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  1. The human papillomavirus E7 oncoprotein

    SciTech Connect

    McLaughlin-Drubin, Margaret E. Muenger, Karl

    2009-02-20

    The human papillomavirus (HPV) E7 oncoprotein shares functional similarities with such proteins as adenovirus E1A and SV40 large tumor antigen. As one of only two viral proteins always expressed in HPV-associated cancers, E7 plays a central role in both the viral life cycle and carcinogenic transformation. In the HPV viral life cycle, E7 disrupts the intimate association between cellular differentiation and proliferation in normal epithelium, allowing for viral replication in cells that would no longer be in the dividing population. This function is directly reflected in the transforming activities of E7, including tumor initiation and induction of genomic instability.

  2. Involvement of aryl hydrocarbon receptor signaling in the development of small cell lung cancer induced by HPV E6/E7 oncoproteins

    PubMed Central

    2011-01-01

    Background Lung cancers consist of four major types that and for clinical-pathological reasons are often divided into two broad categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). All major histological types of lung cancer are associated with smoking, although the association is stronger for SCLC and squamous cell carcinoma than adenocarcinoma. To date, epidemiological studies have identified several environmental, genetic, hormonal and viral factors associated with lung cancer risk. It has been estimated that 15-25% of human cancers may have a viral etiology. The human papillomavirus (HPV) is a proven cause of most human cervical cancers, and might have a role in other malignancies including vulva, skin, oesophagus, head and neck cancer. HPV has also been speculated to have a role in the pathogenesis of lung cancer. To validate the hypothesis of HPV involvement in small cell lung cancer pathogenesis we performed a gene expression profile of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins. Methods Gene expression profile of SCLC has been performed using Agilent whole mouse genome (4 44k) representing ~ 41000 genes and mouse transcripts. Samples were obtained from two HPV16-E6/E7 transgenic mouse models and from littermate's normal lung. Data analyses were performed using GeneSpring 10 and the functional classification of deregulated genes was performed using Ingenuity Pathway Analysis (Ingenuity Systems, http://www.ingenuity.com). Results Analysis of deregulated genes induced by the expression of E6/E7 oncoproteins supports the hypothesis of a linkage between HPV infection and SCLC development. As a matter of fact, comparison of deregulated genes in our system and those in human SCLC showed that many of them are located in the Aryl Hydrocarbon Receptor Signal transduction pathway. Conclusions In this study, the global gene expression of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins led us to identification of several genes involved in SCLC tumor development. Furthermore, our study reveled that the Aryl Hydrocarbon Receptor Signaling is the primarily affected pathway by the E6/E7 oncoproteins expression and that this pathway is also deregulated in human SCLC. Our results provide the basis for the development of new therapeutic approaches against human SCLC. PMID:21205295

  3. Cancerous inhibitor of protein phosphatase 2A contributes to human papillomavirus oncoprotein E7-induced cell proliferation via E2F1

    PubMed Central

    Zhang, Weifang; Chen, Hanxiang; Chen, Yan; Liu, Juan; Wang, Xiao; Yu, Xiuping; Chen, Jason J.; Zhao, Weiming

    2015-01-01

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in many human malignant tumors including cervical cancer. Human papillomavirus (HPV) oncoprotein E7 is the key transformation factor in cervical cancer. Our previous data showed a positive association of CIP2A and HPV-16E7 protein levels; however, how CIP2A is regulated by HPV-E7 and the roles of CIP2A in HPV-E7-mediated cell proliferation are unknown. In this study, we demonstrated that HPV-16E7 protein significantly upregulating CIP2A mRNA and protein expression depended on retinoblastoma protein pRb rather than p130. CIP2A siRNA knockdown in HPV-E7-expressing cells inhibited cell proliferation, DNA synthesis and G1/S cell cycle progression. CIP2A siRNA decreased the protein levels of cyclin-dependent kinase 1 (Cdk1), Cdk2 and their partner cyclin A2, with no change in levels of Cdk4, Cdk6 and their partner cyclin D1. The downregulation of Cdk1 and Cdk2 was independent of c-Myc; instead, E2F1 was the main target of CIP2A in this process, as overexpression of E2F1 rescued the inhibitory effects of CIP2A siRNA knockdown on cell proliferation and G1 arrest of HPV-E7-expressing cells. Our studies reveal a novel function of CIP2A in HPV-16E7-mediated cell proliferation. PMID:25650660

  4. Cancerous inhibitor of protein phosphatase 2A contributes to human papillomavirus oncoprotein E7-induced cell proliferation via E2F1.

    PubMed

    Zhang, Weifang; Chen, Hanxiang; Chen, Yan; Liu, Juan; Wang, Xiao; Yu, Xiuping; Chen, Jason J; Zhao, Weiming

    2015-03-10

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in many human malignant tumors including cervical cancer. Human papillomavirus (HPV) oncoprotein E7 is the key transformation factor in cervical cancer. Our previous data showed a positive association of CIP2A and HPV-16E7 protein levels; however, how CIP2A is regulated by HPV-E7 and the roles of CIP2A in HPV-E7-mediated cell proliferation are unknown. In this study, we demonstrated that HPV-16E7 protein significantly upregulating CIP2A mRNA and protein expression depended on retinoblastoma protein pRb rather than p130. CIP2A siRNA knockdown in HPV-E7-expressing cells inhibited cell proliferation, DNA synthesis and G1/S cell cycle progression. CIP2A siRNA decreased the protein levels of cyclin-dependent kinase 1 (Cdk1), Cdk2 and their partner cyclin A2, with no change in levels of Cdk4, Cdk6 and their partner cyclin D1. The downregulation of Cdk1 and Cdk2 was independent of c-Myc; instead, E2F1 was the main target of CIP2A in this process, as overexpression of E2F1 rescued the inhibitory effects of CIP2A siRNA knockdown on cell proliferation and G1 arrest of HPV-E7-expressing cells. Our studies reveal a novel function of CIP2A in HPV-16E7-mediated cell proliferation. PMID:25650660

  5. Gene expression profile regulated by the HPV16 E7 oncoprotein and estradiol in cervical tissue

    PubMed Central

    Cortés-Malagón, Enoc M.; Bonilla-Delgado, José; Díaz-Chávez, José; Hidalgo-Miranda, Alfredo; Romero-Cordoba, Sandra; Üren, Aykut; Çelik, Haydar; McCormick, Matthew; Munguía-Moreno, José A.; Ibarra-Sierra, Eloisa; Escobar-Herrera, Jaime; Lambert, Paul F.; Mendoza-Villanueva, Daniel; Bermudez-Cruz, Rosa M.; Gariglio, Patricio

    2014-01-01

    The HPV16 E7 oncoprotein and 17β-estradiol are important factors for the induction of premalignant lesions and cervical cancer. The study of these factors is crucial for a better understanding of cervical tumorigenesis. Here, we assessed the global gene expression profiles induced by the HPV16 E7 oncoprotein and/or 17β-estradiol in cervical tissue of FvB and K14E7 transgenic mice. We found that the most dramatic changes in gene expression occurred in K14E7 and FvB groups treated with 17β-estradiol. A large number of differentially expressed genes involved in the immune response were observed in 17β-estradiol treated groups. The E7 oncoprotein mainly affected the expression of genes involved in cellular metabolism. Our microarray data also identified differentially expressed genes that have not previously been reported in cervical cancer. The identification of genes regulated by E7 and 17β-estradiol, provides the basis for further studies on their role in cervical carcinogenesis. PMID:24210110

  6. Human papillomavirus type 16 E7 oncoprotein mediates CCNA1 promoter methylation

    PubMed Central

    Chalertpet, Kanwalat; Pakdeechaidan, Watcharapong; Patel, Vyomesh; Mutirangura, Apiwat; Yanatatsaneejit, Pattamawadee

    2015-01-01

    Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation. PMID:26250467

  7. Human papillomavirus type 16 E7 oncoprotein upregulates the retinoic acid receptor-beta expression in cervical cancer cell lines and K14E7 transgenic mice.

    PubMed

    Gutiérrez, Jorge; García-Villa, Enrique; Ocadiz-Delgado, Rodolfo; Cortés-Malagón, Enoc M; Vázquez, Juan; Roman-Rosales, Alejandra; Alvarez-Rios, Elizabeth; Celik, Haydar; Romano, Marta C; Üren, Aykut; Lambert, Paul F; Gariglio, Patricio

    2015-10-01

    Persistent infection with high-risk human papillomaviruses is the main etiological factor in cervical cancer (CC). The human papillomavirus type 16 (HPV16) E7 oncoprotein alters several cellular processes, regulating the expression of many genes in order to avoid cell cycle control. Retinoic acid receptor beta (RARB) blocks cell growth, inducing differentiation and apoptosis. This tumor suppressor gene is gradually silenced in late passages of foreskin keratinocytes immortalized with HPV16 and in various tumors, including CC, mainly by epigenetic modifications. We investigated the effect of E7 oncoprotein on RARB gene expression. We found that HPV16 E7 increases RARB mRNA and RAR-beta protein expression both in vitro and in the cervix of young K14E7 transgenic mice. In E7-expressing cells, RARB overexpression is further increased in the presence of the tumor suppressor p53 (TP53) R273C mutant. This effect does not change when either C33-A or E7-expressing C33-A cell line is treated with Trichostatin A, suggesting that E7 enhances RARB expression independently of histone deacetylases inhibition. These findings indicate that RARB overexpression is part of the early molecular events induced by the E7 oncoprotein. PMID:26173416

  8. Bovine papillomavirus E7 oncoprotein binds to p600 in naturally occurring equine sarcoids.

    PubMed

    Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

    2011-02-01

    Studies regarding the functions of the bovine papillomavirus (BPV) E7 oncoprotein in vivo are lacking and no E7-mediated mechanism underlying mesenchymal carcinogenesis is known. Here, we show that the interaction between the 600 kDa retinoblastoma protein-associated factor (p600) and BPV E7, described in vitro in cultured cells, takes place in vivo in naturally occurring equine sarcoids. In these cancers we detect the expression of E7 and p600, and demonstrate that E7 and p600 co-localize and physically interact. Furthermore, intracellular signals involved in p600 functional activity are found not to be overexpressed, suggesting a different functional activity of p600 in naturally occurring carcinogenesis. Our results demonstrate, for the first time, that E7-p600 interaction occurs during the natural history of BPV-induced equine tumours, suggesting an important role for E7 in carcinogenesis. Finally, the system provides a suitable animal model of papillomavirus-associated cancer to test therapeutic vaccination against E7. PMID:20965990

  9. HPV E7 Viral Oncoprotein Disrupts Transcriptional Regulation of L1Md Retrotransposon

    PubMed Central

    Montoya-Durango, Diego E.; Ramos, Kenneth S.

    2012-01-01

    Murine L1Md-A5 retrotransposon is a redox-inducible element regulated by Nrf-2/JunD and E2F/Rb-binding sites within its promoter (5′-UTR). Because the human papillomavirus (HPV) oncoprotein E7 interacts with retinoblastoma (pRb) and members of the AP1 family, studies were conducted to examine functional interactions between HPV E7, pRb, and histone deacetylase 2 (HDAC2) in the regulation of L1Md-A5. Using a transient heterologous transcription system we found that HPV E7 alone, or in combination with HDAC2, disrupted pRb-mediated L1MdA-5 transactivation. HPV E7 also ablated the transcriptional response of L1Md-A5 to genotoxic stress, but did not interfere with basal activity. We conclude that HPV E7 associates with proteins involved in the assembly of macromolecular complexes that regulate antioxidant and E2F/Rb sites within L1MdA-5 to regulate biological activity PMID:22172279

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

    SciTech Connect

    Knapp, Alixandra A.; McManus, Patrick M.; Bockstall, Katy; Moroianu, Junona

    2009-01-05

    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.

  11. 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

    Zhou Xiaobo; Muenger, Karl

    2009-03-01

    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.

  12. Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis

    PubMed Central

    Sharma, Sweta; Mandal, Paramita; Sadhukhan, Tamal; Roy Chowdhury, Rahul; Ranjan Mondal, Nidhu; Chakravarty, Biman; Chatterjee, Tanmay; Roy, Sudipta; Sengupta, Sharmila

    2015-01-01

    Human Papillomavirus (HPV) type 16 oncoprotein E7 plays a major role in cervical carcinogenesis by interacting with and functionally inactivating various host regulatory molecules. Long noncoding RNA (lncRNA) HOTAIR is one such regulator that recruits chromatin remodelling complex PRC2, creating gene silencing H3K27 me3 marks. Hence, we hypothesized that HOTAIR could be a potential target of E7, in HPV16 related cervical cancers (CaCx). We identified significant linear trend of progressive HOTAIR down-regulation through HPV negative controls, HPV16 positive non-malignants and CaCx samples. Majority of CaCx cases portrayed HOTAIR down-regulation in comparison to HPV negative controls, with corresponding up-regulation of HOTAIR target, HOXD10, and enrichment of cancer related pathways. However, a small subset had significantly higher HOTAIR expression, concomitant with high E7 expression and enrichment of metastatic pathways. Expression of HOTAIR and PRC2-complex members (EZH2 and SUZ12), showed significant positive correlation with E7 expression in CaCx cases and E7 transfected C33A cell line, suggestive of interplay between E7 and HOTAIR. Functional inactivation of HOTAIR by direct interaction with E7 could also be predicted by in silico analysis and confirmed by RNA-Immunoprecipitation. Our study depicts one of the causal mechanisms of cervical carcinogenesis by HPV16 E7, through modulation of HOTAIR expression and function. PMID:26152361

  13. Regulation of the Wnt/?-Catenin Signaling Pathway by Human Papillomavirus E6 and E7 Oncoproteins

    PubMed Central

    Muoz Bello, Jesus Omar; Olmedo Nieva, Leslie; Contreras Paredes, Adriana; Fuentes Gonzalez, Alma Mariana; Rocha Zavaleta, Leticia; Lizano, Marcela

    2015-01-01

    Cell signaling pathways are the mechanisms by which cells transduce external stimuli, which control the transcription of genes, to regulate diverse biological effects. In cancer, distinct signaling pathways, such as the Wnt/?-catenin pathway, have been implicated in the deregulation of critical molecular processes that affect cell proliferation and differentiation. For example, changes in ?-catenin localization have been identified in Human Papillomavirus (HPV)-related cancers as the lesion progresses. Specifically, ?-catenin relocates from the membrane/cytoplasm to the nucleus, suggesting that this transcription regulator participates in cervical carcinogenesis. The E6 and E7 oncoproteins are responsible for the transforming activity of HPV, and some studies have implicated these viral oncoproteins in the regulation of the Wnt/?-catenin pathway. Nevertheless, new interactions of HPV oncoproteins with cellular proteins are emerging, and the study of the biological effects of such interactions will help to understand HPV-related carcinogenesis. This review addresses the accumulated evidence of the involvement of the HPV E6 and E7 oncoproteins in the activation of the Wnt/?-catenin pathway. PMID:26295406

  14. Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

    SciTech Connect

    Yu, Yueyang; Munger, Karl

    2012-10-10

    The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as well as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.

  15. Human Papillomavirus Type 16 E7 Oncoprotein Causes a Delay in Repair of DNA Damage

    PubMed Central

    Park, Jung Wook; Nickel, Kwangok P.; Torres, Alexandra D.; Lee, Denis; Lambert, Paul F.; Kimple, Randall J.

    2014-01-01

    Background and Purpose Patients with Human papillomavirus related (HPV+) head and neck cancers (HNCs) demonstrate improved clinical outcomes compared to traditional HPV negative (HPV?) HNC patients. We have recently shown that HPV+ HNC cells are more sensitive to radiation than HPV? HNC cells. However, roles of HPV oncogenes in regulating the response of DNA damage repair remain unknown. Material and Methods Using immortalized normal oral epithelial cell lines, HPV+ HNC derived cell lines, and HPV16 E7-transgenic mice we assessed the repair of DNA damage using ?-H2AX foci, single and split dose clonogenic survival assays, and immunoblot. The ability of E7 to modulate expression of proteins associated with DNA repair pathways was assessed by immunoblot. Results HPV16 E7 increased retention of ?-H2AX nuclear foci and significantly decreased sublethal DNA damage repair. While phospho-ATM, phospho-ATR, Ku70, and Ku80 expressions were not altered by E7, Rad51 was induced by E7. Correspondingly, HPV+ HNC cell lines showed retention of Rad51 after ?-radiation. Conclusions Our findings provide further understanding as to how HPV16 E7 manipulates cellular DNA damage responses that may underlie its oncogenic potential and influence the altered sensitivity to radiation seen in HPV+ HNC as compared to HPV? HNC. PMID:25216575

  16. Human Papillomavirus Type 16 E6 and E7 Oncoproteins Act Synergistically to Cause Head and Neck Cancer in Mice

    PubMed Central

    Jabbar, Sean; Strati, Katerina; Shin, Myeong Kyun; Pitot, Henry C.; Lambert, Paul F.

    2010-01-01

    High-risk human papillomaviruses (HPVs) contribute to cervical and other anogenital cancers, and they are also linked etiologically to a subset of head and neck squamous cell carcinomas (HNSCC). We previously established a model for HPV-associated HNSCC in which we treated transgenic mice expressing the papillomaviral oncoproteins with the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO). We found that the HPV-16 E7 oncoprotein was highly potent in causing HNSCC, and its dominance masked any potential oncogenic contribution of E6, a second papillomaviral oncoprotein commonly expressed in human cancers. In the current study, we shortened the duration of treatment with 4-NQO to reduce the incidence of cancers and discovered a striking synergy between E6 and E7 in causing HNSCC. Comparing the oncogenic properties of wild-type versus mutant E6 genes in this model for HNSCC uncovered a role for some but not other cellular targets of E6 previously shown to contribute to cervical cancer. PMID:20797753

  17. Immunoexpression of HPV 16/18 E6 and E7 oncoproteins in high-grade cervical squamous intraepithelial lesions in HIV-positive women.

    PubMed

    Rodrigues, L C; de Gois Speck, N M; de Azevedo Focchi, G R; Schimidt, M A; Marques, R M; Lascasas Ribalta, J C

    2016-01-01

    The aim of this study was to assess the immunoexpression of human papillomavirus genotypes 16 and 18 (E6 and E7) oncoproteins in cervical high-grade squamous intraepithelial lesions (HSIL) of human immunodeficiency virus (HIV)-positive women. These results were also compared to the persistence and/or recurrence of lesions after loop electrosurgical excision procedure. Cervical samples from 158 patients were divided into three groups according to the presence or absence of HSIL in women who were or were not HIV-positive. By using the tissue microarray technique, immunohistochemistry was performed to analyze the expression of HPV 16/18 E6 and E7 oncoproteins. Cervical samples from 95 HIV-positive women and 63 HIV-negative women were studied. A statistically significant difference was found in the immunoexpression of E6 and E7 oncoproteins in samples from HIV-positive women with HSIL and that of women with non-neoplastic tissue (P < 0.001). There was also a statistically significant correlation between the immunoexpression of E6 (P = 0.012) and E7 (P < 0.001) oncoproteins in lesion persistence among HIV-positive women. Within the limitations of this study, the immunoexpression of HPV 16/18 E6 and E7 oncoproteins may have prognostic value regarding lesion persistence in HIV-positive women. PMID:26909984

  18. Delocalization of the microtubule motor Dynein from mitotic spindles by the human papillomavirus E7 oncoprotein is not sufficient for induction of multipolar mitoses.

    PubMed

    Nguyen, Christine L; McLaughlin-Drubin, Margaret E; Mnger, Karl

    2008-11-01

    Dynein is a minus end-directed microtubule motor that transports numerous cargoes throughout the cell. During mitosis, dynein motor activity is necessary for the positioning of spindle microtubules and has also been implicated in inactivating the spindle assembly checkpoint. Mutations in dynein motor and/or accessory proteins are associated with human disease, including cancer, and the delocalization of dynein from mitotic spindles has been correlated with an increased incidence of multipolar spindle formation in some cancer cells that contain supernumerary centrosomes. The high-risk human papillomavirus type 16 (HPV16) E7 oncoprotein induces centrosome overduplication and has been shown to cause multipolar mitotic spindle formation, a diagnostic hallmark of HPV-associated neoplasias. Here, we show that HPV16 E7 expression leads to an increased population of mitotic cells with dynein delocalized from the mitotic spindle. This function maps to sequences of HPV16 E7 that are distinct from the region necessary for centrosome overduplication. However, contrary to previous reports, we provide evidence that dynein delocalization by HPV16 E7 is neither necessary nor sufficient to cause the formation of multipolar mitoses. PMID:18974113

  19. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

    SciTech Connect

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.

  20. Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins

    SciTech Connect

    Onder, Zeynep; Moroianu, Junona

    2014-01-20

    We have previously discovered and characterized the nuclear import pathways for the E7 oncoproteins of mucosal alpha genus HPVs, type 16 and 11. Here we investigated the nuclear import of cutaneous beta genus HPV8 E7 protein using confocal microscopy after transfections of HeLa cells with EGFP-8E7 and mutant plasmids and nuclear import assays in digitonin-permeabilized HeLa cells. We determined that HPV8 E7 contains a nuclear localization signal (NLS) within its zinc-binding domain that mediates its nuclear import. Furthermore, we discovered that a mostly hydrophobic patch {sub 65}LRLFV{sub 69} within the zinc-binding domain is essential for the nuclear import and localization of HPV8 E7 via hydrophobic interactions with the FG nucleoporins Nup62 and Nup153. Substitution of the hydrophobic residues within the {sub 65}LRLFV{sub 69} patch to alanines, and not R66A mutation, disrupt the interactions between the 8E7 zinc-binding domain and Nup62 and Nup153 and consequently inhibit nuclear import of HPV8 E7. - Highlights: • HPV8 E7 has a cNLS within its zinc-binding domain that mediates its nuclear import. • Discovery of a hydrophobic patch that is critical for the nuclear import of HPV8 E7. • HPV8 E7 nuclear import is mediated by hydrophobic interactions with FG-Nups, Nup62 and Nup153.

  1. Locking Src/Abl Tyrosine Kinase Activities Regulate Cell Differentiation and Invasion of Human Cervical Cancer Cells Expressing E6/E7 Oncoproteins of High-Risk HPV.

    PubMed

    Yasmeen, Amber; Alachkar, Amal; Dekhil, Hafedh; Gambacorti-Passerini, Carlo; Al Moustafa, Ala-Eddin

    2010-01-01

    In this study, we compared the effects of SKI-606 with Iressa, Src/Abl and EGF-R kinase inhibitors, respectively, on selected parameters in HeLa and SiHa cervical cancer cell lines, which express E6/E7 oncoproteins of high-risk HPV types 18 and 16, respectively. Our results show that SKI-606 and Iressa inhibit cell proliferation and provoke G(0)-G(1) cell cycle arrest and reduction of S and G(2)-M phase using 2 and 5??M concentrations of these inhibitors. In contrast, SKI-606 induces differentiation to an epithelial phenotype "mesenchymal-epithelial transition"; thus SKI-606 causes a dramatic decrease in cell motility and invasion abilities of HeLa and SiHa cancer cells, in comparison to untreated cells and Iressa-treated cells in which these parameters are only slightly affected. These changes are accompanied by a regulation of the expression patterns of E-cadherin and catenins. The molecular pathway analysis of Src/Abl inhibitor revealed that SKI-606 blocks the phosphorylation of ?-catenin and consequently converts its role from a transcriptional regulator to a cell-cell adhesion molecule. Our findings indicate that SKI-606 inhibits signaling pathways involved in regulating tumor cell migration and invasion genes via ?-catenin alteration, suggesting that Src inhibitor, in comparison to EGF-R, is a promising therapeutic agent for human cervical cancer. PMID:20862378

  2. Direct association of the HPV16 E7 oncoprotein with cyclin A/CDK2 and cyclin E/CDK2 complexes

    PubMed Central

    Nguyen, Christine L.; Mnger, Karl

    2008-01-01

    The human papillomavirus (HPV) E7 oncoprotein has been shown to associate with cyclin/CDK2 complexes. Here we present evidence that HPV E7 proteins can associate with cyclin A/CDK2 and cyclin E/CDK2 complexes in cells that lack retinoblastoma tumor suppressor family members through sequences outside of the core retinoblastoma tumor suppressor binding site. Moreover, we show that HPV16 E7 can directly associate with cyclin A/CDK2 and cyclin E/CDK2 complexes. These results suggest that cyclin/CDK2 complexes may be components of HPV E7-associated cellular complexes that do not contain retinoblastoma tumor suppressor family members. PMID:18718623

  3. The high-risk HPV16 E7 oncoprotein mediates interaction between the transcriptional coactivator CBP and the retinoblastoma protein pRb

    PubMed Central

    Jansma, Ariane L.; Martinez-Yamout, Maria A.; Liao, Rong; Sun, Peiqing; Dyson, H. Jane; Wright, Peter E.

    2014-01-01

    The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the CREB-binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high risk HPV16-E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control. PMID:25451029

  4. Functional interaction between human papillomavirus type 16 E6 and E7 oncoproteins and cigarette smoke components in lung epithelial cells.

    PubMed

    Muoz, Juan Pablo; Gonzlez, Carolina; Parra, Brbara; Corvaln, Alejandro H; Tornesello, Maria Lina; Eizuru, Yoshito; Aguayo, Francisco

    2012-01-01

    The smoking habit is the most important, but not a sufficient cause for lung cancer development. Several studies have reported the human papillomavirus type 16 (HPV16) presence and E6 and E7 transcripts expression in lung carcinoma cases from different geographical regions. The possible interaction between HPV infection and smoke carcinogens, however, remains unclear. In this study we address a potential cooperation between tobacco smoke and HPV16 E6 and E7 oncoproteins for alterations in proliferative and tumorigenic properties of lung epithelial cells. A549 (alveolar, tumoral) and BEAS-2B (bronchial, non-tumoral) cell lines were stably transfected with recombinant pLXSN vectors expressing HPV16 E6 and E7 oncoproteins and exposed to cigarette smoke condensate (CSC) at different concentrations. HPV16 E6 and E7 expression was associated with loss of p53 stability, telomerase (hTERT) and p16(INK4A) overexpression in BEAS-2B cells as demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). In A549 cells we observed downregulation of p53 but not a significant increase of hTERT transcripts. In addition, the HPV16 E6/E7 transfected cell lines showed an increased proliferation rate and anchorage-independent growth in a HPV16 E6 and E7 expression-dependent manner. Moreover, both HPV16 E6/E7 and mock transfected cells showed an increased proliferation rate and anchorage-independent growth in the presence of 0.1 and 10 g/mL CSC. However, this increase was significantly greater in HPV16 E6/E7 transfected cells (p<0.001). Data were confirmed by FCSE proliferation assay. The results obtained in this study are suggestive of a functional interaction between tobacco smoke and HPV16 E6/E7 oncoproteins for malignant transformation and tumorigenesis of lung epithelial cells. More studies are warranted in order to dissect the molecular mechanisms involved in this cooperation. PMID:22662279

  5. Functional Interaction between Human Papillomavirus Type 16 E6 and E7 Oncoproteins and Cigarette Smoke Components in Lung Epithelial Cells

    PubMed Central

    Muoz, Juan Pablo; Gonzlez, Carolina; Parra, Brbara; Corvaln, Alejandro H.; Tornesello, Maria Lina; Eizuru, Yoshito; Aguayo, Francisco

    2012-01-01

    The smoking habit is the most important, but not a sufficient cause for lung cancer development. Several studies have reported the human papillomavirus type 16 (HPV16) presence and E6 and E7 transcripts expression in lung carcinoma cases from different geographical regions. The possible interaction between HPV infection and smoke carcinogens, however, remains unclear. In this study we address a potential cooperation between tobacco smoke and HPV16 E6 and E7 oncoproteins for alterations in proliferative and tumorigenic properties of lung epithelial cells. A549 (alveolar, tumoral) and BEAS-2B (bronchial, non-tumoral) cell lines were stably transfected with recombinant pLXSN vectors expressing HPV16 E6 and E7 oncoproteins and exposed to cigarette smoke condensate (CSC) at different concentrations. HPV16 E6 and E7 expression was associated with loss of p53 stability, telomerase (hTERT) and p16INK4A overexpression in BEAS-2B cells as demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB). In A549 cells we observed downregulation of p53 but not a significant increase of hTERT transcripts. In addition, the HPV16 E6/E7 transfected cell lines showed an increased proliferation rate and anchorage-independent growth in a HPV16 E6 and E7 expression-dependent manner. Moreover, both HPV16 E6/E7 and mock transfected cells showed an increased proliferation rate and anchorage-independent growth in the presence of 0.1 and 10 g/mL CSC. However, this increase was significantly greater in HPV16 E6/E7 transfected cells (p<0.001). Data were confirmed by FCSE proliferation assay. The results obtained in this study are suggestive of a functional interaction between tobacco smoke and HPV16 E6/E7 oncoproteins for malignant transformation and tumorigenesis of lung epithelial cells. More studies are warranted in order to dissect the molecular mechanisms involved in this cooperation. PMID:22662279

  6. E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78

    PubMed Central

    Ajiro, Masahiko

    2015-01-01

    ABSTRACT Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41N-terminal amino acid (aa) residues of E6 and the 38C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (?-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic ?-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy. PMID:25691589

  7. The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

    PubMed Central

    Amellem, O.; Sandvik, J. A.; Stokke, T.; Pettersen, E. O.

    1998-01-01

    We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions. Images Figure 2 Figure 4 Figure 8 PMID:9528826

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

    SciTech Connect

    Zhang Weifang; Li Jing; Kanginakudru, Sriramana; Zhao Weiming; Yu Xiuping; Chen, Jason J.

    2010-02-05

    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.

  9. Idaein chloride induced p53 dependent apoptosis in cervical cancer cells through inhibition of viral oncoproteins.

    PubMed

    Natarajan, Thillainathan; Anandhi, Manickam; Aiswarya, Dilipkumar; Ramkumar, Rajendiran; Kumar, Subramanian; Perumal, Pachiappan

    2016-02-01

    Host dependent expression of early HPV oncoproteins, E6 and E7 play central role in the formation of cervical carcinoma. Presently, we have shown that the cyanidin analog, idaein chloride treatment induced dose dependent apoptosis (IC50=2.579?g/ml) in HPV positive - HeLa cells. Flow cytometric analysis showed arrest of cell cycle at G1 phase with an increased sub G1 cell population on 12th h of exposure. The recorded reduced expression levels of cell cycle proteins - cyclin D, cdk 4 and cdk 6 confirmed the occurrence of cell cycle arrest at G1 phase. In addition, the idaein chloride significantly inhibited the expression of E6 and E7 proteins, resulting in p53 re-expression and hence triggering of p53 dependent apoptosis. This has been further supported by the recorded variations in the expression patterns of p21/WAF, pRb and E2F regulatory proteins. In case of mitochondrial apoptotic markers, the expression of Bax was restored and Bcl-2 level got decreased at 12th h. Cleaved caspases 3 & 9 and PARP were also observed after 3h of treatment. Interestingly, the epigenetic regulatory enzymes (DNMTs) were inhibited by idaein chloride. Thus, idaein chloride could be a potent source for developing a drug against cervical carcinoma. PMID:26586108

  10. The human papillomavirus (HPV) E7 protein antagonises an Imiquimod-induced inflammatory pathway in primary human keratinocytes.

    PubMed

    Richards, Kathryn H; Wasson, Christopher W; Watherston, Oliver; Doble, Rosella; Eric Blair, G; Wittmann, Miriam; Macdonald, Andrew

    2015-01-01

    High-risk human papillomaviruses (HPV) are the etiological pathogen of cervical and a number of ano-genital cancers. How HPVs overcome the significant barriers of the skin immune system has been the topic of intensive research. The E6 and E7 oncoproteins have emerged as key players in the deregulation of host innate immune pathways that are required for the recruitment of effector cells of the immune response. Here we demonstrate that E7, and to a lesser extend E6, strongly reduce NFκB activation in response to the inflammatory mediator imiquimod. Moreover, we establish that undifferentiated keratinocytes do not express the putative receptor for imiquimod, TLR7, and as such are stimulated by imiquimod through a novel pathway. Inhibition of imiquimod induced cytokine production required residues in the CR1 and CR3 regions of E7 and resulted in reduced nuclear translocation and acetylation of the p65 sub-unit of NFκB. The results provide further evidence for a TLR7-independent role of imiquimod in the epithelial immune response and reinforce the ability of the HPV oncoproteins to disrupt the innate immune response, which may have important consequences for establishment of a chronic infection. PMID:26268216

  11. Inducing Oncoprotein Degradation to Improve Targeted Cancer Therapy1

    PubMed Central

    Ray, Dipankar; Cuneo, Kyle C.; Rehemtulla, Alnawaz; Lawrence, Theodore S.; Nyati, Mukesh K.

    2015-01-01

    Over the past decade, inhibition of the kinase activities of oncogenic proteins using small molecules and antibodies has been a mainstay of our anticancer drug development effort, resulting in several Food and Drug Administration–approved cancer therapies. The clinical effectiveness of kinase-targeted agents has been inconsistent, mostly because of the development of resistance. The expression and function of oncoproteins and tumor suppressors are regulated by numerous posttranslational protein modifications including phosphorylation, ubiquitination, and acetylation; hence, targeting specific posttranslational protein modifications provides for an attractive strategy for anticancer drug development. The present review discusses the hypothesis that targeted degradation of an oncoprotein may overcome many of the shortcomings seen with kinase inhibitors and that the approach would enable targeted inhibition of oncogenic proteins previously thought to be undruggable. PMID:26476077

  12. Recombinant Lipidated HPV E7 Induces a Th-1-Biased Immune Response and Protective Immunity against Cervical Cancer in a Mouse Model

    PubMed Central

    Shen, Kuan-Yin; Chang, Li-Sheng; Yeh, Yi-Chen; Chen, I-Hua; Chong, Pele; Liu, Shih-Jen; Leng, Chih-Hsiang

    2012-01-01

    The E7 oncoprotein of human papillomavirus (HPV) is an ideal target for developing immunotherapeutic strategies against HPV-associated tumors. However, because protein-based immunogens alone are poor elicitors of the cytotoxic T-lymphocyte (CTL) responses, they have been difficult to exploit for therapeutic purposes. In this study, we report that a recombinant lipoprotein consisting of inactive E7 (E7m) biologically linked to a bacterial lipid moiety (rlipo-E7m) induces the maturation of mouse bone marrow-derived dendritic cells through toll-like receptor 2 (TLR2), skews the immune responses toward the Th1 responses and induces E7-specific CTL responses. We further studied the ability of rlipo-E7m to provide protection against a TC-1 tumor cell challenge in an animal model. Mice prophylactically immunized with two 10-µg doses of rlipo-E7m were found to be free of TC-1 tumor growth. Experiments in a therapeutic immunization model showed that the tumor volume in mice receiving a single dose of rlipo-E7m was less than 0.01 cm3 on day 40, whereas the tumor volume in mice treated with rE7m was 2.28±1.21 cm3. The tumor volume of the entire control group was over 3 cm3. In addition, we demonstrated that the CD8+ T cells play a major role in anti-tumor immunity when administration of rlipo-E7m. These results demonstrate that rlipo-E7m could be a promising candidate for treating HPV-associated tumors. PMID:22815882

  13. Human papillomavirus oncoproteins and apoptosis (Review)

    PubMed Central

    JIANG, PEIYUE; YUE, YING

    2014-01-01

    The aim of this study was to review the literature and identify the association between human papillomavirus (HPV) oncoproteins and apoptosis. HPV-associated apoptosis may be primarily blocked by a number of oncoproteins, including E5, E6 and E7. E5 protein protects cells from tumor necrosis factor-associated apoptosis; the oncoprotein E6 predominantly inhibits apoptosis through the p53 pathway; and oncoprotein E7 is involved in apoptosis activation and inhibition. In addition, HPV oncoproteins are involved in activating or repressing the transcription of E6/E7. In conclusion, HPV oncoproteins, including E5, E6 and E7 protein, may interfere with apoptosis via certain regulatory principles. PMID:24348754

  14. Nuclear import of high risk HPV16 E7 oncoprotein is mediated by its zinc-binding domain via hydrophobic interactions with Nup62

    SciTech Connect

    Eberhard, Jeremy; Onder, Zeynep; Moroianu, Junona

    2013-11-15

    We previously discovered that nuclear import of high risk HPV16 E7 is mediated by a cNLS located within the zinc-binding domain via a pathway that is independent of karyopherins/importins (Angeline et al., 2003; Knapp et al., 2009). In this study we continued our characterization of the cNLS and nuclear import pathway of HPV16 E7. We find that an intact zinc-binding domain is essential for the cNLS function in mediating nuclear import of HPV16 E7. Mutagenesis of cysteine residues to alanine in each of the two CysXXCys motifs involved in zinc-binding changes the nuclear localization of the EGFP-16E7 and 2xEGFP-16E7 mutants. We further discover that a patch of hydrophobic residues, {sub 65}LRLCV{sub 69}, within the zinc-binding domain of HPV16 E7 mediates its nuclear import via hydrophobic interactions with the FG domain of the central channel nucleoporin Nup62. - Highlights: • An intact zinc-binding domain is essential for the nuclear localization of HPV16 E7. • Identification of a hydrophobic patch that is critical for the nuclear import of HPV16 E7. • HPV16 E7 interacts via its zinc-binding domain with the FG domain of Nup62.

  15. Berberine alters epigenetic modifications, disrupts microtubule network, and modulates HPV-18 E6-E7 oncoproteins by targeting p53 in cervical cancer cell HeLa: a mechanistic study including molecular docking.

    PubMed

    Saha, Santu Kumar; Khuda-Bukhsh, Anisur Rahman

    2014-12-01

    Increased evidence of chemo-resistance, toxicity and carcinogenicity necessitates search for alternative approaches for determining next generation cancer therapeutics and targets. We therefore tested the efficacy of plant alkaloid berberine on human papilloma virus (HPV) -18 positive cervical cancer cell HeLa systematically-involving certain cellular, viral and epigenetic factors. We observed disruptions of microtubule network and changes in membrane topology due to berberine influx through confocal and atomic force microscopies (AFM). We examined nuclear uptake, internucleosomal DNA damages, mitochondrial membrane potential (MMP) alterations and cell migration assays to validate possible mode of cell death events. Analytical data on interactions of berberine with pBR322 through fourier transform infrared (FTIR) and gel migration assay strengthen berberine?s biologically significant DNA binding abilities. We measured cellular uptake, DNA ploidy and DNA strand-breaks through fluorescence activated cell sorting (FACS). To elucidate epigenetic modifications, in support of DNA binding associated processes, if any, we conducted methylation-specific restriction enzyme (RE) assay, methylation specific-PCR (MSP) and expression studies of histone proteins. We also analyzed differential interactions and localization of cellular tumor suppressor p53 and viral oncoproteins HPV-18 E6-E7 through siRNA approach. We further made in-silico approaches to determine possible binding sites of berberine on histone proteins. Overall results indicated cellular uptake of berberine through cell membrane depolarization causing disruption of microtubule networks and its biological DNA binding abilities that probably contributed to epigenetic modifications. Results of modulation in p53 and viral oncoproteins HPV-18 E6-E7 by berberine further proved its potential as a promising chemotherapeutic agent in cervical cancer. PMID:25448308

  16. miR-1226 targets expression of the mucin 1 oncoprotein and induces cell death

    PubMed Central

    JIN, CAINING; RAJABI, HASAN; KUFE, DONALD

    2011-01-01

    The MUC1 oncoprotein is aberrantly overexpressed in human carcinomas and hematologic malignancies. MicroRNAs (miRNAs) have been implicated in the suppression and induction of oncogenesis. The present studies demonstrate that the MUC1 mRNA 3? untranslated region (3?UTR) contains a highly conserved motif for binding of a novel miRNA, miR-1226, that has no known targets. The results show that miR-1226 is expressed in human breast cancer cell lines and non-malignant mammary epithelial cells. We also show that miR-1226 interacts with the MUC1 mRNA 3?UTR and that miR-1226 downregulates endogenous MUC1 protein levels. Consistent with miR-1226-induced downregulation of MUC1 expression, the results demonstrate that miR-1226 induces (i) an increase in reactive oxygen species, (ii) loss of the mitochondrial transmembrane potential, and (iii) a decrease in cell survival. These findings indicate that expression of the MUC1 oncoprotein is downregulated by miR-1226 and that miR-1226 thereby functions as a tumor suppressor by promoting the induction of cell death. PMID:20514397

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

    PubMed

    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

    2012-02-01

    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. PMID:22173032

  18. HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

    PubMed Central

    Cardeal, Laura Beatriz da Silva; Boccardo, Enrique; Termini, Lara; Rabachini, Tatiana; Andreoli, Maria Antonieta; di Loreto, Celso; Filho, Adhemar Longatto; Villa, Luisa Lina; Maria-Engler, Silvya Stuchi

    2012-01-01

    Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome. PMID:22438955

  19. Transcriptional regulation of genes involved in keratinocyte differentiation by human papillomavirus 16 oncoproteins.

    PubMed

    Gyngysi, Eszter; Szalms, Anita; Ferenczi, Annamria; Pliska, Szilrd; Knya, Jzsef; Veress, Gyrgy

    2015-02-01

    The life cycle of human papillomaviruses (HPVs) is strictly linked to the differentiation of their natural host cells. The HPV E6 and E7 oncoproteins can delay the normal differentiation program of keratinocytes; however, the exact mechanisms responsible for this have not yet been identified. The goal of this study was to investigate the effects of HPV16 oncoproteins on the expression of genes involved in keratinocyte differentiation. Primary human keratinocytes transduced by LXSN (control) retroviruses or virus vectors expressing HPV16 E6, E7 or E6/E7 genes were subjected to gene expression profiling. The results of microarray analysis showed that HPV 16 E6 and E7 have the capacity to downregulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm the microarray data. To investigate the effects of the HPV oncoproteins on the promoters of selected keratinocyte differentiation genes, luciferase reporter assays were performed. Our results suggest that the HPV 16 E6 and/or E7 oncogenes are able to downregulate the expression of several genes involved in keratinocyte differentiation (such as desmocollin 1, keratin 4, S100 calcium-binding protein A8 and small proline-rich protein 1A), at least partially by downregulating their promoter activity. This activity of the HPV oncoproteins may have a role in the productive virus life cycle, and also in virus-induced carcinogenesis. PMID:25488293

  20. The HPV-16 E7 oncogene sensitizes malignant cells to IFN-alpha-induced apoptosis

    SciTech Connect

    Wang, Yisong

    2005-10-01

    Interferons (IFNs) exert antitumor effects in several human malignancies, but their mechanism of action is unclear. There is a great variability in sensitivity to IFN treatment depending on both tumor type and the individual patient. The reason for this variable sensitivity is not known. The fact that several IFN-induced anticellular effects are exerted through modulation of proto-oncogenes and tumor suppressor genes may indicate that the malignant genotype may be decisive in the cell's sensitivity to IFN. To determine if a deregulated oncogene could alter the cellular response to IFN, a mouse lymphoma cell line (J3D) was stably transfected with the viral human papillomavirus-16 (HPV-16) E7 oncogene. The E7-transfected cells and their respective mock-transfected sister clones were treated with IFN-{alpha} and examined for possible IFN-induced anticellular effects. We found that the E7-transfected clones were greatly sensitized to IFN-{alpha}-induced apoptosis compared with their mock-transfected counterparts. Induction of apoptosis in the transfected cells correlated with the ability of IFN to activate parts of the proapoptotic machinery specifically in these cells, including activation of caspases and the proapoptotic protein Bak. In summary, our data suggest that transfection of malignant cells with the E7 oncogene can sensitize them to IFN-{alpha}-induced apoptosis. This demonstrates that an oncogenic event may alter the cellular sensitivity to IFN and might also have implications for treatment of HPV related diseases with IFN.

  1. Tetrasomy is induced by human papillomavirus type 18 E7 gene expression in keratinocyte raft cultures.

    PubMed

    Southern, S A; Noya, F; Meyers, C; Broker, T R; Chow, L T; Herrington, C S

    2001-06-15

    We have demonstrated previously that oncogenic human papillomaviruses (HPVs) induce basal cell tetrasomy in low-grade squamous intraepithelial lesions of the cervix. To identify HPV genes and growth conditions involved in this process, we analyzed: (a) organotypic raft cultures of primary human keratinocytes transfected with whole HPV-18 genomes; and (b) organotypic raft cultures acutely infected with recombinant retroviruses expressing the HPV-18 E6, E7, or E6/E7 genes from the differentiation-dependent HPV-18 enhancer-promoter. Cultures were examined for HPV DNA by in situ hybridization and for karyotype by interphase cytogenetics. Tetrasomy occurred in the suprabasal strata of raft cultures expressing E7 and E6/E7 but not in those expressing E6 alone or in a control culture. These data indicate that suprabasal tetrasomy occurs in association with expression of the E7 gene alone. Basal cell tetrasomy was additionally observed in the raft culture transfected with whole HPV-18 genomes, consistent with observations in low-grade squamous intraepithelial lesions. The distribution of tetrasomic cells in these raft cultures may reflect the involvement of additional viral genes or possibly differences in the pattern of viral oncogene and host gene expression. PMID:11406563

  2. HPV16-E7 Expression in skin induces TSLP secretion, type 2 ILC infiltration and atopic dermatitis-like lesions

    PubMed Central

    Bergot, Anne-Sophie; Monnet, Nastasia; Tran, Le Son; Mittal, Deepak; Al-Kouba, Jane; Steptoe, Raymond J.; Grimbaldeston, Michele A.; Frazer, Ian H.; Wells, James W.

    2014-01-01

    Atopic dermatitis is a common pruritic and inflammatory skin disorder with unknown etiology. Most commonly occurring during early childhood, atopic dermatitis is associated with eczematous lesions and lichenification, in which the epidermis becomes hypertrophied resulting in thickening of the skin. In this study, we report an atopic dermatitis-like pathophysiology results in a murine model following the expression of the high-risk Human Papillomavirus (HPV) 16 oncoprotein E7 in keratinocytes under the Keratin 14 promoter. We show that HPV 16 E7 expression in the skin is associated with skin thickening, acanthosis and light spongiosis. Locally, HPV 16 E7 expressing skin secreted high levels of TSLP and contained increased numbers of ILCs. High levels of circulating IgE were associated with increased susceptibility to skin allergy in a model of cutaneous challenge, and to airway bronchiolar inflammation, enhanced airway goblet cell metaplasia and mucus production in a model of atopic march. Surprisingly, skin pathology occurred independently of T-cells and mast cells. Thus, our findings suggest that the expression of a single HPV oncogene in the skin can drive the onset of atopic dermatitis-like pathology through the induction of TSLP and type 2 ILC infiltration. PMID:25601274

  3. Increased sensitivity of HPV-positive head and neck cancer cell lines to x-irradiation Cisplatin due to decreased expression of E6 and E7 oncoproteins and enhanced apoptosis

    PubMed Central

    Ziemann, Frank; Arenz, Andrea; Preising, Stefanie; Wittekindt, Claus; Klussmann, Jens P; Engenhart-Cabillic, Rita; Wittig, Andrea

    2015-01-01

    Squamous cell carcinoma of the head and neck region (HNSCC), which is related to an infection with human papilloma virus (HPV), responds better to simultaneous radio-chemotherapy with Cisplatin based regimens than HPV-negative tumors. The underlying molecular mechanisms for this clinical observation are not fully understood. Therefore, the response of four HPV-positive (HPV+) (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (HPV-) (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) HNSCC cell lines to x-irradiation Cisplatin incubation in terms of clonogenic survival, cell cycle progression, protein expression (cyclin A2, cyclin E2, E6, E7, p53) and induction of apoptosis, was investigated. HPV+ cells were more radio- and chemosensitive and were more effectively sensitized to x-irradiation by simultaneous Cisplatin incubation than HPV- cell lines. HPV+ cell lines revealed an increased and prolonged G2/M arrest after irradiation, whereas Cisplatin induced a blockage of cells in S phase. In comparison to irradiation only, addition of Cisplatin significantly enhanced apoptosis especially in HPV+ cell lines. While irradiation alone increased the amount of HPV E6 and E7 proteins, both were down-regulated by Cisplatin incubation either alone or in combination with x-rays, which however did not increase the expression of endogenous p53. Our results demonstrate that cell cycle deregulation together with downregulation of HPV E6 and E7 proteins facilitating apoptosis after Cisplatin incubation promote the enhanced sensitivity of HPV+ HNSCC cells to simultaneous radio-chemotherapy. Combined effects of irradiation and Cisplatin appear to be relevant in mediating the enhanced therapeutic response of HPV-related HNSCC and are indicative of the benefit of combined modality approaches in future treatment optimization strategies. PMID:26045983

  4. MUC1-C oncoprotein suppresses reactive oxygen speciesinduced terminal differentiation of acute myelogenous leukemia cells

    PubMed Central

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

    2011-01-01

    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. PMID:21422470

  5. Longitudinal assessment of DNA methylation changes during HPVE6E7-induced immortalization of primary keratinocytes

    PubMed Central

    Schütze, Denise M; Kooter, Jan M; Wilting, Saskia M; Meijer, Chris JLM; Quint, Wim; Snijders, Peter JF; Steenbergen, Renske DM

    2015-01-01

    High-risk human papillomavirus (hrHPV)-induced immortalization and malignant transformation are accompanied by DNA methylation of host genes. To determine when methylation is established during cell immortalization and whether it is hrHPV-type dependent, DNA methylation was studied in a large panel of HPVE6E7-immortalized keratinocyte cell lines. These cell lines displayed different growth behaviors, i.e., continuous growth versus crisis period prior to immortalization, reflecting differential immortalization capacities of the 7 HPV-types (16/18/31/33/45/66/70) studied. In this study, cells were monitored for hypermethylation of 14 host genes (APC, CADM1, CYGB, FAM19A4, hTERT, mir124–1, mir124–2, mir124–3, MAL, PHACTR3, PRDM14, RASSF1A, ROBO3, and SFRP2) at 4 different stages during immortalization. A significant increase in overall methylation levels was seen with progression through each stage of immortalization. At stage 1 (pre-immortalization), a significant increase in methylation of hTERT, mir124–2, and PRDM14 was already apparent, which continued over time. Methylation of ROBO3 was significantly increased at stage 2 (early immortal), followed by CYGB (stage 3) and FAM19A4, MAL, PHACTR3, and SFRP2 (stage 4). Methylation patterns were mostly growth behavior independent. Yet, hTERT methylation levels were significantly increased in cells that just escaped from crisis. Bisulfite sequencing of hTERT confirmed increased methylation in immortal cells compared to controls, with the transcription core and known repressor sites remaining largely unmethylated. In conclusion, HPV-induced immortalization is associated with a sequential and progressive increase in promoter methylation of a subset of genes, which is mostly independent of the viral immortalization capacity. PMID:25580631

  6. Human hsp70 and HPV16 oE7 fusion protein vaccine induces an effective antitumor efficacy.

    PubMed

    Zong, Jinbao; Wang, Changyuan; Liu, Bin; Liu, Mingjun; Cao, Yongxian; Sun, Xiufang; Yao, Yuan; Sun, Guirong

    2013-07-01

    The persistent infection by human papilloma virus (HPV) is considered to be the major risk factor of cervical cancer, which is one of the most common cancers in women worldwide. Millions of women are currently infected with high-risk HPV. Thus, it is urgent to develop therapeutic vaccines to eliminate established infection or HPV-related diseases. In the present study, we constructed a very promising therapeutic HPV16 protein vaccine of optimized E7 (oE7)/huhsp70 using human hsp70 linked to HPV16 oE7. Our results demonstrated that vaccination with the oE7/huhsp70 protein vaccine induced a very strong E7-specific CD8(+) T cell immune response and resulted in a significant therapeutic effect against E7-expressing tumor cells. Our study verifies that huhsp70 is an effective immune adjuvant in the development of tumor therapeutic protein vaccines, and emphasizes that homologous huhsp70 is a promising tool in future human clinical applications. PMID:23660931

  7. Papillomavirus E6 oncoproteins

    PubMed Central

    Vande Pol, Scott B.; Klingelhutz, Aloysius J.

    2013-01-01

    Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on the associated cellular proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression. PMID:23711382

  8. Codon Optimization of the Human Papillomavirus E7 Oncogene Induces a CD8+ T Cell Response to a Cryptic Epitope Not Harbored by Wild-Type E7

    PubMed Central

    Lorenz, Felix K. M.; Wilde, Susanne; Voigt, Katrin; Kieback, Elisa; Mosetter, Barbara; Schendel, Dolores J.; Uckert, Wolfgang

    2015-01-01

    Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine. PMID:25799237

  9. Requirement for the SnoN oncoprotein in transforming growth factor beta-induced oncogenic transformation of fibroblast cells.

    PubMed

    Zhu, Qingwei; Pearson-White, Sonia; Luo, Kunxin

    2005-12-01

    Transforming growth factor beta (TGF-beta) was originally identified by virtue of its ability to induce transformation of the AKR-2B and NRK fibroblasts but was later found to be a potent inhibitor of the growth of epithelial, endothelial, and lymphoid cells. Although the growth-inhibitory pathway of TGF-beta mediated by the Smad proteins is well studied, the signaling pathway leading to the transforming activity of TGF-beta in fibroblasts is not well understood. Here we show that SnoN, a member of the Ski family of oncoproteins, is required for TGF-beta-induced proliferation and transformation of AKR-2B and NRK fibroblasts. TGF-beta induces upregulation of snoN expression in both epithelial cells and fibroblasts through a common Smad-dependent mechanism. However, a strong and prolonged activation of snoN transcription that lasts for 8 to 24 h is detected only in these two fibroblast lines. This prolonged induction is mediated by Smad2 and appears to play an important role in the transformation of both AKR-2B and NRK cells. Reduction of snoN expression by small interfering RNA or shortening of the duration of snoN induction by a pharmacological inhibitor impaired TGF-beta-induced anchorage-independent growth of AKR-2B cells. Interestingly, Smad2 and Smad3 play opposite roles in regulating snoN expression in both fibroblasts and epithelial cells. The Smad2/Smad4 complex activates snoN transcription by direct binding to the TGF-beta-responsive element in the snoN promoter, while the Smad3/Smad4 complex inhibits it through a novel Smad inhibitory site. Mutations of Smad4 that render it defective in heterodimerization with Smad3, which are found in many human cancers, convert the activity of Smad3 on the snoN promoter from inhibitory to stimulatory, resulting in increased snoN expression in cancer cells. Thus, we demonstrate a novel role of SnoN in the transforming activity of TGF-beta in fibroblasts and also uncovered a mechanism for the elevated SnoN expression in some human cancer cells. PMID:16314499

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

    PubMed Central

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

    2013-01-01

    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. PMID:23538857

  11. Bag3-Induced Autophagy Is Associated with Degradation of JCV Oncoprotein, T-Ag

    PubMed Central

    Sariyer, Ilker Kudret; Merabova, Nana; Patel, Prem Kumer; Knezevic, Tijana; Rosati, Alessandra; Turco, Maria C.; Khalili, Kamel

    2012-01-01

    JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag), in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag) family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases. PMID:22984599

  12. Arsenic trioxide and all-trans retinoic acid target NPM1 mutant oncoprotein levels and induce apoptosis in NPM1-mutated AML cells.

    PubMed

    Martelli, Maria Paola; Gionfriddo, Ilaria; Mezzasoma, Federica; Milano, Francesca; Pierangeli, Sara; Mulas, Floriana; Pacini, Roberta; Tabarrini, Alessia; Pettirossi, Valentina; Rossi, Roberta; Vetro, Calogero; Brunetti, Lorenzo; Sportoletti, Paolo; Tiacci, Enrico; Di Raimondo, Francesco; Falini, Brunangelo

    2015-05-28

    Nucleophosmin (NPM1) mutations represent an attractive therapeutic target in acute myeloid leukemia (AML) because they are common (?30% AML), stable, and behave as a founder genetic lesion. Oncoprotein targeting can be a successful strategy to treat AML, as proved in acute promyelocytic leukemia by treatment with all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO), which degrade the promyelocytic leukemia (PML)-retinoic acid receptor fusion protein. Adjunct of ATRA to chemotherapy was reported to be beneficial for NPM1-mutated AML patients. Leukemic cells with NPM1 mutation also showed sensibility to ATO in vitro. Here, we explore the mechanisms underlying these observations and show that ATO/ATRA induce proteasome-dependent degradation of NPM1 leukemic protein and apoptosis in NPM1-mutated AML cell lines and primary patients' cells. We also show that PML intracellular distribution is altered in NPM1-mutated AML cells and reverted by arsenic through oxidative stress induction. Interestingly, similarly to what was described for PML, oxidative stress also mediates ATO-induced degradation of the NPM1 mutant oncoprotein. Strikingly, NPM1 mutant downregulation by ATO/ATRA was shown to potentiate response to the anthracyclin daunorubicin. These findings provide experimental evidence for further exploring ATO/ATRA in preclinical NPM1-mutated AML in vivo models and a rationale for exploiting these compounds in chemotherapeutic regimens in clinics. PMID:25795919

  13. RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism.

    PubMed

    Gorla, L; Cant, M; Miccich, F; Patelli, C; Mondellini, P; Pierotti, M A; Bongarzone, I

    2006-12-01

    We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis. PMID:16843637

  14. E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

    SciTech Connect

    Turner, Roberta L.; Wilkinson, John C.; Ornelles, David A.

    2014-05-15

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.

  15. Splicing-factor oncoprotein SRSF1 stabilizes p53 via RPL5 and induces cellular senescence.

    PubMed

    Fregoso, Oliver I; Das, Shipra; Akerman, Martin; Krainer, Adrian R

    2013-04-11

    Splicing and translation are highly regulated steps of gene expression. Altered expression of proteins involved in these processes can be deleterious. Therefore, the cell has many safeguards against such misregulation. We report that the oncogenic splicing factor SRSF1, which is overexpressed in many cancers, stabilizes the tumor suppressor protein p53 by abrogating its MDM2-dependent proteasomal degradation. We show that SRSF1 is a necessary component of an MDM2/ribosomal protein complex, separate from the ribosome, that functions in a p53-dependent ribosomal-stress checkpoint pathway. Consistent with the stabilization of p53, increased SRSF1 expression in primary human fibroblasts decreases cellular proliferation and ultimately triggers oncogene-induced senescence (OIS). These findings underscore the deleterious outcome of SRSF1 overexpression and identify a cellular defense mechanism against its aberrant function. Furthermore, they implicate the RPL5-MDM2 complex in OIS and demonstrate a link between spliceosomal and ribosomal components, functioning independently of their canonical roles, to monitor cellular physiology and cell-cycle progression. PMID:23478443

  16. 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

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

    2014-09-01

    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

  17. Cutaneous Human Papillomavirus Type 38 E7 Regulates Actin Cytoskeleton Structure for Increasing Cell Proliferation through CK2 and the Eukaryotic Elongation Factor 1A?

    PubMed Central

    Yue, Jiping; Shukla, Ruchi; Accardi, Rosita; Zanella-Cleon, Isabelle; Siouda, Maha; Cros, Marie-Pierre; Krutovskikh, Vladimir; Hussain, Ishraq; Niu, Yamei; Hu, Shiqiong; Becchi, Michel; Jurdic, Pierre; Tommasino, Massimo; Sylla, Bakary S.

    2011-01-01

    We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2MEKextracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling. PMID:21697493

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

    SciTech Connect

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

    2014-01-05

    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. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  19. Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain

    PubMed Central

    Boronat, M Assumpci; Garca-Fuster, M Julia; Garca-Sevilla, Jess A

    2001-01-01

    This study was designed to assess the influence of activation and blockade of the endogenous opioid system in the brain on two key proteins involved in the regulation of programmed cell death: the pro-apoptotic Fas receptor and the anti-apoptotic Bcl-2 oncoprotein. The acute treatment of rats with the ?-opioid receptor agonist morphine (3??30?mg?kg?1, i.p., 2?h) did not modify the immunodensity of Fas or Bcl-2 proteins in the cerebral cortex. Similarly, the acute treatment with low and high doses of the antagonist naloxone (1 and 100?mg?kg?1, i.p., 2?h) did not alter Fas or Bcl-2 protein expression in brain cortex. These results discounted a tonic regulation through opioid receptors on Fas and Bcl-2 proteins in rat brain. Chronic morphine (10??100?mg?kg?1, 5 days, and 10?mg?kg?1, 13 days) induced marked increases (47??123%) in the immunodensity of Fas receptor in the cerebral cortex. In contrast, chronic morphine (5 and 13 days) decreased the immunodensity of Bcl-2 protein (15??30%) in brain cortex. Chronic naloxone (10?mg?kg?1, 13 days) did not alter the immunodensities of Fas and Bcl-2 proteins in the cerebral cortex. The concurrent chronic treatment (13 days) of naloxone (10?mg?kg?1) and morphine (10?mg?kg?1) completely prevented the morphine-induced increase in Fas receptor and decrease in Bcl-2 protein immunoreactivities in the cerebral cortex. The results indicate that morphine, through the sustained activation of opioid receptors, can promote abnormal programmed cell death by enhancing the expression of pro-apoptotic Fas receptor protein and damping the expression of anti-apoptotic Bcl-2 oncoprotein. PMID:11704646

  20. Recombinant HPV16 E7 assembled into particles induces an immune response and specific tumour protection administered without adjuvant in an animal model

    PubMed Central

    2011-01-01

    Background The HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a Zn++ ion. The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated. Methods E7 was expressed in E. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM). The Zn++ ion content was analysed by mass-spectrometry. Ten ?g of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-? ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months. Results In western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 ?g without any added adjuvant. Conclusions This report shows that a particulate form of HPV16 E7 is able to induce, without adjuvant, an E7-specific tumour protection in C57BL/6 mice. The protective immunity is sustained by both humoral and cell-mediated immune responses. The E. coli-derived HPV16 E7 assembled in vitro into micro- and nanoparticles represents not only a good substrate for antigen-presenting cell uptake and processing, but also a cost-effective means for the production of a new generation of HPV subunit vaccines. PMID:21592382

  1. The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing.

    PubMed

    Frum, Rebecca A; Singh, Shilpa; Vaughan, Catherine; Mukhopadhyay, Nitai D; Grossman, Steven R; Windle, Brad; Deb, Sumitra; Deb, Swati Palit

    2014-01-01

    Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53-/-:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53-/- mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53. PMID:24163099

  2. Inhibition of the MUC1-C oncoprotein induces multiple myeloma cell death by down-regulating TIGAR expression and depleting NADPH.

    PubMed

    Yin, Li; Kosugi, Michio; Kufe, Donald

    2012-01-19

    The MUC1-C oncoprotein is aberrantly expressed in most multiple myeloma cells. However, the functional significance of MUC1-C expression in multiple myeloma is not known. The present studies demonstrate that treatment of multiple myeloma cells with a MUC1-C inhibitor is associated with increases in reactive oxygen species (ROS), oxidation of mitochondrial cardiolipin, and loss of the mitochondrial transmembrane potential. The MUC1-C inhibitor-induced increases in ROS were also associated with down-regulation of the p53-inducible regulator of glycolysis and apoptosis (TIGAR). In concert with the decrease in TIGAR expression, which regulates the pentose phosphate pathway, treatment with the MUC1-C inhibitor reduced production of NADPH, and in turn glutathione (GSH) levels. TIGAR protects against oxidative stress-induced apoptosis. The suppression of TIGAR and NADPH levels thus contributed to ROS-mediated late apoptosis/necrosis of multiple myeloma cells. These findings indicate that multiple myeloma cells are dependent on MUC1-C and TIGAR for maintenance of redox balance and that targeting MUC1-C activates a cascade involving TIGAR suppression that contributes to multiple myeloma cell death. PMID:22117045

  3. E6 and E7 gene silencing results in decreased methylation of tumor suppressor genes and induces phenotype transformation of human cervical carcinoma cell lines

    PubMed Central

    Long, Jia; Shen, Danbei; Zhou, Wuqing; Zhou, Qiyan; Yang, Jia; Jiang, Mingjun

    2015-01-01

    In SiHa and CaSki cells, E6 and E7-targeting shRNA specifically and effectively knocked down human papillomavirus (HPV) 16 E6 and E7 at the transcriptional level, reduced the E6 and E7 mRNA levels by more than 80% compared with control cells that expressed a scrambled-sequence shRNA. E6 and E7 repression resulted in down-regulation of DNA methyltransferase mRNA and protein expression, decreased DNA methylation and increased mRNA expression levels of tumor suppressor genes, induced a certain apoptosis and inhibited proliferation in E6 and E7 shRNA-infected SiHa and CaSki cells compared with the uninfected cells. Repression of E6 and E7 oncogenes resulted in restoration of DNA methyltransferase suppressor pathways and induced apoptosis in HPV16-positive cervical carcinoma cell lines. Our findings suggest that the potential carcinogenic mechanism of HPV16 through influencing DNA methylation pathway to activate the development of cervical cancer exist, and maybe as a candidate therapeutic strategy for cervical and other HPV-associated cancers. PMID:26329329

  4. Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects

    PubMed Central

    Peng, Shiwen; Trimble, Cornelia; Alvarez, Ronald D.; Huh, Warner K.; Lin, Zhenhua; Monie, Archana; Hung, Chien-Fu; Wu, T.-C.

    2010-01-01

    Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal timeframe. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation. PMID:18401437

  5. Pericentromeric regions are refractory to prompt repair after replication stress-induced breakage in HPV16 E6E7-expressing epithelial cells.

    PubMed

    Deng, Wen; Tsao, Sai Wah; Guan, Xin-Yuan; Cheung, Annie L M

    2012-01-01

    Chromosomal instability is the major form of genomic instability in cancer cells. Amongst various forms of chromosomal instability, pericentromeric or centromeric instability remains particularly poorly understood. In the present study, we found that pericentromeric instability, evidenced by dynamic formation of pericentromeric or centromeric rearrangements, breaks, deletions or iso-chromosomes, was a general phenomenon in human cells immortalized by expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7). In particular, for the first time, we surprisingly found a dramatic increase in the proportion of pericentromeric chromosomal aberrations relative to total aberrations in HPV16 E6E7-expressing cells 72 h after release from aphidicolin (APH)-induced replication stress, with pericentromeric chromosomal aberrations becoming the predominant type of structural aberrations (~70% of total aberrations). In contrast, pericentromeric aberrations accounted for only about 20% of total aberrations in cells at the end of APH treatment. This increase in relative proportion of pericentromeric aberrations after release from APH treatment revealed that pericentromeric breaks induced by replication stress are refractory to prompt repair in HPV16 E6E7-expressing epithelial cells. Telomerase-immortalized epithelial cells without HPV16 E6E7 expression did not exhibit such preferential pericentromeric instability after release from APH treatment. Cancer development is often associated with replication stress. Since HPV16 E6 and E7 inactivate p53 and Rb, and p53 and Rb pathway defects are common in cancer, our finding that pericentromeric regions are refractory to prompt repair after replication stress-induced breakage in HPV16 E6E7-expressing cells may shed light on mechanism of general pericentromeric instability in cancer. PMID:23119062

  6. Design, Immune Responses and Anti-Tumor Potential of an HPV16 E6E7 Multi-Epitope Vaccine

    PubMed Central

    Chaves, Agatha A. Muniz; Cavalher, Aline Marques; Lopes, Aline Soriano; Diniz, Mariana de Oliveira; Schanoski, Alessandra Soares; de Melo, Robson Lopes; Ferreira, Lus Carlos de Souza; de Oliveira, Maria Leonor S.; Demasi, Marilene; Ho, Paulo Lee

    2015-01-01

    Cervical cancer is a common type of cancer among women worldwide and infection with high-risk human papillomavirus (HPVs) types represents the major risk factor for the etiopathogenesis of the disease. HPV-16 is the most frequently identified HPV type in cervical lesions and expression of E6 and E7 oncoproteins is required for the uncontrolled cellular proliferation. In the present study we report the design and experimental testing of a recombinant multi-epitope protein containing immunogenic epitopes of HPV-16 E6 and E7. Tumor preventive assays, based on the engraftment of TC-1 cells in mice, showed that the E6E7 multi-epitope protein induced a full preventive anti-tumor protection in wild-type mice, as well as in mice deficient in expression of CD4+ T cells and TLR4 receptor. Nonetheless, no anti-tumor protection was observed in mice deficient in CD8+ T cells. Also, the vaccine promoted high activation of E6/E7-specific T cells and in a therapeutic-approach, E6E7 protein conferred full anti-tumor protection in mice. These results show a potential use of this E6E7 multi-epitope antigen as a new and promising antigen for the development of a therapeutic vaccine against tumors induced by HPV. PMID:26390407

  7. Mechanistic Analysis of the Role of Bromodomain-containing Protein 4 (BRD4) in BRD4-NUT Oncoprotein-induced Transcriptional Activation*

    PubMed Central

    Wang, Ranran; You, Jianxin

    2015-01-01

    NUT midline carcinoma (NMC) is a rare but highly aggressive cancer typically caused by the translocation t(15;19), which results in the formation of the BRD4-NUT fusion oncoprotein. Previous studies have demonstrated that fusion of the NUT protein with the double bromodomains of BRD4 may significantly alter the cellular gene expression profile to contribute to NMC tumorigenesis. However, the mechanistic details of this BRD4-NUT function remain poorly understood. In this study, we examined the NUT function in transcriptional regulation by targeting it to a LacO transgene array integrated in U2OS 2-6-3 cells, which allow us to visualize how NUT alters the in situ gene transcription dynamic. Using this system, we demonstrated that the NUT protein tethered to the LacO locus recruits p300/CREB-binding protein (CBP), induces histone hyperacetylation, and enriches BRD4 to the transgene array chromatin foci. We also discovered that, in BRD4-NUT expressed in NMC cells, the NUT moiety of the fusion protein anchored to chromatin by the double bromodomains also stimulates histone hyperacetylation, which causes BRD4 to bind tighter to chromatin. Consequently, multiple BRD4-interacting factors are recruited to the NUT-associated chromatin locus to activate in situ transgene expression. This gene transcription function was repressed by either expression of a dominant negative inhibitor of the p300-NUT interaction or treatment with (+)-JQ1, which dissociates BRD4 from the LacO chromatin locus. Our data support a model in which BRD4-NUT-stimulated histone hyperacetylation recruits additional BRD4 and interacting partners to support transcriptional activation, which underlies the BRD4-NUT oncogenic mechanism in NMC. PMID:25512383

  8. HTLV-1 Tax Oncoprotein Inhibits the Estrogen-Induced-ER α-Mediated BRCA1 Expression by Interaction with CBP/p300 Cofactors

    PubMed Central

    Shukrun, Meital; Jabareen, Azhar; Abou-Kandil, Ammar; Chamias, Rachel; Aboud, Mordechai; Huleihel, Mahmoud

    2014-01-01

    BRCA1 is a multifunctional tumor suppressor, whose expression is activated by the estrogen (E2)-liganded ERα receptor and regulated by certain recruited transcriptional co-activators. Interference with BRCA1 expression and/or functions leads to high risk of breast or/and ovarian cancer. Another multifunctional protein, HTLV-1Tax oncoprotein, is widely regarded as crucial for developing adult T-cell leukemia and other clinical disorders. Tax profile reveals that it can antagonize BRCA1 expression and/or functionality. Therefore, we hypothesize that Tax expression in breast cells can sensitize them to malignant transformation by environmental carcinogens. Here we examined Tax effect on BRCA1 expression by testing its influence on E2-induced expression of BRCA1 promoter-driven luciferase reporter (BRCA1-Luc). We found that E2 strongly stimulated this reporter expression by liganding to ERα, which consequently associated with BRCA1 promoter, while ERα concomitantly recruited CBP/p300 to this complex for co-operative enhancement of BRCA1 expression. Introducing Tax into these cells strongly blocked this E2-ERα-mediated activation of BRCA1 expression. We noted, also, that Tax exerted this inhibition by binding to CBP/p300 without releasing them from their complex with ERα. Chip assay revealed that the binding of Tax to the CBP/p300-ERα complex, prevented its link to AP1 site. Interestingly, we noted that elevating the intracellular pool of CBP or p300 to excessive levels dramatically reduced the Tax-mediated inhibition of BRCA1 expression. Exploring the mechanism of this reduction revealed that the excessive co-factors were sufficient to bind separately the free Tax molecules, thus lowering their amount in the CBP/p300-ERα complex and relieving, thereby, the inhibition of BRCA1 expression. PMID:24586743

  9. Oncoprotein mdig contributes to silica-induced pulmonary fibrosis by altering balance between Th17 and Treg T cells

    PubMed Central

    Sun, Jiaying; Zhang, Yadong; Lu, Yongju; Battelli, Lori; Porter, Dale W.; Chen, Fei

    2015-01-01

    Mineral dust-induced gene (mdig, also named Mina53) was first identified from alveolar macrophages of the coal miners with chronic lung inflammation or fibrosis, but how this gene is involved in lung diseases is poorly understood. Here we show that heterozygotic knockout of mdig (mdig+/−) ameliorates silica-induced lung fibrosis by altering the balance between Th17 cells and Treg cells. Relative to the wild type (WT) mice, infiltration of the macrophages and Th17 cells was reduced in lungs from silica-exposed mdig+/− mice. In contrast, an increased infiltration of the T regulatory (Treg) cells to the lung intestitium was observed in the mdig+/− mice treated with silica. Both the number of Th17 cells in the lung lymph nodes and the level of IL-17 in the bronchoalveolar lavage fluids were decreased in the mdig+/− mice in response to silica. Thus, these results suggest that mdig may contribute to silica-induced lung fibrosis by altering the balance between Th17 and Treg cells. Genetic deficiency of mdig impairs Th17 cell infiltration and function, but favors infiltration of the Treg cells, the immune suppressive T cells that are able to limit the inflammatory responses by repressing the Th17 cells and macrophages. PMID:25669985

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

    PubMed

    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

    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

  11. Oncoprotein mdig contributes to silica-induced pulmonary fibrosis by altering balance between Th17 and Treg T cells.

    PubMed

    Thakur, Chitra; Wolfarth, Michael; Sun, Jiaying; Zhang, Yadong; Lu, Yongju; Battelli, Lori; Porter, Dale W; Chen, Fei

    2015-02-28

    Mineral dust-induced gene (mdig, also named Mina53) was first identified from alveolar macrophages of the coal miners with chronic lung inflammation or fibrosis, but how this gene is involved in lung diseases is poorly understood. Here we show that heterozygotic knockout of mdig (mdig+/-) ameliorates silica-induced lung fibrosis by altering the balance between Th17 cells and Treg cells. Relative to the wild type (WT) mice, infiltration of the macrophages and Th17 cells was reduced in lungs from silica-exposed mdig+/- mice. In contrast, an increased infiltration of the T regulatory (Treg) cells to the lung intestitium was observed in the mdig+/- mice treated with silica. Both the number of Th17 cells in the lung lymph nodes and the level of IL-17 in the bronchoalveolar lavage fluids were decreased in the mdig+/- mice in response to silica. Thus, these results suggest that mdig may contribute to silica-induced lung fibrosis by altering the balance between Th17 and Treg cells. Genetic deficiency of mdig impairs Th17 cell infiltration and function, but favors infiltration of the Treg cells, the immune suppressive T cells that are able to limit the inflammatory responses by repressing the Th17 cells and macrophages. PMID:25669985

  12. Novel Functions of the Human Papillomavirus E6 Oncoproteins.

    PubMed

    Wallace, Nicholas A; Galloway, Denise A

    2015-11-01

    Human papillomaviruses (HPVs) infect the epidermis as well as mucous membranes of humans. They are the causative agents of anogenital tract and some oropharyngeal cancers. Infections begin in the basal epithelia, where the viral genome replicates slowly along with its host cell. As infected cells begin to differentiate and progress toward the periphery, the virus drives proliferation in cells that would otherwise be quiescent. To uncouple differentiation from continued cellular propagation, HPVs express two oncoproteins, HPV E6 and E7. This review focuses on high-risk α-HPV E6, which in addition to supporting viral replication has transforming properties. HPV E6 promotes p53 degradation and activates telomerase, but the multifaceted oncoprotein has numerous other functions that are highlighted here. PMID:26958922

  13. N-Benzylcinnamide induces apoptosis in HPV16 and HPV18 cervical cancer cells via suppression of E6 and E7 protein expression.

    PubMed

    Xiong, Yuanhuan; Chen, Lin; Luo, Puying

    2015-05-01

    Seventy percent of all cervical cancers are caused by human papillomavirus (HPV) infections. Natural products are being extensively explored for their potential ability to prevent and treat cervical cancers. N-benzylcinnamide (PT-3) is a natural product purified from Piper submultinerve. Whether or not PT-3 has an effect on cervical cancer cells is as yet unknown. Therefore, we set out to explore the mechanism of action behind PT-3 and how it affects cells that either contain or lack HPV DNA. Our results demonstrate that PT-3 slows the growth kinetics of CaSki (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner, but does not slows HPV-negative cells. Importantly, we also found that PT-3 induces apoptosis by suppressing expression of E6 and E7 viral oncogenes in HPV-infected cervical cancer CaSki and HeLa cells. Moreover, we found that suppression of E6 and E7 expression leads to modulations in p53 and protein retinoblastomas, which are not changed in HPV-negative cervical cancer C33A cells. These findings demonstrate that PT-3 can effectively promote apoptosis by downregulating expression of E6 and E7. PMID:25914202

  14. Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils and Their HPV16 E6/E7-Induced Perturbation.

    PubMed

    Kang, Sung Yoon Catherine; Kannan, Nagarajan; Zhang, Lewei; Martinez, Victor; Rosin, Miriam P; Eaves, Connie J

    2015-12-01

    Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44(+)NGFR(+) and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44(+)NGFR(+) cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44(+)NGFR(+) cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcriptional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation. PMID:26527383

  15. Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils and Their HPV16 E6/E7-Induced Perturbation

    PubMed Central

    Kang, Sung Yoon Catherine; Kannan, Nagarajan; Zhang, Lewei; Martinez, Victor; Rosin, Miriam P.; Eaves, Connie J.

    2015-01-01

    Summary Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44+NGFR+ cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcriptional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation. PMID:26527383

  16. PKN binds and phosphorylates human papillomavirus E6 oncoprotein.

    TOXLINE Toxicology Bibliographic Information

    Gao Q; Kumar A; Srinivasan S; Singh L; Mukai H; Ono Y; Wazer DE; Band V

    2000-05-19

    The high risk human papillomaviruses (HPVs) are associated with carcinomas of 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 has provided a single gene model to study the mechanisms of E6-induced oncogenic transformation. In recent years, it has become clear that in addition to E6-induced degradation of p53 tumor suppressor protein, other targets of E6 are required for mammary epithelial cells immortalization. Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid- and Rho small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HPVs were able to bind PKN. Furthermore, all the immortalization-competent and many immortalization-non-competent E6 mutants bind PKN. These data suggest that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells. Finally, we show that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a phosphoprotein. Our finding suggests a novel link between HPV E6 mediated oncogenesis and regulation of a well known phosphorylation cascade.

  17. PKN binds and phosphorylates human papillomavirus E6 oncoprotein.

    PubMed

    Gao, Q; Kumar, A; Srinivasan, S; Singh, L; Mukai, H; Ono, Y; Wazer, D E; Band, V

    2000-05-19

    The high risk human papillomaviruses (HPVs) are associated with carcinomas of 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 has provided a single gene model to study the mechanisms of E6-induced oncogenic transformation. In recent years, it has become clear that in addition to E6-induced degradation of p53 tumor suppressor protein, other targets of E6 are required for mammary epithelial cells immortalization. Using the yeast two-hybrid system, we have identified a novel interaction of HPV16 E6 with protein kinase PKN, a fatty acid- and Rho small G protein-activated serine/threonine kinase with a catalytic domain highly homologous to protein kinase C. We demonstrate direct binding of high risk HPV E6 proteins to PKN in wheat-germ lysate in vitro and in 293T cells in vivo. Importantly, E6 proteins of high risk HPVs but not low risk HPVs were able to bind PKN. Furthermore, all the immortalization-competent and many immortalization-non-competent E6 mutants bind PKN. These data suggest that binding to PKN may be required but not sufficient for immortalizing normal mammary epithelial cells. Finally, we show that PKN phosphorylates E6, demonstrating for the first time that HPV E6 is a phosphoprotein. Our finding suggests a novel link between HPV E6 mediated oncogenesis and regulation of a well known phosphorylation cascade. PMID:10809724

  18. Tobacco Smoke Activates Human Papillomavirus 16 p97 Promoter and Cooperates with High-Risk E6/E7 for Oxidative DNA Damage in Lung Cells

    PubMed Central

    Muñoz, Juan P.; Chnaiderman, Jonás; Urzúa, Ulises; León, Oscar; Tornesello, Maria L.; Corvalán, Alejandro H.; Soto-Rifo, Ricardo; Aguayo, Francisco

    2015-01-01

    We have previously shown a functional interaction between human papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins and cigarette smoke condensate (CSC) in lung cells suggesting cooperation during carcinogenesis. The molecular mechanisms of such interaction, however, remain to be elucidated. Here we first present evidence showing that cigarette smoke condensate (CSC) has the ability to activate the HPV-16 p97 promoter by acting on the long control region (LCR) in lung epithelial cells. Interestingly, we observed that CSC-induced p97 promoter activation occurs in a dose-dependent manner in both tumor A-549 (lung adenocarcinoma), H-2170 (bronchial carcinoma), SiHa or Hela (cervical carcinoma) cells but not in non-tumor BEAS-2B (bronchial) or NL-20 (alveolar) lung cells unless they ectopically expressed the HPV-16 E6 and E7 oncogenes. In addition, we also observed a significant increase of primary DNA damage in tumor and non-tumor CSC-treated lung cells expressing HPV-16 E6 and E7 oncogenes suggesting a cooperative effect in this process, even though the contribution of E7 was significantly higher. Taken together, our results strongly suggest that tobacco smoke is able to induce the activation of the HPV-16 p97 promoter in cooperation with HPV-16 E6 and E7 oncogenes that, in turn, sensitize lung cells to tobacco smoke-induced DNA damage. PMID:25830243

  19. Disc large 1 expression is altered by human papillomavirus E6/E7 proteins in organotypic cultures of human keratinocytes.

    PubMed

    Valdano, M Bugnon; Cavatorta, A L; Morale, M G; Marziali, F; de Souza Lino, V; Steenbergen, R D M; Boccardo, E; Gardiol, D

    2016-02-01

    Loss of cell polarity is a fundamental process in cell transformation. Among polarity proteins, we focused on human disc large (DLG1), which is localized mainly at adherens junctions and contributes to the control of cell proliferation. We previously demonstrated that its expression is altered in HPV-associated cervical neoplastic lesions, but the mechanisms beyond this remain unknown. In this study, we analysed the contribution of HPV proteins to the changes in DLG1 expression in the squamous epithelium. We observed tissue and intracellular misdistribution of DLG1 when high-risk HPV-18 E7 or E6/E7 proteins were expressed in organotypic raft cultures. The viral oncoproteins induce the loss of DLG1 from the cell borders and an increase in the level of DLG1 protein, reflecting the pattern observed in cervical lesions. These findings were corroborated in cultures bearing the entire HPV-18 genome. Interestingly, changes in tissue distribution and abundance of DLG1 were also detected in organotypic cultures expressing the low-risk HPV-11 E7 or E6/E7 proteins, suggesting a conserved function among different HPV types. However, for low-risk HPVs, the subcellular localization of DLG1 at cell-to-cell contacts was predominantly maintained. This report offers new evidence, we believe, of the involvement of HPV proteins in DLG1 expression pattern and our data support previous observations regarding DLG1 expression in cervical lesions. PMID:26653181

  20. ADXS-HPV: A therapeutic Listeria vaccination targeting cervical cancers expressing the HPV E7 antigen

    PubMed Central

    Cory, Lori; Chu, Christina

    2014-01-01

    Listeria monocytogenes is a bacterium that can be genetically modified to express fusion proteins with antigens specific to certain cancer models. This technology has been harnessed to develop ADXS11-001, a vaccine that aims to elicit an immune response against human papillomavirus (HPV) oncoprotein E7.??Pre-clinical studies assessing the efficacy of recombinant Listeria vaccination targeting this same oncoprotein have consistently demonstrated successful reduction of in vivo tumor burden among animal cancer models. Several clinical trials are underway to assess the efficacy of ADXS11-001 in eliciting both immune and clinical responses against HPV-related human cervical, oropharyngeal and anal cancers. PMID:25483687

  1. Histone Deacetylase Inhibitor AR-42 Enhances E7-Specific CD8+ T Cell-Mediated Antitumor Immunity Induced by Therapeutic HPV DNA Vaccination

    PubMed Central

    Lee, Sung Yong; Huang, Zhuomin; Kang, Tae Heung; Soong, Ruey-Shyang; Knoff, Jayne; Axenfeld, Ellen; Wang, Chenguang; Alvarez, Ronald D.; Chen, Ching-Shih; Hung, Chien-Fu; Wu, T.-C.

    2013-01-01

    We have previously created a potent DNA vaccine encoding calreticulin linked to the HPV oncogenic protein E7 (CRT/E7). While treatment of the CRT/E7 DNA vaccine generates significant tumor-specific immune responses in vaccinated mice, the potency of the DNA vaccine could potentially be improved by co-administration of a histone deacetylase inhibitor (HDACi) as HDACi have been shown to increase the expression of MHC class I and II molecules. Thus, we aimed to determine whether co-administration of a novel HDACi, AR-42, with therapeutic HPV DNA vaccines could improve activation of HPV antigen-specific CD8+ T cells resulting in potent therapeutic antitumor effects. To do so, HPV-16 E7-expressing murine TC-1 tumor-bearing mice were treated orally with AR-42 and/or CRT/E7 DNA vaccine via gene gun. Mice were monitored for E7-specific CD8+ T cell immune responses and antitumor effects. TC-1 tumor-bearing mice treated with AR-42 and CRT/E7 DNA vaccine experienced longer survival, decreased tumor growth, and enhanced E7-specific immune response compared to mice treated with AR-42 or CRT/E7 DNA vaccine alone. Additionally, treatment of TC-1 cells with AR-42 increased surface expression of MHC class I molecules and increased the susceptibility of tumor cells to the cytotoxicity of E7-specific T cells. This study indicates the ability of AR-42 to significantly enhance the potency of the CRT/E7 DNA vaccine by improving tumor-specific immune responses and antitumor effects. Both AR-42 and CRT/E7 DNA vaccine have been used in independent clinical trials and the current study serves as foundation for future clinical trials combining both treatments in cervical cancer therapy. PMID:23715898

  2. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells.

    PubMed

    Zacapala-Gmez, Ana Elvira; Del Moral-Hernndez, Oscar; Villegas-Seplveda, Nicols; Hidalgo-Miranda, Alfredo; Romero-Crdoba, Sandra Lorena; Beltrn-Anaya, Fredy Omar; Leyva-Vzquez, Marco Antonio; Alarcn-Romero, Luz Del Carmen; Illades-Aguiar, Berenice

    2016-01-15

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. PMID:26655236

  3. MUC1-C oncoprotein induces TCF7L2 transcription factor activation and promotes cyclin D1 expression in human breast cancer cells.

    PubMed

    Rajabi, Hasan; Ahmad, Rehan; Jin, Caining; Kosugi, Michio; Alam, Maroof; Joshi, Maya Datt; Kufe, Donald

    2012-03-23

    MUC1 is a heterodimeric glycoprotein that is overexpressed in breast cancers. The present studies demonstrate that the oncogenic MUC1 C-terminal subunit (MUC1-C) associates with the TCF7L2 transcription factor. The MUC1-C cytoplasmic domain (MUC1-CD) binds directly to the TCF7L2 C-terminal region. MUC1-C blocks the interaction between TCF7L2 and the C-terminal-binding protein (CtBP), a suppressor of TCF7L2-mediated transcription. TCF7L2 and MUC1-C form a complex on the cyclin D1 gene promoter and MUC1-C promotes TCF7L2-mediated transcription by the recruitment of ?-catenin and p300. Silencing MUC1-C in human breast cancer cells down-regulated activation of the cyclin D1 promoter and decreased cyclin D1 expression. In addition, a MUC1-C inhibitor blocked the interaction with TCF7L2 and suppressed cyclin D1 levels. These findings indicate that the MUC1-C oncoprotein contributes to TCF7L2 activation and thereby promotes cyclin D1 expression in breast cancer cells. PMID:22318732

  4. MUC1-C Oncoprotein Induces TCF7L2 Transcription Factor Activation and Promotes Cyclin D1 Expression in Human Breast Cancer Cells*

    PubMed Central

    Rajabi, Hasan; Ahmad, Rehan; Jin, Caining; Kosugi, Michio; Alam, Maroof; Joshi, Maya Datt; Kufe, Donald

    2012-01-01

    MUC1 is a heterodimeric glycoprotein that is overexpressed in breast cancers. The present studies demonstrate that the oncogenic MUC1 C-terminal subunit (MUC1-C) associates with the TCF7L2 transcription factor. The MUC1-C cytoplasmic domain (MUC1-CD) binds directly to the TCF7L2 C-terminal region. MUC1-C blocks the interaction between TCF7L2 and the C-terminal-binding protein (CtBP), a suppressor of TCF7L2-mediated transcription. TCF7L2 and MUC1-C form a complex on the cyclin D1 gene promoter and MUC1-C promotes TCF7L2-mediated transcription by the recruitment of ?-catenin and p300. Silencing MUC1-C in human breast cancer cells down-regulated activation of the cyclin D1 promoter and decreased cyclin D1 expression. In addition, a MUC1-C inhibitor blocked the interaction with TCF7L2 and suppressed cyclin D1 levels. These findings indicate that the MUC1-C oncoprotein contributes to TCF7L2 activation and thereby promotes cyclin D1 expression in breast cancer cells. PMID:22318732

  5. Neuroepithelial carcinomas in mice transgenic with human papillomavirus type 16 E6/E7 ORFs.

    PubMed Central

    Arbeit, J. M.; Mnger, K.; Howley, P. M.; Hanahan, D.

    1993-01-01

    The effect of the human papillomavirus (HPV) oncogenes E6 and E7 was examined in transgenic mice with a construct containing the human beta-actin promoter regulating HPV16 E6 and E7 open reading frames. In the sole line of mice that transmitted the transgene, neuroepithelial tumors appeared at 2.5 months of life, and by 10 months, 87 of 122 (71%) of the animals were dead from brain tumors. The most frequent type of tumor (74%) was an anaplastic neuroepithelial tumor associated with the ependyma of the third and fourth ventricles, which locally invaded adjacent brain tissue and spread for considerable distances along the ventricular surface. The other two types of tumor were well-differentiated choroid plexus carcinomas (26%) and rare pituitary carcinomas (8.7%). HPV16 E6 RNA and E7 oncoprotein expression were demonstrated in tumor tissue and primary cell lines derived from the tumors. Examination of two tumor suppressor gene products, the retinoblastoma protein and p53, known to bind to HPV16 E7 and E6 oncoproteins, respectively, showed both were expressed in the primary tumor cell lines. These data support a causative role for the HPV oncoproteins in epithelial carcinogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8386443

  6. Human telomerase reverse transcriptase regulates vascular endothelial growth factor expression via human papillomavirus oncogene E7 in HPV-18-positive cervical cancer cells.

    PubMed

    Li, Fang; Cui, Jinquan

    2015-07-01

    Human papillomavirus (HPV) infection induces chronic and precancerous lesions and results in invasive cervical cancer. Human telomerase as well as inflammatory and angiogenic factors such as telomerase reverse transcriptase (hTERT) or vascular endothelial growth factor (VEGF) could play a role in regulating HPV-induced cervical cancer. This study investigated underlying molecular events in HPV-induced HPV-positive cervical cancer through hTERT and VEGF in vitro. Expressions of hTERT, a rate-limiting subunit of telomerase, and VEGF mRNA and proteins were, respectively, assessed by qRT-PCR, ELISA, and TRAP-ELISA in HPV-positive tissue samples and cervical cancer cell lines. To assess hTERT and VEGF secretion, hTERT overexpression and knockdown were conducted in HPV-18-positive Hela cells by hTERT cDNA and shRNA transfection, respectively. Then, the effect of HPV E6 and E7 on VEGF expressions was assessed in HPV-negative cervical cancer cells. Data have shown that VEGF expression levels are associated with hTERT expressions and telomerase activity in HPV-positive cervical cancer tissues and cells. Knockdown of hTERT expression down-regulated VEGF expressions, whereas overexpression of hTERT up-regulated VEGF expressions in HPV-18-positive Hela cells. Furthermore, HPV E7 oncoprotein was necessary for hTERT to up-regulate VEGF expressions in HPV-negative cervical cancer cells. Data from this current study indicate that HPV oncoproteins up-regulated hTERT and telomerase activity and in turn promoted VEGF expressions, which could be a key mechanism for HPV-induced cervical cancer development and progression. PMID:26067630

  7. Episomal expression of truncated listeriolysin O in LmddA-LLO-E7 vaccine enhances antitumor efficacy by preferentially inducing expansions of CD4+FoxP3- and CD8+ T cells.

    PubMed

    Chen, Zhisong; Ozbun, Laurent; Chong, Namju; Wallecha, Anu; Berzofsky, Jay A; Khleif, Samir N

    2014-09-01

    Studies have shown that Listeria monocytogenes (Lm)-based vaccine expressing a fusion protein comprising truncated listeriolysin O (LLO) and human papilloma virus (HPV) E7 protein (Lm-LLO-E7) induces a decrease in regulatory T cells (Treg) and complete regression of established, transplanted HPV-TC-1 tumors in mice. However, how the Lm-based vaccine causes a decrease in Tregs remains unclear. Using a highly attenuated Lm dal dat ?actA strain (LmddA)-based vaccine, we report here that the vector LmddA was sufficient to induce a decrease in the proportion of Tregs by preferentially expanding CD4(+)FoxP3(-) T cells and CD8(+) T cells by a mechanism dependent on and directly mediated by LLO. Episomal expression of a nonhemolytic truncated LLO in Lm (LmddA-LLO) significantly augmented the expansion, thus further decreasing Treg frequency. Although adoptive transfer of Tregs compromised the antitumor efficacy of the LmddA-LLO-E7 vaccine, a combination of LmddA-LLO and an Lm-based vaccine expressing E7 protein (Lm-E7) induced complete regression against established TC-1 tumors. An engineered LLO-minus Lm expressing perfringolysin O (PFO) that enables the recombinant bacteria to exit from the phagolysosome without LLO confirmed that the adjuvant effect was dependent on LLO. These results suggest that LLO may serve as a promising adjuvant by preferentially inducing the expansions of CD4(+)FoxP3(-) T cells and CD8(+) T cells, thus reducing the ratio of Tregs to CD4(+)FoxP3(-) T cells and to CD8(+) T cells favoring immune responses to eradicate tumor. PMID:24872025

  8. HPV16 E6 and E7 proteins induce a chronic oxidative stress response via NOX2 that causes genomic instability and increased susceptibility to DNA damage in head and neck cancer cells.

    PubMed

    Marullo, Rossella; Werner, Erica; Zhang, Hongzheng; Chen, Georgia Z; Shin, Dong M; Doetsch, Paul W

    2015-11-01

    Human papillomavirus (HPV) is the causative agent of a subgroup of head and neck cancer characterized by an intrinsic radiosensitivity. HPV initiates cellular transformation through the activity of E6 and E7 proteins. E6 and E7 expression is necessary but not sufficient to transform the host cell, as genomic instability is required to acquire the malignant phenotype in HPV-initiated cells. This study reveals a key role played by oxidative stress in promoting genomic instability and radiosensitivity in HPV-positive head and neck cancer. By employing an isogenic human cell model, we observed that expression of E6 and E7 is sufficient to induce reactive oxygen species (ROS) generation in head and neck cancer cells. E6/E7-induced oxidative stress is mediated by nicotinamide adenine dinucleotide phosphate oxidases (NOXs) and causes DNA damage and chromosomal aberrations. This mechanism for genomic instability distinguishes HPV-positive from HPV-negative tumors, as we observed NOX-induced oxidative stress in HPV-positive but not HPV-negative head and neck cancer cells. We identified NOX2 as the source of HPV-induced oxidative stress as NOX2 silencing significantly reduced ROS generation, DNA damage and chromosomal aberrations in HPV-positive cells. Due to their state of chronic oxidative stress, HPV-positive cells are more susceptible to DNA damage induced by ROS and ionizing radiation (IR). Furthermore, exposure to IR results in the formation of complex lesions in HPV-positive cells as indicated by the higher amount of chromosomal breakage observed in this group of cells. These results reveal a novel mechanism for sustaining genomic instability in HPV-positive head and neck tumors and elucidate its contribution to their intrinsic radiosensitivity. PMID:26354779

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

    PubMed

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

    2013-02-01

    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

  10. Attenuated Recombinant Influenza A Virus Expressing HPV16 E6 and E7 as a Novel Therapeutic Vaccine Approach

    PubMed Central

    Jindra, Christoph; Huber, Bettina; Shafti-Keramat, Saeed; Wolschek, Markus; Ferko, Boris; Muster, Thomas; Brandt, Sabine; Kirnbauer, Reinhard

    2015-01-01

    Persistent infection with high-risk human papillomavirus (HPV) types, most often HPV16 and HPV18, causes all cervical and most anal cancers, and a subset of vulvar, vaginal, penile and oropharyngeal carcinomas. Two prophylactic virus-like particle (VLPs)-based vaccines, are available that protect against vaccine type-associated persistent infection and associated disease, yet have no therapeutic effect on existing lesions or infections. We have generated recombinant live-attenuated influenza A viruses expressing the HPV16 oncogenes E6 and E7 as experimental immunotherapeutic vaccine candidates. The influenza A virus life cycle lacks DNA intermediates as important safety feature. Different serotypes were generated to ensure efficient prime and boost immunizations. The immune response to vaccination in C57BL/6 mice was characterized by peptide ELISA and IFN-γ ELISpot, demonstrating induction of cell-mediated immunity to HPV16 E6 and E7 oncoproteins. Prophylactic and therapeutic vaccine efficacy was analyzed in the murine HPV16-positive TC-1 tumor challenge model. Subcutaneous (s.c.) prime and boost vaccinations of mice with recombinant influenza A serotypes H1N1 and H3N2, followed by challenge with TC-1 cells resulted in complete protection or significantly reduced tumor growth as compared to control animals. In a therapeutic setting, s.c. vaccination of mice with established TC-1 tumors decelerated tumor growth and significantly prolonged survival. Importantly, intralesional vaccine administration induced complete tumor regression in 25% of animals, and significantly reduced tumor growth in 50% of mice. These results suggest recombinant E6E7 influenza viruses as a promising new approach for the development of a therapeutic vaccine against HPV-induced disease. PMID:26381401

  11. Design of a highly effective therapeutic HPV16 E6/E7-specific DNA vaccine: optimization by different ways of sequence rearrangements (shuffling).

    PubMed

    Almajhdi, Fahad N; Senger, Tilo; Amer, Haitham M; Gissmann, Lutz; hlschlger, Peter

    2014-01-01

    Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism. PMID:25422946

  12. Design of a Highly Effective Therapeutic HPV16 E6/E7-Specific DNA Vaccine: Optimization by Different Ways of Sequence Rearrangements (Shuffling)

    PubMed Central

    Almajhdi, Fahad N.; Senger, Tilo; Amer, Haitham M.; Gissmann, Lutz; hlschlger, Peter

    2014-01-01

    Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16) is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism. PMID:25422946

  13. Developing Michigan Cancer Foundation 7 Cells with Stable Expression of E7 Gene of Human Papillomavirus Type 16

    PubMed Central

    Ghasemi, Faezeh; Rostami, Sina; Nabavinia, Maryam Sadat; Meshkat, Zahra

    2016-01-01

    Background: Human papillomavirus (HPV) is responsible for the development of cervical neoplasia. Infection with human papillomavirus type 16 (HPV-16) is a major risk factor for the development of cervical cancer. The virus encodes three oncoproteins (E5, E6 and E7), of which, the E7 oncoprotein is the major protein involved in cell immortalization and transformation of the infected cells. The aim of the current study was to develop Michigan Cancer Foundation 7 (MCF7) cells, which could stably express E7 protein of HPV type 16. Methods: E7 gene of HPV type 16 was introduced into MCF7 cells by Lipofectamine 2000 reagent and the transfected cells were treated with G418 antibiotic. After antibiotic selection of the transfected cells, stable expression of E7 gene of HPV16 was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Results: Antibiotic selections of transfected cells were performed and transfected cells were alive in cytotoxic concentration of the antibiotic. RNA was extracted from transfected cells and E7 gene of HPV16 was amplified by RT-PCR method and a 350-bp band corresponds to E7 was observed. Conclusion: Results confirmed the stable transfection of cells. The stably transfected cells can be used as a useful tool in future studies on HPV16 and cancers caused by this virus. PMID:26870142

  14. The epithelial-mesenchymal transition induced by keratinocyte growth conditions is overcome by E6 and E7 from HPV16, but not HPV8 and HPV38: Characterization of global transcription profiles

    SciTech Connect

    Azzimonti, Barbara; Dell'Oste, Valentina; Borgogna, Cinzia; Mondini, Michele; Gugliesi, Francesca; De Andrea, Marco; Chiorino, Giovanna; Scatolini, Maria; Ghimenti, Chiara; Landolfo, Santo; Gariglio, Marisa

    2009-06-05

    The aim of this study was to evaluate the growth properties of primary human keratinocytes expressing E6 and E7 proteins, which are from either the beta- or alpha-genotypes, under different culture conditions. We demonstrated that keratinocytes expressing E6 and E7, from both HPV8 and 38, irreversibly underwent the epithelial-mesenchymal transition (EMT) when grown on plastic with FAD medium (F12/DMEM/5%FBS). Expression of E6/E7 from HPV16 was capable of fully overcoming the FAD-induced EMT. Immortalization was only observed in HPV16-transduced cell lines, while the more proliferating phenotype of both KerHPV8 and 38 was mainly related to FAD-induced EMT. Microarray analysis of exponentially growing cells identified 146 cellular genes that were differentially regulated in HPV16 compared to HPV8- and 38-transduced cells. A large accumulation of transcripts associated with epidermal development and differentiation was observed in HPV16-transduced cells, whereas transcripts of genes involved in the extracellular matrix, multicellular organismal processes, and inflammatory response were affected in HPV8 and 38-transduced cells.

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

    PubMed

    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

    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

  16. E5 and E6/E7 of high-risk HPVs cooperate to enhance cancer progression through EMT initiation.

    PubMed

    Moustafa, Ala-Eddin Al

    2015-09-01

    It is estimated that 10-20% of human carcinogenesis is linked to virus infection including papillomaviruses (HPVs). Moreover, since metastatic cancer disease is a major cause of morbidity and mortality in cancer patients, the role of onco-viruses in cancer progression to a metastatic form is of particular interest. Recent studies reported that E5 and E6/E7 onco-proteins of high-risk HPVs could enhance cancer progression via the initiation of the epithelial-mesenchymal transition (EMT) event. Herein, we discuss the association between E5 as well as E6/E7 of high-risk HPV and cancer progression. PMID:26177717

  17. Enhanced Cytotoxic CD8 T Cell Priming Using Dendritic Cell-Expressing Human Papillomavirus-16 E6/E7-p16INK4 Fusion Protein with Sequenced Anti-Programmed Death-1.

    PubMed

    Garcia-Bates, Tatiana M; Kim, Eun; Concha-Benavente, Fernando; Trivedi, Sumita; Mailliard, Robbie B; Gambotto, Andrea; Ferris, Robert L

    2016-03-15

    The incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma has increased in recent decades, though HPV prevention vaccines may reduce this rise in the future. HPV-related cancers express the viral oncoproteins E6 and E7. The latter inactivates the tumor suppressor protein retinoblastoma (Rb), which leads to the overexpression of p16(INK4) protein, providing unique Ags for therapeutic HPV-specific cancer vaccination. We developed potential adenoviral vaccines that express a fusion protein of HPV-16 E6 and E7 (Ad.E6E7) alone or fused with p16 (Ad.E6E7p16) and also encoding an anti-programmed death (PD)-1 Ab. Human monocyte-derived dendritic cells (DC) transduced with Ad.E6E7 or Ad.E6E7p16 with or without Ad.αPD1 were used to activate autologous CD8 CTL in vitro. CTL responses were tested against naturally HPV-infected head and neck squamous cell carcinoma cells using IFN-γ ELISPOT and [(51)Cr]release assay. Surprisingly, stimulation and antitumor activity of CTL were increased after incubation with Ad.E6E7p16-transduced DC (DC.E6E7p16) compared with Ad.E6E7 (DC.E6E7), a result that may be due to an effect of p16 on cyclin-dependent kinase 4 levels and IL-12 secretion by DC. Moreover, the beneficial effect was most prominent when anti-PD-1 was introduced during the second round of stimulation (after initial priming). These data suggest that careful sequencing of Ad.E6E7.p16 with Ad.αPD1 could improve antitumor immunity against HPV-related tumors and that p16 may enhance the immunogenicity of DC, through cyclin-dependent pathways, Th1 cytokine secretion, and by adding a nonviral Ag highly overexpressed in HPV-induced cancers. PMID:26851223

  18. Species restriction of Herpesvirus saimiri and Herpesvirus ateles: human lymphocyte transformation correlates with distinct signaling properties of viral oncoproteins.

    PubMed

    Katsch, Kristin; de Jong, Sarah Jill; Schmidt, Monika; Mller-Fleckenstein, Ingrid; Fleckenstein, Bernhard; Albrecht, Jens-Christian; Biesinger, Brigitte

    2012-05-01

    The potential of Herpesvirus saimiri (HVS) subgroups A, B and C and Herpesvirus ateles (HVA) to transform primary T cells to permanent growth in vitro is restricted by the primate host species and by viral variability represented by distinct viral oncoproteins. We now addressed the relation between the transforming potential of the different viruses and the signaling pathways activated by transiently expressed oncoproteins. Marmoset lymphocytes were transformed by all HVS subgroups as well as HVA, while transformation of human cells was restricted to HVS-C and, unexpectedly, HVA. NF-?B and Src-family kinase (SFK) activity was required for survival of all transformed lymphocytes. Accordingly, NF-?B was induced by oncoproteins of all viruses. In contrast, SFK-related signaling was detectable only for oncoproteins of HVS-C and HVA. Thus, the restricted transformation of human lymphocytes likely correlates with the specific SFK targeting by these oncoproteins. These results will enable further studies into novel SFK effector mechanisms relevant for T-cell proliferation. PMID:22374337

  19. Serum growth factors and oncoproteins in firefighters.

    PubMed

    Ford, J; Smith, S; Luo, J C; Friedman-Jimenez, G; Brandt-Rauf, P; Markowitz, S; Garibaldi, K; Niman, H

    1992-02-01

    Firefighters are potentially at increased risk for cancer and non-malignant respiratory disease due to their toxic exposures on the job. Growth factors and oncogene proteins are thought to play a role in the development of various malignancies and pulmonary fibrotic diseases. Therefore, a cohort of firefighters and matched controls have been screened for the presence of nine different growth factors and oncoproteins using an immunoblotting assay. Fourteen of the firefighters were found to be positive for beta-transforming growth factor (beta-TGF) related proteins compared to no positives in the controls (P = 0.0017). These results suggest that beta-TGF may be a possible biomarker for monitoring firefighters and other exposed workers for the potential development of cancer or non-malignant respiratory disease. PMID:1533320

  20. Oncoprotein metastasis and its suppression revisited

    PubMed Central

    2010-01-01

    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. PMID:20380702

  1. 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

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

    2013-01-01

    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. PMID:23935506

  2. Karyopherin {beta}3: A new cellular target for the HPV-16 E5 oncoprotein

    SciTech Connect

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

    2008-07-11

    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 {beta}3, a member of the nuclear import receptor family with critical roles in the nuclear import of ribosomal proteins and in the secretory pathway.

  3. The Curcumin Analogue 1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3-one Induces Apoptosis and Downregulates E6 and E7 Oncogene Expression in HPV16 and HPV18-Infected Cervical Cancer Cells.

    PubMed

    Paulraj, Felicia; Abas, Faridah; Lajis, Nordin H; Othman, Iekhsan; Hassan, Sharifah Syed; Naidu, Rakesh

    2015-01-01

    In an effort to study curcumin analogues as an alternative to improve the therapeutic efficacy of curcumin, we screened the cytotoxic potential of four diarylpentanoids using the HeLa and CaSki cervical cancer cell lines. Determination of their EC50 values indicated relatively higher potency of 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one (MS17, 1.03 ± 0.5 μM; 2.6 ± 0.9 μM) and 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one (MS13, 2.8 ± 0.4; 6.7 ± 2.4 μM) in CaSki and HeLa, respectively, with significantly greater growth inhibition at 48 and 72 h of treatment compared to the other analogues or curcumin. Based on cytotoxic and anti-proliferative activity, MS17 was selected for comprehensive apoptotic studies. At 24 h of treatment, fluorescence microscopy detected that MS17-exposed cells exhibited significant morphological changes consistent with apoptosis, corroborated by an increase in nucleosomal enrichment due to DNA fragmentation in HeLa and CaSki cells and activation of caspase-3 activity in CaSki cells. Quantitative real-time PCR also detected significant down-regulation of HPV18- and HPV16-associated E6 and E7 oncogene expression following treatment. The overall data suggests that MS17 treatment has cytotoxic, anti-proliferative and apoptosis-inducing potential in HPV-positive cervical cancer cells. Furthermore, its role in down-regulation of HPV-associated oncogenes responsible for cancer progression merits further investigation into its chemotherapeutic role for cervical cancer. PMID:26132907

  4. Intravaginal HPV DNA vaccination with electroporation induces local CD8+ T-cell immune responses and antitumor effects against cervicovaginal tumors.

    PubMed

    Sun, Y; Peng, S; Qiu, J; Miao, J; Yang, B; Jeang, J; Hung, C-F; Wu, T-C

    2015-07-01

    Therapeutic human papillomavirus (HPV) vaccines have the potential to inhibit the progression of an established HPV infection to precancer and cancer lesions by targeting HPV oncoproteins. We have previously developed a therapeutic DNA vaccine encoding calreticulin (CRT) linked to E7, CRT/E7 DNA vaccine, for use in the treatment of HPV-associated lesions. Since the transfection efficiency of DNA vaccines administered in vivo is typically low, we examined the use of electroporation as well as different routes of administration to enhance antigen-specific tumor control. We tested the effects of the CRT/E7 DNA vaccine administered intramuscularly or intravaginally, with or without electroporation, on the generation of CD8+ T-cell immunity and therapeutic antitumor effects in HPV16 E7-expressing cervicovaginal tumor-bearing mice. We found that intravaginal vaccination of CRT/E7 DNA followed by electroporation-induced potent E7-specific CD8(+) T-cell responses in the cervicovaginal tract, compared with intramuscular injection followed by electroporation. Furthermore, tumor-bearing mice vaccinated intravaginally followed by electroporation had an enhanced survival, antitumor effects and local production of IFN-?+CD8+ T cells compared with those vaccinated intramuscularly with electroporation. Thus, we show that intravaginal CRT/E7 DNA vaccination followed by electroporation generates the most potent therapeutic antitumor effects against an orthotopic E7-expressing tumor model. The current study will have significant clinical implications once a clinically applicable electroporation device for intravaginal use becomes available. PMID:25786869

  5. Intravaginal HPV DNA vaccination with electroporation induces local CD8+ T-cell immune responses and antitumor effects against cervicovaginal tumors

    PubMed Central

    Sun, Y; Peng, S; Qiu, J; Miao, J; Yang, B; Jeang, J; Hung, C-F; Wu, T-C

    2015-01-01

    Therapeutic human papillomavirus (HPV) vaccines have the potential to inhibit the progression of an established HPV infection to precancer and cancer lesions by targeting HPV oncoproteins. We have previously developed a therapeutic DNA vaccine encoding calreticulin (CRT) linked to E7, CRT/E7 DNA vaccine, for use in the treatment of HPV-associated lesions. Since the transfection efficiency of DNA vaccines administered in vivo is typically low, we examined the use of electroporation as well as different routes of administration to enhance antigen-specific tumor control. We tested the effects of the CRT/E7 DNA vaccine administered intramuscularly or intravaginally, with or without electroporation, on the generation of CD8+ T-cell immunity and therapeutic antitumor effects in HPV16 E7-expressing cervicovaginal tumor-bearing mice. We found that intravaginal vaccination of CRT/E7 DNA followed by electroporation-induced potent E7-specific CD8+ T-cell responses in the cervicovaginal tract, compared with intramuscular injection followed by electroporation. Furthermore, tumor-bearing mice vaccinated intravaginally followed by electroporation had an enhanced survival, antitumor effects and local production of IFN-?+CD8+ T cells compared with those vaccinated intramuscularly with electroporation. Thus, we show that intravaginal CRT/E7 DNA vaccination followed by electroporation generates the most potent therapeutic antitumor effects against an orthotopic E7-expressing tumor model. The current study will have significant clinical implications once a clinically applicable electroporation device for intravaginal use becomes available. PMID:25786869

  6. Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator

    PubMed Central

    Gammoh, Noor; Grm, Helena Sterlinko; Massimi, Paola; Banks, Lawrence

    2006-01-01

    In order to ensure a productive life cycle, human papillomaviruses (HPVs) require fine regulation of their gene products. Uncontrolled activity of the viral oncoproteins E6 and E7 results in the immortalization of the infected epithelial cells and thus prevents the production of mature virions. Ectopically expressed E2 has been shown to suppress transcription of the HPV E6 and E7 region in cell lines where the viral DNA is integrated into the host genome, resulting in growth inhibition. However, it has been demonstrated that growth control of these cell lines can also occur independently of HPV E2 transcriptional activity in high-risk HPV types. In addition, E2 is unable to suppress transcription of the same region in cell lines derived from cervical tumors that harbor only episomal copies of the viral DNA. Here we show that HPV type 16 (HPV-16) E2 is capable of inhibiting HPV-16 E7 cooperation with an activated ras oncogene in the transformation of primary rodent cells. Furthermore, we demonstrate a direct interaction between the E2 and E7 proteins which requires the hinge region of E2 and the zinc-binding domain of E7. These viral proteins interact in vivo, and E2 has a marked effect upon both the stability of E7 and its cellular location, where it is responsible for recruiting E7 onto mitotic chromosomes at the later stages of mitosis. These results demonstrate a direct role for E2 in regulating the function of E7 and suggest an important role for E2 in directing E7 localization during mitosis. PMID:16439535

  7. Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo.

    PubMed

    Jung, Hun Soon; Rajasekaran, Nirmal; Song, Sang Yong; Kim, Young Deug; Hong, Sungyoul; Choi, Hyuck Jae; Kim, Young Seok; Choi, Jong-Sun; Choi, Yoon-La; Shin, Young Kee

    2015-01-01

    The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated ?-galactosidase (SA-?-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas. PMID:26035754

  8. E7 properties of mucosal human papillomavirus types 26, 53 and 66 correlate with their intermediate risk for cervical cancer development

    SciTech Connect

    Mansour, Mariam; Touka, Majid; Hasan, Uzma; Bellopede, Angelica; Smet, Anouk; Accardi, Rosita; Gabet, Anne-Sophie; Sylla, Bakary S.; Tommasino, Massimo

    2007-10-10

    Epidemiological studies have demonstrated that 15 different mucosal human papillomavirus (HPV) types of the genus alpha of the HPV phylogetic tree are classified as high risk for cervical cancer development. Three additional HPV types of the same genus, HPV26, 53 and 66, are classified as probable high-risk types. In this study, we have characterized the biological properties of the E7 oncoproteins from these three HPV types. All of the corresponding E7 proteins were able to associate with retinoblastoma protein (pRb) and up-regulated the expression of several positive cell cycle regulators, i.e. CDK2, cyclin A and cylin E. However, HPV26 E7 appears to be more efficient than HPV53 and 66 E7 in up-regulating the transcription of cyclin A. Unlike E7 from the high-risk type HPV16 protein, HPV26, 53 and 66 did not efficiently promote pRb degradation. In addition, E7 from these viruses was able to promote proliferation of primary human keratinocytes and circumvent G1 arrest imposed by overexpression of p16{sup INK4a}, but with less efficiency than the high-risk HPV16 E7. Together, our data show that in vitro properties of these E7 proteins correlate with the epidemiological classification of HPV26, 53 and 66 as HPV types with an intermediate risk for cervical cancer development.

  9. Mucin 1 Oncoprotein Expression Is Suppressed by the miR-125b Oncomir

    PubMed Central

    Rajabi, Hasan; Jin, Caining; Ahmad, Rehan; McClary, Andrew Cain; Joshi, Maya Datt; Kufe, Donald

    2010-01-01

    The MUC1 oncoprotein is overexpressed in most human breast cancers by mechanisms that are incompletely understood. The microRNA, miR-125b, is downregulated in breast cancer cells. The present studies demonstrate that the MUC1 3?UTR contains a site for binding of the miR-125b seed region. The results show that the MUC1 3?UTR suppresses luciferase expression and that this effect is abrogated by mutation or deletion of the miR-125b binding site. Expression of an antisense miR-125b in BT-549 breast cancer cells was associated with induction of MUC1 protein but not MUC1 mRNA levels. The antisense miR-125b also increased BT-549 cell growth by a MUC1-dependent mechanism. In addition, overexpression of exogenous miR-125b downregulated MUC1 protein and not MUC1 transcripts in ZR-75-1 breast cancer cells. Silencing of MUC1 in ZR-75-1 cells with a siRNA has been shown to promote DNA damage-induced apoptosis. In concert with these observations, miR-125b-induced decreases in MUC1 levels increased the apoptotic response of ZR-75-1 cells to cisplatin treatment. These findings indicate that miR-125b suppresses translation of the MUC1 oncoprotein and that miR-125b thereby functions as a tumor suppressor in breast cancer cells. PMID:20729973

  10. Degradation of nuclear oncoproteins by the ubiquitin system in vitro

    SciTech Connect

    Ciechanover, A.; Bercovich, B.; Orian, A. ); DiGiuseppe, J.A.; Schwartz, A.L.; Brodeur, G.M. ); Richter, J.D. )

    1991-01-01

    Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins. In the present study, the authors have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the N-myc, c-myc, c-fos, p53, and E1A genes. Each of these nuclear oncoproteins was synthesized in vitro by transcription of the appropriate cDNA and translation of the resulting mRNA in the presence of ({sup 35}S)methionine. Degradation of labeled proteins was monitored in the ubiquitin cell-free system. ATP stimulated the degradation of all the proteins between 3- and 10-fold. The degradation was completely inhibited by neutralizing antibody directed against the ubiquitin-activating enzyme, E{sub 1}, the first enzyme in the ubiquitin-mediated proteolytic cascade. Morever, degradation in E{sub 1}-depleted lysates could be restored in each case by the addition of affinity-purified E{sub 1}. These data suggest that the ubiquitin system mediates the degradation of these oncoproteins in vitro. Degradation of other proteins, such as superoxide dismutase, cytochrome c, enolase, RNase A, and ornithine decarboxylase, is not mediated by the ubiquitin cell-free system. This suggests that the nuclear oncoproteins studies here possess specific signals that target them for rapid turnover by this proteolytic pathway. Furthermore, the relative sensitivity to degradation of various E1A mutants in vivo is also maintained in the cell-free system, suggesting that the ubiquitin pathway may play a role in the cellular degradation of these proteins as well.

  11. Epstein-Barr Virus oncoprotein super-enhancers control B cell growth

    PubMed Central

    Zhou, Hufeng; Schmidt, Stefanie CS; Jiang, Sizun; Willox, Bradford; Bernhardt, Katharina; Liang, Jun; Johannsen, Eric C; Kharchenko, Peter; Gewurz, Benjamin E; Kieff, Elliott; Zhao, Bo

    2015-01-01

    Summary Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ~1800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals characteristic of super-enhancers, and were designated “EBV super-enhancers”. EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, enabling LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had a high co-occupancy of the transcription factors STAT5 and NFAT. EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor, JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions. PMID:25639793

  12. The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins.

    PubMed

    Li, Ning; Yousefi, Maryam; Nakauka-Ddamba, Angela; Li, Fan; Vandivier, Lee; Parada, Kimberly; Woo, Dong-Hun; Wang, Shan; Naqvi, Ammar S; Rao, Shilpa; Tobias, John; Cedeno, Ryan J; Minuesa, Gerard; Y, Katz; Barlowe, Trevor S; Valvezan, Alexander; Shankar, Sheila; Deering, Raquel P; Klein, Peter S; Jensen, Shane T; Kharas, Michael G; Gregory, Brian D; Yu, Zhengquan; Lengner, Christopher J

    2015-12-22

    Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers. PMID:26673327

  13. Abrogation of the postmitotic checkpoint contributes to polyploidization in human papillomavirus E7-expressing cells.

    PubMed

    Heilman, Susan A; Nordberg, Joshua J; Liu, Yingwang; Sluder, Greenfield; Chen, Jason J

    2009-03-01

    High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint. PMID:19129456

  14. Expressions of E2 and E7-HPV16 proteins in pre-malignant and malignant lesions of the uterine cervix.

    PubMed

    Ramirez, N; Guerra, F; Camporeale, G; Quintana, S; Diaz, L B; Cuneo, N; Villacorta Hidalgo, J; Tatti, S A; Alonso, L G; Borkosky, S S; Prat Gay, G; Palaoro, L

    2015-12-01

    Continuous production of the E7 protein from different types of high risk human papilloma virus (HPV) is required for progression of malignancy. We developed antibodies against HPV type 16 E7 and E2 proteins to evaluate their utility as markers for diagnosis during early stages of cervical cancer. Forty biopsies from uterine cervices were diagnosed as low grade intraepithelial lesion (LSIL), high grade intraepithelial lesion (HSIL), squamous carcinoma (SC), in situ adenocarcinoma (ISA) and invasive adenocarcinoma (AC), all of which were infected with HPV 16. Immunohistochemistry was used to investigate the expressions of E7 and E2 (both from HPV 16) and p16. P16 was expressed in eight of 12 LSILs, in all HSILs, in 16 of 18 SC and in all ACs. E2 was expressed in six of 12 LSILs. E7 was positive in eight of 12 LSILs and in all HSIL and carcinomas. The expressions of E2 and E7 of HPV16 related to p16 expression confirmed the value of the viral oncogenic proteins as complementary to histology and support the carcinogenic model of the uterine cervix, because HPVDNA integration into cellular DNA implies the destruction of the gene encoding E2, which suppresses the expression of the E6 and E7 oncoproteins. E2 from HPV16 could be marker for LSILs, while E7 could be a marker for progression of LSILs to HSILs in patients infected by HPV16, because viral typing has little positive predictive value. PMID:26052817

  15. The association of mammalian DREAM complex and HPV16 E7 proteins

    PubMed Central

    Rashid, Nurshamimi Nor; Rothan, Hussin A; Yusoff, Mohd Shahrizal Mohd

    2015-01-01

    The mammalian DREAM (Drosophila, RB, E2F, and Myb) complex was discovered in 2004 by several research groups. It was initially identified in Drosophila followed by Caenorhaditis elegans and later in mammalian cells. The composition of DREAM is temporally regulated during cell cycle; being associated with E2F-4 and either p107 or p130 in G0/G1 (repressive DREAM complexes) and with B-myb transcription factor in S/G2 (activator DREAM complex). High risk human papillomavirus (HPV) E6 and E7 oncoproteins expression are important for malignant transformation of cervical cancer cells. In particular, the E7 of high risk HPV binds to pRB family members (pRB, p107 and p130) for degradation. It has recently been discovered that the p107 and p130 pocket proteins are members of mammalian DREAM complexes. With this understanding, we would like to hypothesise the mammalian DREAM complex could plays a critical role for malignant transformation in cervical cancer cells. PMID:26885443

  16. Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling.

    PubMed

    Sun, Y; Liu, X; Eaton, E N; Lane, W S; Lodish, H F; Weinberg, R A

    1999-10-01

    TGF-beta treatment of cells induces a variety of physiologic responses, including growth inhibition, differentiation, and induction of apoptosis. TGF-beta induces phosphorylation and nuclear translocation of Smad3. We describe here the association of Smad3 with the nuclear protooncogene protein Ski in response to the activation of TGF-beta signaling. Association with Ski represses transcriptional activation by Smad3, and overexpression of Ski renders cells resistant to the growth-inhibitory effects of TGF-beta. The transcriptional repression as well as the growth resistance to TGF-beta by overexpression of Ski can be overcome by overexpression of Smad3. These results demonstrate that Ski is a novel component of the TGF-beta signaling pathway and shed light on the mechanism of action of the Ski oncoprotein. PMID:10549282

  17. The multiple cellular functions of the oncoprotein Golgi phosphoprotein 3

    PubMed Central

    Belloni, Giorgio; Colotti, Gianni; Giansanti, Maria Grazia

    2015-01-01

    The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein, a component of Trans-Golgi Network (TGN), has been defined as a first-in-class Golgi oncoprotein and characterized as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is commonly amplified in several solid tumors. Furthermore this protein has been associated with poor prognosis in many cancers. Highly conserved from yeast to humans, GOLPH3 provides an essential function in vesicle trafficking and Golgi structure. Recent data have also implicated this oncoprotein in regulation of cytokinesis, modulation of mitochondrial mass and cellular response to DNA damage. A minute dissection of the molecular pathways that require GOLPH3 protein will be helpful to develop new therapeutic cancer strategies. PMID:25691054

  18. Human T-cell leukemia virus type 1 (HTLV-1) Tax1 oncoprotein but not HTLV-2 Tax2 induces the expression of OX40 ligand by interacting with p52/p100 and RelB.

    PubMed

    Motai, Yosuke; Takahashi, Masahiko; Takachi, Takayuki; Higuchi, Masaya; Hara, Toshifumi; Mizuguchi, Mariko; Aoyagi, Yutaka; Terai, Shuji; Tanaka, Yuetsu; Fujii, Masahiro

    2016-02-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a causative retrovirus of adult T-cell leukemia and HTLV-1-associated myelopathy. Unlike HTLV-1, the same group of retrovirus HTLV-2 has not been found to be associated with these diseases. HTLV-1 and HTLV-2 encode transforming proteins Tax1 and Tax2, and a few distinct activities of Tax1 from those of Tax2 have been proposed to contribute to the HTLV-1-specific pathogenesis of disease. One significant difference of Tax1 from Tax2 is the activation of transcription factor NF-κB2/p100/p52. We found that Tax1 but not Tax2 induces the expression of OX40 ligand (OX40L) in a human T-cell line. To induce the OX40L expression, Tax1 but not Tax2 was observed to interact with NF-κB2/p100/p52 and RelB and the distinct interaction activity was mediated by the Tax1 amino acid region of 225-232. In addition, Tax1 but not Tax2 or Tax1/225-232 interacted with p65, p50, and c-Rel; however, the interactions were much less than those noted with NF-κB2/p100/p52 and RelB. OX40L is a T-cell costimulatory molecule of the tumor necrosis factor family, and its signal plays a critical role in establishing adaptive immunity by inducing the polarized differentiation of T-cells to cells such as T helper type 2 and T follicular helper cells. Therefore, the present findings suggest that Tax1 might alter the immune response to HTLV-1 and/or differentiation of HTLV-1-infected T-cells via OX40L induction, thereby acting as a factor mediating the distinct phenotypes and pathogenesis of HTLV-1 from that of HTLV-2. PMID:26739459

  19. NSD3-NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

    PubMed Central

    French, Christopher A.; Rahman, Shaila; Walsh, Erica M.; Kühnle, Simone; Grayson, Adlai R.; Lemieux, Madeleine E.; Grunfeld, Noam; Rubin, Brian P.; Antonescu, Cristina R.; Zhang, Songlin; Venkatramani, Rajkumar; Cin, Paola Dal; Howley, Peter M.

    2014-01-01

    NUT midline carcinoma (NMC) is an aggressive subtype of squamous cell carcinoma that typically harbors BRD4/3-NUT fusion oncoproteins that block differentiation and maintain tumor growth. In 20% of cases NUT is fused to uncharacterized non-BRD gene(s). We established a new patient-derived NMC cell line (1221) and demonstrated that it harbors a novel NSD3-NUT fusion oncogene. We find that NSD3-NUT is both necessary and sufficient for the blockade of differentiation and maintenance of proliferation in NMC cells. NSD3-NUT binds to BRD4, and BRD bromodomain inhibitors induce differentiation and arrest proliferation of 1221 cells. We find further that NSD3 is required for the blockade of differentiation in BRD4-NUT-expressing NMCs. These findings identify NSD3 as a novel critical oncogenic component and potential therapeutic target in NMC. PMID:24875858

  20. MUCIN 1 ONCOPROTEIN EXPRESSION IS SUPPRESSED BY THE miR-125b ONCOMIR.

    PubMed

    Rajabi, Hasan; Jin, Caining; Ahmad, Rehan; McClary, Cain; Joshi, Maya Datt; Kufe, Donald

    2010-01-01

    The MUC1 oncoprotein is overexpressed in most human breast cancers by mechanisms that are incompletely understood. The microRNA, miR-125b, is downregulated in breast cancer cells. The present studies demonstrate that the MUC1 3'UTR contains a site for binding of the miR-125b seed region. The results show that the MUC1 3'UTR suppresses luciferase expression and that this effect is abrogated by mutation or deletion of the miR-125b binding site. Expression of an anti-sense miR-125b in BT-549 breast cancer cells was associated with induction of MUC1 protein, but not MUC1 mRNA, levels. The anti-sense miR-125b also increased BT-549 cell growth by a MUC1-dependent mechanism. In addition, overexpression of exogenous miR-125b downregulated MUC1 protein, and not MUC1 transcripts, in ZR-75-1 breast cancer cells. Silencing of MUC1 in ZR-75-1 cells with a siRNA has been shown to promote DNA damage-induced apoptosis. In concert with these observations, miR-125b-induced decreases in MUC1 levels increased the apoptotic response of ZR-75-1 cells to cisplatin treatment. These findings indicate that miR-125b suppresses translation of the MUC1 oncoprotein and that miR-125b thereby functions as a tumor suppressor in breast cancer cells. PMID:20729973

  1. Induction of cellular immunosuppression by the human papillomavirus type 16 E7 oncogenic protein.

    PubMed

    Le Buanec, H; Lachgar, A; D'Anna, R; Zagury, J F; Bizzini, B; Bernard, J; Ittel, D; Hallez, S; Giannouli, C; Burny, A; Zagury, D

    1999-08-01

    The human papillomavirus type 16 (HPV-16) E7 oncogenic protein is found in the culture supernatant of SiHa cells, a cervical carcinoma cell line. Extracellular E7 protein, acting as a viral toxin in human immune cells, induces the overproduction of the immune suppressive IFN alpha cytokine by APCs, and inhibits the T-cell response to recall and allogenic antigens. These effects should be taken into account for the design of anti-human cervical carcinoma vaccines. PMID:10472433

  2. 29 CFR 2584.8477(e)-7 - Effective date.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... promulgated by the Board appearing at title 5, CFR, chapter VI, 1660.1 through 1660.5 will no longer be... 29 Labor 9 2011-07-01 2011-07-01 false Effective date. 2584.8477(e)-7 Section 2584.8477(e)-7 Labor... FOR THE ALLOCATION OF FIDUCIARY RESPONSIBILITY 2584.8477(e)-7 Effective date. This section...

  3. 29 CFR 2584.8477(e)-7 - Effective date.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... promulgated by the Board appearing at title 5, CFR, chapter VI, 1660.1 through 1660.5 will no longer be... 29 Labor 9 2014-07-01 2014-07-01 false Effective date. 2584.8477(e)-7 Section 2584.8477(e)-7 Labor... FOR THE ALLOCATION OF FIDUCIARY RESPONSIBILITY 2584.8477(e)-7 Effective date. This section...

  4. TRIP-Br1 oncoprotein inhibits autophagy, apoptosis, and necroptosis under nutrient/serum-deprived condition.

    PubMed

    Jung, Samil; Li, Chengping; Duan, Jingjing; Lee, Soonduck; Kim, Kyeri; Park, Yeonji; Yang, Young; Kim, Keun-Il; Lim, Jong-Seok; Cheon, Chung-Il; Kang, Young-Sook; Lee, Myeong-Sok

    2015-10-01

    TRIP-Br1 oncogenic protein has been shown to have multiple biological functions in cells. In this study, we demonstrate that TRIP-Br1 functions as an oncoprotein by inhibiting autophagy, apoptosis, and necroptosis of cancer cells and eventually helping them to survive under the nutrient/serum starved condition. TRIP-Br1 expression level was significantly increased in conditions with low levels of nutrients. Nutrient depleted conditions were induced by culturing cancer cells until they were overcrowded with high cell density or in media deprived of glucose, amino acids, or serum. Among them, serum starvation significantly enhanced the expression of TRIP-Br1 only in all tested breast cancer cell lines (MCF7, MDA-MB-231, T47D, MDA-MB-435, Hs578D, BT549, and MDA-MB-435) but not in the three normal cell lines (MCF10A, HfCH8, and NIH3T3). As compared with the control cells, the introduction of TRIP-Br1 silencing siRNA into MCF7 and MDA-MB-231 cells accelerated cell death by inducing apoptosis and necroptosis. In this process, TRIP-Br1 confers resistance to serum starvation-induced cell deaths by stabilizing the XIAP protein and inhibiting cellular ROS production. Moreover, our data also show that the intracellular increase of TRIP-Br1 protein resulting from serum starvation seems to occur in part through the blockage of PI3K/AKT signaling pathway. PMID:26334958

  5. TRIP-Br1 oncoprotein inhibits autophagy, apoptosis, and necroptosis under nutrient/serum-deprived condition

    PubMed Central

    Duan, Jingjing; Lee, Soonduck; Kim, Kyeri; Park, Yeonji; Yang, Young; Kim, Keun-Il; Lim, Jong-Seok; Cheon, Chung-Il; Kang, Young-Sook; Lee, Myeong-Sok

    2015-01-01

    TRIP-Br1 oncogenic protein has been shown to have multiple biological functions in cells. In this study, we demonstrate that TRIP-Br1 functions as an oncoprotein by inhibiting autophagy, apoptosis, and necroptosis of cancer cells and eventually helping them to survive under the nutrient/serum starved condition. TRIP-Br1 expression level was significantly increased in conditions with low levels of nutrients. Nutrient depleted conditions were induced by culturing cancer cells until they were overcrowded with high cell density or in media deprived of glucose, amino acids, or serum. Among them, serum starvation significantly enhanced the expression of TRIP-Br1 only in all tested breast cancer cell lines (MCF7, MDA-MB-231, T47D, MDA-MB-435, Hs578D, BT549, and MDA-MB-435) but not in the three normal cell lines (MCF10A, HfCH8, and NIH3T3). As compared with the control cells, the introduction of TRIP-Br1 silencing siRNA into MCF7 and MDA-MB-231 cells accelerated cell death by inducing apoptosis and necroptosis. In this process, TRIP-Br1 confers resistance to serum starvation-induced cell deaths by stabilizing the XIAP protein and inhibiting cellular ROS production. Moreover, our data also show that the intracellular increase of TRIP-Br1 protein resulting from serum starvation seems to occur in part through the blockage of PI3K/AKT signaling pathway. PMID:26334958

  6. Adenovirus type 12 E1B 55-kilodalton oncoprotein promotes p53-mediated apoptotic response of ovarian cancer to cisplatin.

    PubMed

    Wang, Junnai; Gao, Qinglei; Li, Qiang

    2015-08-01

    The tumor suppressor p53-mediated apoptotic response plays an important role in cisplatin resistant in ovarian cancer. The adenovirus (Ad) type 12 E1B 55-kDa protein binds to p53 and inactivates its transcriptional transactivation function. In this study, we test the hypothesis that Ad12 E1B 55-kDa oncoprotein promotes p53-mediated apoptotic response of ovarian cancer to cisplatin. First, we observed the upregulation protein level of p53 target genes in cisplatin-resistant or cisplatin-sensitive ovarian cancer by Western blotting. Second, after transfection of Ad12 E1b 55-kDa expression plasmid, the expressions of p53 target genes in A2780 cells were further enhanced. Co-IP experiment demonstrated Ad12 E1b 55 kDa associated with p53. MTT assay confirmed that the cell proliferation was enhanced after transfection, as well as the enhanced cell inhibitory rate in the presence of cisplatin. Using flow cytometry, transfection of Ad12 E1B 55-kDa protein induced apoptosis and promoted S-phase transition in proliferation. Finally, results showed that all these changes promoted by Ad12 E1b 55 kDa were attenuated by the exposure of specific inhibitor of p53 signaling, pifithrin-?. Taken together, we concluded that Ad E1B 55-kDa oncoprotein promotes p53-mediated apoptotic response of ovarian cancer to cisplatin. PMID:25820823

  7. The HBx oncoprotein of hepatitis B virus engages nucleophosmin to promote rDNA transcription and cellular proliferation.

    PubMed

    Ahuja, Richa; Kapoor, Neetu Rohit; Kumar, Vijay

    2015-08-01

    The pleiotropic HBx oncoprotein of hepatitis B virus is well known to promote the expression of ribosomal RNAs and several host proteins that are known to support the development and progression of hepatocellular carcinoma (HCC). While overexpression of the nucleolar phosphoprotein, nucleophosmin (NPM), correlates with HCC progression, its upregulation by viral HBx and the resulting impact on perturbed nucleolar functions remain enigmatic. The present study shows that HBx up-regulates NPM levels and hijacks its functions to promote cellular proliferation. We found that HBx expression stabilizes NPM through post-translational modifications. Enhanced CDK2-mediated phosphorylation of NPM at Thr199 upon HBx expression prevented its proteolytic cleavage and provided resistance to apoptosis. Further, HBx directly interacted with the C-terminal domain of NPM and got translocated into the nucleolus where it facilitated the recruitment of RNA polymerase I transcriptional machinery onto the rDNA promoter. Our results indicate that HBx enhances rDNA transcription via a novel regulatory mechanism involving acetylation of NPM and the subsequent depletion of histones from the rDNA promoter. Enhanced production of ribosomal RNA resulting from co-expression of HBx and NPM promoted ribosome biogenesis, cellular proliferation and transformation. Taken together, our study strongly suggests an important role of NPM in mediating the oncogenic effects of HBx and the corresponding nucleolar perturbations induced by this viral oncoprotein. PMID:25918010

  8. Fusion of CTLA-4 with HPV16 E7 and E6 Enhanced the Potency of Therapeutic HPV DNA Vaccine

    PubMed Central

    Gan, Lili; Jia, Rong; Zhou, Lili; Guo, Jihua; Fan, Mingwen

    2014-01-01

    Preventive anti-HPV vaccines are effective against HPV infection but not against existing HPV-associated diseases, including cervical cancer and other malignant diseases. Therefore, the development of therapeutic vaccines is urgently needed. To improve anti-tumor effects of therapeutic vaccine, we fused cytotoxic T-lymphocyte antigen 4 (CTLA-4) with HPV16 E7 and E6 as a fusion therapeutic DNA vaccine (pCTLA4-E7E6). pCTLA4-E7E6 induced significantly higher anti-E7E6 specific antibodies and relatively stronger specific CTL responses than the nonfusion DNA vaccine pE7E6 in C57BL/6 mice bearing with TC-1 tumors. pCTLA4-E7E6 showed relatively stronger anti-tumor effects than pE7E6 in therapeutic immunization. These results suggest that fusing CTLA-4 with E7E6 is a useful strategy to develop therapeutic HPV DNA vaccines. In addition, fusing the C-terminal of E7 with the N-terminal of E6 impaired the functions of both E7 and E6. PMID:25265018

  9. Influence of single-walled carbon nanotubes (< 0.001 wt %) and/or zwitter-ionic phospholipid (SOPC) surface layer on the behaviour of the gradient flexoelectric and surface induced polarization domains arising in a homeotropic E7 (a mixture of 5CB, 7CB, 8OCB and 5CT) nematic layer

    NASA Astrophysics Data System (ADS)

    Hinov, H. P.; Pavli?, J. I.; Marinov, Y. G.; Petrov, A. G.; Sridevi, S.; Rafailov, P. M.; Dettlaff-Weglikowska, U.

    2010-11-01

    The influence has been studied of single-walled carbon nanotubes with a concentration between 0.0001 and 0.001 wt % and a dried zwitter-ionic phospholipid (SOPC: l-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) layer of thickness, smaller than 0.5 ?m, deposited only on a half of one of the two glass plates, on the behaviour of the gradient flexoelectric and surface polarization induced domains arising in a homeotropic nematic E7 (a mixture of 5CB, 7CB, 8OCB and 5CT) layer. We have observed for the first time different polar on/off formation of the surface polarization induced domains in the region of the liquid crystal cell without surface deposited lipid SOPC layer. On the other hand, the SOPC layer strongly decreases the gradient of the electric field thus leading to less-pronounced flexoelectric domains. However, the SOPC layer does not influence the creation of surface polarization induced domains and of injection induced domains arising at voltages above 4V. Appropriate dynamic light transmitted curves have been recorded and typical microphotographs have been taken.

  10. KIAA1324 Suppresses Gastric Cancer Progression by Inhibiting the Oncoprotein GRP78.

    PubMed

    Kang, Jin Muk; Park, Sujin; Kim, Staci Jakyong; Kim, Hyojung; Lee, Bona; Kim, Junil; Park, Jinah; Kim, Shin Tae; Yang, Han-Kwang; Kim, Woo Ho; Kim, Seong-Jin

    2015-08-01

    Recent advances in genome and transcriptome analysis have contributed to the identification of many potential cancer-related genes. Furthermore, biological and clinical investigations of the candidate genes provide us with a better understanding of carcinogenesis and development of cancer treatment. Here, we report a novel role of KIAA1324 as a tumor suppressor in gastric cancer. We observed that KIAA1324 was downregulated in most gastric cancers from transcriptome sequencing data and found that histone deacetylase was involved in the suppression of KIAA1324. Low KIAA1324 levels were associated with poor prognosis in gastric cancer patients. In the xenograft model, KIAA1324 significantly reduced tumor formation of gastric cancer cells and decreased development of preformed tumors. KIAA1324 also suppressed proliferation, invasion, and drug resistance and induced apoptosis in gastric cancer cells. Through protein interaction analysis, we identified GRP78 (glucose-regulated protein 78 kDa) as a KIAA1324-binding partner. KIAA1324 blocked oncogenic activities of GRP78 by inhibiting GRP78-caspase-7 interaction and suppressing GRP78-mediated AKT activation, thereby inducing apoptosis. In conclusion, our study reveals a tumor suppressive role of KIAA1324 via inhibition of GRP78 oncoprotein activities and provides new insight into the diagnosis and treatment of gastric cancer. PMID:26045166

  11. αB-Crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer

    PubMed Central

    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

    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 α-basic–crystallin (αB-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 αB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, αB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing αB-crystallin by RNA interference inhibited these abnormalities. αB-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 αB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing αB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that αB-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. PMID:16395408

  12. AlphaB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer.

    PubMed

    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

    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 alpha-basic-crystallin (alphaB-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 alphaB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, alphaB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing alphaB-crystallin by RNA interference inhibited these abnormalities. alphaB-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 alphaB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing alphaB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that alphaB-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. PMID:16395408

  13. Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip

    PubMed Central

    2012-01-01

    Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis. PMID:22385615

  14. Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip.

    PubMed

    Katsch, Kristin; de Jong, Sarah Jill; Albrecht, Jens-Christian; Steger, Julia; Genth, Harald; Posern, Guido; Biesinger, Brigitte

    2012-01-01

    Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis. PMID:22385615

  15. MUC1 oncoprotein activates the I?B kinase ? complex and constitutive NF-?B signalling

    PubMed Central

    Ahmad, Rehan; Raina, Deepak; Trivedi, Vishal; Ren, Jian; Rajabi, Hasan; Kharbanda, Surender; Kufe, Donald

    2014-01-01

    Nuclear factor-?B (NF-?B) is constitutively activated in diverse human malignancies by mechanisms that are not understood1,2. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-?B, blocks apoptosis and induces transformation36. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-?B p65. We show that MUC1 interacts with the high-molecular-weight I?B kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKK? and IKK?. Interaction of MUC1 with both IKK? and IKK? is necessary for IKK? activation, resulting in phosphorylation and degradation of I?B?. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKK?IKK? in response to TNF? stimulation. TNF?-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKK? is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKK? and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKK?NF-?B p65 pathway. PMID:18037881

  16. The oncoprotein gankyrin interacts with RelA and suppresses NF-{kappa}B activity

    SciTech Connect

    Higashitsuji, Hiroaki Higashitsuji, Hisako; Liu, Yu; Masuda, Tomoko; Fujita, Takanori; Abdel-Aziz, H. Ismail; Kongkham, Supranee; Dawson, Simon; John Mayer, R.; Itoh, Yoshito; Sakurai, Toshiharu; Itoh, Katsuhiko; Fujita, Jun

    2007-11-23

    Gankyrin is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It interacts with multiple proteins and accelerates degradation of tumor suppressors Rb and p53. Since gankyrin consists of 7 ankyrin repeats and is structurally similar to I{kappa}Bs, we investigated its interaction with NF-{kappa}B. We found that gankyrin directly binds to RelA. In HeLa and 293 cells, overexpression of gankyrin suppressed the basal as well as TNF{alpha}-induced transcriptional activity of NF-{kappa}B, whereas down-regulation of gankyrin increased it. Gankyrin did not affect the NF-{kappa}B DNA-binding activity or nuclear translocation of RelA induced by TNF{alpha} in these cells. Leptomycin B that inhibits nuclear export of RelA suppressed the NF-{kappa}B activity, which was further suppressed by gankyrin. The inhibitory effect of gankyrin was abrogated by nicotinamide as well as down-regulation of SIRT1, a class III histone deacetylase. Thus, gankyrin binds to NF-{kappa}B and suppresses its activity at the transcription level by modulating acetylation via SIRT1.

  17. Human papillomavirus (HPV)-18 E6 oncoprotein interferes with the epithelial cell polarity Par3 protein.

    PubMed

    Facciuto, Florencia; Bugnon Valdano, Marina; Marziali, Federico; Massimi, Paola; Banks, Lawrence; Cavatorta, Ana Laura; Gardiol, Daniela

    2014-05-01

    High-risk human papillomavirus (HPV) infection is the principal risk factor for the development of cervical cancer. The HPV E6 oncoprotein has the ability to target and interfere with several PSD-95/DLG/ZO-1 (PDZ) domain-containing proteins that are involved in the control of cell polarity. This function can be significant for E6 oncogenic activity because a deficiency in cell polarisation is a marker of tumour progression. The establishment and control of polarity in epithelial cells depend on the correct asymmetrical distribution of proteins and lipids at the cell borders and on specialised cell junctions. In this report, we have investigated the effects of HPV E6 protein on the polarity machinery, with a focus on the PDZ partitioning defective 3 (Par3) protein, which is a key component of tight junctions (TJ) and the polarity network. We demonstrate that E6 is able to bind and induce the mislocalisation of Par3 protein in a PDZ-dependent manner without significant reduction in Par3 protein levels. In addition, the high-risk HPV-18 E6 protein promotes a delay in TJ formation when analysed by calcium switch assays. Taken together, the data presented in this study contribute to our understanding of the molecular mechanism by which HPVs induce the loss of cell polarity, with potential implications for the development and progression of HPV-associated tumours. PMID:24462519

  18. HPV16-E7 Expression in Squamous Epithelium Creates a Local Immune Suppressive Environment via CCL2- and CCL5- Mediated Recruitment of Mast Cells

    PubMed Central

    Bergot, Anne-Sophie; Ford, Neill; Leggatt, Graham R.

    2014-01-01

    Human Papillomavirus (HPV) 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions. PMID:25340820

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

    PubMed Central

    Buitrago-Prez, gueda; Hachimi, Mariam; Dueas, Marta; Lloveras, Beln; Santos, Almudena; Holgun, Almudena; Duarte, Blanca; Santiago, Juan Luis; Akgl, Baki; Rodrguez-Peralto, Jos L.; Storey, Alan; Ribas, Catalina; Larcher, Fernando; del Rio, Marcela; Paramio, Jess M.; Garca-Escudero, Ramn

    2012-01-01

    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

  20. COOPERATIVITY OF THE MUC1 ONCOPROTEIN AND STAT1 PATHWAY IN POOR PROGNOSIS HUMAN BREAST CANCER

    PubMed Central

    Khodarev, Nikolai; Ahmad, Rehan; Rajabi, Hasan; Pitroda, Sean; Kufe, Turner; McClary, Cain; Joshi, Maya Datt; MacDermed, Dhara; Weichselbaum, Ralph; Kufe, Donald

    2009-01-01

    Signal transducer and activator of transcription 1 (STAT1) is activated in the inflammatory response to interferons. The MUC1 oncoprotein is overexpressed in human breast cancers. Analysis of genes differentially expressed in MUC1-transformed cells has identified a network linking MUC1 and STAT1 that is associated with cellular growth and inflammation. The results further demonstrate that the MUC1-C subunit associates with STAT1 in cells and that the MUC1-C cytoplasmic domain binds directly to the STAT1 DNA binding domain. The interaction between MUC1-C and STAT1 is inducible by IFN? in non-malignant epithelial cells and constitutive in breast cancer cells. Moreover, the MUC1-STAT1 interaction contributes to the activation of STAT1 target genes, including MUC1 itself. Analysis of two independent databases demonstrated that MUC1 and STAT1 are coexpressed in about 15% of primary human breast tumors. Coexpression of MUC1 and the STAT1 pathway was found to be significantly associated with decreased recurrence-free and overall survival. These findings indicate that (i) MUC1 and STAT1 function in an auto-inductive loop, and (ii) activation of both MUC1 and the STAT1 pathway in breast tumors confers a poor prognosis for patients. PMID:19915608

  1. MUC1 ONCOPROTEIN IS A DRUGGABLE TARGET IN HUMAN PROSTATE CANCER CELLS

    PubMed Central

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

    2009-01-01

    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. PMID:19887552

  2. Cooperativity of the MUC1 oncoprotein and STAT1 pathway in poor prognosis human breast cancer.

    PubMed

    Khodarev, N; Ahmad, R; Rajabi, H; Pitroda, S; Kufe, T; McClary, C; Joshi, M D; MacDermed, D; Weichselbaum, R; Kufe, D

    2010-02-11

    Signal transducer and activator of transcription 1 (STAT1) is activated in the inflammatory response to interferons. The MUC1 oncoprotein is overexpressed in human breast cancers. Analysis of genes differentially expressed in MUC1-transformed cells has identified a network linking MUC1 and STAT1 that is associated with cellular growth and inflammation. The results further show that the MUC1-C subunit associates with STAT1 in cells and the MUC1-C cytoplasmic domain binds directly to the STAT1 DNA-binding domain. The interaction between MUC1-C and STAT1 is inducible by IFNgamma in non-malignant epithelial cells and constitutive in breast cancer cells. Moreover, the MUC1-STAT1 interaction contributes to the activation of STAT1 target genes, including MUC1 itself. Analysis of two independent databases showed that MUC1 and STAT1 are coexpressed in about 15% of primary human breast tumors. Coexpression of MUC1 and the STAT1 pathway was found to be significantly associated with decreased recurrence-free and overall survival. These findings indicate that (i) MUC1 and STAT1 function in an auto-inductive loop, and (ii) activation of both MUC1 and the STAT1 pathway in breast tumors confers a poor prognosis for patients. PMID:19915608

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

    PubMed

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

    2009-11-01

    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

  4. MUC1-C oncoprotein promotes FLT3 receptor activation in acute myeloid leukemia cells

    PubMed Central

    Liu, Suiyang; Yin, Li; Stroopinsky, Dina; Rajabi, Hasan; Puissant, Alexandre; Stegmaier, Kimberly; Avigan, David; Kharbanda, Surender; Kufe, Donald

    2014-01-01

    Blasts from approximately one-third of patients with acute myeloid leukemia (AML) harbor activating mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase that confer a poor prognosis. The Mucin 1-C-terminal subunit (MUC1-C) oncoprotein is aberrantly expressed in AML blasts and stem cells; however, there is no known interaction between MUC1-C and FLT3. The present studies demonstrate that MUC1-C associates with wild-type and mutant FLT3 in AML cells. Targeting MUC1-C with the cell-penetrating peptide inhibitor GO-203 disrupts MUC1-C/FLT3 complexes and downregulates FLT3 activation. GO-203 treatment of AML cells was also associated with inhibition of the FLT3 downstream effectors AKT, extracellular signal-regulated kinase, and STAT5. The results further show that AML cells with FLT3-activating mutations and resistant to the FLT3 inhibitor midostaurin/PKC412 are sensitive to GO-203induced growth arrest and death. Moreover, GO-203 increases sensitivity of mutant FLT3 AML cells to FLT3 inhibitor treatment. These results indicate that MUC1-C contributes to FLT3 activation in AML cells and that targeting MUC1-C inhibits the FLT3 signaling pathway. Our findings support the development of MUC1-C inhibitors alone and in combination with agents that target FLT3 for the treatment of wild-type and mutant FLT3 AML. PMID:24282218

  5. High Expression of the PAX3-FKHR Oncoprotein Is Required to Promote Tumorigenesis of Human Myoblasts

    PubMed Central

    Xia, Shujuan J.; Holder, Dara D.; Pawel, Bruce R.; Zhang, Chune; Barr, Frederic G.

    2009-01-01

    PAX3-FKHR is a fusion oncoprotein generated by the 2;13 chromosomal translocation in alveolar rhabdomyosarcoma (ARMS), a cancer associated with the skeletal muscle lineage. Previous studies determined that high-level PAX3-FKHR expression is a consistent feature in ARMS tumors. To investigate the relationship between expression and phenotype in human myogenic cells, PAX3-FKHR was introduced into immortalized human myoblasts to produce a low overall PAX3-FKHR expression level. Although PAX3-FKHR alone failed to exert transforming activity, a combination of PAX3-FKHR and MYCN induced transforming activity in cell culture assays. Furthermore, myoblasts expressing PAX3-FKHR with or without MYCN formed tumors in SCID mice. These tumors demonstrated invasive features and expressed myogenic markers, consistent with rhabdomyosarcoma. Comparisons of tumor and parental cells revealed that only a subset of parental cells developed into tumors and that tumor cells expressed high PAX3-FKHR levels compared with transduced parental cells. Subcloning of parental PAX3-FKHR/MYCN-transduced myoblasts identified rare high PAX3-FKHR-expressing subclones with high transforming and tumorigenic activity; however, most subclones expressed low PAX3-FKHR and showed neither transforming nor tumorigenic activity. Finally, RNA interference experiments in myoblast-derived tumor and ARMS cells revealed that high PAX3-FKHR expression plays a crucial role in regulating proliferation, transformation, and differentiation. These findings support the premise that high PAX3-FKHR-expressing cells are selected during tumorigenesis. PMID:19893043

  6. Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.

    SciTech Connect

    Li, D.; Zhao, R.; Lilyestrom, W.; Gai, D.; Zhang, R.; DeCaprio, J. A.; Fanning, E.; Joachimiak, A.; Szakonyi, G.; Chen, X. S.; Univ. of Colorado Health Science Center; Dana-Farber Cancer Ins.; Vanderbilt Univ.

    2003-05-29

    The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting alpha-helices to expand/constrict the channel, producing an 'iris' effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.

  7. The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

    PubMed Central

    Antinore, M J; Birrer, M J; Patel, D; Nader, L; McCance, D J

    1996-01-01

    The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. Images PMID:8617242

  8. Unique potential of 4-1BB agonist antibody to promote durable regression of HPV+ tumors when combined with an E6/E7 peptide vaccine.

    PubMed

    Bartkowiak, Todd; Singh, Shailbala; Yang, Guojun; Galvan, Gloria; Haria, Dhwani; Ai, Midan; Allison, James P; Sastry, K Jagannadha; Curran, Michael A

    2015-09-22

    Antibody modulation of T-cell coinhibitory (e.g., CTLA-4) or costimulatory (e.g., 4-1BB) receptors promotes clinical responses to a variety of cancers. Therapeutic cancer vaccination, in contrast, has produced limited clinical benefit and no curative therapies. The E6 and E7 oncoproteins of human papilloma virus (HPV) drive the majority of genital cancers, and many oropharyngeal tumors. We discovered 15-19 amino acid peptides from HPV-16 E6/E7 for which induction of T-cell immunity correlates with disease-free survival in patients treated for high-grade cervical neoplasia. We report here that intranasal vaccination with these peptides and the adjuvant alpha-galactosylceramide elicits systemic and mucosal T-cell responses leading to reduced HPV(+) TC-1 tumor growth and prolonged survival in mice. We hypothesized that the inability of these T cells to fully reject established tumors resulted from suppression in the tumor microenvironment which could be ameliorated through checkpoint modulation. Combining this E6/E7 peptide vaccine with checkpoint blockade produced only modest benefit; however, coadministration with a 4-1BB agonist antibody promoted durable regression of established genital TC-1 tumors. Relative to other therapies tested, this combination of vaccine and ?4-1BB promoted the highest CD8(+) versus regulatory FoxP3(+) T-cell ratios, elicited 2- to 5-fold higher infiltration by E7-specific CTL, and evoked higher densities of highly cytotoxic TcEO (T cytotoxic Eomesodermin) CD8 (>70-fold) and ThEO (T helper Eomesodermin) CD4 (>17-fold) T cells. These findings have immediate clinical relevance both in terms of the direct clinical utility of the vaccine studied and in illustrating the potential of 4-1BB antibody to convert therapeutic E6/E7 vaccines already in clinical trials into curative therapies. PMID:26351680

  9. Unique potential of 4-1BB agonist antibody to promote durable regression of HPV+ tumors when combined with an E6/E7 peptide vaccine

    PubMed Central

    Bartkowiak, Todd; Singh, Shailbala; Yang, Guojun; Galvan, Gloria; Haria, Dhwani; Ai, Midan; Allison, James P.; Sastry, K. Jagannadha; Curran, Michael A.

    2015-01-01

    Antibody modulation of T-cell coinhibitory (e.g., CTLA-4) or costimulatory (e.g., 4-1BB) receptors promotes clinical responses to a variety of cancers. Therapeutic cancer vaccination, in contrast, has produced limited clinical benefit and no curative therapies. The E6 and E7 oncoproteins of human papilloma virus (HPV) drive the majority of genital cancers, and many oropharyngeal tumors. We discovered 1519 amino acid peptides from HPV-16 E6/E7 for which induction of T-cell immunity correlates with disease-free survival in patients treated for high-grade cervical neoplasia. We report here that intranasal vaccination with these peptides and the adjuvant alpha-galactosylceramide elicits systemic and mucosal T-cell responses leading to reduced HPV+ TC-1 tumor growth and prolonged survival in mice. We hypothesized that the inability of these T cells to fully reject established tumors resulted from suppression in the tumor microenvironment which could be ameliorated through checkpoint modulation. Combining this E6/E7 peptide vaccine with checkpoint blockade produced only modest benefit; however, coadministration with a 4-1BB agonist antibody promoted durable regression of established genital TC-1 tumors. Relative to other therapies tested, this combination of vaccine and ?4-1BB promoted the highest CD8+ versus regulatory FoxP3+ T-cell ratios, elicited 2- to 5-fold higher infiltration by E7-specific CTL, and evoked higher densities of highly cytotoxic TcEO (T cytotoxic Eomesodermin) CD8 (>70-fold) and ThEO (T helper Eomesodermin) CD4 (>17-fold) T cells. These findings have immediate clinical relevance both in terms of the direct clinical utility of the vaccine studied and in illustrating the potential of 4-1BB antibody to convert therapeutic E6/E7 vaccines already in clinical trials into curative therapies. PMID:26351680

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

    PubMed

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

    2014-05-15

    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

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

    PubMed Central

    Jin, Caining; Kufe, Donald

    2014-01-01

    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

  12. Mutual reinforcement of inflammation and carcinogenesis by the Helicobacter pylori CagA oncoprotein.

    PubMed

    Suzuki, Nobumi; Murata-Kamiya, Naoko; Yanagiya, Kohei; Suda, Wataru; Hattori, Masahira; Kanda, Hiroaki; Bingo, Atsuhiro; Fujii, Yumiko; Maeda, Shin; Koike, Kazuhiko; Hatakeyama, Masanori

    2015-01-01

    Helicobacter pylori cagA-positive strain delivers the CagA oncoprotein into gastric epithelial cells and at the same time elicits stomach inflammation. To experimentally investigate the pathophysiological interplay between CagA and inflammation, transgenic mice systemically expressing the bacterial cagA gene were treated with a colitis inducer, dextran sulfate sodium (DSS). Compared with control mice, DSS-induced colitis was markedly deteriorated in cagA-transgenic mice. In the colonic epithelia of cagA-transgenic mice, there was a substantial decrease in the level of I?B, which binds and sequesters NF-?B in the cytoplasm. This I?B reduction was due to CagA-mediated inhibition of PAR1, which may stimulate I?B degradation by perturbing microtubule stability. Whereas the CagA-mediated I?B reduction did not automatically activate NF-?B, it lowered the threshold of NF-?B activation by inflammogenic insults, thereby contributing to colitis exacerbation in cagA-transgenic mice. CagA also activates inflammasomes independently of NF-?B signaling, which further potentiates inflammation. The incidence of colonic dysplasia was elevated in DSS-treated cagA-transgenic mice due to a robust increase in the number of pre-cancerous flat-type dysplasias. Thus, CagA deteriorated inflammation, whereas inflammation strengthened the oncogenic potential of CagA. This work revealed that H. pylori CagA and inflammation reinforce each other in creating a downward spiral that instigates neoplastic transformation. PMID:25944120

  13. Mutual reinforcement of inflammation and carcinogenesis by the Helicobacter pylori CagA oncoprotein

    PubMed Central

    Suzuki, Nobumi; Murata-Kamiya, Naoko; Yanagiya, Kohei; Suda, Wataru; Hattori, Masahira; Kanda, Hiroaki; Bingo, Atsuhiro; Fujii, Yumiko; Maeda, Shin; Koike, Kazuhiko; Hatakeyama, Masanori

    2015-01-01

    Helicobacter pylori cagA-positive strain delivers the CagA oncoprotein into gastric epithelial cells and at the same time elicits stomach inflammation. To experimentally investigate the pathophysiological interplay between CagA and inflammation, transgenic mice systemically expressing the bacterial cagA gene were treated with a colitis inducer, dextran sulfate sodium (DSS). Compared with control mice, DSS-induced colitis was markedly deteriorated in cagA-transgenic mice. In the colonic epithelia of cagA-transgenic mice, there was a substantial decrease in the level of I?B, which binds and sequesters NF-?B in the cytoplasm. This I?B reduction was due to CagA-mediated inhibition of PAR1, which may stimulate I?B degradation by perturbing microtubule stability. Whereas the CagA-mediated I?B reduction did not automatically activate NF-?B, it lowered the threshold of NF-?B activation by inflammogenic insults, thereby contributing to colitis exacerbation in cagA-transgenic mice. CagA also activates inflammasomes independently of NF-?B signaling, which further potentiates inflammation. The incidence of colonic dysplasia was elevated in DSS-treated cagA-transgenic mice due to a robust increase in the number of pre-cancerous flat-type dysplasias. Thus, CagA deteriorated inflammation, whereas inflammation strengthened the oncogenic potential of CagA. This work revealed that H. pylori CagA and inflammation reinforce each other in creating a downward spiral that instigates neoplastic transformation. PMID:25944120

  14. Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family

    PubMed Central

    Tompa, Peter; Lee, Kevin A. W.; Chan, Hue Sun

    2013-01-01

    Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined “fuzziness”, often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs. PMID:24086122

  15. The oncoprotein HBXIP promotes glucose metabolism reprogramming via downregulating SCO2 and PDHA1 in breast cancer

    PubMed Central

    You, Xiaona; Liu, Yunxia; Li, Yinghui; Wang, Zhen; Wang, Yue

    2015-01-01

    The glucose metabolism reprogramming is a hallmark of cancer. The oncoprotein hepatitis B X-interacting protein (HBXIP) functions in the development of breast cancer. In this study, we supposed that HBXIP might be involved in the glucose metabolism reprogramming in breast cancer. We showed that HBXIP led to increases in generation of intracellular glucose and lactate, as well as decreases in generation of reactive oxygen species. Expression of synthesis of cytochrome c oxidase 2 (SCO2) and pyruvate dehydrogenase alpha 1 (PDHA1), two factors of metabolic switch from oxidative phosphorylation to aerobic glycolysis, was suppressed by HBXIP. In addition, miR-183/182 and miR-96 directly inhibited the expression of SCO2 and PDHA1 through targeting their mRNA coding sequences (CDSs), respectively. Interestingly, HBXIP elevated the miR-183/96/182 cluster expression through hypoxia-inducible factor 1? (HIF1?). The stability of HIF1? was enhanced by HBXIP through disassociating interaction of von Hippel-Lindau protein (pVHL) with HIF1?. Moreover, miR-183 increased the levels of HIF1? protein through directly targeting CDS of VHL mRNA, forming a feedback loop of HIF1?/miR-183/pVHL/HIF1?. In function, HBXIP-elevated miR-183/96/182 cluster enhanced the glucose metabolism reprogramming in vitro. HBXIP-triggered glucose metabolism reprogramming promoted the growth of breast cancer in vivo. Thus, we conclude that the oncoprotein HBXIP enhances glucose metabolism reprogramming through suppressing SCO2 and PDHA1 in breast cancer. PMID:26309161

  16. Functional characterization of human oncoprotein gankyrin in Zebrafish

    PubMed Central

    Kim, So Yeon; Hur, Wonhee; Choi, Jung-Eun; Kim, Daniel; Wang, Jin Sang; Yoon, Hye-Yeon; Piao, Lian-Shu

    2009-01-01

    Gankyrin is an oncoprotein containing seven ankyrin repeats that is overexpressed in hepatocellular carcinoma (HCC). Gankyrin binds to Mdm2, which results in accelerated ubiquitylation via degradation of p53, and it also plays an important role in cell proliferation. However, little is known about the relationships between p53 levels, cell proliferation, and gankyrin over-expression. In order to investigate the influence of gankyrin protein on p53 and Mdm2 in a zebrafish model, we injected human gankyrin (hgankyrin) containing expression vectors (pCS2-hgankyrin, pCS2-hgankyrin-EGFP) into zebrafish embryos. To measure p53 and Mdm2 expression in hgankyrin-injected embryos, RT-PCR, Northern blot and in-situ hybridization and BrdU immunostaining were used. In addition, to know the effect of hgankyrin on cell proliferation in vitro, cell viability assays such as MTT, trypan blue staining and RT-PCR following transfection of hgankyrin-containing vector into HEK 293 cell line were performed. In vivo results indicated that p53 mRNA levels decreased but those of Mdm2 were not decreased in the presence of hgankyrin. These results suggest that gankyrin downregulates p53 expression and not Mdm2 expression. In the study of cell proliferation, BrdU-positive cells were predominantly increased in the head and tail regions in hgankyrin-injected zebrafish. Additional in vitro studies using trypan blue staining and MTT assay showed that gankyrin-expressing HEK 293 cells proliferated at a faster rate, indicating that gankyrin promotes cell proliferation. Our results demonstrate that hgankyrin overexpression downregulates p53 expression and promotes cell proliferation in zebrafish. Gankyrin may play an important role in tumorigenesis via its effects on p53 and cell proliferation. PMID:19287195

  17. The Subcellular Localisation of the Human Papillomavirus (HPV) 16 E7 Protein in Cervical Cancer Cells and Its Perturbation by RNA Aptamers

    PubMed Central

    Cesur, zlem; Nicol, Clare; Groves, Helen; Mankouri, Jamel; Blair, George Eric; Stonehouse, Nicola J.

    2015-01-01

    Human papillomavirus (HPV) is the most common viral infection of the reproductive tract, affecting both men and women. High-risk oncogenic types are responsible for almost 90% of anogenital and oropharyngeal cancers including cervical cancer. Some of the HPV early genes, particularly E6 and E7, are known to act as oncogenes that promote tumour growth and malignant transformation. Most notably, HPV-16 E7 interacts with the tumour suppressor protein pRb, promoting its degradation, leading to cell cycle dysregulation in infected cells. We have previously shown that an RNA aptamer (termed A2) selectively binds to HPV16 E7 and is able to induce apoptosis in HPV16-transformed cervical carcinoma cell lines (SiHa) through reduction of E7 levels. In this study, we investigated the effects of the A2 aptamer on E7 localisation in order to define its effects on E7 activity. We demonstrate for the first time that E7 localised to the plasma membrane. In addition, we show that A2 enhanced E7 localisation in the ER and that the A2-mediated reduction of E7 was not associated with proteasomal degradation. These data suggest that A2 perturbs normal E7 trafficking through promoting E7 ER retention. PMID:26131956

  18. The contribution of NT-gp96 as an adjuvant for increasing HPV16 E7-specific immunity in C57BL?/6 mouse model.

    PubMed

    Mohit, E; Bolhassani, A; Zahedifard, F; Taslimi, Y; Rafati, S

    2012-01-01

    To control cervical cancer, efficient vaccination against human papillomavirus (HPV) is highly required. Despite the advantages and safety of the protein vaccines, additional strategies to enhance their immunogenicity are needed. E7 is a transforming protein which represents a perfect target antigen for vaccines or immunotherapies. Heat shock proteins (HSPs) facilitate cellular immune responses to antigenic peptides or proteins bound to them. Regarding to previous studies, vaccination with purified HSP/antigen complexes efficiently elicit antigen-specific immune responses in mice model. The N-terminal of glycoprotein 96 (NT-gp96) has adjuvant effect and can induce effective cumulative immune response against clinical disorders, especially cancers. In this study, the recombinant HPV16 E7 and E7 linked to NT-gp96 (E7-NT-gp96) proteins were generated in prokaryotic expression system. Mice were vaccinated twice with this recombinant proteins and the immunogenicity of the fusion protein was determined. The preventive efficacy of E7-NT-gp96 fusion protein was also evaluated and compared to E7 protein after challenging with cancerous TC-1 cell line. In vitro re-stimulated splenocytes of mice vaccinated with rE7-NT-gp96 protein induced higher IFN-? response in comparison with E7 protein immunization. Moreover, immunization with E7-NT-gp96 protein displayed low but stable humoral responses at post-challenge time. The data showed that vaccination with fused E7-NT-gp96 protein delayed the tumour occurrence and growth as compared to protein E7 alone. These results suggest that fused adjuvant-free E7-NT-gp96 protein vaccination could direct the immune responses towards Th1 immunity. Furthermore, the linkage of NT-gp96 to E7 could enhance protective anti-tumour immunity. PMID:21916914

  19. Problem-Solving Test: The Mechanism of Action of a Human Papilloma Virus Oncoprotein

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: human papilloma virus; cervical cancer; oncoproteins; malignant transformation; retinoblastoma protein; cell cycle; quiescent and cycling cells; cyclin/cyclin-dependent kinase (Cdk) complexes; E2F; S-phase genes; enhancer element; proto-oncogenes; tumor suppressor genes; radioactive…

  20. Problem-Solving Test: The Mechanism of Action of a Human Papilloma Virus Oncoprotein

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: human papilloma virus; cervical cancer; oncoproteins; malignant transformation; retinoblastoma protein; cell cycle; quiescent and cycling cells; cyclin/cyclin-dependent kinase (Cdk) complexes; E2F; S-phase genes; enhancer element; proto-oncogenes; tumor suppressor genes; radioactive

  1. ERG oncoprotein expression in prostate carcinoma patients of different ethnicities

    PubMed Central

    KELLY, GREGORY M.; KONG, YINK HEAY; DOBI, ALBERT; SRIVASTAVA, SHIV; SESTERHENN, ISABELL A.; PATHMANATHAN, RAJADURAI; TAN, HUI MENG; TAN, SHYH-HAN; CHEONG, SOK CHING

    2015-01-01

    Overexpression of the erythroblast transformation-specific-related gene (ERG) oncoprotein due to transmembrane protease, serine 2 (TMPRSS2)-ERG fusion, the most prevalent genomic alteration in prostate cancer (CaP), is more frequently observed among Caucasian patients compared to patients of African or Asian descent. To the best of our knowledge, this is the first study to investigate the prevalence of ERG alterations in a multiethnic cohort of CaP patients. A total of 191 formalin-fixed paraffin-embedded sections of transrectal ultrasound-guided prostate biopsy specimens, collected from 120 patients treated at the Sime Darby Medical Centre, Subang Jaya, Malaysia, were analyzed for ERG protein expression by immunohistochemistry using the anti-ERG monoclonal antibody 9FY as a surrogate for the detection of ERG fusion events. The overall frequency of ERG protein expression in the population evaluated in this study was 39.2%. Although seemingly similar to rates reported in other Asian communities, the expression of ERG was distinct amongst different ethnic groups (P=0.004). Malaysian Indian (MI) patients exhibited exceedingly high expression of ERG in their tumors, almost doubling that of Malaysian Chinese (MC) patients, whereas ERG expression was very low amongst Malay patients (12.5%). When collectively analyzing data, we observed a significant correlation between younger patients and higher ERG expression (P=0.04). The prevalence of ERG expression was significantly different amongst CaP patients of different ethnicities. The higher number of ERG-expressing tumors among MI patients suggested that the TMPRSS2-ERG fusion may be particularly important in the pathogenesis of CaP amongst this group of patients. Furthermore, the more frequent expression of ERG among the younger patients analyzed suggested an involvement of ERG in the early onset of CaP. The results of this study underline the value of using ERG status to better understand the differences in the etiology of CaP initiation and progression between ethnic groups. PMID:25469265

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

    PubMed Central

    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

    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. PMID:23708675

  3. Ordered Self-Assembly Mechanism of a Spherical Oncoprotein Oligomer Triggered by Zinc Removal and Stabilized by an Intrinsically Disordered Domain

    PubMed Central

    Smal, Clara; Alonso, Leonardo G.; Wetzler, Diana E.; Heer, Angeles; de Prat Gay, Gonzalo

    2012-01-01

    Background Self-assembly is a common theme in proteins of unrelated sequences or functions. The human papillomavirus E7 oncoprotein is an extended dimer with an intrinsically disordered domain, that can form large spherical oligomers. These are the major species in the cytosol of HPV transformed and cancerous cells. E7 binds to a large number of targets, some of which lead to cell transformation. Thus, the assembly process not only is of biological relevance, but represents a model system to investigate a widely distributed mechanism. Methodology/Principal Findings Using various techniques, we monitored changes in secondary, tertiary and quaternary structure in a time course manner. By applying a robust kinetic model developed by Zlotnik, we determined the slow formation of a monomeric Z-nucleus after zinc removal, followed by an elongation phase consisting of sequential second-order events whereby one monomer is added at a time. This elongation process takes place at a strikingly slow overall average rate of one monomer added every 28 seconds at 20 M protein concentration, strongly suggesting either a rearrangement of the growing complex after binding of each monomer or the existence of a conformation editing mechanism through which the monomer binds and releases until the appropriate conformation is adopted. The oligomerization determinant lies within its small 5 kDa C-terminal globular domain and, remarkably, the E7 N-terminal intrinsically disordered domain stabilizes the oligomer, preventing an insoluble amyloid route. Conclusion We described a controlled ordered mechanism with features in common with soluble amyloid precursors, chaperones, and other spherical oligomers, thus sharing determining factors for symmetry, size and shape. In addition, such a controlled and discrete polymerization reaction provides a valuable tool for nanotechnological applications. Finally, its increased immunogenicity related to its supramolecular structure is the basis for the development of a promising therapeutic vaccine candidate for treating HPV cancerous lesions. PMID:22590549

  4. Targeting the Two Oncogenic Functional Sites of the HPV E6 Oncoprotein with a High-Affinity Bivalent Ligand.

    PubMed

    Ramirez, Juan; Poirson, Juline; Foltz, Clémence; Chebaro, Yassmine; Schrapp, Maxime; Meyer, Amandine; Bonetta, Anaëlle; Forster, Anne; Jacob, Yves; Masson, Murielle; Deryckère, François; Travé, Gilles

    2015-06-26

    The E6 oncoproteins of high-risk mucosal (hrm) human papillomaviruses (HPVs) contain a pocket that captures LxxLL motifs and a C-terminal motif that recruits PDZ domains, with both functions being crucial for HPV-induced oncogenesis. A chimeric protein was built by fusing a PDZ domain and an LxxLL motif, both known to bind E6. NMR spectroscopy, calorimetry and a mammalian protein complementation assay converged to show that the resulting PDZ-LxxLL chimera is a bivalent nanomolar ligand of E6, while its separated PDZ and LxxLL components are only micromolar binders. The chimera binds to all of the hrm-HPV E6 proteins tested but not to low-risk mucosal or cutaneous HPV E6. Adenovirus-mediated expression of the chimera specifically induces the death of HPV-positive cells, concomitant with increased levels of the tumour suppressor P53, its transcriptional target p21, and the apoptosis marker cleaved caspase 3. The bifunctional PDZ-LxxLL chimera opens new perspectives for the diagnosis and treatment of HPV-induced cancers. PMID:26014966

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

    SciTech Connect

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri; Su, Xiong; Liu, Jialiu; Sundaresan, Sinju; Stahl, Philip D.

    2013-05-03

    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.

  6. The high-risk HPV E6 oncoprotein preferentially targets phosphorylated nuclear forms of hDlg

    SciTech Connect

    Narayan, Nisha; Subbaiah, Vanitha Krishna; Banks, Lawrence

    2009-04-25

    High-risk mucosal HPV E6 oncoproteins target a number of PDZ domain-containing substrates for proteasome mediated degradation. One of these, Discs Large (Dlg), is involved in the regulation of cell polarity and proliferation control. Previous studies had suggested that Dlg when hyperphosphorylated by osmotic shock, or when present in the nucleus could be preferentially targeted by E6. In this study we use phospho-specific antibodies directed against Dlg phosphorylated at residues S158 and S442 to show that these two observations are, in fact, linked. Dlg, when phosphorylated on S158 and S442 by CDK1 or CDK2, shows a preferential nuclear accumulation. However, these forms of Dlg are absent in cells derived from HPV-induced cervical cancers. Upon either proteasome inhibition or siRNA ablation of E6 expression, we see specific rescue of these phosphorylated forms of Dlg. These results demonstrate that nuclear forms of Dlg phosphorylated on its CDK phospho-acceptor sites has enhanced susceptibility to E6-induced degradation and place previous studies on the stress-induced phosphorylation of Dlg into a relevant biological context.

  7. Targeting the Two Oncogenic Functional Sites of the HPV E6 Oncoprotein with a High-Affinity Bivalent Ligand**

    PubMed Central

    Ramirez, Juan; Poirson, Juline; Foltz, Clmence; Chebaro, Yassmine; Schrapp, Maxime; Meyer, Amandine; Bonetta, Analle; Forster, Anne; Jacob, Yves; Masson, Murielle; Deryckre, Franois; Trav, Gilles

    2015-01-01

    The E6 oncoproteins of high-risk mucosal (hrm) human papillomaviruses (HPVs) contain a pocket that captures LxxLL motifs and a C-terminal motif that recruits PDZ domains, with both functions being crucial for HPV-induced oncogenesis. A chimeric protein was built by fusing a PDZ domain and an LxxLL motif, both known to bind E6. NMR spectroscopy, calorimetry and a mammalian protein complementation assay converged to show that the resulting PDZ-LxxLL chimera is a bivalent nanomolar ligand of E6, while its separated PDZ and LxxLL components are only micromolar binders. The chimera binds to all of the hrm-HPV E6 proteins tested but not to low-risk mucosal or cutaneous HPV E6. Adenovirus-mediated expression of the chimera specifically induces the death of HPV-positive cells, concomitant with increased levels of the tumour suppressor P53, its transcriptional target p21, and the apoptosis marker cleaved caspase 3. The bifunctional PDZ-LxxLL chimera opens new perspectives for the diagnosis and treatment of HPV-induced cancers. PMID:26014966

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

    PubMed Central

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

    2010-01-01

    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 (E71119) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E71120), 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. PMID:20615877

  9. The levels of epithelial anchor proteins β-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

    PubMed

    Heuser, Sandra; Hufbauer, Martin; Marx, Benjamin; Tok, Ali; Majewski, Slawomir; Pfister, Herbert; Akgül, Baki

    2016-02-01

    Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins β-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that β-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin. PMID:26645068

  10. MUC1 oncoprotein activates the IkappaB kinase beta complex and constitutive NF-kappaB signalling.

    PubMed

    Ahmad, Rehan; Raina, Deepak; Trivedi, Vishal; Ren, Jian; Rajabi, Hasan; Kharbanda, Surender; Kufe, Donald

    2007-12-01

    Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-kappaB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-kappaB p65. We show that MUC1 interacts with the high-molecular-weight IkappaB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKbeta and IKKgamma. Interaction of MUC1 with both IKKbeta and IKKgamma is necessary for IKKbeta activation, resulting in phosphorylation and degradation of IkappaBalpha. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKbeta-IKKgamma in response to TNFalpha stimulation. TNFalpha-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKbeta is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKbeta and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKbeta-NF-kappaB p65 pathway. PMID:18037881

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

    PubMed Central

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

    2013-01-01

    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. PMID:23516355

  12. Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RAR? oncoprotein.

    PubMed

    Wang, Zhuo; Cao, Lizhi; Kang, Rui; Yang, Minghua; Liu, Liying; Zhao, Yiming; Yu, Yan; Xie, Min; Yin, Xiaocheng; Livesey, Kristen M; Tang, Daolin

    2011-04-01

    PML-RAR? oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-? (RAR?) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RAR? degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RAR? degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RAR? degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RAR? degradation and myeloid cell differentiation. Moreover, PML-RAR? co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RAR? degradation and myeloid cell differentiation. The identification of PML-RAR? as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL. PMID:21187718

  13. Casein kinase II phosphorylation of the human papillomavirus-18 E7 protein is critical for promoting S-phase entry.

    PubMed

    Chien, W M; Parker, J N; Schmidt-Grimminger, D C; Broker, T R; Chow, L T

    2000-08-01

    The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase alpha or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry. PMID:10965847

  14. 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

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

    2014-01-01

    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

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

    SciTech Connect

    Manzo-Merino, Joaquin; Lizano, Marcela

    2014-02-15

    The HPV-16 E6 and E6{sup ⁎} 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{sup ⁎}. 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. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

  16. Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

    PubMed Central

    Demers, G W; Espling, E; Harry, J B; Etscheid, B G; Galloway, D A

    1996-01-01

    Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals. PMID:8794328

  17. Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens.

    PubMed

    Fan, Alice C; Deb-Basu, Debabrita; Orban, Mathias W; Gotlib, Jason R; Natkunam, Yasodha; O'Neill, Roger; Padua, Rose-Ann; Xu, Liwen; Taketa, Daryl; Shirer, Amy E; Beer, Shelly; Yee, Ada X; Voehringer, David W; Felsher, Dean W

    2009-05-01

    Current methods of protein detection are insensitive to detecting subtle changes in oncoprotein activation that underlie key cancer signaling processes. The requirement for large numbers of cells precludes serial tumor sampling for assessing a response to therapeutics. Therefore, we have developed a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in nanoliter volumes. Our method can quantify amounts of MYC oncoprotein and B cell lymphoma protein-2 (BCL2) in Burkitt's and follicular lymphoma; identify changes in activation of extracellular signal-related kinases-1 (ERK1) and ERK2, mitogen-activated kinase-1 (MEK), signal transducer and activator of transcription protein-3 (STAT3) and STAT5, c-Jun N-terminal kinase (JNK) and caspase-3 in imatinib-treated chronic myelogeneous leukemia (CML) cells; measure an unanticipated change in the phosphorylation of an ERK2 isomer in individuals with CML who responded to imatinib; and detect a decrease in STAT3 and STAT5 phosphorylation in individuals with lymphoma who were treated with atorvastatin. Therefore, we have described a new and highly sensitive method for determining oncoprotein expression and phosphorylation in clinical specimens for the development of new therapeutics for cancer. PMID:19363496

  18. Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

    PubMed

    Qin, Haiying; Cho, Monica; Haso, Waleed; Zhang, Ling; Tasian, Sarah K; Oo, Htoo Zarni; Negri, Gian Luca; Lin, Yongshun; Zou, Jizhong; Mallon, Barbara S; Maude, Shannon; Teachey, David T; Barrett, David M; Orentas, Rimas J; Daugaard, Mads; Sorensen, Poul H B; Grupp, Stephan A; Fry, Terry J

    2015-07-30

    Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL. PMID:26041741

  19. Genomic instability driven by the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax.

    PubMed

    Lemoine, Francene J; Marriott, Susan J

    2002-10-17

    The importance of maintaining genomic stability is evidenced by the fact that transformed cells often contain a variety of chromosomal abnormalities such as euploidy, translocations, and inversions. Gene amplification is a well-characterized hallmark of genomic instability thought to result from recombination events following the formation of double-strand, chromosomal breaks. Therefore, gene amplification frequency serves as an indicator of genomic stability. The PALA assay is designed to measure directly the frequency with which a specific gene, CAD, is amplified within a cell's genome. We have used the PALA assay to analyse the effects of the human T-cell leukemia virus type I (HTLV-I) oncoprotein, Tax, on genomic amplification. We demonstrate that Tax-expressing cells are five-times more likely to undergo gene amplification than control cells. Additionally, we show that Tax alters the ability of cells to undergo the typical PALA-mediated G(1) phase cell cycle arrest, thereby allowing cells to replicate DNA in the absence of appropriate nucleotide pools. This effect is likely the mechanism by which Tax induces gene amplification. These data suggest that HTLV-I Tax alters the genomic stability of cells, an effect that may play an important role in Tax-mediated, HTLV-I associated cellular transformation. PMID:12370813

  20. Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase.

    PubMed Central

    Salmeron, A; Ahmad, T B; Carlile, G W; Pappin, D; Narsimhan, R P; Ley, S C

    1996-01-01

    The Tpl-2 protein serine/threonine kinase was originally identified, in a C-terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats. The kinase domain of Tpl-2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl-2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl-2 and Tpl-2deltaC (a C-terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK-1 and SEK-1, in vitro. Furthermore, transfection of Tpl-2 into COS-1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK-1 and SAP kinase (JNK), which are substrates for MEK-1 and SEK-1, respectively. Tpl-2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl-2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK-1. Images PMID:8631303

  1. Amino-functionalized poly(l-lactide) lamellar single crystals as a valuable substrate for delivery of HPV16-E7 tumor antigen in vaccine development

    PubMed Central

    Di Bonito, Paola; Petrone, Linda; Casini, Gabriele; Francolini, Iolanda; Ammendolia, Maria Grazia; Accardi, Luisa; Piozzi, Antonella; D’Ilario, Lucio; Martinelli, Andrea

    2015-01-01

    Background Poly(l-lactide) (PLLA) is a biodegradable polymer currently used in many biomedical applications, including the production of resorbable surgical devices, porous scaffolds for tissue engineering, nanoparticles and microparticles for the controlled release of drugs or antigens. The surfaces of lamellar PLLA single crystals (PLLAsc) were provided with amino groups by reaction with a multifunctional amine and used to adsorb an Escherichia coli-produced human papillomavirus (HPV)16-E7 protein to evaluate its possible use in antigen delivery for vaccine development. Methods PLLA single crystals were made to react with tetraethylenepentamine to obtain amino-functionalized PLLA single crystals (APLLAsc). Pristine and amino-functionalized PLLAsc showed a two-dimensional microsized and one-dimensional nanosized lamellar morphology, with a lateral dimension of about 15–20 μm, a thickness of about 12 nm, and a surface specific area of about 130 m2/g. Both particles were characterized and loaded with HPV16-E7 before being administered to C57BL/6 mice for immunogenicity studies. The E7-specific humoral-mediated and cell-mediated immune response as well as tumor protective immunity were analyzed in mice challenged with TC-1 cancer cells. Results Pristine and amino-functionalized PLLAsc adsorbed similar amounts of E7 protein, but in protein-release experiments E7-PLLAsc released a higher amount of protein than E7-APLLAsc. When the complexes were dried for observation by scanning electron microscopy, both samples showed a compact layer, but E7-APLLAsc showed greater roughness than E7-PLLAsc. Immunization experiments in mice showed that E7-APLLAsc induced a stronger E7-specific immune response when compared with E7-PLLAsc. Immunoglobulin G isotyping and interferon gamma analysis suggested a mixed Th1/Th2 immune response in both E7-PLLAsc-immunized and E7-APLLAsc-immunized mice. However, only the mice receiving E7-APLLAsc were fully protected from TC-1 tumor growth after three doses of vaccine. Conclusion Our results show that APLLA single crystals improve the immunogenicity of HPV16-E7 and indicate that E7-APLLAsc could be used for development of an HPV16 therapeutic vaccine against HPV16-related tumors. PMID:26056443

  2. HSP70 and modified HPV 16 E7 fusion gene without the addition of a signal peptide gene sequence as a candidate therapeutic tumor vaccine.

    PubMed

    Zong, Jinbao; Wang, Changyuan; Wang, Qingyong; Peng, Qinglin; Xu, Yufei; Xie, Xixiu; Xu, Xuemei

    2013-12-01

    Millions of women are currently infected with high-risk human papillomavirus (HPV), which is considered to be a major risk factor for cervical cancer. Thus, it is urgent to develop therapeutic vaccines to eliminate the established infections or HPV-related diseases. In the present study, using the mycobacterium tuberculosis heat shock protein 70 (MtHSP70) gene linked to the modified HPV 16 E7 (mE7) gene, we generated two potential therapeutic HPV DNA vaccines, mE7/MtHSP70 and SigmE7/MtHSP70, the latter was linked to the signal peptide gene sequence of human CD33 at the upstream of the fusion gene. We found that vaccination with the mE7/MtHSP70 DNA vaccine induced a stronger E7-specific CD8+ T cell response and resulted in a more significant therapeutic effect against E7-expressing tumor cells in mice. Our results demonstrated that HSP70 can play a more important role in mE7 and MtHSP70 fusion DNA vaccine without the help of a signal peptide. This may facilitate the use of HSP70 and serve as a significant reference for future study. PMID:24065282

  3. API2-MALT1 oncoprotein promotes lymphomagenesis via unique program of substrate ubiquitination and proteolysis

    PubMed Central

    Rosebeck, Shaun; Lim, Megan S; Elenitoba-Johnson, Kojo S J; McAllister-Lucas, Linda M; Lucas, Peter C

    2016-01-01

    Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin’s lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB (NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor (TNF) receptor associated factor (TRAF) 2 and receptor interacting protein 1 (RIP1), two proteins essential for TNF receptor-induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While the intrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for non-canonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase (NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to generate a stable, constitutively active fragment. Thus, API2-MALT1 harnesses multiple unique pathways to achieve deregulated NF-κB activation. Emerging data from our group and others have also detailed additional gain-of-function activities of API2-MALT1 that extend beyond NF-κB activation. Specifically, API2-MALT1 recruits and subverts multiple other signaling factors, including LIM domain and actin-binding protein 1 (LIMA1) and Smac/DIABLO. Like NIK, LIMA1 represents a unique substrate for API2-MALT1 protease activity, but unlike NIK, its cleavage sets in motion a major NF-κB-independent pathway for promoting oncogenesis. In this review, we highlight the most recent results characterizing these unique and diverse gain-of-function activities of API2-MALT1 and how they contribute to lymphomagenesis. PMID:26981201

  4. Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.

    PubMed Central

    Monica, K; LeBrun, D P; Dedera, D A; Brown, R; Cleary, M L

    1994-01-01

    The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions. Images PMID:7969166

  5. Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination.

    PubMed

    Mortensen, Franziska; Schneider, Daniel; Barbic, Tanja; Sladewska-Marquardt, Anna; Kühnle, Simone; Marx, Andreas; Scheffner, Martin

    2015-08-11

    Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein. PMID:26216987

  6. Prothymosin alpha associates with the oncoprotein SET and is involved in chromatin decondensation.

    PubMed

    Karetsou, Zoe; Martic, Goran; Tavoulari, Sotiria; Christoforidis, Savvas; Wilm, Matthias; Gruss, Claudia; Papamarcaki, Thomais

    2004-11-19

    Prothymosin alpha (ProTalpha) is a histone H1-binding protein that interacts with the transcription coactivator CREB-binding protein and potentiates transcription. Based on coimmunoprecipitation and mammalian two-hybrid assays, we show here that ProTalpha forms a complex with the oncoprotein SET. ProTalpha efficiently decondenses human sperm chromatin, while overexpression of GFP-ProTalpha in mammalian cells results in global chromatin decondensation. These results indicate that decondensation of compacted chromatin fibers is an important step in the mechanism of ProTalpha function. PMID:15556635

  7. Casein kinase II motif-dependent phosphorylation of human papillomavirus E7 protein promotes p130 degradation and S-phase induction in differentiated human keratinocytes.

    PubMed

    Genovese, Nicholas J; Banerjee, N Sanjib; Broker, Thomas R; Chow, Louise T

    2008-05-01

    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 and organotypic cultures of primary human keratinocytes, the E7 proteins of the high-risk mucosotrophic HPV-18, the benign cutaneous HPV-1, and, to a lesser extent, the low-risk mucosotropic HPV-11 destabilized p130. This E7 activity depends on an intact pocket protein binding domain and a casein kinase II (CKII) phosphorylation motif. Coimmunoprecipitation experiments showed that both E7 domains were important for binding to p130 in extracts of organotypic cultures. Metabolic labeling in vivo demonstrated that E7 proteins were indeed phosphorylated in a CKII motif-dependent manner. Moreover, the efficiencies of the E7 proteins of various HPV types or mutations to induce S-phase reentry in spinous cells correlated with their relative abilities to bind and to destabilize p130. Collectively, these data support the notion that p130 controls the homeostasis of the differentiated keratinocytes and is therefore targeted by E7 for degradation to establish conditions permissive for viral DNA amplification. PMID:18321970

  8. Role of dual specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1B) in S-phase entry of HPV E7 expressing cells from quiescence

    PubMed Central

    Zhou, Na; Yuan, Shoudao; Wang, Rongchun; Zhang, Weifang; Chen, Jason J.

    2015-01-01

    The high-risk human papillomavirus (HPV) is the causative agent for cervical cancer. The HPV E7 oncogene promotes S-phase entry from quiescent state in the presence of elevated cell cycle inhibitor p27Kip1, a function that may contribute to carcinogenesis. However, the mechanism by which HPV E7 induces quiescent cells to entry into S-phase is not fully understood. Interestingly, we found that Dyrk1B, a dual-specificity kinase and negative regulator of cell proliferation in quiescent cells, was upregulated in E7 expressing cells. Surprisingly and in contrast to what was previously reported, Dyrk1B played a positive role in S-phase entry of quiescent HPV E7 expressing cells. Mechanistically, Dyrk1B contributed to p27 phosphorylation (at serine 10 and threonine 198), which was important for the proliferation of HPV E7 expressing cells. Moreover, Dyrk1B up-regulated HPV E7. Taken together, our studies uncovered a novel function of Dyrk1B in high-risk HPV E7-mediated cell proliferation. Dyrk1B may serve as a target for therapy in HPV-associated cancers. PMID:26307683

  9. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30II accessory protein and the induction of oncogenic cellular transformation by p30II/c-MYC

    PubMed Central

    Romeo, Megan M.; Ko, Bookyung; Kim, Janice; Brady, Rebecca; Heatley, Hayley C.; He, Jeffrey; Harrod, Carolyn K.; Barnett, Braden; Ratner, Lee; Lairmore, Michael D.; Martinez, Ernest; Lüscher, Bernhard; Robson, Craig N.; Henriksson, Marie; Harrod, Robert

    2014-01-01

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30II protein is a multifunctional latency-maintenance factor that negatively regulates viral gene expression and deregulates host signaling pathways involved in aberrant T-cell growth and proliferation. We have previously demonstrated that p30II interacts with the c-MYC oncoprotein and enhances c-MYC-dependent transcriptional and oncogenic functions. However, the molecular and biochemical events that mediate the cooperation between p30II and c-MYC remain to be completely understood. Herein we demonstrate that p30II induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30II in c-myc−/− HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30II is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30II inhibits apoptosis in proliferating cells expressing c-MYC under conditions of genotoxic stress. These findings suggest that c-MYC-acetylation is required for the cooperation between p30II/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. PMID:25569455

  10. SPOP Promotes Ubiquitination and Degradation of the ERG Oncoprotein to Suppress Prostate Cancer Progression.

    PubMed

    Gan, Wenjian; Dai, Xiangpeng; Lunardi, Andrea; Li, Zhen; Inuzuka, Hiroyuki; Liu, Pengda; Varmeh, Shoreh; Zhang, Jinfang; Cheng, Liang; Sun, Yin; Asara, John M; Beck, Andrew H; Huang, Jiaoti; Pandolfi, Pier Paolo; Wei, Wenyi

    2015-09-17

    The ERG gene is fused to TMPRSS2 in approximately 50% of prostate cancers (PrCa), resulting in its overexpression. However, whether this is the sole mechanism underlying ERG elevation in PrCa is currently unclear. Here we report that ERG ubiquitination and degradation are governed by the Cullin 3-based ubiquitin ligase SPOP and that deficiency in this pathway leads to aberrant elevation of the ERG oncoprotein. Specifically, we find that truncated ERG (?ERG), encoded by the ERG fusion gene, is stabilized by evading SPOP-mediated destruction, whereas prostate cancer-associated SPOP mutants are also deficient in promoting ERG ubiquitination. Furthermore, we show that the SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Importantly, we demonstrate that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the SPOP/?ERG interaction and its consequent degradation. Therefore, SPOP functions as a tumor suppressor to negatively regulate the stability of the ERG oncoprotein in prostate cancer. PMID:26344095

  11. Nano-fluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens

    PubMed Central

    Fan, Alice C.; Deb-Basu, Debabrita; Orban, Mathias W.; Gotlib, Jason R.; Natkunam, Yasodha; O’Neill, Roger; Padua, Rose-Ann; Xu, Liwen; Taketa, Daryl; Shirer, Amy E.; Beer, Shelly; Yee, Ada X.; Voehringer, David W.; Felsher, Dean W.

    2014-01-01

    Current methods of protein detection are insensitive to detecting subtle changes in oncoprotein activation that underlie critical cancer signaling processes. The requirement for large numbers of cells precludes serial tumor sampling for assessing a response to therapeutics. Therefore, we have developed a nano-fluidic proteomic immunoassay (NIA) to quantify total and low abundance protein isoforms in 4 nanoliters of lysate. Our method could quantify levels of MYC and BCL2 proteins in Burkitt’s versus follicular lymphoma; identify changes in activation of ERK1/2, MEK1, STAT3/5, JNK and caspase 3 in imatinib-treated chronic myelogeneous leukemia (CML) cells; measure a novel change in phosphorylation of an ERK2 isomer in CML patients who responded to imatinib; and detect a decrease in STAT3/5 phosphorylation in lymphoma patients treated with atorvastatin. Therefore, we have described a novel and highly sensitive method for interrogating oncoprotein expression and phosphorylation in clinical specimens for the development of new therapeutics for cancer. PMID:19363496

  12. Clinicopathological correlation of Bcl-2 oncoprotein expression in oral precancer and cancer

    PubMed Central

    Arya, Vandana; Singh, Subash; Daniel, M. Jonathan

    2016-01-01

    Oral squamous cell carcinoma is the most common malignant tumor of the oral cavity. Normally the death of cell and the growth are active processes and depend not only on external factors but also on the expression of genes such as Bcl-2, which activate and inhibit apoptosis. The term Bcl-2 is an acronym for B-cell lymphoma/leukemia-2 genes. It has been reported that there is deregulation of Bcl-2 expression during progression from oral epithelial dysplasia to squamous cell carcinoma. Expression of this oncoprotein can be detected by immunohistochemistry. Aims and objectives An attempt was made to evaluate Bcl-2 oncoprotein expression in patients with oral precancer and cancer. Materials and methods A selective prospective clinical and immunohistochemical study. Clinicopathological examination was correlated with immunohistochemical findings. The immunolocalization of Bcl-2 protein was performed using the labeled streptavidin biotin method. To visualize the reaction, 3,3-diaminobenzidine was used. Results Bcl-2 expression was positive in 11 [36.66%, low Bcl-2 expression 3 (10.00%), moderate Bcl-2 expression 7 (23.33%), and high Bcl-2 expression 1 (3.33%)] oral cancer cases and 14 [87.50%, low expression 8 (50%), moderate expression 6 (37.50%)] precancer cases. Conclusion On the basis of the results of our study, we conclude that positive Bcl-2 expression may be an indicator of poor prognosis in oral cancer and precancer. PMID:26937364

  13. Seminatural SUSY from the E7 nonlinear sigma model

    NASA Astrophysics Data System (ADS)

    Harigaya, Keisuke; Yanagida, Tsutomu T.; Yokozaki, Norimi

    2015-08-01

    We present a new focus point supersymmetry-breaking scenario based on the supersymmetric E_7 nonlinear sigma model. In this model, squarks and sleptons are identified with (pseudo) Nambu-Goldstone bosons. Their masses are generated only radiatively through gauge and Yukawa interactions, and they are much smaller than the gravitino and gaugino masses at a high energy scale. On the other hand, Higgs doublets belong to matter multiplets and hence may have unsuppressed supersymmetry-breaking soft masses. We consider their masses to be equal to the gravitino mass at the high energy scale, assuming the minimal Kähler potential for Higgs doublets. We show that the fine-tuning measure of the electroweak symmetry-breaking scale is reduced significantly to Δ =30{-}70, if the ratio of the gravitino mass to the gaugino mass is around 5/4. Also, the prospects for the discovery/exclusion of supersymmetric particles at the Large Hadron Collider and dark matter direct detection experiments are discussed.

  14. HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

    PubMed Central

    Di Bonito, Paola; Ridolfi, Barbara; Columba-Cabezas, Sandra; Giovannelli, Andrea; Chiozzini, Chiara; Manfredi, Francesco; Anticoli, Simona; Arenaccio, Claudia; Federico, Maurizio

    2015-01-01

    We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity. PMID:25760140

  15. HPV-E7 delivered by engineered exosomes elicits a protective CD8? T cell-mediated immune response.

    PubMed

    Di Bonito, Paola; Ridolfi, Barbara; Columba-Cabezas, Sandra; Giovannelli, Andrea; Chiozzini, Chiara; Manfredi, Francesco; Anticoli, Simona; Arenaccio, Claudia; Federico, Maurizio

    2015-03-01

    We developed an innovative strategy to induce a cytotoxic T cell (CTL) immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut), which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV)-E7 with that of lentiviral virus-like particles (VLPs) incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity. PMID:25760140

  16. A novel MLL5 isoform that is essential to activate E6 and E7 transcription in HPV16/18-associated cervical cancers.

    PubMed

    Yew, Chow Wenn; Lee, Pei; Chan, Wai Keong; Lim, Vania Kai Jun; Tay, Sun Kuie; Tan, Theresa M C; Deng, Lih-Wen

    2011-11-01

    Human papillomavirus (HPV) is the primary cause of human cervical cancer. The viral proteins E6 and E7 are essential to transform noncancerous epithelial cells into cancerous carcinomas by targeting key tumor suppressors p53 and retinoblastoma (Rb) proteins, respectively, but the cellular factors involved in E6 and E7 transcription themselves are incompletely understood. In this study, we defined a novel isoform of the mixed lineage leukemia 5 gene (MLL5?) as a specific and critical regulator of E6 and E7 transcription in cervical carcinoma cells. MLL5? is present in HPV16/18-positive cells including human primary cervical carcinoma specimens. Interaction of MLL5? with the AP-1-binding site at the distal region of the HPV18 long control region led to activation of E6/E7 transcription. Conversely, RNA interference-mediated knockdown of MLL5? downregulated both E6 and E7 expression. MLL5? downregulation was sufficient to restore p53 protein levels and reduce Rb phosphorylation, thereby reactivating apoptosis and cell-cycle checkpoints. By defining this novel MLL5? isoform and its specific critical role in activating E6/E7 gene transcription in HPV16/18-induced cervical cancers, our work highlights the potential of MLL5? as a biomarker and new therapeutic target in primary HPV-induced cervical cancers. PMID:21908553

  17. Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin.

    PubMed

    Mansilla, Cristina; Berraondo, Pedro; Durantez, Maika; Martínez, Marta; Casares, Noelia; Arribillaga, Laura; Rudilla, Francesc; Fioravanti, Jessica; Lozano, Teresa; Villanueva, Lorea; Sarobe, Pablo; Borrás, Francisco; Leclerc, Claude; Prieto, Jesús; Lasarte, Juan José

    2012-08-01

    Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinoma. PMID:21898393

  18. Cutaneous Human Papillomavirus E7 Type-Specific Effects on Differentiation and Proliferation of Organotypic Skin Cultures

    PubMed Central

    Westphal, K.; Akgl, B.; Storey, A.; Nindl, I.

    2009-01-01

    Background: A role for cutaneous human ?-papillomavirus (HPV) types as co-factors in the development of non-melanoma skin cancer has been postulated. Here we have investigated the effects of E7 expression on keratinocyte differentiation, proliferation and cell-cycle proteins in organotypic skin cultures. Methods: Recombinant retroviruses containing the E7 genes from cutaneous HPV types 1, 4, 5, 8, 20, 38 and RTRX7 were produced that include types associated with benign and malignant lesions. Adult human primary keratinocytes were transduced with these recombinant retroviruses and differentiated into skin-equivalents using de-epidermalised human dermis. Results: Expression patterns of the basal keratinocyte marker cytokeratin 14 (CK14) were not altered by any of the viral E7 types analysed. However, expression of the early and late differentiation markers CK10 and involucrin were markedly altered in HPV 1, 4 and 38 cultures. The highest proliferation rates in basal cell layers, as judged by BrdU and Ki67 staining, were observed in HPV 1, 4 and 38 cultures. Interestingly, co-expression of cyclin E and p16INK4a within the same cell of the suprabasal cell layers was observed only in cultures generated using E7 of HPV 5 or HPV 8. Conclusion: HPV types associated with either benign or malignant lesions perturb keratinocyte proliferation and differentiation in different ways. Moreover, expression of E7 from HPV 5 or HPV 8 seem able to overcome p16INK4a induced cell cycle arrest in a subset of keratinocytes. PMID:19478389

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

    PubMed Central

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

    2014-01-01

    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

  20. MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7.

    PubMed

    Saleh, Tayebeh; Bolhassani, Azam; Shojaosadati, Seyed Abbas; Aghasadeghi, Mohammad Reza

    2015-06-22

    DNA vaccines against human papillomavirus (HPV) type 16 have not been successful in clinical trials, due to the lack of an appropriate delivery system. In this study, a peptide-based gene delivery system, MPG, which forms stable non-covalent nanoparticles with nucleic acids, was used for in vitro and in vivo delivery of HPV16 E7 DNA as a model antigen. The results demonstrated that at Nitrogen/Phosphate (N/P) ratio over 10:1, this peptide can effectively condense plasmid DNA into stable nanoparticles with an average size of 180-210nm and a positive surface charge. The transfection efficiency of MPG-based nanoparticles was shown to be comparable with Polyethyleneimine (PEI). The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent delivery system in DNA vaccine formulations. Immunization with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 induced a stronger Th1 cellular immune response with a predominant interferon-? (IFN-?) profile than those induced by E7DNA alone in a murine tumor model. These findings suggest that MPG peptide as a novel gene delivery system could have promising applications in improving HPV therapeutic vaccines. PMID:26001433

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

    SciTech Connect

    Barrow-Laing, Lisa; Chen Wei; Roman, Ann

    2010-05-10

    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.

  2. Low- and High-Risk Human Papillomavirus E7 Proteins Regulate p130 Differently

    PubMed Central

    Barrow-Laing, Lisa; Chen, Wei; Roman, Ann

    2010-01-01

    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. PMID:20189212

  3. 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

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

    2011-01-29

    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

  4. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine 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.

  5. Therapeutic DNA vaccination against colorectal cancer by targeting the MYB oncoprotein.

    PubMed

    Cross, Ryan S; Malaterre, Jordane; Davenport, Alexander J; Carpinteri, Sandra; Anderson, Robin L; Darcy, Phillip K; Ramsay, Robert G

    2015-01-01

    Cancers can be addicted to continued and relatively high expression of nuclear oncoproteins. This is evident in colorectal cancer (CRC) where the oncoprotein and transcription factor MYB is over expressed and essential to continued proliferation and tumour cell survival. Historically, targeting transcription factors in the context of cancer has been very challenging. Nevertheless, we formulated a DNA vaccine to generate a MYB-specific immune response in the belief MYB peptides might be aberrantly presented on the cell surface of CRC cells. MYB, like many tumour antigens, is weakly immunogenic as it is a 'self' antigen and is subject to tolerance. To break tolerance, a fusion vaccine was generated comprising a full-length MYB complementary DNA (cDNA) flanked by two potent CD4-epitopes derived from tetanus toxoid. Vaccination was achieved against tumours initiated by two distinct highly aggressive, syngeneic cancer cell lines (CT26 and MC38) that express MYB. This was done in BALB/c and C57BL/6 mouse strains respectively. We introduced multiple inactivating mutations into the oncogene sequence for safety and sub-cloned the cDNA into a Food and Drug Administration (FDA)-compliant vector. We used low dose cyclophosphamide (CY) to overcome T-regulatory cell immune suppression, and anti-program cell death receptor 1 (anti-PD-1) antibodies to block T-cell exhaustion. Anti-PD-1 administered alone slightly delayed tumour growth in MC38 and more effectively in CT26 bearing mice, while CY treatment alone did not. We found that therapeutic vaccination elicits protection when MC38 tumour burden is low, mounts tumour-specific cell killing and affords enhanced protection when MC38 and CT26 tumour burden is higher but only in combination with anti-PD-1 antibody or low dose CY, respectively. PMID:25671128

  6. Natural variant of the Helicobacter pylori CagA oncoprotein that lost the ability to interact with PAR1.

    PubMed

    Hashi, Kana; Murata-Kamiya, Naoko; Varon, Christine; Mgraud, Francis; Dominguez-Bello, Maria Gloria; Hatakeyama, Masanori

    2014-03-01

    Helicobacter pylori strains carrying the cagA gene are associated with severe disease outcomes, most notably gastric cancer. CagA protein is delivered into gastric epithelial cells by a type IV secretion system. The translocated CagA undergoes tyrosine phosphorylation at the C-terminal EPIYA motifs by host cell kinases. Tyrosine-phosphorylated CagA acquires the ability to interact with and activate SHP2, thereby activating mitogenic signaling and inducing cell morphological transformation (hummingbird phenotype). CagA also interacts with PAR1b via the CM sequence, resulting in induction of junctional and polarity defects. Furthermore, CagA-PAR1b interaction stabilizes the CagA-SHP2 complex. Because transgenic mice systemically expressing CagA develop gastrointestinal and hematological malignancies, CagA is recognized as a bacterium-derived oncoprotein. Interestingly, the C-terminal region of CagA displays a large diversity among H.pylori strains, which influences the ability of CagA to bind to SHP2 and PAR1b. In the present study, we investigated the biological activity of v225d CagA, an Amerindian CagA of H.pylori isolated from a Venezuelan Piaroa Amerindian subject, because the variant CagA does not possess a canonical CM sequence. We found that v225d CagA interacts with SHP2 but not PAR1b. Furthermore, SHP2-binding activity of v225d CagA was much lower than that of CagA of H.pylori isolated from Western countries (Western CagA). v225d CagA also displayed a reduced ability to induce the hummingbird phenotype than that of Western CagA. Given that perturbation of PAR1b and SHP2 by CagA underlies the oncogenic potential of CagA, the v225d strain is considered to be less oncogenic than other well-studied cagA-positive H.pylori strains. PMID:24354359

  7. The Epstein-Barr virus encoded LMP1 oncoprotein modulates cell adhesion via regulation of activin A/TGF? and ?1 integrin signalling

    PubMed Central

    Morris, Mhairi A.; Dawson, Christopher W.; Laverick, Louise; Davis, Alexandra M.; Dudman, Joe P. R.; Raveenthiraraj, Sathuwarman; Ahmad, Zeeshan; Yap, Lee-Fah; Young, Lawrence S.

    2016-01-01

    Approximately 20% of global cancer incidence is causally linked to an infectious agent. Epstein-Barr virus (EBV) accounts for around 1% of all virus-associated cancers and is associated with nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP1), the major oncoprotein encoded by EBV, behaves as a constitutively active tumour necrosis factor (TNF) receptor activating a variety of signalling pathways, including the three classic MAPKs (ERK-MAPK, p38 MAPK and JNK/SAPK). The present study identifies novel signalling properties for this integral membrane protein via the induction and secretion of activin A and TGF?1, which are both required for LMP1s ability to induce the expression of the extracellular matrix protein, fibronectin. However, it is evident that LMP1 is unable to activate the classic Smad-dependent TGF? signalling pathway, but rather elicits its effects through the non-Smad arm of TGF? signalling. In addition, there is a requirement for JNK/SAPK signalling in LMP1-mediated fibronectin induction. LMP1 also induces the expression and activation of the major fibronectin receptor, ?5?1 integrin, an effect that is accompanied by increased focal adhesion formation and turnover. Taken together, these findings support the putative role for LMP1 in the pathogenesis of NPC by contributing to the metastatic potential of epithelial cells. PMID:26782058

  8. The Epstein-Barr virus encoded LMP1 oncoprotein modulates cell adhesion via regulation of activin A/TGFβ and β1 integrin signalling.

    PubMed

    Morris, Mhairi A; Dawson, Christopher W; Laverick, Louise; Davis, Alexandra M; Dudman, Joe P R; Raveenthiraraj, Sathuwarman; Ahmad, Zeeshan; Yap, Lee-Fah; Young, Lawrence S

    2016-01-01

    Approximately 20% of global cancer incidence is causally linked to an infectious agent. Epstein-Barr virus (EBV) accounts for around 1% of all virus-associated cancers and is associated with nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP1), the major oncoprotein encoded by EBV, behaves as a constitutively active tumour necrosis factor (TNF) receptor activating a variety of signalling pathways, including the three classic MAPKs (ERK-MAPK, p38 MAPK and JNK/SAPK). The present study identifies novel signalling properties for this integral membrane protein via the induction and secretion of activin A and TGFβ1, which are both required for LMP1's ability to induce the expression of the extracellular matrix protein, fibronectin. However, it is evident that LMP1 is unable to activate the classic Smad-dependent TGFβ signalling pathway, but rather elicits its effects through the non-Smad arm of TGFβ signalling. In addition, there is a requirement for JNK/SAPK signalling in LMP1-mediated fibronectin induction. LMP1 also induces the expression and activation of the major fibronectin receptor, α5β1 integrin, an effect that is accompanied by increased focal adhesion formation and turnover. Taken together, these findings support the putative role for LMP1 in the pathogenesis of NPC by contributing to the metastatic potential of epithelial cells. PMID:26782058

  9. Receptor Protein-tyrosine Phosphatase α Regulates Focal Adhesion Kinase Phosphorylation and ErbB2 Oncoprotein-mediated Mammary Epithelial Cell Motility*

    PubMed Central

    Boivin, Benoit; Chaudhary, Fauzia; Dickinson, Bryan C.; Haque, Aftabul; Pero, Stephanie C.; Chang, Christopher J.; Tonks, Nicholas K.

    2013-01-01

    We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ErbB2 oncoprotein in mammary epithelial cells. Using this model, we demonstrated that activation of ErbB2 led to the transient inactivation of PTPα, suggesting that attenuation of PTPα activity may contribute to enhanced ErbB2 signaling. Furthermore, RNAi-induced suppression of PTPα led to increased cell migration in an ErbB2-dependent manner. The ability of ErbB2 to increase cell motility in the absence of PTPα was characterized by prolonged interaction of GRB7 with ErbB2 and increased association of ErbB2 with a β1-integrin-rich complex, which depended on GRB7-SH2 domain interactions. Finally, suppression of PTPα resulted in increased phosphorylation of focal adhesion kinase on Tyr-407, which induced the recruitment of vinculin and the formation of a novel focal adhesion kinase complex in response to ErbB2 activation in mammary epithelial cells. Collectively, these results reveal a new role for PTPα in the regulation of motility of mammary epithelial cells in response to ErbB2 activation. PMID:24217252

  10. [Prokaryotic expression and polyclonal antibody preparation of HPV6b E7 protein].

    PubMed

    Tang, Yi; Zhou, Qiang; Wang, Qi; Cheng, Hao

    2011-09-01

    To express and prepare polyclonal antibody of Human papillomavirus type 6b (HPV6b) E7 protein. a prokaryotic expression vector pGEX-4T-2/HPV6b E7 was constructed and GST-HPV6b E7 fusion protein was expressed as a soluble protein in E. coli. The expressed fusion protein was purified via Glutathione-Sepharose 4B column and thrombin cleavage in order to obtain HPV6b E7 protein. Polyclonal IgG antibody was prepared by immunizing New-Zealand rabbits with HPV6b E7 protein. Western-Blot and immunofluorescence analysis showed that the polyclonal IgG antibody could specifically recognize HPV6b E7 protein and its titer was identified. SDS-PAGE analysis demonstrated that large amounts of soluble GST-HPV6b E7 fusion protein was expressed in E. coli after 3.0-6.0 hours of IPTG induction. Polyclonal IgG antibody successfully prepared from immunized rabbits showed high titer and high specificity as confirmed by Western-Blot and immunofluorescence. The preparation of anti-HPV6b E7 polyclonal antibody will facilitate further research on the biological and immunological functions of HPV6b E7 protein. PMID:21998951

  11. A DNA vaccine encoding mutated HPV58 mE6E7-Fc-GPI fusion antigen and GM-CSF and B7.1

    PubMed Central

    Wang, He; Yu, Jiyun; Li, Li

    2015-01-01

    Background Persistent infection with high-risk human papillomavirus (HPV) is a predominant cause of cervical cancer, and HPV58 is the third most common virus detected in the patients with cervical cancer in Asia. E6 and E7 are the viral oncogenes which are constitutively expressed in HPV-associated tumor cells and can be used as target antigens for related immunotherapy. In this study, we modified the HPV58 E6 and E7 oncogenes to eliminate their oncogenic potential and constructed a recombinant DNA vaccine that coexpresses the sig-HPV58 mE6E7-Fc-GPI fusion antigen in addition to granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7.1 as molecular adjuvants (PVAX1-HPV58 mE6E7FcGB) for the treatment of HPV58 (+) cancer. Methods PVAX1-HPV58 mE6E7FcGB recombinant DNA vaccine was constructed to express a fusion protein containing a signal peptide, a modified HPV58 mE6E7 gene, and human IgG Fc and glycosylphosphatidylinositol (GPI)-anchoring sequences using the modified DNA vaccine vector PVAX1-IRES-GM/B7.1 that coexpresses GM-CSF, and B7.1. C57BL/6 mice were challenged by HPV58 E6E7-expressing B16-HPV58 E6E7 cells, followed by immunization by PVAX1-HPV58 mE6E7FcGB vaccine on days 7, 14, 21 after tumor challenge. The cellular immune responses in immunized mice were assessed by measuring IFN-γ production in splenocytes upon stimulation by HPV58 E6E7-GST protein and the lysis of B16-HPV58 E6E7 target cells by splenocytes after restimulation with HPV58 E6E7-GST protein. The antitumor efficacy was evaluated by monitoring the growth of the tumor. Results PVAX1-HPV58 mE6E7FcGB elicited varying levels of IFN-lsgdB58onn T-cell immune responses and lysis of target cell in mice in response to the recombinant antigen HPV58 E6E7-GST. Furthermore, the vaccine also induced antitumor responses in the HPV58 (+) B16-HPV58 E6E7 tumor challenge model as evidenced by delayed tumor development. Conclusion The recombinant DNA vaccine PVAX1-HPV58 mE6E7FcGB efficiently generates cellular immunity and antitumor efficacy in immunized mice. These data provide a basis for the further study of this recombinant vaccine as a potential candidate vaccine. PMID:26604780

  12. Human papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients.

    PubMed Central

    Bleul, C; Mller, M; Frank, R; Gausepohl, H; Koldovsky, U; Mgaya, H N; Luande, J; Pawlita, M; ter Meulen, J; Viscidi, R

    1991-01-01

    Antibody-reactive regions on the human papillomavirus type 18 (HPV-18) E6 and E7 proteins were identified with rabbit polyclonal anti-fusion protein sera by screening of an fd phage expression library containing subgenomic HPV-18 DNA fragments and by testing of overlapping decapeptides representing the E6 and E7 open reading frames. Peptides comprising the delineated regions (designated E6/1 to E6/4 and E7/1) were synthesized and used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-HPV-18 antibodies in human sera. A total of 232 human serum samples (identical numbers of cervical cancer patients and age-matched controls) collected in Tanzania were tested. Similar prevalences (between 0.8 and 4.3%) of antibodies recognizing the different E6 peptides were found in the sera from tumor patients and controls. With a synthetic 28-mer peptide (designated pepE701) comprising the E7/1 region, a significant difference was found: 10 of 116 tumor serum samples but 0 of 116 control serum samples showed a specific reaction (P less than 0.001). This observation confirms earlier results with HPV-16 E7 fusion proteins (I. Jochmus-Kudielka, A. Schneider, R. Braun, R. Kimmig, U. Koldovsky, K. E. Schneweis, K. Seedorf, and L. Gissmann, J. Natl. Cancer Inst. 81:1698-1704, 1989). A lower prevalence of anti-HPV-18 E7 antibodies was observed when 188 human serum samples collected in Germany from tumor patients and controls were tested (3 of 94 positive in the cancer group; 0 of 94 positive in the control group). The type specificity of anti-HPV-18 E7 antibodies was demonstrated when the HPV type found by Southern hybridization in the cervical cancer biopsies was compared with seroreactivity: 4 of 8 serum samples obtained from HPV-18 DNA-positive but 0 of 16 serum samples from HPV-18 DNA-negative tumor patients reacted in the HPV-18 E7 ELISA. In addition, HPV-18-positive sera failed to react in a peptide ELISA with the homologous HPV-16 E7 region (M. Mller, H. Gausepohl, G. de Martinoff, R. Frank, R. Brasseur, and L. Gissmann, J. Gen. Virol. 71:2709-2717, 1990) and vice versa. PMID:1722219

  13. Flat cells come full sphere: Are mutant cytoskeletal-related proteins oncoprotein-monsters or useful immunogens?

    PubMed

    Parry, Michele L; Blanck, George

    2016-01-01

    Osteogenesis imperfecta is inherited as a dominant disease because if one allele is mutated, it contributes a mutant, destructive subunit polypeptide to collagen, which requires many subunits to form normal, polymeric, collagenous structures. Recent cancer genome atlas (TCGA) data indicate that cytoskeletal-related proteins are among the most commonly mutated proteins in human cancers, in distinct mutation frequency groups, i.e., including low mutation frequency groups. Part of the explanation for this observation is likely to be the fact that many of the coding regions for these proteins are very large, and indeed, it is likely these coding regions are mutated in many cells that never become cancerous. However, it would not be surprising if mutations in cytoskeletal proteins, when combined with oncoprotein or tumor suppressor protein mutations, had significant impacts on cancer development, for a number of reasons, including results obtained almost 5 decades ago indicating that well-spread cells in tissue culture, with well-formed cytoskeletons, were less tumorigenic than spherical cells with disrupted cytoskeletons. This raises the question, are mutant cytoskeletal proteins, which would likely interfere with polymer formation, a new class of oncoproteins, in particular, dominant negative oncoproteins? If these proteins are so commonly mutant, could they be the bases for common cancer vaccines? PMID:26225584

  14. An HPV-E6/E7 immunotherapy plus PD-1 checkpoint inhibition results in tumor regression and reduction in PD-L1 expression.

    PubMed

    Rice, A E; Latchman, Y E; Balint, J P; Lee, J H; Gabitzsch, E S; Jones, F R

    2015-09-01

    We have investigated if immunotherapy against human papilloma virus (HPV) using a viral gene delivery platform to immunize against HPV 16 genes E6 and E7 (Ad5 [E1-, E2b-]-E6/E7) combined with programmed death-ligand 1 (PD-1) blockade could increase therapeutic effect as compared to the vaccine alone. Ad5 [E1-, E2b-]-E6/E7 as a single agent induced HPV-E6/E7 cell-mediated immunity. Immunotherapy using Ad5 [E1-, E2b-]-E6/E7 resulted in clearance of small tumors and an overall survival benefit in mice with larger established tumors. When immunotherapy was combined with immune checkpoint blockade, an increased level of anti-tumor activity against large tumors was observed. Analysis of the tumor microenvironment in Ad5 [E1-, E2b-]-E6/E7 treated mice revealed elevated CD8(+) tumor infiltrating lymphocytes (TILs); however, we observed induction of suppressive mechanisms such as programmed death-ligand 1 (PD-L1) expression on tumor cells and an increase in PD-1(+) TILs. When Ad5 [E1-, E2b-]-E6/E7 immunotherapy was combined with anti-PD-1 antibody, we observed CD8(+) TILs at the same level but a reduction in tumor PD-L1 expression on tumor cells and reduced PD-1(+) TILs providing a mechanism by which combination therapy favors a tumor clearance state and a rationale for pairing antigen-specific vaccines with checkpoint inhibitors in future clinical trials. PMID:26337747

  15. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.

    PubMed Central

    Huibregtse, J M; Scheffner, M; Howley, P M

    1993-01-01

    The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro. Images PMID:8380895

  16. The MUC1-C Oncoprotein Binds to the BH3 Domain of the Pro-apoptotic BAX Protein and Blocks BAX Function*

    PubMed Central

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

    2012-01-01

    The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-CBAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway. PMID:22544745

  17. The MUC1-C oncoprotein binds to the BH3 domain of the pro-apoptotic BAX protein and blocks BAX function.

    PubMed

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

    2012-06-15

    The pro-apoptotic BAX protein contains a BH3 domain that is necessary for its dimerization and for activation of the intrinsic apoptotic pathway. The MUC1 (mucin 1) heterodimeric protein is overexpressed in diverse human carcinomas and blocks apoptosis in the response to stress. In this study, we demonstrate that the oncogenic MUC1-C subunit associates with BAX in human cancer cells. MUC1-CBAX complexes are detectable in the cytoplasm and mitochondria and are induced by genotoxic and oxidative stress. The association between MUC1-C and BAX is supported by the demonstration that the MUC1-C cytoplasmic domain is sufficient for the interaction with BAX. The results further show that the MUC1-C cytoplasmic domain CQC motif binds directly to the BAX BH3 domain at Cys-62. Consistent with binding to the BAX BH3 domain, MUC1-C blocked BAX dimerization in response to (i) truncated BID in vitro and (ii) treatment of cancer cells with DNA-damaging agents. In concert with these results, MUC1-C attenuated localization of BAX to mitochondria and the release of cytochrome c. These findings indicate that the MUC1-C oncoprotein binds directly to the BAX BH3 domain and thereby blocks BAX function in activating the mitochondrial death pathway. PMID:22544745

  18. Hydrophobic Effect Drives Oxygen Uptake in Myoglobin via Histidine E7*

    PubMed Central

    Boechi, Leonardo; Arrar, Mehrnoosh; Mart, Marcelo A.; Olson, John S.; Roitberg, Adrin E.; Estrin, Daro A.

    2013-01-01

    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. PMID:23297402

  19. Fbw7 and its counteracting forces in stem cells and cancer: Oncoproteins in the balance.

    PubMed

    Cremona, Catherine A; Sancho, Rocio; Diefenbacher, Markus E; Behrens, Axel

    2016-02-01

    Fbw7 is well characterised as a stem cell regulator and tumour suppressor, powerfully positioned to control proliferation, differentiation and apoptosis by targeting key transcription factors for ubiquitination and destruction. Evidence in support of these roles continues to accumulate from in vitro studies, mouse models and human patient data. Here we summarise the latest of these findings, highlighting the tumour-suppressive role of Fbw7 in multiple tissues, and the rare circumstances where Fbw7 activity can be oncogenic. We discuss mechanisms that regulate ubiquitination by Fbw7, including ubiquitin-specific proteases such as USP28 that counteract Fbw7 activity and thereby stabilise oncoproteins. Deubiquitination of key Fbw7 substrates to prevent their destruction is beginning to be appreciated as an important pro-tumourigenic mechanism. As the ubiquitin-proteasome system represents a largely untapped field for drug development, the interplay between Fbw7 and its counterpart deubiquitinating enzymes in tumours is likely to attract increasing interest and influence future treatment strategies. PMID:26410034

  20. Defective human retinoblastoma protein identified by lack of interaction with the E1A oncoprotein.

    PubMed

    Paggi, M G; Martelli, F; Fanciulli, M; Felsani, A; Sciacchitano, S; Varmi, M; Bruno, T; Carapella, C M; Floridi, A

    1994-02-15

    Inactivating mutations of the retinoblastoma susceptibility gene (Rb) are involved in the pathogenesis of hereditary and sporadic retinoblastoma. Alterations in the Rb gene have also been found in several other human tumors occurring with epidemiological incidence higher than that of retinoblastoma. Four human malignant glioma cell lines were examined for abnormalities in the retinoblastoma gene product (pRb), using a procedure based on the interaction of pRb with an in vitro-translated adenovirus E1A oncoprotein. In the CRS-A2 cell line, derived from a glioblastoma multiforme, pRb did not bind with the in vitro-translated E1A protein. Restriction analysis of the CRS-A2 Rb gene and Rb mRNA expression provided patterns that could not be distinguished from the other glioma cell lines. Further investigation revealed the presence of a truncated pRb in the CRS-A2 cell line, due to a nucleotide insertion in the coding sequence at position 2550. In addition, this truncated Rb protein was undetectable in phosphorylated form. The binding assay with the in vitro-translated E1A was also used to study other cell lines with known mutations in the Rb gene. This method, which evaluates the interaction between in vitro-translated E1A and the pRb, is proposed as a rapid screening for detecting functional alterations in the retinoblastoma protein. PMID:8313367

  1. Oncoproteins, heterochromatin silencing and microRNAs: a new link for leukemogenesis.

    PubMed

    Nervi, Clara; Fazi, Francesco; Grignani, Francesco

    2008-01-01

    The pathogenesis of acute myeloid leukemias involves complex molecular events triggered by diverse alterations of genomic DNA. A limited number of initiating lesions, such as chromosomal translocations generating fusion genes, are constantly identified in specific forms of leukemia and are critical to leukemogenesis. Leukemia fusion proteins derived from chromosomal translocations can mediate epigenetic silencing of gene expression. Epigenetic deregulation of the DNA methylation status and of the chromatin "histone code" at specific gene sites cooperate in the pathogenesis of leukemias. The neutralization of these crucial oncogenic events can revert the leukemia phenotype. Thus, their identification and the study of their molecular and biological consequences is essential for the development of novel and specific therapeutic strategies. In this context, we recently reported a link between the differentiation block of leukemia and the epigenetic silencing of the microRNA-223 gene by the AML1/ETO oncoprotein, the product of the t(8;21) the commonest AML-associated chromosomal translocation. This finding indicates microRNAs as additional epigenetic targets for leukemogenesis and for therapeutic intervention in leukemias. PMID:18416028

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

    PubMed Central

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

    2011-01-01

    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. PMID:21969598

  3. A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein.

    PubMed

    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

    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. PMID:24737027

  4. A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein

    PubMed Central

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

    2014-01-01

    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's 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 (SMM) screens to identify and characterize drug-like compounds that modulate the biological 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 pharmacological ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins. PMID:24737027

  5. Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2

    PubMed Central

    Bueren-Calabuig, Juan A.; Michel, Julien

    2015-01-01

    Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29) peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2. PMID:26046940

  6. Truncated ERG Oncoproteins from TMPRSS2-ERG Fusions Are Resistant to SPOP-Mediated Proteasome Degradation.

    PubMed

    An, Jian; Ren, Shancheng; Murphy, Stephen J; Dalangood, Sumiya; Chang, Cunjie; Pang, Xiaodong; Cui, Yangyan; Wang, Liguo; Pan, Yunqian; Zhang, Xiaowei; Zhu, Yasheng; Wang, Chenji; Halling, Geoffrey C; Cheng, Liang; Sukov, William R; Karnes, R Jeffrey; Vasmatzis, George; Zhang, Qing; Zhang, Jun; Cheville, John C; Yan, Jun; Sun, Yinghao; Huang, Haojie

    2015-09-17

    SPOP mutations and TMPRSS2-ERG rearrangements occur collectively in up to 65% of human prostate cancers. Although the two events are mutually exclusive, it is unclear whether they are functionally interrelated. Here, we demonstrate that SPOP, functioning as an E3 ubiquitin ligase substrate-binding protein, promotes ubiquitination and proteasome degradation of wild-type ERG by recognizing a degron motif at the N terminus of ERG. Prostate cancer-associated SPOP mutations abrogate the SPOP-mediated degradation function on the ERG oncoprotein. Conversely, the majority of TMPRSS2-ERG fusions encode N-terminal-truncated ERG proteins that are resistant to the SPOP-mediated degradation because of degron impairment. Our findings reveal degradation resistance as a previously uncharacterized mechanism that contributes to elevation of truncated ERG proteins in prostate cancer. They also suggest that overcoming ERG resistance to SPOP-mediated degradation represents a viable strategy for treatment of prostate cancers expressing either mutated SPOP or truncated ERG. PMID:26344096

  7. Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma

    PubMed Central

    Slemmons, Katherine K.; Crose, Lisa E. S.; Rudzinski, Erin; Bentley, Rex C.; Linardic, Corinne M.

    2015-01-01

    Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS) and pleomorphic (pRMS) histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways. PMID:26496700

  8. Role of the YAP Oncoprotein in Priming Ras-Driven Rhabdomyosarcoma.

    PubMed

    Slemmons, Katherine K; Crose, Lisa E S; Rudzinski, Erin; Bentley, Rex C; Linardic, Corinne M

    2015-01-01

    Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle histogenesis, is the most common soft tissue sarcoma of childhood and adolescence. Survival for high-risk groups is less than 30% at 5 years. RMS also occurs during adulthood, with a lower incidence but higher mortality. Recently, mutational profiling has revealed a correlation between activating Ras mutations in the embryonal (eRMS) and pleomorphic (pRMS) histologic variants of RMS, and a poorer outcome for those patients. Independently, the YAP transcriptional coactivator, an oncoprotein kept in check by the Hippo tumor suppressor pathway, is upregulated in eRMS. Here we show that YAP promotes cell proliferation and antagonizes apoptosis and myogenic differentiation of human RMS cells bearing oncogenic Ras mutations in cell culture studies in vitro and in murine xenografts in vivo. Pharmacologic inhibition of YAP by the benzoporphyrin derivative verteporfin decreased cell proliferation and tumor growth in vivo. To interrogate the temporal contribution of YAP in eRMS tumorigenesis, we used a primary human cell-based genetic model of Ras-driven RMS. Constitutively active YAP functioned as an early genetic lesion, permitting bypass of senescence and priming myoblasts to tolerate subsequent expression of hTERT and oncogenic Ras, which were necessary and sufficient to generate murine xenograft tumors mimicking RMS in vivo. This work provides evidence for cooperation between YAP and oncogenic Ras in RMS tumorigenesis, laying the foundation for preclinical co-targeting of these pathways. PMID:26496700

  9. Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms.

    PubMed Central

    Smotkin, D; Prokoph, H; Wettstein, F O

    1989-01-01

    A new promoter located within E6 was mapped in human papillomavirus type 6b (HPV6b)- and HPV11-containing benign genital condylomata (genital warts). The RNA transcribed from this promoter represented the major RNA species colinear with open reading frames E6 and E7 and can encode the E7 protein. No equivalent promoter was active in HPV16-containing cancers and cancer-derived cell lines. In those, the major transcripts contained one of two different introns within E6 and the RNAs could encode two different E6 proteins and E7. Images PMID:2536845

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

    SciTech Connect

    Li, Hui; Zhan, TaiLan; Li, Chang; Liu, Mugen; Wang, Qing K.

    2009-10-16

    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.

  11. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... by subpart Q of 45 CFR part 74. (b) The Director may permit unobligated grant funds remaining in the... HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the...

  12. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... by subpart Q of 45 CFR part 74. (b) The Director may permit unobligated grant funds remaining in the... HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the...

  13. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... by subpart Q of 45 CFR part 74. (b) The Director may permit unobligated grant funds remaining in the... HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the...

  14. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... by subpart Q of 45 CFR part 74. (b) The Director may permit unobligated grant funds remaining in the... HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the...

  15. 42 CFR 52e.7 - What are the terms and conditions of awards?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... by subpart Q of 45 CFR part 74. (b) The Director may permit unobligated grant funds remaining in the... HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.7 What are the...

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

    PubMed Central

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

    1993-01-01

    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 PMID:7688182

  17. c-erbB-2 oncoprotein expression in primary and advanced breast cancer.

    PubMed Central

    Lovekin, C.; Ellis, I. O.; Locker, A.; Robertson, J. F.; Bell, J.; Nicholson, R.; Gullick, W. J.; Elston, C. W.; Blamey, R. W.

    1991-01-01

    Immunoreactivity for c-erbB-2 oncogene product expression has been investigated in patients with breast cancer using the polyclonal antibody 21N. Three series of patients were studied, 602 presenting with primary operable cancer, 57 with stage 3 and 123 with stage 4 disease. Representative tissue sections of each primary tumour were stained using a standard immunoperoxidase technique. Invasive tumour membrane immunoreactivity was assessed and identified in 15% of patients with primary operable cancer and 20% in the advanced breast cancer group. The results demonstrate a relationship between poorer survival and oncogene expression in all three patient groups. Patients in the primary operable cancer group with membrane oncoprotein expression had a poorer outcome, 35% 10-year survival, compared with those in which membrane expression was absent, 55% 10-year survival. The median survival of patients with stage 3 disease with c-erbB-2 membrane positivity was 17 months compared to 24 months with membrane negativity. In stage 4 disease median survival with membrane expression was 8.8 months compared to 19.7 months with no membrane expression. In addition in the series of primary cancers a correlation existed between histological grade and membrane immunoreactivity. Multivariate analysis showed histological grade to be a more powerful prognostic factor than c-erbB-2 protein expression. In conclusion, this study demonstrates, in a large series of patients presenting to one centre, that c-erbB-2 protein expression is a prognostic indicator in patients with primary operable and advanced breast disease. Images Figure 1 PMID:1672254

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

    PubMed Central

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

    2012-01-01

    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. PMID:22473899

  19. Ligand binding to synthetic mutant myoglobin (His-E7----Gly): role of the distal histidine.

    PubMed Central

    Braunstein, D; Ansari, A; Berendzen, J; Cowen, B R; Egeberg, K D; Frauenfelder, H; Hong, M K; Ormos, P; Sauke, T B; Scholl, R

    1988-01-01

    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 site-directed mutagenesis. Substitution of His-E7 with glycine [GlyE7]Mb bound with CO (CO[GlyE7]Mb) results in one broad bound-CO IR stretch band, v(C-O), centered at 1973 cm-1 at 10 K, in contrast to three distinct bands for native and synthetic wild-type MbCO at 1966, 1945, and 1929 cm-1. After flash photolysis at 10 K, the unbound state of CO[GlyE7]Mb exhibits two CO stretch bands, whereas MbCO has three. Fourier transform IR spectroscopy measurements of the linear dichroism after photoselective flash photolysis of CO bound to [GlyE7]Mb at 10 K reveals the bound CO to be oriented at an angle of alpha = 20 degrees +/- 2 degrees with respect to the heme normal. Flash photolysis data from 10 to 300 K provide evidence for a larger distal pocket and a smaller enthalpy barrier (by approximately 4 kJ/mol) for [GlyE7]MbCO as compared with wild-type MbCO. These results reinforce the notion that the dominant control of the binding step at the heme iron comes from the proximal side through the protein structure. PMID:3186740

  20. The HPV16 E6 oncoprotein and UVB irradiation inhibit the tumor suppressor TGFβ pathway in the epidermis of the K14E6 transgenic mouse.

    PubMed

    Popoca-Cuaya, Marco; Diaz-Chavez, Jose; Hernandez-Monge, Jesus; Alvarez-Rios, Elizabeth; Lambert, Paul F; Gariglio, Patricio

    2015-06-01

    High-risk human papillomaviruses (HR-HPVs) are the causative agents of cervical cancer, and they are also associated with a subset of head and neck squamous cell carcinomas. In addition, HPVs have also been postulated in the development of non-melanoma skin cancers (NMSC). In these cancers, the oncogene E6 is best known for its ability to inactivate the tumor suppressor p53 protein. Interestingly, in transgenic mice for HPV16 E6 (K14E6), it was reported that E6 alone induced epithelial hyperplasia and delay in differentiation in skin epidermis independently of p53 inactivation. Transforming growth factor β (TGFβ) is an important regulator of cell growth/differentiation and apoptosis, and this pathway is often lost during tumorigenesis. Ultraviolet radiation B (UVB) exposure activates diverse cellular responses, including DNA damage and apoptosis. In this study, we investigated whether the E6 oncogene alone or in combination with UVB dysregulate some components of the TGFβ pathway in the epidermis of K14E6 mice. We used 8-day-old K14E6 and non-transgenic mice irradiated and unirradiated with a single dose of UVB. We found that the E6 oncogene and UVB irradiation impair the TGFβ pathway in epidermis of K14E6 mice by downregulation of the TGFβ type II receptor (TβRII). This loss of TβRII prevents downstream activation of Smad2 and target genes as p15, an important regulator of cell cycle progression. In summary, the TGFβ signalling in cells of the epidermis is downregulated in our mouse model by both the E6 oncoprotein and the UVB irradiation. PMID:25776923

  1. Cooperative Interaction between the MUC1-C Oncoprotein and the Rab31 GTPase in Estrogen Receptor-Positive Breast Cancer Cells

    PubMed Central

    Jin, Caining; Rajabi, Hasan; Pitroda, Sean; Li, Ailing; Kharbanda, Akriti; Weichselbaum, Ralph; Kufe, Donald

    2012-01-01

    Rab31 is a member of the Ras superfamily of small GTPases that has been linked to poor outcomes in patients with breast cancer. The MUC1-C oncoprotein is aberrantly overexpressed in most human breast cancers and also confers a poor prognosis. The present results demonstrate that MUC1-C induces Rab31 expression in estrogen receptor positive (ER+) breast cancer cells. We show that MUC1-C forms a complex with estrogen receptor ? (ER?) on the Rab31 promoter and activates Rab31 gene transcription in an estrogen-dependent manner. In turn, Rab31 contributes to the upregulation of MUC1-C abundance in breast cancer cells by attenuating degradation of MUC1-C in lysosomes. Expression of an inactive Rab31(S20N) mutant in nonmalignant breast epithelial cells confirmed that Rab31 regulates MUC1-C expression. The functional significance of the MUC1-C/Rab31 interaction is supported by the demonstration that Rab31 confers the formation of mammospheres by a MUC1-C-dependent mechanism. Analysis of microarray databases further showed that (i) Rab31 is expressed at higher levels in breast cancers as compared to that in normal breast tissues, (ii) MUC1+ and ER+ breast cancers have increased levels of Rab31 expression, and (iii) patients with Rab31-positive breast tumors have a significantly decreased ten-year overall survival as compared to those with Rab31-negative tumors. These findings indicate that MUC1-C and Rab31 function in an autoinductive loop that contributes to overexpression of MUC1-C in breast cancer cells. PMID:22792175

  2. Apoptosis of vascular smooth muscle cells. Protein kinase C and oncoprotein Bcl-2 are involved in regulation of apoptosis in non-transformed rat vascular smooth muscle cells.

    PubMed Central

    Leszczynski, D.; Zhao, Y.; Luokkamki, M.; Foegh, M. L.

    1994-01-01

    We examined the effect of several inhibitors/activators of various protein kinases on the proliferation and apoptosis of nontransformed rat coronary vascular smooth muscle cells (SMC). As expected, all the compounds (calphostin C, KT5720, KT5823, verapamil, W7, and dibutyryl-cAMP) inhibited SMC proliferation, as judged by [3H]thymidine incorporation. Three (calphostin C, verapamil and dibutyryl-cAMP) of the six compounds caused occurrence of the classical apoptotic morphology in SMC. The effect of calphostin C, an inhibitor of protein kinase C, was examined in more detail due to the known involvement of this kinase in regulation of apoptosis in a variety of cell types. In SMC cultures exposed for 1, 2, and 3 days to 0.1 mumol/L calphostin C, 7 +/- 1%, 32 +/- 3%, and 29 +/- 3% of cells underwent apoptosis, respectively, as assessed by cell morphology (control cultures had 1 to 3% of apoptotic cells). The effect of calphostin C was transient in that on day 6 following exposure to this compound the number of apoptotic cells declined to control values. Simultaneous with the induction of apoptotic morphology in SMC, a decline was seen (within 24 hours) in expression of the oncoprotein Bcl-2 in morphologically nonapoptotic SMC. An altered distribution of Bcl-2 was seen in the apoptotic cells. The calphostin C-induced generation of apoptotic cells in SMC cultures and the decline/alteration of Bcl-2 expression were not accompanied by degradation of DNA into nucleosomal fragments. In conclusion, normal, nontransformed rat coronary artery vascular SMC undergo apoptosis when exposed to an inhibitor of protein kinase C (calphostin C), to a calcium channel blocker (verapamil), and to a stimulator of cAMP-dependent protein kinase (dibutyryl-cAMP). The induction of apoptosis by the inhibitor of protein kinase C is accompanied by alterations in the Bcl-2 expression but not by DNA fragmentation. Images Figure 3 Figure 2 PMID:7527616

  3. A novel dithiocarbamate derivative induces cell apoptosis through p53-dependent intrinsic pathway and suppresses the expression of the E6 oncogene of human papillomavirus 18 in HeLa cells.

    PubMed

    Li, Yanhong; Qi, Hongxue; Li, Xiaobo; Hou, Xueling; Lu, Xueying; Xiao, Xiangwen

    2015-06-01

    Dithiocarbamates (DTCs) exhibit a broad spectrum of antitumor activities, however, their molecular mechanisms of antitumor have not yet been elucidated. Previously, we have synthesized a series of novel dithiocarbamate derivatives. These DTCs were examined for cytotoxic activities against five human cancer cell lines. In this study, one of dithiocarbamate (DTC1) with higher potential for HeLa cells was chosen to investigate molecular mechanisms for its anti-tumor activities. DTC1 could inhibit proliferation, and highly induce apoptosis in HeLa cells by activating caspase-3, -6 and -9; moreover, activities of caspase-3, -6 and -9 were inhibited by pan-caspase inhibitor, Z-VAD-FMK. Furthermore, DTC1 decreased the levels of Bcl-2 and Bcl-xL, and increased expression of cytosol cytochrome c, Bak, Bax and p53 in a time-dependent manner but had no effect on the level of Rb. It was shown that DTC1 induced HeLa cells apoptosis through a p53-dependent pathway as tested by the wild type p53 inhibitor, pifithrin-?. Additionally, the relative expression of E6 and E7 were evaluated in HPV18-positive (HeLa cells) by real-time PCR and western blotting. The results firstly demonstrated that DTC1 suppressed both expression of E6 mRNA and E6 oncoprotein, but had no effect on the expression of E7 mRNA and protein in HPV18. Our results suggested that DTC1 may serve as novel chemotherapeutic agents in the treatment of cervical cancer and potential anti-HPV virus candidates that merit further studies. PMID:25772545

  4. The Transactivation Domain of Marek's Disease Virus (MDV) Meq Oncoprotein Does Not Affect Tumor Incidence But Plays a Role in Tumor Phenotype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mareks disease virus encoded oncoprotein, Meq, is responsible for the tumorigenic phenotype of the virus. We have previously shown that replacement of the meq gene in the very virulent strain Md5 with that of vaccine strain CVI988/Rispens results in virus attenuation in chickens. To determine the...

  5. Alteration of a single amino acid in the basic domain of Marek's disease virus Meq oncoprotein plays an important role in T-cell transformation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mareks disease virus encoded oncoprotein, Meq, has been shown to play a major role in transformation of T-lymphocytes. We have earlier shown that replacement of the meq gene in the very virulent strain Md5 with that of vaccine strain CVI988/Rispens resulted in virus attenuation in chickens. To dete...

  6. The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein.

    PubMed Central

    Mietz, J A; Unger, T; Huibregtse, J M; Howley, P M

    1992-01-01

    The observed interaction between p53 and the oncoproteins encoded by several DNA tumor viruses suggests that these viruses mediate their transforming activities at least in part by altering the normal growth regulatory function of p53. In this study we examined the effect of viral oncoprotein expression on the transcriptional transactivation function of wild-type p53 in human cells. Plasmids expressing human p53 were cotransfected with either SV40 large T-antigen or human papillomavirus (HPV) type 16 E6 expression plasmids and assayed for transactivation function using a reporter gene driven by a p53-responsive promoter containing multiple copies of the consensus p53 DNA binding motif, TGCCT. Both large T-antigen and E6 were able to inhibit transactivation by wild-type p53. Furthermore, SV40 T-antigen mutants that are defective for p53 binding were not able to inhibit transactivation and HPV E6 proteins that were either mutant or derived from non-oncogenic HPV types and unable to bind p53, had no effect on p53 transactivation. These results demonstrate the physiological relevance of the interaction of SV40 T-antigen and HPV E6 oncoproteins with p53 in vivo and suggest that the transforming functions of these viral oncoproteins may be linked to their ability to inhibit p53-mediated transcriptional activation. Images PMID:1464323

  7. 77 FR 9948 - International Conference on Harmonisation; Guidance on E7 Studies in Support of Special...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ...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......

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

    PubMed Central

    Brylinski, R; Kostant, B

    1994-01-01

    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. PMID:11607467

  9. Molecular mobility of nematic E7 confined to molecular sieves with a low filling degree.

    PubMed

    Brs, A R; Frunza, S; Guerreiro, L; Fonseca, I M; Corma, A; Frunza, L; Dionsio, M; Schnhals, A

    2010-06-14

    The nematic liquid crystalline mixture E7 was confined with similar filling degrees to molecular sieves with constant composition but different pore diameters (from 2.8 to 6.8 nm). Fourier transform infrared analysis proved that the E7 molecules interact via the cyanogroup with the pore walls of the molecular sieves. The molecular dynamics of the system was investigated by broadband dielectric spectroscopy (10(-2)-10(9) Hz) covering a wide temperature range of approximately 200 K from temperatures well above the isotropic-nematic transition down to the glass transition of bulk E7. A variety of relaxation processes is observed including two modes that are located close to the bulk behavior in its temperature dependence. For all confined samples, two relaxation processes, at frequencies lower than the processes observed for the bulk, were detected. At lower temperatures, their relaxation rates have different temperature dependencies whereas at higher temperatures, they seem to collapse into one chart. The temperature dependence of the slowest process (S-process) obeys the Vogel-Fulcher-Tammann law indicating a glassy dynamics of the E7 molecules anchored to the pore surface. The pore size dependence of both the Vogel temperature and fragility revealed a steplike transition around 4 nm pore size, which indicates a transition from a strong to a fragile behavior. The process with a relaxation rate in between the bulklike and the S-process (I-process) shows no dependence on the pore size. The agreement of the I-process with the behavior of a 5CB surface layer adsorbed on nonporous silica leads to the assignment of E7 molecules anchored at the outer surface of the microcrystals of the molecular sieves. PMID:20550409

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

    PubMed Central

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

    1996-01-01

    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. PMID:8970996

  11. The DEK Oncoprotein Is a Critical Component of the EKLF/KLF1 Enhancer in Erythroid Cells.

    PubMed

    Lohmann, Felix; Dangeti, Mohan; Soni, Shefali; Chen, Xiaoyong; Planutis, Antanas; Baron, Margaret H; Choi, Kyunghee; Bieker, James J

    2015-11-01

    Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established. PMID:26303528

  12. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.

    PubMed Central

    Halbert, C L; Demers, G W; Galloway, D A

    1992-01-01

    Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation. Images PMID:1312623

  13. Mutagenic Potential ofBos taurus Papillomavirus Type 1 E6 Recombinant Protein: First Description

    PubMed Central

    Araldi, Rodrigo Pinheiro; Mazzuchelli-de-Souza, Jacqueline; Modolo, Diego Grando; de Souza, Edislane Barreiros; de Melo, Thatiana Corrêa; Spadacci-Morena, Diva Denelle; Magnelli, Roberta Fiusa; de Carvalho, Márcio Augusto Caldas Rocha; de Sá Júnior, Paulo Luis; de Carvalho, Rodrigo Franco; Beçak, Willy; Stocco, Rita de Cassia

    2015-01-01

    Bovine papillomavirus (BPV) is considered a useful model to study HPV oncogenic process. BPV interacts with the host chromatin, resulting in DNA damage, which is attributed to E5, E6, and E7 viral oncoproteins activity. However, the oncogenic mechanisms of BPV E6 oncoprotein per se remain unknown. This study aimed to evaluate the mutagenic potential of Bos taurus papillomavirus type 1 (BPV-1) E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA). Peripheral blood samples of five calves were collected. Samples were subjected to molecular diagnosis, which did not reveal presence of BPV sequences. Samples were treated with 1 μg/mL of BPV-1 E6 oncoprotein and 50 μg/mL of cyclophosphamide (positive control). Negative controls were not submitted to any treatment. The samples were submitted to the CBMNA and CA. The results showed that BPV E6 oncoprotein induces clastogenesis per se, which is indicative of genomic instability. These results allowed better understanding the mechanism of cancer promotion associated with the BPV E6 oncoprotein and revealed that this oncoprotein can induce carcinogenesis per se. E6 recombinant oncoprotein has been suggested as a possible vaccine candidate. Results pointed out that BPV E6 recombinant oncoprotein modifications are required to use it as vaccine. PMID:26783529

  14. Identification of the murine H-2D(b) and human HLA-A*0201 MHC class I-restricted HPV6 E7-specific cytotoxic T lymphocyte epitopes.

    PubMed

    Peng, Shiwen; Mattox, Austin; Best, Simon R; Barbu, Anca M; Burns, James A; Akpeng, Belinda; Jeang, Jessica; Yang, Benjamin; Ishida, Eiichi; Hung, Chien-Fu; Wu, Tzyy-Choou; Pai, Sara I

    2016-03-01

    Recurrent respiratory papillomatosis is caused by human papillomavirus (HPV) infection, most commonly types 6 (HPV-6) and 11 (HPV-11). Due to failed host immune responses, HPV is unable to be cleared from the host, resulting in recurrent growth of HPV-related lesions that can obstruct the lumen of the airway within the upper aerodigestive tract. In our murine model, the HPV-6b and HPV-11 E7 antigens are not innately immunogenic. In order to enhance the host immune responses against the HPV E7 antigen, we linked calreticulin (CRT) to HPV-6b E7 and found that vaccinating C57BL/6 mice with the HPV-6b CRT/E7 DNA vaccine is able to induce a CD8(+) T cell response that recognizes an H-2D(b)-restricted E7aa21-29 epitope. Additionally, vaccination of HLA-A*0201 transgenic mice with HPV-6b CRT/E7 DNA generated a CD8(+) T cell response against the E7aa82-90 epitope that was not observed in the wild-type C57BL/6 mice, indicating this T cell response is restricted to HLA-A*0201. In vivo cytotoxic T cell killing assays demonstrated that the vaccine-induced CD8(+) T cells are able to efficiently kill target cells. Interestingly, the H-2D(b)-restricted E7aa21-29 sequence and the HLA-A*0201-restricted E7aa82-90 sequence are conserved between HPV-6b and HPV-11 and may represent shared immunogenic epitopes. The identification of the HPV-6b/HPV-11 CD8(+) T cell epitopes facilitates the evaluation of various immunomodulatory strategies in preclinical models. More importantly, the identified HLA-A*0201-restricted T cell epitope may serve as a peptide vaccination strategy, as well as facilitate the monitoring of vaccine-induced HPV-specific immunologic responses in future human clinical trials. PMID:26759151

  15. A New E6/P63 Pathway, Together with a Strong E7/E2F Mitotic Pathway, Modulates the Transcriptome in Cervical Cancer Cells▿

    PubMed Central

    Teissier, Sébastien; Ben Khalifa, Youcef; Mori, Marcella; Pautier, Patricia; Desaintes, Christian; Thierry, Françoise

    2007-01-01

    Cervical carcinoma is associated with certain types of human papillomaviruses expressing the E6 and E7 oncogenes, which are involved in carcinogenesis through their interactions with the p53 and pRB pathways, respectively. A critical event on the path to malignant transformation is often manifested by the loss of expression of the viral E2 transcription factor due to the integration into the host genome of the viral DNA. Using microarrays, we have previously shown that reintroduction of a functional E2 in the HeLa cervical carcinoma cell line activates a cluster of p53 target genes while at the same time severely repressing a group of E2F target genes. In the present study, using new high-density microarrays containing more than 22,000 human cDNA sequences, we identified a novel p63 pathway among E2-activated genes and 38 new mitotic genes repressed by E2. We then sought to determine the pathways through which these genes were modulated and used an approach that relies on small interfering RNA to demonstrate that the p63 target genes were activated through silencing of the E6/E6AP pathway while the mitotic genes were mainly repressed through E7 silencing. Importantly, a subset of the mitotic genes was shown to be significantly induced in biopsies of stage IV cervical cancers, which points to a prominent E7 pathway in cervical carcinoma. PMID:17582001

  16. 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

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

    1991-01-01

    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. PMID:1712110

  17. Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

    SciTech Connect

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

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer H-bonding network loop by PheB10Tyr mutation is proposed. Black-Right-Pointing-Pointer The propionate group H-bonding network restricted the flexibility of the heme. Black-Right-Pointing-Pointer The hydrogen bonding interaction modulates the electron density of the iron. Black-Right-Pointing-Pointer Propionate H-bonding network loop explains the heme-ligand stabilization. -- Abstract: 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. {sup 1}H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OH{eta} at 31.00 ppm, GlnE7 N{sub {epsilon}1}H/N{sub {epsilon}2}H at 10.66 ppm/-3.27 ppm, and PheE11 C{sub {delta}}H at 11.75 ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-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 heme-ligand moiety.

  18. Structure of the retinoblastoma protein bound to adenovirus E1A reveals the molecular basis for viral oncoprotein inactivation of a tumor suppressor

    SciTech Connect

    Liu, Xin; Marmorstein, Ronen

    2008-04-02

    The adenovirus (Ad) E1A (Ad-E1A) oncoprotein mediates cell transformation, in part, by displacing E2F transcription factors from the retinoblastoma protein (pRb) tumor suppressor. In this study we determined the crystal structure of the pRb pocket domain in complex with conserved region 1 (CR1) of Ad5-E1A. The structure and accompanying biochemical studies reveal that E1A-CR1 binds at the interface of the A and B cyclin folds of the pRb pocket domain, and that both E1A-CR1 and the E2F transactivation domain use similar conserved nonpolar residues to engage overlapping sites on pRb, implicating a novel molecular mechanism for pRb inactivation by a viral oncoprotein.

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

    SciTech Connect

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

    2013-08-15

    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), {sub 76}IRQLQDLLL{sub 84}, 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 {sub 65}VRLVV{sub 69} 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 {sub 65}VRLVV{sub 69} 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. - Highlights: • HPV11 E7 has a leucine-rich nuclear export signal that mediates its nuclear export via CRM1. • HPV11 E7 interacts via its unique cNLS with the FG domain of Nup62. • Identification of a hydrophobic patch essential for nuclear localization of HPV11 E7.

  20. 17 CFR 240.14e-7 - Unlawful tender offer practices in connection with roll-ups.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Unlawful tender offer practices in connection with roll-ups. 240.14e-7 Section 240.14e-7 Commodity and Securities Exchanges... tender offer practices in connection with roll-ups. In order to implement section 14(h) of the Act (15...

  1. 17 CFR 240.14e-7 - Unlawful tender offer practices in connection with roll-ups.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Unlawful tender offer practices in connection with roll-ups. 240.14e-7 Section 240.14e-7 Commodity and Securities Exchanges... tender offer practices in connection with roll-ups. In order to implement section 14(h) of the Act (15...

  2. Arsenic trioxide amplifies cisplatin toxicity in human tubular cells transformed by HPV-16 E6/E7 for further therapeutic directions in renal cell carcinoma.

    PubMed

    Dogra, Samriti; Bandi, Sriram; Viswanathan, Preeti; Gupta, Sanjeev

    2015-01-28

    Human papillomavirus (HPV) DNA integrations may affect therapeutic responses in cancers through ATM network-related DNA damage response (DDR). We studied whether cisplatin-induced DDR was altered in human HK-2 renal tubular cells immortalized by HPV16 E6/E7 genes. Cytotoxicity assays utilized thiazolyl blue dye and DDR was identified by gene expression differences, double-strand DNA breaks, ATM promoter activity, and analysis of cell cycling and side population cells. After cisplatin, HK-2 cells showed greater ATM promoter activity indicating activation of this network, but DDR was muted, since little ?H2AX was expressed, DNA strand breaks were absent and cells continued cycling. When HK-2 cells were treated with the MDM2 antagonist inducing p53, nutlin-3, or p53 transcriptional activator, tenovin-1, cell growth decreased but cisplatin toxicity was unaffected. By contrast, arsenic trioxide, which by inhibiting wild-type p53-induced phosphatase-1 that serves responses downstream of p53, and by depolymerizing tubulin, synergistically enhanced cisplatin cytotoxicity including loss of SP cells. Our findings demonstrated that HPV16 E6/E7 altered DDR through p53-mediated cell growth controls, which may be overcome by targeting of WIP1 and other processes, and thus should be relevant for treating renal cell carcinoma. PMID:25444910

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

    PubMed Central

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

    2000-01-01

    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. PMID:11000240

  4. Structure and Catalytic Mechanism of the Thioesterase CalE7 in Enediyne Biosynthesis*

    PubMed Central

    Kotaka, Masayo; Kong, Rong; Qureshi, Insaf; Ho, Qin Shi; Sun, Huihua; Liew, Chong Wai; Goh, Lan Pei; Cheung, Peter; Mu, Yuguang; Lescar, Julien; Liang, Zhao-Xun

    2009-01-01

    The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-Å crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg37 plays a crucial role in the hydrolysis of the thioester bond, and that Tyr29 and a hydrogen-bonded water network assist the decarboxylation of the β-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4=C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism. PMID:19357082

  5. Arsenic Trioxide Is a Potent Inhibitor of the Interaction of SMRT Corepressor with Its Transcription Factor Partners, Including the PML-Retinoic Acid Receptor ? Oncoprotein Found in Human Acute Promyelocytic Leukemia

    PubMed Central

    Hong, Suk-Hyun; Yang, Zhihong; Privalsky, Martin L.

    2001-01-01

    The SMRT corepressor complex participates in transcriptional repression by a diverse array of vertebrate transcription factors. The ability to recruit SMRT appears to play a crucial role in leukemogenesis by the PML-retinoic acid receptor ? (RAR?) oncoprotein, an aberrant nuclear hormone receptor implicated in human acute promyelocytic leukemia (APL). Arsenite induces clinical remission of APL through a incompletely understood mechanism. We report here that arsenite is a potent inhibitor of the interaction of SMRT with its transcription factor partners, including PML-RAR?. Arsenite operates, in part, through a mitogen-activated protein (MAP) kinase cascade culminating in phosphorylation of the SMRT protein, dissociation of SMRT from its nuclear receptor partners, and a relocalization of SMRT out of the nucleus into the cytoplasm of the cell. Conversely, inhibition of this MAP kinase cascade attenuates the effects of arsenite on APL cells. Our results implicate SMRT as an important biological target for the actions of arsenite in both normal and neoplastic cells. PMID:11585900

  6. Exosomal sorting of the viral oncoprotein LMP1 is restrained by TRAF2 association at signalling endosomes

    PubMed Central

    Verweij, Frederik J.; de Heus, Cecilia; Kroeze, Stefanie; Cai, Houjian; Kieff, Elliott; Piersma, Sander R.; Jimenez, Connie R.; Middeldorp, Jaap M.; Pegtel, Dirk Michiel

    2015-01-01

    The Epstein–Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1–TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1–TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1–TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1–TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that “signalling endosomes” in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes. PMID:25865256

  7. Establishment of immunoassay for detecting HPV16 E6 and E7 RNA

    PubMed Central

    Ding, Sen; Qian, Steven Y.; Zhang, Yang; Wu, Wenlei; Lu, Gensheng; Lu, Yan; Feng, Xiujing; Li, Li; Shen, Pingping

    2015-01-01

    Cervical carcinoma is the most prevalent malignancy second only to breast cancer among women worldwide. Since more than 99% of cervical cancers are caused by human papilloma virus (HPV), measurement of HPV (HPV test) was commonly used in screening risk and/or early stage of cervical cancer as well as assessing the efficacies of the treatments that can decrease the incidence of cervical cancer. Many approaches that diagnose HPV infections have been developed, while most of them have distinct shortcomings. We here established a novel immunoassay method in which the pairs of unlabeled DNA probes firstly bind to HPV16 E6 and E7 RNAs to form the DNA-RNA hybrids, and the hybrids will subsequently be identified by S9.6 antibody. The sensitivity of this highly specific method can reach ~0.923?pg/mL and ~0.424?pg/mL of in vitro transcribed HPV16 E6 and E7 RNA, respectively, and reverse transcription and polymerase chain reaction (PCR) amplification were no longer needed. Thus, our immunoassay approaches can precisely reflect the actually viral load that is related to the course of HPV infection. In addition, it has also fast and low cost characteristic feature. PMID:26333509

  8. Characterization and evaluation of the immune responses elicited by a novel human papillomavirus (HPV) therapeutic vaccine: HPV 16E7-HBcAg-Hsp65 fusion protein.

    PubMed

    Zhou, Chao-Ming; Zhang, Gao-Xia; Ma, Xin-Xing

    2014-03-01

    Human papillomaviruses (HPV), particularly HPV16, are associated with most cervical cancers. Currently, although prophylactic vaccines have been developed, there is still an urgent need to develop therapeutic HPV vaccines. In this study, a novel fusion protein, HPV 16 E7-HBcAg-Hsp65 (VR111), with the goal of increasing anti-HPV16 cellular immunity was developed. VR111 was analyzed using SDS-PAGE, western-blotting, capillary isoelectric focusing (cIEF), analytical ultracentrifugation (AUC) and dynamic light scattering (DLS). Gamma interferon (IFN-?) secretion assay was performed by enzyme-linked immunospot (ELISPOT) and ELISA to test their ability to induce cellular immune response. Significant correlation between ELISPOT and ELISA was observed (r=0.8680, p<0.0001). It was shown that VR111 could induce a significant increase in E7-specific CD8(+) T cell responses. Humoral immune response was also observed. The antibody titer levels were measured by ELISA. These results indicated that VR111 was a promising therapeutic vaccine for treatment of cervical cancer with possible therapeutic potential in clinical settings. PMID:24291739

  9. A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2

    PubMed Central

    Abdullah, Ammara; Sane, Sanam; Branick, Kate A.; Freeling, Jessica L.; Wang, Hongmin; Zhang, Dong; Rezvani, Khosrow

    2015-01-01

    Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53. PMID:26188124

  10. The Catalytic Efficiency of Lipin 1? Increases by Physically Interacting with the Proto-oncoprotein c-Fos.

    PubMed

    Cardozo Gizzi, Andres M; Prucca, Cesar G; Gaveglio, Virginia L; Renner, Marianne L; Pasquar, Susana J; Caputto, Beatriz L

    2015-12-01

    Phosphatidic acid (PA) is a central precursor for membrane phospholipid biosynthesis. The lipin family is a magnesium-dependent type I PA phosphatase involved in de novo synthesis of neutral lipids and phospholipids. The regulation of lipin activity may govern the pathways by which these lipids are synthesized and control the cellular levels of important signaling lipids. Moreover, the proto-oncoprotein c-Fos has an emerging role in glycerolipid synthesis regulation; by interacting with key synthesizing enzymes it is able to increase overall phospho- and glycolipid synthesis. We studied the lipin 1? enzyme activity in a cell-free system using PA/Triton X-100 mixed micelles as substrate, analyzing it in the presence/absence of c-Fos. We found that lipin 1? kcat value increases around 40% in the presence of c-Fos, with no change in the lipin 1? affinity for the PA/Triton X-100 mixed micelles. We also probed a physical interaction between both proteins. Although the c-Fos domain involved in lipin activation is its basic domain, the interaction domain is mapped to the N-terminal c-Fos. In conclusion, we provide evidence for a novel positive regulator of lipin 1? PA phosphatase activity that is not achieved via altering its subcellular localization or affinity for membranes but rather through directly increasing its catalytic efficiency. PMID:26475860

  11. The role of oncoprotein NM23 gene from Exopalaemon carinicauda is response to pathogens challenge and ammonia-N stress.

    PubMed

    Duan, Yafei; Li, Jitao; Zhang, Zhe; Li, Jian; Ge, Qianqian; Liu, Ping

    2015-12-01

    Oncoprotein NM23, as a family of genes encoding the nucleoside diphosphate (NDP) kinase, plays important roles in bioenergetics, DNA replication, differentiation and tumor metastasis. In this study, a full-length cDNA of NM23 (designated EcNM23) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcNM23 was 755bp, which contains an open reading frame (ORF) of 518bp, encoding a 175 amino-acid polypeptide with the predicted molecular weight of 19.60kDa and estimated isoelectric point of 7.67. The deduced amino acid sequence of EcNM23 shared high identity (86%-93%) with that of other crustaceans. a NDP kinase super family signature was identified in E.carinicauda EcNM23. Quantitative real-time RT-qPCR analysis indicated that EcNM23 was expressed in all the examined tissues with the high expression level in hemocytes and ovary. The EcNM23 expression in immune-related tissues changed rapidly and reached peak at different time after pathogens (Vibrio parahaemolyticus and WSSV) challenge and ammonia-N stress treatment. The results suggested that EcNM23 might be associated with the immune defenses to pathogens infection and ammonia-N stress in E.carinicauda. PMID:26314522

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

    PubMed

    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

    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

  13. Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

    PubMed Central

    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

    2013-01-01

    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

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

    SciTech Connect

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli; Zhang, Xiaodong; Ye, Lihong

    2013-05-03

    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-κB (NF-κB) 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-κB 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.

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

    SciTech Connect

    Kaur, Tranum; Gupta, Rajesh; Vaiphei, Kim; Kapoor, Rakesh; Gupta, N.M.; Khanduja, K.L.

    2008-02-01

    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.

  16. Transforming properties of the cottontail rabbit papillomavirus oncoproteins Le6 and SE6 and of the E8 protein.

    PubMed Central

    Harry, J B; Wettstein, F O

    1996-01-01

    Cottontail rabbit papillomavirus induces on cottontail and domestic rabbits papillomas which progress at a high frequency to carcinoma. The virus encodes three transforming proteins; one is translated from open reading frame (ORF) E7 and binds the retinoblastoma protein, and two, LE6 and SE6, are translated from the first and second ATGs of ORF E6, respectively. Here we show that neither of the E6 proteins coprecipitated with p53 in vitro, nor did they bind to a recently identified E6-binding protein (J. J. Chen, C. E. Reid, V. Band, and E. Androphy, Science 269:529-531, 1995). This protein was shown to bind to the E6 proteins of the high-risk human papillomairus types 16 and 18 but not to the low-risk human papillomavirus types VI and II. In-frame deletions cloned into the pZipNeo vector were used to identify structural features of SE6 and LE6 important for transformation of NIH 3T3 cells. Three deletions covering the amino-terminal half of SE6 did not transform cells. In two of the three deletions, two Cys-X-X-Cys motifs were deleted, each deletion preventing the formation of one of the potential small Zn fingers of SE6. Among the LE6 deletions, only one had a reduced transformation efficiency, while seven transformed cells at least as efficiently as wild-type LE6. In each of three of these seven mutants, two Cys-X-X-Cys motifs were deleted. None of the three amino acid deletions which abolished transformation by SE6 reduced transformation by LE6. Furthermore, transformation did not correlate with the level of SE6 or LE6 proteins detectable. ORF E8 colinear with ORF E6, which could generate a 50-amino-acid protein with a hydrophobic segment, did not transform cells when cloned into the pZipNeo vector. However, mutation of the E8 ATG, which did not alter the amino acid sequence of LE6, increased transformation by LE6 without affecting the level of LE6 expression. The data suggest that transformation by the E6 proteins is not mediated by interfering with p53 function or through binding to the E6-binding protein. Furthermore, different structural features are important to maintain transformation functions and protein stability of LE6 and SE6. Finally, E8 seems not to be a transforming protein but rather appears to modulate transformation bv LE6. PMID:8648665

  17. Identification of RNA aptamers that internalize into HPV-16 E6/E7 transformed tonsillar epithelial cells.

    PubMed

    Gourronc, Francoise A; Rockey, William M; Thiel, William H; Giangrande, Paloma H; Klingelhutz, Aloysius J

    2013-11-01

    Human papillomavirus type 16 (HPV-16) associated oropharyngeal cancers are on a significant increase and better therapeutic strategies are needed. The HPV-16 oncogenes E6 and E7 are expressed in HPV-associated cancers and are able to transform human tonsillar epithelial cells (HTECs). We used cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select for RNA aptamers that entered into HPV-16 E6/E7-HTECs. After 12 rounds of cell-SELEX, a pool of aptamers was obtained that had significantly greater internalization capacity (~5-fold) into E6/E7-HTECs as compared to primary HTECs or fibroblasts. Analysis of individual aptamers from the pool indicated variable internalization into E6/E7-HTECs (1-8-fold as compared to a negative control). Most of the individual aptamers internalized into E6/E7 and primary HTECs with similar efficiency, while one aptamer exhibited ~3-fold better internalization into E6/E7-HTECs. Aptamers that internalize into cells may be useful for delivering therapeutic agents to HPV-16 associated malignancies. PMID:24074596

  18. Interaction of Marek's Disease Virus (MDV) Oncoprotein Meq with Host Proteins: A Proteomic Approach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s Disease is a T cell lymphoma disease of chicken induced by an oncogenic, cell associated alpha herpes virus. Oncogenicity in the Marek’s disease is mostly attributed to a transcription factor termed as Meq. To understand the mechanisms of oncogenicity of Meq, it is necessary to understand it...

  19. Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells.

    PubMed

    Heredia-Rojas, J Antonio; Rodrguez de la Fuente, Abraham Octavio; Alcocer Gonzlez, Juan Manuel; Rodrguez-Flores, Laura E; Rodrguez-Padilla, Cristina; Santoyo-Stephano, Martha A; Castaeda-Garza, Esperanza; Tamz-Guerra, Reyes S

    2010-10-01

    It has been reported that 50-60 Hz magnetic fields (MF) with flux densities ranging from microtesla to millitesla are able to induce heat shock factor or heat shock proteins in various cells. In this study, we investigated the effect of 60 Hz sinusoidal MF at 8 and 80 ?T on the expression of the luciferase gene contained in a plasmid labeled as electromagnetic field-plasmid (pEMF). This gene construct contains the specific sequences previously described for the induction of hsp70 expression by MF, as well as the reporter for the luciferase gene. The pEMF vector was transfected into INER-37 and RMA E7 cell lines that were later exposed to either MF or thermal shock (TS). Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system for evaluate luciferase activity. An increased luciferase gene expression was observed in INER-37 cells exposed to MF and TS compared with controls (p < 0.05), but MF exposure had no effect on the RMA E7 cell line. PMID:20835776

  20. Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region.

    PubMed

    Lavezzo, Enrico; Masi, Giulia; Toppo, Stefano; Franchin, Elisa; Gazzola, Valentina; Sinigaglia, Alessandro; Masiero, Serena; Trevisan, Marta; Pagni, Silvana; Palù, Giorgio; Barzon, Luisa

    2016-01-01

    Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity. PMID:26985902

  1. An Intrinsically Disordered Region in the Proapoptotic ASPP2 Protein Binds to the Helicobacter pylori Oncoprotein CagA.

    PubMed

    Reingewertz, Tali H; Iosub-Amir, Anat; Bonsor, Daniel A; Mayer, Guy; Amartely, Hadar; Friedler, Assaf; Sundberg, Eric J

    2015-06-01

    The leading risk factor for gastric cancer in humans is infection by Helicobacter pylori strains that express and translocate the oncoprotein CagA into host epithelial cells. Once inside host cells, CagA interacts with ASPP2, which specifically stimulates p53-mediated apoptosis and reverses its pro-apoptotic function to promote ASPP2-dependent degradation of p53. The X-ray crystal structure of a complex between the N-terminal domain of CagA and a 56-residue fragment of ASPP2, of which 22 residues were resolved, was recently described. Here, we present biochemical and biophysical analyses of the interaction between the additional regions of CagA and ASPP2 potentially involved in this interaction. Using size exclusion chromatography-multiangle laser light scattering, circular dichroism, and nuclear magnetic resonance analyses, we observed that the ASPP2 region spanning residues 331-692, which was not part of the ASPP2 fragment used for crystallization, is intrinsically disordered in its unbound state. By surface plasmon resonance analysis and isothermal titration calorimetry, we found that a portion of this disordered region in ASPP2, residues 448-692, binds to the N-terminal domain of CagA. We also measured the affinity of the complex between the ASPP2 fragment composed of residues 693-918 and inclusive of the fragment used for crystallization and CagA. Additionally, we mapped the binding regions between ASPP2 and CagA using peptide arrays, demonstrating interactions between CagA and numerous peptides distributed throughout the ASPP2 protein sequence. Our results identify previously uncharacterized regions distributed throughout the protein sequence of ASPP2 as determinants of CagA binding, providing mechanistic insight into apoptosis reprogramming by CagA and potential new drug targets for H. pylori-mediated gastric cancer. PMID:25963096

  2. Transcriptomic analyses of genes differentially expressed by high-risk and low-risk human papilloma virus E6 oncoproteins.

    PubMed

    Ganguly, Pooja; Ganguly, Niladri

    2015-09-01

    Human papilloma virus is the causative agent for cervical cancer with 99% of cervical cancer cases containing HPV. The high risk HPV-16, 18 and 31 are the major causative agents. The low risk HPV-6, 11 have been reported to cause penile, laryngeal, bronchogenic and oesophageal cancer. Since E6 oncoprotein is frequently over expressed in cancers, we did gene expression studies to compare between the E6 genes of high-risk (HPV18) or low-risk (HPV11)stably transfected in epithelial cell line EPC-2 or mock transfected with the basic vector pCDNA3.1. Microarray studies showed a total of 697 genes showing differential expression between the samples. Genes involved in several key cellular processes such as cell adhesion, angiogenesis, transcription regulation, cell cycle regulation and cell division showed altered expression between the samples. Gene Ontology mapping of 44 genes according cellular pathways revealed 13 pathways namely angiogenesis, alzhemier's, Wnt, p53, interleukin, TGF-?, cadherin, integrin, PI3-kinase, catennin, insulin, chemokine and G protein signalling pathways. The microarray results were confirmed by quantitative real-time PCR for some representative genes like IFI27, CTNNA1, OSMR, CYP1B1, TNFSF13, LAMA2 and COL5A3. Analysis of differentially expressed genes by high-risk and low-risk HPV E6 proteins might help in identification of potential biomarkers for diagnosis, progression and therapy of oesophageal cancer. The understanding of mechanisms of activation of these genes as well as the function of gene products will give a further insight into their roles in oesophageal cancer. PMID:26396976

  3. Acetylation at lysine 346 controls the transforming activity of the HTLV-1 Tax oncoprotein in the Rat-1 fibroblast model

    PubMed Central

    2013-01-01

    Background Transformation by the Tax oncoprotein of the human T cell leukemia virus type 1 (HTLV-1) is governed by actions on cellular regulatory signals, including modulation of specific cellular gene expression via activation of signaling pathways, acceleration of cell cycle progression via stimulation of cyclin-dependent kinase activity leading to retinoblastoma protein (pRb) hyperphosphorylation and perturbation of survival signals. These actions control early steps in T cell transformation and development of Adult T cell leukemia (ATL), an aggressive malignancy of HTLV-1 infected T lymphocytes. Post-translational modifications of Tax by phosphorylation, ubiquitination, sumoylation and acetylation have been implicated in Tax-mediated activation of the NF-?B pathway, a key function associated with Tax transforming potential. Results In this study, we demonstrate that acetylation at lysine K346 in the carboxy-terminal domain of Tax is modulated in the Tax nuclear bodies by the acetyltransferase p300 and the deacetylases HDAC5/7 and controls phosphorylation of the tumor suppressor pRb by Tax-cyclin D3-CDK4-p21CIP complexes. This property correlates with the inability of the acetylation deficient K346R mutant, but not the acetylation mimetic K346Q mutant, to promote anchorage-independent growth of Rat-1 fibroblasts. By contrast, acetylation at lysine K346 had no effects on the ability of Tax carboxy-terminal PDZ-binding domain to interact with the tumor suppressor hDLG. Conclusions The identification of the acetyltransferase p300 and the deacetylase HDAC7 as enzymes modulating Tax acetylation points to new therapeutic targets for the treatment of HTLV-1 infected patients at risk of developing ATL. PMID:23880157

  4. Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6.

    PubMed

    Wang, Xiaohong; Wang, Hsu-Kun; McCoy, J Philip; Banerjee, Nilam S; Rader, Janet S; Broker, Thomas R; Meyers, Craig; Chow, Louise T; Zheng, Zhi-Ming

    2009-04-01

    MicroRNAs (miRNA) play pivotal roles in controlling cell proliferation and differentiation. Aberrant miRNA expression in human is becoming recognized as a new molecular mechanism of carcinogenesis. However, the causes for alterations in miRNA expression remain largely unknown. Infection with oncogenic human papillomavirus types 16 (HPV16) and 18 (HPV18) can lead to cervical and other ano-genital cancers. Here, we have demonstrated that cervical cancer tissues and cervical cancer-derived cell lines containing oncogenic HPVs display reduced expression of tumor-suppressive miR-34a. The reduction of miR-34a expression in organotypic tissues derived from HPV-containing primary human keratinocytes correlates with the early productive phase and is attributed to the expression of viral E6, which destabilizes the tumor suppressor p53, a known miR-34a transactivator. Knockdown of viral E6 expression in HPV16(+) and HPV18(+) cervical cancer cell lines by siRNAs leads to an increased expression of p53 and miR-34a and accumulation of miR-34a in G(0)/G(1) phase cells. Ectopic expression of miR-34a in HPV18(+) HeLa cells and HPV(-) HCT116 cells results in a substantial induction of cell growth retardation and a moderate cell apoptosis. Together, this is the first time a viral oncoprotein has been shown to regulate cellular miRNA expression. Our data have provided new insights into mechanisms by which high-risk HPVs contribute to the development of cervical cancer. PMID:19258450

  5. The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities

    PubMed Central

    2010-01-01

    Background Marek's disease virus (MDV) is an oncogenic herpesvirus, which causes malignant lymphoma in chickens. The Meq protein of MDV, which is expressed abundantly in MDV-infected cells and in Marek's disease (MD) tumor cells, functions as a transcriptional activator and has been proposed to play an important role in oncogenic transformation. Preliminary studies demonstrated that Meq is able to bind p53 in vitro, as demonstrated using a protein-binding assay. This observation prompted us to examine whether the interaction between Meq and p53 occurs in cells, and to investigate the biological significance of this interaction. Results We confirmed first that Meq interacted directly with p53 using a yeast two-hybrid assay and an immunoprecipitation assay, and we investigated the biological significance of this interaction subsequently. Exogenous expression of Meq resulted in the inhibition of p53-mediated transcriptional activity and apoptosis, as analyzed using a p53 luciferase reporter assay and a TUNEL assay. The inhibitory effect of Meq on transcriptional activity mediated by p53 was dependent on the physical interaction between these two proteins, because a Meq deletion mutant that lacked the p53-binding region lost the ability to inhibit p53-mediated transcriptional activity and apoptosis. The Meq variants L-Meq and S-Meq, but not VS-Meq and ?Meq, which were expressed in MD tumor cells and MDV-infected cells, exerted an inhibitory effect on p53 transcriptional activity. In addition, ?Meq was found to act as a negative regulator of Meq. Conclusions The Meq oncoprotein interacts directly with p53 and inhibits p53-mediated transcriptional activity and apoptosis. These findings provide valuable insight into the molecular basis for the function of Meq in MDV oncogenesis. PMID:21110861

  6. TARGETING THE MUC1-C ONCOPROTEIN DOWNREGULATES HER2 ACTIVATION AND ABROGATES TRASTUZUMAB RESISTANCE IN BREAST CANCER CELLS

    PubMed Central

    Raina, Deepak; Uchida, Yasumitsu; Kharbanda, Akriti; Rajabi, Hasan; Panchamoorthy, Govind; Jin, Caining; Kharbanda, Surender; Scaltriti, Maurizio; Baselga, Jose; Kufe, Donald

    2014-01-01

    Patients with HER2 positive breast cancer often exhibit intrinsic or acquired resistance to trastuzumab treatment. The transmembrane MUC1-C oncoprotein is aberrantly overexpressed in breast cancer cells and associates with HER2. The present studies demonstrate that silencing MUC1-C in HER2-overexpressing SKBR3 and BT474 breast cancer cells results in downregulation of constitutive HER2 activation. Moreover, treatment with the MUC1-C inhibitor, GO-203, was associated with disruption of MUC1-C/HER2 complexes and decreases in tyrosine phosphorylated HER2 (p-HER2) levels. In studies of trastuzumab-resistant SKBR3R and BT474R cells, we found that the association between MUC1-C and HER2 is markedly increased (~20-fold) as compared to that in sensitive cells. Additionally, silencing MUC1-C in the trastuzumab-resistant cells or treatment with GO-203 decreased p-HER2 and AKT activation. Moreover, targeting MUC1-C was associated with downregulation of phospho-p27 and cyclin E, which confer trastuzumab resistance. Consistent with these results, targeting MUC1-C inhibited the growth and clonogenic survival of both trastuzumab-resistant cells. Our results further demonstrate that silencing MUC1-C reverses resistance to trastuzumab and that the combination of GO-203 and trastuzumab is highly synergistic. These findings indicate that MUC1-C contributes to constitutive activation of the HER2 pathway and that targeting MUC1-C represents a potential approach to abrogate trastuzumab resistance. PMID:23912457

  7. Oncogenic HPV infection interrupts the expression of tumor-suppressive miR-34a through viral oncoprotein E6

    PubMed Central

    Wang, Xiaohong; Wang, Hsu-Kun; McCoy, J. Philip; Banerjee, Nilam S.; Rader, Janet S.; Broker, Thomas R.; Meyers, Craig; Chow, Louise T.; Zheng, Zhi-Ming

    2009-01-01

    MicroRNAs (miRNA) play pivotal roles in controlling cell proliferation and differentiation. Aberrant miRNA expression in human is becoming recognized as a new molecular mechanism of carcinogenesis. However, the causes for alterations in miRNA expression remain largely unknown. Infection with oncogenic human papillomavirus types 16 (HPV16) and 18 (HPV18) can lead to cervical and other ano-genital cancers. Here, we have demonstrated that cervical cancer tissues and cervical cancer-derived cell lines containing oncogenic HPVs display reduced expression of tumor-suppressive miR-34a. The reduction of miR-34a expression in organotypic tissues derived from HPV-containing primary human keratinocytes correlates with the early productive phase and is attributed to the expression of viral E6, which destabilizes the tumor suppressor p53, a known miR-34a transactivator. Knockdown of viral E6 expression in HPV16+ and HPV18+ cervical cancer cell lines by siRNAs leads to an increased expression of p53 and miR-34a and accumulation of miR-34a in G0/G1 phase cells. Ectopic expression of miR-34a in HPV18+ HeLa cells and HPV? HCT116 cells results in a substantial induction of cell growth retardation and a moderate cell apoptosis. Together, this is the first time a viral oncoprotein has been shown to regulate cellular miRNA expression. Our data have provided new insights into mechanisms by which high-risk HPVs contribute to the development of cervical cancer. PMID:19258450

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

    SciTech Connect

    Hayakawa, Kazuo; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto; Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya ; Ikeya, Makoto; Fukuta, Makoto; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto; Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Nagoya City University, Nagoya ; Woltjen, Knut; Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo; Otsuka, Takanobu; Toguchida, Junya; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto; Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto

    2013-03-22

    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.

  9. Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

    SciTech Connect

    Lee, Kiwon; Lee, Ah-Young; Kwon, Yunhee Kim; Kwon, Hyockman

    2011-08-26

    Highlights: {yields} Integration of HPV into host genome critical for activation of E6 and E7 oncogenes. {yields} BAF53 is essential for higher-order chromatin structure. {yields} BAF53 knockdown suppresses E6 and E7 from HPV integrants, but not from episomal HPVs. {yields} BAF53 knockdown decreases H3K9Ac and H4K12Ac on P105 promoter of integrated HPV 18. {yields} BAF53 knockdown restores the p53-dependent signaling pathway in HeLa and SiHa cells. -- Abstract: Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.

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

    PubMed

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

    2013-08-15

    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

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed Central

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

    1996-01-01

    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 PMID:8692833

  13. 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

    SciTech Connect

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

    2008-03-15

    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{sup 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.

  14. c-Jun associates with the oncoprotein Ski and suppresses Smad2 transcriptional activity.

    PubMed

    Pessah, Marcia; Marais, Jacqueline; Prunier, Celine; Ferrand, Nathalie; Lallemand, Francois; Mauviel, Alain; Atfi, Azeddine

    2002-08-01

    The Smad proteins are key intracellular effectors of transforming growth factor-beta (TGF-beta) cytokines. The ability of Smads to modulate transcription results from a functional cooperativity with the coactivators p300/cAMP-response element-binding protein-binding protein (CBP), or the corepressors TGIF and Ski. The c-Jun N-terminal kinase (JNK) pathway, another downstream target activated by TGF-beta receptors, has also been suggested to inhibit TGF-beta signaling through interaction of c-Jun with Smad2 and Smad3. Here we show that c-Jun directly interacts with Ski to enhance the association of Ski with Smad2 in the basal state. Interestingly, TGF-beta signaling induces dissociation of c-Jun from Ski, thereby relieving active repression by c-Jun. Moreover, activation of JNK pathway suppressed the ability of TGF-beta to induce dissociation of c-Jun from ski. Thus, the formation of a c-Jun/Ski complex maintains the repressed state of Smad2-responsive genes in the absence of ligand and participates in negative feedback regulation of TGF-beta signaling by the JNK cascade. PMID:12034730

  15. Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML.

    PubMed

    Zhang, Xiao-Wei; Yan, Xiao-Jing; Zhou, Zi-Ren; Yang, Fei-Fei; Wu, Zi-Yu; Sun, Hong-Bin; Liang, Wen-Xue; Song, Ai-Xin; Lallemand-Breitenbach, Valrie; Jeanne, Marion; Zhang, Qun-Ye; Yang, Huai-Yu; Huang, Qiu-Hua; Zhou, Guang-Biao; Tong, Jian-Hua; Zhang, Yan; Wu, Ji-Hui; Hu, Hong-Yu; de Th, Hugues; Chen, Sai-Juan; Chen, Zhu

    2010-04-01

    Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (As2O3) exerts its therapeutic effect by promoting degradation of an oncogenic protein that drives the growth of APL cells, PML-RARalpha (a fusion protein containing sequences from the PML zinc finger protein and retinoic acid receptor alpha). PML and PML-RARalpha degradation is triggered by their SUMOylation, but the mechanism by which As2O3 induces this posttranslational modification is unclear. Here we show that arsenic binds directly to cysteine residues in zinc fingers located within the RBCC domain of PML-RARalpha and PML. Arsenic binding induces PML oligomerization, which increases its interaction with the small ubiquitin-like protein modifier (SUMO)-conjugating enzyme UBC9, resulting in enhanced SUMOylation and degradation. The identification of PML as a direct target of As2O3 provides new insights into the drug's mechanism of action and its specificity for APL. PMID:20378816

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

    NASA Technical Reports Server (NTRS)

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

    1972-01-01

    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.

  17. Epigenetic silencing of the myelopoiesis regulator microRNA-223 by the AML1/ETO oncoprotein.

    PubMed

    Fazi, Francesco; Racanicchi, Serena; Zardo, Giuseppe; Starnes, Linda M; Mancini, Marco; Travaglini, Lorena; Diverio, Daniela; Ammatuna, Emanuele; Cimino, Giuseppe; Lo-Coco, Francesco; Grignani, Francesco; Nervi, Clara

    2007-11-01

    Hematopoietic transcription factors are involved in chromosomal translocations, which generate fusion proteins contributing to leukemia pathogenesis. Analysis of patient's primary leukemia blasts revealed that those carrying the t(8;21) generating AML1/ETO, the most common acute myeloid leukemia-associated fusion protein, display low levels of a microRNA-223 (miR-223), a regulator of myelopoiesis. Here, we show that miR-223 is a direct transcriptional target of AML1/ETO. By recruiting chromatin remodeling enzymes at an AML1-binding site on the pre-miR-223 gene, AML1/ETO induces heterochromatic silencing of miR-223. Ectopic miR-223 expression, RNAi against AML1/ETO, or demethylating treatment enhances miR-223 levels and restores cell differentiation. Here, we identify an additional action for a leukemia fusion protein linking the epigenetic silencing of a microRNA locus to the differentiation block of leukemia. PMID:17996649

  18. The oncoprotein LMO3 interacts with calcium- and integrin-binding protein CIB.

    PubMed

    Hui, Ling; Ji, Chaoneng; Hui, Bin; Lv, Tongde; Ha, Xiaoqin; Yang, Jinsheng; Cai, Wenqin

    2009-04-10

    The protein LMO3 belongs to the LIM only (LMO) group of transcriptional regulators, which act as molecular adaptors for protein-protein interactions. However, little is known about its interactive proteins and functions. Evaluating LMO3 in a yeast two-hybrid screen, we identified the calcium- and integrin-binding protein CIB as an LMO3-binding protein, which binds via the second LIM domain (LIM2) of LMO3. Cotransfection of LMO3 and CIB resulted in a shift in LMO3 protein from the nucleus to the cytoplasm. In functional assays, LMO3 induced C8 astrocyte proliferation was suppressed by the overexpression of CIB. This study demonstrates one function for LMO3 in C8 cells and suggests that one role of the CIB/LMO3 complex is to inhibit cell proliferation. PMID:19236851

  19. [The study of gene variation and phylogenetic analysis of HPV16 E6 and E7 gene in Hubei, China].

    PubMed

    Ding, Xiao-hua; Zhang, Fan; Cai, Hong-bin; Yang, Zhang-qiu

    2010-09-01

    To study the gene variation and the distribution of HPV16 variant in Hubei, China, DNA was extracted from cervical cancer tissue samples. The E6 and E7 genes of HPV16 were amplified and the PCR products were sequenced using E6- and E7-specific primers. Fortyseven cases were found mutations at nucleotide position 178 of HPV16 E6 gene in 80 cervical cancer samples. This mutation resulted in amino acid change from Asp to Glu. The rate of mutation at nucleotide position 178 of E6 gene was 58. 75%. Twenty two cases were found mutations at nucleotide position 647 of HPV16 E7 gene in 31 cervical cancer samples. This mutation resulted in amino acid change from Asn to Ser. The rate of mutation was 70.97%. These results showed that mutations at nucleotide position 178 of E6 gene, nucleotide position 647 of E7 gene of HPV16 in cerveical cancer samples were prevalent in Hubei, China. Phylogenetic analysis showed that Asian (As) variants of HPV16 are predominated in Hubei, China. European (Ep) varinats were also found in Samples in Hubei areas. None of Asian American (AA), African-1 (Af-1), African-2 (Af-2) variants of HPV16 was found in this region. Whether Asian (As) variants of HPV16 are more oncogenic and play a much more important role in the progress of cervical cancer than European (Ep) variants is not clear. More sequences of E6 and E7 gene in CIN and normal cervical tissue samples and study of the function of E6 and E7 protein of these HPV16 variants are needed to adress above question. PMID:21043136

  20. On section conditions of E 7(+7) exceptional field theory and superparticle in N=8 central charge superspace

    NASA Astrophysics Data System (ADS)

    Bandos, Igor

    2016-01-01

    We study the properties of section conditions of the E 7(+7) exceptional field theory from the perspective of superparticle model in N=8 D = 4 superspace enlarged by additional bosonic coordinates related to the central charge of the maximal supersymmetry superalgebra. In particular, the superparticle model suggests that only a part of the section conditions corresponding to generators of SU(8) subgroup of E 7(+7) is independent, and we show that this part indeed suffices to obtain the (classical counterpart of the) general solution of the strong section condition.

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

    PubMed Central

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

    1996-01-01

    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 PMID:8692997

  2. High risk HPV E6 oncoproteins impair the subcellular distribution of the four and a half LIM-only protein 2 (FHL2).

    PubMed

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

    2015-02-01

    HPVs are the causative agents of approximately 5% of all human cancers, with cervical cancer being the most predominant. To understand the mechanism of action of the viral E6 oncoprotein, we analysed the effects of E6 upon potential cellular target proteins. One candidate is FHL-2, involved in the regulation of signal transduction pathways from the multimeric complexes assembled at focal adhesions. We show that both HPV E6 and E6(?) can interact with FHL-2 in vitro, but unlike most E6 targets, FHL-2 does not appear to be an E6 degradation target. Analysis of the patterns of FHL-2 distribution within HPV-positive tumour-derived cells shows a significant alteration in the pattern of FHL-2 localisation when compared to non-HPV containing cells. This perturbation of FHL-2 distribution is proteasome-dependent and inhibition of E6 expression restores the normal distribution of FHL-2. These results confirm FHL-2 as a new interacting partner of the HPV-E6 oncoproteins. PMID:25540819

  3. The oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells.

    PubMed

    Shi, Hui; Li, Yinghui; Feng, Guoxing; Li, Leilei; Fang, Runping; Wang, Zhen; Qu, Jie; Ding, Peijian; Zhang, Xiaodong; Ye, Lihong

    2016-02-26

    We have reported that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor and is highly expressed in various human cancers. However, the regulatory mechanism of FGF4 in breast cancer remains poorly understood. In the present study, we report that HBXIP is able to up-regulate FGF4 to enhance the migration of breast cancer cells. Immunohistochemistry staining showed that HBXIP and FGF4 were highly expressed in clinical metastatic lymph nodes of breast tumor. The expression levels of HBXIP were positively related to those of FGF4 in clinical breast cancer tissues. Then, we validated that HBXIP up-regulated the expression of FGF4 at the levels of promoter, mRNA and protein by luciferase reporter gene assays, reverse transcription-polymerase chain reaction and Western blot analysis. Moreover, we found that HBXIP was able to activate FGF4 promoter through transcriptional factor Sp1 by luciferase reporter gene assays. Chromatin immunoprecipitation assays confirmed that HBXIP coactivated Sp1 to stimulate FGF4 promoter. In function, we showed that HBXIP promoted breast cancer cell migration through FGF4 by wound healing and transwell cell migration assays. Thus, we conclude that the oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells. Therapeutically, HBXIP may serve as a novel target in breast cancer. PMID:26828265

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

    SciTech Connect

    Piccioli, Zachary; McKee, Courtney H.; Leszczynski, Anna; Onder, Zeynep; Hannah, Erin C.; Mamoor, Shahan; Crosby, Lauren; Moroianu, Junona

    2010-11-10

    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.

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

    PubMed Central

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

    2011-01-01

    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 gp130Janus-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. PMID:21325207

  6. BCR-ABL1 promotes leukemia by converting p27 into a cytoplasmic oncoprotein

    PubMed Central

    Mackenzie, Ryan J.; Besson, Arnaud; Jeng, Sophia; Carey, Alyssa; LaTocha, Dorian H.; Fleischman, Angela G.; Duquesnes, Nicolas; Eide, Christopher A.; Vasudevan, Kavin B.; Loriaux, Marc M.; Firpo, Eduardo; Cortes, Jorge E.; McWeeney, Shannon; OHare, Thomas; Roberts, James M.; Druker, Brian J.; Deininger, Michael W.

    2014-01-01

    Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27CK?) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27T187A) or nuclear retention (p27S10A) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization. PMID:25293778

  7. Dependence on the MUC1-C Oncoprotein in Non-Small Cell Lung Cancer Cells

    PubMed Central

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

    2011-01-01

    Non-small cell lung cancer (NSCLC) cells are often associated with constitutive activation of the phosphatidylinositol 3-kinase (PI3K)->Akt->mTOR pathway. The mucin 1 (MUC1) heterodimeric glycoprotein is aberrantly overexpressed in NSCLC and induces gene signatures that are associated with poor survival of NSCLC patients. The present results demonstrate 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 for activation of the PI3K->Akt pathway and for survival. PMID:21421804

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

    PubMed

    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

    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

  9. MUC1-C oncoprotein promotes STAT3 activation in an autoinductive regulatory loop.

    PubMed

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

    2011-01-01

    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. We found that in breast cancer cells, the MUC1 carboxyl-terminal receptor subunit (MUC1-C) associates 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 loop that may play a role in cancer cell survival. PMID:21325207

  10. Attenuated CagA Oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon*

    PubMed Central

    Suzuki, Masato; Kiga, Kotaro; Kersulyte, Dangeruta; Cok, Jaime; Hooper, Catherine C.; Mimuro, Hitomi; Sanada, Takahito; Suzuki, Shiho; Oyama, Masaaki; Kozuka-Hata, Hiroko; Kamiya, Shigeru; Zou, Quan-Ming; Gilman, Robert H.; Berg, Douglas E.; Sasakawa, Chihiro

    2011-01-01

    Population genetic analyses of bacterial genes whose products interact with host tissues can give new understanding of infection and disease processes. Here we show that strains of the genetically diverse gastric pathogen Helicobacter pylori from Amerindians from the remote Peruvian Amazon contain novel alleles of cagA, a major virulence gene, and reveal distinctive properties of their encoded CagA proteins. CagA is injected into the gastric epithelium where it hijacks pleiotropic signaling pathways, helps Hp exploit its special gastric mucosal niche, and affects the risk that infection will result in overt gastroduodenal diseases including gastric cancer. The Amerindian CagA proteins contain unusual but functional tyrosine phosphorylation motifs and attenuated CRPIA motifs, which affect gastric epithelial proliferation, inflammation, and bacterial pathogenesis. Amerindian CagA proteins induced less production of IL-8 and cancer-associated Mucin 2 than did those of prototype Western or East Asian strains and behaved as dominant negative inhibitors of action of prototype CagA during mixed infection of Mongolian gerbils. We suggest that Amerindian cagA is of relatively low virulence, that this may have been selected in ancestral strains during infection of the people who migrated from Asia into the Americas many thousands of years ago, and that such attenuated CagA proteins could be useful therapeutically. PMID:21757722

  11. Targeting EpsteinBarr virus oncoprotein LMP1-mediated glycolysis sensitizes nasopharyngeal carcinoma to radiation therapy

    PubMed Central

    Xiao, L; Hu, Z-y; Dong, X; Tan, Z; Li, W; Tang, M; Chen, L; Yang, L; Tao, Y; Jiang, Y; Li, J; Yi, B; Li, B; Fan, S; You, S; Deng, X; Hu, F; Feng, L; Bode, A M; Dong, Z; Sun, L-q; Cao, Y

    2014-01-01

    Our goal in this work was to illustrate the Epstein-Barr virus (EBV)-modulated global biochemical profile and provide a novel metabolism-related target to improve the therapeutic regimen of nasopharyngeal carcinoma (NPC). We used a metabolomics approach to investigate EBV-modulated metabolic changes, and found that the exogenous overexpression of the EBV-encoded latent membrane protein 1 (LMP1) significantly increased glycolysis. The deregulation of several glycolytic genes, including hexokinase 2 (HK2), was determined to be responsible for the reprogramming of LMP1-mediated glucose metabolism in NPC cells. The upregulation of HK2 elevated aerobic glycolysis and facilitated proliferation by blocking apoptosis. More importantly, HK2 was positively correlated with LMP1 in NPC biopsies, and high HK2 levels were significantly associated with poor overall survival of NPC patients following radiation therapy. Knockdown of HK2 effectively enhanced the sensitivity of LMP1-overexpressing NPC cells to irradiation. Finally, c-Myc was demonstrated to be required for LMP1-induced upregulation of HK2. The LMP1-mediated attenuation of the PI3-K/Akt-GSK3beta-FBW7 signaling axis resulted in the stabilization of c-Myc. These findings indicate a close relationship between EBV and glycolysis in NPC. Notably, LMP1 is the key regulator of the reprogramming of EBV-mediated glycolysis in NPC cells. Given the importance of EBV-mediated deregulation of glycolysis, anti-glycolytic therapy might represent a worthwhile avenue of exploration in the treatment of EBV-related cancers. PMID:24662831

  12. Expression of the MOZ-TIF2 oncoprotein in mice represses senescence.

    PubMed

    Largeot, Anne; Perez-Campo, Flor Maria; Marinopoulou, Elli; Lie-A-Ling, Michael; Kouskoff, Valerie; Lacaud, Georges

    2016-04-01

    The MOZ-TIF2 translocation, which fuses monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase (HAT) with the nuclear co-activator TIF2, is associated with the development of acute myeloid leukemia. We recently found that in the absence of MOZ HAT activity, p16(INK4a) transcriptional levels are significantly increased, triggering an early entrance into replicative senescence. Because oncogenic fusion proteins must bypass cellular safeguard mechanisms, such as senescence and apoptosis, to induce leukemia, we hypothesized that this repressive activity of MOZ over p16(INK4a) transcription could be preserved, or even reinforced, in MOZ leukemogenic fusion proteins, such as MOZ-TIF2. We describe here that, indeed, MOZ-TIF2 silences expression of the CDKN2A locus (p16(INK4a) and p19(ARF)), inhibits the triggering of senescence and enhances proliferation, providing conditions favorable to the development of leukemia. Furthermore, we describe that abolishing the MOZ HAT activity of the fusion protein leads to a significant increase in expression of the CDKN2A locus and the number of hematopoietic progenitors undergoing senescence. Finally, we report that inhibition of senescence by MOZ-TIF2 is associated with increased apoptosis, suggesting a role for the fusion protein in p53 apoptosis-versus-senescence balance. Our results underscore the importance of the HAT activity of MOZ, preserved in the fusion protein, for repression of the CDKN2A locus transcription and the subsequent block of senescence, a necessary step for the survival of leukemic cells. PMID:26854485

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

    SciTech Connect

    Pang, Ervinna; Delic, Naomi C.; Hong, Angela; Zhang Mei; Rose, Barbara R.; Lyons, J. Guy

    2011-03-01

    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.

  14. Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells

    PubMed Central

    Park, Ji Hyun; Szemes, Marianna; Vieira, Gabriella Cunha; Melegh, Zsombor; Malik, Sally; Heesom, Kate J.; Von Wallwitz-Freitas, Laura; Greenhough, Alexander; Brown, Keith W.; Zheng, Y. George; Catchpoole, Daniel; Deery, Michael J.; Malik, Karim

    2015-01-01

    Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, MannWhitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene. PMID:25475372

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

    PubMed Central

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

    2010-01-01

    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 non-covalently 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 non-covalent interaction between DEK and PAR, which we detail in the present work. 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 non-covalent binding to DEK is dependent on PAR chain length as revealed by an overlay blot technique and PAR EMSA. Intriguingly, DEK promotes the formation of a defined complex with a 54mer PAR (KD=6 × 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 non-covalent interaction does not affect the overall ability of DEK to bind to DNA. Instead the non-covalent interaction interferes with subsequent DNA-dependent multimerization activities of DEK, as seen in South-Western, EMSA, topology and aggregation assays. In particular, non-covalent 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

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

    PubMed

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

    1997-04-01

    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. PMID:9060682

  17. Cleavage of HPV-16 E6/E7 mRNA mediated by modified 10-23 deoxyribozymes.

    PubMed

    Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis M

    2009-09-01

    Deoxyribozymes (DXZs) are small oligodeoxynucleotides capable of mediating phosphodiester bond cleavage of a target RNA in a sequence-specific manner. These molecules are a new generation of artificial catalytic nucleic acids currently used to silence many disease-related genes. The present study describes a DXZ (Dz1023-434) directed against the polycistronic mRNA from the E6 and E7 genes of human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer. Dz1023-434 showed efficient cleavage against a bona fide antisense window at nt 410-445 within HPV-16 E6/E7 mRNA even in low [Mg(2+)] conditions. Using a genetic analysis as guidance, we introduced diverse chemical modifications within Dz1023-434 catalytic core to produce a stable locked nucleic acid (LNA)-modified DXZ (Dz434-LNA) with significant cleavage activity of full E6/E7 transcripts. Cell culture testing of Dz434-LNA produced a sharp decrement of E6/E7 mRNA levels in HPV-16-positive cells resulting in decreased proliferation and considerable cell death in a specific and dose-dependent manner. No significant effects were observed with inactive or scrambled control DXZs nor from using HPV-negative cells, suggesting catalysis-dependent effect and high specificity. The biological effects of Dz434-LNA suggest a potential use for the treatment of cervical cancer. PMID:19732021

  18. The human papillomavirus18 E7 protein inhibits CENP-C binding to α-satellite DNA.

    PubMed

    Yaginuma, Yuji; Yoshimoto, Masafumi; Eguchi, Ayami; Tokuda, Aoi; Takahashi, Shoko

    2015-07-01

    Human papillomavirus (HPV) infection leads to aneuploidy, a numerical chromosomal aberration that is caused by dysregulation of chromosomal segregation. We previously found that the E7 proteins of high-risk HPVs, but not of low-risk HPVs, could bind to centromere protein-C (CENP-C). In this study, we first found that CENP-C could bind centromere α-satellite DNAs using ChIP analysis and HA-tagged CENP-C/nuc transfected 293T cells. We then investigated if HA-CENP-C/nuc binding to α-satellite DNAs was affected by the E7 proteins of high- or low-risk HPVs. We found that transfection of the FLAG tagged HPV18 E7 inhibited the binding of HA-CENP-C/nuc to α-satellite DNAs. This finding was confirmed in HeLa S3 cells transfected with siRNA targeted to HPV18 E7 expression. We therefore speculate that altered function of kinetochores as a result of inhibition of CENP-C and α-satellite DNAs binding may be associated with the chromosomal abnormalities observed in HPV18-positive cancers. PMID:25997930

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

    PubMed

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

    2013-04-01

    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

  20. Chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine generates different anti-tumor effects against tumors expressing the E7 protein of human papillomavirus.

    PubMed

    Khavari, Afshin; Bolhassani, Azam; Alizadeh, Fatemeh; Bathaie, S Zahra; Balaram, Prabha; Agi, Elnaz; Vahabpour, Rouhollah

    2015-02-01

    Saffron and its components have been suggested as promising candidates for cancer prevention. Carotenoids and monoterpene aldehydes are two potent ingredients of saffron. The goal of the current study was to investigate the anti-tumor effect of chemo-immunotherapy using saffron and its ingredients followed by E7-NT (gp96) DNA vaccine against tumors expressing the E7 protein of human papillomavirus. The in vitro cytotoxic and apoptotic effects of aqueous saffron extract and its components were evaluated in malignant TC-1 and non-malignant COS-7 cell lines. Then, multimodality treatments using E7-NT (gp96) DNA vaccine combined with saffron extract and its ingredients as well as single-modality treatments were tested for their efficacy in inhibiting large and bulky tumor growth. Saffron and its components exerted a considerable anti-tumor effect through prevention of cell growth and stimulation of programmed cell death. Furthermore, 100% of mice treated with crocin were tumor-free, in contrast to DNA vaccine alone (~66.7%) and DNA+crocin (~33.3%) indicating the high potency of crocin as a chemotherapeutic agent. Interestingly, the multimodality treatment using DNA vaccine along with picrocrocin augmented the anti-tumor effects of picrocrocin. Thus, the combination of DNA vaccine with saffron extract and crocin at certain concentrations did not potentiate protective and therapeutic effects compared to mono-therapies for the control of TC-1 tumors. PMID:25395243

  1. Regulator role of HPV E7 protein on miR-21 expression in cervical carcinoma cells and its functional implication

    PubMed Central

    Kong, Qingqin; Wang, Wenfeng; Li, Ping

    2015-01-01

    Cervical cancer is the second leading malignant tumor in women. Human papillomavirus 16 (HPV16) is one risk factor for cervical cancer, with its expressed E7 protein can facilitate the transformation of cervical epithelial cells. MicroRNA-21 (miR-21) is one important tumor growth regulatory factor involving in angiogenesis, tumor invasion and metastasis. This study thus aimed to investigate the role of high-risk HPV16 E7 protein in regulating miR-21 expression in cervical carcinoma and its related functions. Hela cells were transfected with pcDNA-HPV16 E7 expressing vectors. The expression level of E7 was determined by Western blotting, while miR-21 level was quantified by real-time PCR. The alternation of tumor cell proliferation is determined by transfecting miR-21 inhibitor into E7-overexpressing Hela cells. Cell apoptosis was studied by caspase-3 assay, while cell invasion was illustrated in Transwell chamber. The overexpression of HPV E7 protein facilitated the expression of miR-21, which potentiated Hela cell proliferation and invasion. The inhibition of miR-21 in E7-overexpressin Hela cells can inhibit both proliferation and invasion, but without significant effects on caspase-3 activity. HPV16 E7 protein can up-regulate host miR-21 expression, thus elevating cervical carcinoma cell growth, proliferation and invasion. Therefore, E7 protein is one critical factor in occurrence and progression of cervical carcinoma. PMID:26884851

  2. SIRT3 inhibits prostate cancer by destabilizing oncoprotein c-MYC through regulation of the PI3K/Akt pathway

    PubMed Central

    Quan, Yizhou; Wang, Naitao; Chen, Qianqian; Xu, Jin; Cheng, Wei; Di, Meijuan; Xia, Weiliang; Gao, Wei-Qiang

    2015-01-01

    SIRT3 is involved in aging-related diseases including cancer, but its role in prostate cancer and detailed regulatory function are not known. We found that SIRT3 was moderately down-regulated in prostate carcinomas. Overexpression of SIRT3 by lentiviral transfection inhibited prostate cancer growth both in vitro and in vivo, whereas knockdown of SIRT3 increased prostate tumor growth. Mechanistically, the tumor suppression effect of SIRT3 was achieved via its inhibition of the PI3K/Akt pathway. Notably, upregulation of SIRT3 suppressed the phosphorylation of Akt, leading to the ubiquitination and degradation of oncoprotein c-MYC; this could be attenuated by constitutive activation of PI3K/Akt signaling. Collectively, our results unveiled SIRT3's tumor suppressive function and the underlying mechanism in prostate cancer, which might provide therapeutic implications for the disease. PMID:26317998

  3. Consideration of Epstein-Barr Virus-Encoded Noncoding RNAs EBER1 and EBER2 as a Functional Backup of Viral Oncoprotein Latent Membrane Protein 1

    PubMed Central

    2016-01-01

    ABSTRACT The Epstein-Barr virus (EBV)-encoded noncoding RNAs EBER1 and EBER2 are highly abundant through all four latency stages of EBV infection (III-II-I-0) and have been associated with an oncogenic phenotype when expressed in cell lines cultured in vitro. In vivo, EBV-infected B cells derived from freshly isolated lymphocytes show that EBER1/2 deletion does not impair viral latency. Based on published quantitative proteomics data from BJAB cells expressing EBER1 and EBER2, we propose that the EBERs, through their activation of AKT in a B-cell-specific manner, are a functionally redundant backup of latent membrane protein 1 (LMP1)an essential oncoprotein in EBV-associated malignancies, with a main role in AKT activation. Our proposed model may explain the lack of effect on viral latency establishment in EBER-minus EBV infection. PMID:26787829

  4. Consideration of Epstein-Barr Virus-Encoded Noncoding RNAs EBER1 and EBER2 as a Functional Backup of Viral Oncoprotein Latent Membrane Protein 1.

    PubMed

    Herbert, Kristina M; Pimienta, Genaro

    2016-01-01

    The Epstein-Barr virus (EBV)-encoded noncoding RNAs EBER1 and EBER2 are highly abundant through all four latency stages of EBV infection (III-II-I-0) and have been associated with an oncogenic phenotype when expressed in cell lines cultured in vitro. In vivo, EBV-infected B cells derived from freshly isolated lymphocytes show that EBER1/2 deletion does not impair viral latency. Based on published quantitative proteomics data from BJAB cells expressing EBER1 and EBER2, we propose that the EBERs, through their activation of AKT in a B-cell-specific manner, are a functionally redundant backup of latent membrane protein 1 (LMP1)-an essential oncoprotein in EBV-associated malignancies, with a main role in AKT activation. Our proposed model may explain the lack of effect on viral latency establishment in EBER-minus EBV infection. PMID:26787829

  5. SET oncoprotein overexpression in B-cell chronic lymphocytic leukemia and non-Hodgkin lymphoma: a predictor of aggressive disease and a new treatment target.

    PubMed

    Christensen, Dale J; Chen, Youwei; Oddo, Jessica; Matta, Karen M; Neil, Jessica; Davis, Evan D; Volkheimer, Alicia D; Lanasa, Mark C; Friedman, Daphne R; Goodman, Barbara K; Gockerman, Jon P; Diehl, Louis F; de Castro, Carlos M; Moore, Joseph O; Vitek, Michael P; Weinberg, J Brice

    2011-10-13

    B-cell chronic lymphocytic leukemia (CLL), an incurable leukemia, is characterized by defective apoptosis. We found that the SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A) tumor suppressor, is overexpressed in primary CLL cells and B-cell non-Hodgkin lymphoma (NHL) cell line cells. In CLL, increased levels of SET correlated significantly with disease severity (shorter time to treatment and overall survival). We developed SET antagonist peptides that bound SET, increased cellular PP2A activity, decreased Mcl-1 expression, and displayed selective cytotoxicity for CLL and NHL cells in vitro. In addition, shRNA for SET was cytotoxic for NHL cells in vitro. The SET antagonist peptide COG449 inhibited growth of NHL tumor xenografts in mice. These data demonstrate that SET is a new treatment target in B-cell malignancies and that SET antagonists represent novel agents for treatment of CLL and NHL. PMID:21844565

  6. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Linn, Anning (La Jolla, CA)

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD 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.

  7. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2002-01-29

    The present invention provides an isolated polynucleotide encoding a c-Jun peptide consisting of about amino acid residues 33 to 79 as set fort in SEQ ID NO: 10 or conservative variations thereof. The invention also provides a method for producing a peptide of SEQ ID NO:1 comprising (a) culturing a host cell containing a polynucleotide encoding a c-Jun peptide consisting of about amino acid residues 33 to 79 as set forth in SEQ ID NO: 10 under conditions which allow expression of the polynucleotide; and (b) obtaining the peptide of SEQ ID NO:1.

  8. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    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.

  9. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    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.

  10. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    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.

  11. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine 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.

  12. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    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.

  13. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine 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.

  14. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    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.

  15. Oncoprotein protein kinase

    DOEpatents

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

    1997-02-25

    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.

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD 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.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD 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.

  18. Proteasomal Degradation of p53 by Human Papillomavirus E6 Oncoprotein Relies on the Structural Integrity of p53 Core Domain

    PubMed Central

    Bernard, Xavier; Robinson, Philip; Nomin, Yves; Masson, Murielle; Charbonnier, Sebastian; Ramirez-Ramos, Juan Ramon; Deryckere, Francois; Trav, Gilles; Orfanoudakis, Georges

    2011-01-01

    The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration. PMID:22046250

  19. An in silico appraisal of azoic and disulphide derivatives for anticancer activity against HPV E6 oncoprotein to medicate cervical cancer.

    PubMed

    Choudhury, Arpita Das; Choudhury, Manabendra Dutta; Chetia, Pankaj; Chowdhury, Abhishek; Talukdar, Anupam Das

    2014-01-01

    Cervical cancer is the second largest form of cancer to infest the leading cause of death in women worldwide. There are many causes of cancer but viruses are the most common among them. Human papillomaviruses (HPVs) are found to be the causative organism in almost 99.7% of the cases. HPV16 is the most frequent HPV type in malignant neoplastic growth in about 60% of cervical carcinoma cases. There is limited success achieved in surgical removal or by immune modulation and more effective therapies are under investigation. Observing the mortality rate we theorize a need for alternative treatment approaches and propose a blueprint of compounds with desirable properties that may lead to the development of drugs to treat HPV-associated neoplasias. E6 oncoprotein of HPV16 has a potential zinc finger domain critical for binding to E6AP, causing p53 degradation and malignancy. Some azoics and disulfides were selected depending on their affinity towards E6 zinc finger and thereby preventing E6-E6AP complex formation. Combinatorial nontoxic derivatives of these azoics and disulfides were docked and validated against the oncoprotein to inhibit E6-E6AP interaction. Among these, two compounds (E)-N-(2-amino-2-oxoethyl)-N-(4-chlorophenyl) diazene-1,2-dicarboxamide and (E)-N-(2- amino-2-methylpropyl)-N-(thiophen-2-yl)diazene-1,2-dicarboxamide showed binding affinity of -23.70, -19.53 and -5.49, -4.65 Kcal/mol respectively in FlexX and Autodock4.2. These compounds are found more effective than those of the approved E6-E6AP binding inhibitors. Pharmacophores of these compounds were generated to confirm it with pharm mapping mechanism. The study may confer the way of design of new mechanism and new compounds to treat cervical cancer. PMID:24164050

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

    SciTech Connect

    Magaldi, Thomas G.; Almstead, Laura L.; Bellone, Stefania; Prevatt, Edward G.; Santin, Alessandro D.; Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028 ; DiMaio, Daniel; Department of Therapeutic Radiology, Yale School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040; Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, P.O. Box 208024; Yale Comprehensive Cancer Center, P.O. Box 208028, New Haven, CT 06520-8028

    2012-01-05

    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.

  1. HPV16-E7 Expression Causes Fluorodeoxyuridine-mediated Radiosensitization in SW620 Human Colon Cancer Cells

    PubMed Central

    Axelson, Michael D; Davis, Mary A; Ethier, Stephen P; Lawrence, Theodore S

    1999-01-01

    Abstract We have reported that HT29 colon cancer cells, which are radiosensitized by fluorodeoxyuridine (FdUrd), exhibit a greater increase in cyclin E-dependent kinase activity and progress further into S phase in the presence of FdUrd than do SW620 colon cancer cells, which are only minimally sensitized by this drug (Cancer Res 56: 3203, 1996). Although these findings suggested that the ability to progress into S phase in the presence of FdUrd permits cells to be radiosensitized, we wished to test this hypothesis by attempting to drive SW620 human colon cells into S phase by transducing them with the HPV16-E7 gene. Two-parameter flow cytometry showed that E7-transduced cells progressed through S phase after radiation and FdUrd treatment more rapidly than SW620 parental cells. We found that E7-transduced SW620 cells were significantly radiosensitized by FdUrd (100 nmol/L, 14 hours) with an enhancement ratio for 2 clones of 1.47±0.03 and 1.51±0.14, compared with 1.24±0.04 in SW620 parental cells. These data strongly support the hypothesis that dysregulation of S-phase progression is an important factor in FdUrd-mediated radiosensitization. PMID:10933053

  2. 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

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

    2012-03-30

    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.

  3. Immunization of protein HPV16 E7 in fusion with mouse HSP70 inhibits the growth of TC-1 cells in tumor bearing mice.

    PubMed

    Li, Yan-Li; Liu, Jie; Liu, Jian-Ning; Zhang, Jing

    2011-08-11

    Human papillomavirus (HPV) 16 is the primary etiologic agent of cervical cancer. Most HPV16 therapeutic vaccines target E7 protein which is consistently expressed in tumor cells. In this study, we cloned mouse autologous heat shock protein 70 (mHSP70) gene from mouse liver cells and then expressed mHSP70 and fused HPV16 E7-mHSP70 (E7 at the N-terminus and mHSP70 at the C-terminus) proteins in E. coli. Then we investigated the inhibition of TC-1 cell growth by using the E7-expressing murine tumor cell line, TC-1, as a model of cervical cancer. In this model, mice were immunized with the fusion protein of E7-mHSP70 without any adjuvant. The results showed that prophylactic immunization of E7-mHSP70 protected mice against challenge with TC-1 cells. In addition, therapeutic immunization with E7-mHSP70 could inhibit TC-1 tumor growth on lungs. Our study demonstrated that immunization with E7-mHSP70 protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells. PMID:21722685

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

    PubMed

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

    2005-09-30

    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. PMID:16051300

  5. O2 and Water Migration Pathways between the Solvent and Heme Pockets of Hemoglobin with Open and Closed Conformations of the Distal HisE7.

    PubMed

    Shadrina, Maria S; Peslherbe, Gilles H; English, Ann M

    2015-09-01

    Hemoglobin transports O2 by binding the gas at its four hemes. Hydrogen bonding between the distal histidine (HisE7) and heme-bound O2 significantly increases the affinity of human hemoglobin (HbA) for this ligand. HisE7 is also proposed to regulate the release of O2 to the solvent via a transient E7 channel. To reveal the O2 escape routes controlled by HisE7 and to evaluate its role in gating heme access, we compare simulations of O2 diffusion from the distal heme pockets of the T and R states of HbA performed with HisE7 in its open (protonated) and closed (neutral) conformations. Irrespective of HisE7's conformation, we observe the same four or five escape routes leading directly from the ?- or ?-distal heme pockets to the solvent. Only 21-53% of O2 escapes occur via these routes, with the remainder escaping through routes that encompass multiple internal cavities in HbA. The conformation of the distal HisE7 controls the escape of O2 from the heme by altering the distal pocket architecture in a pH-dependent manner, not by gating the E7 channel. Removal of the HisE7 side chain in the GlyE7 variant exposes the distal pockets to the solvent, and the percentage of O2 escapes to the solvent directly from the ?- or ?-distal pockets of the mutant increases to 70-88%. In contrast to O2, the dominant water route from the bulk solvent is gated by HisE7 because protonation and opening of this residue dramatically increase the rate of influx of water into the empty distal heme pockets. The occupancy of the distal heme site by a water molecule, which functions as an additional nonprotein barrier to binding of the ligand to the heme, is also controlled by HisE7. Overall, analysis of gas and water diffusion routes in the subunits of HbA and its GlyE7 variant sheds light on the contribution of distal HisE7 in controlling polar and nonpolar ligand movement between the solvent and the hemes. PMID:26226401

  6. Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane

    PubMed Central

    Kim, Hyun-Ju; Ryu, Yang-Hwan; Ahn, Jae-Il; Park, Jeong-Keuk

    2006-01-01

    Purpose To establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM). Methods Primary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively. Results Successful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15±10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV. Conclusions IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells. PMID:16768190

  7. [Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

    PubMed

    Wang, He; Yu, Jiyun; Li, Li

    2013-10-01

    To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations. PMID:24459978

  8. The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the ApcMin/+ mouse small intestine

    PubMed Central

    Patek, Charles E; Arends, Mark J; Rose, Lorraine; Luo, Feijun; Walker, Marion; Devenney, Paul S; Berry, Rachel L; Lawrence, Nicola J; Ridgway, Rachel A; Sansom, Owen J; Hooper, Martin L

    2008-01-01

    Background Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastm?4A/tm?4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastm?4A/tm?4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively. Methods The relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastm?4A/tm?4A mice were generated and the genotypes confirmed by PCR analysis. Survival of stocks was compared by the Mantel-Haenszel test, and tumour number and area compared by Student's t-test in outwardly healthy mice at approximately 106 and 152 days of age. DNA sequencing of codons 12, 13 and 61 was performed to confirm the intestinal tumours did not harbour a K-ras activating mutation. Results The K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology. Conclusion The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations. PMID:18554389

  9. Intron definition and a branch site adenosine at nt 385 control RNA splicing of HPV16 E6*I and E7 expression.

    PubMed

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

    2012-01-01

    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 (91)QYNK(94) to (91)PSFW(94) 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. PMID:23056301

  10. Stabilization of SIRT7 deacetylase by viral oncoprotein HBx leads to inhibition of growth restrictive RPS7 gene and facilitates cellular transformation

    PubMed Central

    Pandey, Vijaya; Kumar, Vijay

    2015-01-01

    Sirtuin-7 (SIRT7) deacetylase exhibits a high selectivity for acetylated H3K18 and has been implicated in the maintenance of malignant phenotype. However, it remains unclear if SIRT7 and H3K18ac play a role in the tumorigenic program driven by oncogenic viruses. We show that ectopically expressed HBx oncoprotein of hepatitis B virus promoted intracellular stability of SIRT7 by salvaging it from ubiquitin-mediated proteasomal degradation. HBx-dependent accumulation of SIRT7 favored H3K18 deacetylation and down-regulated the small ribosomal protein gene, RPS7, involved in cell death and DNA damage response. HBx facilitated the recruitment of SIRT7 to RPS7 promoter thus impeding H3K18ac occupancy and hindering RPS7 transcription. The antagonistic relationship between SIRT7 and RPS7 was also observed in the HBx transgenic mice, where elevated levels of SIRT7 protein were coincident with low levels of H3K18ac and RPS7. Strikingly, inhibition of cellular deubiquitinase activity restored RPS7 gene transcription. Further, depletion of endogenous SIRT7 led to decreased cell viability and transformation. The biological relevance of RPS7 suppression by HBx-SIRT7 axis was evident from ectopic expression of RPS7 which attenuated clonogenicity of cells. Thus, our findings suggest that SIRT7 is a critical regulator of HBx-driven oncogenic program, through its antagonistic impact on growth restrictive ribosomal protein RPS7. PMID:26442981

  11. BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells

    PubMed Central

    Ghaffari, Saghi; Wu, Hong; Gerlach, Melissa; Han, Ying; Lodish, Harvey F.; Daley, George Q.

    1999-01-01

    Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo?/? and EpoR?/? mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210BCR-ABL and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR?/? mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor. PMID:10557295

  12. Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase

    PubMed Central

    Kong, Chen; Samovski, Dmitri; Srikanth, Priya; Wainszelbaum, Marisa J.; Charron, Audra J.; Liu, Jialiu; Lange, Jeffrey J.; Chen, Pin-I; Pan, Zhen-Qiang; Su, Xiong; Stahl, Philip D.

    2012-01-01

    Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8. PMID:23029530

  13. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax

    SciTech Connect

    Mann, Melanie C. Strobel, Sarah Fleckenstein, Bernhard Kress, Andrea K.

    2014-09-15

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. - Highlights: • ELL2, a transcription elongation factor, is upregulated in HTLV-1-positive T-cells. • Tax transactivates the ELL2 promoter. • Tax and ELL2 synergistically activate the HTLV-1 promoter. • Tax and ELL2 interact in vivo.

  14. Stabilization of SIRT7 deacetylase by viral oncoprotein HBx leads to inhibition of growth restrictive RPS7 gene and facilitates cellular transformation.

    PubMed

    Pandey, Vijaya; Kumar, Vijay

    2015-01-01

    Sirtuin-7 (SIRT7) deacetylase exhibits a high selectivity for acetylated H3K18 and has been implicated in the maintenance of malignant phenotype. However, it remains unclear if SIRT7 and H3K18ac play a role in the tumorigenic program driven by oncogenic viruses. We show that ectopically expressed HBx oncoprotein of hepatitis B virus promoted intracellular stability of SIRT7 by salvaging it from ubiquitin-mediated proteasomal degradation. HBx-dependent accumulation of SIRT7 favored H3K18 deacetylation and down-regulated the small ribosomal protein gene, RPS7, involved in cell death and DNA damage response. HBx facilitated the recruitment of SIRT7 to RPS7 promoter thus impeding H3K18ac occupancy and hindering RPS7 transcription. The antagonistic relationship between SIRT7 and RPS7 was also observed in the HBx transgenic mice, where elevated levels of SIRT7 protein were coincident with low levels of H3K18ac and RPS7. Strikingly, inhibition of cellular deubiquitinase activity restored RPS7 gene transcription. Further, depletion of endogenous SIRT7 led to decreased cell viability and transformation. The biological relevance of RPS7 suppression by HBx-SIRT7 axis was evident from ectopic expression of RPS7 which attenuated clonogenicity of cells. Thus, our findings suggest that SIRT7 is a critical regulator of HBx-driven oncogenic program, through its antagonistic impact on growth restrictive ribosomal protein RPS7. PMID:26442981

  15. A non-pathogenic live vector as an efficient delivery system in vaccine design for the prevention of HPV16 E7-overexpressing cancers.

    PubMed

    Hosseinzadeh, Sahar; Bolhassani, Azam; Rafati, Sima; Taheri, Tahereh; Zahedifard, Farnaz; Daemi, Amin; Taslimi, Yasaman; Hashemi, Mehrdad; Memarnejadian, Arash

    2013-01-01

    The attenuated or non-pathogenic live vectors have been evolved specifically to deliver DNA into cells as efficient delivery tools in gene therapy. Recently, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention. In current study, we used Leishmania expression system (LEXSY) for stable expression of HPV16 E7 linked to different mini-chaperones [N-/C-terminal of gp96] and compared their immunogenicity and protective effects in C57BL/6 mice against TC-1 challenge. TC-1 murine model is primary C57BL/6 mice lung epithelial cells co-transformed with HPV16 E6, HPV16 E7 and ras oncogenes. Our results showed that subcutaneous administration of mice with both the recombinant L.tar-E7-NT (gp96) and L.tar-E7-CT (gp96) led to enhance the levels of IFN-? and also IgG2a before and after challenge with TC-1. Furthermore, L.tar-E7-CT (gp96) live vaccine indicated significant protective effects as compared to control groups as well as group vaccinated with L.tar-E7. Indeed, the recombinant live vector is capable of eliciting effective humoral and cellular immune responses in mice, but however, further studies are required to increase their efficacy. PMID:23745741

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

    SciTech Connect

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

    2010-01-01

    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.

  17. Genomic Instability and Cancer: Lessons Learned from Human Papillomaviruses

    PubMed Central

    Korzeniewski, Nina; Spardy, Nicole; Duensing, Anette; Duensing, Stefan

    2010-01-01

    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. PMID:21075512

  18. Cloning of a horn fly cDNA, HialphaE7, encoding an esterase whose transcript concentration is elevated in diazinon-resistant flies.

    PubMed

    Guerrero, F D

    2000-11-01

    Reverse transcriptase-polymerase chain reaction (PCR) was used to clone two esterase cDNAs from a diazinon-resistant field population of horn flies that expresses qualitative and quantitative differences in esterases compared with a susceptible population. The open reading frame from one of the esterase cDNAs, HialphaE7, exhibits substantial amino-acid identity to an esterase associated with diazinon resistance in Lucilia cuprina. RNA Northern blots showed that HialphaE7 mRNA was more abundant in the diazinon-resistant population than the susceptible population. DNA copy number analysis did not reveal major differences in HialphaE7 gene copy number between the two populations. The full-length cDNA to HialphaE7 was cloned and sequenced, and found to contain all of the highly conserved sequence elements associated with carboxyl/cholinesterases. The HialphaE7 homologs in diazinon-resistant strains of L. cuprina and Musca domestica have been shown to possess an amino-acid substitution conferring diazinon hydrolytic activity to the esterase enzyme. This amino-acid substitution was not found in diazinon-resistant horn flies examined by allele-specific PCR. Individual flies from the resistant field population were phenotyped as diazinon-resistant or diazinon-susceptible by topical diazinon application bioassays and total RNA isolated and hybridized to HialphaE7 probe in ribonuclease protection assays. HialphaE7 transcript was expressed at a five-fold higher level in resistant female individual flies than in susceptible female individuals. PMID:10989298

  19. Rearrangement of the distal pocket accompanying E7 His yields Gln substitution in elephant carbonmonoxy- and oxymyoglobin: sup 1 H NMR identification of a new aromatic residue in the heme pocket

    SciTech Connect

    Yu, L.P.; La Mar, G.N. ); Mizukami, H. )

    1990-03-13

    Two-dimensional {sup 1}H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO{sub 2}). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO{sub 2} resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The C{zeta}H proton of the Phe CD4 was found to move toward the iron of the heme by {approximately}4 {angstrom} relative to the position in sperm whale MbCO, requiring minimally a 3-{angstrom} movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His {yields} Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties.

  20. MUC1-C oncoprotein activates the ZEB1/miR-200c regulatory loop and epithelialmesenchymal transition

    PubMed Central

    Rajabi, H; Alam, M; Takahashi, H; Kharbanda, A; Guha, M; Ahmad, R; Kufe, D

    2013-01-01

    The epithelialmesenchymal 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 epithelial-mesenchymal transition (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

  1. MUC1-C oncoprotein activates the ZEB1/miR-200c regulatory loop and epithelial-mesenchymal transition.

    PubMed

    Rajabi, H; Alam, M; Takahashi, H; Kharbanda, A; Guha, M; Ahmad, R; Kufe, D

    2014-03-27

    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

  2. Bovine Papillomavirus Type 2 (BPV-2) E5 Oncoprotein Binds to the Subunit D of the V1-ATPase Proton Pump in Naturally Occurring Urothelial Tumors of the Urinary Bladder of Cattle

    PubMed Central

    Roperto, Sante; Russo, Valeria; Borzacchiello, Giuseppe; Urraro, Chiara; Luc, Roberta; Esposito, Iolanda; Riccardi, Marita Georgia; Raso, Cinzia; Gaspari, Marco; Ceccarelli, Dora Maria; Galasso, Rocco; Roperto, Franco

    2014-01-01

    Background Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. Methods and Findings In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor ? receptor. PDGF?R immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as V1-ATPase subunit D, a component of the central stalk of the V1-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. Conclusion For the first time, a tri-component complex composed of E5/PDGF?R/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V0-ATPase sector. We suggest that the E5/PDGF?R/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses. PMID:24586417

  3. De novo synthesis of (Z)- and (E)-7-hexadecenylitaconic acids by a selective lignin-degrading fungus, Ceriporiopsis subvermispora.

    PubMed

    Nishimura, Hiroshi; Tsuda, Saeko; Shimizu, Hitoe; Ohashi, Yasunori; Watanabe, Takahito; Honda, Yoichi; Watanabe, Takashi

    2008-10-01

    Ceriporic acids are a class of alk(en)ylitaconic acids produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora. Their structural units have similarity with biologically important lichen acids, such as chaetomellic and protolichesterinic acids. The unique function of alkylitaconic acid is the redox silencing of the Fenton reaction system by inhibiting reduction of Fe(3+). As estimated by the catalytic function of Delta9-desaturases, 7-hexadecenyl derivatives bearing a trans configuration have not been reported in the family of alk(en)ylitaconic acids, i.e. the structurally similar lichen acids-alk(en)ylcitraconic and paraconic acids. In this paper, we discuss the isolation of an itaconic acid derivative with an (E)-7-hexadecenyl chain from cultures of C. subvermispora. To identify the natural metabolite, (E)- and (Z)-7-hexadecenylitaconic acids were chemically synthesised. The isolated metabolite was identical to the synthetic (E)-hexadecenylitaconic acid and was designated as ceriporic acid D. Administration of (13)C-[U]-glucose demonstrated that ceriporic acid C and trans-7-hexadecenylitaconic acid (ceriporic acid D) were biosynthesised de novo by C. subvermispora. PMID:18835612

  4. Tattoo Delivery of a Semliki Forest Virus-Based Vaccine Encoding Human Papillomavirus E6 and E7

    PubMed Central

    van de Wall, Stephanie; Walczak, Mateusz; van Rooij, Nienke; Hoogeboom, Baukje-Nynke; Meijerhof, Tjarko; Nijman, Hans W.; Daemen, Toos

    2015-01-01

    The skin is an attractive organ for immunization because of the presence of antigen-presenting cells. Intradermal delivery via tattooing has demonstrated superior vaccine immunogenicity of DNA vaccines in comparison to conventional delivery methods. In this study, we explored the efficacy of tattoo injection of a tumor vaccine based on recombinant Semliki Forest virus replicon particles (rSFV) targeting human papillomavirus (HPV). Tattoo injection of rSFV particles resulted in antigen expression in both the skin and draining lymph nodes. In comparison with intramuscular injection, the overall antigen expression determined at the site of administration and draining lymph nodes was 10-fold lower upon tattoo injection. Delivery of SFV particles encoding the E6 and E7 antigens of human papillomavirus type 16 (SFVeE6,7) via tattooing resulted in HPV-specific cytotoxic T cells and in vivo therapeutic antitumor response. Strikingly, despite the observed lower overall transgene expression, SFVeE6,7 delivered via tattoo injection resulted in higher or equal levels of immune responses as compared to intramuscular injection. The intrinsic immunogenic potential of tattooing provides a benefit for immunotherapy based on an alphavirus. PMID:26343186

  5. The 5' flanking region of the pS2 gene contains a complex enhancer region responsive to oestrogens, epidermal growth factor, a tumour promoter (TPA), the c-Ha-ras oncoprotein and the c-jun protein.

    PubMed Central

    Nunez, A M; Berry, M; Imler, J L; Chambon, P

    1989-01-01

    Expression of the pS2 gene which is transcriptionally controlled by oestrogens in the breast cancer cell line MCF-7 is oestrogen independent in stomach mucosa. We show here that the level of MCF-7 cell pS2 mRNA can also be increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). We further demonstrate, using transient transfection assays, that the -428 to -332 5' flanking sequence of the pS2 gene contains DNA enhancer elements responsive to oestrogens, TPA, EGF, the c-Ha-ras oncoprotein and the c-jun protein. Images PMID:2498085

  6. The Oncogenic MicroRNA OncomiR-21 Overexpressed during Marek's Disease Lymphomagenesis Is Transactivated by the Viral Oncoprotein Meq

    PubMed Central

    Stik, Grégoire; Dambrine, Ginette; Pfeffer, Sébastien

    2013-01-01

    Gallid herpesvirus 2 (GaHV-2) is an oncogenic herpesvirus that causes T lymphoma in chicken. GaHV-2 encodes a basic leucine zipper (bZIP) protein of the AP-1 family, Meq. Upon formation of homo- or heterodimers with c-Jun, Meq may modulate the expression of viral and cellular genes involved in lymphomagenesis. GaHV-2 also encodes viral microRNAs (miRNAs) involved in latency and apoptosis escape. However, little is known about cellular miRNA deregulation during the development of GaHV-2-associated lymphoma. We determined the cellular miRNA expression profiles of chickens infected with a very virulent strain (RB-1B) or a vaccine strain (CVI988) or noninfected. Among the most deregulated cellular miRNAs, we focused our efforts on gga-miR-21, which is upregulated during GaHV-2 infection. We mapped the gga-miR-21 promoter to the 10th intron of the TMEM49 gene and found it to be driven by AP-1- and Ets-responsive elements. We show here that the viral oncoprotein Meq binds to this promoter, thereby transactivating gga-miR-21 expression. We confirmed that this miRNA targets chicken programmed death cell 4 (PDCD4) and promotes tumor cell growth and apoptosis escape. Finally, gga-miR-21 was overexpressed only during infection with a very virulent strain (RB-1B) and not during infection with a nononcogenic strain (CVI988), providing further evidence for its role in GaHV-2 lymphomagenesis. Our data therefore suggest an additional role for Meq in GaHV-2-mediated lymphomagenesis through the induction of miR-21 expression. PMID:23055556

  7. Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

    PubMed Central

    Chiu, K Y; Loke, S L; Ho, F C

    1994-01-01

    AIMS--To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS--Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS--In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS--These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied. Images PMID:7907612

  8. Involvement of the c-Ski oncoprotein in cell cycle arrest and transformation during nurse cell formation after Trichinella spiralis infection.

    PubMed

    Wu, Z; Nagano, I; Boonmars, T; Takahashi, Y

    2006-09-01

    The role of c-Ski, an oncoprotein encoded by the oncogene, c-ski, in Trichinella spiralis-infected muscle tissues during nurse cell formation, was investigated by following the expression kinetics and distribution of c-Ski (both protein and mRNA) in the infected muscle cell, as well as the expression kinetics of the transforming growth factor beta (TGF-beta) signaling pathway factor genes (TGF-beta, Smad2 and Smad4) which cooperate with c-Ski. Immunohistochemical analysis using an anti-c-Ski antibody indicated that in the early stages of infection (13 and 18 days post-infection (p.i.)) the increased expression of the c-Ski protein was limited to the eosinophilic cytoplasm and not the enlarged nuclei or basophilic cytoplasm. At a later stage of infection (23 and 28 days p.i.) the c-Ski protein was limited to the enlarged nuclei in the basophilic cytoplasm, rather than the eosinophilic cytoplasm. At 48 days p.i., the c-Ski protein was barely detectable. Real-time PCR analysis showed that expression of the c-ski gene increased from 13 days p.i., reached a peak at 23-28 days p.i. and then decreased to a low level by 48 days p.i. Expression kinetics for the TGF-beta signaling pathway factor genes (TGF-beta, Smad2 and Smad4) were similar to that of c-ski. These findings provide evidence that the c-Ski protein is involved in nurse cell formation through the TGF-beta signaling pathway process in the host cell nucleus. PMID:16890942

  9. c-erbB-2 oncoprotein detected by automated quantitative immunocytochemistry in breast carcinomas correlates with patients' overall and disease-free survival.

    PubMed Central

    Charpin, C.; Garcia, S.; Bouvier, C.; Martini, F.; Lavaut, M. N.; Allasia, C.; Bonnier, P.; Andrac, L.

    1997-01-01

    The prognostic significance of c-erbB-2 oncoprotein overexpression detected in tumours by immunocytochemical assays (ICAs) was investigated in 148 breast carcinomas. ICAs were performed under optimal technical conditions with frozen tissue sections and included automated immunoperoxidase technique and computer-assisted analysis (densitometry) of digitized coloured microscopic images. Results of quantitative ICAs (expressed in percentages of c-erbB-2-positive surfaces and mean optical densities) were correlated with the patients' follow-up in axillary lymph node-positive (N+) and node-negative (N-) subgroups of patients. Patients' follow-up ranged from 9 months (for the first death) to 101 months (for the 121 alive patients) with a 62.5 months mean overall follow-up. It was shown that marked c-erbB-2 immunocytochemical expression in tumours (cut-off point 35%) significantly correlated with the patients' poor overall survival in N+ and in N- patients (Kaplan-Meier, log-rank test, P = 0.045 and P = 0.015). Also, marked c-erbB-2 immunohistochemical expression correlates with short disease-free (P = 0.005), recurrence-free (P = 0.048) and metastasis-free survival (P = 0.05) (Kaplan-Meier, log-rank test) in N+, but not in N- subgroups. It is concluded that in optimal conditions (automated and quantitative ICAs on frozen sections) c-erbB immunohistochemical expression is a significant prognostic indicator in terms of overall and disease-free survival. The c-erbB-2 protein prognostic significance is independent of node status in terms of overall survival, but not of disease-free survival. Images Figure 1 PMID:9184184

  10. Dependence of intracellular and exosomal microRNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.

    PubMed

    Honegger, Anja; Schilling, Daniela; Bastian, Sandra; Sponagel, Jasmin; Kuryshev, Vladimir; Sltmann, Holger; Scheffner, Martin; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2015-03-01

    Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells. PMID:25760330

  11. Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

    PubMed Central

    Honegger, Anja; Schilling, Daniela; Bastian, Sandra; Sponagel, Jasmin; Kuryshev, Vladimir; Sltmann, Holger; Scheffner, Martin; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2015-01-01

    Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells. PMID:25760330

  12. Distribution of human papilloma virus type 16 E6/E7 gene mutation in cervical precancer or cancer: A case control study in Guizhou Province, China.

    PubMed

    Yang, Yingjie; Ren, Jie; Zhang, Qizhu

    2016-02-01

    HPV-16 varies geographically and is correlated with cervical cancer genesis and progression. This study aimed to determine the distribution of HPV-16 E6/E7 genetic variation in patients with invasive cervical cancer or precancer in Guizhou Province, China. A case-control study was designed, and the distribution of HPV-16 E6/E7 genetic variation was compared among women with cervical cancer, precancer, and sexually active without cervical lesion. HPV infection was detected through flow-through hybridization and gene chip techniques to determine the prevalence of HPV 16 E6/E7 genetic variation. Among 90 specimens (30 cervical cancer, 30 precancer, 30 controls), 81 were subjected to HPV-16 E6/E7 gene sequencing. The rates of DNA sequence mutation and amino acid mutation were 76.5% (62/81) and 66.7% (54/81), respectively. Both E6 and E7 genes showed higher mutation rate than their prototypes. The prevalence of E6/E7 mutation significantly differed between the cervical cancer and the controls (P?E7-M28V (A82G)/L94P (T281C) sites of the amino acid sequence. The most common genetic variation was D32E/M28V/L94P, which accounted for 35.8% of the cases (29/81). D32E/M28V/L94P mutation was higher in the cervical cancer and precancer compared with the prototype. HPV-16 E6/E7 genetic variations, such as D32E/M28V/L94P, are more prevalent in cervical cancer or precancer than those in the controls. The possible correlation between genetic variation and cancerigenesis may be used to design an HPV vaccine for cervical carcinoma. J. Med. Virol. 88:345-350, 2016. 2015 Wiley Periodicals, Inc. PMID:26192265

  13. Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

    SciTech Connect

    Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi; Laimins, Laimonis

    2014-03-15

    Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction of A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.

  14. The Hippo/YAP pathway interacts with EGFR signaling and HPV oncoproteins to regulate cervical cancer progression

    PubMed Central

    He, Chunbo; Mao, Dagan; Hua, Guohua; Lv, Xiangmin; Chen, Xingcheng; Angeletti, Peter C; Dong, Jixin; Remmenga, Steven W; Rodabaugh, Kerry J; Zhou, Jin; Lambert, Paul F; Yang, Peixin; Davis, John S; Wang, Cheng

    2015-01-01

    The Hippo signaling pathway controls organ size and tumorigenesis through a kinase cascade that inactivates Yes-associated protein (YAP). Here, we show that YAP plays a central role in controlling the progression of cervical cancer. Our results suggest that YAP expression is associated with a poor prognosis for cervical cancer. TGF-α and amphiregulin (AREG), via EGFR, inhibit the Hippo signaling pathway and activate YAP to induce cervical cancer cell proliferation and migration. Activated YAP allows for up-regulation of TGF-α, AREG, and EGFR, forming a positive signaling loop to drive cervical cancer cell proliferation. HPV E6 protein, a major etiological molecule of cervical cancer, maintains high YAP protein levels in cervical cancer cells by preventing proteasome-dependent YAP degradation to drive cervical cancer cell proliferation. Results from human cervical cancer genomic databases and an accepted transgenic mouse model strongly support the clinical relevance of the discovered feed-forward signaling loop. Our study indicates that combined targeting of the Hippo and the ERBB signaling pathways represents a novel therapeutic strategy for prevention and treatment of cervical cancer. PMID:26417066

  15. Influence of chromosomal integration on glucocorticoid-regulated transcription of growth-stimulating papillomavirus genes E6 and E7 in cervical carcinoma cells

    SciTech Connect

    Von Knebel Doeberitz, M.; Bauknecht, T.; Bartsch, D.; Zur Hausen, H. )

    1991-02-15

    In most cervical carcinoma cells the E6 and E7 genes of specific human papillomaviruses are transcribed from viral sequences integrated into host cell chromosomes. Glucocorticoids activate the promoter elements of various human papillomaviruses in transient-expression assays. The authors have analyzed the effect of dexamethasone on the transcription rate of human papillomaviruses 18 E6 and E7 genes integrated at different chromosomal sites in four cervical cancer cell lines. Dexamethasone led to an increase in the transcription rate of the integrated E6-E7 sequences in C4-1 and C4-2 cells but led to a decrease in SW 756 cells and did not affect the transcription rate in HeLa cells. It thus appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to steroid hormones. The growth rate of all dexamethasone-treated cell lines correlated consistently with the expression of the papillomavirus E6 and E7 genes, supporting their role in the maintenance of the proliferative phenotype of cervical carcinoma cells. Since human papillomaviruses are integrated into the host cell genome at variable, presumably randomly selected chromosomal loci, regulatory mechanisms that influence viral gene expression, and hence cell growth, may differ among cancers of independent clonal origin.

  16. 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)

    Fernndez Nez, J.; Garca Fuertes, W.; Perelomov, A. M.

    2008-02-01

    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.

  17. The E7 protein of human papillomavirus (HPV) type 16 expressed by recombinant vaccinia virus can be used for detection of antibodies in sera from cervical cancer patients.

    PubMed

    Nindl, I; Gissmann, L; Fisher, S G; Bribiesca, L B; Berumen, J; Mller, M

    1996-10-01

    Sera from 128 Mexican cervical cancer patients (age 30-80; mean 53.6) and from 47 healthy women (age 25-69; mean 49.2) were investigated using a newly developed assay for the detection of serum antibodies to the human papillomavirus (HPV) type 16 early protein E7. This test (CIPA), based upon immunoprecipitation followed by Western blot analysis, uses the complete E7 protein expressed in HeLa cells infected with recombinant vaccinia virus. To determine the sensitivity and specificity of this assay, these results were compared with previous results of the same sera tested by enzyme-linked immunosorbent assay (ELISA; using synthetic peptides derived from HPV 16 E7) and radio-immunoprecipitation (RIPA) using in vitro translated HPV 16 E7 protein. CIPA (45% positives) demonstrated a significant increase in detection rate compared to the peptide-ELISA (30% positives; P = 0.014, chi2-test) and only a slight increase compared to RIPA (38% positives; P = 0.204, chi2-test). Based on the testing of sera from patients with HPV 16 DNA positive tumors the specificity and sensitivity of the CIPA were 0.98 and 0.59, respectively. PMID:8910651

  18. EVI1 oncoprotein interacts with a large and complex network of proteins and integrates signals through protein phosphorylation.

    PubMed

    Bard-Chapeau, Emilie A; Gunaratne, Jayantha; Kumar, Pankaj; Chua, Belinda Q; Muller, Julius; Bard, Frederic A; Blackstock, Walter; Copeland, Neal G; Jenkins, Nancy A

    2013-07-30

    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-β, 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-1α. 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. PMID:23858473

  19. Ponatinib inhibits polyclonal drug-resistant KIT oncoproteins and shows therapeutic potential in heavily pretreated gastrointestinal stromal tumor (GIST) patients

    PubMed Central

    Anjum, Rana; Vodala, Sadanand; Schrock, Alexa; Zhou, Tianjun; Serrano, Cesar; Eilers, Grant; Zhu, Meijun; Ketzer, Julia; Wardwell, Scott; Ning, Yaoyu; Song, Youngchul; Kohlmann, Anna; Wang, Frank; Clackson, Tim; Heinrich, Michael C.; Fletcher, Jonathan A.; Bauer, Sebastian; Rivera, Victor M.

    2014-01-01

    Purpose KIT is the major oncogenic driver of gastrointestinal stromal tumors (GISTs). Imatinib, sunitinib and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here we explored the KIT inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in GIST patients. Experimental Design The cellular and in vivo activities of ponatinib, imatinib, sunitinib and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT co-structure was also determined. The clinical activity of ponatinib was examined in three GIST patients previously treated with all 3 FDA-approved agents. Results In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nM ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nM. This inhibitory profile could be rationalized based on structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three GIST patients. Conclusion Ponatinib possesses potent activity against most major clinically-relevant KIT mutants, and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in GIST patients. PMID:25239608

  20. Multiple aromatic side chains within a disordered structure are critical for transcription and transforming activity of EWS family oncoproteins.

    PubMed

    Ng, King Pan; Potikyan, Gary; Savene, Rupert O V; Denny, Christopher T; Uversky, Vladimir N; Lee, Kevin A W

    2007-01-01

    Chromosomal translocations involving the N-terminal approximately 250 residues of the Ewings sarcoma (EWS) oncogene produce a group of EWS fusion proteins (EFPs) that cause several distinct human cancers. EFPs are potent transcriptional activators and interact with other proteins required for mRNA biogenesis, indicating that EFPs induce tumorigenesis by perturbing gene expression. Although EFPs were discovered more than a decade ago, molecular analysis has been greatly hindered by the repetitive EWS activation domain (EAD) structure, containing multiple degenerate hexapeptide repeats (consensus SYGQQS) with a conserved tyrosine residue. By exploiting total gene synthesis, we have been able to systematically mutagenize the EAD and determine the effect on transcriptional activation by EWS/ATF1 and cellular transformation by EWS/Fli1. In both assays, we find the following requirements for EAD function. First, multiple tyrosine residues are essential. Second, phenylalanine can effectively substitute for tyrosine, showing that an aromatic ring can confer EAD function in the absence of tyrosine phosphorylation. Third, there is little requirement for specific peptide sequences and, thus, overall sequence composition (and not the degenerate hexapeptide repeat) confers EAD activity. Consistent with the above findings, we also report that the EAD is intrinsically disordered. However, a sensitive computational predictor of natural protein disorder (PONDR VL3) identifies potential molecular recognition features that are tyrosine-dependent and that correlate well with EAD function. In summary we have uncovered several molecular features of the EAD that will impact future studies of the broader EFP family and molecular recognition by complex intrinsically disordered proteins. PMID:17202261

  1. Influence of chromosomal integration on glucocorticoid-regulated transcription of growth-stimulating papillomavirus genes E6 and E7 in cervical carcinoma cells.

    PubMed Central

    von Knebel Doeberitz, M; Bauknecht, T; Bartsch, D; zur Hausen, H

    1991-01-01

    In most cervical carcinoma cells the E6 and E7 genes of specific human papillomaviruses are transcribed from viral sequences integrated into host cell chromosomes. Glucocorticoids activate the promoter elements of various human papillomaviruses in transient-expression assays. We have analyzed the effect of dexamethasone on the transcription rate of human papillomavirus 18 E6 and E7 genes integrated at different chromosomal sites in four cervical cancer cell lines. Dexamethasone led to an increase in the transcription rate of the integrated E6-E7 sequences in C4-1 and C4-2 cells but led to a decrease in SW 756 cells and did not affect the transcription rate in HeLa cells. However, when the viral promoter elements derived from HeLa or SW 756 cells, in which dexamethasone does not activate transcription of the integrated E6-E7 sequences, were tested in transient-expression assays within the same cell lines, dexamethasone consistently activated the viral promoter. It thus appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to steroid hormones. The growth rate of all dexamethasone-treated cell lines correlated consistently with the expression of the papillomavirus E6 and E7 genes, supporting their role in the maintenance of the proliferative phenotype of cervical carcinoma cells. Since human papillomaviruses are integrated into the host cell genome at variable, presumably randomly selected chromosomal loci, regulatory mechanisms that influence viral gene expression, and hence cell growth, may differ among cancers of independent clonal origin. Images PMID:1847520

  2. Honeybee venom possesses anticancer and antiviral effects by differential inhibition of HPV E6 andE7 expression on cervical cancer cell line.

    PubMed

    Kim, Yong-Wan; Chaturvedi, Pankaj Kumar; Chun, Sung Nam; Lee, Yang Gu; Ahn, Woong Shick

    2015-04-01

    Bee venom (BV) therapy is a type of alternative medical treatment used to treat various diseases in oriental medicine. The mechanisms underlying the effects of BV remain poorly understood. In the present study, we evaluated the antiviral effect of BV on cervical carcinoma cell lines (CaSki, HeLa, C33A and TC-1). BV treatments resulted in a more significant suppression of cell growth in HPV16-infected cells (CaSki) and a lesser suppression in HPV18-infected cells (HeLa). However, less suppression was observed in HPV-negative C33A cells. In 10g/ml BV-treated CaSki cells, the mRNA expression and protein levels of HPV16 E6 and E7 were significantly decreased by BV, while HPV18 E6 and E7 mRNA expression levels were not significantly altered by 10g/ml BV-treated HeLa cells. The antitumor effects of BV were in accordance with invitro data, in restricting tumor growth invivo and were much more effective on the suppression of tumor growth. Furthermore, the mRNA and protein expression levels of HPV16 E6 and E7 were decreased by BV in TC-1 tumors. These findings demonstrated the antiviral effects of BV in HPV-infected cervical cancer cells and the anticancer effects of BV in HPV16 E6/E7-expressed TC-1 tumors. Collectively, BV plays a differential role in suppressing HPV16-infected cells (CaSki cells) and HPV18-infected cells (HeLa cells) by the downregulation of E6/E7 protein of HPV16/18. PMID:25633640

  3. HPV E6/E7 mRNA versus HPV DNA biomarker in cervical cancer screening of a group of Macedonian women.

    PubMed

    Duvlis, Sotirija; Popovska-Jankovic, Katerina; Arsova, Zorica Sarafinovska; Memeti, Shaban; Popeska, Zaneta; Plaseska-Karanfilska, Dijana

    2015-09-01

    High risk types of human papillomaviruses E6/E7 oncogenes and their association with tumor suppressor genes products are the key factors of cervical carcinogenesis. This study proposed them as specific markers for cervical dysplasia screening. The aim of the study is to compare the clinical and prognostic significance of HPV E6/E7 mRNA as an early biomarker versus HPV DNA detection and cytology in triage of woman for cervical cancer. The study group consists of 413 women: 258 NILM, 26 ASC-US, 81 LSIL, 41 HSIL, and 7 unsatisfactory cytology. HPV4AACE screening, real-time multiplex PCR and MY09/11 consensus PCR primers methods were used for the HPV DNA detection. The real-time multiplex nucleic acid sequence-based assay (NucliSENS EasyQ HPV assay) was used for HPV E6/E7 mRNA detection of the five most common high risk HPV types in cervical cancer (16, 18, 31, 33, and 45). The results show that HPV E6/E7 mRNA testing had a higher specificity 50% (95% CI 32-67) and positive predictive value (PPV) 62% (95% CI 46-76) for CIN2+ compared to HPV DNA testing that had specificity of 18% (95% CI 7-37) and PPV 52% (95% CI 39-76) respectively. The higher specificity and PPV of HPV E6/E7 mRNA testing are valuable in predicting insignificant HPV DNA infection among cases with borderline cytological finding. It can help in avoiding aggressive procedures (biopsies and over-referral of transient HPV infections) as well as lowering patient's anxiety and follow up period. PMID:25880030

  4. Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support

    PubMed Central

    Giri, Shibashish; Bader, Augustinus

    2014-01-01

    Background Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Methods Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. Results After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 57 days. The remaining transfected hepatocytes persisted for 24 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. Conclusion We established a conditional human fetal hepatocytes cell line with mesenchymal characteristics. Thus immortalization of human fetal hepatocytes cell line by telomerase biology offers a great challenge to examine basic biological mechanisms which are directly related to human and best cell source having unlimited population doubling for bioartificial support without any risk of replicative senescence and pathogenic risks. PMID:25755560

  5. Human papillomavirus type 18 DNA and E6-E7 mRNA are detected in squamous cell carcinoma and adenocarcinoma of the lung.

    PubMed Central

    Kinoshita, I.; Dosaka-Akita, H.; Shindoh, M.; Fujino, M.; Akie, K.; Kato, M.; Fujinaga, K.; Kawakami, Y.

    1995-01-01

    To provide an accurate evaluation of the association of human papillomavirus (HPV) with lung cancer, 36 cases of lung cancer were analysed for HPV DNAs by polymerase chain reaction (PCR) with dot-blot and Southern blot analyses, and for the transcripts from the E6-E7 transforming region by in situ hybridisation (ISH). HPV-18 DNA was detected in three (8%) of 36 specimens; histologically, in one (10%) of 10 squamous cell carcinomas and two (9%) of 22 adenocarcinomas. Neither HPV-16 nor -33 DNA was detected in any cases examined. Expression of E6-E7 mRNA was confirmed in the cases which contained, HPV-18 DNA. HPV-18 may play an important role in the development and progression of cancer in some cases of both squamous cell carcinoma and adenocarcinoma of the lung. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7841051

  6. APOBEC3A possesses anticancer and antiviral effects by differential inhibition of HPV E6 and E7 expression on cervical cancer

    PubMed Central

    Chen, Shan; Li, Xiao; Qin, Junpu; Chen, Yuan; Liu, Longyang; Zhang, Dongqing; Wang, Minyi; Wang, Maocai; Zhang, Dikai

    2015-01-01

    Cervical cancer is the second most common cancer among women worldwide and is the leading cause of deaths in developing countries. Persistent infections with a subset of HPVs, called high-risk HPVs, including HPV16 and HPV18, are the primary cause of cervical cancer. The apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of proteins is a group of cellular enzymes that catalyze the deamination of cytidine (C) to uracil (U) in single-stranded DNA/RNA, and functions as antiviral factors in the innate immune system of the host. Recent studies have shown that APOBEC3A could restrict certain DNA viruses, including HPVs. In this study, we confirmed that the expression of APOBEC3A was decreased in cervical cancer tissues. Furthermore, APOBEC3A inhibited the cervical cells proliferation, migration as well as invasion, and promoted apoptosis depend on cytidine deaminase. In addition, APOBEC3A decreased HPV16-E6, HPV16-E7 and HPV18-E6 depend on cytidine deaminase, but no effect on HPV18-E7. Therefore, we believe that, in cervical cancer cells, the expression of APOBEC3A possesses anticancer and antiviral effects by differential inhibition of HPV E6 and E7 expression depend on cytidine deaminase. PMID:26379844

  7. Stereoselective chemo-enzymatic oxidation routes for (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene

    PubMed Central

    Görner, Christian; Hirte, Max; Huber, Stephanie; Schrepfer, Patrick; Brück, Thomas

    2015-01-01

    The diterpene (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene from the marine brown alga Dilophus spiralis belongs to the dolabellanes natural product family and has antimicrobial activity against multi-drug resistant Staphylococcus aureus. Recently, we generated a CotB2 diterpene synthase mutant (W288G), which instead of its native product cyclooctat-9-en-7-ol, generates (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene. In vivo CotB2 W288G reconstitution in an Escherichia coli based terpene production system, allowed efficient production of this olefinic macrocycle. To diversify the 3,7,18-dolabellatriene bioactivity we evaluated chemical and enzymatic methods for selective oxidation. Epoxidation by acetic peracid, which was formed in situ by a lipase catalyzed reaction of acetic acid with H2O2, provided efficient access to two monooxidized dolabellanes and to a novel di-epoxidated dolabellane species. These compounds could act as synthons en-route to new dolabellanes with diversified bioactivities. Furthermore, we demonstrate the almost quantitative 3,7,18-dolabellatriene conversion into the new, non-natural compound (1R,3E,7E,11S,12S,18R)-dolabella-3,7-diene-20-ol by hydroboration–oxidation with an enantiomeric excess of 94%, for the first time. PMID:26528263

  8. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    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.

  9. Oligomerization of oncoprotein p53.

    PubMed Central

    Kraiss, S; Quaiser, A; Oren, M; Montenarh, M

    1988-01-01

    Cellular phosphoprotein p53, which seems to be a multifunctional protein, may be assigned to different structural subclasses. Recently established immortalized or transformed cell lines that overexpress p53 allowed us to perform a detailed analysis of the quaternary structure of p53. By means of sucrose density gradient centrifugation, we found in simian virus 40-transformed cells that overexpress p53, in addition to high-molecular-weight T-p53 complexes, low-molecular-weight forms. The level of T-p53 complexes within simian virus 40-transformed cells seemed to be determined by the intracellular concentration of p53. However, the presence of uncomplexed T antigen and p53 indicated that an appropriate modification of at least one of the two proteins appears to be necessary for complex formation. Using different monoclonal antibodies that distinguish between (i) p53 associated with T antigen or heat shock proteins and (ii) p53 in apparently free form, we found p53 from transformed cells always in high-molecular-weight forms. p53 from normal and immortalized cells, however, was found mainly in low-molecular-weight forms. Pulse-labeling experiments revealed that oligomerization of p53 is a very rapid process. Monomeric forms of p53 which could be detected only by 2 min of pulse-labeling were rapidly converted to stable, high-molecular-weight oligomers. Furthermore, our data indicate a correlation between the occurrence of p53 in high-molecular-weight forms and the transformation state of the cell. Images PMID:3054153

  10. Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells.

    PubMed

    Bauelos, Adriana; Reyes, Elba; Ocadiz, Rodolfo; Alvarez, Elizabeth; Moreno, Martha; Monroy, Alberto; Gariglio, Patricio

    2003-08-01

    Human papillomavirus (HPV) E6 viral oncoprotein plays an important role during cervical carcinogenesis. This oncoprotein binds the tumor suppressor protein p53, leading to its degradation via the ubiquitin-proteasome pathway. Therefore, it is generally assumed that in HPV-positive cancer cells p53 function is completely abolished. Nevertheless, recent findings suggest that p53 activity can be recovered in cells expressing endogenous E6 protein. To investigate whether p53-dependent functions controlling genome integrity, cell proliferation, and apoptosis can be reactivated in cervical cancer cells, we examined the capacity of HeLa, INBL, CaSki, C33A, and ViBo cell lines to respond to neocarzinostatin (NCS), a natural product which induces single- and double-strand breaks in DNA. We found that NCS treatment inhibits cellular proliferation through G2 cell cycle arrest and apoptosis induction. This effect was preceded by nuclear accumulation of p53 protein and by an increase of p21 transcripts. Although apoptosis was blocked in ViBo cells (HPV-negative), nuclear accumulation of transcriptionally active p53 and inhibition of cell proliferation are observed after NCS treatment. These results suggest that HPV-positive cervical cancer cells are capable of responding efficiently to DNA damage provoked by NCS treatment through a p53-dependent pathway in spite of the presence of E6 protein. PMID:12750435

  11. Dose-dependent effects of selenite (Se4+) on arsenite (As3+)-induced apoptosis and differentiation in acute promyelocytic leukemia cells

    PubMed Central

    Wang, S; Geng, Z; Shi, N; Li, X; Wang, Z

    2015-01-01

    To enhance the therapeutic effects and decrease the adverse effects of arsenic on the treatment of acute promyelocytic leukemia, we investigated the co-effects of selenite (Se4+) and arsenite (As3+) on the apoptosis and differentiation of NB4 cells and primary APL cells. A 1.0-?M concentration of Se4+ prevented the cells from undergoing As3+-induced apoptosis by inhibiting As3+ uptake, eliminating As3+-generated reactive oxygen species, and repressing the mitochondria-mediated intrinsic apoptosis pathway. However, 4.0??M Se4+ exerted synergistic effects with As3+ on cell apoptosis by promoting As3+ uptake, downregulating nuclear factor-?B, and activating caspase-3. In addition to apoptosis, 1.0 and 3.2??M Se4+ showed contrasting effects on As3+-induced differentiation in NB4 cells and primary APL cells. The 3.2??M Se4+ enhanced As3+-induced differentiation by promoting the degradation of promyelocytic leukemia proteinretinoic acid receptor-? (PMLRAR?) oncoprotein, but 1.0??M Se4+ did not have this effect. Based on mechanistic studies, Se4+, which is similar to As3+, might bind directly to Zn2+-binding sites of the PML RING domain, thus controlling the fate of PMLRAR? oncoprotein. PMID:25590806

  12. Amolimogene bepiplasmid, a DNA-based therapeutic encoding the E6 and E7 epitopes from HPV, for cervical and anal dysplasia.

    PubMed

    Alvarez-Salas, Luis M

    2008-12-01

    MGI Pharma Biologics is developing amolimogene bepiplasmid as a potential therapy for HPV-associated diseases, including cervical dysplasia. Amolimogene bepiplasmid is a polymer-encapsulated DNA vaccine consisting of a plasmid expressing a chimeric peptide comprising immunogenic hybrid epitopes from HPV-16 and HPV-18 E6 and E7 proteins and an HLA-DRalpha intracellular trafficking peptide. In phase I and I/II clinical trials of ZYC-101 (the precursor of amolimogene bepiplasmid containing a single epitope from HPV-16 E7) in patients with cervical dysplasia and patients with anal dysplasia, ZYC-101 produced significant histological regression and was safe and well tolerated. Results from this trial led to a phase II clinical trial of amolimogene bepiplasmid in patients with cervical dysplasia. This phase II trial demonstrated that treatment with amolimogene bepiplasmid resolution of disease was not significantly superior to placebo except in the predefined group of women who were less than 25 years of age. A phase II/III clinical trial was ongoing at the time of publication examining amolimogene bepiplasmid in this patient population. PMID:19051140

  13. Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein.

    PubMed Central

    Smotkin, D; Wettstein, F O

    1986-01-01

    Human papillomavirus type 16 DNA and RNA were characterized in the cervical cancer-derived CaSki cell line, which contains only integrated DNA, and in a cervical cancer, which contains predominantly plasmid DNA. In both, a major RNA can code for the early open reading frame E7 and a minor one can code for E6. The cervical cancer, but not the CaSki cell line, contains a minor RNA that can code for an intact E2 protein, and this may relate to the continued presence of plasmid DNA. The RNA mapping data suggest that the poly(A)+ RNA is transcribed from a minor fraction of the several hundred gene copies present, and in the cervical cancer these genomes appear to be integrated. The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state. Images PMID:3014503

  14. Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin.

    PubMed Central

    Braunstein, D. P.; Chu, K.; Egeberg, K. D.; Frauenfelder, H.; Mourant, J. R.; Nienhaus, G. U.; Ormos, P.; Sligar, S. G.; Springer, B. A.; Young, R. D.

    1993-01-01

    Fouier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substrate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles alpha > 20 degrees between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the alpha of the CO ligand. PMID:8312483

  15. HBx protein modulates PI3K/Akt pathway to overcome genotoxic stress-induced destabilization of cyclin D1 and arrest of cell cycle.

    PubMed

    Mukherji, A; Janbandhu, V C; Kumar, V

    2009-02-01

    Growth arrest represents an innate barrier to carcinogenesis. DNA damage and replicational stress are known to induce growth arrest and apoptotic death to avert genomic instability and consequently carcinogenesis. In this study, working on the genotoxic stress induced by hydroxyurea and methylmethanesulfone, we observed a growth arrest at G1/S-phase that was mediated by destabilization of cyclin D1. The growth arrest was independent of the stability of cdc25A and preceded transcriptional up-regulation of p21(waf1). Cyclin D1 destabilization involved its phosphorylation by GSK-3beta at threonine-286, since overexpression of the kinase-dead mutant of GSK-3beta or cyclin D1T(286A) Inutant conferred stability to cyclin D1. Further, overexpression of cyclin D1(T286A) also helped in bypassing G1/S phase growth arrest. We also observed a rapid inactivation of Akt/PKB kinase in the presence of hydroxyurea. Enforced expression of the constitutively active Akt or viral oncoprotein HBx (Hepatitis B virus X protein) was sufficient to overcome growth arrest, independent of ATR signaling and stabilized cyclin D1. Thus, the present work not only establishes cyclin D1 to be a novel mediator of genotoxic stress signaling, but also explains how a deregulated mitogenic signaling or a viral oncoprotein can help bypass growth arrest. PMID:19374252

  16. The E7 proteins of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties.

    PubMed Central

    Gage, J R; Meyers, C; Wettstein, F O

    1990-01-01

    The E7 early viral protein of the oncogenic human papillomavirus type 16 (HPV-16) has been strongly implicated in the maintenance of the malignant phenotype in cervical cancers and cancer-derived cell lines. HPV-16 E7 is a nuclear phosphoprotein that can cooperate with ras to transform baby rat kidney cells, transactivates the adenovirus E2 promoter, and binds to the retinoblastoma (RB) protein. The E7 phosphoprotein of the nononcogenic HPV-6b, which is generally associated with benign genital warts, is similar to the HPV-16 E7 in amino acid sequence but differs dramatically in migration in sodium dodecyl sulfate-polyacrylamide gels, sedimentation in nondenaturing glycerol gradients, and the ability to bind the RB protein. Our results indicate that the RB protein preferentially binds the phosphorylated form of HPV-6b E7, which comprises a minor fraction of the total E7 expressed in transiently transfected COS-7 cells. These characteristics may help to explain the difference in the oncogenic potential of the oncogenic and nononcogenic types of genital papillomaviruses. Images PMID:2153238

  17. Successful Rechallenge with Imatinib in a Patient with Chronic Myeloid Leukemia Who Previously Experienced Imatinib Mesylate Induced Pneumonitis

    PubMed Central

    Go, Seong Woo; Kim, Boo Kyeong; Lee, Sung Hak; Kim, Tae-Jung; Huh, Joo Yeon; Lee, Jong Min; Hah, Jick Hwan; Kim, Dong Whi; Cho, Min Jung; Kim, Tae Wan

    2013-01-01

    Imatinib mesylate is a targeted therapy that acts by inhibiting tyrosine kinase of the bcr-abl fusion oncoprotein, which is specific to chronic myeloid leukemia (CML), and the c-transmembrane receptor, which is specific to gastrointestinal stromal tumors. Interstitial pneumonitis is a rare adverse event of imatinib therapy. It is clinically difficult to distinguish from infectious pneumonia, which can frequently occur due to the underlying disease. The standard treatment for imatinib-induced pneumonitis is to discontinue the medication and optionally administer corticosteroids. However, there are a few cases of successful retrial with imatinib. We describe a case of successful rechallenge of imatinib in a patient with imatinib-induced interstitial pneumonitis and CML without a recurrence of the underlying disease after 3 months of follow-up. PMID:24416057

  18. The human papillomavirus 16 European-T350G E6 variant can immortalize but not transform keratinocytes in the absence of E7.

    PubMed

    Togtema, Melissa; Jackson, Robert; Richard, Christina; Niccoli, Sarah; Zehbe, Ingeborg

    2015-11-01

    Human papillomavirus type 16 is commonly implicated in HPV-related cancers. However, only a small number of infected individuals progress to this stage. Epidemiological evidence demonstrated that oncogenic risk is population-specific and variations within the viral oncogene, E6, have been suggested to play a role in these findings. Of focus in this study is the European-T350G variant, which is characterized by an L>V amino acid substitution at residue 83 of the prototype E6 protein. To elucidate the functional effects of this polymorphism, we followed keratinocytes transduced with E-T350G E6 for over 60 passages and compared them to keratinocytes transduced, in parallel, with prototype or Asian-American (Q14H/L83V/H78Y) E6. We found that although E-T350G E6 immortalized transduced keratinocytes in the absence of E7, these cells were not fully transformed. We also found that E-T350G down-regulated E-cadherin compared to the other variants, providing a possible link between its population-based oncogenicity and host genetic variations. PMID:26318249

  19. Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16.

    PubMed Central

    Furukawa, T.; Duguid, W. P.; Rosenberg, L.; Viallet, J.; Galloway, D. A.; Tsao, M. S.

    1996-01-01

    Pancreatic cancer is one of the most lethal cancers in humans. The majority of these cancers arise from the pancreatic duct epithelium. Research into the pathogenesis of pancreatic carcinoma has largely relied on animal models. In vitro models of pancreatic carcinogenesis using propagable cultured epithelial cells derived from the pancreatic ducts of rats and hamsters have been described. A human model, however, has been nonexistent due to the unavailability of propagable cultured duct epithelial cells derived from normal human pancreas. We report here a reproducible method for the long-term culture of pancreatic duct epithelial cells derived from normal and benign adult human pancreata by infection with a retrovirus containing the E6 and E7 genes of the human papilloma virus 16. One of these cell lines has become immortal and has propagated continuously for more than 20 passages. They remain anchorage dependent in their growth and nontumorigenic in nude mice. These cell lines and the methodology described here to establish them may provide new avenues for in vitro studies of the roles played by duct epithelium in human pancreatic diseases and cancers. Images Figure 1 Figure 2 Figure 3 PMID:8669463

  20. Epstein–Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor

    SciTech Connect

    Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit; Kundu, Chanakya N.; Verma, Subhash C.; Choudhuri, Tathagata

    2014-01-05

    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 (ΔNp73). 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 lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C. - Highlights: • EBV-encoded EBNA3C suppresses doxorubicin-induced apoptosis in B-cell lymphomas. • EBNA3C binds to p73 to suppress its apoptotic effect. • EBNA3C maintains latency by regulating downstream mitochondrial pathways.

  1. A natural HIV p17 protein variant up-regulates the LMP-1 EBV oncoprotein and promotes the growth of EBV-infected B-lymphocytes: implications for EBV-driven lymphomagenesis in the HIV setting.

    PubMed

    Martorelli, Debora; Muraro, Elena; Mastorci, Katy; Dal Col, Jessica; Fa, Damiana Antonia; Furlan, Chiara; Giagulli, Cinzia; Caccuri, Francesca; Rusnati, Marco; Fiorentini, Simona; Carbone, Antonino; Caruso, Arnaldo; Dolcetti, Riccardo

    2015-09-15

    Human immunodeficiency virus p17 matrix protein is released by infected cells and may accumulate within lymphoid tissues where it may deregulate the biological activities of different cell populations by binding to CXCR1 and CXCR2 cellular receptors. S75X, a natural p17 variant, was recently shown to enhance the malignant properties of lymphoma cells. We investigated a reference p17 protein and the S75X variant for their ability to bind to Epstein-Barr virus (EBV)-infected primary and fully transformed B-lymphocytes and trigger downstream effects of potential pathogenic relevance. We demonstrate that EBV infection of primary B-lymphocytes or the ectopic expression of the latent membrane protein-1 viral oncoprotein in EBV-negative B-cells up-regulates CXCR2, but not CXCR1. Multispectral imaging flow cytometry showed that EBV-infected primary B-cells more efficiently bind and internalize p17 proteins as compared with activated B-lymphocytes. The S75X variant bound more efficiently to EBV-infected primary and fully transformed B-lymphocytes compared with reference p17, because of a higher affinity to CXCR2, and enhanced the proliferation of these cells, an effect associated with cyclin D2 and D3 up-regulation and increased interleukin-6 production. Notably, the S75X variant markedly up-regulated latent membrane protein-1 expression at both mRNA and protein levels and enhanced the activation of Akt, ERK1/2 and STAT3 signaling, thereby contributing to EBV(+) B-cell growth promotion. These results indicate that EBV infection sensitizes B-lymphocytes to CXCR2-mediated effects of p17 proteins and provide evidence supporting a possible contribution of natural p17 variants to EBV-driven lymphomagenesis in the human immunodeficiency virus setting. PMID:25704763

  2. Crystal Diagnostics Xpress™ E7 STEC Kit for the Rapid Multiplex Detection of E. coli O157 and non-O157 Shiga toxin-producing E. coli.

    PubMed

    Zhao, Weidong; Stumpf, Curtis H; Bullard, Brian; Kuzenko, Stephanie; Niehaus, Gary D

    2015-01-01

    The Crystal Diagnostics (CDx) Xpress E7 STEC kit is a rapid and sensitive detection assay for the detection of Escherichia coli O157 and six non-O157 Shiga toxin-producing E. coli (serogroups O26, O45, O1O3, O111, O121, and O145, collectively referred to as STEC) at 1 CFU/325 g of raw ground beef and raw beef trim, or 200 g of spinach. The system comprises an automatic Crystal Diagnostics Xpress System Reader that integrates immunochemical and optical processes for the liquid crystal-based detection of microorganisms, a CDx BioCassette that incorporates antibody-coupled microspheres and liquid crystal for selective identification of the intended microbe, and additional commercially available components. The Crystal Diagnostics Xpress System(TM) combines proprietary liquid crystal technology with antibody-coated paramagnetic microspheres to selectively capture and detect STEC from food matrixes. The Xpress System expeditiously (9.5 h enrichment) provides the sensitivity and specificity of the U. S. Department of Agriculture Food Safety and Inspection Service and the U. S. Food and Drug Administration reference methods in screening as low as 1 STEC CFU/test portion. The inclusivity validation demonstrated detection of 53 of 54 STEC test strains. Shelf life testing of the antibody-coated microspheres and other Crystal Diagnostic consumables indicated that all materials were stable for a minimum of 3 months (ongoing), and lot-to-lot testing demonstrated consistent results between lots (data not shown). The internal and independent laboratory tests demonstrate that the method is rapid and sensitive for screening of the target STEC. PMID:26651567

  3. MUC1-C Oncoprotein Activates ERK?C/EBP? Signaling and Induction of Aldehyde Dehydrogenase 1A1 in Breast Cancer Cells*

    PubMed Central

    Alam, Maroof; Ahmad, Rehan; Rajabi, Hasan; Kharbanda, Akriti; Kufe, Donald

    2013-01-01

    Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit, we demonstrate that MUC1-C is sufficient for induction of MEK?ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein ? (C/EBP?) transcription factor. The results further show that MUC1-C and C/EBP? form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK?C/EBP??ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells. PMID:24043631

  4. MUC1-C oncoprotein activates ERK?C/EBP? signaling and induction of aldehyde dehydrogenase 1A1 in breast cancer cells.

    PubMed

    Alam, Maroof; Ahmad, Rehan; Rajabi, Hasan; Kharbanda, Akriti; Kufe, Donald

    2013-10-25

    Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is used as a marker of breast cancer stem cells; however, little is known about the regulation of ALDH1A1 expression. Mucin 1 (MUC1) is a heterodimeric protein that is aberrantly overexpressed in most human breast cancers. In studies of breast cancer cells stably silenced for MUC1 or overexpressing the oncogenic MUC1-C subunit, we demonstrate that MUC1-C is sufficient for induction of MEK ? ERK signaling and that treatment with a MUC1-C inhibitor suppresses ERK activation. In turn, MUC1-C induces ERK-mediated phosphorylation and activation of the CCAAT/enhancer-binding protein ? (C/EBP?) transcription factor. The results further show that MUC1-C and C/EBP? form a complex on the ALDH1A1 gene promoter and activate ALDH1A1 gene transcription. MUC1-C-induced up-regulation of ALDH1A1 expression is associated with increases in ALDH activity and is detectable in stem-like cells when expanded as mammospheres. These findings demonstrate that MUC1-C (i) activates a previously unrecognized ERK?C/EBP??ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells. PMID:24043631

  5. Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes.

    PubMed

    Xiang, Di; Yuan, Yunsheng; Chen, Li; Liu, Xin; Belani, Chandra; Cheng, Hua

    2015-08-14

    Adult T cell leukemia and lymphoma (ATL) is a highly aggressive form of hematological malignancy and is caused by chronic infection of human T cell leukemia virus type 1 (HTLV-1). The viral genome encodes an oncogenic protein, Tax, which plays a key role in transactivating viral gene transcription and in deregulating cellular oncogenic signaling to promote survival, proliferation and transformation of virally infected T cells. Hence, Tax is a desirable therapeutic target, particularly at early stage of HTLV-1-mediated oncogenesis. We here show that niclosamide, an anti-helminthic molecule, induced apoptosis of HTLV-1-transformed T cells. Niclosamide facilitated degradation of the Tax protein in proteasome. Consistent with niclosamide-mediated Tax degradation, this compound inhibited activities of MAPK/ERK1/2 and I?B kinases. In addition, niclosamide downregulated Stat3 and pro-survival Bcl-2 family members such as Mcl-1 and repressed the viral gene transcription of HTLV-1 through induction of Tax degradation. Since Tax, Stat3 and Mcl-1 are crucial molecules for promoting survival and growth of HTLV-1-transformed T cells, our findings demonstrate a novel mechanism of niclosamide in inducing Tax degradation and downregulating various cellular pro-survival molecules, thereby promoting apoptosis of HTLV-1-associated leukemia cells. PMID:26116531

  6. The stress oncoprotein LEDGF/p75 interacts with the methyl CpG binding protein MeCP2 and influences its transcriptional activity.

    PubMed

    Leoh, Lai Sum; van Heertum, Bart; De Rijck, Jan; Filippova, Maria; Rios-Colon, Leslimar; Basu, Anamika; Martinez, Shannalee R; Tungteakkhun, Sandy S; Filippov, Valeri; Christ, Frauke; De Leon, Marino; Debyser, Zeger; Casiano, Carlos A

    2012-03-01

    The lens epithelium-derived growth factor p75 (LEDGF/p75) is a transcription coactivator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kDa (DFS70) and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull-down and AlphaScreen assays, coimmunoprecipitation, and nuclear colocalization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS osteosarcoma cells, whereas this effect was more pronounced in PC3 prostate cancer cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75-MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes. PMID:22275515

  7. 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

    Subramaniam, Sugalesini; Johnston, John; Preeyanon, Likit; Brown, C. Titus; Kung, Hsing-Jien

    2013-01-01

    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. PMID:23740999

  8. Interleukin-21 regulates expression of key Epstein-Barr virus oncoproteins, EBNA2 and LMP1, in infected human B cells

    SciTech Connect

    Konforte, Danijela Simard, Nathalie; Paige, Christopher J.

    2008-04-25

    Epstein-Barr virus (EBV) persists for the life of the host by accessing the long-lived memory B cell pool. It has been proposed that EBV uses different combinations of viral proteins, known as latency types, to drive infected B cells to make the transition from resting B cells to memory cells. This process is normally antigen-driven. A major unresolved question is what factors coordinate expression of EBV latency proteins. We have recently described novel type III latency EBV{sup +} B cell lines (OCI-BCLs) that were induced to differentiate into late plasmablasts/early plasma cells in culture with interleukin-21 (IL-21), mimicking normal B cell development. The objective of this study was to determine whether IL-21-mediated signals also regulate the expression of key EBV latent proteins during this window of development. Here we show that IL-21-reduced gene and protein expression of growth-transforming EBV nuclear antigen 2 (EBNA2) in OCI-BCLs. By contrast, the expression of CD40-like, latent membrane protein 1 (LMP1) strongly increased in these cells suggesting an EBNA2-independent mode of regulation. Same results were also observed in Burkitt's lymphoma line Jijoye and B95-8 transformed lymphoblastoid cell lines. The effect of IL-21 on EBNA2 and LMP1 expression was attenuated by a pharmacological JAK inhibitor indicating involvement of JAK/STAT signalling in this process. Our study also shows that IL-21 induced transcription of ebna1 from the viral Q promoter (Qp)

  9. Biosynthesis of the Cyanogenic Glucosides Linamarin and Lotaustralin in Cassava: Isolation, Biochemical Characterization, and Expression Pattern of CYP71E7, the Oxime-Metabolizing Cytochrome P450 Enzyme1[OA

    PubMed Central

    Jrgensen, Kirsten; Morant, Anne Vinther; Morant, Marc; Jensen, Niels Bjerg; Olsen, Carl Erik; Kannangara, Rubini; Motawia, Mohammed Saddik; Mller, Birger Lindberg; Bak, Sren

    2011-01-01

    Cassava (Manihot esculenta) is a eudicotyledonous plant that produces the valine- and isoleucine-derived cyanogenic glucosides linamarin and lotaustralin with the corresponding oximes and cyanohydrins as key intermediates. CYP79 enzymes catalyzing amino acid-to-oxime conversion in cyanogenic glucoside biosynthesis are known from several plants including cassava. The enzyme system converting oxime into cyanohydrin has previously only been identified in the monocotyledonous plant great millet (Sorghum bicolor). Using this great millet CYP71E1 sequence as a query in a Basic Local Alignment Search Tool-p search, a putative functional homolog that exhibited an approximately 50% amino acid sequence identity was found in cassava. The corresponding full-length cDNA clone was obtained from a plasmid library prepared from cassava shoot tips and was assigned CYP71E7. Heterologous expression of CYP71E7 in yeast afforded microsomes converting 2-methylpropanal oxime (valine-derived oxime) and 2-methylbutanal oxime (isoleucine-derived oxime) to the corresponding cyanohydrins, which dissociate into acetone and 2-butanone, respectively, and hydrogen cyanide. The volatile ketones were detected as 2.4-dinitrophenylhydrazone derivatives by liquid chromatography-mass spectrometry. A KS of approximately 0.9 ?m was determined for 2-methylbutanal oxime based on substrate-binding spectra. CYP71E7 exhibits low specificity for the side chain of the substrate and catalyzes the conversion of aliphatic and aromatic oximes with turnovers of approximately 21, 17, 8, and 1 min?1 for the oximes derived from valine, isoleucine, tyrosine, and phenylalanine, respectively. A second paralog of CYP71E7 was identified by database searches and showed approximately 90% amino acid sequence identity. In tube in situ polymerase chain reaction showed that in nearly unfolded leaves, the CYP71E7 paralogs are preferentially expressed in specific cells in the endodermis and in most cells in the first cortex cell layer. In fully unfolded leaves, the expression is pronounced in the cortex cell layer just beside the epidermis and in specific cells in the vascular tissue cortex cells. Thus, the transcripts of the CYP71E7 paralogs colocalize with CYP79D1 and CYP79D2. We conclude that CYP71E7 is the oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in cassava. PMID:21045121

  10. Comprehensive immunohistochemical analysis of Her-2/neu oncoprotein overexpression in breast cancer: HercepTest (Dako) for manual testing and Her-2/neuTest 4B5 (Ventana) for Ventana BenchMark automatic staining system with correlation to results of fluorescence in situ hybridization (FISH).

    PubMed

    Mayr, Doris; Heim, Sibylle; Werhan, Cedric; Zeindl-Eberhart, Evelyn; Kirchner, Thomas

    2009-03-01

    Overexpression of Her-2/neu-oncoprotein is used as marker for Herceptin therapy. To investigate the sensitivity and specificity of automatic immunohistochemistry (Benchmark, Ventana), we compared the results to the manual testing (Dako) in 130 breast carcinomas and validated the results by fluorescence in situ hybridization (FISH). Manual and automatic immunohistochemistry of Her-2/neu-oncoprotein using two different antibodies (HercepTest, Her-2/neuTest 4B5) was analyzed. FISH was performed in all cases with uncertain or strong overexpression in either immunohistochemical stainings or with different immunohistochemical results. Same immunohistochemical results were seen in 73.8%. Two cases with overexpression, detected with Her-2/neuTest 4B5 and confirmed by FISH, showed no overexpression using HercepTest. From 21 cases with 2+ by Her-2/neuTest 4B5, 15 cases had no gene amplification (two of them with 3+ HercepTest); three cases showed a gene amplification (one of them with failing overexpression by HercepTest); two other cases were polysomic; one could not be analyzed. Ventana immunohistochemistry seems to be of same reliability like Dako with a little better concordance to FISH in our study. PMID:19169706

  11. Bcl-3, induced by Tax and HTLV-1, inhibits NF-?B activation and promotes autophagy.

    PubMed

    Wang, Jinheng; Niu, Zhiguo; Shi, Ying; Gao, Cai; Wang, Xia; Han, Jingxian; Li, Junying; Gao, Zhitao; Zhu, Xiaofei; Song, Xiangfeng; Qin, Zhihai; Wang, Hui

    2013-12-01

    The human T cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes an aggressive leukemia known as adult T cell leukemia (ATL). The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-?B (NF-?B) pathway, which is perceived as the primary cause of ATL. Bcl-3, a member of the NF-?B inhibitor (I?B) family, is highly expressed in many HTLV-1-infected T cell lines and ATL cells. However, the role of Bcl-3 in Tax-induced NF-?B activation has not been fully elucidated. Here, we show that Tax induces Bcl-3 expression, which in turn negatively regulates the Tax-induced NF-?B activation. Interestingly, both Bcl-3 up-regulation and NF-?B inhibition promote the autophagy process in HTLV-1-infected cells. Consistent with this, over-expression of Bcl-3 also results in enhancement of rapamycin-, pifithrin-?- or starvation-induced autophagy in control cells. Together, these data demonstrate that Bcl-3 acts as a negative regulator of NF-?B activation and promotes autophagy in HTLV-1-infected cells. PMID:24044922

  12. Sequential Cisplatin Therapy and Vaccination with HPV16 E6E7L2 Fusion Protein in Saponin Adjuvant GPI-0100 for the Treatment of a Model HPV16+ Cancer

    PubMed Central

    Peng, Shiwen; Wang, Joshua W.; Karanam, Balasubramanyam; Wang, Chenguang; Huh, Warner K.; Alvarez, Ronald D.; Pai, Sara I.; Hung, Chien-fu; Wu, T. -C.; Roden, Richard B. S.

    2015-01-01

    Clinical studies suggest that responses to HPV16 E6E7L2 fusion protein (TA-CIN) vaccination alone are modest, and GPI-0100 is a well-tolerated, potent adjuvant. Here we sought to optimize both the immunogenicity of TA-CIN via formulation with GPI-0100 and treatment of HPV16+ cancer by vaccination after cisplatin chemotherapy. HPV16 neutralizing serum antibody titers, CD4+ T cell proliferative and E6/E7-specific CD8+ T cell responses were significantly enhanced when mice were vaccinated subcutaneously (s.c.) or intramuscularly (i.m.) with TA-CIN formulated with GPI-0100. Vaccination was tested for therapy of mice bearing syngeneic HPV16 E6/E7+ tumors (TC-1) either in the lung or subcutaneously. Mice treated with TA-CIN/GPI-0100 vaccination exhibited robust E7-specific CD8+ T cell responses, which were associated with reduced tumor burden in the lung, whereas mice receiving either component alone were similar to controls. Since vaccination alone was not sufficient for cure, mice bearing s.c. TC-1 tumor were first treated with two doses of cisplatin and then vaccinated. Vaccination with TA-CIN/GPI-0100 i.m. substantially retarded tumor growth and extended survival after cisplatin therapy. Injection of TA-CIN alone, but not GPI-0100, into the tumor (i.t.) was similarly efficacious after cisplatin therapy, but the mice eventually succumbed. However, tumor regression and extended remission was observed in 80% of the mice treated with cisplatin and then intra-tumoral TA-CIN/GPI-0100 vaccination. These mice also exhibited robust E7-specific CD8+ T cell and HPV16 neutralizing antibody responses. Thus formulation of TA-CIN with GPI-0100 and intra-tumoral delivery after cisplatin treatment elicits potent therapeutic responses in a murine model of HPV16+ cancer. PMID:25560237

  13. The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein

    PubMed Central

    2012-01-01

    Background Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK) with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90) plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40), is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines. Methods NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP) 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined. Results We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to be due to a decrease in NPM-ALK levels or the ability of this oncoprotein to signal. Conclusions This is the first study demonstrating that the expression of immunophilin family co-chaperones is promoted by an oncogenic tyrosine kinase. Moreover, this is the first report establishing an important role for Cyp40 in lymphoma. PMID:22681779

  14. HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma

    PubMed Central

    Xue, Yuezhen; Toh, Shen Yon; He, Pingping; Lim, Thimothy; Lim, Diana; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

    2015-01-01

    Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo. PMID:26474276

  15. The oncoprotein NPM-ALK of anaplastic large-cell lymphoma induces JUNB transcription via ERK1/2 and JunB translation via mTOR signaling.

    PubMed

    Staber, Philipp B; Vesely, Paul; Haq, Naznin; Ott, Rene G; Funato, Kotaro; Bambach, Isabella; Fuchs, Claudia; Schauer, Silvia; Linkesch, Werner; Hrzenjak, Andelko; Dirks, Wilhelm G; Sexl, Veronika; Bergler, Helmut; Kadin, Marshall E; Sternberg, David W; Kenner, Lukas; Hoefler, Gerald

    2007-11-01

    Anaplastic large cell lymphomas (ALCLs) are highly proliferating tumors that commonly express the AP-1 transcription factor JunB. ALK fusions occur in approximately 50% of ALCLs, and among these, 80% have the t(2;5) translocation with NPM-ALK expression. We report greater activity of JunB in NPM-ALK-positive than in NPM-ALK-negative ALCLs. Specific knockdown of JUNB mRNA using small interfering RNA and small hairpin RNA in NPM-ALK-expressing cells decreases cellular proliferation as evidenced by a reduced cell count in the G2/M phase of the cell cycle. Expression of NPM-ALK results in ERK1/2 activation and transcriptional up-regulation of JUNB. Both NPM-ALK-positive and -negative ALCL tumors demonstrate active ERK1/2 signaling. In contrast to NPM-ALK-negative ALCL, the mTOR pathway is active in NPM-ALK-positive lymphomas. Pharmacological inhibition of mTOR in NPM-ALK-positive cells down-regulates JunB protein levels by shifting JUNB mRNA translation from large polysomes to monosomes and ribonucleic particles (RNPs), and decreases cellular proliferation. Thus, JunB is a critical target of mTOR and is translationally regulated in NPM-ALK-positive lymphomas. This is the first study demonstrating translational control of AP-1 transcription factors in human neoplasia. In conjunction with NPM-ALK, JunB enhances cell cycle progression and may therefore represent a therapeutic target. PMID:17690253

  16. Induced Abortion

    MedlinePLUS

    ... Education & Events Advocacy For Patients About ACOG Induced Abortion Home For Patients Search FAQs Induced Abortion Page ... Induced Abortion FAQ043, May 2015 PDF Format Induced Abortion Special Procedures What is an induced abortion? What ...

  17. JWA enhances As?O?-induced tubulin polymerization and apoptosis via p38 in HeLa and MCF-7 cells.

    PubMed

    Shen, Lianlian; Xu, Wenxia; Li, Aiping; Ye, Jian; Zhou, Jianwei

    2011-11-01

    Arsenic trioxide (As?O?) has potential anti-cancer activity against a wide range of carcinomas via apoptosis induction or oncoprotein degradation. The mechanisms involved are not fully elucidated. Here, we demonstrated that As?O? induced-apoptosis in HeLa and MCF-7 cancer cells was in part triggered by tubulin polymerization. High expression of JWA promoted tubulin polymerization and increased the sensitivity of the cancer cells to As?O?. The activation of the p38 MAPK (mitogen-activated protein kinases) signaling pathway was found to contribute to JWA-promoted tubulin polymerization. Our results suggest that JWA may serve as an effective enhancer of microtubule-targeted As?O? anti-cancer therapy. PMID:21847655

  18. TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy.

    PubMed

    Hu, Zheng; Ding, Wencheng; Zhu, Da; Yu, Lan; Jiang, Xiaohui; Wang, Xiaoli; Zhang, Changlin; Wang, Liming; Ji, Teng; Liu, Dan; He, Dan; Xia, Xi; Zhu, Tao; Wei, Juncheng; Wu, Peng; Wang, Changyu; Xi, Ling; Gao, Qinglei; Chen, Gang; Liu, Rong; Li, Kezhen; Li, Shuang; Wang, Shixuan; Zhou, Jianfeng; Ma, Ding; Wang, Hui

    2015-01-01

    Persistent HPV infection is recognized as the main etiologic factor for cervical cancer. HPV expresses the oncoproteins E6 and E7, both of which play key roles in maintaining viral infection and promoting carcinogenesis. While siRNA-mediated targeting of E6 and E7 transcripts temporarily induces apoptosis in HPV-positive cells, it does not eliminate viral DNA within the host genome, which can harbor escape mutants. Here, we demonstrated that specifically targeting E6 and E7 within host DNA with transcription activator-like effector nucleases (TALENs) induces apoptosis, inhibits growth, and reduces tumorigenicity in HPV-positive cell lines. TALEN treatment efficiently disrupted E6 and E7 oncogenes, leading to the restoration of host tumor suppressors p53 and retinoblastoma 1 (RB1), which are targeted by E6 and E7, respectively. In the K14-HPV16 transgenic mouse model of HPV-driven neoplasms, direct cervical application of HPV16-E7-targeted TALENs effectively mutated the E7 oncogene, reduced viral DNA load, and restored RB1 function and downstream targets transcription factor E2F1 and cycling-dependent kinase 2 (CDK2), thereby reversing the malignant phenotype. Together, the results from our study suggest that TALENs have potential as a therapeutic strategy for HPV infection and related cervical malignancy. PMID:25500889

  19. TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy

    PubMed Central

    Hu, Zheng; Ding, Wencheng; Zhu, Da; Yu, Lan; Jiang, Xiaohui; Wang, Xiaoli; Zhang, Changlin; Wang, Liming; Ji, Teng; Liu, Dan; He, Dan; Xia, Xi; Zhu, Tao; Wei, Juncheng; Wu, Peng; Wang, Changyu; Xi, Ling; Gao, Qinglei; Chen, Gang; Liu, Rong; Li, Kezhen; Li, Shuang; Wang, Shixuan; Zhou, Jianfeng; Ma, Ding; Wang, Hui

    2014-01-01

    Persistent HPV infection is recognized as the main etiologic factor for cervical cancer. HPV expresses the oncoproteins E6 and E7, both of which play key roles in maintaining viral infection and promoting carcinogenesis. While siRNA-mediated targeting of E6 and E7 transcripts temporarily induces apoptosis in HPV-positive cells, it does not eliminate viral DNA within the host genome, which can harbor escape mutants. Here, we demonstrated that specifically targeting E6 and E7 within host DNA with transcription activator–like effector nucleases (TALENs) induces apoptosis, inhibits growth, and reduces tumorigenicity in HPV-positive cell lines. TALEN treatment efficiently disrupted E6 and E7 oncogenes, leading to the restoration of host tumor suppressors p53 and retinoblastoma 1 (RB1), which are targeted by E6 and E7, respectively. In the K14-HPV16 transgenic mouse model of HPV-driven neoplasms, direct cervical application of HPV16-E7–targeted TALENs effectively mutated the E7 oncogene, reduced viral DNA load, and restored RB1 function and downstream targets transcription factor E2F1 and cycling-dependent kinase 2 (CDK2), thereby reversing the malignant phenotype. Together, the results from our study suggest that TALENs have potential as a therapeutic strategy for HPV infection and related cervical malignancy. PMID:25500889

  20. Relationship between HPV 16, 18, 31, 33, 45 DNA detection and quantitation and E6/E7 mRNA detection among a series of cervical specimens with various degrees of histological lesions.

    PubMed

    Baron, Carolina; Henry, Mireille; Tamalet, Catherine; Villeret, Julia; Richet, Herve; Carcopino, Xavier

    2015-08-01

    Better understanding of the correlation between high-risk HPV DNA testing, viral load quantitation, and E6/E7 mRNA detection is required. The aim of this study was to assess the relationship between these markers and the severity of cervical lesions. One-hundred and fifty one directed cervical specimens were analysed (normal, cervical intraepithelial neoplasia, and cancer). HPV types 16, 18, 31, 33, and 45 DNA detection and quantititation and E6/E7 mRNA detection were performed. DNA was detected in 87 (57.6%) samples and increased from 0% (normal) to 93.9% (cancer). E6/E7 mRNA was detected in 65 (43%) samples and increased with the severity of the lesions from 0% (normal) to 78.8% (26/33) (cancers) (P < 0.001). HPV DNA and E6/E7 mRNA detection were compared in the 141 samples harbouring HPV16, 18, 31, 33, or 45 infection: 45.4% (64/141) of specimens were DNA-/mRNA-, 46% (65/141) were DNA + /mRNA+ and 8.5% (12/141) were DNA + /mRNA-. The proportion of DNA + /mRNA+ specimens increased with the severity of the lesions (P < 0.001). All normal cervix specimens were DNA-/mRNA-. Among grade 2 cervical intraepithelial neoplasia, prevalence of DNA was higher than that of mRNA: 41.6% (5/12) versus 25% (3/12), whereas it was 79.3% (46/58) versus 62% (36/58) among grade 3 cervical intraepithelial neoplasia. Full concordance was observed in cancers as all the 26 DNA+ specimens were mRNA +. Median overall HPV load was higher in DNA + /mRNA+ than in DNA + /mRNA- specimens (1.41 × 10(6) vs. 9.1 × 10(2) copies per million cells, P < 0.001). Both E6/E7 mRNA detection and concordant DNA + /mRNA+ detection increases with the severity of the lesions and with the HPV DNA load. PMID:25908062

  1. Diagnostic performance of HPV E6/E7, hTERT, and Ki67 mRNA RT-qPCR assays on formalin-fixed paraffin-embedded cervical tissue specimens from women with cervical cancer.

    PubMed

    Wang, Hye-Young; Kim, Geehyuk; Cho, Hyemi; Kim, Sunghyun; Lee, Dongsup; Park, Sunyoung; Park, Kwang Hwa; Lee, Hyeyoung

    2015-06-01

    Human papillomavirus (HPV) is a major cause of cervical cancer, which is the third most common cancer in women. Human telomerase reverse transcriptase (hTERT) and Ki67 are tumor cell markers indicating cancer cell proliferation in cancer patients, and activation of hTERT and Ki67 leads to progressive cervical carcinogenesis. In the present study, we evaluated the CervicGen HPVE6/E7 mRNA RT-qDx assay, which detects 16 HPV high-risk (HR) genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and 69), and the CervicGen hTERT and Ki67 mRNA RT-qDx assay using 117 formalin-fixed paraffin-embedded (FFPE) cervical cancer tissue samples. The diagnostic validity of the CervicGen HPV RT-qDx assay for detecting histologically proven prevalent squamous cell carcinoma (SCC) was 94% sensitivity, 100% specificity, 77.8% positive predictive value (PPV), and 78.9% negative predictive value (NPV). The most common HPV genotypes detected in FFPE cervical cancer tissue samples were HPV 16 (56%) and HPV 18 (10%). The positivity rate of hTERT and Ki67 mRNA expressions in FFPE cervical cancer tissue samples on RT-qPCR was 65% and 93% respectively. Moreover, the positivity rates were 92% for a combination of HPV E6/E7 and hTERT mRNA expressions, 97% for HPV E6/E7 and Ki67 mRNA expressions, and 99% (99/100) for the combination of HPV E6/E7, hTERT, and Ki67 mRNA expressions. These data showed that SSC FFPE cervical cancer tissue samples correlated more strongly with high Ki67 mRNA expressions than with hTERT mRNA expressions. Notably, hTERT and Ki67 mRNA expression level was increased in high-grade cervical lesions, but was very low in normal samples. Our findings suggest that the combination of HPV E6/E7, hTERT, and Ki67 mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions. Further studies are required to evaluate these assays as a useful predictive tool for screening low-grade cervical lesions. PMID:25835783

  2. Targeting MUC1-C is synergistic with bortezomib in downregulating TIGAR and inducing ROS-mediated myeloma cell death

    PubMed Central

    Yin, Li; Kufe, Turner; Avigan, David

    2014-01-01

    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

  3. Targeting MUC1-C is synergistic with bortezomib in downregulating TIGAR and inducing ROS-mediated myeloma cell death.

    PubMed

    Yin, Li; Kufe, Turner; Avigan, David; Kufe, Donald

    2014-05-01

    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

  4. Dickkopf-1, an inhibitor of the Wnt signaling pathway, is induced by p53.

    PubMed

    Wang, J; Shou, J; Chen, X

    2000-03-30

    Dickkopf-1 (Dkk-1), a secreted glycoprotein, has been found to be necessary and sufficient for inducing amphibian head formation. Interestingly, the mechanism by which Dkk-1 does this is the ability of Dkk-1 to antagonize the Wnt signaling pathway. Wnt, itself a proto-oncoprotein, can promote cell proliferation and transformation when mutated or overexpressed, leading to tumor formation. p53 is a tumor suppressor and loss of p53 function accelerates mammary tumorigenesis by Wnt. In this study, we found that Dkk-1 is induced by wild-type p53 but not mutant p53(R249S). In addition, DNA damage upregulates Dkk-1 in cell lines that harbor an endogenous wild-type p53 gene but not in cell lines that are p53-null or harbor an endogenous mutant p53 gene. We also found a potential p53 responsive element located approximately 2100 nucleotides upstream of the Dkk-1 transcription start site and we show that p53 binds specifically to this element both in vitro and in vivo. Furthermore, we have established several cell lines derived from H1299 lung carcinoma and U118 glioma cells that inducibly express Dkk-1 under a tetracycline-regulated promoter. We found that Dkk-1 has no effect on proliferation of cells that are not transformed by Wnt. Taken together, these results suggest that Dkk-1 may mediate p53 tumor suppression by antagonizing the Wnt signaling pathway. PMID:10777218

  5. Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

    SciTech Connect

    Huai, Lei; Wang, Cuicui; Zhang, Cuiping; Li, Qihui; Chen, Yirui; Jia, Yujiao; Li, Yan; Xing, Haiyan; Tian, Zheng; Rao, Qing; Wang, Min; Wang, Jianxiang

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Metformin induces differentiation in NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces activation of the MEK/ERK signaling pathway in APL cells. Black-Right-Pointing-Pointer Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces the relocalization and degradation of the PML-RAR{alpha} fusion protein. Black-Right-Pointing-Pointer The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RAR{alpha} and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.

  6. Human papillomavirus types detected in skin warts and cancer differ in their transforming properties but commonly counteract UVB induced protective responses in human keratinocytes

    SciTech Connect

    Shterzer, Naama; Heyman, Dariya; Shapiro, Beny; Yaniv, Abraham; Jackman, Anna; Serour, Francis; Chaouat, Malka; Gonen, Pinhas; Tommasino, Massimo; Sherman, Levana

    2014-11-15

    In the present study, E6E7 and E6 proteins of human papillomaviruses (HPVs) associated with skin warts and cancer were compared for their transforming and carcinogenic abilities in primary human keratinocytes (PHKs). We show that E6E7 of cancer associated beta HPV types, notably 49 and 24, were able to extend the life span and enhance the clonogenic efficiency of PHKs when maintained in serum free/low calcium medium. Activities of the beta HPV E6E7 were lower than those of HPV16 E6E7. In contrast, E6 proteins from HPV types detected in skin warts or cancer, notably 10, 49 and 38, attenuated UVB induced protective responses in PHKs including cell death, proliferation arrest and accumulation of the proapoptotic proteins, p53, bax or bak. Together, this investigation revealed functional differences and commonalities between HPVs associated with skin warts and cancer, and allowed the identification of specific properties of beta HPVs supporting their involvement in skin carcinogenesis. - Highlights: • Primary keratinocytes were used to evaluate transforming and carcinogenic abilities of cutaneous HPVs. • E6E7 of cancer associated β HPV types transform primary human keratinocytes. • E6 proteins of cancer and wart associated HPVs inhibit UVB induced cell death. • E6s of cancer and wart associated HPVs attenuate UVB induced proliferation arrest. • E6s of cancer and wart associated HPVs attenuate UVB induced apoptosis signaling.

  7. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RAR? protein degradation

    PubMed Central

    Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-01-01

    Arsenic trioxide (As2O3) is one of the most effective therapeutic agents used for patients with acute promyelocytic leukemia (APL). The probable explanation for As2O3-induced cell differentiation is the direct targeting of PML-RAR? oncoprotein by As2O3, which results in initiation of PML-RARa degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII), therefore, it remains unknown that which arsenic specie is actually responsible for the therapeutic effects against APL. Here we have shown the role of As2O3 (as iAsIII) and its intermediate metabolites (i.e., MMAIII/DMAIII) in NB4 cells. Inorganic iAsIII predominantly showed induction of cell differentiation, while MMAIII and DMAIII specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAsIII, MMAIII showed stronger binding affinity for ring domain of PML recombinant protein, however, could not induce PML protein SUMOylation and ubiquitin/proteasome degradation. In summary, our results suggest that the binding of arsenicals to the ring domain of PML proteins is not associated with the degradation of PML-RARa fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  8. A basal-like breast cancer-specific role for SRF-IL6 in YAP-induced cancer stemness.

    PubMed

    Kim, Tackhoon; Yang, Suk-Jin; Hwang, Daehee; Song, Jinhoi; Kim, Minchul; Kyum Kim, Sang; Kang, Keunsoo; Ahn, Jaebum; Lee, Daeyoup; Kim, Mi-Young; Kim, Seyun; Seung Koo, Ja; Seok Koh, Sang; Kim, Seon-Young; Lim, Dae-Sik

    2015-01-01

    The switch between stem/progenitor cell expansion and differentiation is critical for organ homeostasis. The mammalian Hippo pathway effector and oncoprotein YAP expands undifferentiated stem/progenitor cells in various tissues. However, the YAP-associated transcription factors and downstream targets underlying this stemness-promoting activity are poorly understood. Here we show that the SRF-IL6 axis is the critical mediator of YAP-induced stemness in mammary epithelial cells and breast cancer. Specifically, serum response factor (SRF)-mediated binding and recruitment of YAP to mammary stem cell (MaSC) signature-gene promoters induce numerous MaSC signature genes, among which the target interleukin (IL)-6 is critical for YAP-induced stemness. High SRF-YAP/TAZ expression is correlated with IL6-enriched MaSC/basal-like breast cancer (BLBC). Finally, we show that this high SRF expression enables YAP to more efficiently induce IL6 and stemness in BLBC compared with luminal-type breast cancer. Collectively, our results establish the importance of SRF-YAP-IL6 signalling in promoting MaSC-like properties in a BLBC-specific manner. PMID:26671411

  9. IER3 is a crucial mediator of TAp73?-induced apoptosis in cervical cancer and confers etoposide sensitivity.

    PubMed

    Jin, Hanyong; Suh, Dae-Shik; Kim, Tae-Hyoung; Yeom, Ji-Hyun; Lee, Kangseok; Bae, Jeehyeon

    2015-01-01

    Infection with high-risk human papillomaviruses (HPVs) causes cervical cancer. E6 oncoprotein, an HPV gene product, inactivates the major gatekeeper p53. In contrast, its isoform, TAp73?, has become increasingly important, as it is resistant to E6. However, the intracellular signaling mechanisms that account for TAp73? tumor suppressor activity in cervix are poorly understood. Here, we identified that IER3 is a novel target gene of TAp73?. In particular, TAp73? exclusively transactivated IER3 in cervical cancer cells, whereas p53 and TAp63 failed to do. IER3 efficiently induced apoptosis, and its knockdown promoted survival of HeLa cells. In addition, TAp73?-induced cell death, but not p53-induced cell death, was inhibited upon IER3 silencing. Moreover, etoposide, a DNA-damaging chemotherapeutics, upregulated TAp73? and IER3 in a c-Abl tyrosine kinase-dependent manner, and the etoposide chemosensitivity of HeLa cells was largely determined by TAp73?-induced IER3. Of interest, cervical carcinomas from patients express no observable levels of two proteins. Thus, our findings suggest that IER3 is a putative tumor suppressor in the cervix, and the c-Ab1/p73?/IER3 axis is a novel and crucial signaling pathway that confers etoposide chemosensitivity. Therefore, TAp73? and IER3 induction would be a valuable checkpoint for successful therapeutic intervention of cervical carcinoma patients. PMID:25666857

  10. IER3 is a crucial mediator of TAp73?-induced apoptosis in cervical cancer and confers etoposide sensitivity

    PubMed Central

    Jin, Hanyong; Suh, Dae-Shik; Kim, Tae-Hyoung; Yeom, Ji-Hyun; Lee, Kangseok; Bae, Jeehyeon

    2015-01-01

    Infection with high-risk human papillomaviruses (HPVs) causes cervical cancer. E6 oncoprotein, an HPV gene product, inactivates the major gatekeeper p53. In contrast, its isoform, TAp73?, has become increasingly important, as it is resistant to E6. However, the intracellular signaling mechanisms that account for TAp73? tumor suppressor activity in cervix are poorly understood. Here, we identified that IER3 is a novel target gene of TAp73?. In particular, TAp73? exclusively transactivated IER3 in cervical cancer cells, whereas p53 and TAp63 failed to do. IER3 efficiently induced apoptosis, and its knockdown promoted survival of HeLa cells. In addition, TAp73?-induced cell death, but not p53-induced cell death, was inhibited upon IER3 silencing. Moreover, etoposide, a DNA-damaging chemotherapeutics, upregulated TAp73? and IER3 in a c-Abl tyrosine kinase-dependent manner, and the etoposide chemosensitivity of HeLa cells was largely determined by TAp73?-induced IER3. Of interest, cervical carcinomas from patients express no observable levels of two proteins. Thus, our findings suggest that IER3 is a putative tumor suppressor in the cervix, and the c-Ab1/p73?/IER3 axis is a novel and crucial signaling pathway that confers etoposide chemosensitivity. Therefore, TAp73? and IER3 induction would be a valuable checkpoint for successful therapeutic intervention of cervical carcinoma patients. PMID:25666857

  11. A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

    PubMed Central

    Kim, Tackhoon; Yang, Suk-Jin; Hwang, Daehee; Song, Jinhoi; Kim, Minchul; Kyum Kim, Sang; Kang, Keunsoo; Ahn, Jaebum; Lee, Daeyoup; Kim, Mi-young; Kim, Seyun; Seung Koo, Ja; Seok Koh, Sang; Kim, Seon-Young; Lim, Dae-Sik

    2015-01-01

    The switch between stem/progenitor cell expansion and differentiation is critical for organ homeostasis. The mammalian Hippo pathway effector and oncoprotein YAP expands undifferentiated stem/progenitor cells in various tissues. However, the YAP-associated transcription factors and downstream targets underlying this stemness-promoting activity are poorly understood. Here we show that the SRF–IL6 axis is the critical mediator of YAP-induced stemness in mammary epithelial cells and breast cancer. Specifically, serum response factor (SRF)-mediated binding and recruitment of YAP to mammary stem cell (MaSC) signature-gene promoters induce numerous MaSC signature genes, among which the target interleukin (IL)-6 is critical for YAP-induced stemness. High SRF–YAP/TAZ expression is correlated with IL6-enriched MaSC/basal-like breast cancer (BLBC). Finally, we show that this high SRF expression enables YAP to more efficiently induce IL6 and stemness in BLBC compared with luminal-type breast cancer. Collectively, our results establish the importance of SRF–YAP–IL6 signalling in promoting MaSC-like properties in a BLBC-specific manner. PMID:26671411

  12. Functional inactivation of Rb sensitizes cancer cells to TSC2 inactivation induced cell Death

    PubMed Central

    Danos, Arpad M.; Liao, Yang; Li, Xuan; Du, Wei

    2012-01-01

    We showed previously that inactivation of TSC2 induces death in cancer cells lacking the Retinoblastoma (Rb) tumor suppressor under stress conditions, suggesting that inactivation of TSC2 can potentially be used as an approach to specifically kill cancers that have lost WT Rb. As Rb is often inactivated in cancers by overexpression of cyclin D1, loss of p16ink4a cdk inhibitor, or expression of viral oncoproteins, it will be interesting to determine if such functional inactivation of Rb would similarly sensitize cancer cells to TSC2 inactivation induced cell death. In addition, many cancers lack functional Pten, resulting in increased PI3K/Akt signaling that has been shown to modulate E2F-induced cell death. Therefore it will be interesting to test whether loss of Pten will affect TSC2 inactivation induced killing of Rb mutant cancer cells. Here, we show that overexpression of Cyclin D1 or the viral oncogene E1a sensitizes cancer cells to TSC2 knockdown induced cell death and growth inhibition. On the other hand, knockdown of p16ink4a sensitizes cancer cells to TSC2 knockdown induced cell death in a manner that is likely dependant on serum induction of Cyclin D1 to inactivate the Rb function. Additionally, we demonstrate that loss of Pten does not interfere with TSC2 knockdown induced cell death in Rb mutant cancer cells. Together, these results suggest that TSC2 is potentially a useful target for a large spectrum of cancer types with an inactivated Rb pathway. PMID:23022476

  13. 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

    2011-01-01

    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. PMID:21306638

  14. Phosphoproteome Dynamics in Onset and Maintenance of Oncogene-induced Senescence*

    PubMed Central

    de Graaf, Erik L.; Kaplon, Joanna; Zhou, Houjiang; Heck, Albert J. R.; Peeper, Daniel S.; Altelaar, A. F. Maarten

    2014-01-01

    Expression of the BRAFV600E oncoprotein is known to cause benign lesions, such as melanocytic nevi (moles). Despite the oncogenic function of mutant BRAF, these lesions are arrested by a cell-autonomous mechanism called oncogene-induced senescence. Infrequently, nevi can progress to malignant melanoma, through mechanisms that are incompletely understood. To gain more insight into this vital tumor-suppression mechanism, we performed a mass-spectrometry-based screening of the proteome and phosphoproteome in cycling and senescent cells and in cells with abrogated senescence. Proteome analysis of senescent cells revealed the up-regulation of established senescence biomarkers, including specific cytokines, but also several proteins not previously associated with senescence, including extracellular matrix-interacting. Using both general and targeted phosphopeptide enrichment by Ti4+-IMAC and phosphotyrosine antibody enrichment, we identified over 15,000 phosphorylation sites. Among the regulated phosphorylation sites we encountered components of the interleukin, BRAF/MAPK, and CDK-retinoblastoma pathways and several other factors. The extensive proteome and phosphoproteome dataset of BRAFV600E-expressing senescent cells provides molecular clues as to how oncogene-induced senescence is initiated, maintained, or evaded, serving as a comprehensive proteomic basis for functional validation. PMID:24961811

  15. Pokemon (FBI-1) interacts with Smad4 to repress TGF-β-induced transcriptional responses.

    PubMed

    Yang, Yutao; Cui, Jiajun; Xue, Feng; Zhang, Chuanfu; Mei, Zhu; Wang, Yue; Bi, Mingjun; Shan, Dapeng; Meredith, Alex; Li, Hui; Xu, Zhi-Qing David

    2015-03-01

    Pokemon, an important proto-oncoprotein, is a transcriptional repressor that belongs to the POK (POZ and Krüppel) family. Smad4, a key component of TGF-β pathway, plays an essential role in TGF-β-induced transcriptional responses. In this study, we show that Pokemon can interact directly with Smad4 both in vitro and in vivo. Overexpression of Pokemon decreases TGF-β-induced transcriptional activities, whereas knockdown of Pokemon increases these activities. Interestingly, Pokemon does not affect activation of Smad2/3, formation of Smads complex, or DNA binding activity of Smad4. TGF-β1 treatment increases the interaction between Pokemon and Smad4, and also enhances the recruitment of Pokemon to Smad4-DNA complex. In addition, we also find that Pokemon recruits HDAC1 to Smad4 complex but decreases the interaction between Smad4 and p300/CBP. Taken together, all these data suggest that Pokemon is a new partner of Smad4 and plays a negative role in TGF-β pathway. PMID:25514493

  16. Ethoxysanguinarine Induces Inhibitory Effects and Downregulates CIP2A in Lung Cancer Cells.

    PubMed

    Liu, Zi; Ma, Liang; Wen, Zhe-Sheng; Cheng, Yong-Xian; Zhou, Guang-Biao

    2014-02-13

    Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that is able to stabilize c-Myc oncogenic transcription factor and promote proliferation and transformation of cells. CIP2A is overexpressed in many primary tumors, and pharmacological inactivation of CIP2A is an emerging concept for the development of novel anticancer agents. In this study, we demonstrate that overexpression of CIP2A predicts poor prognosis in lung cancer, and a natural compound, ethoxysanguinarine (ESG), effectively downregulates CIP2A protein and its downstream signaling molecules, c-Myc and pAkt, and induces protein phosphatase 2A (PP2A) activity. ESG inhibits proliferation and induces apoptosis of lung cancer cells, and enhances the effects of cisplatin on malignant cells. Taken together, our findings demonstrate that CIP2A is inversely associated with the clinical outcome of lung cancer, and ESG can serve as a lead compound for the development of CIP2A inhibitor for cancer therapies. PMID:24900782

  17. Human Papillomavirus Type 8 Interferes with a Novel C/EBP?-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration

    PubMed Central

    Walch-Rckheim, Barbara; Wickenhauser, Claudia; Doorbar, John; Pfister, Herbert; Malejczyk, Magdalena; Majewski, S?awomir; Keates, Andrew C.; Smola, Sigrun

    2012-01-01

    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

  18. Pigment Epithelium-Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia.

    PubMed

    Goldberg, Keren; Bar-Joseph, Hadas; Grossman, Hadas; Hasky, Noa; Uri-Belapolsky, Shiri; Stemmer, Salomon M; Chuderland, Dana; Shalgi, Ruth; Ben-Aharon, Irit

    2015-12-01

    Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptor-positive breast cancer. Its significant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamoxifen. Using in vivo mouse models, we demonstrated that tamoxifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uterine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant transformation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demonstrated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-? and c-Myc oncoprotein levels were reduced. Our findings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamoxifen-induced endometrial pathologies. Mol Cancer Ther; 14(12); 2840-9. 2015 AACR. PMID:26450919

  19. Tristetraprolin is a tumor suppressor that impairs Myc-induced lymphoma and abolishes the malignant state

    PubMed Central

    Rounbehler, Robert J.; Fallahi, Mohammad; Yang, Chunying; Steeves, Meredith A.; Li, Weimin; Doherty, Joanne R.; Schaub, Franz X.; Sanduja, Sandhya; Dixon, Dan A.; Blackshear, Perry J.; Cleveland, John L.

    2012-01-01

    SUMMARY Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis. PMID:22863009

  20. Induced Abortion

    MedlinePLUS

    ... AQ FREQUENTLY ASKED QUESTIONS FAQ043 SPECIAL PROCEDURES Induced Abortion What is an induced abortion? What is a first-trimester abortion? How is a first-trimester surgical abortion performed? ...

  1. HCCR-1, a novel oncogene, encodes a mitochondrial outer membrane protein and suppresses the UVC-induced apoptosis

    PubMed Central

    Cho, Goang-Won; Shin, Seung Min; Kim, Hyun Kee; Ha, Seon-Ah; Kim, Sanghee; Yoon, Joo-Hee; Hur, Soo Young; Kim, Tae Eung; Kim, Jin Woo

    2007-01-01

    Background The Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene. Results To investigate the biological function of HCCR-1 in the cell, we predicted biological features using bioinformatic tools, and have identified a LETM1 homologous domain at position 75 to 346 of HCCR-1. This domain contains proteins identified from diverse species predicted to be mitochondrial proteins. Fluorescence microscopy and fractionation experiments showed that HCCR-1 is located in mitochondria in the COS-7, MCF-7 and HEK/293 cell lines, and subcompartamentally at the outer membrane in the HEK/293 cell line. The topological structure was revealed as the NH2-terminus of HCCR-1 oriented toward the cytoplasm. We also observed that the D1-2 region, at position 1 to 110 of HCCR-1, was required and sufficient for posttranslational mitochondrial import. The function of HCCR-1 on mitochondrial membrane is to retard the intrinsic apoptosis induced by UVC and staurosporine, respectively. Conclusion Our experiments show the biological features of HCCR-1 in the cell, and suggest that uncontrolled expression of HCCR-1 may cause mitochondrial dysfunction that can result in resisting the UVC or staurosporine-induced apoptosis and progressing in the tumor formation. PMID:18045496

  2. STAT5 is crucial to maintain leukemic stem cells in acute myelogenous leukemias induced by MOZ-TIF2.

    PubMed

    Tam, Winnie F; Hhnel, Patricia S; Schler, Andrea; Lee, Benjamin H; Okabe, Rachel; Zhu, Nan; Pante, Saskia V; Raffel, Glen; Mercher, Thomas; Wernig, Gerlinde; Bockamp, Ernesto; Sasca, Daniel; Kreft, Andreas; Robinson, Gertraud W; Hennighausen, Lothar; Gilliland, D Gary; Kindler, Thomas

    2013-01-01

    MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2-induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2-transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5(-/-) MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5(+/+) MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option. PMID:23149921

  3. STAT5 Is Crucial to Maintain Leukemic Stem Cells in Acute Myelogenous Leukemias Induced by MOZ-TIF2

    PubMed Central

    Tam, Winnie F.; Hhnel, Patricia S.; Schler, Andrea; Lee, Benjamin H.; Okabe, Rachel; Zhu, Nan; Pante, Saskia V.; Raffel, Glen; Mercher, Thomas; Wernig, Gerlinde; Bockamp, Ernesto; Sasca, Daniel; Kreft, Andreas; Robinson, Gertraud W.; Hennighausen, Lothar; Gilliland, D. Gary; Kindler, Thomas

    2014-01-01

    MOZ-TIF2 is a leukemogenic fusion oncoprotein that confers self-renewal capability to hematopoietic progenitor cells and induces acute myelogenous leukemia (AML) with long latency in bone marrow transplantation assays. Here, we report that FLT3-ITD transforms hematopoietic cells in cooperation with MOZ-TIF2 in vitro and in vivo. Coexpression of FLT3-ITD confers growth factor independent survival/proliferation, shortens disease latency, and results in an increase in the number of leukemic stem cells (LSC). We show that STAT5, a major effector of aberrant FLT3-ITD signal transduction, is both necessary and sufficient for this cooperative effect. In addition, STAT5 signaling is essential for MOZ-TIF2induced leukemic transformation itself. Lack of STAT5 in fetal liver cells caused rapid differentiation and loss of replating capacity of MOZ-TIF2transduced cells enriched for LSCs. Furthermore, mice serially transplanted with Stat5?/? MOZ-TIF2 leukemic cells develop AML with longer disease latency and finally incomplete penetrance when compared with mice transplanted with Stat5+/+ MOZ-TIF2 leukemic cells. These data suggest that STAT5AB is required for the self-renewal of LSCs and represents a combined signaling node of FLT3-ITD and MOZ-TIF2 driven leukemogenesis. Therefore, targeting aberrantly activated STAT5 or rewired downstream signaling pathways may be a promising therapeutic option. PMID:23149921

  4. Association of human papillomavirus with Fanconi anemia promotes carcinogenesis in Fanconi anemia patients.

    PubMed

    Liu, Guang Bin; Chen, Jiezhong; Wu, Zhan He; Zhao, Kong-Nan

    2015-11-01

    Fanconi anemia (FA) is a rare recessive disorder associated with chromosomal fragility. FA patients are at very high risk of cancers, especially head and neck squamous cell carcinomas and squamous cell carcinomas caused by infection of human papillomaviruses (HPVs). By integrating into the host genome, HPV oncogenes E6 and E7 drive the genomic instability to promote DNA damage and gene mutations necessary for carcinogenesis in FA patients. Furthermore, E6 and E7 oncoproteins not only inhibit p53 and retinoblastoma but also impair the FANC/BRCA signaling pathway to prevent DNA damage repair and alter multiple signals including cell-cycle checkpoints, telomere function, cell proliferation, and interference of the host immune system leading to cancer development in FA patients. In this review, we summarize recent advances in unraveling the molecular mechanisms of FA susceptibility to HPV-induced cancers, which facilitate rational preventive and therapeutic strategies. PMID:25776992

  5. The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth

    PubMed Central

    Secombe, Julie; Li, Ling; Carlos, Leni; Eisenman, Robert N.

    2007-01-01

    The Myc oncoprotein is a potent inducer of cell growth, cell cycle progression, and apoptosis. While many direct Myc target genes have been identified, the molecular determinants of Mycs transcriptional specificity remain elusive. We have carried out a genetic screen in Drosophila and identified the Trithorax group protein Little imaginal discs (Lid) as a regulator of dMyc-induced cell growth. Lid binds to dMyc and is required for dMyc-induced expression of the growth regulatory gene Nop60B. The mammalian Lid orthologs, Rbp-2 (JARID1A) and Plu-1 (JARID1B), also bind to c-Myc, indicating that LidMyc function is conserved. We demonstrate that Lid is a JmjC-dependent trimethyl H3K4 demethylase in vivo and that this enzymatic activity is negatively regulated by dMyc, which binds to Lids JmjC domain. Because Myc binding is associated with high levels of trimethylated H3K4, we propose that the LiddMyc complex facilitates Myc binding to, or maintenance of, this chromatin context. PMID:17311883

  6. Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1

    PubMed Central

    Shahbazi, Jeyran; Scarlett, Christopher J.; Norris, Murray D.; Liu, Bing; Haber, Michelle; Tee, Andrew E.; Carrier, Alice; Biankin, Andrew V.; London, Wendy B.; Marshall, Glenn M.; Lock, Richard B.; Liu, Tao

    2014-01-01

    Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. Here we aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis. TP53INP1 was found to be one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma. PMID:24952595

  7. Vanadium-induced apoptosis of HaCaT cells is mediated by c-fos and involves nuclear accumulation of clusterin

    PubMed Central

    Markopoulou, Soultana; Kontargiris, Evangelos; Batsi, Christina; Tzavaras, Theodore; Trougakos, Ioannis; Boothman, David A.; Gonos, Efstathios S.; Kolettas, Evangelos

    2016-01-01

    Vanadium exerts a variety of biological effects, including antiproliferative responses through activation of the respective signaling pathways and the generation of reactive oxygen species. As epidermal cells are exposed to environmental insults, human keratinocytes (HaCaT) were used to investigate the mechanism of the antiproliferative effects of vanadyl(IV) sulfate (VOSO4). Treatment of HaCaT cells with VOSO4 inhibited proliferation and induced apoptosis in a dose-dependent manner. Inhibition of proliferation was associated with downregulation of cyclins D1 and E, E2F1, and the cyclin-dependent kinase inhibitors p21Cip1/Waf1 and p27Kip1. Induction of apoptosis correlated with upregulation of the c-fos oncoprotein, changes in the expression of clusterin (CLU), an altered ratio of antiapoptotic to proapoptotic Bcl-2 protein family members, and poly(ADP-ribose) poly-merase-1 cleavage. Forced overexpression of c-fos induced apoptosis in HaCaT cells that correlated with secretory CLU downregulation and upregulation of nuclear CLU (nCLU), a pro-death protein. Overexpression of Bcl-2 protected HaCaT cells from vanadium-induced apoptosis, whereas secretory CLU overexpression offered no cytoprotection. In contrast, nCLU sensitized HaCaT cells to apoptosis. Our data suggest that vanadium-mediated apoptosis was promoted by c-fos, leading to alterations in CLU isoform processing and induction of the pro-death nCLU protein. PMID:19531052

  8. 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 Central

    Bourgault Villada, I; Moyal Barracco, M; Berville, S; Bafounta, M L; Longvert, C; Prmel, V; Villefroy, P; Jullian, E; Clerici, T; Paniel, B; Maillre, B; Choppin, J; Guillet, J G

    2010-01-01

    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 (1434) and/or E6/4 (4568) peptides, which were identified in the present study as immunodominant among HPV-16 E6 and E7 large peptides. Ex vivo enzyme-linked immunospotinterferon (IFN)-? 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 (1434) and E6/4 (4568) 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 (1434) and E6/4 (4568) fragments thus appear to be good candidates to increase HPV-specific effector T lymphocyte responses and clear classic VIN (BP) disease lesions. PMID:19843089

  9. The Effect of Lactobacillus crispatus and Lactobacillus rhamnosusCulture Supernatants on Expression of Autophagy Genes and HPV E6 and E7 Oncogenes in The HeLa Cell Line

    PubMed Central

    Motevaseli, Elahe; Azam, Rosa; Akrami, Seyed Mohammad; Mazlomy, Mohammadali; Saffari, Mojtaba; Modarressi, Mohammad Hossein; Daneshvar, Maryam; Ghafouri-Fard, Soudeh

    2016-01-01

    Objective The aim of this study was to clarify the mechanism by which lactobacilli exert their cytotoxic effects on cervical cancer cells. In addition, we aimed to evalu- ate the effect of lactobacilli on the expression of human papilloma virus (HPV) onco- genes. Materials and Methods In this experimental study, using quantitative real-time polymer- ase chain reaction (PCR), we analyzed the expression of CASP3 and three autophagy genes [ATG14, BECN1 and alpha 2 catalytic subunit of AMPK (PRKAA2)] along with HPV18 E6 and E7 genes in HeLa cells before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants. Results The expression of CASP3 and autophagy genes in HeLa cells was de- creased after treatment with lactobacilli culture supernatants. However, this de- crease was not significant for PRKAA2 when compared with controls. In addition, expression of HPV E6 was significantly decreased after treatment with lactobacilli culture supernatants. Conclusion Lactobacilli culture supernatants can decrease expression of ATG14 and BECN1 as well as the HPV E6 oncogene. It has been demonstrated that the main changes occurring during cervical carcinogenesis in cell machinery can be reversed by suppression of HPV oncogenes. Therefore, downregulation of HPV E6 by lacto- bacilli may have therapeutic potential for cervical cancer. As the role of autophagy in cancer is complicated, further work is required to clarify the link between downregula- tion of autophagy genes and antiproliferative effects exerted by lactobacilli. PMID:26862519

  10. Electrochemical quartz crystal microbalance, voltammetry, spectroelectrochemical, and microscopic studies of adsorption behavior for (7E,7{prime}Z)-diphenyl-7,7{prime}-diapocarotene electrochemical oxidation product

    SciTech Connect

    Gao, G.; Wurm, D.B.; Kim, Y.T.; Kispert, L.D.

    1997-03-13

    Polymeric products, which are formed by reaction of the dications of (7E,7{prime}Z)-diphenyl-7,7{prime}-diapocarotene (I) generated by electrochemical oxidation in dichloromethane with the neutral carotenoid, are adsorbed on various electrode surfaces. An apparent average molar mass of 5400 g/(mol electrons) was calculated from simultaneous electrochemical quartz crystal microbalance (EQCM) measurements, and the green, fiber-like structure observed by optical microscopy confirms the formation of polymers. X-ray microanalysis of the surface composed of an uneven, layered structure indicates that electrolyte counter anions PF{sub 6}{sup -} are associated with the deposited material. Cathodic stripping voltammetry indicates that the film thickness ranges from 0.16 to 0.84 {mu}m as the charge increases from 10.0 to 51.1 {mu}C. Cation radicals of I show no adsorption behavior nor do the dications of carotenoids terminally substituted with one cyclohexene ring (V) or containing a triple bond at C15 (IV). Apparently a diphenyl-substituted carotenoid containing only double bonds in the backbone is required to observe this unusual behavior. 57 refs., 12 figs., 1 tab.

  11. Synthesis of 4-((1E, 6E)-7-(4-hydroxy-3-methoxyphenyl)-3, 5-dioxohepta-1, 6-dienyl)-2-methoxyphenyl 4-fluorobenzoate, a novel monoester derivative of curcumin, its experimental and theoretical (DFT) studies

    NASA Astrophysics Data System (ADS)

    Srivastava, Sangeeta; Gupta, Preeti; Amandeep; Singh, Ranvijay Pratap

    2016-04-01

    Curcumin (1), isolated as a major component from the chloroform extract of Curcuma longa was converted to its ester derivative 4-((1E, 6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxohepta-1,6-dienyl)-2-methoxyphenyl 4-fluorobenzoate (2). The compound has been characterized with the help of 1H, 13C NMR, UV, IR and mass spectrometry. The molecular geometry of synthesized compound was calculated in ground state by Density functional theory (DFT/B3LYP) using 6-31G (d,p) basis set. 1H and 13C NMR chemical shifts were calculated in ground state by using Gauge-Including Atomic Orbital (GIAO) approach and these values were correlated with experimental observations. The electronic properties such as HOMO and LUMO energies were calculated using time dependent Density Functional Theory (TD-DFT). Stability of the molecule as a result of hyper conjugative interactions and electron delocalization were analysed using Natural bond orbital (NBO) analysis. Intramolecular interactions were analysed by AIM (Atom in molecule) approach. Global reactivity descriptors were calculated to study the reactive site within molecule. The vibrational wavenumbers were calculated using DFT method and assigned with the help of potential energy distribution (PED). First hyperpolarizability values have been calculated to describe the nonlinear optical (NLO) property of the synthesized compounds. Molecular electrostatic potential (MEP) analysis has also been carried out.

  12. Selection of a recombinant Marek's disease virus in vivo through expression of the Marek's EcoRI-Q (Meq)-encoded oncoprotein: characterization of an rMd5-based mutant expressing the Meq of strain RB-1B.

    PubMed

    Kumar, Pankaj; Dong, Huimin; Lenihan, Dawn; Gaddamanugu, Syamsundar; Katneni, Upendra; Shaikh, Shireen; Tavlarides-Hontz, Phaedra; Reddy, Sanjay M; Peters, Wachen; Parcells, Mark S

    2012-06-01

    Marek's disease (MD) is a highly contagious viral disease of chickens (Gallus gallus domesticus) caused by MD virus (MDV), characterized by paralysis, neurologic signs, and the rapid onset of T-cell lymphomas. MDV-induced T-cell transformation requires a basic leucine zipper protein called Marek's EcoRI-Q-encoded protein (Meq). We have identified mutations in the coding sequence of Meq that correlated with virus pathotype (virulent, very virulent, and very virulent plus). The aim of this study was to determine whether recombinant viruses could be isolated based on Meq expression through in vivo selection. Chicken embryo fibroblasts (CEFs) were cotransfected with an rMd5 strain-based Meq deletion virus (rMd5deltaMeq) and meq loci from strains representing different pathotypes of MDV. Transfected CEFs were inoculated into chickens in two independent studies. We were able to isolate a single recombinant virus, rMDV-1137, in a contact-exposed chicken. rMDV-1137 had recombined two copies of the meq gene of RB-1B and was found to have pathogenicity similar to both RB-1B and rMd5 parental strains. We found the RB-1B- and rMd5-induced lymphomas showed differences in composition and that rMDV-1137-induced lymphomas were intermediate in their composition. We were able to establish cell lines from both RB-1B- (MDCC-UD35, -UD37) and rMDV-1137 (MDCC-UD36, -UD38)-induced, but not rMd5-induced, lymphomas. To date, no rMd5- or parent Md5-transformed T-cell lines have been reported. Our results suggest that 1) a recombinant MDV can be selected on the basis of oncogenicity; 2) changes in Meq sequence seem to affect tumor composition and the ability to establish cell lines; and 3) in addition to meq, other genomic loci affect MDV pathogenicity and oncogenicity. PMID:22856190

  13. Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

    SciTech Connect

    Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

    2009-02-05

    Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.

  14. Inhibition of Myc-induced cell transformation by brain acid-soluble protein 1 (BASP1)

    PubMed Central

    Hartl, Markus; Nist, Andrea; Khan, M. Imran; Valovka, Taras; Bister, Klaus

    2009-01-01

    Cell transformation by the Myc oncoprotein involves transcriptional activation or suppression of specific target genes with intrinsic oncogenic or tumor-suppressive potential, respectively. We have identified the BASP1 (CAP-23, NAP-22) gene as a novel target suppressed by Myc. The acidic 25-kDa BASP1 protein was originally isolated as a cortical cytoskeleton-associated protein from rat and chicken brain, but has also been found in other tissues and subcellular locations. BASP1 mRNA and protein expression is specifically suppressed in fibroblasts transformed by the v-myc oncogene, but not in cells transformed by other oncogenic agents. The BASP1 gene encompasses 2 exons separated by a 58-kbp intron and a Myc-responsive regulatory region at the 5? boundary of untranslated exon 1. Bicistronic expression of BASP1 and v-myc from a retroviral vector blocks v-myc-induced cell transformation. Furthermore, ectopic expression of BASP1 renders fibroblasts resistant to subsequent cell transformation by v-myc, and exogenous delivery of the BASP1 gene into v-myc-transformed cells leads to significant attenuation of the transformed phenotype. The inhibition of v-myc-induced cell transformation by BASP1 also prevents the transcriptional activation or repression of known Myc target genes. Mutational analysis showed that the basic N-terminal domain containing a myristoylation site, a calmodulin binding domain, and a putative nuclear localization signal is essential for the inhibitory function of BASP1. Our results suggest that down-regulation of the BASP1 gene is a necessary event in myc-induced oncogenesis and define the BASP1 protein as a potential tumor suppressor. PMID:19297618

  15. B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

    PubMed

    Herr, Ricarda; Khler, Martin; Andrlov, Hana; Weinberg, Florian; Mller, Yvonne; Halbach, Sebastian; Lutz, Lisa; Mastroianni, Justin; Klose, Martin; Bittermann, Nicola; Kowar, Silke; Zeiser, Robert; Olayioye, Monilola A; Lassmann, Silke; Busch, Hauke; Boerries, Melanie; Brummer, Tilman

    2015-01-01

    BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf(V600E) oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf(V600E) decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf(V600E) in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells. PMID:25381152

  16. Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model

    PubMed Central

    Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M.; Lukianov, Stefan; Jenkins, Frank J.; Honda, Kord; Maricich, Stephen M.; Moore, Patrick S.; Chang, Yuan

    2015-01-01

    Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting. PMID:26544690

  17. Human and primate tumour viruses use PDZ binding as an evolutionarily conserved mechanism of targeting cell polarity regulators

    PubMed Central

    Tomai?, V; Gardiol, D; Massimi, P; Ozbun, M; Myers, M; Banks, L

    2013-01-01

    A unique feature of the cancer-causing mucosotropic human papillomaviruses (HPVs) is the ability of their E6 proteins to interact with a number of PDZ domain-containing cellular substrates, including the cell polarity regulators hDlg and hScrib. These interactions are essential for the ability of these viruses to induce malignant progression. Rhesus papillomaviruses (RhPV) are similar to their human counterparts in that they also cause anogenital malignancy in their host, the Rhesus Macaque. However, unlike HPV E6, the RhPV E6 has no PDZ-binding motif. We now show that such a motif is present on the RhPV E7 oncoprotein. This motif specifically confers PDZ-binding activity and directs the interaction of RhPV E7 with the cell polarity regulator Par3, which it targets for proteasome-mediated degradation. These results demonstrate an amazing evolutionary conservation of function between the RhPV and the HPV oncoproteins, where both target proteins of the same cell polarity control network, although through different components and pathways. PMID:18820705

  18. Human and primate tumour viruses use PDZ binding as an evolutionarily conserved mechanism of targeting cell polarity regulators.

    PubMed

    Tomai?, V; Gardiol, D; Massimi, P; Ozbun, M; Myers, M; Banks, L

    2009-01-01

    A unique feature of the cancer-causing mucosotropic human papillomaviruses (HPVs) is the ability of their E6 proteins to interact with a number of PDZ domain-containing cellular substrates, including the cell polarity regulators hDlg and hScrib. These interactions are essential for the ability of these viruses to induce malignant progression. Rhesus papillomaviruses (RhPV) are similar to their human counterparts in that they also cause anogenital malignancy in their host, the Rhesus Macaque. However, unlike HPV E6, the RhPV E6 has no PDZ-binding motif. We now show that such a motif is present on the RhPV E7 oncoprotein. This motif specifically confers PDZ-binding activity and directs the interaction of RhPV E7 with the cell polarity regulator Par3, which it targets for proteasome-mediated degradation. These results demonstrate an amazing evolutionary conservation of function between the RhPV and the HPV oncoproteins, where both target proteins of the same cell polarity control network, although through different components and pathways. PMID:18820705

  19. The presence of high-risk human papillomavirus (HPV) E6/E7 mRNA transcripts in a subset of sinonasal carcinomas is evidence of involvement of HPV in its etiopathogenesis.

    PubMed

    Laco, Jan; Sieglová, Kateřina; Vošmiková, Hana; Dundr, Pavel; Němejcová, Kristýna; Michálek, Jaroslav; Čelakovský, Petr; Chrobok, Viktor; Mottl, Radovan; Mottlová, Alena; Tuček, Luboš; Slezák, Radovan; Chmelařová, Marcela; Sirák, Igor; Vošmik, Milan; Ryška, Aleš

    2015-10-01

    The aim of the study was to investigate prevalence of high-risk human papillomavirus (HR-HPV) infection in sinonasal carcinomas by immunohistochemistry, in situ hybridization, and polymerase chain reaction, detecting p16(INK4a) protein (p16) expression and presence of both HPV DNA and HPV E6/E7 messenger RNA (mRNA). The study comprised 47 males and 26 females, aged 23-83 years (median 62 years), mostly (67 %) with a squamous cell carcinoma (SCC). Of the tumors, 53 % arose in the nasal cavity, 42 % in the maxillary sinus, and 5 % in the ethmoid complex. The follow-up period ranged 1-241 months (median 19 months). HPV16, HPV18, or HPV35 were detected in 18/73 (25 %) tumors, 17 SCCs, and 1 small cell neuroendocrine carcinoma. There was a strong correlation between results of HPV detection methods and p16 expression (p < 0.005). HPV-positive SCCs occurred more frequently in smokers (p = 0.04) and were more frequently p16-positive (p < 0.0001) and nonkeratinizing (p = 0.02), the latter occurring more commonly in nasal cavity (p = 0.025). Median survival for HPV-positive SCC patients was 30 months, while for HPV-negative SCC patients was 14 months (p = 0.23). In summary, we confirm that HR-HPV is actively involved in the etiopathogenesis of a significant subset of sinonasal SCCs. p16 may be used as a reliable surrogate marker for determination of HPV status also in sinonasal SCCs. Although we observed a trend toward better overall survival in HPV-positive SCCs, the prognostic impact of HPV status in sinonasal carcinomas needs to be elucidated by further studies. PMID:26229021

  20. Inducing labor

    MedlinePLUS

    ... method of inducing labor is to "break the bag of waters" or rupture the membranes. Your health care provider will do a pelvic exam and will guide a small plastic probe with a hook on the end through ...

  1. LMP1-Induced Sumoylation Influences the Maintenance of Epstein-Barr Virus Latency through KAP1

    PubMed Central

    Moss, Charles Randall; Whitehurst, Christopher B.; Moody, Cary A.

    2015-01-01

    ABSTRACT As a herpesvirus, Epstein-Barr virus (EBV) establishes a latent infection that can periodically undergo reactivation, resulting in lytic replication and the production of new infectious virus. Latent membrane protein-1 (LMP1), the principal viral oncoprotein, is a latency-associated protein implicated in regulating viral reactivation and the maintenance of latency. We recently found that LMP1 hijacks the SUMO-conjugating enzyme Ubc9 via its C-terminal activating region-3 (CTAR3) and induces the sumoylation of cellular proteins. Because protein sumoylation can promote transcriptional repression, we hypothesized that LMP1-induced protein sumoylation induces the repression of EBV lytic promoters and helps maintain the viral genome in its latent state. We now show that with inhibition of LMP1-induced protein sumoylation, the latent state becomes less stable or leakier in EBV-transformed lymphoblastoid cell lines. The cells are also more sensitive to viral reactivation induced by irradiation, which results in the increased production and release of infectious virus, as well as increased susceptibility to ganciclovir treatment. We have identified a target of LMP1-mediated sumoylation that contributes to the maintenance of latency in this context: KRAB-associated protein-1 (KAP1). LMP1 CTAR