo G; Tatti, Silvio; de Prat-Gay, Gonzalo

2012-04-15

41

Involvement of c-Ski oncoprotein in carcinogenesis of cholangiocacinoma induced by Opisthorchis viverrini and N-nitrosodimethylamine.  

PubMed

Opisthorchiasis is the major public health problem in the endemic areas of Thailand and Laos because Opisthorchis viverrini infection causes serious hepatobiliary diseases including CCA. The molecular mechanism of the CCA carcinogenesis induced by the infection remains obscure. To reveal the potential genes and signaling pathways to involve in the carcinogenesis, the present study investigated the expression of c-Ski, an oncogene, and two TGF-? signaling pathway relative genes, TGF-? and Smad4, during the development of CCA induced by O. viverrini infection in hamster model, and in human opisthorchiasis associated CCA. The results showed that the expression of c-Ski gene was greatly up-regulated during the carcinogenesis of CCA in hamster model. The overexpression of c-Ski was confirmed by immunohistological staining result which showed the increased expression of c-Ski protein in cytoplasm of the epithelial lining of hepatic bile ducts. Moreover, the immunohistological staining of the specimens of human opisthorchiasis associated CCA revealed the up-regulated expression of c-Ski and Smad4 proteins in the cytoplasm of the epithelial lining of hepatic bile ducts and stomal fibrosis respectively. The expression of TGF-? and Smad4 were up-regulated, which expression kinetics was time-dependent of CCA development. These results suggest that c-Ski is likely involved in the carcinogenesis of CCA induced by O. viverrini infection through regulating TGF-? signaling pathway. PMID:20853076

Boonmars, Thidarut; Wu, Zhiliang; Boonjaruspinyo, Sirintip; Puapairoj, Anucha; Kaewsamut, Butsara; Nagano, Isao; Pinlaor, Somchai; Yongvanit, Puangrat; Wonkchalee, Orasa; Juasook, Amornrat; Sudsarn, Pakkayanee; Srisawangwong, Tuanchai

2011-06-01

42

Luteolin induces intrinsic apoptosis via inhibition of E6/E7 oncogenes and activation of extrinsic and intrinsic signaling pathways in HPV-18-associated cells.  

PubMed

Luteolin, a flavonoid extracted from a number of plants with recognized anticancer, anti-inflammatory and anti-oxidative activities, inhibits angiogenic processes and modulates multidrug resistance. However, the efficacy and mechanisms of action of this flavonoid agent are still undergoing study. In order to elucidate whether luteolin exhibits an anticancer effect in cervical cancer cells, HeLa cells were incubated with luteolin and apoptosis was assessed by observing nuclear morphological changes, and performing Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Cell cycle analysis, western blotting, RT-PCR and mitochondrial membrane potential measurements were also carried out. Luteolin showed a significant dose-dependent cytotoxic effect only in human papillomavirus (HPV)-positive cervical cancer cells, when compared to its effect on HPV-negative cervical cancer C33A cells. Expression levels of human papilloma virus E6 and E7 oncogenes were suppressed, those of related factors pRb and p53 were recovered and E2F5 was increased by luteolin treatment. Furthermore, luteolin enhanced the expression of death receptors and death receptor downstream factors such as Fas/FasL, DR5/TRAIL and FADD in HeLa cells, and activated caspase cascades. In particular, luteolin enhanced the activity of caspase-3 and -8 in a dose-dependent manner. Activation of caspase-3 induced caspase-8 activity and vice versa. Luteolin also induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited Bcl-2 and Bcl-xL expression. In conclusion, luteolin exerts anticarcinogenic activity through inhibition of E6 and E7 expression and cross-activation of caspase-3 and -8. Taken together, these results suggest that luteolin induces inactivation of HPV-18 oncogene expression and apoptosis by activating the intrinsic and extrinsic pathways. PMID:24789165

Ham, Sunyoung; Kim, Ki Hong; Kwon, Tae Ho; Bak, Yesol; Lee, Dong Hun; Song, Yong Seok; Park, Su-Ho; Park, Yun Sun; Kim, Man Sub; Kang, Jeong Woo; Hong, Jin Tae; Yoon, Do-Young

2014-06-01

43

Conserved Region 3 of Human Papillomavirus 16 E7 Contributes to Deregulation of the Retinoblastoma Tumor Suppressor  

PubMed Central

The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle.

Todorovic, Biljana; Hung, Katherine; Massimi, Paola; Avvakumov, Nikita; Dick, Frederick A.; Shaw, Gary S.; Banks, Lawrence

2012-01-01

44

A novel mucosal vaccine based on live Lactococci expressing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human papillomavirus type 16-induced tumors.  

PubMed

Current strategies to prevent or treat human papillomavirus type 16 (HPV-16) infection are promising, but remain costly. More economical but efficient vaccines are thus needed. In this study, we evaluated the protective effects of mucosally coadministered live Lactococcus lactis strains expressing cell wall-anchored E7 Ag and a secreted form of IL-12 to treat HPV-16-induced tumors in a murine model. When challenged with lethal levels of tumor cell line TC-1 expressing E7, immunized mice showed full prevention of TC-1-induced tumors, even after a second challenge, suggesting that this prophylactic immunization can provide long-lasting immunity. Therapeutic immunization with L. lactis recombinant strains, i.e., 7 days after TC-1 injection, induced regression of palpable tumors in treated mice. The antitumor effects of vaccination occurred through a CTL response, which is CD4+ and CD8+ dependent. Furthermore, immunized mice developed an E7-specific mucosal immune response. These preclinical results suggest the feasibility of the low-cost mucosal vaccination and/or immunotherapy strategies against HPV-related cervical cancer in humans. PMID:16301635

Bermúdez-Humarán, Luis G; Cortes-Perez, Naima G; Lefèvre, François; Guimarães, Valeria; Rabot, Sylvie; Alcocer-Gonzalez, Juan M; Gratadoux, Jean-Jacques; Rodriguez-Padilla, Cristina; Tamez-Guerra, Reyes S; Corthier, Gérard; Gruss, Alexandra; Langella, Philippe

2005-12-01

45

Identification of Unusual E6 and E7 Proteins within Avian Papillomaviruses: Cellular Localization, Biophysical Characterization, and Phylogenetic Analysis? §  

PubMed Central

Papillomaviruses (PVs) are a large family of small DNA viruses infecting mammals, reptiles, and birds. PV infection induces cell proliferation that may lead to the formation of orogenital or skin tumors. PV-induced cell proliferation has been related mainly to the expression of two small oncoproteins, E6 and E7. In mammalian PVs, E6 contains two 70-residue zinc-binding repeats, whereas E7 consists of a natively unfolded N-terminal region followed by a zinc-binding domain which folds as an obligate homodimer. Here, we show that both the novel francolin bird PV Francolinus leucoscepus PV type 1 (FlPV-1) and the chaffinch bird PV Fringilla coelebs PV contain unusual E6 and E7 proteins. The avian E7 proteins contain an extended unfolded N terminus and a zinc-binding domain of reduced size, whereas the avian E6 proteins consist of a single zinc-binding domain. A comparable single-domain E6 protein may have existed in a common ancestor of mammalian and avian PVs. Mammalian E6 C-terminal domains are phylogenetically related to those of single-domain avian E6, whereas mammalian E6 N-terminal domains seem to have emerged by duplication and subsequently diverged from the original ancestral domain. In avian and mammalian cells, both FlPV-1 E6 and FlPV-1 E7 were evenly expressed in the cytoplasm and the nucleus. Finally, samples of full-length FlPV-1 E6 and the FlPV-1 E7 C-terminal zinc-binding domain were prepared for biophysical analysis. Both constructs were highly soluble and well folded, according to nuclear magnetic resonance spectroscopy measurements.

Van Doorslaer, Koenraad; Ould M'hamed Ould Sidi, Abdellahi; Zanier, Katia; Rybin, Vladimir; Deryckere, Francois; Rector, Annabel; Burk, Robert D.; Lienau, E. Kurt; van Ranst, Marc; Trave, Gilles

2009-01-01

46

E6 and E7 from Beta Hpv38 Cooperate with Ultraviolet Light in the Development of Actinic Keratosis-Like Lesions and Squamous Cell Carcinoma in Mice  

PubMed Central

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21WAF1 and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.

Viarisio, Daniele; Mueller-Decker, Karin; Kloz, Ulrich; Aengeneyndt, Birgit; Kopp-Schneider, Annette; Grone, Hermann-Josef; Gheit, Tarik; Flechtenmacher, Christa; Gissmann, Lutz; Tommasino, Massimo

2011-01-01

47

A Novel Mucosal Vaccine Based on Live Lactococci Expressing E7 Antigen and IL12 Induces Systemic and Mucosal Immune Responses and Protects Mice against Human Papillomavirus Type 16Induced Tumors  

Microsoft Academic Search

Current strategies to prevent or treat human papillomavirus type 16 (HPV-16) infection are promising, but remain costly. More economical but efficient vaccines are thus needed. In this study, we evaluated the protective effects of mucosally coadministered live Lactococcus lactis strains expressing cell wall-anchored E7 Ag and a secreted form of IL-12 to treat HPV-16-induced tumors in a murine model. When

Luis G. Bermudez-Humaran; Naima G. Cortes-Perez; Francois Lefevre; Sylvie Rabot; Juan M. Alcocer-Gonzalez; Jean-Jacques Gratadoux; Cristina Rodriguez-Padilla; Reyes S. Tamez-Guerra; Gerard Corthier; Alexandra Gruss; Philippe Langella

2005-01-01

48

Sequence Evolution of the Intrinsically Disordered and Globular Domains of a Model Viral Oncoprotein  

PubMed Central

In the present work, we have used the papillomavirus E7 oncoprotein to pursue structure-function and evolutionary studies that take into account intrinsic disorder and the conformational diversity of globular domains. The intrinsically disordered (E7N) and globular (E7C) domains of E7 show similar degrees of conservation and co-evolution. We found that E7N can be described in terms of conserved and coevolving linear motifs separated by variable linkers, while sequence evolution of E7C is compatible with the known homodimeric structure yet suggests other activities for the domain. Within E7N, inter-residue relationships such as residue co-evolution and restricted intermotif distances map functional coupling and co-occurrence of linear motifs that evolve in a coordinate manner. Within E7C, additional cysteine residues proximal to the zinc-binding site may allow redox regulation of E7 function. Moreover, we describe a conserved binding site for disordered domains on the surface of E7C and suggest a putative target linear motif. Both homodimerization and peptide binding activities of E7C are also present in the distantly related host PHD domains, showing that these two proteins share not only structural homology but also functional similarities, and strengthening the view that they evolved from a common ancestor. Finally, we integrate the multiple activities and conformations of E7 into a hierarchy of structure-function relationships.

Chemes, Lucia B.; Glavina, Juliana; Alonso, Leonardo G.; Marino-Buslje, Cristina; de Prat-Gay, Gonzalo; Sanchez, Ignacio E.

2012-01-01

49

Oncoprotein metastasis: an expanded topography.  

PubMed

In this survey, the initial insights on the sub- and transcellular process of oncoprotein metastasis (OPM) are linked to recent observations and advances in related fields. The six proteins described here, i.e. insulin, osteopontin, interleukin-6, anterior gradient-2 protein, cellular apoptosis susceptibility protein and hepatoma-derived growth factor, as well as distinct peptide fragments thereof might henceforth serve as pivotal biomarkers for OPM in clinical chemistry, molecular morphology, pathology and oncology and, as a result, guide as potential targets future structure-based interventions in cancer treatment. PMID:23771064

Radulescu, Razvan T

2013-01-01

50

The E6 oncoproteins of high-risk papillomaviruses bind to a novel putative GAP protein, E6TP1, and target it for degradation.  

PubMed

The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mammary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of E6-induced oncogenic transformation. In this system, the E6 protein targets the p53 tumor suppressor protein for degradation, and mutational analyses have shown that E6-induced degradation of p53 protein is required for MEC immortalization. However, the inability of most dominant-negative p53 mutants to induce efficient immortalization of MECs suggests the existence of additional targets of the HPV E6 oncoprotein. Using the yeast two-hybrid system, we have isolated a novel E6-binding protein. This polypeptide, designated E6TP1 (E6-targeted protein 1), exhibits high homology to GTPase-activating proteins for Rap, including SPA-1, tuberin, and Rap1GAP. The mRNA for E6TP1 is widely expressed in tissues and in vitro-cultured cell lines. The gene for E6TP1 localizes to chromosome 14q23.2-14q24.3 within a locus that has been shown to undergo loss of heterozygosity in malignant meningiomas. Importantly, E6TP1 is targeted for degradation by the high-risk but not the low-risk HPV E6 proteins both in vitro and in vivo. Furthermore, the immortalization-competent but not the immortalization-incompetent HPV16 E6 mutants target the E6TP1 protein for degradation. Our results identify a novel target for the E6 oncoprotein and provide a potential link between HPV E6 oncogenesis and alteration of a small G protein signaling pathway. PMID:9858596

Gao, Q; Srinivasan, S; Boyer, S N; Wazer, D E; Band, V

1999-01-01

51

PP2A activity is controlled by methylation and regulates oncoprotein expression in melanoma cells: a mechanism which participates in growth inhibition induced by chloroethylnitrosourea treatment.  

PubMed

Protein phosphatase 2A (PP2A), an Akt pathway inhibitor, is considered to be activated by methylation of its catalytic subunit. Also PP2A downregulation was proposed to take part in carcinogenesis. Recently, PP2A activation was shown to be activated in response to DNA damage. To obtain further information on the role of PP2A in tumors and response to DNA damage, we investigated the relationship between PP2A methylation and activity, cell proliferation, Akt activation, c-Myc expression and PTEN activity in B16 melanoma cells untreated and after chloroethylnitrosourea (CENU) treatment. In untreated cells, okadaic acid, an antagonist of PP2A methylation, inhibited PP2A activity, stimulated cell proliferation, increased Akt activation and c-Myc expression. Xylulose-5-phosphate, an agonist of PP2A methylation, increased PP2A activity, decreased cell proliferation, Akt activation and c-Myc expression. However, both PP2A methylation modulators increased PTEN activity. During the response to CENU treatment, PP2A methylation and activity were strongly increased, Akt activation and c-Myc expression were decreased. However PTEN activity was increased. After tumor cell growth recovery, these modifications were moderately decreased. PP2A methylation was quantified and correlated positively with PP2A activity, and negatively with criteria for cell aggressiveness (cell proliferation, Akt activation, c-Myc expression). Based on these data, PP2A methylation status controls PP2A activity and oncoproteins expression and PP2A is strongly activated after CENU treatment thus partly explaining the growth inhibition in response to this agent. It follows that PP2A promethylating agents are potential candidates for anticancer drugs. PMID:18097542

Guénin, Samuel; Schwartz, Laurent; Morvan, Daniel; Steyaert, Jean Marc; Poignet, Amandine; Madelmont, Jean Claude; Demidem, Aicha

2008-01-01

52

E6 and E7 from Human Papillomavirus Type 16 Cooperate To Target the PDZ Protein Na/H Exchange Regulatory Factor 1 ?  

PubMed Central

Previous studies have shown that the PDZ-binding motif of the E6 oncoprotein from the mucosal high-risk (HR) human papillomavirus (HPV) types plays a key role in HPV-mediated cellular transformation in in vitro and in vivo experimental models. HR HPV E6 oncoproteins have the ability to efficiently degrade members of the PDZ motif-containing membrane-associated guanylate kinase (MAGUK) family; however, it is possible that other PDZ proteins are also targeted by E6. Here, we describe a novel interaction of HPV type 16 (HPV16) E6 with a PDZ protein, Na+/H+ exchange regulatory factor 1 (NHERF-1), which is involved in a number of cellular processes, including signaling and transformation. HPV16 E6 associates with and promotes the degradation of NHERF-1, and this property is dependent on the C-terminal PDZ-binding motif of E6. Interestingly, HPV16 E7, via the activation of the cyclin-dependent kinase complexes, promoted the accumulation of a phosphorylated form of NHERF-1, which is preferentially targeted by E6. Thus, both oncoproteins appear to cooperate in targeting NHERF-1. Notably, HPV18 E6 is not able to induce NHERF-1 degradation, indicating that this property is not shared with E6 from all HR HPV types. Downregulation of NHERF-1 protein levels was also observed in HPV16-positive cervical cancer-derived cell lines, such as SiHa and CaSki, as well as HPV16-positive cervical intraepithelial neoplasia (CIN). Finally, our data show that HPV16-mediated NHERF-1 degradation correlates with the activation of the phosphatidylinositol-3?-OH kinase (PI3K)/AKT signaling pathway, which is known to play a key role in carcinogenesis.

Accardi, Rosita; Rubino, Rosa; Scalise, Mariafrancesca; Gheit, Tarik; Shahzad, Naveed; Thomas, Miranda; Banks, Lawrence; Indiveri, Cesare; Sylla, Bakary S.; Cardone, Rosa A.; Reshkin, Stephan J.; Tommasino, Massimo

2011-01-01

53

HPV16 Oncoproteins Promote Cervical Cancer Invasiveness by Upregulating Specific Matrix Metalloproteinases  

PubMed Central

Production of matrix metalloproteinases (MMPs) for degradation of extracellular matrix is a vital step in cancer metastasis. We investigated the effects of HPV16 oncoproteins (16E6, 16E6*I and 16E7), either individually or combined, on the transcription of 7 MMPs implicated in cervical cancer invasiveness. The levels of 7 MMPs reported to be increased in cervical cancer were determined in C33A stably expressing different HPV16 oncoproteins using quantitative RT-PCR and compared with invasion ability of cell lines using in vitro invasion and wound healing assays. Overexpression of MMP-2 and MT1-MMP was detected in HPV16E6E7 expressing cells which correlated with increased cell invasion. Combination of HPV oncoproteins always showed greater effects than its individual form. Inhibition of cell invasion using a specific MMP-2 inhibitor, OA-Hy, and anti-MT1-MMP antibody confirmed that invasion in these cells was dependent on both MMP-2 and MT1-MMP expression. Depletion of HPV16E6E7 by shRNA-mediated knock-down experiments resulted in decreased MMP-2 and MT1-MMP expression levels as well as reduced invasion ability which strongly suggested specific effects of HPV oncoproteins on both MMPs and on cell invasion. Immunohistochemistry study in invasive cervical cancers confirmed the enhanced in vivo expression of these two MMPs in HPV16-infected cells. In addition, possible sites required by HPV16E6E7 on the MMP-2 and MT1-MMP promoters were investigated and PEA3 (at ?552/?540 for MMP-2, ?303 for MT1-MMP) and Sp1 (at ?91 for MMP-2, ?102 for MT1-MMP) binding sites were shown to be essential for mediating their transactivation activity. In conclusion, our study demonstrated that HPV16E6 and E7 oncoproteins cooperate in promoting cervical cancer invasiveness by specifically upregulating MMP-2 and MT1-MMP transcription in a similar manner.

Kaewprag, Jittranan; Umnajvijit, Wareerat; Ngamkham, Jarunya; Ponglikitmongkol, Mathurose

2013-01-01

54

Ethyl 1,8-Naphthyridone-3-carboxylates Downregulate Human Papillomavirus-16 E6 and E7 Oncogene Expression.  

PubMed

Strong epidemiological and molecular data associate cervical cancer (CC) with high-risk human papillomavirus (HPV) infections. The carcinogenic mechanism depends mainly on the expression of E6 and E7 oncoproteins encoded by the viral genome. Using a cell-based high-throughput assay, an in-house library of compounds was screened identifying the 1,8-naphthyridone 1 that efficiently inhibited the transcription driven by the long control region of the HPV genome. A series of analogues were then synthesized, obtaining more potent derivatives able to downregulate E6 and E7 transcripts in HPV-16-positive CC CaSki cells. An unusual structural insight emerged for the C-3 position of the 1,8-naphthyridone core, where the ethyl carboxylate esters, but not the carboxylic acids, are responsible for the activity. In vitro uptake studies showed that the 3-ethyl carboxylates do not act as prodrugs. The 1,8-naphthyridones emerged as valid starting points for the development of innovative agents potentially useful for the treatment of HPV-induced CC. PMID:24905115

Donalisio, Manuela; Massari, Serena; Argenziano, Monica; Manfroni, Giuseppe; Cagno, Valeria; Civra, Andrea; Sabatini, Stefano; Cecchetti, Violetta; Loregian, Arianna; Cavalli, Roberta; Lembo, David; Tabarrini, Oriana

2014-07-10

55

Interferon-? Induced microRNA-129-5p Down-Regulates HPV-18 E6 and E7 Viral Gene Expression by Targeting SP1 in Cervical Cancer Cells  

PubMed Central

Infection by human papillomavirus (HPV) can cause cervical intraepithelial neoplasia (CIN) and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-? can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-? inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.

Zhang, Jiarong; Li, Shuangdi; Yan, Qin; Chen, Xiaoyue; Yang, Yixia; Liu, Xuelian; Wan, Xiaoping

2013-01-01

56

Expression of a Single, Viral Oncoprotein in Skin Epithelium Is Sufficient to Recruit Lymphocytes  

PubMed Central

Established cancers are frequently associated with a lymphocytic infiltrate that fails to clear the tumour mass. In contrast, the importance of recruited lymphocytes during premalignancy is less well understood. In a mouse model of premalignant skin epithelium, transgenic mice that express the human papillomavirus type 16 (HPV16) E7 oncoprotein under a keratin 14 promoter (K14E7 mice) display epidermal hyperplasia and have a predominant infiltrate of lymphocytes consisting of both CD4 and CD8 T cells. Activated, but not naïve T cells, were shown to preferentially traffic to hyperplastic skin with an increased frequency of proliferative CD8+ T cells and CD4+ T cells expressing CCR6 within the tissue. Disruption of the interaction between E7 protein and retinoblastoma tumour suppressor protein (pRb) led to reduced epithelial hyperplasia and T cell infiltrate. Finally, while K14E7 donor skin grafts are readily accepted onto syngeneic, non-transgenic recipients, these same skin grafts lacking skin-resident lymphocytes were rejected. Our data suggests that expression of a single oncoprotein in the epidermis is sufficient for lymphocyte trafficking (including immunosuppressive lymphocytes) to premalignant skin.

Narayan, Sharmal; Mattarollo, Stephen R.; Liem, Amy; Lambert, Paul F.; Frazer, Ian H.; Leggatt, Graham R.

2013-01-01

57

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes  

Microsoft Academic Search

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins,

CRISTINA BALAGUE; FRANCISCO NOYA; RAMON ALEMANY; LOUISE T. CHOW; D. T. Curiel

2001-01-01

58

Skin Hyperproliferation and Susceptibility to Chemical Carcinogenesis in Transgenic Mice Expressing E6 and E7 of Human Papillomavirus Type 38  

Microsoft Academic Search

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter.

Wen Dong; Ulrich Kloz; Rosita Accardi; Sandra Caldeira; Wei-Min Tong; Zhao-Qi Wang; Lars Jansen; Matthias Durst; Bakary S. Sylla; Lutz Gissmann; Massimo Tommasino

2005-01-01

59

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes  

Microsoft Academic Search

BACKGROUND: Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins,

Mark A Merkley; Ellen Hildebrandt; Robert H Podolsky; Hilal Arnouk; Daron G Ferris; William S Dynan; Hubert Stöppler

2009-01-01

60

A20/TNFAIP3 inhibits NF-?B activation induced by the Kaposi's sarcoma-associated herpesvirus vFLIP oncoprotein  

PubMed Central

Kaposi’s sarcoma-associated herpesvirus (KSHV) K13/vFLIP (viral Flice-inhibitory protein) induces transcription of numerous genes through NF-?B activation, including pro-inflammatory cytokines, which contribute to the pathogenesis of Kaposi’s sarcoma (KS). In this study, we report that KSHV vFLIP induces the expression of the NF-?B regulatory proteins A20, ABIN-1 and ABIN-3 (A20-binding NF-?B inhibitors) in primary human endothelial cells, and that KS spindle cells express A20 in KS tissue. In reporter assays, A20 strongly impaired vFLIP-induced NF-?B activation in 293T cells, but ABIN-1 and ABIN-3 did not. Mutational analysis established that the C-terminal domain (residues 427–790) is critical for A20 modulation of NF-?B, but the ubiquitin-editing OTU (ovarian tumor) domain is not. In functional assays, A20 inhibited vFLIP-induced expression of the chemokine IP-10, reduced vFLIP-induced cell proliferation and increased IKK1 protein levels. Thus, we demonstrate that A20 negatively regulates NF-?B activation directly induced by KSHV vFLIP. By attenuating excessive and prolonged vFLIP-induced NF-?B activation that could be harmful to KSHV-infected cells, A20 likely has an important role in the pathogenesis of KSHV-associated diseases, in which vFLIP is expressed.

Sakakibara, S; Espigol-Frigole, G; Gasperini, P; Uldrick, TS; Yarchoan, R; Tosato, G

2012-01-01

61

Kaposi's Sarcoma-Associated Herpesvirus Oncoprotein K13 Protects against B Cell Receptor-Induced Growth Arrest and Apoptosis through NF-?B Activation  

PubMed Central

Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (MCD). We have characterized the role of KSHV-encoded viral FLICE inhibitory protein (vFLIP) K13 in the modulation of anti-IgM-induced growth arrest and apoptosis in B cells. We demonstrate that K13 protects WEHI 231, an immature B-cell line, against anti-IgM-induced growth arrest and apoptosis. The protective effect of K13 was associated with the activation of the NF-?B pathway and was deficient in a mutant K13 with three alanine substitutions at positions 58 to 60 (K13-58AAA) and a structural homolog, vFLIP E8, both of which lack NF-?B activity. K13 upregulated the expression of NF-?B subunit RelB and blocked the anti-IgM-induced decline in c-Myc and rise in p27Kip1 that have been associated with growth arrest and apoptosis. K13 also upregulated the expression of Mcl-1, an antiapoptotic member of the Bcl2 family. Finally, K13 protected the mature B-cell line Ramos against anti-IgM-induced apoptosis through NF-?B activation. Inhibition of anti-IgM-induced apoptosis by K13 may contribute to the development of KSHV-associated lymphoproliferative disorders.

Graham, Ciaren; Matta, Hittu; Yang, Yanqiang; Yi, Han; Suo, Yulan; Tolani, Bhairavi

2013-01-01

62

Kaposi's sarcoma-associated herpesvirus oncoprotein K13 protects against B cell receptor-induced growth arrest and apoptosis through NF-?B activation.  

PubMed

Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (MCD). We have characterized the role of KSHV-encoded viral FLICE inhibitory protein (vFLIP) K13 in the modulation of anti-IgM-induced growth arrest and apoptosis in B cells. We demonstrate that K13 protects WEHI 231, an immature B-cell line, against anti-IgM-induced growth arrest and apoptosis. The protective effect of K13 was associated with the activation of the NF-?B pathway and was deficient in a mutant K13 with three alanine substitutions at positions 58 to 60 (K13-58AAA) and a structural homolog, vFLIP E8, both of which lack NF-?B activity. K13 upregulated the expression of NF-?B subunit RelB and blocked the anti-IgM-induced decline in c-Myc and rise in p27(Kip1) that have been associated with growth arrest and apoptosis. K13 also upregulated the expression of Mcl-1, an antiapoptotic member of the Bcl2 family. Finally, K13 protected the mature B-cell line Ramos against anti-IgM-induced apoptosis through NF-?B activation. Inhibition of anti-IgM-induced apoptosis by K13 may contribute to the development of KSHV-associated lymphoproliferative disorders. PMID:23236068

Graham, Ciaren; Matta, Hittu; Yang, Yanqiang; Yi, Han; Suo, Yulan; Tolani, Bhairavi; Chaudhary, Preet M

2013-02-01

63

Centrosome amplification induced by hydroxyurea leads to aneuploidy in pRB deficient human and mouse fibroblasts  

Microsoft Academic Search

Alterations in the number and\\/or morphology of centrosomes are frequently observed in human tumours. However, it is still debated if a direct link between supernumerary centrosomes and tumorigenesis exists and if centrosome amplification could directly cause aneuploidy. Here, we report that hydroxyurea treatment induced centrosome amplification in both human fibroblasts expressing the HPV16 -E6-E7 oncoproteins, which act principally by targeting

Laura Lentini; Flora Iovino; Angela Amato; Aldo Di Leonardo

2006-01-01

64

Comparative Analysis of Transforming Properties of E6 and E7 from Different Beta Human Papillomavirus Types  

PubMed Central

The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.

Cornet, Iris; Bouvard, Veronique; Campo, Maria Saveria; Thomas, Miranda; Banks, Lawrence; Gissmann, Lutz; Lamartine, Jerome; Sylla, Bakary S.; Accardi, Rosita

2012-01-01

65

The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal.  

PubMed

The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Here we show that Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, we conclude that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation. PMID:8887656

Grimes, H L; Chan, T O; Zweidler-McKay, P A; Tong, B; Tsichlis, P N

1996-11-01

66

Therapeutic vaccination with papillomavirus E6 and E7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model.  

PubMed

This study was performed to test the therapeutic efficacy of overlapping long E6 and E7 peptides, containing both CD4+ T-helper and CD8+ CTL epitopes, on CRPV-induced lesions, which is an appropriate pre-clinical model for HPV diseases, including recurrent respiratory papillomatosis (RRP). Therapeutic peptide vaccination was able to significantly control wart growth (p < 0.01) and abrogate latent CRPV infection (p = 0.0006) compared to controls. Vaccination was associated with a T(H)1 T cell response, as suggested by a strong DTH skin test, antigen-specific proliferation of PBMC and a minimal IgG antibody response. Thus, this study shows promise for treatment of RRP by vaccination with long peptides. PMID:16054734

Vambutas, A; DeVoti, J; Nouri, M; Drijfhout, J W; Lipford, G B; Bonagura, V R; van der Burg, S H; Melief, C J M

2005-11-01

67

Andrographolide downregulates the v-Src and Bcr-Abl oncoproteins and induces Hsp90 cleavage in the ROS-dependent suppression of cancer malignancy.  

PubMed

Andrographolide is a diterpenoid compound isolated from Andrographis paniculata that exhibits anticancer activity. We previously reported that andrographolide suppressed v-Src-mediated cellular transformation by promoting the degradation of Src. In the present study, we demonstrated the involvement of Hsp90 in the andrographolide-mediated inhibition of Src oncogenic activity. Using a proteomics approach, a cleavage fragment of Hsp90? was identified in andrographolide-treated cells. The concentration- and time-dependent induction of Hsp90 cleavage that accompanied the reduction in Src was validated in RK3E cells transformed with either v-Src or a human truncated c-Src variant and treated with andrographolide. In cancer cells, the induction of Hsp90 cleavage by andrographolide and its structural derivatives correlated well with decreased Src levels, the suppression of transformation, and the induction of apoptosis. Moreover, the andrographolide-induced Hsp90 cleavage, Src degradation, inhibition of transformation, and induction of apoptosis were abolished by a ROS inhibitor, N-acetyl-cysteine. Notably, Hsp90 cleavage, decreased levels of Bcr-Abl (another known Hsp90 client protein), and the induction of apoptosis were also observed in human K562 leukemia cells treated with andrographolide or its active derivatives. Together, we demonstrated a novel mechanism by which andrographolide suppressed cancer malignancy that involved inhibiting Hsp90 function and reducing the levels of Hsp90 client proteins. Our results broaden the molecular basis of andrographolide-mediated anticancer activity. PMID:24161787

Liu, Sheng-Hung; Lin, Chao-Hsiung; Liang, Fong-Ping; Chen, Pei-Fen; Kuo, Cheng-Deng; Alam, Mohd Mujahid; Maiti, Barnali; Hung, Shih-Kai; Chi, Chin-Wen; Sun, Chung-Ming; Fu, Shu-Ling

2014-01-15

68

Autocrine CCL3 and CCL4 Induced by the Oncoprotein LMP1 Promote Epstein-Barr Virus-Triggered B Cell Proliferation  

PubMed Central

Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.

Tsai, Shu-Chun; Lin, Sue-Jane; Lin, Cheau-Jye; Chou, Ya-Ching; Lin, Jiun-Han; Yeh, Te-Huei; Chen, Mei-Ru; Huang, Li-Min; Lu, Meng-You; Huang, Ya-Chi; Chen, Huan-Yun

2013-01-01

69

The leukemia-associated Mll-Ell oncoprotein induces fibroblast growth factor 2 (Fgf2)-dependent cytokine hypersensitivity in myeloid progenitor cells.  

PubMed

The subset of acute myeloid leukemias (AML) with chromosomal translocations involving the MLL gene have a poor prognosis (referred to as 11q23-AML). The MLL fusion proteins that are expressed in 11q23-AML facilitate transcription of a set of HOX genes, including HOXA9 and HOXA10. Because Hox proteins are transcription factors, this suggests the possibility that Hox target genes mediate the adverse effects of MLL fusion proteins in leukemia. Identifying such Hox target genes might provide insights to the pathogenesis and treatment of 11q23-AML. In the current study we found that Mll-Ell (an MLL fusion protein) induced transcriptional activation of the FGF2 gene in a HoxA9- and HoxA10-dependent manner. FGF2 encodes fibroblast growth factor 2 (also referred to as basic fibroblast growth factor). Fgf2 influences proliferation and survival of hematopoietic stem cells and myeloid progenitor cells, and increased Fgf2-expression has been described in AMLs. We determined that expression of Mll-Ell in myeloid progenitor cells resulted in autocrine production of Fgf2 and Fgf2-dependent cytokine hypersensitivity. Therefore, our results implicated increased Fgf2 expression in progenitor proliferation and expansion in 11q23-AML. Because small molecule inhibitors of Fgf-receptors are in human clinical trials, this suggested a potential therapeutic approach to this treatment refractory leukemia. PMID:24089521

Shah, Chirag A; Bei, Ling; Wang, Hao; Platanias, Leonidas C; Eklund, Elizabeth A

2013-11-01

70

Understanding the targeting of the RB family proteins by viral oncoproteins to defeat their oncogenic machinery.  

PubMed

The retinoblastoma (RB) family consists of three genes, RB1, RBL1, and RBL2, that code for the pRb, p107, and pRb2/p130 proteins, respectively. All these factors have pivotal roles in controlling fundamental cellular mechanisms such as cell cycle, differentiation and apoptosis. The founder and the most investigated RB family protein is pRb, which is considered to be the paradigm of tumor suppressors. However, p107 and pRb2/p130 clearly display a high degree of structural and functional homology with pRb. Interestingly, these factors were first identified as physical targets of the Adenovirus E1A oncoprotein. Indeed, RB family proteins are the most important and widely investigated targets of small DNA virus oncoproteins, such as Adenovirus E1A, human papillomavirus E7 and Simian virus 40 large T antigen. By interacting with pRb and with other RB family members, these oncoproteins neutralize their growth suppressive properties, thus stimulating proliferation of the infected cells, de-differentiation, and resistance to apoptosis. All these acquired features strongly favor the rise and selection of immortalized and mutation-prone cells, leading to a higher propensity in undergoing transformation. Our present work aims to illustrate and delve into these protein-protein interactions. Considering that these viral oncoproteins are dispensable for normal cellular functions, they can create "oncogene addiction" in the infected/transformed cells. This makes the possibility to dismantle these interactions extremely attractive, thus promoting the development of highly specific smart molecules capable of targeting only the infected/transformed cells that express these viral factors. PMID:22718244

Bellacchio, Emanuele; Paggi, Marco G

2013-02-01

71

Stable expression of anti-HPV 16 E7-ribozyme in CV-1 cell lines.  

PubMed

The HPV16 (human papilloma virus type 16) E7 gene product, an oncoprotein, has been considered to be involved in the pathogenesis of anogenital cancer, particularly of cervical cancer. In order to evaluate the effect of suppression of the expression of the E7 gene in CV-1 cells by ribozyme, Rz523 with a transacting ribozyme targeted to the E7 RNA and two processing ribozyme genes at the 5' and 3' flank was cloned into the eukaryotic expression plasmid pREP9 under the control of RSV-LTR promoter. The resultant plasmid pRSV-Rz523 was transfected into CV-1 cells by calcium phosphate coprecipitation. The expression of the ribozyme in G418-resistant cells was detected by dot-blot hybridization. Ribozymes stably expressed in the CV-1 cells were at a level of 9.0 pmol per 10(6) cells, in which the active ribozyme molecules were more than 50 fmol per 10(6) cells. The result of RNase protection assay showed that the steady-state level of the E7 RNA fragment in CV-1 cell lines was significantly reduced by about 90% in ribozyme-expressing cells. In contrast, the antisense control plasmid pRSV-AE7 only exhibited about 20%. This result implicated the possibility of reversing the malignant phenotype of cervical cancer by means of suppressing the expression of the E7 gene with ribozyme. PMID:9187492

Huang, Y; Kong, Y; Wang, Y; Qi, G; Lu, C

1996-01-01

72

Functionally distinct monomers and trimers produced by a viral oncoprotein  

PubMed Central

While the process of homo-oligomer formation and disassembly into subunits represents a common strategy to regulate protein activity, reports of proteins in which the subunit and homo-oligomer perform independent functions are scarce. Tumorigenesis induced by the adenovirus E4-ORF1 oncoprotein depends on its binding to a select group of cellular PDZ proteins, including MUPP1, MAGI-1, ZO-2 and Dlg1. We report here that in cells E4-ORF1 exists as both a monomer and trimer and that monomers specifically bind and sequester MUPP1, MAGI-1 and ZO-2 within insoluble complexes whereas trimers specifically bind Dlg1 and promote its translocation to the plasma membrane. This work exposes a novel strategy wherein the oligomerization state of a protein not only determines the capacity to bind separate related targets but also couples the interactions to different functional consequences.

Chung, S-H; Weiss, RS; Frese, KK; Prasad, BVV; Javier, RT

2012-01-01

73

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes?  

PubMed Central

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes.

McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

2014-01-01

74

Molecular cloning and expression of Ehf, a new member of the ets transcription factor/oncoprotein gene family.  

PubMed

The ets family is a large multigene family of transcription factors that share a conserved DNA-binding "ETS" domain and include several oncoproteins that induce tumorigenesis when overexpressed. Here we report the cDNA cloning from mouse pituitary somatotroph tumors, sequence characterization and tissue-specific expression pattern in mice of a novel ets family gene, "Ehf" ("ets homologous factor"). The putative 300 amino acid Ehf protein is a highly divergent ets family member, but is most related to the recently identified oncoprotein ESX (36% overall and 84% ETS domain amino acid identity). Thus, Ehf and ESX comprise a new ets subfamily. Ehf is a single-copy gene, but produces four distinct mRNA transcripts. Ehf transcripts are abundant in mouse kidney and lung, less so in muscle and liver, and not detected in brain, spleen or testes. Because of its presence in somatotroph tumors and its relationship to ESX, Ehf may represent a new oncoprotein. PMID:9600089

Bochert, M A; Kleinbaum, L A; Sun, L Y; Burton, F H

1998-05-01

75

Hsp90-associated oncoproteins: multiple targets of geldanamycin and its analogs  

Microsoft Academic Search

Geldanamycin (GA), herbimycin A and radicicol bind heat-shock protein-90 (Hsp90) and destabilize its client proteins including v-Src, Bcr-Abl, Raf-1, ErbB2, some growth factor receptors and steroid receptors. Thus, Hsp90-active agents induce ubiquitination and proteasomal degradation of numerous oncoproteins. Depending on the cellular context, HSP90-active agents cause growth arrest, differentiation and apoptosis, or can prevent apoptosis. HSP-active agents are undergoing clinical

MV Blagosklonny

2002-01-01

76

Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain  

PubMed Central

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.

Mischo, Andre; Ohlenschlager, Oliver; Hortschansky, Peter; Ramachandran, Ramadurai; Gorlach, Matthias

2013-01-01

77

Cutaneous Papillomavirus E6 oncoproteins associate with MAML1 to repress transactivation and NOTCH signaling  

PubMed Central

Papillomavirus E6 oncoproteins associate with LXXLL motifs on target cellular proteins to alter their function. Using a proteomic approach, we found the E6 oncoproteins of cutaneous papillomaviruses Bovine Papillomavirus Type 1 (BE6) and HPV types 1 and 8 (1E6 and 8E6) associated with the MAML1 transcriptional co-activator. All three E6 proteins bind to an acidic LXXLL motif at the carboxy-terminus of MAML1 and repress transactivation by MAML1. MAML1 is best known as the co-activator and effector of NOTCH induced transcription, and BPV-1 E6 represses synthetic NOTCH responsive promoters, endogenous NOTCH responsive promoters, and is found in a complex with MAML1 in stably transformed cells. BPV-1 induced papillomas show characteristics of repressed NOTCH signal transduction, including suprabasal expression of integrins, talin, and basal type keratins, and delayed expression of the NOTCH dependent HES1 transcription factor. These observations give rise to a model whereby papillomavirus oncoproteins including BPV-1 E6 and the cancer associated HPV-8 E6 repress Notch induced transcription, thereby delaying keratinocyte differentiation.

Brimer, Nicole; Lyons, Charles; Wallberg, Annika E.; Vande Pol, Scott B.

2011-01-01

78

Fowlpox virus recombinants expressing HPV16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits  

Microsoft Academic Search

BACKGROUND: Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPV-related precancerous lesions. METHODS: Three different immunisation

Antonia Radaelli; Eleana Pozzi; Sole Pacchioni; Carlo Zanotto; Carlo De Giuli Morghen

2010-01-01

79

The E6 Oncoproteins of High-Risk Papillomaviruses Bind to a Novel Putative GAP Protein, E6TP1, and Target It for Degradation  

Microsoft Academic Search

The high-risk human papillomaviruses (HPVs) are associated with carcinomas of the cervix and other genital tumors. Previous studies have identified two viral oncoproteins, E6 and E7, which are expressed in the majority of HPV-associated carcinomas. The ability of high-risk HPV E6 protein to immortalize human mam- mary epithelial cells (MECs) has provided a single-gene model to study the mechanisms of

QINGSHEN GAO; SEETHA SRINIVASAN; SARAH N. BOYER; DAVID E. WAZER; VIMLA BAND

1999-01-01

80

Oncoprotein metastasis and its suppression revisited  

PubMed Central

The past two decades have witnessed an increasing appreciation of the role of the tumor microenvironment, of genetic and epigenetic alterations in normal cells adjacent to tumors and of the migration of normal cells with aberrant intrinsic properties in cancer pathophysiology. Aside from these insights, a novel concept termed "oncoprotein metastasis" (OPM) has recently been advanced and proposed to reflect protein-based neoplastic phenomena that might occur even before any modifications relating to the morphology, location or (epi)genetic outfit of cells during the malignant process. Here, evidence is presented that supports the OPM perception and thus should contribute not only to further rethink the definition of a normal cell, but also the treatment of cancer disease in the years to come.

2010-01-01

81

Structure-Function Analysis of the v-Myc Oncoprotein.  

National Technical Information Service (NTIS)

Oncoproteins of the Myc family are important regulators or cellular proliferation, differentiation, transformation, and apoptosis. Overwhelming evidence suggests the transcription activation domain, or TAD, of Myc is responsible for mediating all of Myc's...

D. Echlin

1998-01-01

82

The E6 Oncoprotein from HPV16 Enhances the Canonical Wnt/?-catenin Pathway in Skin Epidermis in vivo  

PubMed Central

The contribution of the Wnt signaling pathway to HPV-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk human papilloma viruses (HR-HPVs), ?-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the E6 protein’s PDZ-binding domain. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of ?-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of ?-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2+/LacZ mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of ?-catenin, the accumulation of cellular?-catenin-responsive genes and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6?PDZ mice) or in combination with Axin2+/LacZ. Conversely, co-transfection with either E6 or E6?PDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that utilized a luciferase Wnt/?-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only.

Bonilla-Delgado, Jose; Bulut, Gulay; Liu, Xuefeng; Cortes-Malagon, Enoc M.; Schlegel, Richard; Flores-Maldonado, Catalina; Contreras, Ruben G.; Chung, Sang-Hyuk; Lambert, Paul F.; Uren, Aykut; Gariglio, Patricio

2012-01-01

83

Karyopherin ?3: a new cellular target for the HPV-16 E5 oncoprotein  

PubMed Central

Epidemiological and experimental studies have shown that high-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer worldwide, and that HPV-16 is associated with more than half of these cases. In addition to the well-characterized E6 and E7 oncoproteins of HPV-16, recent evidence increasingly has implicated the HPV-16 E5 protein (16E5) as an important mediator of oncogenic transformation. Since 16E5 has no known intrinsic enzymatic activity, its effects on infected cells are most likely mediated by interactions with various cellular proteins and/or its documented association with lipid rafts. In the present study, we describe a new cellular target that binds to 16E5 in COS cells and in stable human ectocervical cell lines. This target is karyopherin ?3, a member of the nuclear import receptor family with critical roles in the nuclear import of ribosomal proteins and in the secretory pathway.

Krawczyk, Ewa; Hanover, John A.; Schlegel, Richard; Suprynowicz, Frank A.

2009-01-01

84

Post-Translational Control of IL-1? via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53  

PubMed Central

Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1?) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1? production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1? processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1? regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1? was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1? that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1? is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1? levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1? regulation which ultimately inhibits the secretion of IL-1? in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1? towards cervical cancer could be discerned. Hence, attenuation of IL-1? by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy.

Niebler, Martina; Qian, Xu; Hofler, Daniela; Kogosov, Vlada; Kaewprag, Jittranan; Kaufmann, Andreas M.; Ly, Regina; Bohmer, Gerd; Zawatzky, Rainer; Rosl, Frank; Rincon-Orozco, Bladimiro

2013-01-01

85

An underlying prognosis predictor of hepatocellular carcinoma: Oncoprotein 18  

PubMed Central

Recent studies have reported the association between the expression of oncoprotein 18 (op18) and hepatocellular carcinoma (HCC). However, any underlying mechanistic connection between op18 expression and hepatocarcinogenesis is poorly understood. In the present study, Flag-pcDNA3.1 vector and Flag-pcDNA3.1-op18 plasmid were stably transfected in SMMC7721 cells, respectively. Stable SMMC7721 control and op18 overexpression SMMC7721 cell lines were constructed and identified by western blot analysis. Using a cell counting kit-8 (CCK8), it was shown that cell proliferation was significantly increased in the op18 overexpression SMMC7721 cell group (0.60±0.05), compared with the control group (0.29±0.03) at an absorbance of 450 nm (P<0.01). Flow cytometry was used to analyze cell apoptosis by FITC-Annexin V and propidium iodide (PI) apoptosis assay kit. The results demonstrated that the percentage of apoptotic cells was inhibited to 5.80±0.33% in the op18 overexpression group, compared with 11.79±1.09% in the control group. Using FACS, single cell analysis data showed that op18 overexpression induced cell cycle arrest by inhibiting progression from G2 to M phase. The results suggest that op18 expression is closely associated with SMMC7721 cell proliferation and apoptosis, which appears to be a potential predictor of prognosis in HCC.

GONG, SHU; TAO, ZHONGHUA; LIU, XIAOYAN; GAN, LIN

2014-01-01

86

Cooperative Interactions of HER-2 and HPV-16 Oncoproteins in the Malignant Transformation of Human Mammary Epithelial Cells1  

PubMed Central

Abstract To better understand the mechanisms of transformation by the oncogene HER-2, we transduced the human mammary epithelial (HME) cell line MCF-10A with HER-2 and developed a cell line that appeared to moderately overexpress HER-2. These MCF-10HER-2 cells were unable to grow in the absence of epidermal growth factor (EGF). However, coexpression of HER-2 with the HPV-16 oncoproteins E6 and E7 resulted in EGF-independent cells that expressed very high levels of constitutively activated HER-2. Interestingly, coexpression of E7 with HER-2 resulted in cells that were EGF-independent for growth but did not express HER-2 to high levels, and coexpression of E6 with HER-2 resulted in cells expressing higher levels of HER-2, which were still dependent on EGF for growth and survival. The MCF-10HER-2E7 and HER-2/E6E7 cells exhibited constitutive activation of a form of epidermal growth factor receptor (EGFR) that had a faster electrophoretic mobility than EGFR activated by exogenous growth factors. Exposure of cells with EGFR activation to ZD1839 (Iressa), at concentrations specific for EGFR, had little or no influence on proliferation of cells with amplified HER-2 but little or no EGFR. These results indicate that HER-2, E6, and E7 cooperate with endogenous EGFR to yield fully transformed cells.

Woods Ignatoski, Kathleen M; Dziubinski, Michele L; Ammerman, Cheryl; Ethier, Stephen P

2005-01-01

87

Quantitative proteomic analysis of Myc oncoprotein function  

PubMed Central

This study applies a new quantitative proteomics technology to the analysis of the function of the Myc oncoprotein in mammalian cells. Employing isotope-coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry, the global pattern of protein expression in rat myc-null cells was compared with that of myc-plus cells (myc-null cells in which myc has been introduced) to generate a differential protein expression catalog. Expression differences among many functionally related proteins were identified, including reduction of proteases, induction of protein synthesis pathways and upregulation of anabolic enzymes in myc-plus cells, which are predicted to lead to increased cell mass (cell growth). In addition, reduction in the levels of adhesion molecules, actin network proteins and Rho pathway proteins were observed in myc-plus cells, leading to reduced focal adhesions and actin stress fibers as well as altered morphology. These effects are dependent on the highly conserved Myc Box II region. Our results reveal a novel cytoskeletal function for Myc and indicate the feasibility of quantitative whole-proteome analysis in mammalian cells.

Shiio, Yuzuru; Donohoe, Sam; Yi, Eugene C.; Goodlett, David R.; Aebersold, Ruedi; Eisenman, Robert N.

2002-01-01

88

Activation of human papillomavirus type 18 E6-E7 oncogene expression by transcription factor Sp1.  

PubMed Central

The human papillomavirus 18 (HPV18) E6 and E7 proteins are considered to be primarily responsive for the transforming activity of the virus. In order to analyse the molecular mechanisms resulting in viral oncoprotein expression, it is necessary to identify the factors involved in the transcriptional regulation of the E6/E7 genes. Here we define by gel retardation experiments a sequence aberrant Sp1 binding site present in the promoter proximal part of the viral transcriptional control region (Upstream Regulatory Region, URR). Functional analyses employing transient reporter assays reveal that this Sp1 element is required for an efficient stimulation of the HPV18 E6/E7-promoter. Mutation of the Sp1 element in the natural context of the HPV18 URR leads to a strong decrease in the activity of the E6/E7-promoter in several cell lines. The magnitude of reduction varies between different cell types and is higher in cell lines of epithelial origin when compared with nonepithelial cells. Cotransfection assays using Sp1 expression vector systems further define the promoter proximal HPV18 Sp1 binding motif as a functional Sp1 element in vivo and show that its integrity is essential for the stimulation of the E6/E7-promoter by augmented levels of Sp1. These results indicate, that the cellular transcription factor Sp1 plays an important role for the stimulation of the E6/E7-promoter by the viral URR and represents a major determinant for the expression of HPV18 transforming genes E6 and E7. Images

Hoppe-Seyler, F; Butz, K

1992-01-01

89

Identification of Key Amino Acid Residues That Determine the Ability of High Risk HPV16-E7 to Dysregulate Major Histocompatibility Complex Class I Expression*  

PubMed Central

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.

Heller, Corina; Weisser, Tanja; Mueller-Schickert, Antje; Rufer, Elke; Hoh, Alexander; Leonhardt, Ralf M.; Knittler, Michael R.

2011-01-01

90

A Molecular Model for the Differential Activation of STAT3 and STAT6 by the Herpesviral Oncoprotein Tip  

PubMed Central

Constitutive STAT signaling provides growth promoting signals in many forms of malignancy. We performed molecular modeling and molecular dynamics studies of the interaction between the regulatory Src homology 2 (SH2) domains of STAT3 and 6 with phosphorylated peptides of the herpesviral oncoprotein Tip, which facilitates Src kinase mediated STAT-activation and T cell proliferation. The studies give insight into the ligand binding specificity of the STAT SH2 domains and provide the first model for the differential activation of STAT3 or STAT6 by two distinct regions of the viral Tip protein. The biological relevance of the modeled interactions was then confirmed by activation studies using corresponding recombinant oncoproteins, and finally by respective recombinant viruses. The functional data give experimental validation of the molecular dynamics study, and provide evidence for the involvement of STAT6 in the herpesvirus induced T cell proliferation.

Vogel, Benjamin; Heck, Elke; Scholz, Brigitte; Lengenfelder, Doris; Sticht, Heinrich; Ensser, Armin

2012-01-01

91

New Approach to Identify Novel Regulators of Myc Oncoprotein Stability.  

National Technical Information Service (NTIS)

The Myc oncoprotein is deregulated in the majority of breast cancers yet it has not been possible to develop a therapeutic to target Myc using traditional approaches. It has recently been shown that targeting Myc for degradation may offer a new therapeuti...

L. Z. Penn

2012-01-01

92

Combination of hTERT and bmi-1, E6, or E7 Induces Prolongation of the Life Span of Bone Marrow Stromal Cells from an Elderly Donor without Affecting Their Neurogenic Potential†  

PubMed Central

Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.

Mori, Taisuke; Kiyono, Tohru; Imabayashi, Hideaki; Takeda, Yukiji; Tsuchiya, Kohei; Miyoshi, Shunichirou; Makino, Hatsune; Matsumoto, Kenji; Saito, Hirohisa; Ogawa, Satoshi; Sakamoto, Michiie; Hata, Jun-Ichi; Umezawa, Akihiro

2005-01-01

93

Curcumin counteracts the proliferative effect of estradiol and induces apoptosis in cervical cancer cells.  

PubMed

Cervical cancer is the most common cancer in Indian females and is associated with infection with high-risk Human papilloma viruses (HPVs) which encode viral oncoprotein E6 and E7. Estradiol has been established as a risk factor for cervical cancer and has been shown to play a synergistic role with viral oncoproteins. Curcumin (Diferuloyl methane), a chemopreventive agent, is a natural compound extracted from Curcuma longa that allows suppression and retardation of carcinogenesis in many types of cancer and is currently being tested in various human clinical trials as it has been found to be well tolerated at higher doses with a relatively well established safety profile. The objective of this study was to test the effect of curcumin on HPV-positive and negative cervical cancer cell lines HeLa, SiHa, CaSki, and C33A pretreated with estradiol. It was found that HPV-positive cells pretreated with estradiol show reduced apoptosis as compared to curcumin by itself. However, curcumin was able to counteract the proliferative response of estradiol, and induce apoptosis. There was no difference in percentage apoptosis as compared to estradiol pretreatment in HPV-negative cell line C33A. Molecular studies showed elevation of Telomerase, viral oncoproteins E6 and E7, PCNA, p16, Cyclin D1 in HPV-positive cell lines on treatment with estradiol but after treatment with curcumin the level of E7, PCNA, and Cyclin D1 was reduced but the level of E6, Telomerase, and p16 was unaltered. Furthermore, estradiol-pretreated HPV-negative cell line C33A showed reduction in level of Telomerase, PCNA, p16, and activation of both p53 and p73 tumor suppressor proteins, thus, demonstrating the importance of E6 in estradiol-mediated protective effect. PMID:20941532

Singh, Mayank; Singh, Neeta

2011-01-01

94

Selective PDZ protein-dependent stimulation of phosphatidylinositol 3-kinase by the adenovirus E4-ORF1 oncoprotein  

PubMed Central

While PDZ domain-containing proteins represent cellular targets for several different viral oncoproteins, including human papillomavirus E6, human T-cell leukemia virus type 1 Tax, and human adenovirus E4-ORF1, the functional consequences for such interactions have not been elucidated. Here we report that, at the plasma membrane of cells, the adenovirus E4-ORF1 oncoprotein selectively and potently stimulates phosphatidylinositol 3-kinase (PI3K), triggering a downstream cascade of events that includes activation of both protein kinase B and p70S6-kinase. This activity of E4-ORF1 could be abrogated by overexpression of its PDZ-protein targets or by disruption of its PDZ domain-binding motif, which was shown to mediate complex formation between E4-ORF1 and PDZ proteins at the plasma membrane of cells. Furthermore, E4-ORF1 mutants unable to activate the PI3K pathway failed to transform cells in culture or to promote tumors in animals, and drugs that block either PI3K or p70S6-kinase inhibited E4-ORF1-induced transformation of cells. From these results, we propose that the transforming and tumorigenic potentials of the adenovirus E4-ORF1 oncoprotein depend on its capacity to activate PI3K through a novel PDZ protein-dependent mechanism of action.

Frese, Kristopher K; Lee, Siu Sylvia; Thomas, Darby L; Latorre, Isabel J; Weiss, Robert S; Glaunsinger, Britt A; Javier, Ronald T

2012-01-01

95

Cleavage of NIK by the API2MALT1 Fusion Oncoprotein Leads to Noncanonical NF-kappaB Activation  

Microsoft Academic Search

Proper regulation of nuclear factor kappaB (NF-kappaB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-kappaB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-kappaB-inducing kinase (NIK) at arginine 325. NIK cleavage requires the concerted actions of both fusion partners

Shaun Rosebeck; Lisa Madden; Xiaohong Jin; Shufang Gu; Ingrid J. Apel; Alex Appert; Rifat A. Hamoudi; Heidi Noels; Xavier Sagaert; Peter Van Loo; Mathijs Baens; Ming-Qing Du; Peter C. Lucas; Linda M. McAllister-Lucas

2011-01-01

96

RNA interference against HPV16 E7 oncogene leads to viral E6 and E7 suppression in cervical cancer cells and apoptosis via upregulation of Rb and p53.  

PubMed

The simultaneous expression of human papillomavirus type 16 (HPV16) E6 and E7 oncogenes is pivotal for malignant transformation and maintenance of malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether HPV16 E6 and E7 could be both silenced to obtain most efficient antitumor activity by using RNA interference (RNAi) technology. Herein, we designed a small interfering RNA (siRNA) targeting HPV16-E7 region to degrade either E6, or truncated E6 (E6*) and E7 mRNAs and to simultaneously knockdown both E6 and E7 expression. Firstly, the sequence targeting HPV16-E7 region was inserted into the shRNA packing vector pSIREN-DNR, yielding pSIREN-16E7 to stably express corresponding shRNA. HPV16-transformed SiHa and CaSki cells were used as a model system; RT-PCR, Western Blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay and flow cytometry were applied to examine the effects of pSIREN-16E7. Our results indicated that HPV16-E7 specific shRNA (16E7-shRNA) induced selective degradation of E6 and E7 mRNAs and proteins. E6 silencing induced accumulation of cellular p53 and p21. In contrast, E7 silencing induced hypophosphorylation of retinoblastoma (Rb) protein. The loss of E6 and E7 reduced cell growth and ultimately resulted in massive apoptotic cell death selectively in HPV-positive cancer cells, compared with the HPV-negative ones. We demonstrated that 16E7-shRNA can induce simultaneous E6 and E7 suppression and lead to striking apoptosis in HPV16-related cancer cells by activating cellular p53, p21 and Rb. Therefore, RNAi using E7 shRNA may have the gene-specific therapy potential for HPV16-related cancers. PMID:18060502

Sima, Ni; Wang, Wei; Kong, Debo; Deng, Dongrui; Xu, Qian; Zhou, Jianfeng; Xu, Gang; Meng, Li; Lu, Yunping; Wang, Shixuan; Ma, Ding

2008-02-01

97

Structural basis for hijacking of cellular LxxLL motifs by papillomavirus E6 oncoproteins  

PubMed Central

E6 viral oncoproteins are key players in epithelial tumors induced by Papillomaviruses in vertebrates, including cervical cancer in humans. E6 proteins target many host proteins by specifically interacting with acidic LxxLL motifs. Here, we solved the crystal structures of Bovine (BPV1) and Human (HPV16) Papillomavirus E6 proteins bound to LxxLL peptides from the focal adhesion protein paxillin and the ubiquitin ligase E6AP, respectively. In both E6 proteins, two zinc domains and a linker helix form a basic-hydrophobic pocket, which captures helical LxxLL motifs in a way compatible with other interaction modes. Mutational inactivation of the LxxLL binding pocket disrupts the oncogenic activities of both E6 proteins. This work reveals the structural basis of both the multifunctionality and the oncogenicity of E6 proteins.

Zanier, Katia; Charbonnier, Sebastian; Sidi, Abdellahi Ould M'hamed Ould; McEwen, Alastair G.; Ferrario, Maria Giovanna; Poussin, Pierre; Cura, Vincent; Brimer, Nicole; Babah, Khaled Ould; Ansari, Tina; Muller, Isabelle; Stote, Roland H.; Cavarelli, Jean; Pol, Scott Vande; Trave, Gilles

2014-01-01

98

The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF.  

PubMed

Tumor angiogenesis plays an important role in the development of cancer. Previously, we reported that hepatitis B X-interacting protein (HBXIP) functioned as an oncoprotein in breast cancer. However, the role of HBXIP in angiogenesis in breast cancer remains poorly understood. In the present study, we show that the oncoprotein HBXIP plays crucial roles in the event. We observed that the expression levels of HBXIP were positively correlated with those of fibroblast growth factor 8 (FGF8) or vascular endothelial growth factor (VEGF) in clinical breast cancer tissues. Then, we demonstrated that HBXIP was able to upregulate FGF8 through activation of its promoter involving direct binding to cAMP response element-binding protein (CREB) in breast cancer cells and thereby increased its secretion. Strikingly, we identified another pathway that HBXIP upregulated FGF8 and VEGF through inhibiting miRNA-503, which directly targeted 3' untranslated region of FGF8 or VEGF mRNA in the cells. Moreover, we revealed that HBXIP-induced FGF8 could upregulate VEGF expression through activating phosphoinositide 3-kinase (PI3K)/Akt/hypoxia-inducible factor 1-alpha (HIF1?) signaling and increase its secretion. In function, matrigel angiogenesis assay and hemoglobin content analysis uncovered that HBXIP-enhanced FGF8/VEGF boosted tumor angiogenesis and growth in breast cancer in vitro and in vivo in a paracrine/autocrine manner. Thus, we conclude that HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Our finding provides new insights into the mechanism of tumor angiogenesis in breast cancer. Therapeutically, HBXIP may serve as a novel target of tumor angiogenesis. PMID:24464787

Liu, Fabao; You, Xiaona; Wang, Yue; Liu, Qian; Liu, Yunxia; Zhang, Shuqin; Chen, Lingyi; Zhang, Xiaodong; Ye, Lihong

2014-05-01

99

E7(7) on the light cone  

Microsoft Academic Search

We use the Cremmer-Julia E7(7) non-linear symmetry of Script N = 8 Supergravity to derive its order kappa2 on-shell Hamiltonian in terms of one chiral light-cone superfield. By requiring that E7(7) commute with the super-Poincaré group, we deduce to lowest non-trivial order in kappa, the light cone E7(7) transformations of all fields of the theory, including the graviton. We then

Lars Brink; Sung-Soo Kim; Pierre Ramond

2008-01-01

100

[Prokaryotic expression and identification of HPV16 E7 protein].  

PubMed

HPV16 E7 fusion protein was expressed in E. coli BL21, and its applied value for HPV was evaluated. HPV16 E7 gene was amplified by PCR, and cloned into prokaryotic expression vector pGEX6p-1. The recombinant plasmid was transformed into E. coli BL21, and HPV16 E7 fusion was expressed through IPTG induction. The expressed product was analyzed by SDS-PAGE and Western blot, subsequently purified according to Glutathione Sepharose 4B purification procedure. An indirect ELISA with the purified fusion protein as the coating antigen was then established to detect E7 serum antibodies from mice immunized with recombinant Listeria monocytogenes delivering HPV16 E7. The results demonstrated that the soluble fusion protein was highly expressed at 25 degrees C after induction with 0.5 mM IPTG. Furthermore, the result of Western blot analysis showed that the fusion protein had good specific reaction with an anti-E7 monoclonal antibody. Indirect ELISA result confirmed that the fusion protein could detect the serum antibodies against E7 with a titer of 1:200. The expressed GST-E7 fusion protein was immunocompetent, which was useful in the research of E7 biological function and therapeutic vaccine. PMID:22416350

Jia, Yan-Yan; Yin, Yue-Lan; Bai, Chun-Guang; Fu, Hong; Gao, Yun-Fei; Pan, Zhi-Ming; Jiao, Xin-An

2012-01-01

101

Constitutive Activation of Stat3 in Fibroblasts Transformed by Diverse Oncoproteins and in Breast Carcinoma Cells  

Microsoft Academic Search

Signal transducers and activators of transcription (STATs) were originally identified as key components of signaling pathways involved in mediating responses to IFNs. Previous studies showed that the Src oncoprotein constitutively activates one STAT family member, Stat3. In this study, we investigated STAT activation in a panel of rodent fibroblast cell lines stably transformed by diverse viral oncoproteins. Using a temperature-sensitive

Roy Garcia; Chao-Lan Yu; Anne Hudnall; Robyn Catlett; Knstie L Nelson; Thomas Smithgall; Donald J. Fujita; Stephen P. Ethier; Richard Jove

102

A humanized mouse model of HPV-associated pathology driven by E7 expression.  

PubMed

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies. PMID:22911850

Buitrago-Pérez, Águeda; Hachimi, Mariam; Dueñas, Marta; Lloveras, Belén; Santos, Almudena; Holguín, Almudena; Duarte, Blanca; Santiago, Juan Luis; Akgül, Baki; Rodríguez-Peralto, José L; Storey, Alan; Ribas, Catalina; Larcher, Fernando; del Rio, Marcela; Paramio, Jesús M; García-Escudero, Ramón

2012-01-01

103

A Humanized Mouse Model of HPV-Associated Pathology Driven by E7 Expression  

PubMed Central

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPV-associated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPV-associated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies.

Buitrago-Perez, Agueda; Hachimi, Mariam; Duenas, Marta; Lloveras, Belen; Santos, Almudena; Holguin, Almudena; Duarte, Blanca; Santiago, Juan Luis; Akgul, Baki; Rodriguez-Peralto, Jose L.; Storey, Alan; Ribas, Catalina; Larcher, Fernando; del Rio, Marcela; Paramio, Jesus M.; Garcia-Escudero, Ramon

2012-01-01

104

The receptor-type protein tyrosine phosphatase J antagonizes the biochemical and biological effects of RET-derived oncoproteins.  

PubMed

Thyroid cancer is frequently associated with the oncogenic conversion of the RET receptor tyrosine kinase. RET gene rearrangements, which lead to the generation of chimeric RET/papillary thyroid carcinoma (PTC) oncogenes, occur in PTC, whereas RET point mutations occur in familial multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinomas (MTC). We showed previously that the expression of the receptor-type protein tyrosine phosphatase J (PTPRJ) is suppressed in neoplastically transformed follicular thyroid cells. We now report that PTPRJ coimmunoprecipitates with wild-type RET and with the MEN2A-associated RET(C634R) oncoprotein but not with the RET/PTC1 and RET-MEN2B isoforms. Using mutated forms of PTPRJ and RET-MEN2A, we show that the integrity of the respective catalytic domains is required for the PTPRJ/RET-MEN2A interaction. PTPRJ expression induces dephosphorylation of the RET(C634R) and, probably via an indirect mechanism, RET/PTC1 oncoproteins on two key RET autophosphorylation sites (Tyr1062 and Tyr905). This results in a significant decrease of RET-induced Shc and extracellular signal-regulated kinase 1/2 phosphorylation levels. In line with this finding, adoptive PTPRJ expression reduced the oncogenic activity of RET(C634R) in an in vitro focus formation assay of NIH3T3 cells. As expected from the coimmunoprecipitation results, the RET(M918T) oncoprotein, which is associated to MEN2B and sporadic MTC, was resistant to the dephosphorylating activity of PTPRJ. Taken together, these findings identify RET as a novel substrate of PTPRJ and suggest that PTPRJ expression levels may affect tumor phenotype associated with RET/PTC1 and RET(C634R) mutants. On the other hand, resistance to PTPRJ may be part of the mechanism of RET oncogenic conversion secondary to the M918T mutation. PMID:16778204

Iervolino, Angela; Iuliano, Rodolfo; Trapasso, Francesco; Viglietto, Giuseppe; Melillo, Rosa Marina; Carlomagno, Francesca; Santoro, Massimo; Fusco, Alfredo

2006-06-15

105

Human papillomavirus (HPV) type 16 E7 protein bodies cause tumour regression in mice  

PubMed Central

Background Human papillomaviruses (HPV) are the causative agents of cervical cancer in women, which results in over 250 000 deaths per year. Presently there are two prophylactic vaccines on the market, protecting against the two most common high-risk HPV types 16 and 18. These vaccines remain very expensive and are not generally affordable in developing countries where they are needed most. Additionally, there remains a need to treat women that are already infected with HPV, and who have high-grade lesions or cervical cancer. Methods In this paper, we characterize the immunogenicity of a therapeutic vaccine that targets the E7 protein of the most prevalent high-risk HPV - type 16 – the gene which has previously been shown to be effective in DNA vaccine trials in mice. The synthetic shuffled HPV-16 E7 (16E7SH) has lost its transforming properties but retains all naturally-occurring CTL epitopes. This was genetically fused to Zera®, a self-assembly domain of the maize ?-zein able to induce the accumulation of recombinant proteins into protein bodies (PBs), within the endoplasmic reticulum in a number of expression systems. Results High-level expression of the HPV 16E7SH protein fused to Zera® in plants was achieved, and the protein bodies could be easily and cost-effectively purified. Immune responses comparable to the 16E7SH DNA vaccine were demonstrated in the murine model, with the protein vaccine successfully inducing a specific humoral as well as cell mediated immune response, and mediating tumour regression. Conclusions The fusion of 16E7SH to the Zera® peptide was found to enhance the immune responses, presumably by means of a more efficient antigen presentation via the protein bodies. Interestingly, simply mixing the free PBs and 16E7SH also enhanced immune responses, indicating an adjuvant activity for the Zera® PBs.

2014-01-01

106

Normal human fibroblasts immortalized by introduction of human papillomavirus type 16 (HPV-16) E6-E7 genes.  

PubMed

This report demonstrates that normal human fibroblasts can be immortalized by the introduction of HPV-16 E6-E7 genes. We designed zinc-inducible expression plasmids with HPV-16 E6, E7 or both. Each plasmid was introduced into normal human fibroblasts (TIG-3 cells) using lipofection methods. Only transfectants with the HPV-16 E6-E7 zinc-inducible expression plasmid, which were cultured in medium supplemented with 100 microM ZnSO4, overcame crisis and could be cultured over 200 population doubling levels (PDLs). These cell lines showed the reactivation of telomerase after crisis, and morphological alterations were also observed. PMID:9159405

Shiga, T; Shirasawa, H; Shimizu, K; Dezawa, M; Masuda, Y; Simizu, B

1997-01-01

107

Concomitant Oncoprotein Detection with Fluorescence in Situ Hybridization (CODFISH)  

PubMed Central

We sought the validation of a three-color fluorescence-based system that simultaneously profiles Her-2/neu oncogene copy by fluorescence in situ hybridization (FISH) and Her-2/neu encoded protein by the use of a versatile alkaline phosphatase chromogen fast red K in either fluorescence or bright-field mode. Nuclei were counterstained with DAPI. Nineteen infiltrating ductal carcinomas of breast were comprehensively evaluated for Her-2/neu amplification/overexpression by direct and indirect FISH using digoxigenin (DigFISH) and direct fluorescently labeled probes, autoradiographic RNA:RNA in situ hybridization, and immunohistochemistry using monoclonal antibody CB11. CODFISH results correlated well with DigFISH, direct-label FISH, mRNA expression, and oncoprotein expression as assessed with CB11, and enabled simultaneous visualization of gene copy and protein. In addition, qualitative immunohistochemistry may be followed by CODFISH gene copy enumeration to clarify ambiguous cases.

Tubbs, Raymond R.; Pettay, James; Roche, Pat; Stoler, Mark H.; Jenkins, Robert; Myles, Jon; Grogan, Thomas

2000-01-01

108

Prognostic significance of immunohistochemical expression of the HER2\\/neu oncoprotein in bone metastatic prostate cancer  

Microsoft Academic Search

ObjectivesTo investigate the usefulness of the overexpression of the human epidermal growth factor receptor (HER-2) oncoprotein in patients with bone metastatic prostate cancer as a marker for the time to recurrence and outcome after endocrine therapy.

Yoshitaka Nishio; Yoshiaki Yamada; Hiroto Kokubo; Kogenta Nakamura; Shigeyuki Aoki; Tomohiro Taki; Nobuaki Honda; Atsuko Nakagawa; Shinsuke Saga; Kazuo Hara

2006-01-01

109

University of Pittsburgh Cancer Institute researchers identify key oncoprotein found in Merkel Cell Carcinoma:  

Cancer.gov

Researchers at the University of Pittsburgh Cancer Institute (UPCI) have identified the oncoprotein that allows a common and usually harmless virus to transform healthy cells into a rare but deadly skin cancer called Merkel Cell Carcinoma (MCC).

110

Chimeric Infectious Bursal Disease Virus-Like Particles as Potent Vaccines for Eradication of Established HPV-16 E7-Dependent Tumors  

PubMed Central

Cervical cancer is caused by persistent high-risk human papillomavirus (HR-HPV) infection and represents the second most frequent gynecological malignancy in the world. The HPV-16 type accounts for up to 55% of all cervical cancers. The HPV-16 oncoproteins E6 and E7 are necessary for induction and maintenance of malignant transformation and represent tumor-specific antigens for targeted cytotoxic T lymphocyte–mediated immunotherapy. Therapeutic cancer vaccines have become a challenging area of oncology research in recent decades. Among current cancer immunotherapy strategies, virus-like particle (VLP)–based vaccines have emerged as a potent and safe approach. We generated a vaccine (VLP-E7) incorporating a long C-terminal fragment of HPV-16 E7 protein into the infectious bursal disease virus VLP and tested its therapeutic potential in HLA-A2 humanized transgenic mice grafted with TC1/A2 tumor cells. We performed a series of tumor challenge experiments demonstrating a strong immune response against already-formed tumors (complete eradication). Remarkably, therapeutic efficacy was obtained with a single dose without adjuvant and against two injections of tumor cells, indicating a potent and long-lasting immune response.

Gonzalez-Cintado, Leticia; Kowalczyk, Wioleta; Jimenez Torres, Ignacio; Calderita, Gloria; Rodriguez, Margarita; Gondar, Virginia; Bernal, Juan Jose; Ardavin, Carlos; Andreu, David; Zurcher, Thomas; von Kobbe, Cayetano

2012-01-01

111

Papillomavirus E6 oncoprotein up-regulates occludin and ZO-2 expression in ovariectomized mice epidermis.  

PubMed

We have studied the expression of the tight junction proteins (TJ) occludin, claudin-1 and ZO-2 in the epidermis of female mice. We observed a peak of expression of these proteins at postnatal day 7 and a decrease in 6 week-old mice to values similar to those found in newborn animals. We explored if the expression of the E6 oncoprotein from high-risk human papilloma virus type 16 (HPV16) in the skin of transgenic female mice (K14E6), altered TJ protein expression in a manner sensitive to ovarian hormones. We observed that in ovariectomized mice E6 up-regulates the expression of occludin and ZO-2 in the epidermis and that this effect was canceled by 17?-estradiol. Progesterone instead induced occludin and ZO-2 over-expression. However, the decreased expression of occludin and ZO-2 induced by 17?-estradiol in the epidermis was not overturned by E6 or progesterone. In addition, we employed MDCK cells transfected with E6, and observed that ZO-2 delocalizes from TJs and accumulates in the cell nuclei due to a decrease in the turnover rate of the protein. These results reinforce the view of 17?-estradiol and E6 as risk factors for the development of cancer through effects on expression and mislocalization of TJ proteins. PMID:23948304

Hernández-Monge, Jesús; Garay, Erika; Raya-Sandino, Arturo; Vargas-Sierra, Orlando; Díaz-Chávez, José; Popoca-Cuaya, Marco; Lambert, Paul F; González-Mariscal, Lorenza; Gariglio, Patricio

2013-10-15

112

Koilocytosis: a cooperative interaction between the human papillomavirus E5 and E6 oncoproteins.  

PubMed

A long-recognized, pathognomonic feature of human papillomavirus (HPV) infection is the appearance of halo or koilocytotic cells in the differentiated layers of the squamous epithelium. These koilocytes are squamous epithelial cells that contain an acentric, hyperchromatic nucleus that is displaced by a large perinuclear vacuole. However, the genesis of the cytoplasmic vacuole has remained unclear, particularly because both HPV DNA replication and virion assembly occur exclusively in the nucleus. In clinical biopsies, koilocytosis is observed in both low- and high-risk HPV infections; therefore, in this study, we demonstrated that the E5 and E6 proteins from both low- and high-risk HPVs cooperate to induce koilocyte formation in human cervical cells in vitro, using both stable and transient assays. Both E5 and E6 also induce koilocytosis in human foreskin keratinocytes but not in primate COS cells. Deletion of the 20 C-terminal amino acids of E5 completely abrogates koilocytosis, whereas a 10-amino acid-deletion mutant retains approximately 50% of its activity. Because the E6 protein from both the low- and high-risk HPVs is capable of potentiating koilocytosis with E5, it is apparent that the targeting of both p53 and PDZ proteins by E6 is not involved. Our data suggest new, cooperative functions for both the E5 and E6 proteins, hinting at additional targets and roles for these oncoproteins in the viral life cycle. PMID:18688031

Krawczyk, Ewa; Suprynowicz, Frank A; Liu, Xuefeng; Dai, Yuhai; Hartmann, Dan P; Hanover, John; Schlegel, Richard

2008-09-01

113

?B-Crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer  

PubMed Central

Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein ?-basic–crystallin (?B-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of ?B-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, ?B-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing ?B-crystallin by RNA interference inhibited these abnormalities. ?B-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by ?B-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing ?B-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that ?B-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.

Moyano, Jose V.; Evans, Joseph R.; Chen, Feng; Lu, Meiling; Werner, Michael E.; Yehiely, Fruma; Diaz, Leslie K.; Turbin, Dmitry; Karaca, Gamze; Wiley, Elizabeth; Nielsen, Torsten O.; Perou, Charles M.; Cryns, Vincent L.

2006-01-01

114

Oct4 Is Required ~E7.5 for Proliferation in the Primitive Streak  

PubMed Central

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ?E6.0–E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ?E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ?E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ?E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

DeVeale, Brian; Brokhman, Irina; Mohseni, Paria; Babak, Tomas; Yoon, Charles; Lin, Anthony; Onishi, Kento; Tomilin, Alexey; Pevny, Larysa; Zandstra, Peter W.; Nagy, Andras; van der Kooy, Derek

2013-01-01

115

High incidence of female reproductive tract cancers in FA-deficient HPV16-transgenic mice correlates with E7's induction of DNA damage response, an activity mediated by E7's inactivation of pocket proteins.  

PubMed

Fanconi anemia (FA) is a rare genetic disorder caused by defects in a DNA damage repair system, the FA pathway. FA patients frequently develop squamous cell carcinoma (SCC) at sites that are associated with human papillomavirus (HPV)-driven cancer including the female reproductive tract. To assess experimentally whether FA deficiency increases susceptibility to HPV-associated cervical/vaginal cancer, we monitored cancer incidence in the female lower reproductive tract of FA-deficient mice expressing HPV16 oncogenes, E6 and/or E7. FA deficiency specifically increased the incidence of cancers in mice expressing E7; but this effect was not observed in mice just expressing E6. We also observed that E7, but not E6, induced DNA damage as scored by induction of ?-H2AX and 53BP1 (p53 binding protein 1) nuclear foci, and this induction was heightened in FA-deficient tissue. Finally, we discovered that this induction of DNA damage responses was recapitulated in mice deficient in expression of 'pocket' proteins, pRb, p107 and p130, which are established targets of E7. Our findings support the hypothesis that E7 induces cancer by causing DNA damage at least in part through the inactivation of pocket proteins. This hypothesis explains why a deficiency in DNA damage repair would increase susceptibility to E7-driven cancer. PMID:24013229

Park, J W; Shin, M-K; Lambert, P F

2014-06-26

116

Codon Modified Human Papillomavirus Type 16 E7 DNA Vaccine Enhances Cytotoxic T-Lymphocyte Induction and Anti-tumour Activity  

Microsoft Academic Search

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells

Wen Jun Liu; Fengguang Gao; Kong Nan Zhao; Weiming Zhao; Germain J. G. Fernando; Ranjeny Thomas; Ian H. Frazer

2002-01-01

117

MUC1 oncoprotein is a druggable target in human prostate cancer cells.  

PubMed

Human prostate cancers are dependent on the androgen receptor for their progression. The MUC1 heterodimeric oncoprotein is aberrantly overexpressed in prostate cancers; however, it is not known if MUC1 is of functional importance to these tumors. To assess dependence on MUC1, we synthesized an inhibitor, designated GO-201, which interacts directly with the MUC1-C subunit at its oligomerization domain. Treatment of MUC1-positive DU145 and PC3 prostate cancer cells with GO-201, and not an altered version, resulted in inhibition of proliferation. GO-201 also induced necrotic cell death that was associated with increases in reactive oxygen species, loss of mitochondrial transmembrane potential, and depletion of ATP. By contrast, GO-201 had no effect against MUC1-negative LNCaP, CWR22Rv1, and MDA-PCa-2b prostate cancer cells. Significantly, GO-201 treatment of DU145 and PC3 xenografts growing in nude mice resulted in complete tumor regression and prolonged lack of recurrence. These findings indicate that certain prostate cancer cells are dependent on MUC1-C for growth and survival and that directly targeting MUC1-C results in their death in vitro and in tumor models. PMID:19887552

Joshi, Maya Datt; Ahmad, Rehan; Yin, Li; Raina, Deepak; Rajabi, Hasan; Bubley, Glenn; Kharbanda, Surender; Kufe, Donald

2009-11-01

118

MUC1 ONCOPROTEIN IS A DRUGGABLE TARGET IN HUMAN PROSTATE CANCER CELLS  

PubMed Central

Human prostate cancers are dependent on the androgen receptor for their progression. The MUC1 heterodimeric oncoprotein is aberrantly overexpressed in prostate cancers; however, it is not known if MUC1 is of functional importance to these tumors. To assess dependence on MUC1, we synthesized an inhibitor, designated GO-201, that interacts directly with the MUC1-C subunit at its oligomerization domain. Treatment of MUC1-positive DU145 and PC3 prostate cancer cells with GO-201, and not an altered version, resulted in inhibition of proliferation. GO-201 also induced necrotic cell death that was associated with increases in reactive oxygen species, loss of mitochondrial transmembrane potential and depletion of ATP. By contrast, GO-201 had no effect against MUC1-negative LNCaP, CWR22Rv1 and MDA PCa 2b prostate cancer cells. Significantly, GO-201 treatment of DU145 and PC3 xenografts growing in nude mice resulted in complete tumor regression and prolonged lack of recurrence. These findings indicate that certain prostate cancer cells are dependent on MUC1-C for growth and survival and that directly targeting MUC1-C results in their death in vitro and in tumor models.

Joshi, Maya Datt; Ahmad, Rehan; Yin, Li; Raina, Deepak; Rajabi, Hasan; Bubley, Glenn; Kharbanda, Surender; Kufe, Donald

2009-01-01

119

Ha-ras and ?-catenin oncoproteins orchestrate metabolic programs in mouse liver tumors.  

PubMed

The process of hepatocarcinogenesis in the diethylnitrosamine (DEN) initiation/phenobarbital (PB) promotion mouse model involves the selective clonal outgrowth of cells harboring oncogene mutations in Ctnnb1, while spontaneous or DEN-only-induced tumors are often Ha-ras- or B-raf-mutated. The molecular mechanisms and pathways underlying these different tumor sub-types are not well characterized. Their identification may help identify markers for xenobiotic promoted versus spontaneously occurring liver tumors. Here, we have characterized mouse liver tumors harboring either Ctnnb1 or Ha-ras mutations via integrated molecular profiling at the transcriptional, translational and post-translational levels. In addition, metabolites of the intermediary metabolism were quantified by high resolution (1) H magic angle nuclear magnetic resonance. We have identified tumor genotype-specific differences in mRNA and miRNA expression, protein levels, post-translational modifications, and metabolite levels that facilitate the molecular and biochemical stratification of tumor phenotypes. Bioinformatic integration of these data at the pathway level led to novel insights into tumor genotype-specific aberrant cell signaling and in particular to a better understanding of alterations in pathways of the cell intermediary metabolism, which are driven by the constitutive activation of the ?-Catenin and Ha-ras oncoproteins in tumors of the two genotypes. PMID:24535843

Unterberger, Elif B; Eichner, Johannes; Wrzodek, Clemens; Lempiäinen, Harri; Luisier, Raphaëlle; Terranova, Rémi; Metzger, Ute; Plummer, Simon; Knorpp, Thomas; Braeuning, Albert; Moggs, Jonathan; Templin, Markus F; Honndorf, Valerie; Piotto, Martial; Zell, Andreas; Schwarz, Michael

2014-10-01

120

Apoptosis Inhibition by the Human DEK Oncoprotein Involves Interference with p53 Functions  

Microsoft Academic Search

The DEK proto-oncogene has been associated with human carcinogenesis—either as a fusion with the CAN nucleoporin protein or when transcriptionally upregulated. Mechanisms of intracellular DEK functions, however, have remained relatively unexplored. We have recently demonstrated that DEK expression is induced by the high-risk human papillomavirus (HPV) E7 protein in a manner which is dependent upon retinoblastoma protein function and have

Trisha M. Wise-Draper; Hillary V. Allen; Elizabeth E. Jones; Kristen B. Habash; Hiroshi Matsuo; Susanne I. Wells

2006-01-01

121

The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein.  

PubMed

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal/anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal/anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 3T3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin-sensitive compartment, most likely the cell surface. In contrast, NIH 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin. PMID:8227125

Xu, Y F; Meyer, A N; Webster, M K; Lee, B A; Donoghue, D J

1993-11-01

122

SA-4-1BBL as the immunomodulatory component of a HPV-16 E7 protein based vaccine shows robust therapeutic efficacy in a mouse cervical cancer model  

PubMed Central

Cervical cancer is the leading cause of cancer-related deaths among women worldwide. Current prophylactic vaccines based on HPV (Human papillomavirus) late gene protein, L1 are ineffective in therapeutic settings. Therefore, there is an acute need for the development of therapeutic vaccines for HPV associated cancers. The HPV E7 oncoprotein is expressed in cervical cancer and has been associated with the cellular transformation and maintenance of the transformed phenotype. As such, E7 protein represents an ideal target for the development of therapeutic subunit vaccines against cervical cancer. However, the low antigenicity of this protein may require potent adjuvants for therapeutic efficacy. We recently generated a novel chimeric form of the 4-1BBL costimulatory molecule engineered with core streptavidin (SA-4-1BBL) and demonstrated its safe and pleiotropic effects on various cells of the immune system. We herein tested the utility of SA-4-1BBL as the immunomodulatory component of HPV-16 E7 recombinant protein based therapeutic vaccine in the E7 expressing TC-1 tumor as a model of cervical cancer in mice. A single subcutaneous vaccination was effective in eradicating established tumors in approximately 70% of mice. The therapeutic efficacy of the vaccine was associated with robust primary and memory CD4+ and CD8+ T cell responses, Th1 cytokine response, infiltration of CD4+ and CD8+ T cells into the tumor, and enhanced NK cell killing. Importantly, NK cells played an important role in vaccine mediated therapy since their physical depletion compromised vaccine efficacy. Collectively, these data demonstrate the utility of SA-4-1BBL as a new class of multifunctional immunomodulator for the development of therapeutic vaccines against cancer and chronic infections.

Sharma, Rajesh K.; Srivastava, Abhishek K.; Yolcu, Esma S.; MacLeod, Kathryn J.; Schabowsky, Rich-Henry; Madireddi, Shravan; Shirwan, Haval

2010-01-01

123

Molecular genetic characterization of p53 mutated oropharyngeal squamous cell carcinoma cells transformed with human papillomavirus E6 and E7 oncogenes  

PubMed Central

Patients with HPV-positive oropharyngeal cancer show better tumor response to radiation or chemotherapy than patients with HPV-negative cancer. HPV oncoprotein E6 binds and degrades a typically wild-type p53 protein product. However, HPV16 infection and p53 mutation infrequently coexist in a subset of HNSCCs. The purpose of this study was to investigate the mechanisms through which tumor biology and molecular genetic mechanisms change when two HPV-negative, p53-mutated oropharyngeal cell lines (YD8, non-disruptive p53 mutation; YD10B, disruptive p53 mutation) derived from patients with a history of heavy smoking are transfected with HPV E6 and E7 oncogenes in vitro. Transfection with HPV E6 and E7 oncogenes in YD8, reduced the abundance of proteins encoded by tumor suppressor genes, such as p-p53 and p-Rb. Cell proliferative activity was increased in the cells transfected with E6E7 compared to cells transfected with vector alone (P=0.09), whereas the invasiveness of E6E7-transfected cells was significantly reduced (P=0.02). cDNA microarray of the transfected cells with E6E7 showed significant changes in mRNA expression in several signaling pathways, including focal adhesion, JAK-STAT signaling pathway, cell cycle and p53 signaling pathway. Regarding the qPCR array for the p53 signaling pathway, the mRNA expression of STAT1 was remarkably upregulated by 6.47-fold (P<0.05); in contrast, IGF-1R was significantly downregulated by 2.40-fold in the YD8-vector compared toYD8-E6E7 (P<0.01). Finally, data collected from these two array experiments enabled us to select two genes, STAT1 and IGF-1R, for further study. In immunohistochemical study, nuclear STAT1 expression was slightly higher in HPV-positive compared to HPV-negative oropharyngeal tumors (P=0.18); however, cytoplasmic STAT1 was significantly lower in HPV-positive cases (P=0.03). IGF-1R expression levels were remarkably lower in HPV-positive compared to HPV-negative cases (P=0.01). Our data suggest that upregulated STAT1 and interferon signals by HPV16 E6 and E7 genes may play a major role in the relatively favorable prognosis for HPV-positive oropharyngeal squamous cell carcinoma cases with non-disruptive p53 mutations.

OH, JI-EUN; KIM, JEONG-OH; SHIN, JUNG-YOUNG; ZHANG, XIANG-HUA; WON, HYE-SUNG; CHUN, SANG-HOON; JUNG, CHAN-KWON; PARK, WON-SANG; NAM, SUK-WOO; EUN, JUNG-WOO; KANG, JIN-HYOUNG

2013-01-01

124

Groups of type E_7 over arbitrary fields  

Microsoft Academic Search

Freudenthal triple systems come in two flavors, degenerate and nondegenerate.\\u000aThe best criterion for distinguishing between the two which is available in the\\u000aliterature is by descent. We provide an identity which is satisfied only by\\u000anondegenerate triple systems. We then use this to define algebraic structures\\u000awhose automorphism groups produce all adjoint algebraic groups of type E_7 over\\u000aan

R. Skip Garibaldi

1998-01-01

125

A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers  

PubMed Central

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFN? ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS3 Poisson detection mass spectrometry. Only one epitope (E711–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E711–20), previously used as a vaccine candidate, was neither detected by MS3 on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.

Riemer, Angelika B.; Keskin, Derin B.; Zhang, Guanglan; Handley, Maris; Anderson, Karen S.; Brusic, Vladimir; Reinhold, Bruce; Reinherz, Ellis L.

2010-01-01

126

Serum oncoproteins and growth factors in asbestosis and silicosis patients.  

PubMed

Levels of 9 different oncoproteins and growth factors were assayed by immunoblotting with monoclonal antibodies in 91 serum samples collected between March 1983 and August 1987 from 46 pneumoconiosis patients (36 asbestosis, 10 silicosis) at high risk for the development of cancer. Follow-up of these patients through June 1991 showed that 18 had developed cancer (11 lung, 2 pleural mesothelioma, 2 transitional-cell carcinomas of the urinary bladder, 1 osteosarcoma, 1 non-Hodgkin's lymphoma, 1 adenocarcinoma of the gallbladder). Increased serum levels of ras oncogene-related protein (p21) were found in 7 of the 18 patients who developed cancer (5 lung, 2 pleural mesothelioma) versus 2 of the 28 patients without cancer, a statistically significant difference (p = 0.012). In addition, 6 of the 7 p21-positive cancer cases had positive serum samples prior to clinical diagnosis of disease (average = 16.3 months, range = 3-26 months prior to diagnosis), suggesting that elevated serum p21 levels may be a useful marker for earlier detection in a significant percentage of respiratory malignancies. Finally, elevated serum levels of PDGF-related protein were detected significantly more frequently in advanced pneumoconiosis cases (ILO radiographic classification of 2/1 or greater) than in less advanced cases (80% vs. 41.9%; p = 0.016), and there was a tendency for these PDGF-positive patients to have progression of their disease (68.2% vs. 41.7%; p = 0.065), suggesting that elevated serum PDGF levels may be a marker for the development of severe and progressive pneumoconioses. PMID:1313398

Brandt-Rauf, P W; Smith, S; Hemminki, K; Koskinen, H; Vainio, H; Niman, H; Ford, J

1992-04-01

127

Targeting the MUC1-C oncoprotein inhibits self-renewal capacity of breast cancer cells.  

PubMed

The capacity of breast cancer cells to form mammospheres in non-adherent serum-free culture is used as a functional characteristic of the self-renewing stem-like cell population. The present studies demonstrate that silencing expression of the MUC1-C oncoprotein inhibits growth of luminal MCF-7 and HER2-overexpressing SKBR3 breast cancer cells as mammospheres. We also show that triple-negative MDA-MB-468 breast cancer cells are dependent on MUC1-C for growth as mammospheres and tumor xenografts. Similar results were obtained when MUC1-C function was inhibited by expression of a MUC1-C(CQC?AQA) mutant. Moreover, treatment with the MUC1-C inhibitor GO-203, a cell penetrating peptide that binds to the MUC1-C cytoplasmic domain and blocks MUC1-C function, confirmed the importance of this target for self-renewal. The mechanistic basis for these findings is supported by the demonstration that MUC1-C activates NF-?B, occupies the IL-8 promoter with NF-?B, and induces IL-8 transcription. MUC1-C also induces NF-?B-dependent expression of the IL-8 receptor, CXCR1. In concert with these results, targeting MUC1-C with GO-203 suppresses IL-8/CXCR1 expression and disrupts the formation of established mammospheres. Our findings indicate that MUC1-C contributes to the self-renewal of breast cancer cells by activating the NF-?B?IL-8/CXCR1 pathway and that targeting MUC1-C represents a potential approach for the treatment of this population. PMID:24770886

Alam, Maroof; Rajabi, Hasan; Ahmad, Rehan; Jin, Caining; Kufe, Donald

2014-05-15

128

Targeting the MUC1-C oncoprotein inhibits self-renewal capacity of breast cancer cells  

PubMed Central

The capacity of breast cancer cells to form mammospheres in non-adherent serum-free culture is used as a functional characteristic of the self-renewing stem-like cell population. The present studies demonstrate that silencing expression of the MUC1-C oncoprotein inhibits growth of luminal MCF-7 and HER2-overexpressing SKBR3 breast cancer cells as mammospheres. We also show that triple-negative MDA-MB-468 breast cancer cells are dependent on MUC1-C for growth as mammospheres and tumor xenografts. Similar results were obtained when MUC1-C function was inhibited by expression of a MUC1-C(CQC?AQA) mutant. Moreover, treatment with the MUC1-C inhibitor GO-203, a cell penetrating peptide that binds to the MUC1-C cytoplasmic domain and blocks MUC1-C function, confirmed the importance of this target for self-renewal. The mechanistic basis for these findings is supported by the demonstration that MUC1-C activates NF-?B, occupies the IL-8 promoter with NF-?B, and induces IL-8 transcription. MUC1-C also induces NF-?B-dependent expression of the IL-8 receptor, CXCR1. In concert with these results, targeting MUC1-C with GO-203 suppresses IL-8/CXCR1 expression and disrupts the formation of established mammospheres. Our findings indicate that MUC1-C contributes to the self-renewal of breast cancer cells by activating the NF-?B?IL-8/CXCR1 pathway and that targeting MUC1-C represents a potential approach for the treatment of this population.

Jin, Caining; Kufe, Donald

2014-01-01

129

Induction of tetrasomy by human papillomavirus type 16 E7 protein is independent of pRb binding and disruption of differentiation  

Microsoft Academic Search

We have demonstrated previously that high-risk human papillomaviruses (HPVs) induce tetrasomy in low-grade squamous intraepithelial lesions of the cervix. In this study we show that the E6 and E7 genes of high-risk HPV-16, but not those of low-risk HPV-6, are independently able to induce tetrasomy when constitutively expressed in proliferating monolayer cultures of primary human keratinocytes. Of seven HPV-16 E7

M H Lewis; C S Herrington

2004-01-01

130

Positive selection on a bacterial oncoprotein associated with gastric cancer  

PubMed Central

Background Helicobacter pylori is a vertically inherited gut commensal that is carcinogenic if it possesses the cag pathogenicity island (cag PaI); infection with H.pylori is the major risk factor for gastric cancer, the second leading cause of death from cancer worldwide (WHO). The cag PaI locus encodes the cagA gene, whose protein product is injected into stomach epithelial cells via a Type IV secretion system, also encoded by the cag PaI. Once there, the cagA protein binds to various cellular proteins, resulting in dysregulation of cell division and carcinogenesis. For this reason, cagA may be described as an oncoprotein. A clear understanding of the mechanism of action of cagA and its benefit to the bacteria is lacking. Results Here, we reveal that the cagA gene displays strong signatures of positive selection in bacteria isolated from amerindian populations, using the Ka/Ks ratio. Weaker signatures are also detected in the gene from bacteria isolated from asian populations, using the Ka/Ks ratio and the more sensitive branches-sites model of the PAML package. When the cagA gene isolated from amerindian populations was examined in more detail it was found that the region under positive selection contains the EPIYA domains, which are known to modulate the carcinogenicity of the gene. This means that the carcinogenicity modulating region of the gene is undergoing adaptation. The results are discussed in relation to the high incidences of stomach cancer in some latin american and asian populations. Conclusion Positive selection on cagA indicates antagonistic coevolution between host and bacteria, which appears paradoxical given that cagA is detrimental to the human host upon which the bacteria depends. This suggests several non-exclusive possibilities; that gastric cancer has not been a major selective pressure on human populations, that cagA has an undetermined benefit to the human host, or that horizontal transmission of H.pylori between hosts has been more important in the evolution of H.pylori than previously recognized, reducing the selective pressure to lower the pathogenicity of the bacteria. The different patterns of adaptation of the gene in different human populations indicates that there are population specific differences in the human gut environment - due either to differences in host genetics or diet and other lifestyle features.

2011-01-01

131

Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein  

PubMed Central

Infection by oncogenic viruses is a frequent cause for tumor formation as observed in cervical cancer. Viral oncoproteins cause inactivation of p53 function and false transcriptional regulation of central cell cycle genes. Here we analyze the regulation of Plk4, serving as an example of many cell cycle- and p53-regulated genes. Cell cycle genes are often repressed via CDE and CHR elements in their promoters and activated by NF-Y binding to CCAAT-boxes. In contrast, general activation of Plk4 depends on NRF1 and CRE sites. Bioinformatic analyses imply that NRF1 and CRE are central elements of the transcriptional network controlling cell cycle genes. We identify CDE and CHR sites in the Plk4 promoter, which are necessary for binding of the DREAM (DP, RB-like, E2F4 and MuvB) complex and for mediating repression in G0/G1. When cells progress to G2 and mitosis, DREAM is replaced by the MMB (Myb-MuvB) complex that only requires the CHR element for binding. Plk4 expression is downregulated by the p53-p21WAF1/CIP1-DREAM signaling pathway through the CDE and CHR sites. Cell cycle- and p53-dependent repression is abrogated by HPV E7 oncoprotein. Together with genome-wide analyses our results imply that many cell cycle genes upregulated in tumors by viral infection are bound by DREAM through CDE/CHR sites.

Fischer, Martin; Quaas, Marianne; Wintsche, Axel; Muller, Gerd A.; Engeland, Kurt

2014-01-01

132

Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein.  

PubMed

Infection by oncogenic viruses is a frequent cause for tumor formation as observed in cervical cancer. Viral oncoproteins cause inactivation of p53 function and false transcriptional regulation of central cell cycle genes. Here we analyze the regulation of Plk4, serving as an example of many cell cycle- and p53-regulated genes. Cell cycle genes are often repressed via CDE and CHR elements in their promoters and activated by NF-Y binding to CCAAT-boxes. In contrast, general activation of Plk4 depends on NRF1 and CRE sites. Bioinformatic analyses imply that NRF1 and CRE are central elements of the transcriptional network controlling cell cycle genes. We identify CDE and CHR sites in the Plk4 promoter, which are necessary for binding of the DREAM (DP, RB-like, E2F4 and MuvB) complex and for mediating repression in G0/G1. When cells progress to G2 and mitosis, DREAM is replaced by the MMB (Myb-MuvB) complex that only requires the CHR element for binding. Plk4 expression is downregulated by the p53-p21(WAF1/CIP1)-DREAM signaling pathway through the CDE and CHR sites. Cell cycle- and p53-dependent repression is abrogated by HPV E7 oncoprotein. Together with genome-wide analyses our results imply that many cell cycle genes upregulated in tumors by viral infection are bound by DREAM through CDE/CHR sites. PMID:24071582

Fischer, Martin; Quaas, Marianne; Wintsche, Axel; Müller, Gerd A; Engeland, Kurt

2014-01-01

133

Identification and Characterization of Mechanism of Action of P61-E7, a Novel Phosphine Catalysis-Based Inhibitor of Geranylgeranyltransferase-I  

PubMed Central

Small molecule inhibitors of protein geranylgeranyltransferase-I (GGTase-I) provide a promising type of anticancer drugs. Here, we first report the identification of a novel tetrahydropyridine scaffold compound, P61-E7, and define effects of this compound on pancreatic cancer cells. P61-E7 was identified from a library of allenoate-derived compounds made through phosphine-catalyzed annulation reactions. P61-E7 inhibits protein geranylgeranylation and blocks membrane association of geranylgeranylated proteins. P61-E7 is effective at inhibiting both cell proliferation and cell cycle progression, and it induces high p21CIP1/WAF1 level in human cancer cells. P61-E7 also increases p27Kip1 protein level and inhibits phosphorylation of p27Kip1 on Thr187. We also report that P61-E7 treatment of Panc-1 cells causes cell rounding, disrupts actin cytoskeleton organization, abolishes focal adhesion assembly and inhibits anchorage independent growth. Because the cellular effects observed pointed to the involvement of RhoA, a geranylgeranylated small GTPase protein shown to influence a number of cellular processes including actin stress fiber organization, cell adhesion and cell proliferation, we have evaluated the significance of the inhibition of RhoA geranylgeranylation on the cellular effects of inhibitors of GGTase-I (GGTIs). Stable expression of farnesylated RhoA mutant (RhoA-F) results in partial resistance to the anti-proliferative effect of P61-E7 and prevents induction of p21CIP1/WAF1 and p27Kip1 by P61-E7 in Panc-1 cells. Moreover, stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation caused by P61-E7. Taken together, these findings suggest that P61-E7 is a promising GGTI compound and that RhoA is an important target of P61-E7 in Panc-1 pancreatic cancer cells.

Chan, Lai N.; Fiji, Hannah D. G.; Watanabe, Masaru; Kwon, Ohyun; Tamanoi, Fuyuhiko

2011-01-01

134

Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation  

SciTech Connect

Highlights: •Hominoid-specific oncogene TBC1D3 is targeted to plasma membrane by palmitoylation. •TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. •TBC1D3 palmitoylation governs growth factors-induced TBC1D3 degradation. •Post-translational modifications may regulate oncogenic properties of TBC1D3. -- Abstract: Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States)] [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Su, Xiong [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States)] [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Liu, Jialiu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States)] [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States); Sundaresan, Sinju [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States)] [Department of Internal Medicine, Center for Human Nutrition Washington University School of Medicine, St. Louis, MO 63110 (United States); Stahl, Philip D., E-mail: pstahl@wustl.edu [Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110 (United States)

2013-05-03

135

Oncoprotein Bmi-1 Renders Apoptotic Resistance to Glioma Cells through Activation of the IKK-Nuclear Factor-?B Pathway  

PubMed Central

One of the features of malignant gliomas is their deviant resistance to cellular apoptosis induced by cytotoxic reagents. Bmi-1, an oncoprotein, has been linked to oncogenesis and cancer progression in various types of human cancers including gliomas. However, the mechanisms underlying Bmi-1 antiapoptotic function remain largely unknown. In this study, we report that Bmi-1 renders apoptotic resistance to glioma cells through nuclear factor-?B (NF-?B). In glioma cells, ectopic expression of Bmi-1 significantly inhibits doxorubicin-, BCNU-, or UV irradiation- induced apoptosis through reduction of activated caspase-3 and PARP, and induction of Bcl-XL. Cellular depletion of Bmi-1 enhances the sensitivity of glioma cells to apoptosis induced by doxorubicin, BCNU, or UV irradiation. Bmi-1 activates NF-?B through stimulation of I?B phosphorylation, nuclear translocation, and transcriptional activity of NF-?B and expression of downstream genes of NF-?B including caspase-3, PARP, Bcl-XL, and c-Myc. Inhibition of the IKK-NF-?B pathway abrogates the antiapoptotic effect of Bmi-1 on glioma cells. In high-grade gliomas, Bmi-1 and NF-?B are co-expressed in the cell nucleus. Up-regulation of Bmi-1 also correlates with tumor progression and poor survival of patients with gliomas. Together, our data demonstrate that Bmi-1 bestows apoptotic resistance to glioma cells through the IKK-NF-?B pathway and suggest Bmi-1 as a useful indicator for glioma prognosis.

Li, Jun; Gong, Li-Yun; Song, Li-Bing; Jiang, Li-Li; Liu, Li-Ping; Wu, Jueheng; Yuan, Jie; Cai, Jun-Chao; He, Mian; Wang, Lan; Zeng, Musheng; Cheng, Shi-Yuan; Li, Mengfeng

2010-01-01

136

Human Papillomavirus Type 16 E6/E7-Specific Cytotoxic T Lymphocytes for Adoptive Immunotherapy of HPV-Associated Malignancies  

PubMed Central

Vaccines prevent HPV-associated cancer but, although these tumors express foreign, viral antigens (E6 and E7 proteins), they have little benefit in established malignancies, likely due to negative environmental cues that block tumor recognition and induce T cell anergy in vivo. We postulated that we could identify mechanisms by which ex vivo stimulation of T cells could reactivate and expand tumor-directed T-cell lines from HPV-positive cancer patients for subsequent adoptive immunotherapy. A total of 68 patients with HPV-associated cancers were studied. Peripheral blood T cells were stimulated with monocyte-derived dendritic cells loaded with pepmixes (peptide libraries of 15-mers overlapping by 11 amino-acids) spanning E6/E7, in the presence or absence of specific accessory cytokines. The resulting T-cell lines were further expanded with pepmix-loaded activated B-cell blasts. IFN? release and cytotoxic responses to E6/E7 were assessed. We successfully reactivated and expanded (>1200-fold) E6/E7-specific T cells from 8/16 cervical and 33/52 oropharyngeal cancer patients. The presence of the cytokines IL-6, -7, -12 and -15 is critical for this process. These T cell lines possess the desirable characteristics of polyclonality, multiple T-cell subset representation (including the memory compartment) and a TH1 bias, and may eliminate E6/E7-positive targets. In conclusion, we have shown it is possible to robustly generate HPV16 E6/E7-directed T-cell lines from patients with HPV16-associated cancers. Because our technique is scalable and good-manufacturing-procedures compliant, these lines could be used for adoptive cellular immunotherapy of patients with HPV16-positive cancers.

Ramos, Carlos A.; Narala, Neeharika; Vyas, Gayatri M.; Leen, Ann M.; Gerdemann, Ulrike; Sturgis, Erich M.; Anderson, Matthew L.; Savoldo, Barbara; Heslop, Helen E.; Brenner, Malcolm K.; Rooney, Cliona M.

2012-01-01

137

Characterization of the gene for a proliferation-related phosphoprotein (oncoprotein 18) expressed in high amounts in acute leukemia.  

PubMed

The oncoprotein 18 (Op18) gene encodes a proliferation-related cytosolic phosphoprotein, which is induced in normal lymphocytes following mitogenic stimulation. Studies of the Op18 gene are of particular interest because of the proposed role of Op18 protein in signal transduction and because of its occurrence in markedly increased amounts in acute leukemia cells. We have recently reported the cloning and sequencing of two cDNA clones for Op18 (1 and 1.5 kilobases). Both clones code for the same 149-amino acid polypeptide; however, they differ in their 3'-region as a result of alternative polyadenylation. We report here the sequencing of the Op18 gene and describe its expression in leukemia. The Op18 gene, which is 6.3 kilobases in length, is comprised of five exons and four introns and exhibits features that are common to other genes involved in cellular growth and proliferation. The increase in Op18 polypeptide in leukemia is associated with increased RNA transcription without gene amplification or rearrangement. Treatment of K562 leukemia cell line with hemin that induces terminal differentiation resulted in decreased expression of Op18. Our findings suggest that the high amount of Op18 protein in acute leukemia results from increased expression of a structurally unaltered gene. PMID:1917919

Melhem, R F; Zhu, X X; Hailat, N; Strahler, J R; Hanash, S M

1991-09-25

138

Epstein - Barr Virus Transforming Protein LMP-1 Alters B Cells Gene Expression by Promoting Accumulation of the Oncoprotein ?Np73?  

PubMed Central

Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ?Np73?, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ?Np73? promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ?Np73? mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ?Np73? accumulation. The recruitment of p73 to the ?Np73? promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ?Np73? expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ?Np73? down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.

Accardi, Rosita; Fathallah, Ikbal; Gruffat, Henri; Mariggio, Giuseppe; Le Calvez-Kelm, Florence; Voegele, Catherine; Bartosch, Birke; Hernandez-Vargas, Hector; McKay, James; Sylla, Bakary S.; Manet, Evelyne; Tommasino, Massimo

2013-01-01

139

BCL6 Oncoprotein in Breast Cancer: Loss of Expression in Disease Progression  

Microsoft Academic Search

Objective: To investigate the biological role of BCL-6 oncoprotein in breast cancer disease progression (recurrence and metastasis). Methods: The series consisted of 93 consecutive female patients with primary breast cancer and median follow-up of 10 years. BCL-6 expression was assessed in vivo by immunohistochemistry and real-time PCR. Breast cancer cell lines and some metastasis-related genes (CXCR4, Itg?-3 and FLT-1) were

António E. Pinto; Saudade André; Giovani Silva; Sara Vieira; Ana C. Santos; Sérgio Dias; Jorge Soares

2009-01-01

140

Circulating oncoproteins HER2\\/neu, EGFR and CAIX (MN) as novel cancer biomarkers  

Microsoft Academic Search

Pharmaceutical companies have developed targeted therapies such as trastuzumab and lapatinib for human epidermal growth factor receptor (HER)2\\/neu-positive tumors, while others have developed antiepidermal growth factor receptor (EGFR) therapies, such as tarceva and erbitux for EGFR-positive tumors. A drug called rencarex is targeted to an oncoprotein designated carbonic anhydrase IX (CAIX), which is being evaluated in renal cell carcinoma patients.

Walter P Carney

2007-01-01

141

Structure of the MDM2 Oncoprotein Bound to the p53 Tumor Suppressor Transactivation Domain  

Microsoft Academic Search

The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor in that it can bind the transactivation domain of p53 and downregulate its ability to activate transcription. In certain cancers, MDM2 amplification is a common event and contributes to the inactivation of p53. The crystal structure of the 109-residue amino-terminal domain of MDM2 bound to a 15-residue transactivation

Paul H. Kussie; Svetlana Gorina; Vincent Marechal; Brian Elenbaas; Jacque Moreau; Arnold J. Levine; Nikola P. Pavletich

1996-01-01

142

The Bovine Papillomavirus E6 Oncoprotein Interacts with Paxillin and Disrupts the Actin Cytoskeleton  

Microsoft Academic Search

The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) has been shown to transform cells through a p53-independent pathway, but its transforming mechanism is unknown. Here we demonstrate in vitro and in vivo interactions between BPV-1 E6 and the focal adhesion protein paxillin. The ability of BPV-1 E6 to complex with paxillin correlated with its ability to transform; E6 mutant

Xiao Tong; Peter M. Howley

1997-01-01

143

Mechanism of Fas Signaling Regulation by Human Herpesvirus 8 K1 Oncoprotein  

PubMed Central

Background Herpesvirus 8 (HHV-8) oncoprotein K1 is linked to lymphoproliferation and suppression of apoptosis mediated by the Fas death receptor. Expression of K1 in transgenic mice induces accumulation of lymphoid tissue cells and lymphoma. Methods To examine how K1 and Fas interact to suppress apoptosis, K1–Fas binding was studied in human embryonic kidney (HEK) and lymphoma (BJAB) cells that expressed wild-type K1 or a K1 Ig domain deletion mutant and were treated with Fas ligand (FasL) or an agonistic Fas antibody, using immunoprecipitation and Western blot analysis. Cleavage of caspase-3 and apoptosis was compared in liver samples from mice that were transfected with empty vector vs with plasmids expressing wild-type K1 or a K1 Ig deletion mutant and treated with agonistic Fas antibody for 7 hours. These studies used immunohistochemical staining and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay. All statistical tests were two-sided. Results Immunoprecipitation and Western blot analysis of transfected HEK and BJAB cells revealed that wild-type K1 but not Ig-deleted K1 binds to Fas and prevents Fas activation by FasL or by an agonistic Fas antibody. More mice that were transfected with wild-type K1 (7 of 10) than mice transfected with empty vector (3 of 13) or the K1 Ig deletion mutant (0 of 6) survived treatment with the agonistic Fas antibody. Compared with vector-transfected mice, livers of wild-type K1-transfected mice contained fewer cells in which caspase-3 was cleaved (87.6% vs 58.0%, difference =?29.6%, 95% confidence interval [CI] = 19.2% to 40.0%; P = .003) and fewer apoptotic cells (83.7% vs 34.2%, difference = 49.5%, 95% CI = 39.8% to 59.2%; P = .003). Conclusions K1 blocks Fas signaling by directly binding to Fas through the Ig-like domain of K1 and preventing binding of FasL. The relative resistance of cancer cells to Fas-mediated apoptosis may be due to the inhibition of Fas by Ig domain–containing proteins.

Berkova, Zuzana; Wang, Shu; Wise, Jillian F.; Maeng, Hoyoung; Ji, Yuan

2009-01-01

144

The splicing factor SRSF6 is amplified and is an oncoprotein in lung and colon cancers.  

PubMed

An increasing body of evidence connects alterations in the process of alternative splicing with cancer development and progression. However, a direct role of splicing factors as drivers of cancer development is mostly unknown. We analysed the gene copy number of several splicing factors in colon and lung tumours, and found that the gene encoding for the splicing factor SRSF6 is amplified and over-expressed in these cancers. Moreover, over-expression of SRSF6 in immortal lung epithelial cells enhanced proliferation, protected them from chemotherapy-induced cell death and converted them to be tumourigenic in mice. In contrast, knock-down of SRSF6 in lung and colon cancer cell lines inhibited their tumourigenic abilities. SRSF6 up- or down-regulation altered the splicing of several tumour suppressors and oncogenes to generate the oncogenic isoforms and reduce the tumour-suppressive isoforms. Our data suggest that the splicing factor SRSF6 is an oncoprotein that regulates the proliferation and survival of lung and colon cancer cells. PMID:23132731

Cohen-Eliav, Michal; Golan-Gerstl, Regina; Siegfried, Zahava; Andersen, Claus L; Thorsen, Kasper; Ørntoft, Torben F; Mu, David; Karni, Rotem

2013-03-01

145

Colocalization of the p185HER2 oncoprotein and integrin alpha 6 beta 4 in Calu-3 lung carcinoma cells.  

PubMed

Anti-p185HER2 monoclonal antibodies often show intense reactivity with the basement membrane of tumor cells that overexpress the HER2/neu gene product (p185HER2). To evaluate a possible interaction between p185HER2 and adhesion molecules or their receptors, the polarity of p185HER2 was tested in lung carcinoma cell line Calu-3, which overexpresses this protein, in cultures grown as confluent monolayers or as aggregates. MAb immunostaining patterns indicated that p185HER2 is concentrated on the baso-lateral membrane of cells and that it colocalizes with the integrin alpha 6 beta 4 at the cell-cell junctions where laminin is also found. The same membrane region showed intense reactivity with antiphosphotyrosine antibodies. Furthermore, integrin clustering induced by the specific antibody was accompanied by the clustering of p185HER2, as indicated by immunoelectron microscopy, and by a subsequent increase in p185HER2 tyrosine phosphorylation. Treatment with exogenous laminin also resulted in increased basal levels of p185HER2 phosphorylation. These data suggest a physical interaction between the integrin and the oncoprotein that might be functionally relevant in directly controlling the tyrosine phosphorylation of the catalytic domain of p185HER2. PMID:7962174

Campiglio, M; Tagliabue, E; Srinivas, U; Pellegrini, R; Martignone, S; Ménard, S; Colnaghi, M I; Lombardi, L; Marchisio, P C

1994-08-01

146

Retroviral oncoprotein Tax deregulates NF-?B by activating Tak1 and mediating the physical association of Tak1-IKK  

PubMed Central

The Tax oncoprotein of human T-cell leukaemia virus type I (HTLV-I) persistently activates nuclear factor-?B (NF-?B), which is required for HTLV-I-mediated T-cell transformation. Tax activates NF-?B by stimulating the activity of I?B kinase (IKK), but the underlying mechanism remains elusive. Here, we show that Tax functions as an intracellular stimulator of an IKK-activating kinase, Tak1 (TGF-?-activating kinase 1). In addition, Tax physically interacts with Tak1 and mediates the recruitment of IKK to Tak1. In HTLV-I-infected T cells, Tak1 is constitutively activated and complexed with both Tax and IKK. We provide genetic evidence that Tak1 is essential for Tax-induced IKK activation. Furthermore, unlike cellular stimuli, the Tax-specific NF-?B signalling does not require the ubiquitin-binding function of IKK?. These findings show a pathological mechanism of IKK activation by Tax and provide an example for how IKK is persistently activated in cancer cells.

Wu, Xuefeng; Sun, Shao-Cong

2007-01-01

147

Adenoviral oncoprotein E1B55K mediates colocalization of SSBP2 and PML in response to stress  

PubMed Central

Transient expression of adenoviral oncoprotein E1B55K in normal cells induces aggresome formation and sequestration of critical host proteins in aggresomes. Our previous studies reported that Sequence Specific Binding Protein 2 (SSBP2), a candidate tumor suppressor is recruited to aggresomes in adenovirally transformed human embryonal kidney 293 (HEK293) cells. To understand the extent and significance of the E1B55K-SSBP2 interactions in these cells, we have examined SSBP2 localization under conditions of stress in HEK293 cells. SSBP2 localizes to PML- Nuclear Bodies (PML-NBs) in response to inhibition of nuclear export, treatment with etoposide, hydroxyurea or gamma irradiation only in HEK293 cells. Furthermore, the PML-NBs grow in size and number in response to radiation over a 24 hour period in HEK293 cells analogous to previous findings for other cell types. Nonetheless, we conclude that E1B55K subverts SSBP2 function in HEK293 cells. These findings demonstrate the limitations in using HEK293 cells to study DNA damage response and other cellular processes since SSBP2 and similar regulatory proteins are aberrantly localized due to constitutive E1B55K expression.

2010-01-01

148

Novel perspective: exercise training stimulus triggers the expression of the oncoprotein human double minute-2 in human skeletal muscle  

PubMed Central

High expression levels of human double minute-2 (Hdm2) are often associated with increased risk of cancer. Hdm2 is well established as an oncoprotein exerting various tumorigenic effects. Conversely, the physiological functions of Hdm2 in nontumor cells and healthy tissues remain largely unknown. We previously demonstrated that exercise training stimulates expression of murine double minute-2 (Mdm2), the murine analog of Hdm2, in rodent skeletal muscle and Mdm2 was required for exercise-induced muscle angiogenesis. Here we showed that exercise training stimulated the expression of Hdm2 protein in human skeletal muscle from +38% to +81%. This robust physiological response was observed in 60–70% of the subjects tested, in both young and senior populations. Similarly, exercise training stimulated the expression of platelet endothelial cell adhesion molecule-1, an indicator of the level of muscle capillarization. Interestingly, a concomitant decrease in the tumor suppressor forkhead box O-1 (FoxO1) transcription factor levels did not occur with training although Mdm2/Hdm2 is known to inhibit FoxO1 expression in diseased skeletal muscle. This could suggest that Hdm2 has different targets when stimulated in a physiological context and that exercise training could be considered therapeutically in the context of cancer in combination with anti-Hdm2 drug therapies in order to preserve Hdm2 physiological functions in healthy tissues.

Roudier, Emilie; Aiken, Julian; Slopack, Dara; Gouzi, Fares; Mercier, Jacques; Haas, Tara L; Gustafsson, Thomas; Hayot, Maurice; Birot, Olivier

2013-01-01

149

Low- and high-risk human papillomavirus E7 proteins regulate p130 differently  

SciTech Connect

The E7 protein of high-risk human papillomaviruses (HR HPVs) targets pRb family members (pRb, p107 and p130) for degradation; low-risk (LR) HPV E7 only targets p130 for degradation. The effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. These results suggest that there may be divergent mechanisms by which LR and HR HPV E7 target p130 for degradation.

Barrow-Laing, Lisa; Chen Wei [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Roman, Ann, E-mail: aroman@iupui.ed [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States)

2010-05-10

150

Design of a colicin E7 based chimeric zinc-finger nuclease.  

PubMed

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity-offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region. PMID:24952471

Németh, Eszter; Schilli, Gabriella K; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

2014-08-01

151

HPV16E7 silencing enhances susceptibility of CaSki cells to natural killer cells.  

PubMed

The aim of the present study was to investigate the cytotoxicity of natural killer (NK) cells to CaSki cells following knockdown of the E7 protein of the human papillomavirus type 16 (HPV16E7). Recombinant adenovirus-short hairpin-E7 protein of the human panillomavirus type 16 (Ad?sh?HPV16E7) was constructed and used to infect CaSki cells. The expression of HPV16E7 in CaSki cells was assessed using western blot analysis. The expression of cell surface molecule major histocompatibility complex?I (MHC?I) in CaSki cells infected with Ad?sh?HPV16E7 was examined using flow cytometry. The cytotoxicity of NK cells isolated and expanded from healthy volunteers on Ad?sh?HPV16E7?infected CaSki cells was assessed using the lactate dehydrogenase (LDH) release assay. Ad?sh?HPV16E7 was successfully constructed and able to inhibit HPV16E7 the expression in CaSki cells. The expression of major histocompa-tibility complex I (MHC?I), a surface molecule, in CaSki cells was increased after infection with Ad?sh?HPV16E7. Compared with the controls, the cytotoxicity of NK cells on CaSki cells, which were infected with Ad?sh?HPV16E7, was decreased (p<0.05). In conclusion, HPV16E7 suppresses the expression of MHC?I on CaSki cells to evade cytotoxic T?cell (CTL) response. However, it was possible to enhance the cytotoxicity of expanded NK cells to cervical cancer cells or HPV16?infected cells in vitro, indicating that NK cells may be used for immunotherapy of cervical cancer. PMID:24566606

Guo, Huimin; Hu, Ruili; Guan, Xinlei; Guo, Fang; Zhao, Shuzhen; Zhang, Xueying

2014-04-01

152

Type-Specific Human Papillomavirus E6/E7 mRNA Detection by Real-Time PCR Improves Identification of Cervical Neoplasia ?  

PubMed Central

DNA-based human papillomavirus (HPV) assays show high sensitivity but poor specificity in detecting high-grade cervical lesions. Assays detecting mRNA of the oncoproteins E6 and E7 show higher specificity but lack either detection of all high-risk HPV genotypes or the capacity to specify the detected genotypes. Therefore, a real-time PCR assay detecting type-specific E6/E7 mRNA was developed and the clinical performance evaluated. A total of 210 cervical LBC (liquid-based cytology) samples from 204 women were analyzed for HPV DNA and mRNA with the in-house real-time PCR as well as PreTect HPV-Proofer. The sensitivity of real-time PCR mRNA detection to identify histologically confirmed CIN2+ (cervical intraepithelial neoplasia, grade 2 or higher) was 0.91, compared to 0.95 for DNA analysis. The specificity was 0.68 compared to 0.38, and the positive predictive value (PPV) was higher for mRNA (0.67 versus 0.52) without any loss in negative predictive value (NPV). The sensitivity of the real-time PCR mRNA test was somewhat higher than that for PreTect HPV-Proofer (0.83 versus 0.75) in analyses for the same genotypes. The specificities were similar (0.76 versus 0.77). In analyses for mRNA of the eight most common genotypes in cervical cancer (HPV16, -18, -31, -33, -35, -45, -52, and -58), the sensitivity of detection of CIN2+ lesions was 0.87 and the specificity 0.74, with a PPV of 0.70. In conclusion, real-time PCR for detection of HPV E6/E7 mRNA transcripts can be a sensitive and specific tool in screening and investigation of cervical neoplasia. The composition of HPV types in mRNA testing needs to be further investigated to optimize sensitivity and specificity.

Andersson, Elin; Karrberg, Cecilia; Radberg, Thomas; Blomqvist, Lennart; Zetterqvist, Britt-Marie; Ryd, Walter; Lindh, Magnus; Horal, Peter

2011-01-01

153

Different Isoforms of HPV-16 E7 Protein are Present in Cytoplasm and Nucleus  

PubMed Central

The E7 protein of high risk HPV types has been found with different molecular weights, mainly because of phosphorylation, an event that changes protein charge and mobility in SDS-PAGE. Distribution of E7 protein in the cellular compartments has also been subject of debate as some groups report the protein in nucleus and others in cytoplasm. The different subcellular distribution and molecular weights reported for the E7 protein suggest the presence of isoforms. We examined this possibility by using several antibodies that recognize different epitopes on the HPV-16 E7 protein. We showed that E7 is processed in 3 isoforms with different molecular weights and isoelectric points (IEP), and described as E7a1 (17.5 kDa, IEP 4.68), E7a (17 kDa, IEP 6.18) and E7b (16 kDa, IEP 6.96). The immunofluorescense results also showed that E7 is distributed into different compartments (ER, Golgi and nucleus), which suggest the presence of other posttranslational modifications, besides phosphorylation.

Valdovinos-Torres, H; Orozco-Morales, M; Pedroza-Saavedra, A; Padilla-Noriega, L; Esquivel-Guadarrama, F; Gutierrez-Xicotencatl, L

2008-01-01

154

The Human DEK Proto-Oncogene Is a Senescence Inhibitor and an Upregulated Target of High-Risk Human Papillomavirus E7  

PubMed Central

The human DEK proto-oncogene is a nucleic acid binding protein with suspected roles in human carcinogenesis, autoimmune disease, and viral infection. Intracellular DEK functions, however, are poorly understood. In papillomavirus-positive cervical cancer cells, downregulation of viral E6/E7 oncogene expression results in cellular senescence. We report here the specific repression of DEK message and protein levels in senescing human papillomavirus type 16- (HPV16-) and HPV18-positive cancer cell lines as well as in primary cells undergoing replicative senescence. Cervical cancer cell senescence was partially overcome by DEK overexpression, and DEK overexpression was sufficient for extending the life span of primary keratinocytes, supporting critical roles for this molecule as a senescence regulator. In order to determine whether DEK is a bona fide HPV oncogene target in primary cells, DEK expression was monitored in human keratinocytes transduced with HPV E6 and/or E7. The results identify high-risk HPV E7 as a positive DEK regulator, an activity that is not shared by low-risk HPV E7 protein. Experiments in mouse embryo fibroblasts recapitulated the observed E7-mediated DEK induction and demonstrated that both basal and E7-induced regulation of DEK expression are controlled by the retinoblastoma protein family. Taken together, our results suggest that DEK upregulation may be a common event in human carcinogenesis and may reflect its senescence inhibitory function.

Wise-Draper, Trisha M.; Allen, Hillary V.; Thobe, Megan N.; Jones, Elizabeth E.; Habash, Kristen B.; Munger, Karl; Wells, Susanne I.

2005-01-01

155

Methods for determining Myc-induced apoptosis.  

PubMed

Although many oncoproteins promote cell growth and proliferation, some also possess the potential to induce cell death by apoptosis. Deregulated expression of the myc oncogene promotes apoptosis in both cultured cells and in some tissues in vivo. Here we describe techniques to detect Myc-induced apoptosis in vitro using flow cytometry and microscopy and in vivo using immunohistochemical staining. PMID:24006060

Lu, Dan; Littlewood, Trevor D

2013-01-01

156

A novel fusion protein-based vaccine comprising a cell penetrating and immunostimulatory peptide linked to human papillomavirus (HPV) type 16 E7 antigen generates potent immunologic and anti-tumor responses in mice.  

PubMed

The ultimate success of cancer vaccination is dependent upon the generation of tumor-specific CTLs. In this study, we designed and evaluated a novel fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32-51 of the Limulus polyphemus protein (LALF(32-51)) linked to human papillomavirus (HPV) 16 E7 antigen (LALF(32-51)-E7). We demonstrated that LALF(32-51) penetrates the cell membrane and delivers E7 into cells. In a preclinical model of HPV16-induced cervical carcinoma we showed that vaccination with adjuvant-free LALF(32-51)-E7 fusion protein significantly improves the presentation of E7-derived peptides to T-cells in vitro and induces suppression of tumor growth. PMID:21145912

Granadillo, Milaid; Vallespi, Maribel G; Batte, Aileen; Mendoza, Osmany; Soria, Yordanka; Lugo, Victoria M; Torrens, Isis

2011-01-29

157

Phosphatidylinositol 3-kinase (PI3K): The Oncoprotein  

PubMed Central

The catalytic and regulatory subunits of class I phosphoinositide 3-kinase (PI3K) have oncogenic potential. The catalytic subunit p110? and the regulatory subunit p85 undergo cancer-specific gain-of-function mutations that lead to enhanced enzymatic activity, ability to signal constitutively and oncogenicity. The ?, ? and ? isoforms of p110 are cell-transforming as overexpressed wild-type proteins. Class I PI3Ks have the unique ability to generate phosphoinositide 3,4,5 trisphosphate (PIP3). Class II and class III PI3Ks lack this ability. Genetic and cell biological evidence suggests that PIP3 is essential for PI3K-mediated oncogenicity, explaining why class II and class III enzymes have not been linked to cancer. Mutational analysis reveals the existence of at least two distinct molecular mechanisms for the gain of function seen with cancer-specific mutations in p110?, one causing independence from upstream receptor tyrosine kinases, the other inducing independence from Ras. An essential component of the oncogenic signal that is initiated by PI3K is the TOR (target of rapamycin) kinase. TOR is an integrator of growth and of metabolic inputs. In complex with the raptor protein (TORC1), it controls cap-dependent translation, and this function is essential for PI3K-initiated oncogenesis.

Vogt, Peter K.; Hart, Jonathan R.; Gymnopoulos, Marco; Jiang, Hao; Kang, Sohye; Bader, Andreas G.; Zhao, Li; Denley, Adam

2010-01-01

158

GLIPR1 Suppresses Prostate Cancer Development through Targeted Oncoprotein Destruction  

PubMed Central

Downregulation of the proapoptotic p53 target gene GLIPR1 occurs frequently in prostate cancer (PCa), but the functional meaning of this event is obscure. Here we report the discovery of functional relationship between GLIPR1 and c-Myc in PCa where c-Myc is often upregulated. We found that the expression of GLIPR1 and c-Myc were inversely correlated in human PCa. Restoration of GLIPR1 expression in PCa cells downregulated c-myc levels, inhibiting cell cycle progression. Downregulation was linked to a reduction in ?-catenin/TCF4-mediated transcription of the c-myc gene, which were caused by GLIPR1-mediated redistribution of casein kinase 1? (CK1?) from the Golgi apparatus to the cytoplasm where CK1? could phosphorylate ?-catenin and mediate its destruction. In parallel, GLIPR1 also promoted c-Myc protein ubiquitination and degradation by glycogen synthase kinase-3?- and/or CK1?-mediated c-Myc phosphorylation. Notably, genetic ablation of the mouse homolog of Glipr1 cooperated with c-myc overexpression to induce prostatic intraepithelial neoplasia (PIN) and PCa. Together, our findings provide evidence for CK1?-mediated destruction of c-Myc and identify c-Myc S252 as a crucial CK1? phosphorylation site for c-Myc degradation. Further, they reveal parallel mechanisms of c-myc downregulation by GLIPR1 that when ablated in the prostate are sufficient to drive c-Myc expression and malignant development.

Li, Likun; Ren, Chengzhen; Yang, Guang; Fattah, Elmoataz Abdel; Goltsov, Alexei A.; Kim, Soo Mi; Lee, Ju-Seog; Park, Sanghee; Demayo, Francesco J.; Ittmann, Michael M.; Troncoso, Patricia; Thompson, Timothy C.

2013-01-01

159

Hydrophobic Effect Drives Oxygen Uptake in Myoglobin via Histidine E7*  

PubMed Central

Since the elucidation of the myoglobin (Mb) structure, a histidine residue on the E helix (His-E7) has been proposed to act as a gate with an open or closed conformation controlling access to the active site. Although it is believed that at low pH, the His-E7 gate is in its open conformation, the full relationship between the His-E7 protonation state, its conformation, and ligand migration in Mb is hotly debated. We used molecular dynamics simulations to first address the effect of His-E7 protonation on its conformation. We observed the expected shift from the closed to the open conformation upon protonation, but more importantly, noted a significant difference between the conformations of the two neutral histidine tautomers. We further computed free energy profiles for oxygen migration in each of the possible His-E7 states as well as in two instructive Mb mutants: Ala-E7 and Trp-E7. Our results show that even in the closed conformation, the His-E7 gate does not create a large barrier to oxygen migration and permits oxygen entry with only a small rotation of the imidazole side chain and movement of the E helix. We identify, instead, a hydrophobic site in the E7 channel that can accommodate an apolar diatomic ligand and enhances ligand uptake particularly in the open His-E7 conformation. This rate enhancement is diminished in the closed conformation. Taken together, our results provide a new conceptual framework for the histidine gate hypothesis.

Boechi, Leonardo; Arrar, Mehrnoosh; Marti, Marcelo A.; Olson, John S.; Roitberg, Adrian E.; Estrin, Dario A.

2013-01-01

160

Prevention and Inhibition of TC-1 Cell Growth in Tumor Bearing Mice by HPV16 E7 Protein in Fusion with Shiga Toxin B-Subunit from shigella dysenteriae  

PubMed Central

Objective: For immunotherapy of human papillomavirus (HPV) -16-associated cervical cancers the E7 protein is considered a prime candidate. However it is a poor inducer of cytotoxic T-cell response, when being used as a singular antigen in protein vaccination. Hence, in this study we focused on the utilization of a vaccine delivery system for prevention or treatment of cervical cancer. Materials and Methods: In this experimental study, we designed and evaluated a novel fusion protein comprising HPV16 E7 antigen fused to Shiga toxin B-subunit (STxB) as both an antigen vector and an adjuvant. Then we designed two preventive and therapeutic tumor models to investigate the prevention and inhibition of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-STxB or E7 without any adjuvant. Results: We demonstrated that prophylactic immunization of E7-STxB protected mice against TC-1 cells. Also in the therapeutic model, E7-STxB inhibited TC-1 tumor growth inlungs. The results were significant when compared with the immunization of E7 singularly. Conclusion: We concluded that immunization with the E7-STxB protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells.This research verifies the clinical applications and the future prospects of developing HPV16 E7 therapeutic vaccines fused to immunoadjuvants.

Sadraeian, Mohammad; Khoshnood Mansoorkhani, Mohammad Javad; Mohkam, Milad; Rasoul-Amini, Sara; Hesaraki, Mahdi; Ghasemi, Younes

2013-01-01

161

Arginine methylation controls the subcellular localization and functions of the oncoprotein splicing factor SF2/ASF.  

PubMed

Alternative splicing and posttranslational modifications (PTMs) are major sources of protein diversity in eukaryotic proteomes. The SR protein SF2/ASF is an oncoprotein that functions in pre-mRNA splicing, with additional roles in other posttranscriptional and translational events. Functional studies of SR protein PTMs have focused exclusively on the reversible phosphorylation of Ser residues in the C-terminal RS domain. We confirmed that human SF2/ASF is methylated at residues R93, R97, and R109, which were identified in a global proteomic analysis of Arg methylation, and further investigated whether these methylated residues regulate the properties of SF2/ASF. We show that the three arginines additively control the subcellular localization of SF2/ASF and that both the positive charge and the methylation state are important. Mutations that block methylation and remove the positive charge result in the cytoplasmic accumulation of SF2/ASF. The consequent decrease in nuclear SF2/ASF levels prevents it from modulating the alternative splicing of target genes, results in higher translation stimulation, and abrogates the enhancement of nonsense-mediated mRNA decay. This study addresses the mechanisms by which Arg methylation and the associated positive charge regulate the activities of SF2/ASF and emphasizes the significance of localization control for an oncoprotein with multiple functions in different cellular compartments. PMID:20308322

Sinha, Rahul; Allemand, Eric; Zhang, Zuo; Karni, Rotem; Myers, Michael P; Krainer, Adrian R

2010-06-01

162

Elucidating Molecular Interactions of Natural Inhibitors with HPV-16 E6 Oncoprotein through Docking Analysis  

PubMed Central

Human papillomavirus (HPV) infection is the leading cause of cancer mortality among women worldwide. The life-threatening infection caused by HPV demands the need for designing anticancerous drugs. In the recent years, different compounds from natural origins, such as carrageenan, curcumin, epigallocatechin gallate, indole-3-carbinol, jaceosidin, and withaferin, have been used as a hopeful source of anticancer therapy. These compounds have been shown to suppress HPV infection by different researchers. In the present study, we explored these natural inhibitors against E6 oncoprotein of high-risk HPV-16, which is known to inactivate the p53 tumor suppressor protein. A robust homology model of HPV-16 E6 was built to anticipate the interaction mechanism of E6 oncoprotein with natural inhibitory molecules using a structure-based drug designing approach. Docking analysis showed the interaction of these natural compounds with the p53-binding site of E6 protein residues 113-122 (CQKPLCPEEK) and helped the restoration of p53 functioning. Docking analysis, besides helping in silico validation of natural compounds, also helps understand molecular mechanisms of protein-ligand interactions.

Jena, Lingaraja; Galande, Sneha; Daf, Sangeeta; Mohod, Kanchan; Varma, Ashok K.

2014-01-01

163

The TrkAIII Oncoprotein Inhibits Mitochondrial Free Radical ROS-Induced Death of SH-SY5Y Neuroblastoma Cells by Augmenting SOD2 Expression and Activity at the Mitochondria, within the Context of a Tumour Stem Cell-like Phenotype  

PubMed Central

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.

Di Ianni, Natalia; Cappabianca, Lucia; Ragone, Marzia; Ianni, Giulia; Gulino, Alberto; Mackay, Andrew R.

2014-01-01

164

Induction of robust cellular immunity against HPV6 and HPV11 in mice by DNA vaccine encoding for E6/E7 antigen  

PubMed Central

Due to the strong relationship between the Human Papillomavirus (HPV) “high-risk” subtypes and cervical cancers, most HPV-related studies have been focusing on the “high-risk” HPV subtypes 16 and 18. However, it has been suggested that the “low-risk” subtypes of HPV, HPV6 and HPV11, are the major cause of recurrent respiratory papillomatosis and genital warts. In addition, HPV 6 and 11 are also associated with otolaryngologic malignancies, carcinoma of the lung, tonsil, larynx and low-grade cervical lesions. Therefore, development of HPV therapeutic vaccines targeting on subtypes 6 and 11 E6 or E7 are in great need. In this report, we describe two novel engineered DNA vaccines that encode HPV 6 and 11 consensus E6/E7 fusion proteins (p6E6E7 and p11E6E7) by utilizing a multi-phase strategy. Briefly, after generating consensus sequences, several modifications were performed to increase the expression of both constructs, including codon/RNA optimization, addition of a Kozak sequence and a highly efficient leader sequence. An endoproteolytic cleavage site was also introduced between E6 and E7 protein for proper protein folding and for better CTL processing. The expressions of both constructs were confirmed by western blot analysis and immunofluorescence assay. Vaccination with these DNA vaccines could elicit robust cellular immune responses. The epitope mapping assay was performed to further characterize the cellular immune responses induced by p6E6E7 and p11E6E7. The HPV 6 and 11 E6 or E7-specific immunodominant and subdominant epitopes were verified, respectively. The intracellular cytokine staining revealed that the magnitude of IFN-? and TNF-? secretion in antigen-specific CD8+ cells was significantly enhanced, indicating that the immune responses elicited by p6E6E7 and p11E6E7 was heavily skewed toward driving CD8+ T cells. Such DNA immunogens are interesting candidates for further studies on HPV 6 and 11-associated diseases.

Shin, Thomas; Pankhong, Panyupa; Yan, Jian; Khan, Amir S.; Sardesai, Niranjan Y.; Weiner, David B.

2012-01-01

165

An RNA Aptamer Targets the PDZ-Binding Motif of the HPV16 E6 Oncoprotein.  

PubMed

Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus which is the primary causative agent of cervical cancer. Cell transformation arises from deregulated expression of the E6 and E7 oncogenes. E6 has been shown to bind a number of cellular proteins, including p53 and proteins containing a PDZ domain. This study reports the first RNA aptamers to E6. These have been employed as molecular tools to further investigate E6-p53 and E6-PDZ interactions. This study is focussed on two aptamers (termed F2 and F4) which induced apoptosis in cells derived from an HPV16-transformed cervical carcinoma. The molecules were able to inhibit the interaction between E6 and PDZ1 from Magi1, with F2 being the most effective inhibitor. Neither of the aptamers inhibited E6-p53 interaction or p53 degradation. This study shows the specificity of this approach and highlights the potential benefits of the E6 aptamers as potential therapeutic or diagnostic agents in the future. PMID:25062098

Belyaeva, Tamara A; Nicol, Clare; Cesur, Ozlem; Travé, Gilles; Blair, George Eric; Stonehouse, Nicola J

2014-01-01

166

Generalized E7(7) coset dynamics and D = 11 supergravity  

Microsoft Academic Search

The hidden on-shell E7(7) symmetry of maximal supergravity is usually discussed in a truncation from D = 11 to four dimensions. In this article, we reverse the logic and start from a theory with manifest off-shell E7(7) symmetry inspired by West's coset construction. Following de Wit's and Nicolai's idea that a 4+56 dimensional ``exceptional geometry'' underlies maximal supergravity, we construct

Christian Hillmann

2009-01-01

167

COX-2/PGE2: molecular ambassadors of Kaposi's sarcoma-associated herpes virus oncoprotein-v-FLIP  

PubMed Central

Kaposi's sarcoma herpesvirus (KSHV) latent oncoprotein viral FLICE (FADD-like interferon converting enzyme)-like inhibitory protein (v-FLIP) or K13, a potent activator of NF-?B, has well-established roles in KSHV latency and oncogenesis. KSHV-induced COX-2 represents a novel strategy employed by KSHV to promote latency and inflammation/angiogenesis/invasion. Here, we demonstrate that v-FLIP/K13 promotes tumorigenic effects via the induction of host protein COX-2 and its inflammatory metabolite PGE2 in an NF-?B-dependent manner. In addition to our previous studies demonstrating COX-2/PGE2's role in transcriptional regulation of KSHV latency promoter and latent gene expression, the current study adds to the complexity that though LANA-1 (latency associated nuclear antigen) is utilizing COX-2/PGE2 as critical factors for its transcriptional regulation, it is the v-FLIP/K13 gene in the KSHV latency cluster that maintains continuous COX-2/PGE2 levels in the infected cells. We demonstrate that COX-2 inhibition, via its chemical inhibitors (NS-398 or celecoxib), reduced v-FLIP/K13-mediated NF-?B induction, and extracellular matrix (ECM) interaction-mediated signaling, mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) levels, and subsequently downregulated detachment-induced apoptosis (anoikis) resistance. vFLIP expression mediated the secretion of cytokines, and spindle cell differentiation activated the phosphorylation of p38, RSK, FAK, Src, Akt and Rac1-GTPase. The COX-2 inhibition in v-FLIP/K13-HMVECs reduced inflammation and invasion/metastasis-related genes, along with reduced anchorage-independent colony formation via modulating ‘extrinsic' as well as ‘intrinsic' cell death pathways. COX-2 blockade in v-FLIP/K13-HMVEC cells drastically augmented cell death induced by removal of essential growth/survival factors secreted in the microenvironment. Transformed cells obtained from anchorage-independent colonies of COX-2 inhibitor-treated v-FLIP/K13-HMVEC cells expressed lower levels of endothelial–mesenchymal transition genes such as slug, snail and twist, and higher expression of the tumor-suppressor gene, E-cadherin. Taken together, our study provides strong evidences that FDA-approved COX-2 inhibitors have great potential in blocking tumorigenic events linked to KSHV's oncogenic protein v-FLIP/K13.

Sharma-Walia, N; Patel, K; Chandran, K; Marginean, A; Bottero, V; Kerur, N; Paul, A G

2012-01-01

168

Human Papillomavirus E7 Repression in Cervical Carcinoma Cells Initiates a Transcriptional Cascade Driven by the Retinoblastoma Family, Resulting in Senescence? †  

PubMed Central

This work demonstrates a central role for the retinoblastoma (Rb) family in driving the transcriptional program of induced and replicative senescence. HeLa cervical carcinoma cells rapidly undergo senescence when the human papillomavirus (HPV) type 18 E7 gene in these cells is repressed by the bovine papillomavirus (BPV) E2 protein. This senescence response requires the endogenous Rb pathway but not the p53 pathway. Microarray analysis 6 days after BPV E2 introduction into HeLa cells identified 224 cellular genes induced by E7 repression and 354 repressed genes. Many repressed genes were involved in cell cycle progression, and numerous induced genes encoded lysosomal proteins. These gene expression changes were blocked by constitutive expression of the wild-type HPV16 E7 or adenovirus E1A gene, but not by E7 or E1A mutants defective for Rb binding. Short hairpin RNAs targeting the Rb family also inhibited these gene expression changes and blocked senescence. Therefore, surprisingly, the transcriptional response to BPV E2 expression was entirely dependent on E7 repression and activation of the Rb family, and the BPV E2 protein did not directly affect the expression of cellular genes. Activation of the Rb family repressed E2F-responsive genes and stimulated transcriptional activators, thereby mobilizing multiple signals, such as repression of B-MYB and DEK, that were independently sufficient to induce senescence. There was extensive overlap between the transcriptional profiles of senescent, late-passage primary human fibroblasts and senescent cervical carcinoma cells, suggesting that this Rb family-mediated transcriptional cascade also plays a central role in replicative senescence.

Johung, Kimberly; Goodwin, Edward C.; DiMaio, Daniel

2007-01-01

169

Human papillomavirus 16 E7 protein is associated with the nuclear matrix.  

PubMed Central

The cellular localization of the human papillomavirus (HPV)-16 E7 gene product in the cell lines CaSki and SiHa has been determined by both biochemical and immunocytochemical methods. These measurements show E7 to be localized in the cell nucleus, specifically with the nonchromatin nuclear structure or nuclear matrix. This localization of E7 required an unambiguous fractionation of the nuclear constituents. This was achieved by using a gentle sequential fractionation procedure to prepare the scaffold consisting of the nuclear matrix and intermediate filaments (NM-IF). Chromatin was cleaved with nuclease and the resulting nucleosomes eluted with 0.25 M ammonium sulfate. Immunostaining of cells after this extraction procedure with monoclonal antibodies (mAbs) to E7 revealed a fine grained, punctate nuclear fluorescence in CaSki and SiHa, which was absent in normal cervical keratinocytes and the HPV-negative cell line C33.1. Western blots of cell fractions with these mAbs showed that E7 was localized in the NM-IF fraction in SiHa and CaSki but was not detected in HPV-negative cells. A second protein of slightly higher mobility is identified by these antisera in HPV-16-containing cells. The data suggest that the previous inability to directly visualize E7 by immunocytology is due to the masking of epitopes by cellular components and not to low levels of protein. Images

Greenfield, I; Nickerson, J; Penman, S; Stanley, M

1991-01-01

170

Apoptosis Inhibition by the Human DEK Oncoprotein Involves Interference with p53 Functions?  

PubMed Central

The DEK proto-oncogene has been associated with human carcinogenesis—either as a fusion with the CAN nucleoporin protein or when transcriptionally upregulated. Mechanisms of intracellular DEK functions, however, have remained relatively unexplored. We have recently demonstrated that DEK expression is induced by the high-risk human papillomavirus (HPV) E7 protein in a manner which is dependent upon retinoblastoma protein function and have implicated DEK in the inhibition of cellular senescence. Additionally, overexpression of DEK resulted in significant life span extension of primary human keratinocytes. In order to determine whether DEK expression is required for cellular proliferation and/or survival, we monitored cellular responses to the knockdown of DEK in cancer and primary cells. The results indicate that DEK expression protects both HPV-positive cancer and primary human cells from apoptotic cell death. Cell death in response to DEK depletion was accompanied by increased protein stability and transcriptional activity of the p53 tumor suppressor and consequent upregulation of known p53 target genes such as p21CIP and Bax. Consistent with a possible role for p53 in DEK-mediated cell death inhibition, the p53-negative human osteosarcoma cell line SAOS-2 was resistant to the knockdown of DEK. Finally, expression of a dominant negative p53 miniprotein inhibited DEK RNA interference-induced p53 transcriptional induction, as well as cell death, thus directly implicating p53 activation in the observed apoptotic phenotype. These findings suggest a novel role for DEK in cellular survival, involving the destabilization of p53 in a manner which is likely to contribute to human carcinogenesis.

Wise-Draper, Trisha M.; Allen, Hillary V.; Jones, Elizabeth E.; Habash, Kristen B.; Matsuo, Hiroshi; Wells, Susanne I.

2006-01-01

171

RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts  

Microsoft Academic Search

BACKGROUND: Incorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. The correct centrosome duplication, assuring assembly of a bipolar mitotic spindle, is essential for chromosome segregation fidelity and preventing aneuploidy. Alteration of p53 and pRb functions by expression of HPV16-E6 and E7 oncoproteins has been associated with centrosome amplification. However, these last findings

Flora Iovino; Laura Lentini; Angela Amato; Aldo Di Leonardo

2006-01-01

172

CRPV genomes with synonymous codon optimizations in the CRPV E7 gene show phenotypic differences in growth and altered immunity upon E7 vaccination.  

PubMed

Papillomaviruses use rare codons relative to their hosts. Recent studies have demonstrated that synonymous codon changes in viral genes can lead to increased protein production when the codons are matched to those of cells in which the protein is being expressed. We theorized that the immunogenicity of the virus would be enhanced by matching codons of selected viral genes to those of the host. We report here that synonymous codon changes in the E7 oncogene are tolerated in the context of the cottontail rabbit papillomavirus (CRPV) genome. Papilloma growth rates differ depending upon the changes made indicating that synonymous codons are not necessarily neutral. Immunization with wild type E7 DNA yielded significant protection from subsequent challenge by both wild type and codon-modified genomes. The reduction in growth was most dramatic with the genome containing the greatest number of synonymous codon changes. PMID:18698362

Cladel, Nancy M; Hu, Jiafen; Balogh, Karla K; Christensen, Neil D

2008-01-01

173

The DEK oncoprotein is a Su(var) that is essential to heterochromatin integrity.  

PubMed

Heterochromatin integrity is crucial for genome stability and regulation of gene expression, but the factors involved in mammalian heterochromatin biology are only incompletely understood. Here we identify the oncoprotein DEK, an abundant nuclear protein with a previously enigmatic in vivo function, as a Suppressor of Variegation [Su(var)] that is crucial to global heterochromatin integrity. We show that DEK interacts directly with Heterochromatin Protein 1 ? (HP1?) and markedly enhances its binding to trimethylated H3K9 (H3K9me3), which is key for maintaining heterochromatic regions. Loss of Dek in Drosophila leads to a Su(var) phenotype and global reduction in heterochromatin. Thus, these findings show that DEK is a key factor in maintaining the balance between heterochromatin and euchromatin in vivo. PMID:21460035

Kappes, Ferdinand; Waldmann, Tanja; Mathew, Veena; Yu, Jindan; Zhang, Ling; Khodadoust, Michael S; Chinnaiyan, Arul M; Luger, Karolin; Erhardt, Sylvia; Schneider, Robert; Markovitz, David M

2011-04-01

174

Differences in structural elements of Bcr-Abl oncoprotein isoforms in Chronic Myelogenous Leukemia  

PubMed Central

in silico modeling, using Psipred and ExPASy servers was employed to determine the structural elements of Bcr-Abl oncoprotein (p210BCR-ABL) isoforms, b2a2 and b3a2, expressed in Chronic Myelogenous Leukemia (CML). Both these proteins are tyrosine kinases having masses of 210-kDa and differing only by 25 amino acids coded by the b3 exonand an amino acidsubstitution (Glu903Asp). The secondary structure elements of the two proteins show differences in five ?-helices and nine ?-strands which relates to differences in the SH3, SH2, SH1 and DNA-binding domains. These differences can result in different roles played by the two isoforms in mediating signal transduction during the course of CML.

Hai, Abdul; Kizilbash, Nadeem A; Zaidi, Syeda Huma H; Alruwaili, Jamal; Shahzad, Khuram

2014-01-01

175

Differences in structural elements of Bcr-Abl oncoprotein isoforms in Chronic Myelogenous Leukemia.  

PubMed

in silico modeling, using Psipred and ExPASy servers was employed to determine the structural elements of Bcr-Abl oncoprotein (p210(BCR-ABL)) isoforms, b2a2 and b3a2, expressed in Chronic Myelogenous Leukemia (CML). Both these proteins are tyrosine kinases having masses of 210-kDa and differing only by 25 amino acids coded by the b3 exonand an amino acidsubstitution (Glu903Asp). The secondary structure elements of the two proteins show differences in five ?-helices and nine ?-strands which relates to differences in the SH3, SH2, SH1 and DNA-binding domains. These differences can result in different roles played by the two isoforms in mediating signal transduction during the course of CML. PMID:24748748

Hai, Abdul; Kizilbash, Nadeem A; Zaidi, Syeda Huma H; Alruwaili, Jamal; Shahzad, Khuram

2014-01-01

176

The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8.  

PubMed

The HPV-16 E6 and E6(?) proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6(?). Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. PMID:24503077

Manzo-Merino, Joaquin; Massimi, Paola; Lizano, Marcela; Banks, Lawrence

2014-02-01

177

Repression of MHC class I transcription by HPV16E7 through interaction with a putative RXR{beta} motif and NF-{kappa}B cytoplasmic sequestration  

SciTech Connect

Down-regulation of transcription of the MHC class I genes in HPV16 tumorigenic cells is partly due to HPV16E7 associated with the MHC class I promoter and repressed chromatin activation. In this study, we further demonstrated that HPV16E7 is physically associated with a putative RXR{beta} binding motif (GGTCA) of the proximal promoter of the MHC class I genes by using reporter transcriptional assays and chromatin immunoprecipitation assays. Our data also provide evidence that HPV16E7 inhibits TNF-{alpha}-induced up-regulation of MHC class I transcription by impaired nuclear translocation of NF-{kappa}B. More importantly, CaSki tumor cells treated with TSA and transfected with the constitutively active mutant form of IKK-{alpha} (which can activate NF-{kappa}B directly) showed a maximal level of up-regulation of MHC-I expression. Taken together, our results suggest that HPV16E7 may employ two independent mechanisms to ensure that either the constitutive or inducible transcription of MHC class I genes is down-regulated.

Li, Hui; Zhan, TaiLan; Li, Chang [Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan (China)] [Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan (China); Liu, Mugen, E-mail: lium@mail.hust.edu.cn [Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan (China)] [Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan (China); Wang, Qing K., E-mail: qkwang@mail.hust.edu.cn [Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan (China); Center for Cardiovascular Genetics, Cleveland Clinic, Cleveland, OH 44195 (United States)

2009-10-16

178

Downstream and Intermediate Interactions of Synovial Sarcoma-Associated Fusion Oncoproteins and Their Implication for Targeted Therapy  

PubMed Central

Synovial sarcoma (SS), an aggressive type of soft tissue tumor, occurs mostly in adolescents and young adults. The origin and molecular mechanism of the development of SS remain only partially known. Over 90% of SS cases are characterized by the t(X;18)(p11.2;q11.2) translocation, which results mainly in the formation of SS18-SSX1 or SS18-SSX2 fusion genes. In recent years, several reports describing direct and indirect interactions of SS18-SSX1/SSX2 oncoproteins have been published. These reports suggest that the fusion proteins particularly affect the cell growth, cell proliferation, TP53 pathway, and chromatin remodeling mechanisms, contributing to SS oncogenesis. Additional research efforts are required to fully explore the protein-protein interactions of SS18-SSX oncoproteins and the pathways that are regulated by these partnerships for the development of effective targeted therapy.

Przybyl, Joanna; Jurkowska, Monika; Rutkowski, Piotr; Debiec-Rychter, Maria; Siedlecki, Janusz A.

2012-01-01

179

Mmip-2, a novel RING finger protein that interacts with mad members of the Myc oncoprotein network  

Microsoft Academic Search

Mad proteins are basic – helix – loop – helix – leucine zipper (bHLH-ZIP)-containing members of the myc oncoprotein network. They interact with the bHLH-ZIP protein max, compete for the same DNA binding sites as myc-max heterodimers and down-regulate myc-responsive genes. Using the bHLH-ZIP domain of mad1 as a yeast two-hybrid `bait', we identified Mmip-2, a novel RING finger protein

Xiao-Ying Yin; Kalpana Gupta; Wei Ping Han; Edwin S Levitan; Edward V Prochownik

1999-01-01

180

Human T Cell Leukemia Virus Type 1 Oncoprotein Tax Targets the Human Mitotic Checkpoint Protein MAD1  

Microsoft Academic Search

In searching for cellular targets of the HTLV-I oncoprotein Tax, we identified TXBP181, which we characterized as the human homolog of yeast mitotic checkpoint MAD1 protein. Evidence supporting TXBP181 as HsMAD1 includes sequence conservation with yeast MAD1, hyperphosphorylation during S\\/G2\\/M phases and upon treatment of cells with nocodazole, and binding to HsMAD2. HsMAD1 functions as a homodimer. It localizes to

Dong-Yan Jin; Forrest Spencer; Kuan-Teh Jeang

1998-01-01

181

The ENTPD5/mt-PCPH oncoprotein is a catalytically inactive member of the ectonucleoside triphosphate diphosphohydrolase family  

PubMed Central

Expression of the ENTPD5/mt-PCPH onco-protein and overexpression of the normal ENTPD5/PCPH protein contribute to the malignant transformation of diverse mammalian cell types, and PCPH is mutated and/or deregulated in various human tumor types. Expression of PCPH or mt-PCPH caused similar phenotypes, yet the effects promoted by mt-PCPH expression were consistently and substantially greater. ATP depletion and increased stress-resistance are phenotypes commonly associated with PCPH and mt-PCPH expression. It was suggested that the intrinsic nucleoside triphosphate diphosphohydrolase (NTPDase) activity of PCPH and mt-PCPH may be responsible for these phenotypes, but direct supporting evidence remains to be established. Results from experiments designed to test such hypothesis demonstrate that, as expected, mt-PCPH expression in human colorectal carcinoma (CRC) cells decreased their ATP levels and conferred resistance to oxaliplatin, a colorectal cancer-relevant chemotherapeutic agent. Using a combination of site-directed mutagenesis, immunoprecipitation methods, in vitro enzyme activity assays and in situ enzyme activity determinations in live cells, this report also demonstrates that the mt-PCPH oncoprotein lacks detectable NTPDase activity, indicating that direct ATP cleavage by mt-PCPH did not cause the ATP depletion observed in mt-PCPH-expressing CRC cells. These results strongly suggest that the mt-PCPH oncoprotein may regulate the cellular energy levels and subsequent chemoresistance by an NTPDase-independent mechanism. Understanding possible alternative mechanisms will be essential to devise strategies for the successful treatment of predictably therapeutically resistant tumors expressing either increased PCPH levels or, particularly, the mt-PCPH oncoprotein.

MacCARTHY, CAITLIN M.; NOTARIO, VICENTE

182

c-Fos Proto-Oncoprotein Is Degraded by the Proteasome Independently of Its Own Ubiquitinylation In Vivo  

Microsoft Academic Search

Prior ubiquitinylation of the unstable c-Fos proto-oncoprotein is thought to be required for recognition and degradation by the proteasome. Contradicting this view, we report that, although c-Fos can form conjugates with ubiquitin in vivo, nonubiquitinylatable c-Fos mutants show regulated degradation identical to that of the wild-type protein in living cells under two classical conditions of study: transient c-fos gene expression

Guillaume Bossis; Patrizia Ferrara; Claire Acquaviva; Isabelle Jariel-Encontre; Marc Piechaczyk

2003-01-01

183

Association of cottontail rabbit papillomavirus E6 oncoproteins with the hDlg/SAP97 tumor suppressor.  

PubMed

Papillomaviruses are small DNA viruses that infect epithelial tissues and cause warts. Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The E6 and E7 oncogenes are the only genes consistently expressed in HPV-positive cervical cancer cells. Cottontail rabbit papillomavirus (CRPV) induces papillomas and carcinomas on cottontail and domestic rabbits and provides an excellent animal model of HPV infection and vaccine development. CRPV encodes three transforming proteins; LE6, SE6, and E7. Each of these proteins is required for papilloma formation. Like HPV E7, the CRPV E7 protein binds to the tumor suppressor pRB. In contrast, unlike HPV E6, the CRPV E6 proteins do not bind the tumor suppressor p53. Although more than a dozen cellular proteins have been identified as HPV E6 interacting proteins, nothing is known about the cellular interacting proteins of CRPV E6s. Here we describe the association of CRPV E6s with hDlg/SAP97, the mammalian homolog of the Drosophila discs large tumor suppressor protein. HPV E6 has previously shown to bind and target hDlg/SAP97 for degradation. Our results demonstrate that both LE6 and SE6 interact with hDlg/SAP97, although their association does not lead to the degradation of hDlg/SAP97. The PDZ domains of hDlg were shown to be sufficient for interaction with CRPV E6 proteins while the C-terminus of CRPV E6 is essential for the interaction with hDlg. The association of hDlg with SE6 may be important but not sufficient for the transformation of NIH 3T3 cells by SE6. Importantly, a CRPV SE6 mutant defective for papilloma formation did not interact with hDlg. These results suggest that interaction with hDlg/SAP97 plays a role in the biological function of CRPV E6s. PMID:15669058

Du, Minjie; Fan, Xueli; Hanada, Toshihiko; Gao, Hua; Lutchman, Mohini; Brandsma, Janet L; Chishti, Athar H; Chen, Jason J

2005-04-01

184

c-erbB-2 oncoprotein is not an independent prognostic parameter in primary breast carcinoma. An immunohistochemical study.  

PubMed

The prognostic significance of c-erbB-2 oncoprotein expression was studied using the monoclonal antibody, anti-c-erbB-2 (CB-11, BioGenex) and the avidin-biotin-complex (ABC) technique. Four hundred and ninety patients with primary breast carcinoma diagnosed at Gentofte Hospital in the period 1980-1985 were included. Information about treatment, relapse-free period and overall survival was obtained from the Danish Breast Cancer Co-operative Group (DBCG). The mean follow-up period was more than 10 years. Fifteen per cent of the tumours showed positive immunoreactivity for c-erbB-2. Oncoprotein expression was correlated with presence of lymph node metastases, type of tumour, high number of mitoses, severe nuclear pleomorphism, high histological grade (poor differentiation), and absence of steroid hormone receptors. By univariate analysis, expression of c-erbB-2 oncoprotein, was correlated with poorer overall survival and shorter disease-free period in the entire cohort and in patients with lymph metastases, but not in the group of patients without lymph node metastases. By multivariate analysis, c-erbB-2 failed to be an independent prognostic marker for either disease-free period or overall survival, whereas classical histopathological parameters such as presence of lymph node metastases, high number of mitoses, high histological grade (poor differentiation) and absence of progesterone receptors turned out to be of independent prognostic significance. PMID:7946263

Haerslev, T; Jacobsen, G K

1994-08-01

185

Minimal representations of E6, E7, and E8 and the generalized Capelli identity.  

PubMed Central

We explicitly construct, in a uniform fashion, the (unique) minimal and spherical representation pi0 of the split real Lie group of exceptional type E6, E7, or E8. We obtain several algebraic and analytic results about pi0.

Brylinski, R; Kostant, B

1994-01-01

186

A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein.  

PubMed Central

TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.

Jousset, C; Carron, C; Boureux, A; Quang, C T; Oury, C; Dusanter-Fourt, I; Charon, M; Levin, J; Bernard, O; Ghysdael, J

1997-01-01

187

A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein.  

PubMed

TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators. PMID:9009269

Jousset, C; Carron, C; Boureux, A; Quang, C T; Oury, C; Dusanter-Fourt, I; Charon, M; Levin, J; Bernard, O; Ghysdael, J

1997-01-01

188

C-erbB-2 onco-protein expression in breast cancer: relationship to tumour characteristics and short-term survival in Universiti Kebansaan Malaysia Medical Centre.  

PubMed

Breast cancer is the commonest cancer affecting females in Malaysia, contributing 31% of all newly diagnosed cases amongst Malaysian women. The present retrospective cohort study evaluated the relationship between cerbB- 2 onco-protein overexpression with various tumour characteristics and survival rate of breast cancer patients treated at the Universiti Kebangsaan Malaysia Medical Centre (UKMMC) between 1996-2000. CerbB- 2 oncoprotein overexpression was determined by immunohistochemistry (IHC) and tumors showing 2+ positivity were verified by Fluorescence In Situ Hybridization (FISH). One hundred and seventy two patients were eligible for the study with a short-term follow-up (median) of 5.1 years. C-erbB-2 oncoprotein overexpression correlated with lymph node positivity, oestrogen receptor (ER) and progesterone receptor (PR) negativity. Univariate analyses showed shorter disease free survival (DFS) and overall survival (OS) in patients with cerbB- 2 oncoprotein overexpression, Malay ethnicity, higher tumour grade, lymph node positivity, ER and PR negativity. In a subgroup of patients with c-erbB-2 oncoprotein overexpression, a shorter OS was observed in those with lymph node positivity, ER and PR negativity. In multivariate prognostic analysis, lymph node status, ER status and tumour grading were the strongest independent prognostic factors for both OS and DFS. However, c-erbB-2 status was not a significantly independent prognostic factor, even in subsets with lymph node positive or negative group. C-erbB-2 oncoprotein overexpression correlated well with lymph node status, ER and PR. Shorter OS and DFS were significantly observed in patients with c-erbB-2 oncoprotein overexpression. Lymph node status, ER status and tumour grading were the only three independent prognostic factors for OS and DFS in this study. Although c-erbB-2 expression is obviously important from a biological standpoint, multivariate analysis showed that it is not an independent prognostic indicator in breast carcinoma in the local population. PMID:19271345

Sharifah, N A; Lee, B R; Clarence-Ko, C H; Tan, G C; Shiran, M S; Naqiyah, I; Rohaizak, M; Fuad, I; Tamil, A M

2008-01-01

189

Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with Glutamine E7 moiety  

PubMed Central

Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. 1H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OH? at 31.00ppm, GlnE7 N?1H/N?2H at 10.66ppm/?3.27ppm, and PheE11 C?H at 11.75ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between Gln E7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen bonding network loop between the 6-propionate, the heme ligand and nearby amino acids, tailoring in this way the electron density in the hemeligand moiety.

Ramos-Santana, Brenda J.; Lopez-Garriga, Juan

2012-01-01

190

Recapitulation of the Effects of the Human Papillomavirus Type 16 E7 Oncogene on Mouse Epithelium by Somatic Rb Deletion and Detection of pRb-Independent Effects of E7 In Vivo  

PubMed Central

Although the human papillomavirus (HPV) E7 oncogene is known to contribute to the development of human cervical cancer, the mechanisms of its carcinogenesis are poorly understood. The first identified and most recognized function of E7 is its binding to and inactivation of the retinoblastoma tumor suppressor (pRb), but at least 18 other biological activities have also been reported for E7. Thus, it remains unclear which of these many activities contribute to the oncogenic potential of E7. We used a Cre-lox system to abolish pRb expression in the epidermis of transgenic mice and compared the outcome with the effects of E7 expression in the same tissue at early ages. Mice lacking pRb in epidermis showed epithelial hyperplasia, aberrant DNA synthesis, and improper differentiation. In addition, Rb-deleted epidermis (i.e., epidermis composed of cells with Rb deleted) exhibited centrosomal abnormalities and failed to arrest the cell cycle in response to ionizing radiation. Transgenic mice expressing E7 in skin display the same range of phenotypes. In sum, few differences were detected between Rb-deleted epidermis and E7-expressing epidermis in young mice. However, when both E7 was expressed and Rb was deleted in the same tissue, increased hyperplasia and dysplasia were observed. These findings indicate that inactivation of the Rb pathway can largely account for E7's phenotypes at an early age, but that pRb-independent activities of E7 are detectable in vivo.

Balsitis, Scott J.; Sage, Julien; Duensing, Stefan; Munger, Karl; Jacks, Tyler; Lambert, Paul F.

2003-01-01

191

Correlation of oncoprotein 18/stathmin expression in human breast cancer with established prognostic factors  

PubMed Central

Oncoprotein 18/stathmin (Op18) is a conserved cytosolic phosphoprotein that regulates microtubule dynamics. The microtubule destabilizing activity is regulated by phosphorylation, mediated by both growth factor stimulated- and cell-cycle regulating kinases. The protein is highly expressed in a variety of human malignancies. In human breast carcinoma, Op18 has previously been shown to be up-regulated in a subset of the tumours, however, no correlation with clinicopathologic characteristics has been reported so far. In the present study we have examined Op18 protein expression by quantitative Western blot analysis in a panel of 151 semi-consecutive breast carcinoma samples. Op18 levels were negatively correlated with oestrogen receptor (OR) expression and positively correlated with a high fraction of aneuploid cells, proliferation measured by proliferating cell nuclear antigen (PCNA) expression, tumour size and histopathologic grade. Taken together, and in contrast to what has been previously reported, the present study shows that high Op18 expression correlates with general predictive factors and is not restricted to a specific sub-group of breast carcinoma. © 2000 Cancer Research Campaign

Brattsand, G

2000-01-01

192

A Small Molecule That Binds and Inhibits the ETV1 Transcription Factor Oncoprotein.  

PubMed

Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed "undruggable" by conventional methods. Here, we used small-molecule microarray screens to identify and characterize drug-like compounds that modulate the biologic function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacologic ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins. Mol Cancer Ther; 13(6); 1492-502. ©2014 AACR. PMID:24737027

Pop, Marius S; Stransky, Nicolas; Garvie, Colin W; Theurillat, Jean-Philippe; Hartman, Emily C; Lewis, Timothy A; Zhong, Cheng; Culyba, Elizabeth K; Lin, Fallon; Daniels, Douglas S; Pagliarini, Raymond; Ronco, Lucienne; Koehler, Angela N; Garraway, Levi A

2014-06-01

193

The oncoprotein BCL11A binds to orphan nuclear receptor TLX and potentiates its transrepressive function.  

PubMed

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain. PMID:22675500

Estruch, Sara B; Buzón, Víctor; Carbó, Laia R; Schorova, Lenka; Lüders, Jens; Estébanez-Perpiñá, Eva

2012-01-01

194

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex  

PubMed Central

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics.

Andrejka, Laura; Wen, Hong; Ashton, Jonathan; Grant, Megan; Iori, Kevin; Wang, Amy; Manak, J. Robert; Lipsick, Joseph S.

2011-01-01

195

Ligand Binding to Synthetic Mutant Myoglobin (His-E7 --> Gly): Role of the Distal Histidine  

Microsoft Academic Search

Low-temperature flash photolysis with IR and visible spectroscopy was used to probe the influence of the distal histidine His-64(E7) of sperm-whale myoglobin (Mb) on the orientation of bound carbon monoxide (CO) and on the kinetics of CO rebinding. The synthesis and high-level expression of a sperm-whale myoglobin gene in Escherichia coli permits the efficient substitution of the distal histidine through

D. Braunstein; A. Ansari; J. Berendzen; B. R. Cowen; K. D. Egeberg; H. Frauenfelder; M. K. Hong; P. Ormos; T. B. Sauke; R. Scholl; A. Schulte; S. G. Sligar; B. A. Springer; P. J. Steinbach; R. D. Young

1988-01-01

196

Expression of mucosa-related integrin ?E?7 on alveolar T cells in interstitial lung diseases  

PubMed Central

The expression of ?E?7 integrin has been related to the selective retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. To identify potential disease-associated ?E?7 expression patterns on cells accounting for lymphocytic alveolitis in interstitial lung disease (ILD), ?E expression on CD4+ and CD8+ T cell subsets was evaluated by dual-colour flow cytometry in peripheral blood and bronchoalveolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF; n = 18), hypersensitivity pneumonitis (HP; n = 20) and sarcoidosis (n = 44) in comparison with healthy controls (n = 15). In both healthy individuals and all patient groups the proportion of ?E-bearing T cells in peripheral blood was < 2%, whereas the vast majority of alveolar CD8+ T cells consistently co-expressed ?E. Absolute alveolar CD8+?E+ cell numbers/ml were up to 30-fold increased in HP patients. Proportions of ?E-bearing CD4+ cells in BALF were significantly elevated in IPF (74.0 ± 2.7%) and HP (70.0 ± 2.4%) compared with normals (30.0 ± 1.8%) (mean ± s.e.m.; P < 0.01). In sarcoidosis, the ?E expression on BALF CD4+ cells displayed subgroup dependency: proportions significantly lower than normal were noted in chest radiographic stage I (14.3 ± 1.5%), but increased proportions in stages II (50.0 ± 3.8%) and III (64.0 ± 4.8%). Correlations between common markers of T cell activation or BALF transforming growth factor-beta (TGF-?) bioactivity and ?E expression were not noted. We conclude that the vast majority of alveolar CD8+ T cells consistently express ?E?7 and that distinct patterns of ?E?7 expression on alveolar CD4+ lymphocytes in sarcoidosis are related to the diverse manifestations of the sarcoid inflammatory process in the lung.

Lohmeyer, J; Friedrich, J; Grimminger, F; Maus, U; Tenter, R; Morr, H; Velcovsky, H G; Seeger, W; Rosseau, S

1999-01-01

197

Conditioned medium derived from mesenchymal stem cells overexpressing HPV16 E6E7 dramatically improves ischemic limb.  

PubMed

Mesenchymal stem cells (MSCs) have been shown to secrete cytokines and growth factors required for angiogenesis. Previously, we demonstrated that MSCs expressing HPV16 E6E7 mRNA (E6E7-MSCs) increase life span and differentiation potential and maintain without neoplastic transformation. Whether E6E7-MSCs are sources of molecules for enhancing angiogenesis is unknown. We demonstrated that E6E7-MSC-derived conditioned medium (E6E7-CM) enhanced endothelial cell migration and tube formation compared to primary MSC-derived conditioned medium (primary-CM). Moreover, E6E7-MSCs increased AKT activation and enhanced the release of Interleukin-1? (IL-1?) and vascular endothelial growth factor A (VEGFA). Neutralization of E6E7-CM with antibodies against IL-1? or VEGFA abrogated its effect in enhancing endothelial migration and tube formation. Primary-CM, added with IL-1? and VEGFA, enhanced its ability to increase endothelial migration and tube formation. E6E7-CM was shown to increase the ability to improve blood perfusion in a mouse limb ischemia model. Histological analysis revealed that E6E7-CM prohibited muscle loss or fibrosis and increased endothelial cell counts compared to primary-CM. Similarly, the effects of E6E7-CM in improving perfusion in ischemic limb were also contributed by the increase of IL-1? or VEGFA levels. These results suggest that E6E7-MSCs increase the ability to secrete angiogenic factors via AKT activation, and E6E7-CM is abundant in IL-1? and VEGFA levels and thereby increases the ability to improve blood perfusion and prohibit muscle loss or fibrosis in a mouse limb ischemia model. PMID:24786397

Chang, Ming-Chau; Tsao, Ching-Hua; Huang, Wei-Hua; Chih-Hsueh Chen, Paul; Hung, Shih-Chieh

2014-07-01

198

Cloning, purification and metal binding of the HNH motif from colicin E7.  

PubMed

The HNH family of endonucleases is characterized by a ??? metal-finger structural motif. Colicin E7 is a representative member of this family containing the strictly conserved HNH motif at its C-terminus. Structural and biochemical studies suggested that the HNH motif could contain all the residues necessary for metal ion binding and nuclease activity. In this work a 43 amino acid peptide extending from V534 to K576 of colicin E7 and encompassing the HNH motif was cloned and expressed in Escherichia coli as a ubiquitin fusion protein. The N-terminal fusion tag was cleaved off by a specific protease, and the HNH peptide was purified free of ubiquitin. Circular dichroism, fluorescence and mass spectrometry showed that the zinc-ion binding affinity of the purified HNH peptide was much weaker than that of the intact nuclease domain suggesting that the N-terminal part of the nuclease domain is essential for stabilizing the structure of the HNH motif. The coordination sphere of the metal ion was found to be not fully equipped by the ligand - leaving a free coordination site for the substrate. Neither DNA binding nor DNAse activity of the purified HNH peptide was detected. Comparison of the glutathion-S-transferase-fused N-terminal deletion mutants of the colicin E7 nuclease domain suggested that the presence of the DNA-binding site is still not sufficient for the catalytic activity. PMID:23563167

Gyurcsik, Béla; Czene, Anikó; Jankovics, Hajnalka; Jakab-Simon, Noémi I; ?laska-Kiss, Krystyna; Kiss, Antal; Kele, Zoltán

2013-06-01

199

Detection of the apoptosis-suppressing oncoprotein bc1-2 in hormone-refractory human prostate cancers.  

PubMed Central

The oncoprotein encoded by bc1-2 is unique because of its intracellular location (a mitochondrial membrane protein) and apparent mode of action (suppression of apoptosis). To date, this oncogene has been associated only with the development of certain forms of human B-cell lymphoma. In this report, we describe our experience with a monoclonal antibody made against a synthetic peptide for bc1-2 that can recognize the bc1-2 protein and identify cells in human prostate glands expressing this proto-oncogene with in situ immunohistochemical procedures. These procedures were utilized to survey a series of 62 human tissues to evaluate whether bc1-2 might have a role in the developing prostate gland or in prostate oncogenesis. While all primordial epithelial cells in a fetal prostate gland immunostain for bc1-2, normal and hypertrophic prostate glands of the adult show bc1-2 expression restricted to the basal cells. All epithelial cells in areas of prostatic intraepithelial neoplasia were stained by this antibody, as were most (62%) localized invasive prostatic carcinomas. In contrast, all primary prostatic carcinomas and metastases obtained from metastatic prostate cancer patients after hormone treatment (hormone-refractory tumors) stained positive for bc1-2. This study demonstrates that the oncoprotein encoded by bc1-2 can be detected at sequential stages in the natural history of human prostate cancer. Since the bc1-2 oncoprotein is known to suppress the cellular response to apoptotic stimuli, it will be important to determine whether bc1-2 expression is a factor in the development of prostate cancers and in the survival of hormone-refractory prostate cancer cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

Colombel, M.; Symmans, F.; Gil, S.; O'Toole, K. M.; Chopin, D.; Benson, M.; Olsson, C. A.; Korsmeyer, S.; Buttyan, R.

1993-01-01

200

MUC1-C ONCOPROTEIN CONFERS ANDROGEN-INDEPENDENT GROWTH OF HUMAN PROSTATE CANCER CELLS  

PubMed Central

Background The mucin 1 (MUC1) heterodimeric oncoprotein is overexpressed in human prostate cancers with aggressive pathologic and clinical features. However, few insights are available regarding the functional role of MUC1 in prostate cancer. Methods Effects of MUC1-C on AR expression were determined by RT-PCR, immunoblotting and AR promoter activation. Coimmunoprecipitations, direct binding assays and chromatin immunoprecipitation (ChIP) studies were performed to assess the interaction between MUC1-C and AR. Cells were analyzed for invasion, growth in androgen-depleted medium and sensitivity to MUC1-C inhibitors. Results The present studies in androgen-dependent LNCaP and LAPC4 prostate cancer cells demonstrate that the oncogenic MUC1-C subunit suppresses AR expression. The results show that MUC1-C activates a posttranscriptional mechanism involving miR-135b-mediated downregulation of AR mRNA levels. The results further demonstrate that MUC1-C forms a complex with AR through a direct interaction between the MUC1-C cytoplasmic domain and the AR DNA-binding domain. In addition, MUC1-C associates with AR in a complex that occupies the PSA promoter. The interaction between MUC1-C and AR is associated with induction of the epithelial-mesenchymal transition (EMT) and increased invasion. MUC1-C also conferred growth in androgen-depleted medium and resistance to bicalutamide treatment. Moreover, expression of MUC1-C resulted in sensitivity to the MUC1-C inhibitor GO-203 with inhibition of growth in vitro. GO-203 treatment also inhibited growth of established tumor xenografts in nude mice. Conclusions These findings indicate that MUC1-C suppresses AR expression in prostate cancer cells and confers a more aggressive androgen-independent phenotype that is sensitive to MUC1-C inhibition.

Rajabi, Hasan; Ahmad, Rehan; Jin, Caining; Joshi, Maya Datt; Guha, Minakshi; Alam, Maroof; Kharbanda, Surender; Kufe, Donald

2012-01-01

201

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein.  

PubMed

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8? cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-? by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types. PMID:24233342

Jochems, Caroline; Tucker, Jo A; Vergati, Matteo; Boyerinas, Benjamin; Gulley, James L; Schlom, Jeffrey; Tsang, Kwong-Yok

2014-02-01

202

Expression of activated STAT5 in neoplastic mast cells in systemic mastocytosis: subcellular distribution and role of the transforming oncoprotein KIT D816V.  

PubMed

Recent data suggest that the signal transducer and activator of transcription (STAT)5 contributes to differentiation and growth of mast cells. It has also been described that constitutively phosphorylated STAT5 (pSTAT5) plays a pro-oncogenic role in various myeloid neoplasms. We examined the expression of pSTAT5 in neoplastic mast cells in systemic mastocytosis and asked whether the disease-related oncoprotein KIT D816V is involved in STAT5 activation. As assessed by immunohistochemistry using the anti-pSTAT5 antibody AX1, neoplastic mast cells were found to display pSTAT5 in all SM patients examined (n = 40). Expression of pSTAT5 was also demonstrable in the KIT D816V-positive mast cell leukemia cell line HMC-1. Using various staining-protocols, pSTAT5 was found to be located in both the cytoplasmic and nuclear compartment of mast cells. To define the functional role of KIT D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of KIT D816V were used. In these cells, induction of KIT D816V resulted in an increased expression of pSTAT5 without substantial increase in total STAT5. Moreover, the KIT D816V-targeting kinase-inhibitor PKC412 was found to counteract expression of pSTAT5 in HMC-1 cells as well as doxycycline-induced expression of pSTAT5 in Ba/F3 cells. Finally, a dominant negative STAT5-construct was found to inhibit growth of HMC-1 cells. Together, our data show that neoplastic mast cells express cytoplasmic and nuclear pSTAT5, that KIT D816V promotes STAT5-activation, and that STAT5-activation contributes to growth of neoplastic mast cells. PMID:19893034

Baumgartner, Christian; Cerny-Reiterer, Sabine; Sonneck, Karoline; Mayerhofer, Matthias; Gleixner, Karoline V; Fritz, Richard; Kerenyi, Marc; Boudot, Cedric; Gouilleux, Fabrice; Kornfeld, Jan-Wilhelm; Sillaber, Christian; Moriggl, Richard; Valent, Peter

2009-12-01

203

A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes.  

PubMed Central

We have identified a major T-cell epitope, amino acids 48-54 (DRAHYNI, in one-letter code) in the E7 open reading frame protein of human papillomavirus (HPV) type 16. Lymph node cells from mice immunized with synthetic peptides containing DRAHYNI proliferated and produced interleukin when challenged in vitro with peptide or whole HPV-16 E7 fusion protein. The T epitope was recognized in association with all five major histocompatibility complex class II I-A and I-E alleles tested. Synthetic peptides consisting of DRAHYNI linked to major B-cell epitopes on the E7 molecule formed immunogens capable of eliciting strong antibody responses to HPV-16 E7. The T epitope could provide help for the production of antibody to several B epitopes simultaneously, including a B epitope of HPV-18 E7 protein. Mice immunized with a peptide containing DRAHYNI and B epitope and, at a later date, infected with recombinant vaccinia E7 virus, displayed secondary antibody responses to E7. Because E7 has a role in cell transformation and is the most abundant viral protein in HPV-associated neoplastic cervical epithelial cells, the data have implications for vaccine strategies.

Tindle, R W; Fernando, G J; Sterling, J C; Frazer, I H

1991-01-01

204

The oncoprotein hepatitis B X-interacting protein promotes the migration of ovarian cancer cells through the upregulation of S-phase kinase-associated protein 2 by Sp1.  

PubMed

Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein. We have previously reported that HBXIP promotes the proliferation and migration of breast cancer cells. S-phase kinase-associated protein 2 (Skp2) is another oncoprotein which is important for migration. In this study, we investigated whether Skp2 is involved in the migration enhanced by HBXIP in ovarian cancer. The expression of HBXIP and Skp2 in ovarian cancer tissues was examined by immunohistochemistry using tissue microarrays. The role of HBXIP and Skp2 in the migration of ovarian cancer cells was investigated by wound-healing assay and Transwell migration assay. The effect of HBXIP on Skp2 was assessed by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis, luciferase reporter gene assays and chromatin immunoprecipitation in ovarian cancer cells (SKOV3 and CAOV3). We found that both HBXIP and Skp2 were highly expressed in ovarian cancer tissues. We observed that the overexpression of HBXIP enhanced the migration of ovarian cancer cells, while Skp2 siRNAs decreased the cell migration enhanced by HBXIP. The HBXIP siRNAs inhibited ovarian cancer cell migration and Skp2 rescued the migration inhibition induced by HBXIP siRNA. HBXIP could upregulate Skp2 at the levels of mRNA and protein in ovarian cancer cells. Moreover, HBXIP increased the activity of Skp2 promoter via binding to the transcription factor Sp1. HBXIP is highly expressed in ovarian cancer tissues. HBXIP enhances the migration of ovarian cancer cells. HBXIP was able to stimulate the activity of Skp2 promoter via transcription factor Sp1 thus promoting the migration of ovarian cancer cells. PMID:24788380

Xu, Fuqiang; Zhu, Xiaoming; Han, Tao; You, Xiaona; Liu, Fabao; Ye, Lihong; Zhang, Xiaodong; Wang, Xiaohong; Yao, Yuanqing

2014-07-01

205

Increase of human papillomavirus-16 E7-specific T helper type 1 response in peripheral blood of cervical cancer patients after radiotherapy  

PubMed Central

It has been suggested that tumour cell lysis by gamma-radiation induces a tumoral antigen release eliciting an immune response. It is not clear how a specific immune response in cervical cancer patients is developed after radiotherapy. This study is an attempt to investigate the role of the human papillomavirus type 16 (HPV-16) E7-specific T helper response before and after radiotherapy. Lymphocytes were isolated from 32 cervical cancer patients before and after radiotherapy and from 16 healthy women. They were stimulated for 12 hr with autologous HPV-16 E7-pulsed monocyte-derived dendritic cells or directly with HPV-16 E7 synthetic peptides: E751–70, E765–84 and E779–98. The cells were stained for CD4, CD69, intracellular interferon-? (IFN-?) and interleukin-4 (IL-4) cytokines and analysed by flow cytometry. A specific CD4+ CD69+ IFN-?+ immune response against HPV-16 E779–98 peptide was observed in 10 of 14 patients (71·4%) after treatment, compared with 4 of 14 (28·5%) before radiotherapy (P = 0·039); however, this response was not associated with a successful clinical response. Before treatment, 5 of 31 patients showed a HPV-16 E779–98-specific T helper type 2 (Th2) response. Interestingly, this response was significantly associated with a decrease in disease-free survival (P = 0·027). These results suggest that a Th2-type cellular response could be useful as a predictor of recurrence and poor prognosis. An increase of the HPV-specific immune response was observed after radiotherapy; however, it is not enough to control completely the disease after treatment. Our results support that the E7-specific T-cell IFN-? response in cervical cancer patients, rather than reflecting the host’s capability of controlling tumour growth, might be an indicator for disease severity.

Delgado, Felix Giovanni; Martinez, Elizabeth; Cespedes, Maria Angelica; Bravo, Maria Mercedes; Navas, Maria Cristina; Rojas, Alba Lucia Combita

2009-01-01

206

Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity.  

PubMed

A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 microg) with 50microg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon gamma producing CD8(+) T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5x10(4) HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 microg of TA-CIN and 1000 microg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested. PMID:19095032

Karanam, Balasubramanyam; Gambhira, Ratish; Peng, Shiwen; Jagu, Subhashini; Kim, Dae-Jin; Ketner, Gary W; Stern, Peter L; Adams, Robert J; Roden, Richard B S

2009-02-11

207

Simian Virus 40 Sequences and Expression of the Viral Large T Antigen Oncoprotein in Human Pleomorphic Adenomas of Parotid Glands  

PubMed Central

Simian virus 40 (SV40) sequences of the early region coding for the large T antigen (Tag) oncoprotein were investigated in DNA samples from human pleomorphic adenoma (PA) of parotid glands. Specific SV40 sequences were detected, by PCR and filter hybridization with an internal oligoprobe, in 28 of 45 (62%) human PA specimens. None of the DNA samples from 11 normal salivary gland tissues was SV40-positive. DNA sequence analysis, carried out in all PCR amplified products from SV40-positive PA specimens, confirmed the SV40 specificity and indicated that PCR products had a sequence not distinguishable from SV40 DNA wild-type strain 776. SV40 Tag expression was revealed by immunohistochemistry with the specific monoclonal antibody Pab 101 in PA thin sections with a highly sensitive technical approach which retrieved the nuclear viral oncoprotein in 26 out of 28 (93%) samples previously found SV40-positive by PCR. Detection of SV40 sequences and Tag expression in human PA suggests that this oncogenic virus may play a role as a cofactor in the onset and/or progression of this benign neoplasm, or that SV40 DNA could replicate and express the Tag in PA cells.

Martinelli, Marcella; Martini, Fernanda; Rinaldi, Eliana; Caramanico, Laura; Magri, Eros; Grandi, Enrico; Carinci, Francesco; Pastore, Antonio; Tognon, Mauro

2002-01-01

208

Lysyl Oxidase Is Downregulated by the EWS/FLI1 Oncoprotein and Its Propeptide Domain Displays Tumor Supressor Activities in Ewing Sarcoma Cells  

PubMed Central

Ewing sarcoma is the second most common bone malignancy in children and young adults. It is driven by oncogenic fusion proteins (i.e. EWS/FLI1) acting as aberrant transcription factors that upregulate and downregulate target genes, leading to cellular transformation. Thus, identificating these target genes and understanding their contribution to Ewing sarcoma tumorigenesis are key for the development of new therapeutic strategies. In this study we show that lysyl oxidase (LOX), an enzyme involved in maintaining structural integrity of the extracellular matrix, is downregulated by the EWS/FLI1 oncoprotein and in consequence it is not expressed in Ewing sarcoma cells and primary tumors. Using a doxycycline inducible system to restore LOX expression in an Ewing sarcoma derived cell line, we showed that LOX displays tumor suppressor activities. Interestingly, we showed that the tumor suppressor activity resides in the propeptide domain of LOX (LOX-PP), an N-terminal domain produced by proteolytic cleavage during the physiological processing of LOX. Expression of LOX-PP reduced cell proliferation, cell migration, anchorage-independent growth in soft agar and formation of tumors in immunodeficient mice. By contrast, the C-terminal domain of LOX, which contains the enzymatic activity, had the opposite effects, corroborating that the tumor suppressor activity of LOX is mediated exclusively by its propeptide domain. Finally, we showed that LOX-PP inhibits ERK/MAPK signalling pathway, and that many pathways involved in cell cycle progression were significantly deregulated by LOX-PP, providing a mechanistic explanation to the cell proliferation inhibition observed upon LOX-PP expression. In summary, our observations indicate that deregulation of the LOX gene participates in Ewing sarcoma development and identify LOX-PP as a new therapeutic target for one of the most aggressive paediatric malignancies. These findings suggest that therapeutic strategies based on the administration of LOX propeptide or functional analogues could be useful for the treatment of this devastating paediatric cancer.

Garcia-Garcia, Laura; de la Parra, Juan; Alonso, Javier

2013-01-01

209

Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein.  

PubMed Central

Marek's disease virus is a highly oncogenic herpesvirus that can cause T lymphomas and peripheral nerve demyelination in chickens. meq, a candidate oncogene of Marek's disease virus, encodes a basic leucine zipper (bZIP) transcription factor which contains a large proline-rich domain in its C terminus. On the basis of its bZIP structural homology, meq is perhaps the only member of the jun-fos gene family completely viral in origin. We previously showed that Meq's C-terminal domain has potent transactivation activity and that its bZIP domain can dimerize with itself and with c-Jun also. In an effort to identify viral and cellular targets of Meq, we have determined the optimal binding sites for Meq-Jun heterodimers and Meq-Meq homodimers. By a PCR-based approach using cyclic amplification of selected targets, Meq-Jun heterodimers were found to optimally bind tetradecanoylphorbol acetate response element (TRE) and cyclic AMP response element (CRE) consensus sequences. This result was consistent with the results of our previous functional analysis implicating Meq-Jun heterodimers in the transactivation of the Meq promoter through a TRE- or CRE-like sequence. Interestingly, Meq-Meq homodimers were found to bind two distinct motif elements. The first [GAGTGATG AC(G)TCATC] has a consensus which includes a TRE or CRE core flanked by additional nucleotides critical for tight binding. Methylation interference and mutational analyses confirmed the importance of the flanking residues. The sequences of a subset of TRE and CRE sites selected by Meq-Meq are closely related to the binding motif of Maf, another bZIP oncoprotein. The second putative Meq binding site (RACACACAY) bears a completely different consensus not shared by other bZIP proteins. Binding to this consensus sequence also requires secondary structure characteristics associated with DNA bending. CACA motifs are known to promote DNA curvature and function in a number of special biological processes. Our results lend further weight to the increasing importance of DNA bending in transcriptional regulation and provide a baseline for the identification of Meq-responsive targets.

Qian, Z; Brunovskis, P; Lee, L; Vogt, P K; Kung, H J

1996-01-01

210

Correlation of cell cycle regulatory proteins (p53 and p16(ink)?(a)) and bcl-2 oncoprotein with mitotic index and thickness of primary cutaneous malignant melanoma.  

PubMed

The purpose of the study was to determine the frequency of expression p53 and p16INK4a proteins and bcl-2 oncoprotein in malignant skin melanoma and to determine their correlation with the proliferative index and tumor thickness. The study involved 53 patients: 27 (51%) male and 26 (49%) female. Mitotic index showed a correlation with p53 protein expression, a negative correlation with p16INK4a protein expression. Statistically significant correlations were determined between the Breslow tumor thickness, Clark invasion level and p53 protein expression, as well as Breslow tumor thickness and bcl-2 oncoprotein expression (p<0.05), whereas there was no correlation between the p16INK4a protein expression and melanoma thicknes and Clark invasion level. Overexpression p53 protein and bcl-2 oncoprotein, with the loss p16INK4a protein of expression in the nodular melanoma, confirms a frequent loss of function of these tumor suppressor gene and oncogene, and indicates a vertical tumor growth phase. The loss of tumor suppression function the p53 protein and bcl-2 oncoprotein overexpression in cutaneous melanoma correlates with larger tumor thickness, whereas the overexpression of mutated p53 protein and loss p16INK4a protein of expression indicate a higher proliferative tumour potential. Therefore, these evaluated proteins may be the aggressive biological tumour activity markers. PMID:21108607

Kostov, Miloš; Mijovi?, Zaklina; Mihailovi?, Dragan; Cerovi?, Snežana; Stojanovi?, Miroslav; Jeli?, Marija

2010-11-01

211

Examination of ras oncoproteins in human plasma from healthy controls and workers exposed to petroleum emissions, including benzene-related compounds  

Microsoft Academic Search

Ras oncoproteins in blood plasma from workers exposed to petroleum emissions and unexposed controls were examined from Polish and Estonian samples. Twenty-four workers and 35 unexposed controls were examined from Poland and 97 exposed and 40 unexposed controls from Estonia. Of the Estonian workers, 50 were exposed to benzene in a benzene production plant and 47 to polyaromatic hydrocarbons and

D Anderson; J. A Hughes; M. H Brinkworth; A Cebulska-Wasilewska; E Nizankowska; B Graca; T Veidebaum; K Peltonen; M Sorsa

1999-01-01

212

Temperature dependence of pyrene fluorescence spectra in aqueous solutions of C n E m (C 14E 7, C 16E 7, and C 16E 6) nonionic surfactant micelles  

Microsoft Academic Search

Apparent hydrodynamic radii (Rhapp) of nonionic surfactant micelles in aqueous solutions of hepta- and hexa-ethyleneoxide monoalkylether (C14E7, C16E7, and C16E6) were measured at 20–40°C. Fluorescence spectra of pyrene in aqueous solutions of the nonionic surfactants were measured as a function of surfactant concentration and temperature. The cloud point of C16E6 was about 35°C at concentration range of 10?3 to 10?2M.

Chikako Honda; Yumi Katsumata; Risa Yasutome; Sanae Yamazaki; Shigeaki Ishii; Keisuke Matsuoka; Kazutoyo Endo

2006-01-01

213

HPV18 E6 and E7 genes affect cell cycle, pRB and p53 of cervical tumor cells and represent prominent candidates for intervention by use peptide nucleic acids (PNAs).  

PubMed

Approximately 100% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early viral genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for tumor therapy. We established an application controlling the E6/E7 expression of the HPV type 18, by using viral gene directed peptide nucleic acids (PNAs). One consequence was the complete change in growth to a stagnated behavior of the HPV 18 positive HeLa-S cells. With flow cytometry, we investigated changes in the cell cycle and expression of the pRB (retinoblastoma) and p53 genes acting as antagonists to E6 and E7. We realized that application of PNAs via intracellular cleavable conjugated peptide carriers mediates specific inhibitory effects and we showed that the combined E6/E7-directed PNA-application mediated a clear morphological change from suspension to adherend state and the cells stopped growth. These data could demonstrate a promising approach for development of new 'anti-gene therapeutics' against papillomavirus-induced human cancers. PMID:15145519

Braun, Klaus; Ehemann, Volker; Waldeck, Waldemar; Pipkorn, Rüdiger; Corban-Wilhelm, Heike; Jenne, Jürgen; Gissmann, Lutz; Debus, Jürgen

2004-06-01

214

Human cytomegalovirus mtrII oncoprotein binds to p53 and down-regulates p53-activated transcription.  

PubMed Central

The 79-amino-acid (79-aa) open reading frame (UL111a) gene within morphological transforming region II (mtrII) of human cytomegalovirus strain Towne has been shown to transform rodent cells in vitro (J. Thompson, J. Doniger, and L. J. Rosenthal, Arch. Virol. 136:161-172, 1994). Moreover, a translation termination linker (TTL) mutant of mtrII that coded for the first 49 aa of mtrII oncoprotein (designated TTL49) was sufficient for malignant transformation, whereas a TTL mutant that coded for the first 24 aa (designated TTL24) was not. The current study demonstrates the binding of mtrII oncoprotein to the tumor suppressor protein p53 both in vivo using transiently transfected cells and in vitro using labeled proteins. Furthermore, the C-terminally truncated mtrII protein TTL49, but not truncated protein TTL24, bound to p53. The mtrII binding domain mapped to the N-terminal region of p53, residues 1 to 106, with a critical region from aa 27 to 44, whereas the p53 binding domain of mtrII protein was the first 49 aa. Furthermore, mtrII inhibited p53-activated transcription, indicating its ability to alter p53-directed cellular regulatory mechanisms. mtrII oncoprotein was detected both in stably transfected NIH 3T3 cell lines and human cytomegalovirus-infected HEL 299 cells (as early as 12 h after infection) in the perinuclear region and in the nucleus. mtrII-transformed cell lines, at both early and late passage, exhibited high levels of p53 with a 15-fold-extended half-life. However, p53-activated transcription was suppressed in these cells in spite of the increased p53 levels. Finally, the results with wild-type mtrII and its TTL mutants with respect to p53 binding, p53-activated transcription, and transforming ability suggest that the mechanism of mtrII transformation is linked to both p53 binding and disruption of p53 cell regulation.

Muralidhar, S; Doniger, J; Mendelson, E; Araujo, J C; Kashanchi, F; Azumi, N; Brady, J N; Rosenthal, L J

1996-01-01

215

The role of the N-terminal loop in the function of the colicin E7 nuclease domain.  

PubMed

Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX(14)NX(8)HX(3)H ???-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease domain of ColE7 (NColE7, 446-576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7. The effect of the N-terminal truncation on the Zn(2+) ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement was simulated by molecular dynamics. Semiempirical quantum chemical calculations were performed to obtain better insight into the structure of the active centre. The longer protein interacted with both Zn(2+) ion and DNA more strongly than its shorter counterpart. The results were explained by the structural stabilization effect of the N-terminal amino acids on the catalytic centre. In agreement with this, the absence of the N-terminal sequences resulted in significantly increased movement of the backbone atoms compared with that in the native NColE7: in ?N25-NColE7 the amino acid strings between residues 485-487, 511-515 and 570-571, and in ?N4-NColE7 those between residues 467-468, 530-535 and 570-571. PMID:23334162

Czene, Anikó; Németh, Eszter; Zóka, István G; Jakab-Simon, Noémi I; Körtvélyesi, Tamás; Nagata, Kyosuke; Christensen, Hans E M; Gyurcsik, Béla

2013-03-01

216

Immunohistochemically detectable p53 and mdm-2 oncoprotein expression in astrocytic gliomas and their correlation to cell proliferation.  

PubMed

The aim of this report was to investigate the expression of the p53 and mdm-2 oncoproteins in astrocytic gliomas and to assess their interrelation to proliferating activities. Using monoclonal antibodies directed against p53 and mdm-2, these proteins were stained immunohistochemically in 60 astrocytic brain tumors with different histologic grade. Positive p53 stained nuclei were detected in 25.4% of the tumor cases. Mdm-2 staining products were only localized in 10.5% of specimens. Significant correlations could be found between p53, MIB-1, PCNA and mitotic index on the one hand, and tumor grade on the other hand. There were no clear relations between mdm-2 expression and proliferation markers. The grade of ploidy has a lower priority for the proliferating activity. In most cases mdm-2 immunoreactivity was strongly associated with a low or negative p53 expression. PMID:8780933

Dietzmann, K; von Bossanyi, P; Sallaba, J; Kirches, E; Synowitz, H J; Warich-Kirches, M

1996-05-01

217

A set of Hox proteins interact with the Maf oncoprotein to inhibit its DNA binding, transactivation, and transforming activities.  

PubMed

Maf oncoprotein is a basic-leucine zipper (bZip) type of transcriptional activator. Since many transcription factors are known to form functional complexes, we searched for proteins that interact with the DNA-binding domain of Maf using the phage display method and identified two homeodomain-containing proteins, Hoxd12 and MHox/Prx1/Phox1/Pmx1. Studies with mutants of Hox and Maf proteins showed that they associate through their DNA-binding domains; the homeodomain of Hox and the bZip domain of Maf, respectively. Reflecting the high similarity of the bZip domain, all other Maf family members tested (c-/v-Maf, MafB, MafK, MafF, and MafG) also associated with the Hox proteins. Pax6, whose homeodomain is relatively similar to MHox, also could interact with Maf. However, two other bZip oncoproteins, Fos and Jun, failed to associate with the Hox proteins, while a distantly related Hox family member, Meis1, could not interact with Maf. Through interactions with the bZip domain, the Hox proteins inhibited the DNA binding activity of Maf, whereas the binding of Hox proteins to their recognition sequences was not abrogated by Maf. We further showed that coexpression of the Hox proteins repressed transcriptional activation and transforming activity of Maf. These results suggested that the interaction of a set of Hox proteins with Maf family members may interfere not only with their oncogenicity but also with their physiological roles. PMID:11036080

Kataoka, K; Yoshitomo-Nakagawa, K; Shioda, S; Nishizawa, M

2001-01-01

218

The Aqueous Extract of Ficus religiosa Induces Cell Cycle Arrest in Human Cervical Cancer Cell Lines SiHa (HPV-16 Positive) and Apoptosis in HeLa (HPV-18 Positive)  

PubMed Central

Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq) bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca2+ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.

Choudhari, Amit S.; Suryavanshi, Snehal A.; Kaul-Ghanekar, Ruchika

2013-01-01

219

Velocity dispersions in galaxies. I - The E7 galaxy NGC 7332.  

NASA Technical Reports Server (NTRS)

A coude spectrum of the E7 galaxy NGC 7332 with 0.9 A-resolution from 4186 to 4364 A was obtained with the Princeton SEC vidicon television camera and the Hale telescope. Comparisons with spectra of G and K giant stars, numerically broadened for various Maxwellian velocity distributions, give a dispersion velocity in the line of sight of 160 (plus or minus 20) km/sec with the best fit at G8 III. The dispersion appears to be constant within plus or minus 35 km/sec out to 1.4 kpc. After correction for projection, the rotation curve has a slope of 0.18 km/sec per pc at the center and a velocity of 130 km/sec at 1.4 kpc where it is still increasing. For an estimated effective radius of 3.5 kpc enclosing half the light, the virial theorem gives a mass of 140 billion solar masses if the mass-to-light ratio is constant throughout the galaxy.

Morton, D. C.; Chevalier, R. A.

1972-01-01

220

The crystal structure of the immunity protein of colicin E7 suggests a possible colicin-interacting surface.  

PubMed Central

The immunity protein of colicin E7 (ImmE7) can bind specifically to the DNase-type colicin E7 and inhibit its bactericidal activity. Here we report the 1.8-angstrom crystal structure of the ImmE7 protein. This is the first x-ray structure determined in the superfamily of colicin immunity proteins. The ImmE7 protein consists of four antiparallel alpha-helices, folded in a topology similar to the architecture of a four-helix bundle structure. A region rich in acidic residues is identified. This negatively charged area has the greatest variability within the family of DNase-type immunity proteins; thus, it seems likely that this area is involved in specific binding to colicin. Based on structural, genetic, and kinetic data, we suggest that all the DNase-type immunity proteins, as well as colicins, share a "homologous-structural framework" and that specific interaction between a colicin and its cognate immunity protein relies upon how well these two proteins' charged residues match on the interaction surface, thus leading to specific immunity of the colicin. Images Fig. 1 Fig. 2 Fig. 5

Chak, K F; Safo, M K; Ku, W Y; Hsieh, S Y; Yuan, H S

1996-01-01

221

Characterization of the transport signals that mediate the nucleocytoplasmic traffic of low risk HPV11 E7.  

PubMed

We previously discovered that nuclear import of low risk HPV11 E7 is mediated by its zinc-binding domain via a pathway that is independent of karyopherins/importins (Piccioli et al., 2010. Virology 407, 100-109). In this study we mapped and characterized a leucine-rich nuclear export signal (NES), 76IRQLQDLLL84, within the zinc-binding domain that mediates the nuclear export of HPV11 E7 in a CRM1-dependent manner. We also identified a mostly hydrophobic patch 65VRLVV69 within the zinc-binding domain that mediates nuclear import of HPV11 E7 via hydrophobic interactions with the FG-repeats domain of Nup62. Substitutions of hydrophobic residues to alanine within the 65VRLVV69 sequence disrupt the nuclear localization of 11E7, whereas the R66A mutation has no effect. Overall the data support a model of nuclear entry of HPV11 E7 protein via hydrophobic interactions with FG nucleoporins at the nuclear pore complex. PMID:23725695

McKee, Courtney H; Onder, Zeynep; Ashok, Aditya; Cardoso, Rebeca; Moroianu, Junona

2013-08-15

222

Human Papillomavirus Type 18 E6 and E7 Genes Integrate into Human Hepatoma Derived Cell Line Hep G2  

PubMed Central

Background and Objectives Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study.

Ma, Tianzhong; Su, Zhongjing; Chen, Ling; Liu, Shuyan; Zhu, Ningxia; Wen, Lifeng; Yuan, Yan; Lv, Leili; Chen, Xiancai; Huang, Jianmin; Chen, Haibin

2012-01-01

223

Association of Bovine Papillomavirus Type 1 E6 oncoprotein with the focal adhesion protein paxillin through a conserved protein interaction motif  

Microsoft Academic Search

We have found that the E6 oncoprotein of Bovine Papillomavirus Type 1 (BE6) as well as the E6 protein of the cancer associated HPV-16 (16E6) interact with the focal adhesion protein paxillin. Mutational analysis of paxillin revealed that BE6 binds paxillin through small protein interaction motifs called LD motifs that have been previously identified as important in regulating association of

Scott B Vande Pol; Michael C Brown; Christopher E Turner; SB Vande Pol

1998-01-01

224

Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia  

Microsoft Academic Search

The vascular endothelial growth factor (VEGF) family has recently been expanded by the isolation of two additional growth factors, VEGF-B and VEGF-C. Here we compare the regulation of steady-state levels of VEGF, VEGF-B and VEGF-C mRNAs in cultured cells by a variety of stimuli implicated in angiogenesis and endothelial cell physiology. Hypoxia, Ras oncoprotein and mutant p53 tumor suppressor, which

Berndt Enholm; Karri Paavonen; Ari Ristimäki; Vijay Kumar; Yuji Gunji; Juha Klefstrom; Laura Kivinen; Marikki Laiho; Birgitta Olofsson; Vladimir Joukov; Ulf Eriksson; Kari Alitalo

1997-01-01

225

Cytotoxic T-Lymphocyte Responses to Human Papillomavirus Type 16 E5 and E7 Proteins and HLA-A*0201-Restricted T-Cell Peptides in Cervical Cancer Patients?  

PubMed Central

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated naïve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.

Liu, Dai-Wei; Yang, Yuh-Cheng; Lin, Ho-Fan; Lin, Mei-Fang; Cheng, Ya-Wen; Chu, Chen-Chung; Tsao, Yeou-Ping; Chen, Show-Li

2007-01-01

226

Combining integrated genomics and functional genomics to dissect the biology of a cancer-associated, aberrant transcription factor, the ASPSCR1-TFE3 fusion oncoprotein  

PubMed Central

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1–TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1–TFE3, PRCC-TFE3, SFPQ-TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1–TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1–TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1–TFE3, identified 2193 genes bound by ASPSCR1–TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1–TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1–TFE3, including MET (a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1–TFE3. As validation of this approach to identify genuine ASPSCR1–TFE3 target genes, two up-regulated genes bound by ASPSCR1–TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1–TFE3. As the results indicated that ASPSCR1–TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1–TFE3 target genes contribute to the growth of ASPSCR1–TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors.

Merl, Man Yee; Saito, Tsuyoshi; Lae, Marick; Mo, Qianxing; Olshen, Adam; Lianoglou, Steven; Leslie, Christina; Ostrovnaya, Irina; Antczak, Christophe; Djaballah, Hakim; Ladanyi, Marc

2014-01-01

227

Combining integrated genomics and functional genomics to dissect the biology of a cancer-associated, aberrant transcription factor, the ASPSCR1-TFE3 fusion oncoprotein.  

PubMed

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1-TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1-TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1-TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1-TFE3, identified 2193 genes bound by ASPSCR1-TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1-TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1-TFE3, including MET (a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1-TFE3. As validation of this approach to identify genuine ASPSCR1-TFE3 target genes, two up-regulated genes bound by ASPSCR1-TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1-TFE3. As the results indicated that ASPSCR1-TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1-TFE3 target genes contribute to the growth of ASPSCR1-TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors. PMID:23288701

Kobos, Rachel; Nagai, Makoto; Tsuda, Masumi; Merl, Man Yee; Saito, Tsuyoshi; Laé, Marick; Mo, Qianxing; Olshen, Adam; Lianoglou, Steven; Leslie, Christina; Ostrovnaya, Irina; Antczak, Christophe; Djaballah, Hakim; Ladanyi, Marc

2013-04-01

228

Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins  

PubMed Central

The oncoproteins of small DNA tumor viruses promote tumorigenesis by complexing with cellular factors intimately involved in the control of cell proliferation. The major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 proteins, respectively. These seemingly unrelated viral oncoproteins are similar in that their transforming activities in cells depend, in part, on a carboxyl-terminal PDZ domain-binding motif which mediates interactions with the cellular PDZ-protein DLG. Here we demonstrated that both Ad9 E4-ORF1 and high-risk HPV E6 proteins also bind to the DLG-related PDZ-protein MAGI-1. These interactions resulted in MAGI-1 being aberrantly sequestered in the cytoplasm by the Ad9 E4-ORF1 protein or being targeted for degradation by high-risk HPV E6 proteins. Transformation-defective mutant viral proteins, however, were deficient for these activities. Our findings indicate that MAGI-1 is a member of a select group of cellular PDZ proteins targeted by both adenovirus E4-ORF1 and high-risk HPV E6 proteins and, in addition, suggest that the tumorigenic potentials of these viral oncoproteins depend, in part, on an ability to inhibit the function of MAGI-1 in cells.

Glaunsinger, Britt A; Lee, Siu Sylvia; Thomas, Miranda; Banks, Lawrence

2011-01-01

229

Multi-PDZ Domain Protein MUPP1 Is a Cellular Target for both Adenovirus E4-ORF1 and High-Risk Papillomavirus Type 18 E6 Oncoproteins  

PubMed Central

A general theme that has emerged from studies of DNA tumor viruses is that otherwise unrelated oncoproteins encoded by these viruses often target the same important cellular factors. Major oncogenic determinants for human adenovirus type 9 (Ad9) and high-risk human papillomaviruses (HPV) are the E4-ORF1 and E6 oncoproteins, respectively, and although otherwise unrelated, both of these viral proteins possess a functional PDZ domain-binding motif that is essential for their transforming activity and for binding to the PDZ domain-containing and putative tumor suppressor protein DLG. We report here that the PDZ domain-binding motifs of Ad9 E4-ORF1 and high-risk HPV-18 E6 also mediate binding to the widely expressed cellular factor MUPP1, a large multi-PDZ domain protein predicted to function as an adapter in signal transduction. With regard to the consequences of these interactions in cells, we showed that Ad9 E4-ORF1 aberrantly sequesters MUPP1 within the cytoplasm of cells whereas HPV-18 E6 targets this cellular protein for degradation. These effects were specific because mutant viral proteins unable to bind MUPP1 lack these activities. From these results, we propose that the multi-PDZ domain protein MUPP1 is involved in negatively regulating cellular proliferation and that the transforming activities of two different viral oncoproteins depend, in part, on their ability to inactivate this cellular factor.

Lee, Siu Sylvia; Glaunsinger, Britt; Mantovani, Fiamma; Banks, Lawrence; Javier, Ronald T.

2000-01-01

230

Human Papillomavirus Type 8 E6 Oncoprotein Inhibits Transcription of the PDZ Protein Syntenin-2  

PubMed Central

The E6 proteins from high-risk alpha human papillomavirus (HPV) types (e.g., HPV16) are characterized by the presence of a PDZ-binding motif through which they interact with a number of cellular PDZ domain-containing substrates and cooperate in their degradation. The ability of these E6 proteins to bind to PDZ domain proteins correlates with the oncogenic potential of the virus. The E6 proteins of oncogenic HPV from the genus Betapapillomavirus (betaPV, e.g., HPV8) do not encode a PDZ-binding motif. We found that the PDZ domain protein syntenin-2 is transcriptionally downregulated in primary human epidermal keratinocytes (PHEK) by HPV8 E6. The mRNA levels of the known HPV16 E6 PDZ protein targets Dlg, Scribble, Magi-1, Magi-3, PSD95, and Mupp1 were not changed by HPV8 E6. Decreased protein levels of syntenin-2 were observed in cell extracts from PHEK expressing HPV5, -8, -16, -20, and -38 E6 but not in HPV1 and -4 E6-positive keratinocytes. Surprisingly, HPV16 E6 also repressed transcription of syntenin-2 but with a much lower efficiency than HPV8 E6. In healthy human skin, syntenin-2 expression is localized in suprabasal epidermal layers. In organotypic skin cultures, the differentiation-dependent expression of syntenin-2 was absent in HPV8 E6- and E6E7-expressing cells. In basal cell carcinomas of the skin, syntenin-2 was not detectable, whereas in squamous cell carcinomas, expression was located in differentiated areas. Short hairpin RNA-mediated knockdown of syntenin-2 led to an inhibition of differentiation and an increase in the proliferation capacity in PHEK. These results identified syntenin-2 as the first PDZ domain protein controlled by HPV8 and HPV16 at the mRNA level.

Lazic, Daliborka; Hufbauer, Martin; Zigrino, Paola; Buchholz, Stephanie; Kazem, Siamaque; Feltkamp, Mariet C. W.; Mauch, Cornelia; Steger, Gertrud; Pfister, Herbert

2012-01-01

231

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP.  

PubMed

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19(ARF) induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53. PMID:18067942

Ganzenmueller, Tina; Matthaei, Markus; Muench, Peter; Scheible, Michael; Iftner, Angelika; Hiller, Thomas; Leiprecht, Natalie; Probst, Sonja; Stubenrauch, Frank; Iftner, Thomas

2008-03-15

232

The nuclear localization of low risk HPV11 E7 protein mediated by its zinc binding domain is independent of nuclear import receptors  

SciTech Connect

We investigated the nuclear import of low risk HPV11 E7 protein using 1) transfection assays in HeLa cells with EGFP fusion plasmids containing 11E7 and its domains and 2) nuclear import assays in digitonin-permeabilized HeLa cells with GST fusion proteins containing 11E7 and its domains. The EGFP-11E7 and EGFP-11cE7{sub 39-98} localized mostly to the nucleus. The GST-11E7 and GST-11cE7{sub 39-98} were imported into the nuclei in the presence of either Ran-GDP or RanG19V-GTP mutant and in the absence of nuclear import receptors. This suggests that 11E7 enters the nucleus via a Ran-dependent pathway, independent of nuclear import receptors, mediated by a nuclear localization signal located in its C-terminal domain (cNLS). This cNLS contains the zinc binding domain consisting of two copies of Cys-X-X-Cys motif. Mutagenesis of Cys residues in these motifs changed the localization of the EGFP-11cE7/-11E7 mutants to cytoplasmic, suggesting that the zinc binding domain is essential for nuclear localization of 11E7.

Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona, E-mail: moroianu@bc.ed

2010-11-10

233

A novel endogenous induction of ColE7 expression in a csrA mutant of Escherichia coli.  

PubMed

Carbon storage regulator A (CsrA) is an important regulator that controls central metabolic pathways and a variety of physiological functions. We found that disruption of csrA in cells containing the ColE7 operon caused a 12-fold increase in colicin E7 production. Moreover, real-time RT-PCR demonstrated a decrease of around 50 % in the lexA mRNA of the csrA mutant. However, the cellular level of RecA protein and its mRNA were not significantly different from the wild type strain. Our results suggest that a novel induction mechanism might exist in E. coli that allows the expression of ColE7 operon in response to a metabolic shift. Proteomic analysis suggested that csrA deficient mutant may adapt PEP-glyoxylate cycle for energy production. Thus, the physiological changes in the csrA mutant may be similar to carbon source limitation for initiating the expression of ColE7 operon in response to stringent environmental conditions. PMID:23247769

Chang, Hao-Wei; Yang, Tsung-Yeh; Lei, Guang-Sheng; Chak, Kin-Fu

2013-04-01

234

Identification of relevant conformational epitopes on the HER2 oncoprotein by using Large Fragment Phage Display (LFPD).  

PubMed

We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines. PMID:23555577

Gabrielli, Federico; Salvi, Roberto; Garulli, Chiara; Kalogris, Cristina; Arima, Serena; Tardella, Luca; Monaci, Paolo; Pupa, Serenella M; Tagliabue, Elda; Montani, Maura; Quaglino, Elena; Stramucci, Lorenzo; Curcio, Claudia; Marchini, Cristina; Amici, Augusto

2013-01-01

235

The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells  

SciTech Connect

Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-?B (NF-?B) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-?B through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)] [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China)] [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong, E-mail: yelihong@nankai.edu.cn [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)] [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)

2013-05-03

236

Interplay Between Oncoproteins and Antioxidant Enzymes in Esophageal Carcinoma Treated Without and With Chemoradiotherapy: A Prospective Study  

SciTech Connect

Purpose: To analyze p53, bcl-2, c-myc, and cyclooxygenase-2 protein expression changes and examine their relationship with various antioxidant enzymes in esophageal carcinoma patients. Methods and Materials: Patients in Group 1 underwent transhiatal esophagectomy and those in Group 2 were administered chemoradiotherapy followed by surgery after 4 weeks of neoadjuvant therapy. Results: The relationship analysis among the various protein markers and antioxidant enzymes showed an inverse correlation between bcl-2 and superoxide dismutase/catalase in tumor tissues, irrespective of the treatment arm followed. An important positive association was observed between bcl-2 and reduced glutathione levels in the tumor tissue of patients receiving neoadjuvant therapy. Another apoptosis-modulating marker, c-myc, in the tumor tissue of Group 2 patients showed similar pattern levels (high and low) as that of superoxide dismutase/catalase. The association of cyclooxygenase-2 and p53 with various antioxidant enzymes showed a significant positive correlation between cyclooxygenase-2 expression and catalase activity and an inverse trend between p53 expression and superoxide dismutase and catalase activity in the tumor tissue of patients given neoadjuvant therapy. In addition, patients with overexpressed p53 protein levels had lower glutathione peroxidase enzyme levels and vice versa in the tumor tissue of patients who had undergone surgery as their main mode of treatment. Conclusion: The results of this study broaden the insight into the relationships shared among oncoproteins and the antioxidant defense system, and this could be helpful in the clinical management of esophageal carcinoma.

Kaur, Tranum [Department of Biophysics, Postgraduate Institute of Medical Education and Research, Chandigarh (India); Gupta, Rajesh [Department of General Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh (India); Vaiphei, Kim [Department of Histopathology, Postgraduate Institute of Medical Education and Research, Chandigarh (India); Kapoor, Rakesh [Department of Radiotherapy, Postgraduate Institute of Medical Education and Research, Chandigarh (India); Gupta, N.M. [Department of General Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh (India); Khanduja, K.L. [Department of Biophysics, Postgraduate Institute of Medical Education and Research, Chandigarh (India); Department of Biophysics, Postgraduate Institute of Medical Education and Research, Chandigarh (India)], E-mail: klkhanduja@gmail.com

2008-02-01

237

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.  

PubMed Central

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.

Straight, S W; Herman, B; McCance, D J

1995-01-01

238

Diabetes induced in male transgenic mice by expression of human H-ras oncoprotein in pancreatic beta cells.  

PubMed Central

Transgenic mice expressing an insulin-promoted H-ras hybrid gene in pancreatic beta cells developed beta-cell degeneration and diabetes. The disease was manifested in male mice by hyperglycemia, glycosuria, and reduced plasma insulin levels, which appeared around 5 months of age and led to premature death. Histological analyses revealed large holes within the islets of Langerhans and a reduced number of beta cells. The destruction of the islets was not associated with an obvious inflammatory activity. Ultrastructural analysis showed extensive engorgement in the endoplasmic reticulum of the residual beta cells from diabetic males. The females carrying the insulin-promoted ras gene did not manifest any of the physiological abnormalities observed in males and showed only minor histological and ultrastructural changes, even at much greater ages. Images

Efrat, S; Fleischer, N; Hanahan, D

1990-01-01

239

The Ewing's sarcoma oncoprotein EWS\\/FLI induces a p53-dependent growth arrest in primary human fibroblasts  

Microsoft Academic Search

Ewing's sarcoma is associated with a fusion between the EWS and FLI1 genes, forming an EWS\\/FLI fusion protein. We developed a system for the identification of cooperative mutations in this tumor through expression of EWS\\/FLI in primary human fibroblasts. Gene expression profiling demonstrated that this system recapitulates many features of Ewing's sarcoma. EWS\\/FLI-expressing cells underwent growth arrest, suggesting that growth

Stephen L. Lessnick; Caroline S. Dacwag; Todd R. Golub

2002-01-01

240

Photochemical destruction of the Bcl2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4  

Microsoft Academic Search

Photodynamic therapy (PDT), utilizing a photosensitizer and visible light, causes localized oxidative damage. With the mitochondrial photosensitizer Pc 4, PDT induces apoptosis, yet its molecular targets are not known. Here, the anti-apoptotic protein Bcl-2 is shown to be highly sensitive to PDT, as judged on Western blots by the disappearance of anti-Bcl-2-reactive material from the position of the native 26

Liang-yan Xue; Song-mao Chiu; Nancy L Oleinick

2001-01-01

241

Is there an exceptional group in your future? E7 and the travails of the symmetry breaking  

Microsoft Academic Search

We present the group theoretical analysis of a likely symmetry breaking for the vector-like unified theory based on E7. The (proton stabilizing) breaking is effected by means of a set of Higgs fields transforming according to the 912, leaving an effective SU3 × SU3c theory. We note that a nilpotent breaking for the quark mass matrix yields the SU3 limit,

P. Ramond

1977-01-01

242

Unified theory of strong, electromagnetic, and weak interactions based on the vector-like group E(7)  

Microsoft Academic Search

We present a vector-like theory based on the unifying (exceptional) group E(7). The model has six quarks, u and c of charge 2\\/3 and d,s,b,h of chage -1\\/3, as well as two (heavy) leptons of charge -1. These heavy leptons decay right-handedly into the usual antineutrinos and radiatively into the charged light leptons. The quark weak current contains uR -

P. Ramond

1976-01-01

243

Transformation of hematopoietic cells by the Ski oncoprotein involves repression of retinoic acid receptor signaling.  

PubMed

The Ski oncogene has dramatic effects on the differentiation of several different cell types. It induces the differentiation of quail embryo cells into myoblasts and arrests the differentiation of chicken hematopoietic cells. The mechanism that Ski uses to carry out these disparate biological activities is unknown. However, we were struck by the similarity of these effects to those of certain members of the nuclear hormone receptor family. Both Ski and the thyroid hormone receptor-derived oncogene v-ErbA can arrest the differentiation of avian erythroblasts, and v-Ski-transformed avian multipotent progenitor cells resemble murine hematopoietic cells that express a dominant-negative form of the retinoic acid receptor, RARalpha. In this paper, we have tested the hypothesis that v-Ski and its cellular homologue c-Ski exert their effects by interfering with nuclear hormone receptor-induced transcription. We demonstrate that Ski associates with the RAR complex and can repress transcription from a retinoic acid response element. The physiological significance of this finding is demonstrated by the ability of high concentrations of a RARalpha-specific ligand to abolish v-Ski-induced transformation of the multipotent progenitors. These results strongly suggest that the ability of Ski to alter cell differentiation is caused in part by the modulation of RAR signaling pathways. PMID:9736711

Dahl, R; Kieslinger, M; Beug, H; Hayman, M J

1998-09-15

244

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation.  

PubMed

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells. PMID:22056390

Magaldi, Thomas G; Almstead, Laura L; Bellone, Stefania; Prevatt, Edward G; Santin, Alessandro D; DiMaio, Daniel

2012-01-01

245

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation  

SciTech Connect

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.

Magaldi, Thomas G.; Almstead, Laura L. [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States)] [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States); Bellone, Stefania [Department of Obstetrics and Gynecology and Reproductive Sciences, Yale School of Medicine, P.O. Box 208063, New Haven, CT 06520-8063 (United States)] [Department of Obstetrics and Gynecology and Reproductive Sciences, Yale School of Medicine, P.O. Box 208063, New Haven, CT 06520-8063 (United States); Prevatt, Edward G. [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States)] [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States); Santin, Alessandro D. [Department of Obstetrics and Gynecology and Reproductive Sciences, Yale School of Medicine, P.O. Box 208063, New Haven, CT 06520-8063 (United States) [Department of Obstetrics and Gynecology and Reproductive Sciences, Yale School of Medicine, P.O. Box 208063, New Haven, CT 06520-8063 (United States); Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028 (United States); DiMaio, Daniel, E-mail: daniel.dimaio@yale.edu [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States) [Department of Genetics, Yale School of Medicine, P.O. Box 208005, New Haven, CT 06520-8005 (United States); Department of Therapeutic Radiology, Yale School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040 (United States); Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, P.O. Box 208024 (United States); Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028 (United States)

2012-01-05

246

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation  

PubMed Central

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.

Magaldi, Thomas G.; Almstead, Laura L.; Bellone, Stefania; Prevatt, Edward G.; Santin, Alessandro D.; DiMaio, Daniel

2011-01-01

247

Identification of target genes of synovial sarcoma-associated fusion oncoprotein using human pluripotent stem cells  

SciTech Connect

Highlights: ? We tried to identify targets of synovial sarcoma (SS)-associated SYT–SSX fusion gene. ? We established pluripotent stem cell (PSC) lines with inducible SYT–SSX gene. ? SYT–SSX responsive genes were identified by the induction of SYT–SSX in PSC. ? SS-related genes were selected from database by in silico analyses. ? 51 genes were finally identified among SS-related genes as targets of SYT–SSX in PSC. -- Abstract: Synovial sarcoma (SS) is a malignant soft tissue tumor harboring chromosomal translocation t(X; 18)(p11.2; q11.2), which produces SS-specific fusion gene, SYT–SSX. Although precise function of SYT–SSX remains to be investigated, accumulating evidences suggest its role in gene regulation via epigenetic mechanisms, and the product of SYT–SSX target genes may serve as biomarkers of SS. Lack of knowledge about the cell-of-origin of SS, however, has placed obstacle in the way of target identification. Here we report a novel approach to identify SYT–SSX2 target genes using human pluripotent stem cells (hPSCs) containing a doxycycline-inducible SYT–SSX2 gene. SYT–SSX2 was efficiently induced both at mRNA and protein levels within three hours after doxycycline administration, while no morphological change of hPSCs was observed until 24 h. Serial microarray analyses identified genes of which the expression level changed more than twofold within 24 h. Surprisingly, the majority (297/312, 95.2%) were up-regulated genes and a result inconsistent with the current concept of SYT–SSX as a transcriptional repressor. Comparing these genes with SS-related genes which were selected by a series of in silico analyses, 49 and 2 genes were finally identified as candidates of up- and down-regulated target of SYT–SSX, respectively. Association of these genes with SYT–SSX in SS cells was confirmed by knockdown experiments. Expression profiles of SS-related genes in hPSCs and human mesenchymal stem cells (hMSCs) were strikingly different in response to the induction of SYT–SSX, and more than half of SYT–SSX target genes in hPSCs were not induced in hMSCs. These results suggest the importance of cellular context for correct understanding of SYT–SSX function, and demonstrated how our new system will help to overcome this issue.

Hayakawa, Kazuo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan) [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ikeya, Makoto [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan)] [Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Fukuta, Makoto [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan) [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Woltjen, Knut [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan)] [Department of Reprogramming Sciences, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan)] [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Otsuka, Takanobu [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan)] [Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Toguchida, Junya, E-mail: togjun@frontier.kyoto-u.ac.jp [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan) [Department of Tissue Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Kyoto (Japan); Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto (Japan); Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto (Japan)

2013-03-22

248

Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein  

PubMed Central

The 9ORF1 gene encodes an adenovirus E4 region oncoprotein that requires a C-terminal region for transforming activity. Screening a ?gt11 cDNA expression library with a 9ORF1 protein probe yielded a novel cellular PDZ domain-containing protein, 9BP-1, which binds to wild-type, but not a transformation-defective, C-terminal, mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contained such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. It was found that the 9ORF1 protein interacts directly, in vitro and in vivo, with the PDZ domain-containing protein hDlg/SAP97 (DLG), which is a mammalian homolog of the Drosophila discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. Of interest, in forming complexes, the 9ORF1 protein preferentially associated with the second PDZ domain of DLG, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possessed PDZ domain-binding motifs at their C termini and, significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bound DLG in vitro. Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins.

Lee, Siu Sylvia; Weiss, Robert S.; Javier, Ronald T.

1997-01-01

249

The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification ?  

PubMed Central

The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A1 (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.

Suprynowicz, Frank A.; Krawczyk, Ewa; Hebert, Jess D.; Sudarshan, Sawali R.; Simic, Vera; Kamonjoh, Christopher M.; Schlegel, Richard

2010-01-01

250

C/EBP?, C/EBP? Oncoproteins, or C/EBP? Preferentially Bind NF-?B p50 Compared with p65 Focusing Therapeutic Targeting on the C/EBP:p50 Interaction  

PubMed Central

Canonical NF-?B activation signals stimulate nuclear translocation of p50:p65, replacing inhibitory p50:p50 with activating complexes on chromatin. C/EBP interaction with p50 homodimers provides an alternative pathway for NF-?B target gene activation, and interaction with p50:p65 may enhance gene activation. We previously found that C/EBP? cooperates with p50 but not p65 to induce Bcl-2 transcription and that C/EBP? induces Nfkb1/p50 but not RelA/p65 transcription. Using p50 and p65 variants containing the FLAG epitope at their N- or C-termini, we now demonstrate that C/EBP?, C/EBP? myeloid oncoproteins, or the LAP1, LAP2, or LIP isoforms of C/EBP? have markedly higher affinity for p50 in comparison to p65. Deletion of the p65 trans-activation domain did not increase p65 affinity for C/EBPs, suggesting that unique residues in p50 account for specificity, and clustered mutation of HSDL in the “p50 insert” lacking in p65 weakens interaction. Also, in contrast to Nfkb1 gene deletion, absence of the RelA gene does not reduce Bcl-2 or Cebpa RNA in unstimulated cells or prevent interaction of C/EBP? with the Bcl-2 promoter. Saturating mutagenesis of the C/EBP? basic region identifies R300 and nearby residues, identical in C/EBP?, as critical for interaction with p50. These findings support the conclusion that C/EBPs activate NF-?B target genes via contact with p50 even in the absence of canonical NF-?B activation and indicate that targeting C/EBP:p50 rather than C/EBP:p65 interaction in the nucleus will prove effective for inflammatory or malignant conditions, alone or synergistically with agents acting in the cytoplasm to reduce canonical NF-?B activation.

Dooher, Julia E.; Paz-Priel, Ido; Houng, Simone; Baldwin, Albert S.; Friedman, Alan D.

2011-01-01

251

Human apolipoprotein E7:lysine mutations in the carboxy-terminal domain are directly responsible for preferential binding to very low density lipoproteins.  

PubMed

Apolipoprotein E7 (apoE7) (apoE3 E244K/E245K) is a naturally occurring mutant in humans that is associated with increased plasma lipid levels and accelerated atherosclerosis. It is reported to display defective binding to low density lipoprotein (LDL) receptors, high affinity binding for heparin, and like apoE4, preferential association with very low density lipoproteins (VLDL). There are two potential explanations for the preference of apoE7 for VLDL: lysine mutations, which occur in the major lipid-binding region (residues 244-272) of the carboxy-terminal domain of apoE7, could either directly determine the lipoprotein-binding preference or could interact with negatively charged residues in the amino-terminal domain, resulting in a domain interaction similar to that in apoE4 (interaction of Arg-61 with Glu-255), which is responsible for the apoE4 VLDL preference. To distinguish between these possibilities, we determined the binding preferences of recombinant apoE7 and two amino-terminal domain mutants, apoE7 (E49Q/E50Q) and apoE7 (D65N/E66Q), to VLDL-like emulsion particles. ApoE7 and both mutants displayed a higher preference for the emulsion particles than did apoE3, indicating that the carboxy-terminal lysine mutations in apoE7 are directly responsible for its preference for VLDL. Supporting this conclusion, the carboxy-terminal domain 12-kDa fragment of apoE7 (residues 192;-299) displayed a higher preference for VLDL emulsions than did the wild-type fragment. In addition, lipid-free apoE7 had a higher affinity for heparin than did apoE. However, when apoE7 was complexed with dimyristoylphosphatidylcholine or VLDL emulsions, the affinity difference was eliminated. In contrast to previous studies, we found that apoE7 does not bind defectively to the LDL receptor, as determined in both cell culture and solid-phase assays. We conclude that the two additional lysine residues in the carboxy-terminal domain of apoE7 directly alter its lipid- and heparin-binding affinities. These characteristics of apoE7 could contribute to its association with increased plasma lipid levels and atherosclerosis. PMID:11060347

Dong, J; Balestra, M E; Newhouse, Y M; Weisgraber, K H

2000-11-01

252

Targeting Oncoprotein Stability Overcomes Drug Resistance Caused by FLT3 Kinase Domain Mutations  

PubMed Central

FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML.

Duyster, Justus

2014-01-01

253

Oncoprotein v-Myb and retinoic acid receptor alpha are mutual antagonists.  

PubMed

The process of hematopoiesis is critically dependent on correct interactions of multiple regulatory molecules and transcription factors. We have studied the interactions of the v-Myb and retinoic acid receptor proteins which have opposing effects on hematopoiesis. While v-myb acts as a transforming oncogene preventing differentiation of monoblasts to macrophages, RAR alpha functions as an anti-oncogene arresting the growth of v-myb-transformed cells and allowing their final myeloid differentiation steps to occur. We found that the retinoic acid receptor alpha inhibits v-Myb transformation by suppressing the expression of v-Myb target genes typified by the mim-1 gene. Conversely, v-Myb protein interferes with RAR alpha transactivation function as well as with retinoic acid-induced apoptosis of HL-60 cells. These results demonstrate that retinoic acid receptor and v-Myb proteins act in antagonistic ways and reciprocally modify each other's functions. PMID:9714701

Zemanová, K; Smarda, J

1998-06-01

254

Targeting Oncoprotein Stability Overcomes Drug Resistance Caused by FLT3 Kinase Domain Mutations.  

PubMed

FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML. PMID:24849514

Yu, Chuanjiang; Kancha, Rama Krishna; Duyster, Justus

2014-01-01

255

Amplification of the chromosome 20q region is associated with expression of HPV-16 E7 in human airway and anogenital epithelial cells  

PubMed Central

To study the role of human papillomavirus (HPV) infection in the development of genetic instability, we transduced normal human airway and anogenital epithelial cells with various combinations of HPV-16 E6, E7, and the reverse transcriptase component of telomerase (hTERT). Cell lines generated by co-expression of E7 with E6 and/or hTERT (i.e., E6/E7, E7/hTERT, and E6/E7/hTERT) exhibited extra copies of chromosome 20 and specific amplification of the 20q12-ter region, whereas those generated without E7 (i.e., hTERT alone or E6/hTERT) did not. Co-expression of hTERT and a dominant-negative version of cdk4 that has been shown to inactivate the retinoblastoma (pRb) pathway also resulted in 20q amplification. Interestingly, extra copies of chromosome 20 were observed in early passage keratinocytes that expressed E7 alone, and microarray expression analysis revealed that genes in the 20q region and on chromosome 5 were specifically upregulated in these cells. Our results indicate that chromosome 20q amplification is an early event that may be specifically caused by expression of E7 through inactivation of the pRb pathway in human epithelial cells.

Klingelhutz, Aloysius J.; Qian, Qining; Phillips, Stacia L.; Gourronc, Francoise A.; Darbro, Benjamin W.; Patil, Shivanand R.

2008-01-01

256

Antibody Response for L1, E6 and E7 HPV 16 and HPV 18 Antigens in Tunisian Women with Cervical Cancer and Controls  

Microsoft Academic Search

Results obtained in the present work indicated that the Luminex assay is more sensitive than ELISA. The reactivity to the early antigens E6 and E7 was 37% versus 42% for HPV 16 and 21% versus 20% for HPV 18 among cervical cancer cases using ELISA. However, these ratios were 44% and 61%, respectively, for E6 and E7 HPV 16 versus

M. Achour; D. Zeghal; L. Kochbati; S. Kahla; F. Zouari; M. Maâlej; R. Oueslati

2008-01-01

257

Antibody Response for L1, E6, E7 HPV 16, and HPV 18 Antigens in Tunisian Women with Cervical Cancer  

Microsoft Academic Search

Results obtained in the present work indicated that the Luminex assay is more sensitive than ELISA. The reactivity to the early antigens E6 and E7 was 37% versus 42% for HPV 16 and 21% versus 20% for HPV 18 among cervical cancer cases using ELISA. However, these ratios were 44% and 61%, respectively, for E6 and E7 HPV 16 versus

M. Achour; D. Zeghal; L. Kochbati; S. Kahla; F. Zouari; M. Maâlej; R. Oueslati

2008-01-01

258

Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization  

SciTech Connect

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M. [Department of Biology, Clarkson University, Potsdam, NY 13699-5805 (United States)] [Department of Biology, Clarkson University, Potsdam, NY 13699-5805 (United States); Mondal, Sumona [Department of Mathematics, Clarkson University, Potsdam, NY 13699-5805 (United States)] [Department of Mathematics, Clarkson University, Potsdam, NY 13699-5805 (United States); Woodworth, Craig D., E-mail: woodworth@clarkson.edu [Department of Biology, Clarkson University, Potsdam, NY 13699-5805 (United States)

2012-03-30

259

Mucin 1 C-Terminal Subunit Oncoprotein Is a Target for Small-Molecule Inhibitors  

PubMed Central

Mucin 1 (MUC1) is a heterodimeric protein that is overexpressed in diverse human carcinomas. The oncogenic function of the MUC1 C-terminal subunit (MUC1-C) subunit is dependent on the formation of dimers through its cytoplasmic domain; however, it is not known whether MUC1-C can be targeted with small-molecule inhibitors. In the present work, an assay using the MUC1-C cytoplasmic domain (MUC1-CD) was established to screen small-molecule libraries for compounds that block its dimerization. Using this approach, the flavone apigenin was identified as an inhibitor of MUC1-CD dimerization in vitro and in cells. By contrast, the structurally related flavone baicalein was ineffective in blocking the formation of MUC1-CD dimers. In concert with these results, apigenin, and not baicalein, blocked the localization of MUC1-C to the nucleus. MUC1-C activates MUC1 gene expression in an autoinductive loop, and apigenin, but not baicalein, treatment was associated with down-regulation of MUC1 mRNA levels and MUC1-C protein. The results also demonstrate that apigenin-induced suppression of MUC1-C expression is associated with apoptotic cell death and loss of clonogenic survival. These findings represent the first demonstration that the MUC1-C cytoplasmic domain is a target for the development of small-molecule inhibitors.

Zhou, Yongchun; Rajabi, Hasan

2011-01-01

260

MUC1-C Oncoprotein Promotes STAT3 Activation in an Autoinductive Regulatory Loop  

PubMed Central

Signal transducer and activator of transcription 3 (STAT3) is activated in human breast cancer and other malignancies. Mucin 1 (MUC1) is a heterodimeric cell surface glycoprotein that is overexpressed in human carcinomas and, like STAT3, promotes cell survival and induces transformation. Here, we showed that in breast cancer cells, the MUC1 carboxyl-terminal receptor subunit (MUC1-C) associated with the gp130–Janus-activated kinase 1 (JAK1)–STAT3 complex. The MUC1-C cytoplasmic domain interacted directly with JAK1 and STAT3, and MUC1-C was necessary for JAK1-mediated STAT3 activation. In turn, MUC1-C and activated STAT3 occupied the promoter of MUC1, and MUC1-C contributed to STAT3-mediated activation of MUC1 transcription. The MUC1-C inhibitor GO-201 blocked the MUC1-C interaction with STAT3, thereby decreasing MUC1-C and STAT3 occupancy on the MUC1 and STAT3 promoters and activation of STAT3 target genes, including MUC1 itself. These findings indicate that MUC1-C promotes STAT3 activation and that MUC1-C and STAT3 function in an autoinductive lopp that may play a role in cancer cell survival.

Ahmad, Rehan; Rajabi, Hasan; Kosugi, Michio; Joshi, Maya Datt; Alam, Maroof; Vasir, Baldev; Kawano, Takeshi; Kharbanda, Surender; Kufe, Donald

2011-01-01

261

An Oncoprotein from the Plant Pathogen Agrobacterium Has Histone Chaperone-Like Activity[W  

PubMed Central

Protein 6b, encoded by T-DNA from the pathogen Agrobacterium tumefaciens, stimulates the plant hormone–independent division of cells in culture in vitro and induces aberrant cell growth and the ectopic expression of various genes, including genes related to cell division and meristem-related class 1 KNOX homeobox genes, in 6b-expressing transgenic Arabidopsis thaliana and Nicotiana tabacum plants. Protein 6b is found in nuclei and binds to several plant nuclear proteins. Here, we report that 6b binds specifically to histone H3 in vitro but not to other core histones. Analysis by bimolecular fluorescence complementation revealed an interaction in vivo between 6b and histone H3. We recovered 6b from a chromatin fraction from 6b-expressing plant cells. A supercoiling assay and digestion with micrococcal nuclease indicated that 6b acts as a histone chaperone with the ability to mediate formation of nucleosomes in vitro. Mutant 6b, lacking the C-terminal region that is required for cell division–stimulating activity and interaction with histone H3, was deficient in histone chaperone activity. Our results suggest a relationship between alterations in nucleosome structure and the expression of growth-regulating genes on the one hand and the induction of aberrant cell proliferation on the other.

Terakura, Shinji; Ueno, Yoshihisa; Tagami, Hideaki; Kitakura, Saeko; Machida, Chiyoko; Wabiko, Hiroetsu; Aiba, Hiroji; Otten, Leon; Tsukagoshi, Hironaka; Nakamura, Kenzo; Machida, Yasunori

2007-01-01

262

Dependence on the MUC1-C oncoprotein in non-small cell lung cancer cells.  

PubMed

Non-small cell lung cancer (NSCLC) cells are often associated with constitutive activation of the phosphoinositide 3-kinase (PI3K) ? Akt ? mTOR pathway. The mucin 1 (MUC1) heterodimeric glycoprotein is aberrantly overexpressed in NSCLC cells and induces gene signatures that are associated with poor survival of NSCLC patients. The present results show that the MUC1 C-terminal subunit (MUC1-C) cytoplasmic domain associates with PI3K p85 in NSCLC cells. We show that inhibition of MUC1-C with cell-penetrating peptides blocks this interaction with PI3K p85 and suppresses constitutive phosphorylation of Akt and its downstream effector, mTOR. In concert with these results, treatment of NSCLC cells with the MUC1-C peptide inhibitor GO-203 was associated with downregulation of PI3K ? Akt signaling and inhibition of growth. GO-203 treatment was also associated with increases in reactive oxygen species (ROS) and induction of necrosis by a ROS-dependent mechanism. Moreover, GO-203 treatment of H1975 (EGFR L858R/T790M) and A549 (K-Ras G12S) xenografts growing in nude mice resulted in tumor regressions. These findings indicate that NSCLC cells are dependent on MUC1-C both for activation of the PI3K ? Akt pathway and for survival. PMID:21421804

Raina, Deepak; Kosugi, Michio; Ahmad, Rehan; Panchamoorthy, Govind; Rajabi, Hasan; Alam, Maroof; Shimamura, Takeshi; Shapiro, Geoffrey I; Supko, Jeffrey; Kharbanda, Surender; Kufe, Donald

2011-05-01

263

Reexpression of oncoprotein MafB in proliferative ?-cells and Men1 insulinomas in mouse.  

PubMed

MafB, a member of the large Maf transcription factor family, is essential for the embryonic and terminal differentiation of pancreatic ?- and ?-cells. However, the role of MafB in the control of adult islet-cell proliferation remains unknown. Considering its oncogenic potential in several other tissues, we investigated the possible alteration of its expression in adult mouse ?-cells under different conditions of proliferation. We found that MafB, in general silenced in these cells, was reexpressed in ?30% of adaptive ?-cells both in gestational female mice and in mice fed with a high-fat diet. Importantly, reactivated MafB expression was also observed in the early ?-cell lesions and insulinomas that developed in ?-cell specific Men1 mutant mice, appearing in >80% of ?-cells in hyperplasic or dysplastic islets from the mutant mice >4 months of age. Moreover, MafB expression could be induced by glucose stimulation in INS-1 rat insulinoma cells. The induction was further reinforced following Men1 knockdown by siRNA. Furthermore, MafB overexpression in cultured ?TC3 cells enhanced cell foci formation both in culture medium and on soft agar, accompanied with the increased expression of Cyclin B1 and D2. Conversely, MafB downregulation by siRNA transfection reduced BrdU incorporation in INS-1E cells. Taken together, our data reveal that Men1 inactivation leads to MafB reexpression in mouse ?-cells in vivo, and provides evidence that deregulated ectopic MafB expression may have a hitherto unknown role in adult ?-cell proliferation and Men1-related tumorigenesis. PMID:22120711

Lu, J; Hamze, Z; Bonnavion, R; Herath, N; Pouponnot, C; Assade, F; Fontanière, S; Bertolino, P; Cordier-Bussat, M; Zhang, C X

2012-08-01

264

Radiosensitization of Oropharyngeal Squamous Cell Carcinoma Cells by Human Papillomavirus 16 Oncoprotein E6*I  

SciTech Connect

Purpose: Patients with oropharyngeal squamous cell carcinoma (OSCC) whose disease is associated with high-risk human papillomavirus (HPV) infection have a significantly better outcome than those with HPV-negative disease, but the reasons for the better outcome are not known. We postulated that they might relate to an ability of HPV proteins to confer a better response to radiotherapy, a commonly used treatment for OSCC. Methods and Materials: We stably expressed the specific splicing-derived isoforms, E6*I and E6*II, or the entire E6 open reading frame (E6total), which gives rise to both full length and E6*I isoforms, in OSCC cell lines. Radiation resistance was measured in clonogenicity assays, p53 activity was measured using transfected reporter genes, and flow cytometry was used to analyze cell cycle and apoptosis. Results: E6*I and E6total sensitized the OSCC cells to irradiation, E6*I giving the greatest degree of radiosensitization (approximately eightfold lower surviving cell fraction at 10 Gy), whereas E6*II had no effect. In contrast to radiosensitivity, E6*I was a weaker inhibitor than E6total of tumor suppressor p53 transactivator activity in the same cells. Flow cytometric analyses showed that irradiated E6*I expressing cells had a much higher G2M:G1 ratio than control cells, indicating that, after G2, cells were diverted from the cell cycle to programmed cell death. Conclusion: This study supports a role for E6*I in the enhanced responsiveness of HPV-positive oropharyngeal carcinomas to p53-independent radiation-induced death.

Pang, Ervinna [Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, NSW (Australia); Discipline of Infectious Diseases and Immunology, University of Sydney, NSW (Australia); Delic, Naomi C. [Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, NSW (Australia); Discipline of Dermatology, University of Sydney, NSW (Australia); Hong, Angela; Zhang Mei [Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, NSW (Australia); Department of Radiation Oncology, Royal Prince Alfred Hospital, NSW (Australia); Rose, Barbara R. [Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, NSW (Australia); Discipline of Infectious Diseases and Immunology, University of Sydney, NSW (Australia); Lyons, J. Guy, E-mail: guy.lyons@sydney.edu.a [Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital, NSW (Australia); Discipline of Dermatology, University of Sydney, NSW (Australia)

2011-03-01

265

Regulation of the oncoprotein KLF8 by a switch between acetylation and sumoylation.  

PubMed

KLF8 regulates target genes by recruiting the p300 and PCAF co-activators via glutamines (Q) 118 and 248, the CtBP co-repressor to 86PVDLS90 or SUMO to lysine (K) 67. Here we examined how these interactions coordinate to regulate KLF8 transactivity. Mass spectrometry and immunoprecipitations determined that p300 and/or PCAF promoted KLF8 acetylation at K67, K93, and K95 and this acetylation was abolished in lysine-to-arginine (R) mutants. Treatment with HDAC inhibitors or expression of co-activators inhibited sumoylation at K67. K93R or K95R mutation exerted high levels of sumoylation while the acetylation mimetic mutations K93Q and K95Q blocked the sumoylation. Interestingly, CtBP promoted sumoylation at K67 of wild-type but not AVALF mutant KLF8, and KLF8 interaction with CtBP was inhibited by treatment with the HDAC inhibitors, ectopic expression of the co-activators, or K93Q or K95Q mutation. Promoter reporter assays showed that CtBP inhibited KLF8 transactivity which was rescued by PCAF or p300 expresson. Finally, KLF8-mediated cyclin D1 protein expression and cell cycle progression were significantly decreased in the K93R and K95R but increased in the K93Q, K95Q, K67R or K67Q mutant. Taken together, these results identified a novel mechanism by which co-activators promote KLF8 transactivity by competing with SUMO for K67 modification and by acetylating K93 and K95 to inhibit CtBP-induced K67 sumoylation. PMID:21416054

Urvalek, Alison M; Lu, Heng; Wang, Xianhui; Li, Tianshu; Yu, Lin; Zhu, Jinghua; Lin, Qishan; Zhao, Jihe

2011-02-01

266

Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma.  

PubMed Central

We describe molecular and clinical findings in an immunocompetent patient with an oligoastrocytoma and the concomitant presence of the human papovavirus, JC virus (JCV), which is the etiologic agent of the subacute, debilitating demyelinating disease, progressive multifocal leukoencephalopathy. Histologic review revealed a glial neoplasm consisting primarily of a moderately cellular oligodendroglioma with distinct areas of a fibrillary astrocytoma. Immunohistochemical analysis revealed nuclear staining of tumor cells with antibodies against the viral oncoprotein [tumor antigen (T antigen)], the proliferation marker (Ki67), and the cellular proliferation regulator (p53). Using primers specific to the JCV control region, PCR yielded amplified DNA that was identical to the control region of the Mad-4 strain of the virus. PCR analysis demonstrated the presence of the genome for the viral oncoprotein, T antigen, and results from primer extension studies revealed synthesis of the viral early RNA for T antigen in the tumor tissues. The presence of viral T antigen in the tumor tissue was further demonstrated by immunoblot assay. To our knowledge, this is the first report of the presence of JCV DNA, RNA, and T antigen in tissue in which viral T antigen is localized to tumor cell nuclei and suggests the possible association of JCV with some glial neoplasms. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

Rencic, A; Gordon, J; Otte, J; Curtis, M; Kovatich, A; Zoltick, P; Khalili, K; Andrews, D

1996-01-01

267

Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprotein, MEQ.  

PubMed Central

Marek's disease virus (MDV) is one of the most oncogenic herpesviruses and induces T lymphomas in chickens within weeks after infection. Only a limited number of viral transcripts are detected in MDV tumor samples and cell lines. One of the major transcripts encodes MEQ, a 339-amino-acid bZIP protein which is homologous to the Jun/Fos family of transcription factors. The C-terminal half of MEQ contains proline-rich repeats and, when fused to the DNA-binding domain of a yeast transcription factor, Gal4 (residues 1 to 147), exhibits transactivation function. MEQ can dimerize with itself and with c-Jun. The MEQ-c-Jun heterodimers bind to an AP-1-like enhancer within the MEQ promoter region with greater affinity than do homodimers of either protein, and they transactivate MEQ expression. Here we show that MEQ is expressed in the nucleus but, interestingly, with a predominant fraction in the nucleoli and coiled bodies. This makes MEQ the first bZIP protein to be identified in the nucleoli. MEQ contains two stretches of basic residues, designated basic region 1 (BR1) and basic region 2 (BR2). Using a series of deletion mutants, we have mapped the primary nuclear localization signal (NLS) and the sole nucleolar localization signal (NoLS) to the BR2 region. BR1 was shown to provide an auxiliary signal in nuclear translocation. To demonstrate that BR2 is an authentic NoLS, BR2 was fused to cytoplasmic v-Raf (delta gag) kinase. The BR2-Raf fusion protein was observed to migrate into the nucleoplasm and the nucleolus. The BR2 region can be further divided into two long arginine-lysine stretches, BR2N and BR2C, which are separated by the five amino acids Asn-Arg-Asp-Ala-Ala (NRDAA). We provide evidence that the requirement for nuclear translocation is less stringent than that for nucleolar translocation, as either BR2N or BR2C alone is sufficient to translocate the cytoplasmic v-Raf (delta gag) into the nucleus, but only in combination can they translocate v-Raf (delta gag) into the nucleolus. Our studies demonstrate that MEQ is both a nuclear and nucleolar protein, adding MEQ to the growing list of transactivators which localize to the nucleolus.

Liu, J L; Lee, L F; Ye, Y; Qian, Z; Kung, H J

1997-01-01

268

Kinetic screening of antibody-Im7 conjugates by capture on a colicin E7 DNase domain using optical biosensors.  

PubMed

Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark V(NAR) single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors. PMID:19073134

Hosse, Ralf J; Tay, Leigh; Hattarki, Meghan K; Pontes-Braz, Luisa; Pearce, Lesley A; Nuttall, Stewart D; Dolezal, Olan

2009-02-15

269

Polymorphism of Interleukin-6 Is Not Associated with the Presence or Absence of High HPV E6/E7.  

PubMed

The present study evaluated the frequency of the polymorphism of Interleukin-6 (IL6) in women positive for E6/E7 Human Papillomavirus (HPV) (n=152) and women negative for HPV (n=238), 390 women in total. Material for analysis was obtained at the Federal University of São Paulo. Interleukin-6 polymorphism was detected by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and analyzed in 3% agarose gel. Results: No significant associations between the frequency of the polymorphism of IL6 in patients expressing E6 and E7 with HPV-positive and -negative reactions were found. There was no statistically significant difference between the case and control group for genotype distribution (p=0.280). Conclusion: Genotypic analysis showed a striking similarity of IL6 polymorphisms in both cases and controls. The allelic distribution in cases and controls for G and C of IL6 were very similar (p=0.186), which could point to similar IL6 functionality for both groups. PMID:24982360

Porto, Claudia Regina Cinti; DE Oliveira Kleine, João Paulo Ferreira; Filho, Adhemar Longatto; DA Silva, Ismael Dale Cotrim Guerreiro

2014-07-01

270

High-affinity interaction of poly(ADP-ribose) and the human DEK oncoprotein depends upon chain length.  

PubMed

Poly(ADP-ribose) polymerase-1 (PARP-1) is a molecular DNA damage sensor that catalyzes the synthesis of the complex biopolymer poly(ADP-ribose) (PAR) under consumption of NAD(+). PAR engages in fundamental cellular processes such as DNA metabolism and transcription and interacts noncovalently with specific binding proteins involved in DNA repair and regulation of chromatin structure. A factor implicated in DNA repair and chromatin organization is the DEK oncoprotein, an abundant and conserved constituent of metazoan chromatin, and the only member of its protein class. We have recently demonstrated that DEK, under stress conditions, is covalently modified with PAR by PARP-1, leading to a partial release of DEK into the cytoplasm. Additionally, we have also observed a noncovalent interaction between DEK and PAR, which we detail here. Using sequence alignment, we identify three functional PAR-binding sites in the DEK primary sequence and confirm their functionality in PAR binding studies. Furthermore, we show that the noncovalent binding to DEK is dependent on PAR chain length as revealed by an overlay blot technique and a PAR electrophoretic mobility shift assay. Intriguingly, DEK promotes the formation of a defined complex with a 54mer PAR (K(D) = 6 x 10(-8) M), whereas no specific interaction is detected with a short PAR chain (18mer). In stark contrast to covalent poly(ADP-ribosyl)ation of DEK, the noncovalent interaction does not affect the overall ability of DEK to bind to DNA. Instead the noncovalent interaction interferes with subsequent DNA-dependent multimerization activities of DEK, as seen in South-Western, electrophoretic mobility shift, topology, and aggregation assays. In particular, noncovalent attachment of PAR to DEK promotes the formation of DEK-DEK complexes by competing with DNA binding. This was seen by the reduced affinity of PAR-bound DEK for DNA templates in solution. Taken together, our findings deepen the molecular understanding of the DEK-PAR interplay and support the existence of a cellular "PAR code" represented by PAR chain length. PMID:20669926

Fahrer, Jörg; Popp, Oliver; Malanga, Maria; Beneke, Sascha; Markovitz, David M; Ferrando-May, Elisa; Bürkle, Alexander; Kappes, Ferdinand

2010-08-24

271

PP2AB56? controls oncogene-induced senescence in normal and tumor human melanocytic cells  

Microsoft Academic Search

Oncoprotein C-MYC is overexpressed in human metastatic melanomas and melanoma-derived cells where it is required for the suppression of oncogene-induced senescence (OIS). The genetic events that maintain high levels of C-MYC in melanoma cells and their role in OIS are unknown. Here we report that C-MYC in cells from several randomly chosen melanoma lines was upregulated at the protein level,

S Mannava; A R Omilian; J A Wawrzyniak; E E Fink; D Zhuang; J C Miecznikowski; J R Marshall; M S Soengas; R C Sears; C D Morrison; M A Nikiforov

2012-01-01

272

International Conference on Harmonisation; Guidance on E7 Studies in Support of Special Populations; Geriatrics; Questions and Answers; availability. Notice.  

PubMed

The Food and Drug Administration (FDA) is announcing the availability of a guidance entitled ``E7 Studies in Support of Special Populations: Geriatrics; Questions and Answers.'' The guidance was prepared under the auspices of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The questions and answers (Q&A) guidance addresses special considerations for the design and conduct of clinical trials of drugs likely to have significant use in the elderly. The Q&As are intended to provide guidance on the use of geriatric data to adequately characterize and represent the safety and efficacy of a drug for a marketing application, including data collected postmarketing. PMID:22379685

2012-02-21

273

Oncoprotein protein kinase  

DOEpatents

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

Karin, M.; Hibi, M.; Lin, A.

1997-02-25

274

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression  

PubMed Central

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5? splice sites (5? ss) and three 3? splice sites (3? ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5? ss (nt 221 5? ss and nt 191 5? ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5? ss and nt 409 3? ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3? ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3? ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.

Ajiro, Masahiko; Jia, Rong; Zhang, Lifang; Liu, Xuefeng; Zheng, Zhi-Ming

2012-01-01

275

Analysis of human papillomavirus type 16 E6-E7 transcription in cervical carcinomas and normal cervical epithelium using the polymerase chain reaction  

Microsoft Academic Search

Cervical biopsies were collected from Birmingham women having cervical intraepithelial neoplasia or invasive cervical carcinoma and normal controls, and examined for the presence of human papillomavirus type 16 (HPV-16) E6-E7 DNA and mRNA using an adaptationof the polymerase chain reaction. HPV-16 E6-E7 sequences were detected in all abnormal biopsies and in 90% of the normal biopsies examined, confirming previous studies

M. A. Johnson; P. I. Blomfield; I. S. Bevan; C. B. J. Woodman; L. S. Young

1990-01-01

276

A C-terminal Hydrophobic, Solvent-protected Core and a Flexible N-terminus are Potentially Required for Human Papillomavirus 18 E7 Protein Functionality  

SciTech Connect

The oncogenic potential of the high-risk human papillomavirus (HPV) relies on the expression of genes specifying the E7 and E6 proteins. To investigate further the variation in oligomeric structure that has been reported for different E7 proteins, an HPV-18 E7 cloned from a Hispanic woman with cervical intraepithelial neoplasia was purified to homogeneity most probably as a stable monomeric protein in aqueous solution. We determined that one zinc ion is present per HPV-18 E7 monomer by amino acid and inductively coupled plasma-atomic emission spectroscopy analysis. Intrinsic fluorescence and circular dichroism spectroscopic results indicate that the zinc ion is important for the correct folding and thermal stability of HPV-18 E7. Hydroxyl radical mediated protein footprinting coupled to mass spectrometry and other biochemical and biophysical data indicate that near the C-terminus, the four cysteines of the two Cys-X{sub 2}-Cys motifs that are coordinated to the zinc ion form a solvent inaccessible core. The N-terminal LXCXE pRb binding motif region is hydroxyl radical accessible and conformationally flexible. Both factors, the relative flexibility of the pRb binding motif at the N-terminus and the C-terminal metal-binding hydrophobic solvent-protected core, combine together and facilitate the biological functions of HPV-18 E7.

Liu, S.; Tian, Y; Greenaway, F; Sun, M

2010-01-01

277

A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides.  

PubMed Central

Human adenovirus type 9 (Ad9) is unique among oncogenic adenoviruses in that it elicits exclusively mammary tumors in rats and requires the viral E4 region open reading frame 1 (9ORF1) gene for tumorigenicity. The 9ORF1 oncogenic determinant codes for a 14-kDa transforming protein, and three separate regions of this polypeptide, including one at the extreme C terminus, are necessary for transforming activity. In this study, we investigated whether the 9ORF1 transforming protein interacts with cellular factors. Following incubation with cell extracts, a glutathione S-transferase (GST)-9ORF1 fusion protein associated with several cellular phosphoproteins (p220, p180, p160, p155), whereas GST fusion proteins of transformation-defective 9ORF1 C-terminal mutants did not. Similar interactions requiring the 9ORF1 C terminus were revealed with protein-blotting assays, in which a GST-9ORF1 protein probe reacted specifically with cellular polypeptides having gel mobilities resembling those of the 9ORF1-associated cellular phosphoproteins, as well as with additional cellular polypeptides designated p140/p130. In addition, GST fusion proteins containing 9ORF1 C-terminal fragments associated with some of the 9ORF1-associated cellular polypeptides, as did GST fusion proteins of full-length wild-type Ad5 and Ad12 E4 ORF1 transforming proteins. Significantly, the results of coimmunoprecipitation analyses suggested that the same cellular polypeptides also associate with wild-type but not C-terminal-mutant 9ORF1 proteins in vivo. Together, these findings suggest that the 9ORF1 C terminus, which is essential for transformation, participates in specific and direct binding of the 9ORF1 oncoprotein to multiple cellular polypeptides. We propose that interactions with these cellular factors may be responsible, at least in part, for the transforming activity of the 9ORF1 viral oncoprotein.

Weiss, R S; Javier, R T

1997-01-01

278

Transcription of the E6 and E7 genes of human papillomavirus type 6 in anogenital condylomata is restricted to undifferentiated cell layers of the epithelium.  

PubMed Central

The E6 and E7 genes of human genital papillomaviruses (HPVs) appear to transform cells by different mechanisms. They seem to act synergistically but are not equally important when tested under diverse experimental conditions. We were therefore tempted to investigate the E6- and E7-specific transcription pattern in HPV6-infected condylomas separately, by in situ hybridization. Recent studies have identified three promoters within the E6-E7 region of HPV6 and HPV11 by applying S1, exonuclease VII, and cDNA analyses. On the basis of these data, we cloned subgenomic fragments of HPV6 into plasmid pBS to obtain riboprobes that differentiated between transcripts starting upstream of the E6 and E7 open reading frames, respectively. These different species of mRNAs were analyzed in serial thin sections of eight HPV6-positive anogenital condylomas. The E6 probe (nucleotides 7862 to 241) led to weak signals within the basal layer. In three cases, rather strong signals were confined to a few basal cells. The E7 probe (nucleotides 242 to 534) gave rise to a more pronounced labeling of all cells within the two to three lowest epidermal layers. In situ hybridization with a riboprobe for human c-fos revealed an expression pattern similar to that observed with the E7 probe. In contrast to the preferential expression of the transforming E6 and E7 genes in the lower epithelium, the major transcriptional activity of the virus was detected in the middle and upper third by probes colinear with the 3' moiety of the early region. Images

Iftner, T; Oft, M; Bohm, S; Wilczynski, S P; Pfister, H

1992-01-01

279

Expression and localization of human papillomavirus type 16 E6 and E7 open reading frame proteins in human epidermal keratinocyte.  

PubMed

Over 60 different types of human papillomavirus (HPV) have been identified, and they are classified into high and low risk groups based on the risk for malignant progression of HPV associated lesions. HPVs belonging to a high risk group have been shown to express two major transforming proteins, E6 and E7. With respect to the transforming activity of these proteins, many investigators have reported the location of these proteins in the cell, but their results are still controversial. In the present study, HPV type 16 E6 or E7 open reading frame (ORF) proteins were expressed and localized in human epidermal keratinocytes (RHEK-1) using the vaccinia virus as an expression vector. Immunofluorescence detection using monoclonal antibodies against E6 or E7 ORF proteins revealed that E6 or E7 proteins of HPV type 16 were located in the cytoplasm of RHEK-1 cells. These results suggest that E6 and E7 proteins bind to the tumor suppressor counterparts, thereby preventing transport of these proteins into the nucleus. These antioncogene products that fail to be rapidly transported out of the cytosol may be degraded by certain proteases such as the ubiquitin dependent system. In this way, the precise function of antioncogene products in the regulation of cell growth could be destroyed, and abnormal cell growth could occur. PMID:8009891

Kim, K H; Yoon, D J; Moon, Y A; Kim, Y S

1994-03-01

280

Studying high-spin ferric heme proteins by pulsed EPR spectroscopy: Analysis of the ferric form of the E7Q mutant of human neuroglobin  

Microsoft Academic Search

In this work, the high-spin ferric form of the E7Q mutant of human neuroglobin (E7Q-NGB) is studied by X-band continuous-wave\\u000a electron paramagnetic resonance (CW EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. It is shown that the use\\u000a of matched pulses in the HYSCORE experiment is essential to observe the nitrogen spectral contributions. The validity of approximating\\u000a the high-spin Fe(III) system

F. Trandafir; P. Heerdt; M. Fittipaldi; E. Vinck; S. Dewilde; L. Moens; S. Van Doorslaer

2007-01-01

281

Some Novel Insights on HPV16 Related Cervical Cancer Pathogenesis Based on Analyses of LCR Methylation, Viral Load, E7 and E2/E4 Expressions  

PubMed Central

This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1?E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1?E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 CT/E4 CT. Relative quantification based on comparative CT (theshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 CT (p?=?0.007) and E2 CT (p<0.0001), respectively, each normalized with ACTB CT, among episomal cases only. The k-means clustering analysis considering E7 CT from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

Mukhopadhyay, Indranil; Chowdhury, Rahul Roy; Roy, Sudipta; Sengupta, Sharmila

2012-01-01

282

Explicit computations of low-lying eigenfunctions for the quantum trigonometric Calogero-Sutherland model related to the exceptional algebra E 7  

NASA Astrophysics Data System (ADS)

In a previous paper, we studied the characters and Clebsch-Gordan series for the exceptional Lie algebra E7 by relating them to the quantum trigonometric Calogero-Sutherland Hamiltonian with the coupling constant ? = 1. We now extend that approach to the case of an arbitrary coupling constant.

Fernández Núñez, J.; García Fuertes, W.; Perelomov, A. M.

2008-02-01

283

Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia.  

PubMed

It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV-16 E7 specific immune responses in patients with different grades of cervical intra-epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T-cell responses were measured by IFN-gamma ELISPOT analysis, after stimulation with recombinant HPV-16 E7 protein. As expected, HPV-16 E7 specific IFN-gamma T cell responses were more frequent in HPV-16 DNA positive patients compared with that in HPV-16 DNA negative patients (39/50 vs. 16/36, (p=0.006, chi2 test). After invasive procedures, a small number of HPV-16 DNA positive CIN patients, but a considerable proportion of HPV-16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV-16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4+ and CD8+ T cells showed E7 specific IFN-gamma production upon in-vitro stimulation. Our study shows that invasive procedures may enhance HPV-specific cell-mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV-specific immune responses is used as intermediate end-point. PMID:16353143

Visser, Jeroen; van Baarle, Debbie; Hoogeboom, Baukje Nynke; Reesink, Nathalie; Klip, Harry; Schuuring, Ed; Nijhuis, Esther; Pawlita, Michael; Bungener, Laura; de Vries-Idema, Jacqueline; Nijman, Hans; Miedema, Frank; Daemen, Toos; van der Zee, Ate

2006-05-15

284

Targeting of N-CoR and histone deacetylase 3 by the oncoprotein v-erbA yields a chromatin infrastructure-dependent transcriptional repression pathway.  

PubMed

Transcriptional repression by nuclear hormone receptors is thought to result from a unison of targeting chromatin modification and disabling the basal transcriptional machinery. We used Xenopus oocytes to compare silencing effected by the thyroid hormone receptor (TR) and its mutated version, the oncoprotein v-ErbA, on partly and fully chromatinized TR-responsive templates in vivo. Repression by v-ErbA was not as efficient as that mediated by TR, was significantly more sensitive to histone deacetylase (HDAC) inhibitor treatment and, unlike TR, v-ErbA required mature chromatin to effect repression. We find that both v-ErbA and TR can recruit the corepressor N-CoR, but, in contrast to existing models, show a concomitant enrichment for HDAC3 that occurs without an association with Sin3, HDAC1/RPD3, Mi-2 or HDAC5. We propose a requirement for chromatin infrastructure in N-CoR/HDAC3-effected repression and suggest that the inability of v-ErbA to silence on partly chromatinized templates may stem from its impaired capacity to interfere with basal transcriptional machinery function. In support of this notion, we find v-ErbA to be less competent than TR for binding to TFIIB in vitro and in vivo. PMID:10921888

Urnov, F D; Yee, J; Sachs, L; Collingwood, T N; Bauer, A; Beug, H; Shi, Y B; Wolffe, A P

2000-08-01

285

Cyclin-dependent kinase subunit (Cks) 1 or Cks2 overexpression overrides the DNA damage response barrier triggered by activated oncoproteins.  

PubMed

Cyclin-dependent kinase subunit (Cks) proteins are small cyclin-dependent kinase-interacting proteins that are frequently overexpressed in breast cancer, as well as in a broad spectrum of other human malignancies. However, the mechanistic link between Cks protein overexpression and oncogenesis is still unknown. In this work, we show that overexpression of Cks1 or Cks2 in human mammary epithelial and breast cancer-derived cells, as well as in other cell types, leads to override of the intra-S-phase checkpoint that blocks DNA replication in response to replication stress. Specifically, binding of Cks1 or Cks2 to cyclin-dependent kinase 2 confers partial resistance to the effects of inhibitory tyrosine phosphorylation mediated by the intra-S-phase checkpoint, allowing cells to continue replicating DNA even under conditions of replicative stress. Because many activated oncoproteins trigger a DNA damage checkpoint response, which serves as a barrier to proliferation and clonal expansion, Cks protein overexpression likely constitutes one mechanism whereby premalignant cells can circumvent this DNA damage response barrier, conferring a proliferative advantage under stress conditions, and therefore contributing to tumor development. PMID:21697511

Liberal, Vasco; Martinsson-Ahlzén, Hanna-Stina; Liberal, Jennifer; Spruck, Charles H; Widschwendter, Martin; McGowan, Clare H; Reed, Steven I

2012-02-21

286

The TBC1D15 Oncoprotein Controls Stem Cell Self-Renewal through Destabilization of the Numb-p53 Complex  

PubMed Central

Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 tumor suppressor, in conjunction with its interacting partner protein Numb, preserves this asymmetry and functions as a vital barrier against the unchecked expansion of tumor stem cell pools; however, little is known about the biological control of the Numb-p53 interaction. We show here that Numb and p53 are the constituents of a high molecular mass complex, which is disintegrated upon activation of aPKC?, a Numb kinase. Using large-scale affinity purification and tandem mass spectrometry, we identify TBC1D15 as a Numb-associated protein and demonstrate that its amino-terminal domain disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency. Cellular levels of TBC1D15 are diminished upon acute nutrient deprivation through autophagy-mediated degradation, indicating that TBC1D15 serves as a conduit through which cellular metabolic status is linked to self-renewal. The profound deregulation of TBC1D15 expression exhibited in a diverse array of patient tumors underscores its proposed function as an oncoprotein.

Feldman, Douglas E.; Chen, Chialin; Punj, Vasu; Machida, Keigo

2013-01-01

287

Distinct regulatory effect of the p34SEI-1 oncoprotein on cancer metastasis in HER2/neu-positive and -negative cells.  

PubMed

The p34SEI-1 oncoprotein is involved in a transcriptional regulation, cell cycle regulation, apoptosis, development and many other important cellular functions. Our present study suggests that p34SEI-1 can promote metastasis by enhancing migration and invasion of cancer cells. Consistently, p34SEI-1 expression was found to be increased as the tumor invasiveness progressed in human breast tissues. p34SEI-1 may promote cancer metastasis by activating the PI3K/AKT signaling pathway. In this process, p34SEI-1 activates two different serine/threonine kinases, AKT or ILK, depending on the expression status of HER2/neu oncogene. In HER2/neu suppressed cancer cells, p34SEI-1 promoted metastasis mainly by activating AKT via phosphorylation of the 473 serine residue. In HER2/neu expressing cancer cells, p34SEI-1 overexpression downregulates HER2/neu expression, leading to the activation of another crucial serine/threonine kinase ILK due to phosphorylation of the 178 threonine residue instead of AKT. These results suggest that p34SEI-1 affects cancer metastasis by regulating two different signaling pathways depending on the HER2/neu expression level, in which AKT and ILK modulation can be stimulated by p34SEI-1 overexpression. PMID:24789658

Jung, Samil; Ohk, Jiyeon; Jeong, Dongjun; Li, Chengping; Lee, Soonduck; Duan, Jingjing; Kim, Changjin; Lim, Jong-Seok; Yang, Young; Kim, Keun-Il; Lee, Myeong-Sok

2014-07-01

288

Intracellular localization of the tumor suppressor HtrA1/Prss11 and its association with HPV16 E6 and E7 proteins.  

PubMed

We have a long-standing interest in a nuclear protease which appears to be involved in carcinogenesis. We recently identified the protease as high temperature requirement factor A 1 (HtrA1), also known as Prss11, which is member of an oxidative stress-response family of proteases. HtrA1 has been classified as a secreted protease involved in TGFbeta signaling, but recent work has shown HtrA1 to be a tumor suppressor. Here we show that processed forms of HtrA1 are found intracellularly and intranuclearly, and the active intranuclear form of HtrA1 shows an approximately Mr 29,000. Further, expression of HPV E6/E7 proteins is associated with a post-transcriptional up-regulation of HtrA1 (most notably the nuclear form), and HtrA1 is found associated with both HPV E6 and E7 proteins. PMID:18452160

Clawson, Gary A; Bui, Vuong; Xin, Ping; Wang, Ning; Pan, Weihua

2008-09-01

289

MUC1-C oncoprotein activates the ZEB1/miR-200c regulatory loop and epithelial-mesenchymal transition.  

PubMed

The epithelial-mesenchymal transition (EMT) is activated in cancer cells by ZEB1, a member of the zinc finger/homeodomain family of transcriptional repressors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in human carcinoma cells. The present studies in breast cancer cells demonstrate that the oncogenic MUC1-C subunit induces expression of ZEB1 by a NF-?B (nuclear factor kappa B) p65-dependent mechanism. MUC1-C occupies the ZEB1 promoter with NF-?B p65 and thereby promotes ZEB1 transcription. In turn, ZEB1 associates with MUC1-C and the ZEB1/MUC1-C complex contributes to the transcriptional suppression of miR-200c, an inducer of epithelial differentiation. The co-ordinate upregulation of ZEB1 and suppression of miR-200c has been linked to the induction of EMT. In concert with the effects of MUC1-C on ZEB1 and miR-200c, we show that MUC1-C induces EMT and cellular invasion by a ZEB1-mediated mechanism. These findings indicate that (i) MUC1-C activates ZEB1 and suppresses miR-200c with the induction of EMT and (ii) targeting MUC1-C could be an effective approach for the treatment of breast and possibly other types of cancers that develop EMT properties. PMID:23584475

Rajabi, H; Alam, M; Takahashi, H; Kharbanda, A; Guha, M; Ahmad, R; Kufe, D

2014-03-27

290

HPV16 E2 is an immediate early marker of viral infection, preceding E7 expression in precursor structures of cervical carcinoma.  

PubMed

The viral E2 gene product plays a crucial role in the human papillomavirus (HPV) vegetative cycle by regulating both transcription and replication of the viral genome. E2 is a transcriptional repressor of the E6 and E7 viral oncogenes for HPV types 16 and 18, which are involved in cervical cancers. Using new polyclonal antibodies against the HPV16 E2 protein, we showed that E2 is expressed at various precursor stages of cervical carcinoma by immunohistochemistry on paraffin-embedded clinical samples. E2 was found to be highly expressed in the nuclei and cytoplasm of cells forming the intermediate and upper layers of cervical intraepithelial neoplasia (CIN). We could show that the expressions of E2 and p16(INK4a) (surrogate marker for oncogenic E7 expression) were exclusive in most of the cases, thus implying that E2 is not expressed together with high levels of E7. Moreover, we found that E2 is expressed in a subset of columnar cells adjacent to the CIN. We could show that expression of E2 is topologically distinct from the proliferation markers p63 and Ki67, whereas it coincides with the expression of cytokeratin K13, a marker of squamous cell differentiation. Expression of E2 also topologically coincides with episomal amplification of viral genomes in the upper layers of CIN1. These in vivo data thus validate previous assumptions of the crucial role of E2 in the early steps of HPV infection and of its negative link with expression of the viral E6 and E7 oncogenes. PMID:20530671

Xue, Yuezhen; Bellanger, Sophie; Zhang, Wenying; Lim, Diana; Low, Jeffrey; Lunny, Declan; Thierry, Françoise

2010-07-01

291

Vesicular stomatitis virus-based therapeutic vaccination targeted to the E1, E2, E6, and E7 proteins of cottontail rabbit papillomavirus.  

PubMed

Persistent human papillomavirus (HPV)-associated benign and malignant lesions are a major cause of morbidity and mortality worldwide. Vaccination against HPV early proteins could provide an effective means of treating individuals with established infections. Recombinant vesicular stomatitis virus (VSV) vectors have been used previously to elicit strong humoral and cellular immune responses and develop prophylactic vaccines. We have shown that VSV vectors also can be used to elicit therapeutic immunity in the cottontail rabbit papillomavirus (CRPV)-rabbit model of high-risk HPV infection. In the present study, three new VSV vectors expressing the CRPV E1, E2, or E7 protein were produced and compared to the previously generated VSV-E6 vector for therapeutic efficacy. To determine whether vaccine efficacy could be augmented by simultaneous vaccination against two CRPV proteins, the four vaccines were delivered individually and in all possible pairings to rabbits 1 week after CRPV infection. Control rabbits received the recombinant wild-type VSV vector or medium only. Cumulative papilloma volumes were computed for analysis of the data. The analyses showed that VSV-based vaccination against the E1, E2, E6, or E7 protein significantly reduced papilloma volumes relative to those of the controls. Furthermore, VSV-based CRPV vaccination cured all of the papillomas in 5 of 30 rabbits. Of the individual vaccines, VSV-E7 was the most effective. The VSV-E7 vaccine alone was the most effective, as it reduced cumulative papilloma volumes by 96.9% overall, relative to those of the controls, and ultimately eliminated all of the disease in all of the vaccinees. Vaccine pairing was not, however, found to be beneficial, suggesting antigenic competition between the coexpressed CRPV proteins. These preclinical results, obtained in a physiologically relevant animal model of HPV infection, demonstrate that VSV vectors deserve serious consideration for further development as therapeutic antitumor vaccines. PMID:17392369

Brandsma, Janet L; Shylankevich, Mark; Su, Yuhua; Roberts, Anjeanette; Rose, John K; Zelterman, Daniel; Buonocore, Linda

2007-06-01

292

The Human DEK ProtoOncogene Is a Senescence Inhibitor and an Upregulated Target of High-Risk Human Papillomavirus E7  

Microsoft Academic Search

The human DEK proto-oncogene is a nucleic acid binding protein with suspected roles in human carcino- genesis, autoimmune disease, and viral infection. Intracellular DEK functions, however, are poorly understood. In papillomavirus-positive cervical cancer cells, downregulation of viral E6\\/E7 oncogene expression results in cellular senescence. We report here the specific repression of DEK message and protein levels in senescing human papillomavirus

Trisha M. Wise-Draper; Hillary V. Allen; Megan N. Thobe; Elizabeth E. Jones; Kristen B. Habash; Karl Munger; Susanne I. Wells

2005-01-01

293

Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility?†  

PubMed Central

Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.

Morrison, Monique A.; Morreale, Richard J.; Akunuru, Shailaja; Kofron, Matthew; Zheng, Yi; Wells, Susanne I.

2011-01-01

294

Casein Kinase II Motif-Dependent Phosphorylation of Human Papillomavirus E7 Protein Promotes p130 Degradation and S-Phase Induction in Differentiated Human Keratinocytes  

Microsoft Academic Search

The E7 proteins of human papillomaviruses (HPVs) promote S-phase reentry in differentiated keratinocytes of the squamous epithelia to support viral DNA amplification. In this study, we showed that nuclear p130 was present in the differentiated strata of several native squamous epithelia susceptible to HPV infection. In contrast, p130 was below the level of detection in HPV-infected patient specimens. In submerged

Nicholas J. Genovese; N. Sanjib Banerjee; Thomas R. Broker; Louise T. Chow

2008-01-01

295

Addendum: Why There Are No Elliptical Galaxies More Flattened Than E7. Thirty Years Later, Published in Serb. Astron. J. No. 173 (2006), 13-33  

NASA Astrophysics Data System (ADS)

The existence of some sort of instability with regard to prolate configurations has been advocated in an earlier paper (Caimmi 2006) in order to explain the observed lack of elliptical galaxies more elongated than E7. It is recognized that a viable physical process might be the occurrence of bending instabilities, which were fully investigated long time ago, both analytically (Polyachenko and Shukhman 1979) and numerically (Merritt and Hernquist 1991).

Caimmi, R.

2007-06-01

296

Dendritic cells treated with HPV16mE7 in a three-dimensional model promote the secretion of IL-12p70 and IFN-?.  

PubMed

Although the human papillomavirus (HPV) DNA therapeutic vaccine represents a promising approach to the prevention and treatment of cervical cancer, the mechanism of the HPV DNA vaccine is poorly understood. Moreover, current strategies have met with only limited success in preclinical and dendritic cell-based (DC-based) clinical research. In addition, two-dimensional (2-D) DC monolayers poorly mimic the physiology function in vivo. We used a three-dimensional (3-D) DC culture model in vitro to explore the immune mechanism of the HPV DNA vaccine. DCs were generated from peripheral blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cells, growing in 3-D collagen gel, were treated with pcDNA3.1-HPV16mE7 in vitro for 48 h. Compared to DCs treated with E7 in a 2-D culture model, the expression of co-stimulatory molecules CD80 and CD40 were significantly increased in the 3-D model (p<0.05), and a remarkable increase of IL-12 p70 was observed. However, we did not detect any obvious change in IL-10 in 3-D culture. In addition, we found that IFN-? expression increased when HPV16mE7-DC cells were co-cultured with T-cells for 96 h in the 3-D model, and HPV16mE7-DCs stimulated the proliferation of T lymphocytes more efficiently in the 3-D model than in the 2-D model (p<0.05). These results suggest that DCs in 3-D culture model have a notable effect on the enhancement of the HPV16 DNA vaccine's immune reaction and indicate that the DC-based 3-D model is a novel approach to study the HPV vaccine. PMID:21463625

Wang, Ya Ting; Li, WenSheng; Liu, QinShe; Guan, XiaoYing; Hu, Jun

2011-08-01

297

Genomic Instability and Cancer: Lessons Learned from Human Papillomaviruses  

PubMed Central

High-risk HPV E6 and E7 oncoproteins cooperate to subvert critical host cell cycle checkpoint control mechanisms in order to promote viral genome replication. This results not only in aberrant proliferation but also in host cellular changes that can promote genomic instability. The HPV-16 E7 oncoprotein was found to induce centrosome abnormalities thereby disrupting mitotic fidelity and increasing the risk for chromosome missegregation and aneuploidy. In addition, expression of the high-risk HPV E7 oncoprotein stimulates DNA replication stress as a potential source of DNA breakage and structural chromosomal instability. Proliferation of genomically unstable cells is sustained by several mechanisms including the accelerated degradation of claspin by HPV-16 E7 and the degradation of p53 by the high-risk HPV E6 oncoprotein. These results highlight the oncogenic potential of aberrant proliferation and opens new avenues for prevention of malignant progression, not only in HPV-associated cervical cancer but also in non-virally associated malignancies with disrupted cell cycle checkpoint control mechanisms.

Korzeniewski, Nina; Spardy, Nicole; Duensing, Anette; Duensing, Stefan

2010-01-01

298

The myxoid liposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-jB target genes by interaction with NFKBIZ  

Microsoft Academic Search

FUS (also called TLS), EWSR1 and TAF15 (also called TAF2N) are related genes involved in tumor type-specific fusion oncogenes in human malignancies. The FUS- DDIT3 fusion oncogene results from a t(12;16)(q13;p11) chromosome translocation and has a causative role in the initiation of myxoid\\/round cell liposarcomas (MLS\\/ RCLS). The FUS-DDIT3 protein induces increased expression of the CAAT\\/enhancer-binding protein (C\\/EBP) and nuclear

MK Andersson; C Forni; A Stahlberg; C Andersson; A Olofsson; R Mantovani; P Aman

2008-01-01

299

Discovery of multiple interacting partners of gankyrin, a proteasomal chaperone and an oncoprotein-Evidence for a common hot spot site at the interface and its functional relevance.  

PubMed

Gankyrin, a non-ATPase component of the proteasome and a chaperone of proteasome assembly, is also an oncoprotein. Gankyrin regulates a variety of oncogenic signaling pathways in cancer cells and accelerates degradation of tumor suppressor proteins p53 and Rb. Therefore gankyrin may be a unique hub integrating signaling networks with the degradation pathway. To identify new interactions that may be crucial in consolidating its role as an oncogenic hub, crystal structure of gankyrin-proteasome ATPase complex was used to predict novel interacting partners. EEVD, a four amino acid linear sequence seems a hot spot site at this interface. By searching for EEVD in exposed regions of human proteins in PDB database, we predicted 34 novel interactions. Eight proteins were tested and seven of them were found to interact with gankyrin. Affinity of four interactions is high enough for endogenous detection. Others require gankyrin overexpression in HEK 293 cells or occur endogenously in breast cancer cell line- MDA-MB-435, reflecting lower affinity or presence of a deregulated network. Mutagenesis and peptide inhibition confirm that EEVD is the common hot spot site at these interfaces and therefore a potential polypharmacological drug target. In MDA-MB-231 cells in which the endogenous CLIC1 is silenced, trans-expression of Wt protein (CLIC1_EEVD) and not the hot spot site mutant (CLIC1_AAVA) resulted in significant rescue of the migratory potential. Our approach can be extended to identify novel functionally relevant protein-protein interactions, in expansion of oncogenic networks and in identifying potential therapeutic targets. Proteins 2014; 82:1283-1300. © 2013 Wiley Periodicals, Inc. PMID:24338975

Nanaware, Padma P; Ramteke, Manoj P; Somavarapu, Arun K; Venkatraman, Prasanna

2014-07-01

300

Human oncoprotein MDM2 activates the Akt signaling pathway through an interaction with the repressor element-1 silencing transcription factor conferring a survival advantage to cancer cells  

PubMed Central

The current paradigm states that the Akt signaling pathway phosphorylates the human oncoprotein mouse double minute 2 (MDM2), leading to its nuclear translocation and degradation of the tumor suppressor p53. Here we report a novel Akt signaling pathway elicited by MDM2. Upregulation of endogenous MDM2 promotes, whereas its downregulation diminishes, Akt phosphorylation irrespective of p53 status. MDM2 requires phosphatidylinositol (PI)3-kinase activity for enhancing Akt phosphorylation and upregulates this activity by repressing transcription of the regulatory subunit p85 of PI3-kinase. MDM2 interacts with the repressor element-1 silencing transcription factor (REST), a tumor suppressor that functions by downregulating PI3-kinase activity and Akt phosphorylation, prevents localization of REST on the p85 promoter and represses p85 expression. The deletion mutant of MDM2 capable of upregulating Akt phosphorylation represses p85 expression and interferes with localization of REST on the p85 promoter, whereas the deletion mutant of MDM2 that does not increase Akt phosphorylation cannot perform these functions. Silencing of REST abrogates the ability of MDM2 to upregulate Akt phosphorylation and downregulate p85 expression, implicating the ability of MDM2 to interact with REST in its ability to inhibit p85 expression and activate Akt phosphorylation. Inhibition of MDM2-mediated Akt phosphorylation with an Akt-phosphorylation-specific inhibitor abrogates its ability to improve cell survival. Consistently, the Akt phosphorylation function of MDM2 was required for its ability to improve cell survival after treatment with a chemotherapeutic drug. Our report not only unravels a novel signaling pathway that contributes to cell survival but also implicates a p53-independent transcription regulatory function of MDM2 in Akt signaling.

Singh, S; Ramamoorthy, M; Vaughan, C; Yeudall, W A; Deb, S; Palit Deb, S

2013-01-01

301

Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms.  

PubMed Central

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.

Fisher, R. J.; Fivash, M.; Casas-Finet, J.; Erickson, J. W.; Kondoh, A.; Bladen, S. V.; Fisher, C.; Watson, D. K.; Papas, T.

1994-01-01

302

Vaccination with synthetic analog peptides derived from WT1 oncoprotein induces T-cell responses in patients with complete remission from acute myeloid leukemia  

PubMed Central

A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 ?g each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-? release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-?–secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia. This study was registered at www.clinicaltrials.gov as #NCT00398138.

Dao, Tao; Krug, Lee M.; Chanel, Suzanne; Korontsvit, Tatyana; Zakhaleva, Victoria; Zhang, Ronghua; Wolchok, Jedd D.; Yuan, Jianda; Pinilla-Ibarz, Javier; Berman, Ellin; Weiss, Mark; Jurcic, Joseph; Frattini, Mark G.; Scheinberg, David A.

2010-01-01

303

Serum- and oncoprotein-mediated induction of a gene with sequence similarity to the gene encoding carcinoembryonic antigen.  

PubMed Central

We describe the molecular identification of a gene, designated T1, whose expression in mouse NIH 3T3 cells is strongly induced by the Ha-ras(EJ) and v-mos oncogenes and by serum. The T1 gene encodes a 38.5-kDa protein, as predicted from its primary sequence and shown by in vitro translation. The protein was processed at its amino terminus and extensively modified by N-linked glycosylation in vitro in the presence of microsomal vesicles. Sequence comparison of T1 with the MIPSX data base (Max-Planck-Institut für Biochemie, Martinsried/Munich) revealed similarity to the human carcinoembryonic antigen, a tumor marker which is overexpressed in colon adenocarcinomas and in fetal tissues. Considerable sequence similarity has also been observed to the short conserved region of other proteins which, like carcinoembryonic antigen, are encoded by members of the immunoglobulin gene superfamily. Images

Klemenz, R; Hoffmann, S; Werenskiold, A K

1989-01-01

304

A New Crucial Protein Interaction Element That Targets the Adenovirus E4-ORF1 Oncoprotein to Membrane Vesicles?  

PubMed Central

Human adenovirus type 9 exclusively elicits mammary tumors in experimental animals, and the primary oncogenic determinant of this virus is the E4-ORF1 oncogene, as opposed to the well-known E1A and E1B oncogenes. The tumorigenic potential of E4-ORF1, as well as its ability to oncogenically stimulate phosphatidylinositol 3-kinase (PI3K), depends on a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with several different membrane-associated cellular PDZ proteins, including MUPP1, PATJ, MAGI-1, ZO-2, and Dlg1. Nevertheless, because certain E4-ORF1 mutations that alter neither the sequence nor the function of the PBM abolish E4-ORF1-induced PI3K activation and cellular transformation, we reasoned that E4-ORF1 must possess an additional crucial protein element. In the present study, we identified seven E4-ORF1 amino acid residues that define this new element, designated domain 2, and showed that it mediates binding to a 70-kDa cellular phosphoprotein. We also discovered that domain 2 or the PBM independently promotes E4-ORF1 localization to cytoplasmic membrane vesicles and that this activity of domain 2 depends on E4-ORF1 trimerization. Consistent with the latter observation, molecular-modeling analyses predicted that E4-ORF1 trimerization brings together six out of seven domain 2 residues at each of the three subunit interfaces. These findings importantly demonstrate that PI3K activation and cellular transformation induced by E4-ORF1 require two separate protein interaction elements, domain 2 and the PBM, each of which targets E4-ORF1 to vesicle membranes in cells.

Chung, Sang-Hyuk; Frese, Kristopher K.; Weiss, Robert S.; Prasad, B. V. Venkataram; Javier, Ronald T.

2007-01-01

305

Probing into the biological processes influenced by ESC factor and oncoprotein HMGA2 using iPSCs.  

PubMed

Induced pluripotent stem cells (iPSCs) are rapidly evolving into an important research tool due to their close resemblance with pluripotent embryonic stem cells (ESCs). Of particular interest at this point are iPSC applications in disease modeling and drug discovery/testing. The high mobility group AT-hook 2 (HMGA2) protein is a nonhistone chromatin factor normally expressed in ESCs and during early developmental stages. Aberrant HMGA2 expression is associated, for example, with abnormal body stature, diabetes mellitus, heart development and uterine leiomyomas. Furthermore, the protein is re-expressed in many primary tumor cells and plays an important role in metastasis. Here we used iPSC formation in conjunction with exogenous human HMGA2 expression to gain insight into biological functions of HMGA2. Gene expression profiling and gene ontology analyses showed that anatomical development and cell adhesion/differentiation processes are strongly affected by HMGA2. This could help to uncover, at the molecular level, some of the known phenotypic consequences of aberrant HMGA2 expression. Furthermore, our data showed that expression of key diabetes susceptibility genes is influenced by HMGA2, which revealed an interesting link to the recently indentified Lin28/let-7 pathway regulating mammalian glucose metabolism. Contrary to a previous report, our results indicate that HMGA2 is not involved in the regulation of telomerase gene expression. Finally, our data support a model in which tight regulation of intracellular HMGA2 levels is important both to maintain a pluripotent ESC state and to induce differentiation into certain cell lineages during later developmental stages. PMID:22547345

Morshedi, Amir; Ren, Zhonglu; Li, Jinming; Dröge, Peter

2013-08-01

306

Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits.  

PubMed

The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity. PMID:21167863

Bounds, Callie E; Hu, Jiafen; Cladel, Nancy M; Balogh, Karla; Christensen, Neil D

2011-02-01

307

Human papillomavirus 16 E2-, E6- and E7-specific T-cell responses in children and their mothers who developed incident cervical intraepithelial neoplasia during a 14-year follow-up of the Finnish Family HPV cohort  

PubMed Central

Background Human papillomavirus (HPV) infection has traditionally been regarded as a sexually transmitted disease (STD), but recent evidence implicates that an infected mother can transmit HPV to her newborn during pregnancy, at delivery, perinatal period or later. Given the lack of any studies on HPV-specific immune responses in children, we conducted HPV16-specific cell-mediated immune (CMI) monitoring of the mother-child pairs with known oral and genital HPV follow-up (FU) data since the delivery. In the Finnish Family HPV Study, 10 out of 331 mothers developed incident cervical intraepithelial neoplasia (CIN) during their 14-year FU. Our hypothesis according to the common dogma is that there is no HPV16 specific immune response in offspring of the CIN mother as she/he has not started the sexual life yet. Methods We used overlapping 30–35 mer peptides covering the entire HPV16 E2, E6 and E7 protein sequences. Assays for lymphocyte proliferation capacity, cytokine production and HPV16-specific Foxp3?+?CD25?+?CD4+ regulatory T-cells were performed. Results HPV16-specific proliferative T-cell responses were broader in children than in their mothers. Nine of 10 children had responses against both E2 peptide pools compared to only 4 of the 10 mothers. Six of the 10 children and only 2 mothers displayed reactivity to E6 and/or E7. The cytokine levels of IL-2 (p?=?0.023) and IL-5 (p?=?0.028) induced by all peptide pools, were also higher among children than their mothers. The children of the mothers with incident CIN3 had significantly higher IFN-? (p?=?0.032) and TNF-? (p?=?0.008) levels than other children. Conclusions Our study is the first to show that also children could have HPV-specific immunity. These data indicate that the children have circulating HPV16-specific memory T-cells which might have been induced by previous HPV16 exposure or ongoing HPV 16 infection.

2014-01-01

308

Prognostic Value of DNA and mRNA E6/E7 of Human Papillomavirus in the Evolution of Cervical Intraepithelial Neoplasia Grade 2  

PubMed Central

OBJECTIVE This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL). METHODS This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months. RESULTS Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant. CONCLUSIONS The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution.

Discacciati, Michelle G; da Silva, Ismael DCG; Villa, Luisa L; Reis, Leandro; Hayashi, Priscila; Costa, Maria C; Rabelo-Santos, Silvia H; Zeferino, Luiz C

2014-01-01

309

EVI1 oncoprotein interacts with a large and complex network of proteins and integrates signals through protein phosphorylation  

PubMed Central

Ecotropic viral integration site-1 (EVI1) is an oncogenic zinc finger transcription factor whose expression is frequently up-regulated in myeloid leukemia and epithelial cancers. To better understand the mechanisms underlying EVI1-associated disease, we sought to define the EVI1 interactome in cancer cells. By using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we could confidently assign 78 proteins as EVI1-interacting partners for FLAG-tagged EVI1. Subsequently, we showed that 22 of 27 tested interacting proteins could coimmunoprecipitate with endogenous EVI1 protein, which represented an 81.5% validation rate. Additionally, by comparing the stable isotope labeling by amino acids in cell culture (SILAC) data with high-throughput yeast two hybrid results, we showed that five of these proteins interacted directly with EVI1. Functional classification of EVI1-interacting proteins revealed associations with cellular transcription machinery; modulators of transcription; components of WNT, TGF-?, and RAS pathways; and proteins regulating DNA repair, recombination, and mitosis. We also identified EVI1 phosphorylation sites by MS analysis and showed that Ser538 and Ser858 can be phosphorylated and dephosphorylated by two EVI1 interactome proteins, casein kinase II and protein phosphatase-1?. Finally, mutations that impair EVI1 phosphorylation at these sites reduced EVI1 DNA binding through its C-terminal zinc finger domain and induced cancer cell proliferation. Collectively, these combinatorial proteomic approaches demonstrate that EVI1 interacts with large and complex networks of proteins, which integrate signals from various different signaling pathways important for oncogenesis. Comprehensive analysis of the EVI1 interactome has thus provided an important resource for dissecting the molecular mechanisms of EVI1-associated disease.

Bard-Chapeau, Emilie A.; Gunaratne, Jayantha; Kumar, Pankaj; Chua, Belinda Q.; Muller, Julius; Bard, Frederic A.; Blackstock, Walter; Copeland, Neal G.; Jenkins, Nancy A.

2013-01-01

310

Expression of E6 and E7 papillomavirus oncogenes in the outer root sheath of hair follicles extends the growth phase and bypasses resting at telogen.  

PubMed

Hair follicle growth cycle proceeds through a series of stages in which strict control of cell proliferation, differentiation, and cell death occurs. Transgenic mice expressing human papillomavirus type 16 E6/E7 papillomavirus oncogenes in the outer root sheath (ORS) display a fur phenotype characterized by lower hair density and the ability to regenerate hair much faster than wild-type mice. Regenerating hair follicles of transgenic mice show a longer growth phase (anagen), and although bulb regression (catagen) occurs, rest at telogen was not observed. No abnormalities were detected during the first cycle of hair follicle growth, but by the second cycle, initiation of catagen was delayed, and rest at telogen was again not attained, even in the presence of estradiol, a telogen resting signal. In conclusion, expression of E6/E7 in the ORS delays entrance to catagen and makes cells of the ORS insensitive to telogen resting signals bearing to a continuous hair follicle cycling in transgenic mice. PMID:11063126

Escalante-Alcalde, D; Recillas-Targa, F; Valencia, C; Santa-Olalla, J; Chávez, P; Marroquín, A; Gutiérrez-X; Gariglio, P; Covarrubias, L

2000-10-01

311

Comparison of oncogenic HPV type-specific viral DNA load and E6/E7 mRNA detection in cervical samples: results from a multicenter study.  

PubMed

High-risk human papillomavirus (HR-HPV) genotype viral load and E6/E7 mRNA detection are proposed as surrogate markers of malignant cervical lesion progression. Currently, the use of commercially available DNA-based or mRNA-based tests is under investigation. In this study, the viral DNA load and E6/E7 mRNA detection of the five most common HR-HPV types detected in cervical cancer worldwide were compared in 308 cervical samples by using in-house type-specific quantitative real-time PCR assays and PreTect HPV-Proofer test, respectively. Sensitivity and negative predictive values were higher for the HPV-DNA assays combined (95.0% and 96.0%, respectively) than the RNA assays (77.0% and 88.0%, respectively); conversely, the mRNA test showed a higher specificity and higher positive predictive value (81.7% and 66.9%, respectively) than the DNA test (58.6% and 52.5%, respectively) for detecting histology-confirmed high-grade cervical intraepithelial neoplasia. A significantly higher association between viral DNA load and severity of disease was observed for HPV 16 and 31 (? = 0.62 and ? = 0.40, respectively) than for the other HPV types screened. A good degree of association between the two assays was found for detection of HPV 16 (k = 0.83), HPV 18 (k = 0.72), HPV 33 (k = 0.66), and HPV 45 (k = 0.60) but not for HPV 31 (k = 0.24). Sequence analysis in L1 and E6-LCR regions of HPV 31 genotypes showed a high level of intra-type variation. HR-HPV viral DNA load was significantly higher in E6/E7 mRNA positive than negative samples (P < 0.001), except for HPV 31. These findings suggest that transcriptional and replicative activities can coexist within the same sample. PMID:23280876

Broccolo, Francesco; Fusetti, Lisa; Rosini, Sandra; Caraceni, Donatella; Zappacosta, Roberta; Ciccocioppo, Lucia; Matteoli, Barbara; Halfon, Philippe; Malnati, Mauro S; Ceccherini-Nelli, Luca

2013-03-01

312

Therapeutic vaccination of rabbits with a ubiquitin-fused papillomavirus E1, E2, E6 and E7 DNA vaccine.  

PubMed

Previously, we showed that intracutaneous vaccination of rabbits with DNA vectors encoding ubiquitin-fused versions of the cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, E6 and E7 protected against subsequent challenge with CRPV. Here, we tested the immunotherapeutic activity of a vaccine composed of the four CRPV DNA vectors (designated UbE1267) in rabbits. The results show that the UbE1267 DNA vaccine, relative to empty vector DNA, virtually eliminated papilloma growth in rabbits with subclinical infection and greatly reduced papilloma volumes in rabbits bearing papillomas at the time of vaccination. These results in a physiologically relevant animal model of high-risk human papillomavirus (HPV) infection indicate that DNA vaccines targeting the early papillomavirus proteins may have a role in the treatment of HPV-associated lesions in humans. PMID:17630050

Brandsma, Janet L; Shlyankevich, Mark; Zelterman, Daniel; Su, Yuhua

2007-08-14

313

In Vivo Processing of DNase Colicins E2 and E7 Is Required for Their Import into the Cytoplasm of Target Cells  

PubMed Central

DNase colicins E2 and E7, both of which appropriate the BtuB/Tol translocation machinery to cross the outer membrane, undergo a processing step as they enter the cytoplasm. This endoproteolytic cleavage is essential for their killing action. A processed form of the same size, 18.5 kDa, which corresponds to the C-terminal catalytic domain, was detected in the cytoplasm of bacteria treated with either of the two DNase colicins. The inner-membrane protease FtsH is necessary for the processing that allows the translocation of the colicin DNase domain into the cytoplasm. The processing occurs near residue D420, at the same position as the FtsH-dependent cleavage in RNase colicins E3 and D. The cleavage site is located 30 amino acids upstream of the DNase domain. In contrast, the previously reported periplasm-dependent colicin cleavage, located at R452 in colicin E2, was shown to be generated by the outer-membrane protease OmpT and we show that this cleavage is not physiologically relevant for colicin import. Residue R452, whose mutated derivatives led to toxicity defect, was shown to have no role in colicin processing and translocation, but it plays a key role in the catalytic activity, as previously reported for other DNase colicins. Membrane associated forms of colicins E2 and E7 were detected on target cells as proteinase K resistant peptides, which include both the receptor-binding and DNase domains. A similar, but much less proteinase K-resistant form was also detected with RNase colicin E3. These colicin forms are not relevant for colicin import, but their detection on the cell surface indicates that whole nuclease-colicin molecules are found in a stable association with the outer-membrane receptor BtuB of the target cells.

Mora, Liliana; de Zamaroczy, Miklos

2014-01-01

314

Oncoprotein protein kinase antibody kit  

DOEpatents

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA)

2008-12-23

315

Comparison of peptide enzyme-linked immunosorbent assay and radioimmunoprecipitation assay with in vitro-translated proteins for detection of serum antibodies to human papillomavirus type 16 E6 and E7 proteins.  

PubMed Central

Antibodies to human papilloma virus (HPV) type 16 (HPV-16) E6 and E7 proteins in serum are markers for HPV-associated invasive cervical carcinoma. We compared two assays, a radioimmunoprecipitation assay with in vitro-translated HPV-16 E6 and E7 proteins and an enzyme-linked immunosorbent assay (ELISA) with E6 and E7 synthetic peptides, for their abilities to discriminate serologically between patients with invasive cervical cancer and controls. Among the patients, antibody prevalences were higher by the E6 radioimmunoprecipitation assay (55.7%) than by the E6 peptide ELISA (15.5%), but among the controls, they were lower by the radioimmunoprecipitation assay (1.7%) than by the E6 peptide ELISA (5%). For E7, antibody prevalences among the patients were comparable by the radioimmunoprecipitation assay (43%) and the peptide ELISA (41%), but among the controls they were higher by the E7 peptide ELISA (17.4%) than by the radioimmunoprecipitation assay (4.1%). There was good agreement between the E7 radioimmunoprecipitation assay and the E7 peptide ELISA among patients but not among controls. In tests with representative sera, heat denaturation of the translated proteins resulted in a complete loss of reactivity to the E6 protein and a marked decrease in reactivity to the E7 protein. Our study showed that the radioimmunoprecipitation assay discriminates better than the peptide ELISA between patients with invasive cervical cancer and controls and that this is related to the ability of the radioimmunoprecipitation assay to detect conformational epitopes. Images

Sun, Y; Shah, K V; Muller, M; Munoz, N; Bosch, X F; Viscidi, R P

1994-01-01

316

HPV E6/E7 RNA in situ hybridization signal patterns as biomarkers of three-tier cervical intraepithelial neoplasia grade.  

PubMed

Cervical lesion grading is critical for effective patient management. A three-tier classification (cervical intraepithelial neoplasia [CIN] grade 1, 2 or 3) based on H&E slide review is widely used. However, for reasons of considerable inter-observer variation in CIN grade assignment and for want of a biomarker validating a three-fold stratification, CAP-ASCCP LAST consensus guidelines recommend a two-tier system: low- or high-grade squamous intraepithelial lesions (LSIL or HSIL). In this study, high-risk HPV E6/E7 and p16 mRNA expression patterns in eighty-six CIN lesions were investigated by RNAscope chromogenic in situ hybridization (CISH). Specimens were also screened by immunohistochemistry for p16INK4a (clone E6H4), and by tyramide-based CISH for HPV DNA. HPV genotyping was performed by GP5+/6+ PCR combined with cycle-sequencing. Abundant high-risk HPV RNA CISH signals were detected in 26/32 (81.3%) CIN 1, 22/22 (100%) CIN 2 and in 32/32 (100%) CIN 3 lesions. CIN 1 staining patterns were typified (67.7% specimens) by abundant diffusely staining nuclei in the upper epithelial layers; CIN 2 lesions mostly (66.7%) showed a combination of superficial diffuse-stained nuclei and multiple dot-like nuclear and cytoplasmic signals throughout the epithelium; CIN 3 lesions were characterized (87.5%) by multiple dot-like nuclear and cytoplasmic signals throughout the epithelial thickness and absence/scarcity of diffusely staining nuclei (trend across CIN grades: P<0.0001). These data are consistent with productive phase HPV infections exemplifying CIN 1, transformative phase infections CIN 3, whereas CIN 2 shows both productive and transformative phase elements. Three-tier data correlation was not found for the other assays examined. The dual discernment of diffuse and/or dot-like signals together with the assay's high sensitivity for HPV support the use of HPV E6/E7 RNA CISH as an adjunct test for deciding lesion grade when CIN 2 grading may be beneficial (e.g. among young women) or when 'LSIL vs. HSIL' assignment is equivocal. PMID:24625757

Evans, Mark F; Peng, Zhihua; Clark, Kelli M; Adamson, Christine S-C; Ma, Xiao-Jun; Wu, Xingyong; Wang, Hongwei; Luo, Yuling; Cooper, Kumarasen

2014-01-01

317

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli.  

PubMed

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused approximately 5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem-loop distal to cei, and the putative CsrA binding site overlapping the Shine-Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions. PMID:20378712

Yang, Tsung-Yeh; Sung, Yun-Min; Lei, Guang-Sheng; Romeo, Tony; Chak, Kin-Fu

2010-07-01

318

Epithelium and fibroblast-like phenotypes derived from HPV16 E6/E7-immortalized human gingival keratinocytes following chronic ethanol treatment.  

PubMed

Epithelial-mesenchymal transition (EMT) may be critical for neoplastic progression and its eventual tumorigenicity of epithelia. In this context, we investigated whether EMT and EMT-associated features occurred after chronic ethanol treatment of human gingival keratinocytes immortalized with the E6/E7 oncogenes of human papillomavirus (HPV) type 16. Following a nine-week treatment of cells with 30 mM ethanol in keratinocyte growth medium, they were cultured in normal DMEM with 10% serum. These cell populations were able to proliferate in this medium gradually exhibiting elongated morphology indicating that these cells underwent EMT. Control cells without ethanol treatment did not survive subcultures in DMEM. Upon long-term subcultures of ethanol-treated cells, two phenotypes were obtained exhibiting epithelium-like and spindle-shape fibroblast-like morphology (respectively, termed as EPI and FIB cells), the latter indicating EMT. In comparison to EPI cells, the phenotypic transition to FIB cells was concomitant with a decrease in the expression of keratins, desmoplakins and a complete loss of K14. Moreover, FIB cell transition strongly correlates with an increase in the expression of vimentin and simple epithelial keratin K18. These alterations in FIB cells were associated with the ability of these cells to exhibit anchorage-independent growth, while EPI cells exhibited anchorage-dependent growth. Concerning the transformation stage, FIB cells represent a progressively more advanced transformed phenotype which may reflect an early step during HPV- and ethanol-dependent multi-step carcinogenesis. PMID:12868599

Chamulitrat, Walee; Schmidt, Rainer; Chunglok, Warangkana; Kohl, Annette; Tomakidi, Pascal

2003-06-01

319

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli  

PubMed Central

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused ?5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem–loop distal to cei, and the putative CsrA binding site overlapping the Shine–Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions.

Yang, Tsung-Yeh; Sung, Yun-Min; Lei, Guang-Sheng; Romeo, Tony; Chak, Kin-Fu

2010-01-01

320

Degradation of p53 only is not sufficient for the growth stimulatory effect of human papillomavirus 16 E6 oncoprotein in human embryonic fibroblasts.  

PubMed

Certain types of human papillomavirus (HPV), such as types 16 and 18, are thought to be responsible for the development of cervical carcinomas. The E6 and E7 genes of these viruses have transforming activities in various cultured cells and their mRNAs and proteins are expressed in almost all cervical carcinoma cells. Inactivation of the tumor suppressor p53 protein by the E6 gene is believed to be critical for transformation by these oncogenic HPVs. To determine whether degradation of the p53 protein is, in fact, sufficient for cellular transformation by the E6 gene, the E6 gene of HPV16 was introduced into human embryonic fibroblasts (HEF) using recombinant murine retrovirus and examined whether reduction of the p53 protein could substitute for the E6 function. It was found that HEF cells transfected with the E6 gene showed an increased saturation density and degraded the p53 protein. However, when expression of the p53 protein in normal HEF cells was suppressed by the antisense oligonucleotide of the p53 gene, growth stimulation was not observed. These results show that the E6 gene stimulates growth of HEF cells, but that this activity involves some other E6 gene-mediated functions than degradation of the p53 protein. PMID:7852968

Ishiwatari, H; Hayasaka, N; Inoue, H; Yutsudo, M; Hakura, A

1994-11-01

321

Isolation of flat revertants from human papillomavirus type 18 E6E7 transformed 3Y1 cells by transfection with a rat embryo fibroblast cDNA expression library.  

PubMed

A rat embryo fibroblast (REF) cDNA expression library was transfected into 3Y1 cells transformed by human papillomavirus type 18 E6 and E7 genes and 10 flat revertants were isolated. These revertants expressed the same levels of E6 and E7 mRNA as the parent cells, but had greatly reduced ability to form colonies in soft agar. Suppression of transformation was dominant in cell hybrids generated by fusing each revertant with the parental transformed cells. Furthermore, loss of transfected cDNA was observed in re-transformed cell hybrids derived from one flat revertant. Overexpression of the cDNA suppresses the colony-forming efficiency of the cells transformed by E6 and E7 genes. PMID:8033227

Nakanishi, K; Yong-Il, H; Ishiwatari, H; Takami, Y; Hayasaka, N; Yutsudo, M; Nojima, H; Hakura, A

1993-12-01

322

The bmi-1 oncoprotein is differentially expressed in non-small cell lung cancer and correlates with INK4A-ARF locus expression  

Microsoft Academic Search

Genes of the polycomb group function by silencing homeotic selector genes that regulate embryogenesis. In mice, downregulation of one of the polycomb genes, bmi-1, leads to neurological alterations and severe proliferative defects in lymphoid cells, whilst bmi-1 overexpression, together with upregulation of myc-1, induces lymphoma. An oncogenic function has been further supported in primary fibroblast studies where bmi-1 overexpression induces

S Vonlanthen; J Heighway; H J Altermatt; M Gugger; A Kappeler; M M Borner; M van Lohuizen; D C Betticher

2001-01-01

323

Suppression of tumor growth by the 3' untranslated region of mel-18 in 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18.  

PubMed

By introducing a cDNA library derived from rat embryonic fibroblast cells, we isolated several morphologically flat revertants of rat 3Y1 cells transformed by the E6 and E7 genes of human papillomavirus type 18 (HPV18). From one of the revertants, we recovered a 0.2-kb cDNA, N56, that suppresses the tumor growth of the transformed 3Y1 cells irrespective of the expression of the E6 and E7 genes. The nucleotide sequence of the cDNA was shown to be identical to that of the 3' untranslated region of a putative mammalian polycomb group gene, mel-18. PMID:9233832

Ishiwatari, H; Nakanishi, K; Kondoh, G; Hayasaka, N; Li, Q; Yamashita, A; Inoue, H; Hakura, A

1997-07-15

324

Signaling of DNA damage is not sufficient to induce p53 response: (re)activation of wt p53 protein strongly depends on cellular context.  

PubMed

It is generally accepted that exposure of cells to a variety of DNA-damaging agents leads to up-regulation and activation of wild-type (wt) p53 protein. We investigated the (re)-activation of p53 protein in two human cancer cell lines in which the gene for this tumor suppressor is not mutated: HeLaS(3) cervix carcinoma and MCF-7 breast cancer cells, by induction via different genotoxic and cytotoxic stimuli. Treatment of human cells with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or different anti-cancer drugs resulted in a strong DNA damage as evidenced by Comet assay and a marked increase in site-specific phosphorylation of H2AX. Unlike in MCF-7 cells, in HeLaS(3) cells the expression of p53 protein did not increase after MNNG treatment despite a strong DNA damage. However, other agents for example doxorubicin markedly induced p53 response in HeLaS(3) cells. After exposure of these cells to MNNG, the ATM-dependent effector proteins Chk2 and NBS1 were phosphorylated, thereby evidencing that MNNG-induced DNA breakage was recognized and properly signaled. In HeLaS(3) cells wt p53 protein is not functional due to E6-mediated targeting for accelerated ubiquitylation and degradation. Therefore, the activation of a p53 response to genotoxic stress in HeLaS(3) cells seems to depend on the status of E6 oncoprotein. Indeed, the induction of p53 protein in HeLaS(3) cells in response to distinct agents inversely correlates with the cellular level of E6 oncoprotein. This implicates that the capability of different agents to activate p53 in HeLaS(3) cells primarily depends on their inhibitory effect on expression of E6 oncoprotein. PMID:17879942

Wesierska-Gadek, Józefa; Gueorguieva, Marieta; Komina, Oxana; Schmid, Gerald; Kramer, Matthias P

2008-04-01

325

Human Papillomavirus 16 E6 Contributes HIF-1? Induced Warburg Effect by Attenuating the VHL-HIF-1? Interaction  

PubMed Central

Cervical cancer is still one of the leading causes of cancer deaths in women worldwide, especially in the developing countries. It is a major metabolic character of cancer cells to consume large quantities of glucose and derive more energy by glycolysis even in the presence of adequate oxygen, which is called Warburg effect that can be exaggerated by hypoxia. The high risk subtype HPV16 early oncoprotein E6 contributes host cell immortalization and transformation through interacting with a number of cellular factors. Hypoxia-inducible factor 1? (HIF-1?), a ubiquitously expressed transcriptional regulator involved in induction of numerous genes associated with angiogenesis and tumor growth, is highly increased by HPV E6. HIF-1? is a best-known target of the von Hippel-Lindau tumor suppressor (VHL) as an E3 ligase for degradation. In the present work, we found that HPV16 E6 promotes hypoxia induced Warburg effect through hindering the association of HIF-1? and VHL. This disassociation attenuates VHL-mediated HIF-1? ubiquitination and causes HIF-1? accumulation. These results suggest that oncoprotein E6 plays a major role in the regulation of Warburg effect and can be a valuable therapeutic target for HPV-related cancer.

Guo, Yi; Meng, Xiangkai; Ma, Jiaming; Zheng, Yahong; Wang, Qian; Wang, Yanan; Shang, Hong

2014-01-01

326

Human Papillomavirus 16 E6 Contributes HIF-1? Induced Warburg Effect by Attenuating the VHL-HIF-1? Interaction.  

PubMed

Cervical cancer is still one of the leading causes of cancer deaths in women worldwide, especially in the developing countries. It is a major metabolic character of cancer cells to consume large quantities of glucose and derive more energy by glycolysis even in the presence of adequate oxygen, which is called Warburg effect that can be exaggerated by hypoxia. The high risk subtype HPV16 early oncoprotein E6 contributes host cell immortalization and transformation through interacting with a number of cellular factors. Hypoxia-inducible factor 1? (HIF-1?), a ubiquitously expressed transcriptional regulator involved in induction of numerous genes associated with angiogenesis and tumor growth, is highly increased by HPV E6. HIF-1? is a best-known target of the von Hippel-Lindau tumor suppressor (VHL) as an E3 ligase for degradation. In the present work, we found that HPV16 E6 promotes hypoxia induced Warburg effect through hindering the association of HIF-1? and VHL. This disassociation attenuates VHL-mediated HIF-1? ubiquitination and causes HIF-1? accumulation. These results suggest that oncoprotein E6 plays a major role in the regulation of Warburg effect and can be a valuable therapeutic target for HPV-related cancer. PMID:24810689

Guo, Yi; Meng, Xiangkai; Ma, Jiaming; Zheng, Yahong; Wang, Qian; Wang, Yanan; Shang, Hong

2014-01-01

327

DDIT3\\/CHOP and the sarcoma fusion oncoprotein FUS-DDIT3\\/TLS-CHOP bind cyclin-dependent kinase 2  

Microsoft Academic Search

BACKGROUND: The DDIT3 gene encodes a transcription factor belonging to the CCAAT\\/enhancer binding protein (C\\/EBP) family. It is normally expressed at very low levels but is activated by cellular stress conditions and induces G1 arrest and, in some cell types, apoptosis. DDIT3 is found as a part of the fusion oncogene FUS-DDIT3 that is causal for the development of myxoid\\/round-cell

Christoffer Bento; Mattias K Andersson; Pierre Åman

2009-01-01

328

Epstein-Barr virus oncoprotein LMP1 mediates survivin upregulation by p53 contributing to G1/S cell cycle progression in nasopharyngeal carcinoma  

PubMed Central

Latent membrane protein 1 (LMP1) is an important oncogenic protein encoded by Epstein-Barr virus (EBV) and plays an important role in the development of nasopharyngeal carcinoma (NPC). Our previous study has shown that p53 protein was accumulated and phosphorylated in NPC, implying its transcription factor activity in NPC tumorigenesis. However, the biological function and potential downstream target of p53 mediated by LMP1 in NPC remain unknown. In this study, we found that LMP1 simultaneously induced upregulation of both p53 and survivin at the protein level, as well as their phosphorylation. Knockdown of p53 by siRNA revealed that LMP1 increased survivin expression by p53 directly. Furthermore, we found that LMP1 upregulated survivin by p53 at the transcriptional level by increasing p53-mediated survivin promoter activity and DNA binding activity. Moreover, LMP1 induced the co-localization of p53 and survivin in the nucleus, conferring to their related functions in NPC tumorigenesis. We further found that p53 promoted G1/S cell cycle progression, but did not induce apoptosis in LMP1-positive NPC cells. Collectively, these findings suggest that p53 acting as a transcription factor promotes the transcriptional activity of survivin, and further increases its protein expression and phosphorylation in the regulation of LMP1, thus, leading to G1/S cell cycle progression with no effect on apoptosis in NPC tumorigenesis.

GUO, LILI; TANG, MIN; YANG, LIFANG; XIAO, LANBO; BODE, ANN M.; LI, LILI; DONG, ZIGANG; CAO, YA

2012-01-01

329

Differences in signaling through the B-cell leukemia oncoprotein CRLF2 in response to TSLP and through mutant JAK2  

PubMed Central

Approximately 10% of B-cell acute lymphoblastic leukemias (B-ALLs) overexpress the cytokine receptor subunit CRLF2, which may confer a poor prognosis. CRLF2 binds its ligand thymic stromal lymphopoietin (TSLP) as a heterodimer with IL7R. Subsets of CRLF2-overexpressing B-ALLs also have a gain-of-function CRLF2 F232C mutation or activating mutations in JAK2. Whether these mutant alleles confer differences in signaling has not been addressed. Through a domain mutation analysis, we demonstrate a distinct dependence on the CRLF2 intracellular tyrosine Y368 in signaling by CRLF2 F232C, but not signaling induced by TSLP or through CRLF2/mutant JAK2. In contrast, CRLF2 signaling in each context is strictly dependent on both the CRLF2 box1 domain and the intracellular tryptophan W286. Using a global quantitative analysis of tyrosine phosphorylation induced by TSLP, we previously identified TSLP-induced phosphorylation of multiple kinases implicated in B-cell receptor signaling, including Lyn, Btk, Hck, Syk, MAPK8, MAPK9, and MAPK10. We now demonstrate that cells dependent on CRLF2/mutant JAK2 have reduced phosphorylation at these targets, suggesting that the kinases promote TSLP-mediated proliferation but serve as negative regulators of CRLF2/mutant JAK2 signaling. Thus, targetable nodes downstream of CRLF2 differ based on the presence or absence of additional mutations in CRLF2 signaling components.

van Bodegom, Diederik; Zhong, Jun; Kopp, Nadja; Dutta, Chaitali; Kim, Min-Sik; Bird, Liat; Weigert, Oliver; Tyner, Jeffrey; Pandey, Akhilesh; Yoda, Akinori

2012-01-01

330

Regression of Cervical Intraepithelial Neoplasia and Loss of Human Papillomavirus (HPV) Infection Is Associated with Cell-mediated Immune Responses to an HPV Type 16 E7 Peptide1  

Microsoft Academic Search

Most human papillomavirus (HPV)-associated cervical intraepithelial neoplasia (CIN) lesions in normal women regress spontaneously, but a small number persist and may progress to invasive cancer. To evaluate the role of immunity to HPV and the outcome of CIN and associated HPV infection, we examined cell-mediated immune (CMI) responses to HPV 16 E6 and E7 peptides. One hundred thirty-six women with

Anna S. Kadish; Patrick Timmins; Yuexian Wang; Gloria Y. F. Ho; Robert D. Burk; John Ketz; Seymour L. Romney; Anne Johnson; Ruth Angeletti; Maria Abadi

331

Comparison of the detection of HPV-16, 18, 31, 33, and 45 by type-specific DNA- and E6/E7 mRNA-based assays of HPV DNA positive women with abnormal Pap smears.  

PubMed

This study compares the type-specific human papillomavirus (HPV) DNA test with E6/E7 mRNA detection assay because of their importance in cervical cancer screening programs. A total of 105 women with positive high-risk Hybrid Capture 2 or Abbott RealTime High Risk HPV screening test and an abnormal cervical Pap smear were enrolled in the study. HPV typing was performed by multiplex real-time PCR (HPV High Risk Typing Real-TM test). HPV-16, 18, 31, 33, and 45 E6/E7 mRNAs were determined by type-specific real-time NASBA assay (NucliSENS EasyQ HPV v1.1). Infections caused by HPV-16, 18, 31, 33, and 45 types increased with severity of cervical cytology (p=0.008). Global positivity of five HPV E6/E7 mRNAs was lower than DNA positivity within women with atypical squamous cells of undetermined significance (p=0.016; p=0.008). High agreement of the tests was found in the groups of women with low-grade (p=1.000; p=0.063) and high-grade squamous intraepithelial lesion (p=0.250; p=0.125). Type-specific agreement of both diagnostic approaches was high regardless of cytology. Based on the found differences between HPV-16, 18, 31, 33, and 45 E6/E7 mRNA and DNA positivity, further study is needed to test the role of mRNA testing in the triage of women with atypical squamous cells of undetermined significance in Pap smear. PMID:24036071

Salimovi?-Beši?, Irma; Tomi?-?i?a, Anja; Smailji, Admir; Huki?, Mirsada

2013-12-01

332

Adenovirus E1A, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product.  

PubMed Central

The adenovirus E1A gene product, the simian virus 40 large tumor antigen, and the human papillomavirus E7 protein share a short amino acid sequence that constitutes a domain required for the transforming activity of these proteins. These sequences are also required for these proteins to bind to the retinoblastoma gene product (pRb). Recent experiments have shown that E1A can dissociate complexes containing the transcription factor E2F bound to pRb, dependent on this conserved sequence element. We now show that the E7 protein and the simian virus 40 large tumor antigen can dissociate the E2F-pRb complex, dependent on this conserved sequence element. We also find that the E2F-pRb complex is absent in various human cervical carcinoma cell lines that either express the E7 protein or harbor an RB1 mutation, suggesting that the loss of the E2F-pRb interaction may be an important aspect in human cervical carcinogenesis. We suggest that the ability of E1A, the simian virus 40 large tumor antigen, and E7 to dissociate the E2F-pRb complex may be a common activity of these viral proteins that has evolved to stimulate quiescent cells into a proliferating state so that viral replication can proceed efficiently. In circumstances in which a lytic infection does not proceed, the consequence of this action may be to initiate the oncogenic process in a manner analogous to the mutation of the RB1 gene. Images

Chellappan, S; Kraus, V B; Kroger, B; Munger, K; Howley, P M; Phelps, W C; Nevins, J R

1992-01-01

333

High risk HPV DNA subtypes and E6/E7 mRNA expression in a cohort of colposcopy patients from Northern Italy with high-grade histologically verified cervical lesions  

PubMed Central

To evaluate the prevalence of HPV DNA genotypes in women diagnosed with cervical intraepithelial neoplasia grade 2 or greater (CIN 2+), together with the detection of mRNA transcripts from HPV 16/18/31/33/45. In 1113 women referred to our colposcopy unit for abnormal cytology, colposcopic assessment was followed by histologic examination for final diagnosis and by presence of HPV DNA and E6/E7 mRNA transcripts. A total of 134 CIN 2+ cases were identified. Out of the 134 women with CIN 2+ cervical lesions, 115 cases (85.8%) tested positive by PCR to HR HPV DNA types, and 19 (14.2%) were HR HPV DNA negative. 68 cases (50.7%) were positive for HPV DNA 16/18/31/33/45 and of them 50 cases were E6/E7 positive, and 18 were E6/E7 negative. 47 cases (35.1%) were positive for high risk types other than 16/18/31/33/45. HPV 16 is the most frequent genotype found in histologically confirmed high grade cervical lesions in our series; HPV 31 is the second most frequent type, contributing significantly to the proportion of women with CIN 2+ lesions in our population and shows a higher prevalence than HPV 18. Out of the 979 women with lesions less than CIN 2, 588 cases tested positive by PCR to high risk HPV DNA types (60.1%), and 98 cases were E6/E7 positive from HPV 16/18/31/33/45 (10.1%). Although HPV DNA and mRNA negative results should be evaluated with caution, since they could represent “false negatives”, high risk HPV DNA positivity should be assessed carefully with colposcopy before performing excisional treatments, particularly in adolescents but also in patients who want child, since they may reflect transient situations.

CA, Liverani; A, Ciavattini; E, Monti; D, Puglia; S, Mangano; J, DI Giuseppe; A, Zizzi; G, Goteri; G, Bolis

2012-01-01

334

High risk HPV DNA subtypes and E6/E7 mRNA expression in a cohort of colposcopy patients from Northern Italy with high-grade histologically verified cervical lesions.  

PubMed

To evaluate the prevalence of HPV DNA genotypes in women diagnosed with cervical intraepithelial neoplasia grade 2 or greater (CIN 2+), together with the detection of mRNA transcripts from HPV 16/18/31/33/45. In 1113 women referred to our colposcopy unit for abnormal cytology, colposcopic assessment was followed by histologic examination for final diagnosis and by presence of HPV DNA and E6/E7 mRNA transcripts. A total of 134 CIN 2+ cases were identified. Out of the 134 women with CIN 2+ cervical lesions, 115 cases (85.8%) tested positive by PCR to HR HPV DNA types, and 19 (14.2%) were HR HPV DNA negative. 68 cases (50.7%) were positive for HPV DNA 16/18/31/33/45 and of them 50 cases were E6/E7 positive, and 18 were E6/E7 negative. 47 cases (35.1%) were positive for high risk types other than 16/18/31/33/45. HPV 16 is the most frequent genotype found in histologically confirmed high grade cervical lesions in our series; HPV 31 is the second most frequent type, contributing significantly to the proportion of women with CIN 2+ lesions in our population and shows a higher prevalence than HPV 18. Out of the 979 women with lesions less than CIN 2, 588 cases tested positive by PCR to high risk HPV DNA types (60.1%), and 98 cases were E6/E7 positive from HPV 16/18/31/33/45 (10.1%). Although HPV DNA and mRNA negative results should be evaluated with caution, since they could represent "false negatives", high risk HPV DNA positivity should be assessed carefully with colposcopy before performing excisional treatments, particularly in adolescents but also in patients who want child, since they may reflect transient situations. PMID:23145213

Ca, Liverani; A, Ciavattini; E, Monti; D, Puglia; S, Mangano; J, D I Giuseppe; A, Zizzi; G, Goteri; G, Bolis

2012-01-01

335

Epstein-Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor.  

PubMed

The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (?Np73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein-Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein-Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, a commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the low?Np73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73?-induced apoptosis through EBNA3C. PMID:24314664

Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit; Kundu, Chanakya N; Verma, Subhash C; Choudhuri, Tathagata

2014-01-01

336

Bcl-2- and CrmA-inhibitable dephosphorylation and cleavage of retinoblastoma protein during etoposide-induced apoptosis.  

PubMed

Cell numbers are regulated by a balance between proliferation and apoptosis (programmed cell death). Recent evidence suggests that proteins regulating cell proliferation also mediate apoptosis. Therefore, cellular fate might be determined by cross talk between regulators of cell cycle progression and apoptosis. Previously, we had found that during DNA damage-induced apoptosis, retinoblastoma protein (RB), an important G1/S regulator and tumor suppressor, became dephosphorylated and then immediately cleaved into p48 and p68 fragments. Here, we report that expression of the Bcl-2 oncoprotein, an inhibitor of caspases (interleukin 1 -converting enzyme-like proteases), blocked RB dephosphorylation, RB cleavage and apoptosis in etoposide-treated human Jurkat T cells. In addition, expression of the cowpox virus CrmA protein, a direct inhibitor of caspases, also inhibited both RB changes and apoptosis. Taken together, our findings demonstrate important roles for caspases in the processes of etoposide-induced RB dephosphorylation, RB proteolysis and apoptosis. PMID:9852210

An, B; Johnson, D E; Jin, J R; Antoku, K; Dou, Q P

1998-01-01

337

Photodynamic therapy with recombinant adenovirus AdmIL-12 enhances anti-tumour therapy efficacy in human papillomavirus 16 (E6/E7) infected tumour model  

PubMed Central

Immunotherapy with photodynamic therapy (PDT) offers great promise as a new alternative for cancer treatment; however, its use remains experimental. Here we investigated the utility of adenoviral delivery of interleukin-12 (AdmIL-12) as an adjuvant for PDT in mouse tumour challenge model. PDT was performed by irradiating Radachlorin in C57BL/6 mice transplanted with TC-1 cells. PDT plus AdmIL-12 treatment for tumour suppression as well as specific immune responses were evaluated with the following tests: in vitro and in vivo tumour growth inhibition, interferon-? (IFN-?) and tumour necrosis factor-? (TNF-?) assay, and cytotoxic T lymphocyte (CTL) assay. Direct intratumoral injection of AdmIL-12 resulted in a significant suppression of tumour growth compared to the control group. Treatment of PDT along with AdmIL-12 further enhanced antitumour effects significantly higher than either AdmIL-12 or PDT alone. This combined treatment resulted in complete regression of 9-mm sized tumour in every animal. We also evaluated immune responses induced by these treatments. Combined treatment significantly increased the production level of IFN-? and TNF-? compared with that by AdmIL-12 or PDT alone. PDT plus AdmIL-12 enhanced antitumour immunity through increased expansion of the CTL subset mediated by CD8+ T cells. Taken together, these results indicate that the high anti-cancer activity of PDT with AdmIL-12 is a powerful tool against cancer therapy and is a promising subject for further investigation.

Park, Eun Kyung; Bae, Su-Mi; Kwak, Sun-Young; Lee, Sung Jong; Kim, Yong-Wook; Han, Chan-Hee; Cho, Hyun-Jung; Kim, Kyung Tae; Kim, Young-Jae; Kim, Hyun-Jung; Ahn, Woong Shick

2008-01-01

338

Integrated Analyses of Genome-Wide DNA Occupancy and Expression Profiling Identify Key Genes and Pathways Involved in Cellular Transformation by a Marek's Disease Virus Oncoprotein, Meq  

PubMed Central

Marek's disease (MD) is an economically significant disease in chickens that is caused by the highly oncogenic Marek's disease virus (MDV). A major unanswered question is the mechanism of MDV-induced tumor formation. Meq, a bZIP transcription factor discovered in the 1990s, is critically involved in viral oncogenicity, but only a few of its host target genes have been described, impeding our understanding of MDV-induced tumorigenesis. Using chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray analysis, a high-confidence list of Meq binding sites in the chicken genome and a global transcriptome of Meq-responsive genes were generated. Meq binding sites were found to be enriched in the promoter regions of upregulated genes but not in those of downregulated genes. ChIP-seq was also performed for c-Jun, a known heterodimeric partner of Meq. The close location of binding sites of Meq and c-Jun was noted, suggesting cooperativity between these two factors in modulating transcription. Pathway analysis indicated that Meq transcriptionally regulates many genes that are part of several signaling pathways including the extracellular signal-regulated kinase /mitogen-activated protein kinase (ERK/MAPK), Jak-STAT, and ErbB pathways, which are critical for oncogenesis and/or include signaling mediators involved in apoptosis. Meq activates oncogenic signaling cascades by transcriptionally activating major kinases in the ERK/MAPK pathway and simultaneously repressing phosphatases, as verified using inhibitors of MEK and ERK1/2 in a cell proliferation assay. This study provides significant insights into the mechanistic basis of Meq-dependent cell transformation.

Subramaniam, Sugalesini; Johnston, John; Preeyanon, Likit; Brown, C. Titus; Kung, Hsing-Jien

2013-01-01

339

Oncogenicity of human papillomavirus- or adenovirus-transformed cells correlates with resistance to lysis by natural killer cells.  

PubMed Central

The reasons for the dissimilar oncogenicities of human adenoviruses and human papillomaviruses (HPV) in humans are unknown but may relate to differences in the capacities of the E1A and E7 proteins to target cells for rejection by the host natural killer (NK) cell response. As one test of this hypothesis, we compared the abilities of E1A- and E7-expressing human fibroblastic or keratinocyte-derived human cells to be selectively killed by either unstimulated or interferon (IFN)-activated NK cells. Cells expressing the E1A oncoprotein were selectively killed by unstimulated NK cells, while the same parental cells but expressing the HPV type 16 (HPV-16) or HPV-18 E7 oncoprotein were resistant to NK cell lysis. The ability of IFN-activated NK cells to selectively kill virally transformed cells depends on IFN's ability to induce resistance to NK cell lysis in normal (i.e., non-viral oncogene-expressing) but not virally transformed cells. E1A blocked IFN's induction of cytolytic resistance, resulting in the selective lysis of adenovirus-transformed cells by IFN-activated NK cells. The extent of IFN-induced NK cell killing of E1A-expressing cells was proportional to the level of E1A expression and correlated with the ability of E1A to block IFN-stimulated gene expression in target cells. In contrast, E7 blocked neither IFN-stimulated gene expression nor IFN's induction of cytolytic resistance, thereby precluding the selective lysis of HPV-transformed cells by IFN-activated NK cells. In conclusion, E1A expression marks cells for destruction by the host NK cell response, whereas the E7 oncoprotein lacks this activity.

Routes, J M; Ryan, S

1995-01-01

340

Acetylation of the human T-cell leukemia virus type 1 Tax oncoprotein by p300 promotes activation of the NF-{kappa}B pathway  

SciTech Connect

The oncogenic potential of the HTLV-1 Tax protein involves activation of the NF-{kappa}B pathway, which depends on Tax phosphorylation, ubiquitination and sumoylation. We demonstrate that the nuclei of Tax-expressing cells, including HTLV-1 transformed T-lymphocytes, contain a pool of Tax molecules acetylated on lysine residue at amino acid position 346 by the transcriptional coactivator p300. Phosphorylation of Tax on serine residues 300/301 was a prerequisite for Tax localization in the nucleus and correlated with its subsequent acetylation by p300, whereas sumoylation, resulting in the formation of Tax nuclear bodies in which p300 was recruited, favored Tax acetylation. Overexpression of p300 markedly increased Tax acetylation and the ability of a wild type HTLV-1 provirus, -but not of a mutant provirus carrying an acetylation deficient Tax gene-, to activate gene expression from an integrated NF-{kappa}B-controlled promoter. Thus, Tax acetylation favors NF-{kappa}B activation and might play an important role in HTLV-1-induced cell transformation.

Lodewick, Julie; Lamsoul, Isabelle; Polania, Angela; Lebrun, Sylvie [Institute for Microbiological Research J-M Wiame and Laboratory of Microbiology, Universite Libre de Bruxelles, 1, Avenue Emile Gryson, B-1070 Brussels (Belgium); Burny, Arsene [Faculte des Sciences Agronomiques de Gembloux, Gembloux (Belgium); Ratner, Lee [Division of Molecular Oncology, Washington University School of Medicine, St Louis (United States); Bex, Francoise [Institute for Microbiological Research J-M Wiame and Laboratory of Microbiology, Universite Libre de Bruxelles, 1, Avenue Emile Gryson, B-1070 Brussels (Belgium)], E-mail: fbex@ulb.ac.be

2009-03-30

341

Comparison of the performance of the NucliSENS EasyQ HPV E6/E7 mRNA assay and HPV DNA chip for testing squamous cell lesions of the uterine cervix.  

PubMed

This study aims to evaluate the clinical performance of the NucliSENS EasyQ assay and compare it with HPV DNA genotyping for the detection of high-grade squamous intraepithelial lesions (HSIL) and cancer in a Korean population. In 188 total thin prep samples, the remaining fluid after cytology slide preparation was tested with Goodgene HPV DNA chips and the NucliSENS EasyQ HPV E6/E7 messenger RNA (mRNA) assay. The sensitivity and specificity of each test were calculated with HSIL and squamous cell carcinoma (SCC) as the disease endpoint. Out of the 188 samples, 139 (74%) were positive for DNA of 14 HPV types, while 57 (30%) cases were positive for E6/E7 mRNA. The DNA test was positive in cytology cases of SCC, HSIL, and atypical squamous cell. The mRNA test yielded results of 75%, 74%, 60%, 56%, and 29% positivity in abnormal cytology cases of SCC, HSIL, atypical squamous cells - cannot exclude HSIL, atypical squamous cells of undetermined significance, and low-grade squamous intraepithelial lesion, respectively. In normal cytology cases, the positivity rates were 9% and 53% for the mRNA and DNA tests, respectively. For detection of HSIL and SCC, the sensitivity of the mRNA test was 74.36% and that of the DNA test was 100%, while the specificities of the tests were 85% and 40.83%, respectively. These findings suggest that the HPV E6/E7 mRNA assay can overcome the shortcoming of low specificity of DNA assays for clinical detection of high-grade cervical lesions and malignancies. PMID:24856365

Munkhdelger, Jijgee; Choi, Yeonim; Lee, Dongsup; Kim, Sunghyun; Kim, Geehyuk; Park, Sangjung; Choi, Eunhee; Jin, Hyunwoo; Jeon, Bo-Young; Lee, Hyeyoung; Park, Kwang Hwa

2014-08-01

342

Chromogenic In Situ Hybridization and p16/Ki67 Dual Staining on Formalin-Fixed Paraffin-Embedded Cervical Specimens: Correlation with HPV-DNA Test, E6/E7 mRNA Test, and Potential Clinical Applications  

PubMed Central

Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

Zappacosta, Roberta; Colasante, Antonella; Viola, Patrizia; D'Antuono, Tommaso; Lattanzio, Giuseppe; Capanna, Serena; Gatta, Daniela Maria Pia; Rosini, Sandra

2013-01-01

343

A novel HPV 16 L1-based chimeric virus-like particle containing E6 and E7 seroreactive epitopes permits highly specific detection of antibodies in patients with CIN 1 and HPV-16 infection  

PubMed Central

Background The presence of IgG antibodies to HPV-16 L1-virus like particles (VLPs) in serum has been reported as a result of persistent exposure to the virus and as a marker of disease progression. However, detection of VLP-specific antibodies in sera does not always indicate a malignant lesion as positive results may also be due to a nonmalignant viral infection. Furthermore, malignant lesions are associated with an increased antibody titer for E6 and E7 proteins. The aim of this study was to develop an ELISA using a novel chimeric virus-like particle (cVLP) encoding an L1 protein fused with a string of HPV-16 E6 and E7 seroreactive epitopes to its C-terminus to be used for detection of HPV-16 specific antibodies in patients with cervical intraepithelial lesion grade 1 (CIN 1). Results The sera of 30 patients with CIN 1 who also tested positive for HPV-16 DNA and of 30 age-matched normal donors negative for HPV infection were tested for the presence of IgG antibodies specific for either VLP-L1 (HPV-16 L1), gVLP (derived from Gardasil), or cVLP by ELISA. The cVLP-reactive sera yielded two distinct groups of results: (H) reactivity levels that presented very strong cVLP-specific titers, and (L) reactivity levels with significantly lower titers similar to those obtained with VLP-L1 and gVLP antigens. Additionally, the sera that presented the higher cVLP titers closely matched those that had significantly stronger reactivity to E6 and E7 epitopes. Interestingly, the samples with the highest titers corresponded to patients with the higher numbers of sexual partners and pregnancies. On the other hand only 4 out of the 12 sera that harbored antibodies with VLP neutralizing ability corresponded to the group with high cVLP antibody titers. Conclusion We report for the first time that chimeric particles containing HPV-16 L1 protein fused with E6 and E7 seroreactive epitopes enable much better detection of IgG antibodies in the sera of CIN 1 patients positive for HPV-16 infection than those obtained with VLPs containing only the HPV-16 L1 protein. We also found that the sera with higher cVLP antibody titers corresponded to patients with more sexual partners and pregnancies, and not always with to those with a high neutralizing activity. This novel assay could help in the development of a tool to evaluate cervical cancer risk.

2011-01-01

344

Differential Regulation of Cutaneous Oncoprotein HPVE6 by wtp53, Mutant p53R248W and ?Np63? is HPV Type Dependent  

PubMed Central

UV exposure and p53 mutations are major factors in non-melanoma skin cancer, whereas a role for HPV infections has not been defined. Previous data demonstrated the wtp53-mediated degradation of cutaneous HPV20E6 by caspase-3. ?Np63? and hot-spot mutant p53R248W conveyed a protective effect on HPV20E6 under these conditions. We demonstrate a differential regulation by wtp53 of the E6 genes of cutaneous types HPV4, HPV5, HPV7, HPV27, HPV38, HPV48, HPV60 and HPV77. Caspase- or proteasome-mediated down-regulation was HPV type dependent. Mutant p53R248W up-regulated expression of all these E6 proteins as did ?Np63? except for HPV38E6 which was down-regulated by the latter. None of these cellular proteins affected HPV41E6 expression. Ectopic expression of both mutp53R248W and ?Np63? in the normal NIKS keratinocyte cell line harbouring endogenous p53 and p63however led to a down-regulation of HPV20E6. We demonstrate that HPV20E6 expression in these cells is modulated by additional, yet unidentified, cellular protein(s), which are not necessarily involved in apoptosis or autophagy. We further demonstrate proliferation of HPV20E6-expressing keratinocytes. Levels of proteins involved in cell cycle control, cyclin-D1, cdk6 and p16INK4a, phosphorylated pRB, as well as c-Jun and p-c-Jun, were all increased in these cells. HPV20E6 did not compete for the interaction between p16INK4a with cyclin-D1 or cdk6. Phosphorylation of pRB in the HPV20E6 expressing cells seems to be sufficient to override the cytokenetic block induced by the p16INK4a/pRB pathway. The present study demonstrates the diverse influence of p53 family members on individual cutaneous HPVE6 proteins. HPV20E6 expression also resulted in varying protein levels of factors involved in proliferation and differentiation.

Fei, Jian-Wei; de Villiers, Ethel-Michele

2012-01-01

345

The ING4 Binding with p53 and Induced p53 Acetylation were Attenuated by Human Papillomavirus 16 E6  

PubMed Central

High risk subtype HPV16 early oncoprotein E6 contributes host cell immortalization and transformation through interacting with a number of cellular factors. ING4 is one member of the inhibitor of growth (ING) family of type II tumor suppressors and it has been shown to be involved in regulating p53 function. However, the effect and mechanism of HPV16 E6 on ING4 function remain elusive. In this study, we report HPV16 E6 combines with ING4 in vivo and in vitro. The ING4 induced p53 acetylation and its combining with p53 were attenuated by HPV16 E6 independent of p53 degradation. The enhancing function of ING4 on p53 mediated apoptosis was diminished when HPV16 E6 existed. These findings reveal that ING4 may be a common target of oncogenic viruses for driving host cell carcinogenesis.

Guo, Yi; Meng, Xiangkai; Wang, Qian; Wang, Yanan; Shang, Hong

2013-01-01

346

Intercellular trafficking of the nuclear oncoprotein DEK.  

PubMed

DEK is a biochemically distinct, conserved nonhistone protein that is vital to global heterochromatin integrity. In addition, DEK can be secreted and function as a chemotactic, proinflammatory factor. Here we show that exogenous DEK can penetrate cells, translocate to the nucleus, and there carry out its endogenous nuclear functions. Strikingly, adjacent cells can take up DEK secreted from synovial macrophages. DEK internalization is a heparan sulfate-dependent process, and cellular uptake of DEK into DEK knockdown cells corrects global heterochromatin depletion and DNA repair deficits, the phenotypic aberrations characteristic of these cells. These findings thus unify the extracellular and intracellular activities of DEK, and suggest that this paracrine loop involving DEK plays a role in chromatin biology. PMID:23569252

Saha, Anjan K; Kappes, Ferdinand; Mundade, Amruta; Deutzmann, Anja; Rosmarin, David M; Legendre, Maureen; Chatain, Nicolas; Al-Obaidi, Zeina; Adams, Barbara S; Ploegh, Hidde L; Ferrando-May, Elisa; Mor-Vaknin, Nirit; Markovitz, David M

2013-04-23

347

Transcriptional Activation by the Myc Oncoprotein  

Microsoft Academic Search

The Myc transcription factor functions as a downstream effector of most mitogenic signals. Myc is synthesized rapidly in response to extracellular mitogenic signals, and blocking Myc induction abolishes or at least severely attenuates any mitogenic response. Furthermore, ectopic Myc expression can often bypass the requirement for extracellular signals for entry into S phase. Thus, the Myc transcription factor is both

M. D. Cole; M. A. Nikiforov

348

Oncoprotein metastasis and its suppression revisited  

Microsoft Academic Search

The past two decades have witnessed an increasing appreciation of the role of the tumor microenvironment, of genetic and epigenetic\\u000a alterations in normal cells adjacent to tumors and of the migration of normal cells with aberrant intrinsic properties in\\u000a cancer pathophysiology. Aside from these insights, a novel concept termed \\

Razvan T Radulescu

2010-01-01

349

EAPB0503, a novel imidazoquinoxaline derivative, inhibits growth and induces apoptosis in chronic myeloid leukemia cells.  

PubMed

Imatinib, the first-generation tyrosine kinase inhibitor, revolutionized the therapeutic management of chronic myeloid leukemia (CML) and is highly effective in inducing remissions and prolonging the survival of CML patients. However, one-third of patients develop intolerance or resistance to treatment, and CML stem cells remain insensitive to this therapy, leading almost inevitably to relapse upon treatment discontinuation. Imidazoquinoxalines are imiquimod derivatives that induce growth inhibition and induction of caspase-dependent apoptosis in melanoma and T-cell lymphoma cells. We investigated the effects of EAPB0203 and EAPB0503, two novel imidazoquinoxaline derivatives, on human CML cell lines and showed that they induced a dose-dependent and time-dependent cell growth inhibition. EAPB0503 proved more potent and induced a specific cell cycle arrest in mitosis in CML cells and direct activation of apoptosis as evidenced by increased pre-G0 population, breakdown of mitochondrial membrane potential, PARP cleavage, and DNA breakage. Interestingly, EAPB0503 decreased BCR-ABL oncoprotein levels. The combination of EAPB0503 with imatinib synergized to inhibit the proliferation of CML cells, and most importantly, EABP0503 inhibited the proliferation of imatinib-resistant CML cells, offering promising therapeutic modalities that would circumvent resistance to tyrosine kinase inhibitors and improve the prognosis of CML. PMID:24463483

Saliba, Jessica; Deleuze-Masquéfa, Carine; Iskandarani, Ahmad; El Eit, Rabab; Hmadi, Raed; Mahon, François-Xavier; Bazarbachi, Ali; Bonnet, Pierre-Antoine; Nasr, Rihab

2014-07-01

350

RhoB controls the 24 kDa FGF-2-induced radioresistance in HeLa cells by preventing post-mitotic cell death.  

PubMed

Farnesylated Ras oncoprotein induces a cellular resistance to ionizing radiation that can be reversed by farnesyltransferase inhibitors (FTI). We previously demonstrated that, expression of the 24 kDa FGF2 isoform in wild type ras bearing HeLa cells, induced radioresistance which was also reversed by FTI. We tested the hypothesis that wild type Ras or RhoB, which has been proposed as a potential FTI target, could control the FGF-2-induced radioresistance mechanisms. For this, we expressed inducible dominant negative forms of Ras (RasN17) and Rho (RhoBN19) in 24 kDa FGF2 transfected HeLa cells and analysed their survival after irradiation. While no cell survival modification was observed after RasN17 induction, the expression of RhoBN19 induced a radiosensitization of FGF2 radioresistant HeLa cells in the same range as the one observed after a 48 h treatment with the specific FTI, R115777. Moreover, we showed that activated RhoB but not RhoA induced radioresistance in NIH3T3 cells. The radiosensitizer effect of RhoBN19 expression was due to the induction of the radiation induced post-mitotic cell death. Taken together, these data demonstrate that 24 kDa FGF-2-induced radioresistance is controlled by Rho pathways and suggest that RhoB should be a major determinant in cellular resistance to ionizing radiation. PMID:12203112

Ader, Isabelle; Toulas, Christine; Dalenc, Florence; Delmas, Caroline; Bonnet, Jacques; Cohen-Jonathan, Elizabeth; Favre, Gilles

2002-09-01

351

Serum antibody response to Human papillomavirus (HPV) infections detected by a novel ELISA technique based on denatured recombinant HPV16 L1, L2, E4, E6 and E7 proteins  

PubMed Central

Background Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours (skin, head and neck). Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies. Methods The HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine-tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-PathogenISS study in Italy, assessing the HPV-related pathogenetic mechanisms of progression of cervical cancer precursor lesions. Negative human sera were collected from patients affected by other infectious agents. All the HPV-positive sera were also subjected to an avidity test to assess the binding strength in the antigen-antibody complexes. Results Most of the sera showed a positive reactivity to the denatured HPV16 proteins: 82% of the sera from HPV16 infected women and 89% of the sera from women infected by other HPV genotypes recognised at least one of the HPV16 proteins. The percentages of samples showing reactivity to L1, L2 and E7 were similar, but only a few serum samples reacted to E6 and E4. Most sera bound the antigens with medium and high avidity index, suggesting specific antigen-antibody reactions. Conclusion This novel ELISA, based on multiple denatured HPV16 antigens, is able to detect antibodies in women infected by HPV16 and it is not genotype-specific, as it detects antibodies also in women infected by other genital HPVs. The assay is easy to perform and has low cost, making it suitable for monitoring the natural history of HPV infections as well as for detecting pre-existing HPV antibodies in women who receive VLP-based HPV vaccination.

Di Bonito, Paola; Grasso, Felicia; Mochi, Stefania; Accardi, Luisa; Dona, Maria Gabriella; Branca, Margherita; Costa, Silvano; Mariani, Luciano; Agarossi, Alberto; Ciotti, Marco; Syrjanen, Kari; Giorgi, Colomba

2006-01-01

352

Hematopoietic Development from Human Induced Pluripotent Stem Cells  

PubMed Central

A decade of research on human embryonic stem cells (ESC) has paved the way for the discovery of alternative approaches to generating pluripotent stem cells.Combinatorial overexpression of a limited number of proteins linked to pluripotency in ESC was recently found to reprogram differentiated somatic cells back to a pluripotent state, enabling the derivation of isogenic (patient-specific) pluripotent stem cell lines. Current research is focusing on improving reprogramming protocols (e.g. circumventing the use of retroviral technology and oncoproteins), and on methods for differentiation into transplantable tissues of interest. In mouse ESC, we have previously shown that the embryonic morphogens BMP4 and Wnt3a direct blood formation via activation of Cdx and Hox genes. Ectopic expression of Cdx4 and HoxB4 enables the generation of mouse ESC-derived hematopoietic stem cells (HSC) capable of multilineage reconstitution of lethally irradiated adult mice. Here, we explore hematopoietic development from human induced pluripotent stem (iPS) cells generated in our laboratory. Our data show robust differentiation of iPS cells to mesoderm and to blood lineages, as shown by generation of CD34+CD45+ cells, hematopoietic colony activity and gene expression data, and suggest conservation of blood patterning pathways between mouse and human hematopoietic development.

Lengerke, Claudia; Grauer, Matthias; Niebuhr, Nina I.; Riedt, Tamara; Kanz, Lothar; Park, In-Hyun; Daley, George Q.

2010-01-01

353

Towards a "Sample-In, Answer-Out" Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System  

PubMed Central

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1?:?10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Gr?nn, Petter; Karlgard, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

2012-01-01

354

Human papillomavirus 16-specific T cell responses in classic HPV-related vulvar intra-epithelial neoplasia. Determination of strongly immunogenic regions from E6 and E7 proteins.  

PubMed

Cell-mediated immunity directed against human papillomavirus 16 (HPV-16) antigens was studied in 16 patients affected with classic vulvar intra-epithelial neoplasia (VIN), also known as bowenoid papulosis (BP). Ten patients had blood lymphocyte proliferative T cell responses directed against E6/2 (14-34) and/or E6/4 (45-68) peptides, which were identified in the present study as immunodominant among HPV-16 E6 and E7 large peptides. Ex vivo enzyme-linked immunospot-interferon (IFN)-gamma assay was positive in three patients who had proliferative responses. Twelve months later, proliferative T cell responses remained detectable in only six women and the immunodominant antigens remained the E6/2 (14-34) and E6/4 (45-68) peptides. The latter large fragments of peptides contained many epitopes able to bind to at least seven human leucocyte antigen (HLA) class I molecules and were strong binders to seven HLA-DR class II molecules. In order to build a therapeutic anti-HPV-16 vaccine, E6/2 (14-34) and E6/4 (45-68) fragments thus appear to be good candidates to increase HPV-specific effector T lymphocyte responses and clear classic VIN (BP) disease lesions. PMID:19843089

Bourgault Villada, I; Moyal Barracco, M; Berville, S; Bafounta, M L; Longvert, C; Prémel, V; Villefroy, P; Jullian, E; Clerici, T; Paniel, B; Maillère, B; Choppin, J; Guillet, J G

2010-01-01

355

Targeting MUC1-C is synergistic with bortezomib in downregulating TIGAR and inducing ROS-mediated myeloma cell death.  

PubMed

The proteosome inhibitor bortezomib (BTZ) induces endoplasmic reticulum and oxidative stress in multiple myeloma (MM) cells. The mucin 1 C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in most MM cells, and targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is effective in inducing reactive oxygen species (ROS)-mediated MM cell death. The present results demonstrate that GO-203 and BTZ synergistically downregulate expression of the p53-inducible regulator of glycolysis and apoptosis (TIGAR), which promotes shunting of glucose-6-phosphate into the pentose phosphate pathway to generate reduced glutathione (GSH). In turn, GO-203 blocks BTZ-induced increases in GSH and results in synergistic increases in ROS and MM cell death. The results also demonstrate that GO-203 is effective against BTZ-resistant MM cells. We show that BTZ resistance is associated with BTZ-induced increases in TIGAR and GSH levels, and that GO-203 resensitizes BTZ-resistant cells to BTZ treatment by synergistically downregulating TIGAR and GSH. The GO-203/BTZ combination is thus highly effective in killing BTZ-resistant MM cells. These findings support a model in which targeting MUC1-C is synergistic with BTZ in suppressing TIGAR-mediated regulation of ROS levels and provide an experimental rationale for combining GO-203 with BTZ in certain settings of BTZ resistance. PMID:24632713

Yin, Li; Kufe, Turner; Avigan, David; Kufe, Donald

2014-05-01

356

Alteration of the lipid profile in lymphomas induced by MYC overexpression.  

PubMed

Overexpression of the v-myc avian myelocytomatosis viral oncogene homolog (MYC) oncogene is one of the most commonly implicated causes of human tumorigenesis. MYC is known to regulate many aspects of cellular biology including glucose and glutamine metabolism. Little is known about the relationship between MYC and the appearance and disappearance of specific lipid species. We use desorption electrospray ionization mass spectrometry imaging (DESI-MSI), statistical analysis, and conditional transgenic animal models and cell samples to investigate changes in lipid profiles in MYC-induced lymphoma. We have detected a lipid signature distinct from that observed in normal tissue and in rat sarcoma-induced lymphoma cells. We found 104 distinct molecular ions that have an altered abundance in MYC lymphoma compared with normal control tissue by statistical analysis with a false discovery rate of less than 5%. Of these, 86 molecular ions were specifically identified as complex phospholipids. To evaluate whether the lipid signature could also be observed in human tissue, we examined 15 human lymphoma samples with varying expression levels of MYC oncoprotein. Distinct lipid profiles in lymphomas with high and low MYC expression were observed, including many of the lipid species identified as significant for MYC-induced animal lymphoma tissue. Our results suggest a relationship between the appearance of specific lipid species and the overexpression of MYC in lymphomas. PMID:24994904

Eberlin, Livia S; Gabay, Meital; Fan, Alice C; Gouw, Arvin M; Tibshirani, Robert J; Felsher, Dean W; Zare, Richard N

2014-07-22

357

Human Papillomavirus Type 8 Interferes with a Novel C/EBP?-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration  

PubMed Central

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein ? (C/EBP?) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBP? converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBP? as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBP? in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBP?-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity.

Walch-Ruckheim, Barbara; Wickenhauser, Claudia; Doorbar, John; Pfister, Herbert; Malejczyk, Magdalena; Majewski, Slawomir; Keates, Andrew C.; Smola, Sigrun

2012-01-01

358

Human papillomavirus type 8 interferes with a novel C/EBP?-mediated mechanism of keratinocyte CCL20 chemokine expression and Langerhans cell migration.  

PubMed

Infection with genus beta human papillomaviruses (HPV) is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and 8 in patients with epidermodysplasia verruciformis (EV), a genetic skin disease. So far, it has been unknown how these viruses overcome cutaneous immune control allowing their persistence in lesional epidermis of these patients. Here we demonstrate that Langerhans cells, essential for skin immunosurveillance, are strongly reduced in HPV8-positive lesional epidermis from EV patients. Interestingly, the same lesions were largely devoid of the important Langerhans cells chemoattractant protein CCL20. Applying bioinformatic tools, chromatin immunoprecipitation assays and functional studies we identified the differentiation-associated transcription factor CCAAT/enhancer binding protein ? (C/EBP?) as a critical regulator of CCL20 gene expression in normal human keratinocytes. The physiological relevance of this finding is supported by our in vivo studies showing that the expression patterns of CCL20 and nuclear C/EBP? converge spatially in the most differentiated layers of human epidermis. Our analyses further identified C/EBP? as a novel target of the HPV8 E7 oncoprotein, which co-localizes with C/EBP? in the nucleus, co-precipitates with it and interferes with its binding to the CCL20 promoter in vivo. As a consequence, the HPV8 E7 but not E6 oncoprotein suppressed C/EBP?-inducible and constitutive CCL20 gene expression as well as Langerhans cell migration. In conclusion, our study unraveled a novel molecular mechanism central to cutaneous host defense. Interference of the HPV8 E7 oncoprotein with this regulatory pathway allows the virus to disrupt the immune barrier, a major prerequisite for its epithelial persistence and procarcinogenic activity. PMID:22911498

Sperling, Tanya; O?dak, Monika; Walch-Rückheim, Barbara; Wickenhauser, Claudia; Doorbar, John; Pfister, Herbert; Malejczyk, Magdalena; Majewski, S?awomir; Keates, Andrew C; Smola, Sigrun

2012-01-01

359

Performance of ProEx C and PreTect HPV-Proofer E6/E7 mRNA tests in comparison with the hybrid capture 2 HPV DNA test for triaging ASCUS and LSIL cytology.  

PubMed

The clinical usefulness of the ProEx C (Becton Dickinson) and PreTect HPV-Proofer E6/E7 mRNA tests (Proofer; Norchip) for the triage of ASCUS and LSIL cytology was determined in comparison with the Hybrid Capture 2 HPV DNA test (HC2; Qiagen). The study population consisted of women with a history of abnormal cytology referred to colposcopy. Histology-confirmed CIN 2+ served as the disease endpoint. The study was based on 1,360 women (mean age 30.7 years), of whom 380 had CIN 2+. Among 315 with ASCUS (CIN 2+, n = 67), the sensitivities of ProEx C, Proofer, and HC2 to detect CIN 2+ were, 71.6, 71.6, and 95.5%, respectively, with a corresponding specificity of 74.6, 74.2, and 35.1%. Among 363 with LSIL (CIN 2+, n = 108), the sensitivities of ProEx C, Proofer, and HC2 were, 67.6, 74.1, and 96.3%, respectively, with a corresponding specificity of 60, 68.2, and 18.4%. Among 225 HC2-positive ASCUS (CIN 2+, n = 64), 105 tested positive by ProEx C, reducing colposcopy referral by 53.3% and detecting 71.9% of CIN 2+; Proofer was positive in 112/225, reducing colposcopy referral by 50.2% and detecting 75.0% of CIN 2+. Among 312 HC2-positive LSIL (CIN 2+, n = 104), 160 tested positive by ProEx C, reducing coloposcopy referral by 48.7% and detecting 66.3% of CIN 2+; Proofer was positive in 159/312, reducing colposcopy referral by 49.0% and detecting 75.0% of CIN 2+. In conclusion, both ProEx C and Proofer have a similar performance profile with a significantly higher specificity but lower sensitivity than HC2 for the detection of CIN 2+. Consequently, although they can reduce colposcopy referral, they will miss a proportion of CIN 2+ cases. This is a major limitation and should be taken into account if these tests are considered for ASCUS or LSIL triage. PMID:23341349

Alaghehbandan, Reza; Fontaine, Daniel; Bentley, James; Escott, Nicholas; Ghatage, Prafull; Lear, Adrian; Coutlee, Francois; Ratnam, Samuel

2013-09-01

360

Acetylshikonin induces apoptosis of hepatitis B virus X protein-expressing human hepatocellular carcinoma cells via endoplasmic reticulum stress.  

PubMed

Since it has been known that shikonin derived from a medicinal plant possesses anti-cancer activity, we wonder whether acetylshikonin (ASK), a derivate of shikonin, can be used to treat hepatocellular carcinoma cells expressing hepatitis B virus X protein (HBX), an oncoprotein from hepatitis B virus. When ASK was added to Hep3B cells stably expressing HBX, it induced apoptosis in a dose-dependent manner. ASK induced upregulation and export of Nur77 to the cytoplasm and activation of JNK. Likewise, suppression of Nur77 and JNK inactivation protected the cells from ASK-induced apoptosis, indicating that Nur77 upregulation and JNK activation were required for ASK-mediated apoptosis. Furthermore, ASK increased the expression of Bip and ubiquitination levels of cellular proteins, features of endoplasmic reticulum (ER) stress, via the production of reactive oxygen species in a dose-dependent manner. Suppression of reactive oxygen species with N-acetylcysteine reduced levels of Bip protein and ubiquitination levels of cellular proteins during ASK treatment, leading to protection of cells from apoptosis. Cycloheximide treatment reduced ASK-