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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. The HPV16 E7 Oncoprotein Disrupts Dendritic Cell Function and Induces the Systemic Expansion of CD11b+Gr1+ Cells in a Transgenic Mouse Model

    PubMed Central

    Damian-Morales, Gabriela; Serafín-Higuera, Nicolás; Moreno-Eutimio, Mario Adán; Cortés-Malagón, Enoc M.; Bonilla-Delgado, José; Rodríguez-Uribe, Genaro; Ocadiz-Delgado, Rodolfo; Lambert, Paul F.

    2016-01-01

    Objective. The aim of this study was to analyze the effects of the HPV16 E7 oncoprotein on dendritic cells (DCs) and CD11b+Gr1+ cells using the K14E7 transgenic mouse model. Materials and Methods. The morphology of DCs was analyzed in male mouse skin on epidermal sheets using immunofluorescence and confocal microscopy. Flow cytometry was used to determine the percentages of DCs and CD11b+Gr1+ cells in different tissues and to evaluate the migration of DCs. Results. In the K14E7 mouse model, the morphology of Langerhans cells and the migratory activity of dendritic cells were abnormal. An increase in CD11b+Gr1+ cells was observed in the blood and skin of K14E7 mice, and molecules related to CD11b+Gr1+ chemoattraction (MCP1 and S100A9) were upregulated. Conclusions. These data suggest that the HPV16 E7 oncoprotein impairs the function and morphology of DCs and induces the systemic accumulation of CD11b+Gr1+ cells. PMID:27478837

  3. The HPV16 E7 Oncoprotein Disrupts Dendritic Cell Function and Induces the Systemic Expansion of CD11b(+)Gr1(+) Cells in a Transgenic Mouse Model.

    PubMed

    Damian-Morales, Gabriela; Serafín-Higuera, Nicolás; Moreno-Eutimio, Mario Adán; Cortés-Malagón, Enoc M; Bonilla-Delgado, José; Rodríguez-Uribe, Genaro; Ocadiz-Delgado, Rodolfo; Lambert, Paul F; Gariglio, Patricio

    2016-01-01

    Objective. The aim of this study was to analyze the effects of the HPV16 E7 oncoprotein on dendritic cells (DCs) and CD11b(+)Gr1(+) cells using the K14E7 transgenic mouse model. Materials and Methods. The morphology of DCs was analyzed in male mouse skin on epidermal sheets using immunofluorescence and confocal microscopy. Flow cytometry was used to determine the percentages of DCs and CD11b(+)Gr1(+) cells in different tissues and to evaluate the migration of DCs. Results. In the K14E7 mouse model, the morphology of Langerhans cells and the migratory activity of dendritic cells were abnormal. An increase in CD11b(+)Gr1(+) cells was observed in the blood and skin of K14E7 mice, and molecules related to CD11b(+)Gr1(+) chemoattraction (MCP1 and S100A9) were upregulated. Conclusions. These data suggest that the HPV16 E7 oncoprotein impairs the function and morphology of DCs and induces the systemic accumulation of CD11b(+)Gr1(+) cells. PMID:27478837

  4. E6 and E7 oncoproteins from human papillomavirus type 16 induce activation of human transforming growth factor beta1 promoter throughout Sp1 recognition sequence.

    PubMed

    Peralta-Zaragoza, Oscar; Bermúdez-Morales, Víctor; Gutiérrez-Xicotencatl, Lourdes; Alcocer-González, Juan; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2006-01-01

    Human Papillomavirus (HPV) infection is the main etiologic agent of cervical cancer and HPV E6 and E7 oncogenes trans-regulate many cellular genes. An association between TGF-beta1 gene expression and cervical cancer development has been suggested; however, the mechanisms by which HPV influences TGF-beta1 expression remain unclear. In the present study we analyzed the mechanism through which HPV-16 E6 and E7 oncoproteins regulate the TGF-beta1 promoter in cervical tumor cells. Our results showed that E6 and E7 increased TGF-beta1 promoter activity. Furthermore, we identified a specific DNA sequence motif in the TGF-beta1 core promoter that is responsible for trans-activation and that corresponds to the Sp1e-binding site associated with HPV-16 E6 and E7 oncoproteins. Mutational analysis showed that the Sp1e recognition site abolished the trans-activation caused by E6 and E7. These results suggest a physical interaction and functional cooperation between viral oncoproteins and cellular regulatory elements of the TGF-beta1 promoter, and may explain the contribution of HPV-16 to TGF-beta1 gene expression in cervical cancer. PMID:16987065

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

    PubMed Central

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

    2013-01-01

    Our previous studies on cutaneous beta human papillomavirus 38 (HPV38) E6 and E7 oncoproteins highlighted a novel activity of IκB kinase beta (IKKβ) in the nucleus of human keratinocytes, where it phosphorylates and stabilizes ΔNp73α, an antagonist of p53/p73 functions. Here, we further characterize the role of the IKKβ nuclear form. We show that IKKβ nuclear translocation and ΔNp73α accumulation are mediated mainly by HPV38 E7 oncoprotein. Chromatin immunoprecipitation (ChIP)/Re-ChIP experiments showed that ΔNp73α and IKKβ are part, together with two epigenetic enzymes DNA methyltransferase 1 (DNMT1) and the enhancer of zeste homolog 2 (EZH2), of a transcriptional regulatory complex that inhibits the expression of some p53-regulated genes, such as PIG3. Recruitment to the PIG3 promoter of EZH2 and DNMT1 resulted in trimethylation of histone 3 on lysine 27 and in DNA methylation, respectively, both events associated with gene expression silencing. Decreases in the intracellular levels of HPV38 E7 or ΔNp73α strongly affected the recruitment of the inhibitory transcriptional complex to the PIG3 promoter, with consequent restoration of p53-regulated gene expression. Finally, the ΔNp73α/IKKβ/DNMT1/EZH2 complex appears to bind a subset of p53-regulated promoters. In fact, the complex is efficiently recruited to several promoters of genes encoding proteins involved in DNA repair and apoptosis, whereas it does not influence the expression of the prosurvival factor Survivin. In summary, our data show that HPV38 via E7 protein promotes the formation of a multiprotein complex that negatively regulates the expression of several p53-regulated genes. PMID:24006445

  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 E7–Dnmt1 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. Intranasal Immunization with synthetic peptides corresponding to the E6 and E7 oncoproteins of Human Papillomavirus type 16 induces systemic and mucosal cellular immune responses and tumor protection

    PubMed Central

    Manuri, Pallavi R.; Nehete, Bharti; Nehete, Pramod N.; Reisenauer, Rose; Wardell, Seth; Courtney, Amy N.; Gambhira, Ratish; Lomada, Dakshyani; Chopra, Ashok K.; Sastry, K. Jagannadha

    2007-01-01

    The E6 and E7 oncoproteins of the high-risk HPV type16 represent ideal targets for HPV vaccine development, they being consistently expressed in cervical cancer lesions. Since HPV-16 is primarily transmitted through genital mucosal route, mucosal immune responses constitute an essential feature for vaccination strategies against HPV-associated lesions. We present here evidence showing that mucosal immunization of mice by the intranasal route with a mixture of peptides E744–62 and E643–57 from the E7 and E6 oncoproteins of HPV-16, respectively, using a mutant cholera toxin adjuvant (CT-2*), primed strong antigen-specific cellular immune responses in systemic and mucosal tissues. Significant levels of IFN-γ production by both CD4 and CD8 cells were observed along with CTL responses that were effective against both peptide-pulsed targets as well as syngeneic tumor cells (TC-1) expressing the cognate E6 and E7 proteins. Furthermore, mice immunized with the peptide mixture and CT-2* effectively resisted TC-1 tumor challenge. These results together with our earlier observations that T cell responses to these peptides correlate with recurrence-free survival in women after ablative treatment for HPV-associated cervical intraepithelial neoplasia, support the potential of these E6 and E7 peptides for inclusion in vaccine formulations. PMID:17291642

  9. 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.

  10. 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.

  11. Phylogenetic and functional analysis of sequence variation of human papillomavirus type 31 E6 and E7 oncoproteins.

    PubMed

    Ferenczi, Annamária; Gyöngyösi, Eszter; Szalmás, Anita; László, Brigitta; Kónya, József; Veress, György

    2016-09-01

    High-risk human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as a subset of head and neck cancers. The E6 and E7 oncoproteins of HPV contribute to oncogenesis by associating with the tumour suppressor protein p53 and pRb, respectively. For HPV types 16 and 18, intratypic sequence variation was shown to have biological and clinical significance. The functional significance of sequence variation among HPV 31 variants was studied less intensively. HPV 31 variants belonging to different variant lineages were found to have differences in persistence and in the ability to cause high grade cervical intraepithelial neoplasia. In the present study, we started to explore the functional effects of natural sequence variation of HPV 31 E6 and E7 oncoproteins. The E6 variants were tested for their effects on p53 protein stability and transcriptional activity, while the E7 variants were tested for their effects on pRb protein level and also on the transcriptional activity of E2F transcription factors. HPV 31 E7 variants displayed uniform effects on pRb stability and also on the activity of E2F transcription factors. HPV 31 E6 variants had remarkable differences in the ability to inhibit the trans-activation function of p53 but not in the ability to induce the in vivo degradation of p53. Our results indicate that natural sequence variation of the HPV 31 E6 protein may be involved in the observed differences in the oncogenic potential between HPV 31 variants. PMID:27197052

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

    PubMed Central

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

    2013-01-01

    Chronic infection of anogenital epithelium with human papillomavirus (HPV) promotes development of cancer. Many pathogens evoke immunosuppressive mechanisms to enable persistent infection. We have previously shown that grafted skin expressing HPV16 E7 oncoprotein from a keratin-14 promoter (K14E7) is not rejected by a syngeneic, immunocompetent host. In this study we show that indoleamine 2, 3-dioxygenase (IDO) 1, an IFN-γ inducible immunoregulatory molecule, is more highly expressed by langerin−ve dermal dendritic cells from K14E7 skin than nontransgenic control skin. Furthermore, inhibiting IDO activity using 1-D/L-methyl tryptophan promotes K14E7 skin graft rejection. Increased IDO1 expression and activity in K14E7 skin requires IFN-γ and iNKT cells, both of which have been shown to negatively regulate T-cell effector function and suppress K14E7 graft rejection. Further, dendritic cells from K14E7 skin express higher level of IFN-γ receptor (IFN-γR) than dendritic cells from control skin. K14E7 transgenic skin recruits significantly higher number of dendritic cells, independent of IFN-γ and IFN-γR expression. Consistent with these observations in a murine model, we found higher expression of IDO1 and IFN-γ but not IDO2 in the cervical epithelium of patients with HPV-associated cervical intraepithelial neoplasia (CIN) 2/3. Our data support a hypothesis that induction of IDO1 in HPV infected skin contributes to evasion of host immunity. PMID:23652797

  13. Bovine papillomavirus E5 and E7 oncoproteins in naturally occurring tumors: are two better than one?

    PubMed

    Corteggio, Annunziata; Altamura, Gennaro; Roperto, Franco; Borzacchiello, Giuseppe

    2013-01-01

    Bovine papillomaviruses (BPVs) are oncogenic DNA viruses, which mainly induce benign lesions of cutaneous and/or mucosal epithelia in cattle. Thirteen (BPV 1-13) different viral genotypes have been characterized so far. BPVs are usually species-specific but BPV 1/2 may also infect equids as well as buffaloes and bison and cause tumors in these species. BPV-induced benign lesions usually regress, however occasionally they develop into cancer particularly in the presence of environmental carcinogenic co-factors. The major transforming protein of BPV is E5, a very short hydrophobic, transmembrane protein with many oncogenic activities. E5 contributes to cell transformation through the activation of the cellular β receptor for the platelet-derived growth factor (PDGFβ-r), it also decreases cell surface expression of major histocompatibility complex class I (MHCI) causing viral escape from immunosurveillance, and plays a role in the inhibition of the intracellular communication by means of aberrant connexin expression. E7 is considered as a weak transforming gene, it synergies with E5 in cell transformation during cancer development. E7 expression correlates in vivo with the over-expression of β1-integrin, which plays a role in the regulation of keratinocytes proliferation and differentiation. Additionally, E7 is involved in cell-mediated immune responses leading to tumour rejection, in anoikis process by direct binding to p600, and in invasion process by upregulation of Matrix metalloproteinase1 (MMP-1) expression. Studies on the role of BPV E5 and E7 oncoproteins in naturally occurring tumours are of scientific value, as they may shed new light on the biological role of these two oncogenes in cell transformation. PMID:23302179

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

    PubMed Central

    Muñoz 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/β-catenin pathway, have been implicated in the deregulation of critical molecular processes that affect cell proliferation and differentiation. For example, changes in β-catenin localization have been identified in Human Papillomavirus (HPV)-related cancers as the lesion progresses. Specifically, β-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/β-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/β-catenin pathway. PMID:26295406

  15. Human Papillomavirus Type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

    PubMed Central

    Yu, Yueyang; Munger, Karl

    2012-01-01

    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. PMID:22748180

  16. 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.

  17. 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 γ-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 γ-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 γ-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

  18. Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation.

    PubMed

    Rawls, J A; Pusztai, R; Green, M

    1990-12-01

    The human papillomavirus type 16 E7 protein belongs to a family of nuclear oncoproteins that share amino acid sequences and functional homology. To localize biochemical activities associated with E7, we chemically synthesized the full-length 98-amino-acid polypeptide and several deletion mutant peptides. We show that the E7 polypeptide is biologically active and possesses at least two functional domains; the first induces cellular DNA synthesis in quiescent rodent cells, and the second trans activates the adenovirus E1A-inducible early E2 promoter and binds zinc. Further, each domain is autonomous and can function on separate peptides. DNA synthesis induction activity maps within the N-terminal portion of the molecule, which contains sequences related to adenovirus E1A conserved domains 1 and 2 required for cell transformation and binding of the retinoblastoma gene product. trans-Activation and Zn-binding activities map within the C-terminal portion of the molecule, a region which contains Cys-X-X-Cys motifs. trans Activation does not require protein synthesis, implying a mechanism that involves interaction with a preexisting cellular factor(s). E7 trans activates the adenovirus E2 promoter but not other E1A-inducible viral promoters, suggesting the possibility that E7 trans activation involves interaction, directly or indirectly, with cellular transcription factor E2F. PMID:2173783

  19. Naive and radiolabeled antibodies to E6 and E7 HPV-16 oncoproteins show pronounced antitumor activity in experimental cervical cancer

    PubMed Central

    Phaëton, R; Gutierrez, J; Jiang, Z; Karabakhtsian, RG; Albanese, J; Sunkara, J; Fisher, DR; Goldberg, GL; Dadachova, E

    2015-01-01

    Background In spite of profound reduction in incidence, cervical cancer claims >275,000 lives annually. Previously we demonstrated efficacy and safety of radioimmunotherapy directed at HPV16 E6 oncoprotein in experimental cervical cancer. Materials & methods We undertook a direct comparison of targeting E7 and E6 oncoproteins with specific 188Rhenium-labeled monoclonal antibodies in CasKi subcutaneous xenografts of cervical cancer cells in mice. Results The most significant tumor inhibition was seen in radioimmunotherapy-treated mice, followed by the unlabeled monoclonal antibodies to E6 and E7. No hematological toxicity was observed. Immunohistochemistry suggests that the effect of unlabeled antibodies is C3 complement mediated. Conclusion We have demonstrated for the first time that radioimmunotherapy directed toward E7 oncoprotein inhibits experimental tumors growth, decreases E7 expression and may offer a novel approach to cervical cancer therapy. PMID:26098137

  20. Disruption of repressive p130-DREAM complexes by human papillomavirus 16 E6/E7 oncoproteins is required for cell-cycle progression in cervical cancer cells.

    PubMed

    Nor Rashid, Nurshamimi; Yusof, Rohana; Watson, Roger J

    2011-11-01

    Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130-DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130-DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130-DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130-DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130-DREAM complex. PMID:21813705

  1. Delocalization of the Microtubule Motor Dynein from Mitotic Spindles by the Human Papillomavirus E7 Oncoprotein is Not Sufficient for Induction of Multipolar Mitoses

    PubMed Central

    Nguyen, Christine L.; McLaughlin-Drubin, Margaret E.; Münger, Karl

    2008-01-01

    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

  2. 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.

  3. The expression of miR-21 and miR-143 is deregulated by the HPV16 E7 oncoprotein and 17β-estradiol.

    PubMed

    Gómez-Gómez, Yazmín; Organista-Nava, Jorge; Ocadiz-Delgado, Rodolfo; García-Villa, Enrique; Leyva-Vazquez, Marco Antonio; Illades-Aguiar, Berenice; Lambert, Paul F; García-Carrancá, Alejandro; Gariglio, Patricio

    2016-08-01

    MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate their target mRNAs at a posttranscriptional level, thereby affecting crucial processes in cancer development. However, little is known about the molecular events that control expression of miRNAs in cervical cancer (CC). HPV16 E7 oncoprotein in conjunction with estrogen are sufficient to produce high grade cervical dysplasia and invasive cervical malignancies in a mouse model. In the present study, we determined the potential role that the E7 oncoprotein and 17β-estradiol (E2) play in the deregulation of miR-21 and miR-143 expression levels by these two risk factors. We found that, while the expression of miR-21 was upregulated and the expression of miR-143 was downregulated by the HPV16 E7 oncoprotein in vivo, and in vitro and that E2 treatment is also implicated in the deregulation of these important miRNAs in vivo. Sustained upregulation of miR-21 resulted in suppression of PTEN expression, and repression of miR-143 increased the mRNA and protein levels from Bcl-2. These results suggested that HPV type 16 E7 oncoprotein and E2 play an important role in regulating miR-21 and miR-143 expression. We have observed similar results in CC patients containing HPV16 sequences, suggesting that these miRNAs could serve as diagnostic biomarkers in CC. The present study highlights the roles of miRNAs in cervical tissue and implicates these important molecules in cervical carcinogenesis. PMID:27278606

  4. 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.

  5. Human Papillomavirus Type 16 E7 oncoprotein inhibits the anaphase promoting complex/cyclosome activity by dysregulating EMI1 expression in mitosis

    PubMed Central

    Yu, Yueyang; Munger, Karl

    2013-01-01

    The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase complex that orchestrates mitotic progression by targeting key mitotic regulators for proteasomal degradation. APC/C dysfunction is a frequent event during cancer development and can give rise to genomic instability. Here we report that the HPV16 E7 oncoprotein interferes with the degradation of APC/C substrates and that the APC/C inhibitor, EMI1, is expressed at higher levels in HPV16 E7-expressing mitotic cells. HPV16 E7 expression causes increased EMI1 mRNA expression and also inhibits EMI1 degradation. The resulting abnormally high EMI1 levels in HPV16 E7-expressing mitotic cells may inhibit degradation of APC/C substrates and cause the prometaphase delay that we have previously observed in such cells. PMID:24074588

  6. 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

  7. Transcriptional regulation of E-cadherin and oncoprotein E7 by valproic acid in HPV positive cell lines

    PubMed Central

    Faghihloo, Ebrahim; Akbari, Abolfazl; Adjaminezhad-Fard, Fatemeh; Mokhtari-Azad, Talat

    2016-01-01

    Objective(s): Valproic acid (VPA) has proven to be as one of the most promising useful drug with anticancer properties. In this study, we investigate the VPA effects on E-cadherin expression in HeLa, TC1, MKN45, and HCT116 cell lines. This study assesses the effects of VPA on human papillomavirus E7 expression in HPV positive cell lines. Materials and Methods: Cell lines were treated by 2 mmol/l VPA and expression of E-cadherin and E7 was analyzed by quantitative real-time PCR. Student’s t test and ANOVA were used to determine changes in expression levels. Results: The results revealed that mean of E-cadherin expression is increased by VPA 1.8 times in HCT116 and MKN45 cell lines, also the mean of E-cadherin mRNA levels is up-regulated 2.9 times in HeLa and TC1 cell lines. So, E-cadherin augmentation induced by VPA in HeLa and TC-1, HPV positive cell lines, is higher than HPV negative cell lines MKN45 and HCT116. The mean of HPV E7 expression is decreased by VPA, 4.6 times in in HeLa and TC-1 cell lines. Conclusion: This study demonstrates that re-expression of E-cadherin by VPA in HPV positive cell lines is more than HPV negative cell lines. Whereas, HPV E7 reduces the expression of E-cadherin, reduction of HPV E7 expression by VPA is related to more augmentation of E-cadherin in HPV positive cell lines. So, this study demonstrates that VPA has more anticancer properties in HPV positive cell lines, and could potentially be a promising candidate for cervical cancer treatment. PMID:27482340

  8. 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 41 N-terminal amino acid (aa) residues of E6 and the 38 C-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 (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-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

  9. 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.

  10. High Risk HPV E6/E7 Oncoprotein Expression in Women with High Grade Squamous Intraepithelial Lesion.

    PubMed

    Valença, Jefferson Elias Cordeiro; Gonçalves, Ana Katherine; Guerreiro da Silva, Ismael Dale Cotrim; Eleutério Junior, José; Tenório da Silva, Terezinha; Bruneska, Danyelly; Ximenes, Ricardo Arraes de Alencar

    2016-03-01

    Purpose To correlate the expression of high-risk HPV E6 mRNA with pap smear, colposcopy, and biopsy results in women with high grade squamous intraepithelial lesion (HSIL). Methods A cross-sectional study was performed on women referred for primary care services after cytological diagnosis of HSIL. We evaluated the expression of E6/E7 mRNA of HPV types 16,18,31,33, and 45 and correlated the results with those of Pap smear, colposcopy, and biopsy. For amplification/detection of mRNA E6 / E7 we used NucliSENSEasyQ kit to detect HPV mRNA by polymerase chain reaction with primers/probes for HPV types 16, 18, 31, 33, and 45. Results Out of 128 valid tests, the results of 30 (23.4%) tests were negative and 98 (70%) tests were positive. Only one type of HPV was detected in 87.7% of the E6/E7 mRNA positive cases. HPV16 was detected in 61.2% of the cases, followed by HPV33 (26.5%), HPV31 (17.3%), HPV18 (10%), and HPV45 (4.08%). Pap smear tests revealed that the E6/E7 test was positive in 107 (83.8%) women with atypical squamous cells - high grade (ASC-H), HSIL, or higher. The E6/E7 test was positive in 69 (57.5%) specimens presenting negative cytology results. When analyzing the association with colposcopy results, the frequency of positive E6/E7 results increased with the severity of the injury, ranging from 57.1% in women without colposcopy-detected injury to 86.5% in those with higher levels of colposcopy findings. Of the 111 women who underwent biopsy and E6/E7 testing, the E6/E7 test was positive in 84.7% of the women who presented with lesions of cervical intraepithelial neoplasia (CIN) grade 2 or higher. Finally, 41.2% of women with a negative biopsy presented a positive E6/E7 test. Conclusions E6/E7 mRNA expression was higher in women with HSIL and CIN grade 2 or higher. PMID:27022787

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

    PubMed Central

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

    2013-01-01

    Background Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions. Methodology/Principal Findings This study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting. Conclusions/Significance This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future. PMID:23738000

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

    PubMed Central

    Gonzalez, Sonia L.; Stremlau, Matt; He, Xi; Basile, John R.; Münger, Karl

    2001-01-01

    The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does not directly contribute to pRB binding but is required for transformation is also necessary for E7-mediated pRB degradation. Treatment with inhibitors of the 26S proteasome not only blocks E7-mediated pRB degradation but also causes the stabilization of E7. Mutagenic analyses, however, reveal that the processes of proteasomal degradation of E7 and pRB are not linked processes. HPV type 16 E7 also targets the pRB-related proteins p107 and p130 for destabilization by a proteasome-dependent mechanism. Using the SAOS2 flat-cell assay as a biological indicator for pRB function, we demonstrate that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRB. PMID:11462030

  13. 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; Blair, G Eric; 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

  14. Low-Dose Adenovirus Vaccine Encoding Chimeric Hepatitis B Virus Surface Antigen-Human Papillomavirus Type 16 E7 Proteins Induces Enhanced E7-Specific Antibody and Cytotoxic T-Cell Responses

    PubMed Central

    Báez-Astúa, Andrés; Herráez-Hernández, Elsa; Garbi, Natalio; Pasolli, Hilda A.; Juárez, Victoria; zur Hausen, Harald; Cid-Arregui, Angel

    2005-01-01

    Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (106 infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8+-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein. PMID:16188983

  15. 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

  16. HUMAN PAPILLOMAVIRUS E7 ENHANCES HYPOXIA-INDUCIBLE FACTOR 1 MEDIATED TRANSCRIPTION BY INHIBITING BINDING OF HISTONE DEACETYLASES

    PubMed Central

    Bodily, Jason M.; Mehta, Kavi P. M.; Laimins, Laimonis A.

    2010-01-01

    Infection by human papillomaviruses (HPVs) leads to the formation of benign lesions, warts, and in some cases, cervical cancer. The formation of these lesions is dependent upon increased expression of pro-angiogenic factors. Angiogenesis is linked to tissue hypoxia through the activity of the oxygen sensitive hypoxia inducible factor 1α (HIF-1α). Our studies indicate that the HPV E7 protein enhances HIF-1 transcriptional activity while E6 functions to counteract the repressive effects of p53. Both high and low risk HPV E7 proteins were found to bind to HIF-1α through a domain located in the the N terminus. Importantly, the ability of E7 to enhance HIF-1 activity mapped to the C terminus and correlated with the displacement of the histone deacetylases HDAC1, HDAC4, and HDAC7 from HIF-1α by E7. Our findings describe a novel role of the E7 oncoprotein in activating the function of a key transcription factor mediating hypoxic responses by blocking the binding of HDACs. PMID:21148070

  17. Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.

    PubMed Central

    Yu, C L; Prochownik, E V; Imperiale, M J; Jove, R

    1993-01-01

    Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells. Images PMID:8384301

  18. 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.

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

    PubMed

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

    2013-05-01

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

  20. Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity

    PubMed Central

    Chandra, Janin; Miao, Yan; Romoff, Natasha; Frazer, Ian H.

    2016-01-01

    Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs. PMID:27031095

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

    PubMed

    Gyöngyösi, Eszter; Szalmás, Anita; Ferenczi, Annamária; Póliska, Szilárd; Kónya, József; Veress, György

    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

  2. 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.

  3. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt

    PubMed Central

    Aoki, Masahiro; Blazek, Erik; Vogt, Peter K.

    2001-01-01

    The oncoproteins P3k (homolog of the catalytic subunit of class IA phosphoinositide 3-kinase) and Akt (protein kinase B) induce oncogenic transformation of chicken embryo fibroblasts. The transformed cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase (S6K) and of the eukaryotic initiation factor 4E-BP1 binding protein (4E-BP1), a negative regulator of translation. Phosphorylation activates S6K and inactivates 4E-BP1. A mutant of Akt that retains kinase activity but does not induce phosphorylation of S6K or of 4E-BP1 fails to transform chicken embryo fibroblasts, suggesting a correlation between the oncogenicity of Akt and phosphorylation of S6K and 4E-BP1. The macrolide antibiotic rapamycin effectively blocks oncogenic transformation induced by either P3k or Akt but does not reduce the transforming activity of 11 other oncoproteins. Rapamycin inhibits the kinase mTOR, an important regulator of translation, and this inhibition requires binding of the antibiotic to the immunophilin FKBP12. Displacement of rapamycin from FKBP12 relieves the inhibition of mTOR and also restores P3k-induced transformation. These data are in accord with the hypothesis that transformation by P3k or Akt involves intervention in translational controls. PMID:11134523

  4. 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

  5. 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

  6. Inhibitory role of TRIP-Br1 oncoprotein in hypoxia-induced apoptosis in breast cancer cell lines.

    PubMed

    Li, Chengping; Jung, Samil; Yang, Young; Kim, Keun-Il; Lim, Jong-Seok; Cheon, Chung-Il; Lee, Myeong-Sok

    2016-06-01

    TRIP-Br1 oncoprotein is known to be involved in many vital cellular functions. In this study, we examined the role of TRIP-Br1 in hypoxia-induced cell death. Exposure to the overcrowded and CoCl2-induced hypoxic conditions increased TRIP-Br1 expression at the protein level in six breast cancer cell lines (MCF7, MDA-MB-231, T47D, Hs578D, BT549, and MDA-MB-435) but resulted in no significant change in three normal cell lines (MCF10A, MEF and NIH3T3). Our result revealed that CoCl2-induced hypoxia stimulated apoptosis and autophagy, in which TRIP-Br1 expression was found to be upregulated. Interestingly, TRIP-Br1 silencing in the MCF7 and MDA-MB-231 cancer cells accelerated apoptosis and destabilization of XIAP under the CoCl2-induced hypoxic condition, implying that TRIP-Br1 may render cancer cells resistant to apoptosis through the stabilization of XIAP. We also propose that TRIP-Br1 seems to be upregulated at least partly as a result of the inhibition of PI3K/AKT signaling pathway and the overexpression of HIF-1α. In conclusion, our findings suggest that TRIP-Br1 functions as an oncogenic protein by providing cancer cells resistance to the hypoxia-induced cell death. PMID:27035851

  7. Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes.

    PubMed

    Liu, Yu-Chen; Cai, Zhi-Ming; Zhang, Xue-Jun

    2016-01-01

    The persistence infection of low-risk type (type 6 or type 11) of human papillomavirus (HPV) is the main cause of genital warts. Given the high rate of recurrence after treatment, the use of a new molecular agent is certain to be of value. The aim of this study was to achieve targeted inactivation of viral E 7 gene in keratinocytes using the reprogrammed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system. To accomplish this, a universal CRISPR-Cas9 system for targeting both HPV6/11 E 7 genes was constructed by using a dual guide RNA vector. After transfection of the vector into E 7-transfromed keratinocytes, the expression level of E 7 protein was measured using western-blot analysis and the sequence of the E 7 gene was determined using Sanger sequencing. Cell proliferation was analyzed by CCK-8 assay, and cell apoptosis was evaluated by Hoechst 33258 staining, flow cytometry analysis and ELISA assay. The results indicated that both HPV6/11 E 7 genes can be inactivated by the single CRISPR-Cas9 system. Furthermore, silencing of E 7 led to inhibition of cell proliferation and induction of apoptosis in E 7-transfromed keratinocytes but not in normal keratinocytes. Our data suggested that the reprogrammed CRISPR-Cas9 system has the potential for the development of an adjuvant therapy for genital warts. PMID:26228041

  8. The heat shock protein 90-binding geldanamycin inhibits cancer cell proliferation, down-regulates oncoproteins, and inhibits epidermal growth factor-induced invasion in thyroid cancer cell lines.

    PubMed

    Park, Jin-Woo; Yeh, Michael W; Wong, Mariwil G; Lobo, Margaret; Hyun, William C; Duh, Quan-Yang; Clark, Orlo H

    2003-07-01

    Heat shock protein 90 (HSP90) serves as a chaperone protein and plays a critical role in tumor cell growth and/or survival. Geldanamycin, a specific inhibitor of HSP90, is cytotoxic to several human cancer cell lines, but its effect in thyroid cancer is unknown. We, therefore, investigated the effect of geldanamycin on cell proliferation, oncoprotein expression, and invasion in human thyroid cancer cell lines. We used six thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO (anaplastic). We used the dimethyl-thiazol-diphenyltetrazolium bromide assay, a clonogenic assay, an apoptotic assay, and a Matrigel invasion assay. We evaluated oncoprotein expression using Western blots and flow cytometry. After 6 d of treatment with 50 nM geldanamycin, the percent inhibition of growth was 29.4% in TPC-1, 97.5% in FTC-133, 96.7% in FTC-236, 10.8% in FTC-238, 70.9% in XTC-1, and 45.5% in ARO cell lines. In the FTC-133 cell line, geldanamycin treatment decreased clonogenicity by 21% at a concentration of 50 nM; geldanamycin induced apoptosis and down-regulated c-Raf-1, mutant p53, and epidermal growth factor (EGF) receptor expression; geldanamycin inhibited EGF-stimulated invasion. In conclusion, geldanamycin inhibited cancer cell proliferation, down-regulated oncoproteins, and inhibited EGF-induced invasion in thyroid cancer cell lines. PMID:12843186

  9. 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

    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

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

    PubMed Central

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

    1995-01-01

    In this study, we analysed immunocytochemically p53 expression in first primary and second primary cancers from 25 head and neck cancer patients (HNCPs) with multiple malignancies in comparison with oncoprotein expression in tumour tissues from 25 historical HNCP controls with single cancer in a match-paired analysis. Moreover, we investigated bleomycin-induced chromosome fragility in both groups of HNCPs and in 21 additional healthy controls. Thirty-nine out of 75 tumour specimens analysed (52%) showed positive p53 immunostaining. Eleven out of 25 (44%) from single cancer patients and 28 out of 50 (56%) tumours from HNCPs with multiple malignancies were p53 positive. In the group of multiple primary cancers, nine patients (36%) showed positive staining of both first and second primaries, whereas six (24%) had positive labelling of first primary cancer but not of the subsequent second primary, four (16%) patient showed p53 expression only in the second primary cancer and six (24%) patients showed no p53 immunoreactivity in both tumours. Chromosomal analysis demonstrated a higher sensitivity to clastogens of HNCPs with multiple tumours than of HNCPs with a single cancer (P < 0.01), and a significant correlation between chromosome fragility and p53 overexpression (P < 0.01) only in HNCPs with multiple malignancies more than in those with single head and neck cancer (P = 0.11). Moreover, we found that patients with p53-positive staining of both first and second primaries showed a statistically significant higher mutagen sensitivity than those with a single p53 immunoreactive tumour or those in whom both cancers were p53 negative (P < 0.01). Our data suggest that subjects with increased susceptibility to carcingogens after exposure to tobacco or alcohol are at higher risk for multiple cancers in which one of the most common genetic events is aberrant p53 expression. Images Figure 1 PMID:7734291

  11. Human Papillomavirus (HPV) E7 Induces Prolonged G2 following S Phase Reentry in Differentiated Human Keratinocytes*

    PubMed Central

    Banerjee, N. Sanjib; Wang, Hsu-Kun; Broker, Thomas R.; Chow, Louise T.

    2011-01-01

    The productive program of human papillomaviruses occurs in differentiated squamous keratinocytes. We have previously shown that HPV-18 DNA amplification initiates in spinous cells in organotypic cultures of primary human keratinocytes during prolonged G2 phase, as signified by abundant cytoplasmic cyclin B1 (Wang, H. K., Duffy, A. A., Broker, T. R., and Chow, L. T. (2009) Genes Dev. 23, 181–194). In this study, we demonstrated that the E7 protein, which induces S phase reentry in suprabasal cells by destabilizing the p130 pocket protein (Genovese, N. J., Banerjee, N. S., Broker, T. R., and Chow, L. T. (2008) J. Virol. 82, 4862–4873), also elicited extensive G2 responses. Western blots and indirect immunofluorescence assays were used to probe for host proteins known to control G2/M progression. E7 expression induced cytoplasmic accumulation of cyclin B1 and cdc2 in the suprabasal cells. The elevated cdc2 had inactivating phosphorylation on Thr14 or Tyr15, and possibly both, due to an increase in the responsible Wee1 and Myt1 kinases. In cells that harbored cytoplasmic cyclin B1 or cdc2, there was also an accumulation of the phosphatase-inactive cdc25C phosphorylated on Ser216, unable to activate cdc2. Moreover, E7 expression induced elevated expression of phosphorylated ATM (Ser1981) and the downstream phosphorylated Chk1, Chk2, and JNKs, kinases known to inactivate cdc25C. Similar results were observed in primary human keratinocyte raft cultures in which the productive program of HPV-18 took place. Collectively, this study has revealed the mechanisms by which E7 induces prolonged G2 phase in the differentiated cells following S phase induction. PMID:21321122

  12. Reprogramming of plant cells induced by 6b oncoproteins from the plant pathogen Agrobacterium.

    PubMed

    Ito, Masaki; Machida, Yasunori

    2015-05-01

    Reprogramming of plant cells is an event characterized by dedifferentiation, reacquisition of totipotency, and enhanced cell proliferation, and is typically observed during formation of the callus, which is dependent on plant hormones. The callus-like cell mass, called a crown gall tumor, is induced at the sites of infection by Agrobacterium species through the expression of hormone-synthesizing genes encoded in the T-DNA region, which probably involves a similar reprogramming process. One of the T-DNA genes, 6b, can also by itself induce reprogramming of differentiated cells to generate tumors and is therefore recognized as an oncogene acting in plant cells. The 6b genes belong to a group of Agrobacterium T-DNA genes, which include rolB, rolC, and orf13. These genes encode proteins with weakly conserved sequences and may be derived from a common evolutionary origin. Most of these members can modify plant growth and morphogenesis in various ways, in most cases without affecting the levels of plant hormones. Recent studies have suggested that the molecular function of 6b might be to modify the patterns of transcription in the host nuclei, particularly by directly targeting the host transcription factors or by changing the epigenetic status of the host chromatin through intrinsic histone chaperone activity. In light of the recent findings on zygotic resetting of nucleosomal histone variants in Arabidopsis thaliana, one attractive idea is that acquisition of totipotency might be facilitated by global changes of epigenetic status, which might be induced by replacement of histone variants in the zygote after fertilization and in differentiated cells upon stimulation by plant hormones as well as by expression of the 6b gene. PMID:25694001

  13. 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

  14. E1B and E4 Oncoproteins of Adenovirus Antagonize the Effect of Apoptosis Inducing Factor

    PubMed Central

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

    2014-01-01

    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. PMID:24889240

  15. 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.

  16. Role of Cdc6 in re-replication in cells expressing human papillomavirus E7 oncogene.

    PubMed

    Fan, Xueli; Zhou, Yunying; Chen, Jason J

    2016-08-01

    The E7 oncoprotein of high-risk human papillomavirus (HPV) types induces DNA re-replication that contributes to carcinogenesis; however, the mechanism is not fully understood. To better understand the mechanism by which E7 induces re-replication, we investigated the expression and function of cell division cycle 6 (Cdc6) in E7-expressing cells. Cdc6 is a DNA replication initiation factor and exhibits oncogenic activities when overexpressed. We found that in E7-expressing cells, the steady-state level of Cdc6 protein was upregulated and its half-life was increased. Cdc6 was localized to the nucleus and associated with chromatin, especially upon DNA damage. Importantly, downregulation of Cdc6 reduced E7-induced re-replication. Interestingly, the level of Cdc6 phosphorylation at serine 54 (S54P) was increased in E7-expressing cells. S54P was associated with an increase in the total amount of Cdc6 and chromatin-bound Cdc6. DNA damage-enhanced upregulation and chromatin binding of Cdc6 appeared to be due to downregulation of cyclin-dependent kinase 1 (Cdk1) as Cdk1 knockdown increased Cdc6 levels. Furthermore, Cdk1 knockdown or inhibition led to re-replication. These findings shed light on the mechanism by which HPV induces genomic instability and may help identify potential targets for drug development. PMID:27207654

  17. 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.

  18. RNA polymerase II transcription is required for human papillomavirus type 16 E7- and hydroxyurea-induced centriole overduplication.

    PubMed

    Duensing, A; Liu, Y; Spardy, N; Bartoli, K; Tseng, M; Kwon, J-A; Teng, X; Duensing, S

    2007-01-11

    Aberrant centrosome numbers are detected in virtually all human cancers where they can contribute to chromosomal instability by promoting mitotic spindle abnormalities. Despite their widespread occurrence, the molecular mechanisms that underlie centrosome amplification are only beginning to emerge. Here, we present evidence for a novel regulatory circuit involved in centrosome overduplication that centers on RNA polymerase II (pol II). We found that human papillomavirus type 16 E7 (HPV-16 E7)- and hydroxyurea (HU)-induced centriole overduplication are abrogated by alpha-amanitin, a potent and specific RNA pol II inhibitor. In contrast, normal centriole duplication proceeded undisturbed in alpha-amanitin-treated cells. Centriole overduplication was significantly reduced by siRNA-mediated knock down of CREB-binding protein (CBP), a transcriptional co-activator. We identified cyclin A2 as a key transcriptional target of RNA pol II during HU-induced centriole overduplication. Collectively, our results show that ongoing RNA pol II transcription is required for centriole overduplication whereas it may be dispensable for normal centriole duplication. Given that many chemotherapeutic agents function through inhibition of transcription, our results may help to develop strategies to target centrosome-mediated chromosomal instability for cancer therapy and prevention. PMID:16819507

  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 Assumpció; García-Fuster, M Julia; García-Sevilla, Jesús 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 μ-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. Reactive oxygen species mediate N-(4-hydroxyphenyl)retinamide-induced cell death in malignant T cells and are inhibited by the HTLV-I oncoprotein Tax.

    PubMed

    Darwiche, N; Abou-Lteif, G; Bazarbachi, A

    2007-02-01

    N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth of many human tumor cells, including those resistant to natural retinoids. HPR is an effective chemopreventive agent for prostate, cervix, breast, bladder, skin and lung cancers, and has shown promise for the treatment of neuroblastomas. We have previously shown that HPR inhibits proliferation and induces apoptosis of human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia (ATL) and HTLV-I-negative malignant T cells, whereas no effect is observed on normal lymphocytes. In this report, we identified HPR-induced reactive oxygen species (ROS) generation as the key mediator of cell cycle arrest and apoptosis of malignant T cells. HPR treatment of HTLV-I-negative malignant T cells was associated with a rapid and progressive ROS accumulation. Pre-treatment with the antioxidants vitamin C and dithiothreitol inhibited ROS generation, prevented HPR-induced ceramide accumulation, cell cycle arrest, cytochrome c release, caspase-activation and apoptosis. Therefore, anti-oxidants protected malignant T cells from HPR-induced growth inhibition. The expression of the HTLV-I oncoprotein Tax abrogated HPR-induced ROS accumulation in HTLV-I-infected cells, which explains their lower sensitivity to HPR. Defining the mechanism of free radical induction by HPR may support a potential therapeutic role for this synthetic retinoid in ATL and HTLV-I-negative T-cell lymphomas. PMID:17122865

  1. Carboxyl-terminal fusion of E7 into Flagellin shifts TLR5 activation to NLRC4/NAIP5 activation and induces TLR5-independent anti-tumor immunity

    PubMed Central

    Lin, Kuo-Hsing; Chang, Li-Sheng; Tian, Chun-Yuan; Yeh, Yi-Chen; Chen, Yu-Jie; Chuang, Tsung-Hsien; Liu, Shih-Jen; Leng, Chih-Hsiang

    2016-01-01

    Flagellin has the capacity to activate both Toll-like receptor 5 (TLR5) and Nod-like receptor C4 (NLRC4)/neuronal apoptosis inhibitory protein 5 (NAIP5) inflammasome signaling. We fused E7m (the inactivated E7 of human papillomavirus) to either end of the flagellin protein, and the resulting recombinant flagellin-E7m proteins (rFliCE7m and rE7mFliC) were used as immunogens. Both fusion proteins activated receptor signaling to different degrees. rE7mFliC-induced TLR5 activity was 10-fold higher than that of rFliCE7m, whereas rFliCE7m activated the NLRC4/NAIP5 pathway more strongly. Therefore, these recombinant proteins provided a tool to investigate which signaling pathway is critical for the induction of antigen-specific T cell responses and anti-tumor immunity. We demonstrated that rFliCE7m induced higher levels of E7-specific IFN-gamma-secreting cells and cytotoxic T lymphocytes (CTLs) than rE7mFliC, and a single injection with rFliCE7m but not rE7mFliC inhibited E7-expressing tumor growth in vivo. Furthermore, we confirmed that CD8+ T cells played a major role in the anti-tumor immunity induced by rFliCE7m. These findings suggested that the NLRC4/NAIP5 intracellular signaling pathway was critical for the induction of anti-tumor immunity. These observations provide important information for the rational design of flagellin-based immunotherapy. PMID:27063435

  2. 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, Luís 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

  3. 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

  4. Use of a rapid, efficient inoculation method to induce papillomas by cottontail rabbit papillomavirus DNA shows that the E7 gene is required.

    PubMed Central

    Brandsma, J L; Yang, Z H; Barthold, S W; Johnson, E A

    1991-01-01

    A simple inoculation method to induce papillomas efficiently with cottontail rabbit papillomavirus (CRPV) DNA is described. Using a jet injector, recombinant CRPV DNA is easily delivered to 100 or more sites per rabbit and induces typical epithelial papillomas in approximately 50% of those sites. Papillomas begin to form by 3 weeks and continue to develop for up to 7 weeks, a pattern similar to that reported following infection with intact virus. This system readily lends itself to investigation of viral gene function by delivering mutant viral genomes into an immunologically intact host. Two mutations in the E7 open reading frame were introduced into the complete CRPV genome and analyzed by this method. One was a frameshift mutation encoding just nine amino-terminal amino acids of the E7 protein; the other was an in-frame insertion mutation at position 9. Both E7 mutations were in a region of homology to the 300-kDa protein binding domain of adenovirus E1A protein. Neither mutant construct was able to induce papillomas, thereby demonstrating that the E7 gene participates in this biologic function. Exploitation of this approach, which demonstrates that a papillomavirus E7 gene is involved in the induction of papillomas in vivo, should permit detailed studies into molecular mechanisms involved in papilloma induction, malignant conversion, and host immune response. The high efficiency of papilloma induction with recombinant CRPV DNA suggests that the jet injector can also be used to study the biologic effects of other genetic elements in rabbits or in other species. Images PMID:1647019

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

    PubMed

    Scheffner, Martin; Whitaker, Noel J

    2003-02-01

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

  6. 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

  7. Interleukin 24 is induced by the RET/PTC3 oncoprotein and is an autocrine growth factor for epithelial cells.

    PubMed

    Shinohara, Shogo; Rothstein, Jay L

    2004-09-30

    Thyroid cancers, like hematological malignancies, are commonly associated with chromosomal translocations leading to the formation of fusion proteins. Through altered signaling by fusion proteins, cell death and survival pathways are disrupted and the physiological balance of cell-cell communication may be lost. A consequence of this disruption is the release of factors by stressed cells that alert the host. One type of host response is leukocytic infiltration that may develop into chronic inflammation or autoimmune disease. Although inflammation can be associated with neoplastic tissue, the mechanism driving this process is largely unknown. Therefore, to address the mechanism of cancer inflammation we investigated the effects of an oncogene in a murine model system. A comprehensive genetic analysis revealed several soluble factors that were induced by RET/papillary thyroid carcinoma (PTC)3 gene expression including several proinflammatory cytokines, chemokines and immunologically relevant costimulatory molecules. Following a large genetic screen using RP3-expressing thyroid cells, we identified a highly abundant transcript and later identified it as interleukin 24 (Il24), a cytokine with diverse tumor suppressor and inflammatory activities. We show that RET/PTC3 induces Il24 expression in rat thyrocytes and that this expression is dependent on the signaling properties of its tyrosine kinase. Likewise, RET/PTC3 induces large amounts of Il24 following expression in murine thyrocytes, but its expression is dramatically reduced in poorly differentiated carcinomas, a finding that parallels the loss of RET/PTC3 expression. Consistent with its behavior as a tumor suppressor, the loss of Il24 coincided with the loss of RET/PTC3 in poorly differentiated mouse tumors. A functional role of Il24 in the autocrine growth/survival of RET/PTC3-expressing thyroid cells was identified helping to support its role in cellular transformation. These data suggest that the induction of

  8. Histopathological changes induced by selective inactivation of menin on the thyroid gland in RET÷PTC3 and E7 transgenic mice. A study of 77 cases.

    PubMed

    Căpraru, Oana Maria; Berger, Nicole; Gadot, Nicolas; Decaussin-Petrucci, Myriam; Zhang, Chang; Borda, Angela; Szilágyi, Tibor; Borson-Chazot, Françoise; Selmi-Ruby, Samia

    2016-01-01

    Multiple Endocrine Neoplasia Type 1 (MEN1) does not involve the thyroid gland, but animal studies have shown that mice with inactivation of menin could develop thyroid pathologies. The objective was to evaluate if the selective inactivation of menin in murine thyroid glands expressing RET÷PTC3 and E7 oncogenes, might induce an increased index of proliferation and a more rapid development of thyroid hyperplasia and÷or tumors. The thyroid glands of 77 mice aged 4-18 months (31 expressing the E7 oncogene and 25 the RET÷PTC3 oncogene) were analyzed for histological changes and Ki67 proliferation index. Fifty-two mice had selective inactivation of menin in the thyroid gland (16 mice with RET÷PTC3 oncogene and 19 mice with E7 oncogene). As compared to wild type, mice with inactivation of menin presented an increased Ki67 proliferation index. Mice presenting the E7 oncogene showed larger thyroid glands with a pattern of diffuse hyperplasia. Mice expressing the RET÷PTC3 oncogene presented larger thyroid glands compared to the wild type mice but smaller compared to E7 mice. The lesions in the RET÷PTC3 group were "proliferative papillary cystic changes" (60%), "cribriform" (16%), "solid" (8%) and a combination of these patterns in the rest of the thyroid glands. The inactivation of menin in the thyroid gland of young mice does not seem to change the histological pattern, but it influences the proliferation of follicular cells. Further molecular studies especially in aged mice are needed to better understand the correlation between certain oncogenes and the inactive status of menin. PMID:27151693

  9. 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

  10. Effect of human papillomavirus 16 oncoproteins on oncostatin M upregulation in oral squamous cell carcinoma.

    PubMed

    Chuerduangphui, Jureeporn; Pientong, Chamsai; Chaiyarit, Ponlatham; Patarapadungkit, Natcha; Chotiyano, Apinya; Kongyingyoes, Bunkerd; Promthet, Supannee; Swangphon, Piyawut; Wongjampa, Weerayut; Ekalaksananan, Tipaya

    2016-08-01

    Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development. PMID:27349249

  11. 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

  12. Protein vaccination with HPV16 E7/Pep-1 nanoparticles elicits a protective T-helper cell-mediated immune response.

    PubMed

    Mardani, Golnaz; Bolhassani, Azam; Agi, Elnaz; Shahbazi, Sepideh; Mehdi Sadat, Seyed

    2016-06-01

    Two human papillomavirus (HPV) viral oncoproteins, E6 and E7 represent ideal targets for development of a therapeutic HPV vaccine. It is important to reduce the rate of HPV-associated malignancies through improvement of vaccine modalities. In this study, we used a short amphipathic peptide carrier, Pep-1, for delivery of the full-length HPV16 E7 protein into mammalian cells and evaluated immune responses and protective effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7/Pep-1 protein form stable nanoparticles through noncovalent binding with an average size of 120 to 250 nm. The efficient delivery of E7 protein by Pep-1 at molar ratio of 1:20 was detected in HEK-293T cell line for 1 h and 3 h post-transfection. Immunization with E7/Pep-1 nanoparticles at a ratio of 1:20 induced a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein in a murine tumor model. These data suggest that Pep-1 peptide would indicate promising applications for improvement of HPV therapeutic vaccines. © 2016 IUBMB Life, 68(6):459-467, 2016. PMID:27094221

  13. 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

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

    PubMed Central

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

    2012-01-01

    The human papillomavirus (HPV) E7 oncoprotein binds cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. A key target of E7 is the product of the retinoblastoma susceptibility locus (pRb). This interaction results in the release of E2F transcription factors and drives the host cell into the S phase of the cell cycle. E7 binds pRb via a high-affinity binding site in conserved region 2 (CR2) and also targets a portion of cellular pRb for degradation via the proteasome. Evidence suggests that a secondary binding site exists in CR3, and that this interaction influences pRb deregulation. Additionally, evidence suggests that CR3 also participates in the degradation of pRb. We have systematically analyzed the molecular mechanisms by which CR3 contributes to deregulation of the pRb pathway by utilizing a comprehensive series of mutations in residues predicted to be exposed on the surface of HPV16 E7 CR3. Despite differences in the ability to interact with cullin 2, all CR3 mutants degrade pRb comparably to wild-type E7. We identified two specific patches of residues on the surface of CR3 that contribute to pRb binding independently of the high-affinity CR2 binding site. Mutants within CR3 that affect pRb binding are less effective than the wild-type E7 in overcoming pRb-induced cell cycle arrest. This demonstrates that the interaction between HPV16 E7 CR3 and pRb is functionally important for alteration of the cell cycle. PMID:23015707

  15. 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

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

    PubMed Central

    Gao, Qingshen; Srinivasan, Seetha; Boyer, Sarah N.; Wazer, David E.; Band, Vimla

    1999-01-01

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

  17. Quercetin induces structural chromosomal aberrations and uncommon rearrangements in bovine cells transformed by the E7 protein of bovine papillomavirus type 4.

    PubMed

    Leal, A M; Ferraz, O P; Carvalho, C; Freitas, A C; Beniston, R G; Beçak, W; Campo, M S; Stocco dos Santos, R C

    2003-03-01

    Bovine papillomavirus type 4 (BPV-4) and bracken fern are cofactors in the carcinogenesis of the upper gastrointestinal (GI) tract of cattle. An experimental in vitro model system has been developed to analyse the co-operation between the viral transforming protein E7, the cellular ras oncogene and quercetin, one of the mutagens of bracken fern, during neoplastic progression of primary bovine cells. We now report cytogenetic studies of these cells at different stages of malignant transformation: parental primary non-transformed PalF cells; E7R cells transformed by BPV-4 E7 and activated ras but not tumorigenic, and tumorigenic E7Q cells derived from E7R cells after treatment with quercetin. All cell lines presented increased numbers of aneuploid cells. The rate of structural chromosomal aberrations observed was increased in transformed cells. In addition, E7Q cells showed chromosomes with peculiar rearrangements, which resulted in metacentric and submetacentric marker chromosomes, with an increase in the mean chromosome arm number. These markers were the products of possible centric fusions. These aberrations and rearrangements were distributed throughout the karyotype, no specific chromosome was involved and the heterochromatic centromeric regions appeared to be preserved. PMID:19379326

  18. Oncoprotein stability after tumour resection.

    PubMed Central

    Ong, G.; Gullick, W.; Sikora, K.

    1990-01-01

    The means by which oncogenes and their products activate malignant transformation are currently under intense investigation. However, published papers on experiments using human tumour material do not always report in detail their methods of collection or storage of the specimens. In order to assess the stability of oncogene encoded proteins following collection or storage of human tumour biopsies, we have examined the rate of decay of the c-myc, neu and EGF-receptor proteins. Solid tumours, containing amplified copies of each oncogene, were established in nude mice and the stability of the oncogene protein in portions of each tumour, left in phosphate buffered saline at room temperature for varying time intervals, was examined by immunoblotting. Intact EGF-receptor and neu oncoproteins were present even after 24 h under these conditions while the c-myc protein was apparently rapidly degraded after 20 min. These data demonstrate that oncogene products decay at different rates after tumour resection and that collection of human biopsies should take this into account in order to provide the basis for consistent measurements of protein expression. Images Figure 1 Figure 2 Figure 3 PMID:2139576

  19. 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; Öhlschläger, 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

  20. 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.

  1. Interleukin-12-secreting human papillomavirus type 16-transformed cells provide a potent cancer vaccine that generates E7-directed immunity.

    PubMed

    Hallez, S; Detremmerie, O; Giannouli, C; Thielemans, K; Gajewski, T F; Burny, A; Leo, O

    1999-05-01

    The development of a vaccine that would be capable of preventing or curing the (pre)cancerous lesions induced by genital oncogenic human papillomaviruses (HPVs) is the focus of much research. Many studies are presently evaluating vaccines based on the viral E6 and E7 oncoproteins, both of which are continually expressed by tumor cells. The success of a cancer vaccine relies, in large part, on the induction of a tumor-specific Th1-type immunity. In this study, we have evaluated the ability of B7-related and/or interleukin-12 (IL-12)-expressing, non-immunogenic murine HPV16-transformed BMK-16/myc cells, to achieve this goal. BMK-16/myc cells engineered to express surface B7-1 or B7-2 molecules remain tumorigenic in syngeneic BALB/c mice, suggesting that expression of these molecules alone is not sufficient to induce tumor regression. In contrast, mice injected with tumor cells engineered to secrete IL-12 remained tumor-free, demonstrating that IL-12 expression is sufficient to induce tumor rejection. IL-12-secreting BMK-16/myc cells were further shown to induce potent and specific long-term tumor resistance, even after irradiation. B7-1 was found to slightly but systematically improve anti-tumor immunity elicited by IL-12-secreting BMK-16/myc cells. Injection of irradiated B7-1/IL-12+ BMK-16/myc cells generates long-lasting, Th1-type, BMK-16/myc-directed immunity in tumor-resistant mice. These mice display a memory-type, E7-specific, cell-mediated immune response, which is potentially significant for clinical applications. PMID:10209958

  2. Molecular Targeting of the Oncoprotein PLK1 in Pediatric Acute Myeloid Leukemia: RO3280, a Novel PLK1 Inhibitor, Induces Apoptosis in Leukemia Cells

    PubMed Central

    Wang, Na-Na; Li, Zhi-Heng; Zhao, He; Tao, Yan-Fang; Xu, Li-Xiao; Lu, Jun; Cao, Lan; Du, Xiao-Juan; Sun, Li-Chao; Zhao, Wen-Li; Xiao, Pei-Fang; Fang, Fang; Su, Guang-Hao; Li, Yan-Hong; Li, Gang; Li, Yi-Ping; Xu, Yun-Yun; Zhou, Hui-Ting; Wu, Yi; Jin, Mei-Fang; Liu, Lin; Ni, Jian; Wang, Jian; Hu, Shao-Yan; Zhu, Xue-Ming; Feng, Xing; Pan, Jian

    2015-01-01

    Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined. PMID:25574601

  3. A LONGITUDINAL STUDY OF HPV16 L1, E6 AND E7 SEROPOSITIVITY AND ORAL HPV16 INFECTION

    PubMed Central

    Beachler, Daniel C.; Viscidi, Raphael; Sugar, Elizabeth A.; Minkoff, Howard; Strickler, Howard D.; Cranston, Ross D.; Wiley, Dorothy J.; Jacobson, Lisa P.; Weber, Kathleen M.; Margolick, Joseph B.; Reddy, Susheel; Gillison, Maura L.; D’Souza, Gypsyamber

    2014-01-01

    Background Individuals with HPV infections can develop IgG antibodies to HPV proteins including the L1 capsid and E6 and E7 oncoproteins. Evidence on whether L1 antibodies reduce the risk of cervical HPV infection is mixed, but this has not been explored for oral HPV infections. Antibodies to HPV16’s E6 oncoprotein have been detected in some oropharyngeal cancer cases years prior to cancer diagnosis, but it is unknown if these antibodies are associated with oral HPV16 DNA. Methods Enzyme linked immunosorbent assays tested for serum antibodies to HPV16’s L1 capsid in 463 HIV-infected and 293 HIV-uninfected adults, and for antibodies to recombinantly expressed E6 and E7 oncoproteins to HPV16 in 195 HIV-infected and 69 HIV-uninfected cancer-free participants at baseline. Oral rinse samples were collected semi-annually for up to three years and tested for HPV DNA using PGMY 09/11 primers. Adjusted Poisson, logistic, and Wei-Lin-Weissfeld regression models were utilized. Results HPV16 L1 seroreactivity did not reduce the subsequent risk of incident oral HPV16 infection in unadjusted (HR=1.4, 95%CI=0.59–3.3) or adjusted (aHR=1.1, 95%CI=0.41–3.0) analysis. Antibodies to HPV16 E6 and E7 oncoproteins were detected in 7.6% and 3.4% of participants respectively, but they were not associated with baseline oral HPV16 DNA prevalence or oral HPV16 persistence (each p-value>0.40). Conclusions Naturally acquired HPV16 L1 antibodies did not reduce the risk of subsequent oral HPV16 infection. HPV16 E6 and E7 seropositivity was not a marker for oral HPV16 infection in this population without HPV-related cancer. PMID:25585068

  4. 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.

  5. Mutual regulation between deubiquitinase CYLD and retroviral oncoprotein Tax

    PubMed Central

    2011-01-01

    Background Oncoprotein Tax, encoded by the human T-cell leukemia virus type 1 (HTLV1), persistently induces NF-κB activation, which contributes to HTLV1-mediated T-cell transformation. Recent studies suggest that the signaling function of Tax requires its ubiquitination, although how the Tax ubiquitination is regulated remains unclear. Results We show here that the deubiquitinase CYLD physically interacts with Tax and negatively regulates the ubiquitination of this viral protein. This function of CYLD is associated with inhibition of Tax-mediated activation of IKK although not that of Tak1. Interestingly, CYLD undergoes constitutive phosphorylation in HTLV1-transformed T cells, a mechanism known to inactivate the catalytic activity of CYLD. Consistently, a phospho-mimetic CYLD mutant fails to inhibit Tax ubiquitination. Conclusion These findings suggest that CYLD negatively regulates the signaling function of Tax through inhibition of Tax ubiquitination. Conversely, induction of CYLD phosphorylation may serve as a mechanism by which HTLV1 overrides the inhibitory function of CYLD, leading to the persistent activation of NF-κB. PMID:21824392

  6. Functional diversity and mutational analysis of Agrobacterium 6B oncoproteins.

    PubMed

    Helfer, A; Pien, S; Otten, L

    2002-07-01

    Many Agrobacterium T-DNA genes belong to a diverse family of T-DNA genes, the rolB family. These genes cause various growth abnormalities but their modes of action remain largely unknown. So far, none of the RolB-like proteins has been subjected to mutational analysis. The RolB-like oncoprotein 6B, which induces tumours on species such as Nicotiana glauca and Kalanchoe tubiflora, was chosen to investigate the role of the most conserved amino acid residues within the RolB family. We first determined which of the natural 6B variants had the strongest oncogenic activity; to this end, six 6b coding sequences (A- 6b, AB- 6b, C- 6b, CG- 6b, S- 6b and T- 6b) were placed under the control of the strong constitutive 2x35S promoter and compared for tumour induction on N. glauca, N. tabacum and K. daigremontiana. Oncogenicity increased in the order C- 6b/CG- 6b, A- 6b/AB- 6b, and S- 6b/T- 6b. The most conserved amino acid residues in the strongly oncogenic T-6B protein were mutated and shown to be required for oncogenicity and accumulation of the T-6B protein in planta but not in bacteria. Hybrids between T-6B and the weakly oncogenic A-6B protein revealed an additional oncogenic determinant required for the formation of large tumours. PMID:12172796

  7. The Viral Oncoprotein HBx of Hepatitis B virus Promotes the Growth of Hepatocellular Carcinoma through Cooperating with the Cellular Oncoprotein RMP

    PubMed Central

    Wang, Qi; Xu, Yi; Zhou, Wei; Zhong, Lei; Wen, Zengqing; Yu, Huijun; Chen, Shaomu; Shen, Jian; Chen, Han; She, Qinying; Jiang, Jingting; Miao, Jingcheng; Wei, Wenxiang

    2014-01-01

    The smallest gene HBx of Hepatitis B virus (HBV) is recognized as an important viral oncogene (V-oncogene) in the hepatocarcinogenesis. Our previous work demonstrated that RMP is a cellular oncogene (C-oncogene) required for the proliferation of hepatocellular carcinoma (HCC) cells. Here we presented the collaboration between V-oncogene HBx and C-oncogene RMP in the development of HCC. The coexpression of HBx and RMP resulted in the cooperative effect of antiapoptosis and proliferation of HCC cells. In vivo, overexpression of RMP accelerated the growth of HBx-induced xenograft tumors in nude mice and vice versa HBx promoted the growth of RMP-driven xenograft tumors. Although HBx didn't regulate the expression of RMP, HBx and RMP interact with each other and collocalized in the cytoplasm of HCC cells. HBx and RMP collaboratively inhibited the expression of apoptotic factors and promoted the expression of antiapoptotic factors. This finding suggests that HBV may induce, or at least partially contributes to the carcinogenesis of HCC, through its V-oncoprotein HBx interacting with the C-oncoprotein RMP. PMID:25516716

  8. Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells

    PubMed Central

    Smith, Stephen P.; Scarpini, Cinzia G.; Groves, Ian J.; Odle, Richard I.; Coleman, Nicholas

    2016-01-01

    Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of ‘master regulators’ for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins. PMID:27457222

  9. Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells.

    PubMed

    Smith, Stephen P; Scarpini, Cinzia G; Groves, Ian J; Odle, Richard I; Coleman, Nicholas

    2016-01-01

    Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of 'master regulators' for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins. PMID:27457222

  10. The fibronectin/α3β1 integrin axis serves as molecular basis for keratinocyte invasion induced by βHPV.

    PubMed

    Heuser, S; Hufbauer, M; Steiger, J; Marshall, J; Sterner-Kock, A; Mauch, C; Zigrino, P; Akgül, B

    2016-08-25

    Organ-transplant-recipients exhibit cancerization of the skin from which multiple human papillomavirus (HPV)-positive squamous cell carcinomas (SCCs) arise. However, the molecular basis for HPV-induced invasion of skin keratinocytes is not known. We generated a transgenic mouse model expressing the E7 oncoprotein of HPV8 in the murine epidermis under the control of the keratin-14 promoter and showed that E7 is carcinogenic in mice. We further showed that both, the E7-expressing keratinocyte and mesenchymal components of the extracellular matrix as critical in eliciting the invasive behavior. E7 expression in basal keratinocytes, grown on fibronectin, led to epithelial-mesenchymal transition mediated by a cadherin switch. E7-positive keratinocytes displayed enhanced EDA-fibronectin expression and secretion and stimulated dermal fibroblasts to express EDA-fibronectin. Deposition of fibronectin was also detected in the peritumoral stroma of HPV8-positive skin SCC. When grown on fibronectin, E7-positive keratinocytes, in particular stem cell-like cells, exhibited increased cell surface levels of the α3-integrin chain. Functional blocking confirmed α3 as a critical molecule sufficient to induce E7-mediated invasion. This mechanistic link is further supported by expression of an E7-mutant, impaired in targeting α3 to the cell surface. These findings highlight the importance of epithelial-extracellular matrix interaction required for keratinocyte invasion and provide further mechanistic evidence for a role of HPV in skin carcinogenesis. PMID:26804167

  11. 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-γ+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

  12. MicroRNA-331-3p Suppresses Cervical Cancer Cell Proliferation and E6/E7 Expression by Targeting NRP2

    PubMed Central

    Fujii, Tomomi; Shimada, Keiji; Asano, Aya; Tatsumi, Yoshihiro; Yamaguchi, Naoko; Yamazaki, Masaharu; Konishi, Noboru

    2016-01-01

    Aberrant expression of microRNAs (miRNAs) is involved in the development and progression of various types of cancers. In this study, we investigated the role of miR-331-3p in cell proliferation and the expression of keratinocyte differentiation markers of uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) are putative target molecules that regulate the human papillomavirus (HPV) related oncoproteins E6 and E7. Cell proliferation in the human cervical cancer cell lines SKG-II, HCS-2, and HeLa was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cellular apoptosis was measured using the TdT-mediated dUTP nick end labeling (TUNEL) and Annexin V assays. Quantitative RT-PCR was used to measure the messenger RNA (mRNA) expression of the NRP2, E6, E7, p63, and involucrin (IVL) genes. A functional assay for cell growth was performed using cell cycle analyses. Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II, HCS-2 and HeLa cells. The luciferase reporter assay of the NRP2 3′-untranslated region revealed the direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in SKG-II, HCS-2, and HeLa cells overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA. Moreover, miR-331-3p overexpression was suppressed NRP2 expression in protein level. We showed that miR-331-3p and NRP2 were key effectors of cell proliferation by regulating the cell cycle, apoptosis. NRP-2 also regulates the expression of E6/E7 and keratinocyte differentiation markers. Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and that miR-331-3p may contribute to keratinocyte differentiation through NRP2 suppression. miR-331-3p and NRP2 may contribute to anti-cancer effects

  13. MicroRNA-331-3p Suppresses Cervical Cancer Cell Proliferation and E6/E7 Expression by Targeting NRP2.

    PubMed

    Fujii, Tomomi; Shimada, Keiji; Asano, Aya; Tatsumi, Yoshihiro; Yamaguchi, Naoko; Yamazaki, Masaharu; Konishi, Noboru

    2016-01-01

    Aberrant expression of microRNAs (miRNAs) is involved in the development and progression of various types of cancers. In this study, we investigated the role of miR-331-3p in cell proliferation and the expression of keratinocyte differentiation markers of uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) are putative target molecules that regulate the human papillomavirus (HPV) related oncoproteins E6 and E7. Cell proliferation in the human cervical cancer cell lines SKG-II, HCS-2, and HeLa was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cellular apoptosis was measured using the TdT-mediated dUTP nick end labeling (TUNEL) and Annexin V assays. Quantitative RT-PCR was used to measure the messenger RNA (mRNA) expression of the NRP2, E6, E7, p63, and involucrin (IVL) genes. A functional assay for cell growth was performed using cell cycle analyses. Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II, HCS-2 and HeLa cells. The luciferase reporter assay of the NRP2 3'-untranslated region revealed the direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in SKG-II, HCS-2, and HeLa cells overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA. Moreover, miR-331-3p overexpression was suppressed NRP2 expression in protein level. We showed that miR-331-3p and NRP2 were key effectors of cell proliferation by regulating the cell cycle, apoptosis. NRP-2 also regulates the expression of E6/E7 and keratinocyte differentiation markers. Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and that miR-331-3p may contribute to keratinocyte differentiation through NRP2 suppression. miR-331-3p and NRP2 may contribute to anti-cancer effects. PMID

  14. HPV16 oncoprotein regulates the translocation of β-catenin via activation of EGFR

    PubMed Central

    Hu, Zhongliang; Müller, Susan; Qian, Guoqin; Xu, Jing; Kim, Sungjin; Chen, Zhengjia; Jiang, Ning; Wang, Dongsheng; Zhang, Hongzheng; Saba, Nabil F.; Shin, Dong M.; Chen, Zhuo Georgia

    2014-01-01

    Background To understand the mechanism of frequent and early lymph node metastasis in high risk human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC), we investigated whether β-catenin is regulated by HPV oncoprotein and contributes to OPSCC metastasis. Methods Expression levels of p16, β-catenin, and epidermal growth factor receptor (EGFR) were examined in OPSCC samples (n=208) by immunohistochemistry. Expression and subcellular localization of β-catenin and EGFR activation were also studied in HPV-positive and -negative head and neck SCC cell lines by Western blot analysis. HPV16 E6 siRNA was used to elucidate the effect of HPV oncoprotein on β-catenin translocation. The involvement of EGFR in β-catenin translocation was confirmed by treatment with erlotinib. Moreover, the invasive capacity was evaluated after HPV16 E6/E7 repression. Results Our results showed that membrane weighted index (WI) of β-catenin was inversely correlated with p16 positivity (p<0.001) and lymph node metastasis (p=0.026), while nuclear staining of β-catenin was associated with p16-positive OPSCC (p<0.001). A low level of membrane β-catenin expression was significantly associated with disease free and overall survival (p<0.0001 in both cases). Furthermore, the membrane WI of EGFR was inversely correlated with p16 positivity (p<0.001) and positively correlated with membrane β-catenin (p<0.001). Our in vitro study showed that HPV16 E6 repression led to reductions of phosphoEGFR, and nuclear β-catenin, which were also observed after erlotinib treatment, and inhibition of invasion. Conclusions Our findings suggest that HPV16 E6 mediates the translocation of β-catenin to the nucleus, which may be regulated by activated EGFR. PMID:25209444

  15. Systematic Analysis of the Amino Acid Residues of Human Papillomavirus Type 16 E7 Conserved Region 3 Involved in Dimerization and Transformation ▿

    PubMed Central

    Todorovic, Biljana; Massimi, Paola; Hung, Katherine; Shaw, Gary S.; Banks, Lawrence; Mymryk, Joe S.

    2011-01-01

    The human papillomavirus (HPV) E7 oncoprotein exists as a dimer and acts by binding to many cellular factors, preventing or retargeting their function and thereby making the infected cell conducive for viral replication. Dimerization of E7 is attributed primarily to the C-terminal domain, referred to as conserved region 3 (CR3). CR3 is highly structured and is necessary for E7's transformation ability. It is also required for binding of numerous E7 cellular targets. To systematically analyze the molecular mechanisms by which HPV16 E7 CR3 contributes to carcinogenesis, we created a comprehensive panel of mutations in residues predicted to be exposed on the surface of CR3. We analyzed our novel collection of mutants, as well as mutants targeting predicted hydrophobic core residues of the dimer, for the ability to dimerize. The same set of mutants was also assessed functionally for transformation capability in a baby rat kidney cell assay in conjugation with activated ras. We show that some mutants of HPV16 E7 CR3 failed to dimerize yet were still able to transform baby rat kidney cells. Our results identify several novel E7 mutants that abrogate transformation and also indicate that E7 does not need to exist as a stable dimer in order to transform cells. PMID:21775462

  16. 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.

  17. The Ski oncoprotein interacts with the Smad proteins to repress TGFbeta signaling.

    PubMed

    Luo, K; Stroschein, S L; Wang, W; Chen, D; Martens, E; Zhou, S; Zhou, Q

    1999-09-01

    Smad proteins are critical signal transducers downstream of the receptors of the transforming growth factor-beta (TGFbeta) superfamily. On phosphorylation and activation by the active TGFbeta receptor complex, Smad2 and Smad3 form hetero-oligomers with Smad4 and translocate into the nucleus, where they interact with different cellular partners, bind to DNA, regulate transcription of various downstream response genes, and cross-talk with other signaling pathways. Here we show that a nuclear oncoprotein, Ski, can interact directly with Smad2, Smad3, and Smad4 on a TGFbeta-responsive promoter element and repress their abilities to activate transcription through recruitment of the nuclear transcriptional corepressor N-CoR and possibly its associated histone deacetylase complex. Overexpression of Ski in a TGFbeta-responsive cell line renders it resistant to TGFbeta-induced growth inhibition and defective in activation of JunB expression. This ability to overcome TGFbeta-induced growth arrest may be responsible for the transforming activity of Ski in human and avian cancer cells. Our studies suggest a new paradigm for inactivation of the Smad proteins by an oncoprotein through transcriptional repression. PMID:10485843

  18. 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

  19. Epstein-Barr virus oncoprotein super-enhancers control B cell growth.

    PubMed

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

    2015-02-11

    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 ∼1,800 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, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). 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

  20. How do oncoprotein mutations rewire protein-protein interaction networks?

    PubMed

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  1. Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation.

    PubMed Central

    Griep, A E; Herber, R; Jeon, S; Lohse, J K; Dubielzig, R R; Lambert, P F

    1993-01-01

    The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression. Images PMID:8382301

  2. Cell Cycle Regulatory Functions of the KSHV Oncoprotein LANA

    PubMed Central

    Wei, Fang; Gan, Jin; Wang, Chong; Zhu, Caixia; Cai, Qiliang

    2016-01-01

    Manipulation of cell cycle is a commonly employed strategy of viruses for achieving a favorable cellular environment during infection. Kaposi’s sarcoma-associated herpesvirus (KSHV), the primary etiological agent of several human malignancies including Kaposi’s sarcoma, and primary effusion lymphoma, encodes several oncoproteins that deregulate normal physiology of cell cycle machinery to persist with endothelial cells and B cells and subsequently establish a latent infection. During latency, only a small subset of viral proteins is expressed. Latency-associated nuclear antigen (LANA) is one of the latent antigens shown to be essential for transformation of endothelial cells in vitro. It has been well demonstrated that LANA is critical for the maintenance of latency, episome DNA replication, segregation and gene transcription. In this review, we summarize recent studies and address how LANA functions as an oncoprotein to steer host cell cycle-related events including proliferation and apoptosis by interacting with various cellular and viral factors, and highlight the potential therapeutic strategy of disrupting LANA-dependent signaling as targets in KSHV-associated cancers. PMID:27065950

  3. The Sumo-targeted ubiquitin ligase RNF4 regulates the localization and function of the HTLV-1 oncoprotein Tax

    PubMed Central

    Fryrear, Kimberly A.; Guo, Xin

    2012-01-01

    The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)–modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB–mediated and decreased cAMP Response Element-Binding (CREB)–mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage–induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes. PMID:22106342

  4. The Sumo-targeted ubiquitin ligase RNF4 regulates the localization and function of the HTLV-1 oncoprotein Tax.

    PubMed

    Fryrear, Kimberly A; Guo, Xin; Kerscher, Oliver; Semmes, O John

    2012-02-01

    The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)-modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB-mediated and decreased cAMP Response Element-Binding (CREB)-mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage-induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes. PMID:22106342

  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. MUC1 oncoprotein suppresses activation of the ARF-MDM2-p53 pathway.

    PubMed

    Raina, Deepak; Ahmad, Rehan; Chen, Dongshu; Kumar, Shailendra; Kharbanda, Surender; Kufe, Donald

    2008-12-01

    The MUC1 oncoprotein interacts with the c-Abl tyrosine kinase and blocks nuclear targeting of c-Abl in the apoptotic response to DNA damage. Mutation of the MUC1 cytoplasmic domain at Tyr-60 disrupts the MUC1-c-Abl interaction. The present results demonstrate that the MUC1(Y60F) mutant is a potent inducer of the ARF tumor suppressor. MUC1(Y60F) induces transcription of the ARF locus by a c-Abl-dependent mechanism that promotes CUL-4A-mediated nuclear export of the replication protein Cdc6. The functional significance of these findings is that MUC1(Y60F)-induced ARF expression and thereby inhibition of MDM2 results in the upregulation of p53 and the homeodomain interacting protein kinase 2 (HIPK2) serine/threonine kinase. HIPK2-mediated phosphorylation of p53 on Ser-46 was further associated with a shift from expression of the cell cycle arrest-related p21 gene to the apoptosis-related PUMA gene. We also show that the MUC1(Y60F) mutant functions as dominant negative inhibitor of tumorigenicity. These findings indicate that the oncogenic function of MUC1 is conferred by suppressing activation of the ARF-MDM2-p53 pathway. PMID:18981727

  7. Disruption of the G1/S Transition in Human Papillomavirus Type 16 E7-Expressing Human Cells Is Associated with Altered Regulation of Cyclin E

    PubMed Central

    Martin, Larry G.; Demers, G. William; Galloway, Denise A.

    1998-01-01

    The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F. PMID:9444990

  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. Clastogenic effect of the human T-cell leukemia virus type I Tax oncoprotein correlates with unstabilized DNA breaks.

    PubMed

    Majone, F; Jeang, K T

    2000-10-20

    Expression of the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein rapidly engenders DNA damage as reflected in a significant increase of micronuclei (MN) in cells. To understand better this phenomenon, we have investigated the DNA content of MN induced by Tax. Using an approach that we termed FISHI, fluorescent in situ hybridization and incorporation, we attempted to characterize MN with centric or acentric DNA fragments for the presence or absence of free 3'-OH ends. Free 3'-OH ends were defined as those ends accessible to in situ addition of digoxigenin-dUTP using terminal deoxynucleotidyl transferase. MN were also assessed for centromeric sequences using standard fluorescent in situ hybridization (FISH). Combining these results, we determined that Tax oncoprotein increased the frequency of MN containing centric DNA with free 3'-OH and decreased the frequency of MN containing DNA fragments that had incorporation-inaccessible 3'-ends. Recently, it has been suggested that intracellular DNA breaks without detectable 3'-OH ends are stabilized by the protective addition of telomeric caps, while breaks with freely detectable 3'-OH are uncapped and are labile to degradation, incomplete replication, and loss during cell division. Accordingly, based on increased detection of free 3'-OH-containing DNA fragments, we concluded that HTLV-I Tax interferes with protective cellular mechanism(s) used normally for stabilizing DNA breaks. PMID:10969065

  10. NUP98 fusion oncoproteins interact with the APC/C(Cdc20) as a pseudosubstrate and prevent mitotic checkpoint complex binding.

    PubMed

    Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

    2016-09-01

    NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function. PMID:27097363

  11. The Viral Oncoprotein LMP1 Exploits TRADD for Signaling by Masking Its Apoptotic Activity

    PubMed Central

    Schneider, Frank; Neugebauer, Julia; Griese, Janine; Liefold, Nicola; Kutz, Helmut; Briseño, Cinthia; Kieser, Arnd

    2008-01-01

    The tumor necrosis factor (TNF)-receptor 1–associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-κB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-κB kinase β (IKKβ). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1′s TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-κB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function. PMID:18198944

  12. Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1

    PubMed Central

    Dao, Tao; Pankov, Dmitry; Scott, Andrew; Korontsvit, Tatyana; Zakhaleva, Victoriya; Xu, Yiyang; Xiang, Jingyi; Yan, Su; de Morais Guerreiro, Manuel Direito; Veomett, Nicholas; Dubrovsky, Leonid; Curcio, Michael; Doubrovina, Ekaterina; Ponomarev, Vladimir; Liu, Cheng; O’Reilly, Richard J; Scheinberg, David A

    2015-01-01

    Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell–based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE. PMID:26389576

  13. 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

  14. 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...

  15. 29 CFR 2584.8477(e)-7 - Effective date.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... promulgated by the Board appearing at title 5, CFR, chapter VI, §§ 1660.1 through 1660.5 will no longer be... 29 Labor 9 2010-07-01 2010-07-01 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...

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

    PubMed Central

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

    2012-01-01

    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

  17. Detection of human papillomavirus DNA and oncoprotein overexpression are associated with distinct morphological patterns of tonsillar squamous cell carcinoma.

    PubMed Central

    Wilczynski, S. P.; Lin, B. T.; Xie, Y.; Paz, I. B.

    1998-01-01

    Human papillomavirus (HPV) DNA has been detected in approximately 15% of squamous cell carcinomas (SCCs) of the head and neck. Recent studies have shown a predilection of HPV for certain anatomical sites, especially the tonsillar region, with viral DNA identified in approximately 60% of SCCs of the Waldeyer's tonsillar ring. This study was undertaken to determine whether there are differences in morphology or in oncogene expression in SCC of the tonsil with and without detectable HPV DNA. Twenty-two SCCs of the tonsil were analyzed for the presence of HPV DNA by polymerase chain reaction (PCR) using both a consensus primer set (My09/My11) and type-specific primers. Viral transcription was established in both primary and metastatic tumors by RNA in situ hybridization. The morphology of invasive SCC was classified into three subtypes: well keratinized (K-SCC), intermediate keratinized (I-SCC), and poorly keratinized (P-SCC). Expression of p53, pRB, and cyclin D1 (bcl-1) were studied by immunohistochemistry. In these cases (6 K-SCCs, 2 I-SCCs, and 14 P-SCCs), HPV DNA was detected in 14 (64%), with 11 containing HPV-16 (10 P-SCCs, 1 I-SCCs, and 0 K-SCCs) and 1 each containing HPV-33, HPV-59, and an unclassified HPV type (all P-SCCs). Viral oncoprotein E6/E7 transcription was demonstrated in 7 of 7 HPV-16-positive tumors. Cyclin D1 protein overexpression was detected in the majority of HPV-negative tumors (7 of 8 cases), whereas it was minimal or absent in 13 HPV-positive tumors. Overexpression of p53 protein was detected in 3 HPV-negative K-SCCs. In the HPV-positive tumors, fewer malignant cells expressed pRB and the staining was less intense than in the HPV-negative cancers. HPV DNA and E6/E7 expression, especially HPV-16, is detected in the majority of tonsillar SCCs and is almost exclusively associated with a poorly keratinized tumor histology. Decreased expression of cyclin D1, pRB, and p53 in tumors with HPV DNA is consistent with the known effects of the viral

  18. Cellular localization of BARF1 oncoprotein and its cell stimulating activity in human epithelial cell.

    PubMed

    Sakka, Emna; Zur Hausen, Axel; Houali, Karim; Liu, Haying; Fiorini, Sylvie; Ooka, Tadamasa

    2013-06-01

    BARF1 gene encoded by Epstein-Barr virus is capable of immortalizing the primary monkey epithelial cells and of inducing malignant transformation in human EBV-negative B cell lines as well as rodent fibroblast. This oncoprotein is a secreted protein capable of acting as a powerful mitogen. We have studied the effect of BARF1 protein in transfected or BARF1 protein treated human HaCaT epithelial cells. In BARF1-transfected cells, cell growth was activated and its protein was found both in culture medium and cellular compartment (membrane, cytoplasm and nuclei). When purified BARF1 protein was exogenously added in the cell culture medium of HaCaT cells in absence of fetal calf serum led to its entrance into cells and its intracellular localization in cytoplasm, nuclear periphery and nuclei at 14h treatment, determined by confocal and immunoelectron microscopy. Cell fractionation confirmed its nuclear localization. Nuclear localization was observed in both systems. More interestingly, purified BARF1 protein p29 exogenously added in the cell culture medium activated cell passage of G1 to S phase. S phase activation by its autocrine activity and its tumorigenic activity would be associated with the development of EBV-associated carcinomas. PMID:23458996

  19. LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding

    PubMed Central

    Layer, Justin H.; Alford, Catherine E.; McDonald, W. Hayes

    2015-01-01

    LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R320LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens. PMID:26598604

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

    PubMed

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

    2014-10-01

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

  1. 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-203–induced 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

  2. Interferon regulatory factor 7 is activated by a viral oncoprotein through RIP-dependent ubiquitination.

    PubMed

    Huye, Leslie E; Ning, Shunbin; Kelliher, Michelle; Pagano, Joseph S

    2007-04-01

    As a key mediator of type I interferon (IFN) (IFN-alpha/beta) responses, IFN regulatory factor 7 (IRF7) is essential to host immune defenses. Activation of IRF7 generally requires virus-induced C-terminal phosphorylation, which leads to its nuclear accumulation and activation of target genes. Here we use the Epstein-Barr virus (EBV) oncoprotein LMP1, which activates IRF7, to identify factors involved in IRF7 activation. We demonstrate for the first time that RIP activates IRF7 and that RIP and IRF7 interact under physiological conditions in EBV-positive Burkitt's lymphoma cells. We provide evidence that both RIP and IRF7 are ubiquitinated in these cells and that IRF7 preferentially interacts with ubiquitinated RIP. RIP is required for full activation of IRF7 by LMP1, with LMP1 stimulating the ubiquitination of RIP and its interaction with IRF7. Moreover, LMP1 stimulates RIP-dependent K63-linked ubiquitination of IRF7, which regulates protein function rather than proteasomal degradation of proteins. We suggest that RIP may serve as a general activator of IRF7, responding to and transmitting the signals from various stimuli, and that ubiquitination may be a general mechanism for enhancing the activity of IRF7. PMID:17296724

  3. [Inactivation of failsafe programs by Twist oncoproteins and tumor progression].

    PubMed

    Puisieux, A

    2008-01-01

    Multicellular organisms have developed innate defense mechanisms to prevent the expansion of abnormal cells with significant proliferative potential. The two major safeguard mechanisms are premature senescence, which is characterized by definitive cell cycle arrest, and apoptosis, the most common form of programmed cell death. In normal and premalignant cells, the control of these processes is coupled to the regulation of cell proliferation, mainly through the p16 (Ink4A) -Rb and ARF-p53 intracellular signaling pathways. Hence, in benign tumors, aberrant mitogenic activity is counterbalanced by the induction of these oncosuppressive pathways, leading to either apoptosis or senescence which both limit tumor outgrowth. Progression towards malignant and potentially metastatic tumors requires the inhibition of these failsafe programs. Based on our work on Twist oncoproteins, we propose a presentation of recent data on cellular mechanisms by which cancer cells override the surveillance machinery and escape senescence and apoptosis, and we will describe the biological impact of this process on tumor metastasis. PMID:19061727

  4. Small-Molecule Inhibitors of the Myc Oncoprotein

    PubMed Central

    Fletcher, Steven; Prochownik, Edward V.

    2014-01-01

    The c-Myc (Myc) oncoprotein is among the most attractive of cancer targets given that is deregulated in the majority of tumors and that its inhibition profoundly affects their growth and/or survival. However, its role as a seldom-mutated transcription factor, its lack of enzymatic activity for which suitable pharmaceutical inhibitors could be crafted and its expression by normal cells have largely been responsible for its being viewed as “undruggable”. Work over the past several years, however, has begun to reverse this idea by allowing us to view Myc within the larger context of global gene regulatory control. Thus, Myc and its obligate heterodimeric partner, Max, are integral to the coordinated recruitment and post-translational modification of components of the core transcriptional machinery. Moreover, Myc over-expression re-programs numerous critical cellular functions and alters the cell’s susceptibility to their inhibition. This new knowledge has therefore served as a framework upon which to develop new pharmaceutical approaches. These include the continuing development of small molecules which act directly to inhibit the critical Myc-Max interaction, those which act indirectly to prevent Myc-directed post-translational modifications necessary to initiate productive transcription and those which inhibit vital pathways upon which the Myc-transformed cell is particularly reliant. PMID:24657798

  5. The HTLV-1 Tax oncoprotein represses Ku80 gene expression.

    PubMed

    Ducu, Razvan I; Dayaram, Tajhal; Marriott, Susan J

    2011-07-20

    The HTLV-I oncoprotein Tax interferes with DNA double strand break repair. Since non-homologous end joining (NHEJ) is a major pathway used to repair DNA double strand breaks we examined the effect of Tax on this pathway, with particular interest in the expression and function of Ku80, a critical component of the NHEJ pathway. Tax expression decreased Ku80 mRNA and protein levels, and repressed transcription from the Ku80 promoter. Conversely, Ku80 mRNA increased following siRNA knockdown of Tax in HTLV-I infected cells. Tax expression was associated with an elevated number of micronuclei and nucleoplasmic bridges, hallmarks of improper DNA double strand break repair. Our studies identified Tax as a transcriptional repressor of Ku80 that correlates with decreased DNA repair function. The reduction of Ku80 transcription by Tax may deplete the cell of an essential DNA break binding protein, resulting in reduced repair of DNA double strand breaks and accumulation genomic mutations. PMID:21571351

  6. E7(7) on the light cone

    NASA Astrophysics Data System (ADS)

    Brink, Lars; Kim, Sung-Soo; Ramond, Pierre

    2008-06-01

    We use the Cremmer-Julia E7(7) non-linear symmetry of Script N = 8 Supergravity to derive its order κ2 on-shell Hamiltonian in terms of one chiral light-cone superfield. By requiring that E7(7) commute with the super-Poincaré group, we deduce to lowest non-trivial order in κ, the light cone E7(7) transformations of all fields of the theory, including the graviton. We then derive the dynamical supersymmetry transformation to order κ2, and express the Hamiltonian as a quadratic form in the chiral superfield.

  7. Chimeric infectious bursal disease virus-like particles as potent vaccines for eradication of established HPV-16 E7-dependent tumors.

    PubMed

    Martin Caballero, Juan; Garzón, Ana; González-Cintado, Leticia; Kowalczyk, Wioleta; Jimenez Torres, Ignacio; Calderita, Gloria; Rodriguez, Margarita; Gondar, Virgínia; Bernal, Juan Jose; Ardavín, Carlos; Andreu, David; Zürcher, Thomas; von Kobbe, Cayetano

    2012-01-01

    Cervical cancer is caused by persistent high-risk human papillomavirus (HR-HPV) infection and represents the second most frequent gynecological malignancy in the world. The HPV-16 type accounts for up to 55% of all cervical cancers. The HPV-16 oncoproteins E6 and E7 are necessary for induction and maintenance of malignant transformation and represent tumor-specific antigens for targeted cytotoxic T lymphocyte-mediated immunotherapy. Therapeutic cancer vaccines have become a challenging area of oncology research in recent decades. Among current cancer immunotherapy strategies, virus-like particle (VLP)-based vaccines have emerged as a potent and safe approach. We generated a vaccine (VLP-E7) incorporating a long C-terminal fragment of HPV-16 E7 protein into the infectious bursal disease virus VLP and tested its therapeutic potential in HLA-A2 humanized transgenic mice grafted with TC1/A2 tumor cells. We performed a series of tumor challenge experiments demonstrating a strong immune response against already-formed tumors (complete eradication). Remarkably, therapeutic efficacy was obtained with a single dose without adjuvant and against two injections of tumor cells, indicating a potent and long-lasting immune response. PMID:23300838

  8. 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

  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 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 α4-1BB 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. 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 1α (HIF1α). The stability of HIF1α was enhanced by HBXIP through disassociating interaction of von Hippel-Lindau protein (pVHL) with HIF1α. Moreover, miR-183 increased the levels of HIF1α protein through directly targeting CDS of VHL mRNA, forming a feedback loop of HIF1α/miR-183/pVHL/HIF1α. 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

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

    PubMed Central

    Cesur, Özlem; 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

  12. 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

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

    PubMed

    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

  14. 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…

  15. 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.

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

    PubMed Central

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

    2015-01-01

    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

  17. 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.

  18. 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

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

    PubMed Central

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

    2013-01-01

    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. PMID:23578663

  20. 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.

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

    PubMed Central

    Fischer, Martin; Quaas, Marianne; Wintsche, Axel; Müller, 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-p21WAF1/CIP1-DREAM signaling pathway through the CDE and CHR sites. Cell cycle- and p53-dependent repression is abrogated by HPV E7 oncoprotein. Together with genome-wide analyses our results imply that many cell cycle genes upregulated in tumors by viral infection are bound by DREAM through CDE/CHR sites. PMID:24071582

  2. 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

  3. The Human Papillomavirus Type 16 E6 Oncoprotein Can Down-Regulate p53 Activity by Targeting the Transcriptional Coactivator CBP/p300

    PubMed Central

    Zimmermann, Holger; Degenkolbe, Roland; Bernard, Hans-Ulrich; O’Connor, Mark J.

    1999-01-01

    The transforming proteins of the small DNA tumor viruses, simian virus 40 (SV40), adenovirus, and human papillomavirus (HPV) target a number of identical cellular regulators whose functional abrogation is required for transformation. However, while both adenovirus E1A and SV40 large T transforming properties also depend on the targeting of the transcriptional coactivator CBP/p300, no such interaction has been described for the HPV oncoprotein E6 or E7. Here, we demonstrate that the HPV-16 E6 protein, previously shown to facilitate the degradation of p53 in a complex with E6-associated protein (E6AP), also targets CBP/p300 in an interaction involving the C-terminal zinc finger of E6 and CBP residues 1808 to 1826. Furthermore, this interaction is limited to E6 proteins of high-risk HPVs associated with cervical cancer that have the capacity to repress p53-dependent transcription. An HPV-16 E6 mutant (L50G) that binds CBP/p300, but not E6AP, is still capable of down-regulating p53 transcriptional activity. Thus, HPV E6 proteins possess two distinct mechanisms by which to abrogate p53 function: the repression of p53 transcriptional activity by targeting the p53 coactivator CBP/p300, and the removal of cellular p53 protein through the proteosome degradation pathway. PMID:10400710

  4. 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

  5. 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

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

    PubMed Central

    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.

    2015-01-01

    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

  7. 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

  8. Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells.

    PubMed

    Francis, D A; Schmid, S I; Howley, P M

    2000-03-01

    The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA. PMID:10684283

  9. A Drosophila Model of HPV E6-Induced Malignancy Reveals Essential Roles for Magi and the Insulin Receptor

    PubMed Central

    Padash Barmchi, Mojgan; Gilbert, Mary; Thomas, Miranda; Banks, Lawrence; Zhang, Bing; Auld, Vanessa J.

    2016-01-01

    Cervical cancer is one of the leading causes of cancer death in women worldwide. The causative agents of cervical cancers, high-risk human papillomaviruses (HPVs), cause cancer through the action of two oncoproteins, E6 and E7. The E6 oncoprotein cooperates with an E3 ubiquitin ligase (UBE3A) to target the p53 tumour suppressor and important polarity and junctional PDZ proteins for proteasomal degradation, activities that are believed to contribute towards malignancy. However, the causative link between degradation of PDZ proteins and E6-mediated malignancy is largely unknown. We have developed an in vivo model of HPV E6-mediated cellular transformation using the genetic model organism, Drosophila melanogaster. Co-expression of E6 and human UBE3A in wing and eye epithelia results in severe morphological abnormalities. Furthermore, E6, via its PDZ-binding motif and in cooperation with UBE3A, targets a suite of PDZ proteins that are conserved in human and Drosophila, including Magi, Dlg and Scribble. Similar to human epithelia, Drosophila Magi is a major degradation target. Magi overexpression rescues the cellular abnormalities caused by E6+UBE3A coexpression and this activity of Magi is PDZ domain-dependent. Drosophila p53 was not targeted by E6+UBE3A, and E6+UBE3A activity alone is not sufficient to induce tumorigenesis, which only occurs when E6+UBE3A are expressed in conjunction with activated/oncogenic forms of Ras or Notch. Finally, through a genetic screen we have identified the insulin receptor signaling pathway as being required for E6+UBE3A induced hyperplasia. Our results suggest a highly conserved mechanism of HPV E6 mediated cellular transformation, and establish a powerful genetic model to identify and understand the cellular mechanisms that underlie HPV E6-induced malignancy. PMID:27537218

  10. A Drosophila Model of HPV E6-Induced Malignancy Reveals Essential Roles for Magi and the Insulin Receptor.

    PubMed

    Padash Barmchi, Mojgan; Gilbert, Mary; Thomas, Miranda; Banks, Lawrence; Zhang, Bing; Auld, Vanessa J

    2016-08-01

    Cervical cancer is one of the leading causes of cancer death in women worldwide. The causative agents of cervical cancers, high-risk human papillomaviruses (HPVs), cause cancer through the action of two oncoproteins, E6 and E7. The E6 oncoprotein cooperates with an E3 ubiquitin ligase (UBE3A) to target the p53 tumour suppressor and important polarity and junctional PDZ proteins for proteasomal degradation, activities that are believed to contribute towards malignancy. However, the causative link between degradation of PDZ proteins and E6-mediated malignancy is largely unknown. We have developed an in vivo model of HPV E6-mediated cellular transformation using the genetic model organism, Drosophila melanogaster. Co-expression of E6 and human UBE3A in wing and eye epithelia results in severe morphological abnormalities. Furthermore, E6, via its PDZ-binding motif and in cooperation with UBE3A, targets a suite of PDZ proteins that are conserved in human and Drosophila, including Magi, Dlg and Scribble. Similar to human epithelia, Drosophila Magi is a major degradation target. Magi overexpression rescues the cellular abnormalities caused by E6+UBE3A coexpression and this activity of Magi is PDZ domain-dependent. Drosophila p53 was not targeted by E6+UBE3A, and E6+UBE3A activity alone is not sufficient to induce tumorigenesis, which only occurs when E6+UBE3A are expressed in conjunction with activated/oncogenic forms of Ras or Notch. Finally, through a genetic screen we have identified the insulin receptor signaling pathway as being required for E6+UBE3A induced hyperplasia. Our results suggest a highly conserved mechanism of HPV E6 mediated cellular transformation, and establish a powerful genetic model to identify and understand the cellular mechanisms that underlie HPV E6-induced malignancy. PMID:27537218

  11. Expression and In Silico Analysis of the Recombinant Bovine Papillomavirus E6 Protein as a Model for Viral Oncoproteins Studies

    PubMed Central

    Mazzuchelli-de-Souza, J.; Carvalho, R. F.; Ruiz, R. M.; Melo, T. C.; Araldi, R. P.; Carvalho, E.; Thompson, C. E.; Sircili, M. P.; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomaviruses (BPVs) are recognized as the causal agents of economical relevant diseases in cattle, associated with the development of tumors in skin and mucosa. The oncogenesis process is mainly associated with different viral oncoprotein expressions, which are involved in cell transformation. The expression and characterization of recombinant viral oncoproteins represent an attractive strategy to obtain biotechnological products as antibodies and potential vaccines, Thus, the aim of this work was to clone and express the BPV-1 and BPV-2 E6 recombinant proteins and perform in silico analysis in order to develop a strategy for the systematic study of other papillomaviruses oncoproteins. The results demonstrated that BPV-1 and BPV-2 E6 recombinant proteins were expressed and purified from bacterial system as well as its in silico analysis was performed in order to explore and predict biological characteristics of these proteins. PMID:23878806

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

    PubMed Central

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

    2015-01-01

    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

  13. Intracellular Analysis of the Interaction between the Human Papillomavirus Type 16 E6 Oncoprotein and Inhibitory Peptides.

    PubMed

    Stutz, Christina; Reinz, Eileen; Honegger, Anja; Bulkescher, Julia; Schweizer, Johannes; Zanier, Katia; Travé, Gilles; Lohrey, Claudia; Hoppe-Seyler, Karin; Hoppe-Seyler, Felix

    2015-01-01

    Oncogenic types of human papillomaviruses (HPVs) cause cervical cancer and other malignancies in humans. The HPV E6 oncoprotein is considered to be an attractive therapeutic target since its inhibition can lead to the apoptotic cell death of HPV-positive cancer cells. The HPV type 16 (HPV16) E6-binding peptide pep11, and variants thereof, induce cell death specifically in HPV16-positive cancer cells. Although they do not encompass the LxxLL binding motif found in cellular HPV16 E6 interaction partners, such as E6AP, the pep11 variants strongly bind to HPV16 E6 by contacting the recently identified E6AP binding pocket. Thus, these peptides can serve as prototype E6-inhibitory molecules which target the E6AP pocket. We here analyzed their intracellular interaction with HPV16 E6. By comprehensive intracellular binding studies and GST pull-down assays, we show that E6-binding competent pep11 variants induce the formation of a trimeric complex, consisting of pep11, HPV16 E6 and p53. These findings indicate that peptides, which do not contain the LxxLL motif, can reshape E6 to enable its interaction with p53. The formation of the trimeric HPV16 E6 / peptide / p53 complex was associated with an increase of endogenous HPV16 E6 protein amounts. Yet, total cellular p53 amounts were also increased, indicating that the E6 / E6AP-mediated degradation of p53 is blocked. These findings suggest that inhibition of oncogenic activities by targeting the E6AP pocket on HPV16 E6 could be a strategy for therapeutic intervention. PMID:26151636

  14. Alternative splicing and caspase-mediated cleavage generate antagonistic variants of the stress oncoprotein LEDGF/p75.

    PubMed

    Brown-Bryan, Terry A; Leoh, Lai S; Ganapathy, Vidya; Pacheco, Fabio J; Mediavilla-Varela, Melanie; Filippova, Maria; Linkhart, Thomas A; Gijsbers, Rik; Debyser, Zeger; Casiano, Carlos A

    2008-08-01

    There is increasing evidence that an augmented state of cellular oxidative stress modulates the expression of stress genes implicated in diseases associated with health disparities such as certain cancers and diabetes. Lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 autoantigen, is emerging as a survival oncoprotein that promotes resistance to oxidative stress-induced cell death and chemotherapy. We previously showed that LEDGF/p75 is targeted by autoantibodies in prostate cancer patients and is overexpressed in prostate tumors, and that its stress survival activity is abrogated during apoptosis. LEDGF/p75 has a COOH-terminally truncated splice variant, p52, whose role in stress survival and apoptosis has not been thoroughly investigated. We observed unbalanced expression of these proteins in a panel of tumor cell lines, with LEDGF/p75 generally expressed at higher levels. During apoptosis, caspase-3 cleaved p52 to generate a p38 fragment that lacked the NH(2)-terminal PWWP domain and failed to transactivate the Hsp27 promoter in reporter assays. However, p38 retained chromatin association properties and repressed the transactivation potential of LEDGF/p75. Overexpression of p52 or its variants with truncated PWWP domains in several tumor cell lines induced apoptosis, an activity that was linked to the presence of an intron-derived COOH-terminal sequence. These results implicate the PWWP domain of p52 in transcription function but not in chromatin association and proapoptotic activities. Consistent with their unbalanced expression in tumor cells, LEDGF/p75 and p52 seem to play antagonistic roles in the cellular stress response and could serve as targets for novel antitumor therapies. PMID:18708362

  15. 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

  16. 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

  17. 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

  18. Oxymatrine Downregulates HPV16E7 Expression and Inhibits Cell Proliferation in Laryngeal Squamous Cell Carcinoma Hep-2 Cells In Vitro

    PubMed Central

    Ying, Xin-Jiang; Jin, Bin; Chen, Xin-Wei; Xie, Jin; Xu, Hong-Ming; Dong, Pin

    2015-01-01

    Objective. To investigate the possible mechanisms of oxymatrine's role in anti laryngeal squamous cell carcinoma. Methods. We examined the effects of oxymatrine on the proliferation, cell cycle phase distribution, apoptosis, and the protein and mRNA expression levels of HPV16E7 gene in laryngeal carcinoma Hep-2 cells in vitro. The HPV16E7 siRNA inhibition was also done to confirm the effect of downregulating HPV16E7 on the proliferation in Hep-2 cells. Results. Oxymatrine significantly inhibited the growth and proliferation of Hep-2 cells in a dose-dependence and time-dependence manner. Oxymatrine blocked Hep-2 cells in G0/G1 phase, resulting in an obvious accumulation of G0/G1 phase cells while decreasing S phase cells. Oxymatrine induced apoptosis of Hep-2 cells, whose apoptotic rate amounted to about 42% after treatment with 7 mg/mL oxymatrine for 72 h. Oxymatrine also downregulated the expression of HPV16E7 gene, as determined by the western blotting and reverse transcription-polymerase chain reaction analysis. Knockdown of HPV16E7 effectively inhibited the proliferation of Hep-2 cells. Conclusions. Oxymatrine inhibits the proliferation and induces apoptosis of laryngeal carcinoma Hep-2 cells, which might be mediated by a significant cell cycle arrest in G0/G1 phase and downregulation of HPV16E7 gene. Oxymatrine is considered to be a likely preventive and curative candidate for laryngeal cancer. PMID:25811021

  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. 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.

  1. 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.

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

    PubMed Central

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

    2012-01-01

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

  3. 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-γ (IFN-γ) 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

  4. 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

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

    PubMed Central

    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. Deletion analysis of BMI1 oncoprotein identifies its negative regulatory domain

    PubMed Central

    2010-01-01

    Background The polycomb group (PcG) protein BMI1 is an important regulator of development. Additionally, aberrant expression of BMI1 has been linked to cancer stem cell phenotype and oncogenesis. In particular, its overexpression has been found in several human malignancies including breast cancer. Despite its established role in stem cell maintenance, cancer and development, at present not much is known about the functional domains of BMI1 oncoprotein. In the present study, we carried out a deletion analysis of BMI1 to identify its negative regulatory domain. Results We report that deletion of the C-terminal domain of BMI1, which is rich in proline-serine (PS) residues and previously described as PEST-like domain, increased the stability of BMI1, and promoted its pro-oncogenic activities in human mammary epithelial cells (HMECs). Specifically, overexpression of a PS region deleted mutant of BMI1 increased proliferation of HMECs and promoted an epithelial-mesenchymal transition (EMT) phenotype in the HMECs. Furthermore, when compared to the wild type BMI1, exogenous expression of the mutant BMI1 led to a significant downregulation of p16INK4a and an efficient bypass of cellular senescence in human diploid fibroblasts. Conclusions In summary, our data suggest that the PS domain of BMI1 is involved in its stability and that it negatively regulates function of BMI1 oncoprotein. Our results also suggest that the PS domain of BMI1 could be targeted for the treatment of proliferative disorders such as cancer and aging. PMID:20569464

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

    PubMed

    Hashi, Kana; Murata-Kamiya, Naoko; Varon, Christine; Mégraud, 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

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

  10. Kaposi sarcoma herpesvirus (KSHV) vFLIP oncoprotein induces B cell transdifferentiation and tumorigenesis in mice

    PubMed Central

    Ballon, Gianna; Chen, Kang; Perez, Rocio; Tam, Wayne; Cesarman, Ethel

    2011-01-01

    Kaposi sarcoma herpesvirus (KSHV) is specifically associated with Kaposi sarcoma (KS) and 2 B cell lymphoproliferative diseases, namely primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). KS, PEL, and MCD are largely incurable and poorly understood diseases most common in HIV-infected individuals. Here, we have revealed the role of viral FLICE-inhibitory protein (vFLIP) in the initiation of PEL and MCD by specifically expressing vFLIP at different stages of B cell differentiation in vivo. Mice showed MCD-like abnormalities and immunological defects including lack of germinal centers (GCs), impaired Ig class switching, and affinity maturation. In addition, they showed increased numbers of cells expressing cytoplasmic IgM-λ, a thus far enigmatic feature of the KSHV-infected cells in MCD. B cell–derived tumors arose at high incidence and displayed Ig gene rearrangement with downregulated expression of B cell–associated antigens, which are features of PEL. Interestingly, these tumors exhibited characteristics of transdifferentiation and acquired expression of histiocytic/dendritic cell markers. These results define immunological functions for vFLIP in vivo and reveal what we believe to be a novel viral-mediated tumorigenic mechanism involving B cell reprogramming. Additionally, the robust recapitulation of KSHV-associated diseases in mice provides a model to test inhibitors of vFLIP as potential anticancer agents. PMID:21339646

  11. Transforming activity of a novel mutant of HPV16 E6E7 fusion gene.

    PubMed

    Xie, Qiang; Zhou, Zhi-Xiang; Li, Ze-Lin; Zeng, Yi

    2011-06-01

    An optimized recombinant HPV16 E6E7 fusion gene (HPV16 ofE6E7) was constructed according to codon usage for mammalian cell expression, and a mutant of HPV16 ofE6E7 fusion gene (HPV16 omfE6E7) was generated by site-directed mutagenesis at L57G, C113R for the E6 protein and C24G, E26G for the E7 protein for HPV16 ofE6E7 [patent pending (CN 101100672)]. The HPV16 omfE6E7 gene constructed in this work not only lost the transformation capability to NIH 3T3 cells and tumorigenicity in SCID mice, but also maintained very good stability and antigenicity. These results suggests that the HPV16 omfE6E7 gene should undergo further study for application as a safe antigen-specific therapeutic vaccine for HPV16-associated tumors. PMID:21667341

  12. In Silico Profiling of the Potentiality of Curcumin and Conventional Drugs for CagA Oncoprotein Inactivation.

    PubMed

    Srivastava, Akhileshwar K; Tewari, Mallika; Shukla, Hari S; Roy, Bijoy K

    2015-08-01

    The oncoprotein cytotoxic associated gene A (CagA) of Helicobacter pylori plays a pivotal role in the development of gastric cancer, so it has been an important target for anti-H. pylori drugs. Conventional drugs are currently being implemented against H. pylori. The inhibitory role of plant metabolites like curcumin against H. pylori is still a major scientific challenge. Curcumin may represent a novel promising drug against H. pylori infection without producing side effects. In the present study, a comparative analysis between curcumin and conventional drugs (clarithromycin, amoxicillin, pantoprazole, and metronidazole) was carried out using databases to investigate the potential of curcumin against H. pylori targeting the CagA oncoprotein. Curcumin was filtered using Lipinski's rule of five and the druglikeness property for evaluation of pharmacological properties. Subsequently, molecular docking was employed to determine the binding affinities of curcumin and conventional drugs to the CagA oncoprotein. According to the results obtained from FireDock, the binding energy of curcumin was higher than those of amoxicillin, pantoprazole, and metronidazole, except for clarithromycin, which had the highest binding energy. Accordingly, curcumin may become a promising lead compound against CagA+ H. pylori infection. PMID:25996140

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

    PubMed Central

    Boechi, Leonardo; Arrar, Mehrnoosh; Martí, Marcelo A.; Olson, John S.; Roitberg, Adrián E.; Estrin, Darío 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

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

    PubMed Central

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

    2012-01-01

    Due to the strong relationship between the Human Papillomavirus (HPV) “high-risk” subtypes and cervical cancers, most HPV-related studies have been focusing on the “high-risk” HPV subtypes 16 and 18. However, it has been suggested that the “low-risk” subtypes of HPV, HPV6 and HPV11, are the major cause of recurrent respiratory papillomatosis and genital warts. In addition, HPV 6 and 11 are also associated with otolaryngologic malignancies, carcinoma of the lung, tonsil, larynx and low-grade cervical lesions. Therefore, development of HPV therapeutic vaccines targeting on subtypes 6 and 11 E6 or E7 are in great need. In this report, we describe two novel engineered DNA vaccines that encode HPV 6 and 11 consensus E6/E7 fusion proteins (p6E6E7 and p11E6E7) by utilizing a multi-phase strategy. Briefly, after generating consensus sequences, several modifications were performed to increase the expression of both constructs, including codon/RNA optimization, addition of a Kozak sequence and a highly efficient leader sequence. An endoproteolytic cleavage site was also introduced between E6 and E7 protein for proper protein folding and for better CTL processing. The expressions of both constructs were confirmed by western blot analysis and immunofluorescence assay. Vaccination with these DNA vaccines could elicit robust cellular immune responses. The epitope mapping assay was performed to further characterize the cellular immune responses induced by p6E6E7 and p11E6E7. The HPV 6 and 11 E6 or E7-specific immunodominant and subdominant epitopes were verified, respectively. The intracellular cytokine staining revealed that the magnitude of IFN-γ and TNF-α secretion in antigen-specific CD8+ cells was significantly enhanced, indicating that the immune responses elicited by p6E6E7 and p11E6E7 was heavily skewed toward driving CD8+ T cells. Such DNA immunogens are interesting candidates for further studies on HPV 6 and 11-associated diseases. PMID:22336879

  15. Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells.

    PubMed Central

    Bosch, F X; Schwarz, E; Boukamp, P; Fusenig, N E; Bartsch, D; zur Hausen, H

    1990-01-01

    The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells. Images PMID:2168962

  16. Acetylation of the c-MYC oncoprotein is required for cooperation with the HTLV-1 p30{sup II} accessory protein and the induction of oncogenic cellular transformation by p30{sup II}/c-MYC

    SciTech Connect

    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

    2015-02-15

    The human T-cell leukemia retrovirus type-1 (HTLV-1) p30{sup II} 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 p30{sup II} 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 p30{sup II} and c-MYC remain to be completely understood. Herein we demonstrate that p30{sup II} induces lysine-acetylation of the c-MYC oncoprotein. Acetylation-defective c-MYC Lys→Arg substitution mutants are impaired for oncogenic transformation with p30{sup II} in c-myc{sup −/−} HO15.19 fibroblasts. Using dual-chromatin-immunoprecipitations (dual-ChIPs), we further demonstrate that p30{sup II} is present in c-MYC-containing nucleoprotein complexes in HTLV-1-transformed HuT-102 T-lymphocytes. Moreover, p30{sup II} 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 p30{sup II}/c-MYC which could promote proviral replication and contribute to HTLV-1-induced carcinogenesis. - Highlights: • Acetylation of c-MYC is required for oncogenic transformation by HTLV-1 p30{sup II}/c-MYC. • Acetylation-defective c-MYC mutants are impaired for foci-formation by p30{sup II}/c-MYC. • The HTLV-1 p30{sup II} protein induces lysine-acetylation of c-MYC. • p30{sup II} is present in c-MYC nucleoprotein complexes in HTLV-1-transformed T-cells. • HTLV-1 p30{sup II} inhibits apoptosis in c-MYC-expressing proliferating cells.

  17. Specific recognition of four-way DNA junctions by the C-terminal zinc-binding domain of HPV oncoprotein E6.

    PubMed

    Ristriani, T; Nominé, Y; Masson, M; Weiss, E; Travé, G

    2001-01-26

    E6 is an oncoprotein implicated in cervical cancers produced by " high risk " human papillomaviruses. E6 binds specifically to several cellular proteins, including the tumour suppressor p53 and the ubiquitin ligase E6-AP. However, E6 is also a DNA-binding protein which recognizes a structural motive present in four-way junctions. Here, we demonstrate that the C-terminal zinc-binding domain of E6, expressed separately from the rest of the protein, fully retains the selective four-way junction recognition activity. The domain can bind to two identical and independent sites on a single junction, whereas full-length E6 can only bind to one site. The junction bound to either one or two domains adopts an extended square conformation. These results allow us to assign the structure-dependent DNA recognition activity of E6 to its C-terminal domain, which therefore represents a new class of zinc-stabilized DNA-binding module. Comparison with the binding characteristics of other junction-specific proteins enlightens the rules which govern protein-induced deformation of four-way DNA junctions. PMID:11162088

  18. Functional interactions of the cystine/glutamate antiporter, CD44v and MUC1-C oncoprotein in triple-negative breast cancer cells

    PubMed Central

    Hasegawa, Masanori; Takahashi, Hidekazu; Rajabi, Hasan; Alam, Maroof; Suzuki, Yozo; Yin, Li; Tagde, Ashujit; Maeda, Takahiro; Hiraki, Masayuki; Sukhatme, Vikas P.; Kufe, Donald

    2016-01-01

    The xCT light chain of the cystine/glutamate transporter (system XC−) is of importance for the survival of triple-negative breast cancer (TNBC) cells. The MUC1-C transmembrane oncoprotein is aberrantly overexpressed in TNBC and, like xCT, has been linked to maintaining glutathione (GSH) levels and redox balance. However, there is no known interaction between MUC1-C and xCT. Here we show that silencing MUC1-C is associated with decreases in xCT expression in TNBC cells. The results demonstrate that MUC1-C forms a complex with xCT and the CD44 variant (CD44v), which interacts with xCT and thereby controls GSH levels. MUC1-C binds directly with CD44v and in turn promotes stability of xCT in the cell membrane. The interaction between MUC1-C and xCT is further supported by the demonstration that targeting xCT with silencing or the inhibitor sulfasalazine suppresses MUC1 gene transcription by increasing histone and DNA methylation on the MUC1 promoter. In terms of the functional significance of the MUC1-C/xCT interaction, we show that MUC1-C protects against treatment with erastin, an inhibitor of XC− and inducer of ferroptosis, a form of non-apoptotic cell death. These findings indicate that targeting this novel MUC1-C/xCT pathway could represent a potential therapeutic approach for promoting TNBC cell death. PMID:26930718

  19. 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

  20. 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

  1. 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

  2. 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

  3. Impact of Ser17 Phosphorylation on the Conformational Dynamics of the Oncoprotein MDM2.

    PubMed

    Bueren-Calabuig, Juan A; Michel, Julien

    2016-05-01

    MDM2 is an important oncoprotein that downregulates the activity of the tumor suppressor protein p53 via binding of its N-terminal domain to the p53 transactivation domain. The first 24 residues of the MDM2 N-terminal domain form an intrinsically disordered "lid" region that interconverts on a millisecond time scale between "open" and "closed" states in unliganded MDM2. While the former conformational state is expected to facilitate p53 binding, the latter competes in a pseudo-substrate manner with p53 for its binding site. Phosphorylation of serine 17 in the MDM2 lid region is thought to modulate the equilibrium between "open" and "closed" lid states, but contradictory findings on the favored lid conformational state upon phosphorylation have been reported. Here, the nature of the conformational states of MDM2 pSer17 and Ser17Asp variants was addressed by means of enhanced sampling molecular dynamics simulations. Detailed analyses of the computed lid conformational ensembles indicate that both lid variants stabilize a "closed" state, with respect to wild type. Nevertheless, the nature of the closed-state conformational ensembles differs significantly between the pSer17 and Ser17Asp variants. Thus, care should be applied in the interpretation of biochemical experiments that use phosphomimetic variants to model the effects of phosphorylation on the structure and dynamics of this disordered protein region. PMID:27050388

  4. 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

  5. Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein

    PubMed Central

    King, Cason R.; Cohen, Michael J.; Fonseca, Gregory J.; Dirk, Brennan S.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2016-01-01

    The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs. PMID:27137912

  6. Aggregation In Heavy Water Micellar Dilute Solutions Of Three Nonionic Classic Surfactants: C10E7 AND C12E7 And C14E7 Study By SANS Method

    NASA Astrophysics Data System (ADS)

    Aldona, Rajewska

    2010-01-01

    Three nonionic classic surfactants C10E7 (heptaethylene glycol monodecyl ether) and C12E7 (heptaethylene glycol monododecyl ether) and C14E7 (heptaethylene glycol monotetradecyl ether) in water solutions were investigated for concentration c = 0.5% (dilute regime) at temperatures t = 6°, 10°, 15°, 20°, 25°, 30° and 35° C with two methods—tensiometric and small-angle neutron scattering ( SANS ) on SANS diffractometer "Yellow Submarine" at Budapest Neutron Center, Budapest ( Hungary ) and SANS spectrometer ("YuMO") of the IBR-2 on pulsed neutron source at FLNP, JINR in Dubna ( Russia ). Measurements have covered Q range from 8 10-3 to 0.4 Å-1. The micellar solutions were prepared in D2O since the contrast between the micelles and the solvent in neutron experiments is better with D2O than with H2O. It was obtained as the result that the shape of micelles changes depending on surfactant concentration and temperature. At lower concentrations and temperatures micelles are ellipsoids but at higher concentrations and temperature are rather cylinders. For calculation and approximation results from SANS experiments was used program PCG 2.0 of Glatter O. and co-workers from University of Graz (Austria).

  7. 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...

  8. 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...

  9. 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...

  10. 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...

  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. The footprint of E7(7) in amplitudes of Script N = 8 supergravity

    NASA Astrophysics Data System (ADS)

    Kallosh, Renata; Kugo, Taichiro

    2009-01-01

    We study the low energy theorems associated with the non-linearly realized continuous E7(7)(Bbb R) symmetry of the on-shell Script N = 8 supergravity. For Nambu-Goldstone bosons we evaluate the one-soft-scalar-boson emission amplitudes by computing the E7(7) current matrix element on the one-particle external lines. We use the explicit form of the conserved E7(7) Noether current and prove that all such matrix elements vanish in the soft momentum limit, assuming the SU(8) symmetry of the S-matrix. This implies that all tree amplitudes vanish in the one-soft-boson limit. We also discuss the implications of unbroken E7(7)(Bbb R) symmetry for higher-order amplitudes.

  13. Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

    PubMed Central

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  14. Combined stimulation of IL-2 and 4-1BB receptors augments the antitumor activity of E7 DNA vaccines by increasing Ag-specific CTL responses.

    PubMed

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  15. Detection of Human Papillomavirus-16 E6-Oncoprotein in Epithelial Ovarian Tumors Samples of Iraqi Patients

    PubMed Central

    Mahmood, Fahem Mohsin; Kadhim, Haider Sabah; Mousa Al Khuzaee, Liqaa Riadh

    2014-01-01

    Background: Human papillomavirus (HPV) is the causal factor for cervical cancer. However, the role of HPV infection in ovarian cancer is unclear. Objectives: This study aimed to determine the presence of human papillomavirus-16 (HPV-16) in ovarian tumor tissues. Patients and Methods: This was a retrospective study, which included 61 Archived human ovarian tumor tissues embedded in paraffin blocks. The ovarian tumor tissues were divided into four groups. The first group was the malignant ovarian epithelial tumor group; it included 31 cases with invasive surface epithelial ovarian tumors. The second group was the borderline epithelial ovarian tumor group: it included four cases with borderline intermediate malignancy. The third group was the benign epithelial ovarian tumors group: it included 18 cases with benign epithelial ovarian tumors. The fourth group had functional ovarian cystic lesions: it included eight cases with non-neoplastic functional ovarian cysts. Sections were made from each of the paraffin embedded blocks and examined using immunohistochemistry to detect HPV 16-E6-oncoprotein in ovarian tumor tissues. Results: Out of the eight cases with functional cysts only one case (12.5%) expressed HPV. No HPV expression was seen in cases with benign and borderline tumors. Out of the 31 cases with one malignant surface epithelial ovarian tumor only three (9.67%) cases expressed HPV. There was no significant statistical difference in HPV expression among neoplastic and non-neoplastic ovarian tumors included in the present study (P= 0.476). Conclusions: HPV type 16 was detected in only 9.67% of malignant epithelial tumors. It appears that HPV infection plays a relatively minor role in the pathogenesis of ovarian carcinomas. PMID:25485061

  16. A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models

    PubMed Central

    Amici, Carla; Visintin, Michela; Verachi, Francesca; Paolini, Francesca; Percario, Zulema; Di Bonito, Paola; Mandarino, Angela; Affabris, Elisabetta; Venuti, Aldo; Accardi, Luisa

    2016-01-01

    Single-chain variable fragments (scFvs) expressed as “intracellular antibodies” (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients. A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells. Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors. PMID:26788990

  17. E4BP4 expression is regulated by the t(17;19)-associated oncoprotein E2A-HLF in pro-B cells.

    PubMed

    Yeung, Jenny; O'Sullivan, Elaine; Hubank, Mike; Brady, Hugh J M

    2004-06-01

    The E4BP4 basic leucine zipper (bZIP) transcription factor is regulated by interleukin-3 (IL-3) in pro-B cells and has been reported to promote survival of the murine IL-3-dependent pro-B cell lines, FL5.12 and Baf-3. The E2A-HLF oncoprotein arises from a t(17;19) translocation in childhood pro-B cell acute lymphoblastic leukaemia and acts as an anti-apoptotic factor in FL5.12 and Baf-3 cells. To assess the functions of E2A-HLF and E4BP4 in cell survival, a tetracycline-inducible system was established in Baf-3 cells to express E4BP4 or E2A-HLF. Upon IL-3 withdrawal, expression of E2A-HLF conferred resistance to apoptosis whereas overexpression of E4BP4 did not. E4BP4 and E2A-HLF both recognized the same DNA sequence in reporter gene assays, but had opposite effects on transcription. E2A-HLF acts as a transcriptional activator and E4BP4 as a transcriptional repressor. Furthermore, E4BP4 is a downstream transcriptional target of E2A-HLF. Our data suggests that the overexpression of E4BP4 is unable to block apoptosis induced by IL-3 withdrawal and that the expression of E2A-HLF does not replace the function of E4BP4 in mediating survival. PMID:15147370

  18. A novel intracellular antibody against the E6 oncoprotein impairs growth of human papillomavirus 16-positive tumor cells in mouse models.

    PubMed

    Amici, Carla; Visintin, Michela; Verachi, Francesca; Paolini, Francesca; Percario, Zulema; Di Bonito, Paola; Mandarino, Angela; Affabris, Elisabetta; Venuti, Aldo; Accardi, Luisa

    2016-03-29

    Single-chain variable fragments (scFvs) expressed as "intracellular antibodies" (intrabodies) can target intracellular antigens to hamper their function efficaciously and specifically. Here we use an intrabody targeting the E6 oncoprotein of Human papillomavirus 16 (HPV16) to address the issue of a non-invasive therapy for HPV cancer patients.A scFv against the HPV16 E6 was selected by Intracellular Antibody Capture Technology and expressed as I7nuc in the nucleus of HPV16-positive SiHa, HPV-negative C33A and 293T cells. Colocalization of I7nuc and recombinant E6 was observed in different cell compartments, obtaining evidence of E6 delocalization ascribable to I7nuc. In SiHa cells, I7nuc expressed by pLNCX retroviral vector was able to partially inhibit degradation of the main E6 target p53, and induced p53 accumulation in nucleus. When analyzing in vitro activity on cell proliferation and survival, I7nuc was able to decrease growth inducing late apoptosis and necrosis of SiHa cells.Finally, I7nuc antitumor activity was demonstrated in two pre-clinical models of HPV tumors. C57BL/6 mice were injected subcutaneously with HPV16-positive TC-1 or C3 tumor cells, infected with pLNCX retroviral vector expressing or non-expressing I7nuc. All the mice injected with I7nuc-expressing cells showed a clear delay in tumor onset; 60% and 40% of mice receiving TC-1 and C3 cells, respectively, remained tumor-free for 17 weeks of follow-up, whereas 100% of the controls were tumor-bearing 20 days post-inoculum. Our data support the therapeutic potential of E6-targeted I7nuc against HPV tumors. PMID:26788990

  19. The Canine Papillomavirus and Gamma HPV E7 Proteins Use an Alternative Domain to Bind and Destabilize the Retinoblastoma Protein

    PubMed Central

    Wang, Jingang; Zhou, Dan; Prabhu, Anjali; Schlegel, Richard; Yuan, Hang

    2010-01-01

    The high-risk HPV E6 and E7 proteins cooperate to immortalize primary human cervical cells and the E7 protein can independently transform fibroblasts in vitro, primarily due to its ability to associate with and degrade the retinoblastoma tumor suppressor protein, pRb. The binding of E7 to pRb is mediated by a conserved Leu-X-Cys-X-Glu (LXCXE) motif in the conserved region 2 (CR2) of E7 and this domain is both necessary and sufficient for E7/pRb association. In the current study, we report that the E7 protein of the malignancy-associated canine papillomavirus type 2 encodes an E7 protein that has serine substituted for cysteine in the LXCXE motif. In HPV, this substitution in E7 abrogates pRb binding and degradation. However, despite variation at this critical site, the canine papillomavirus E7 protein still bound and degraded pRb. Even complete deletion of the LXSXE domain of canine E7 failed to interfere with binding to pRb in vitro and in vivo. Rather, the dominant binding site for pRb mapped to the C-terminal domain of canine E7. Finally, while the CR1 and CR2 domains of HPV E7 are sufficient for degradation of pRb, the C-terminal region of canine E7 was also required for pRb degradation. Screening of HPV genome sequences revealed that the LXSXE motif of the canine E7 protein was also present in the gamma HPVs and we demonstrate that the gamma HPV-4 E7 protein also binds pRb in a similar way. It appears, therefore, that the type 2 canine PV and gamma-type HPVs not only share similar properties with respect to tissue specificity and association with immunosuppression, but also the mechanism by which their E7 proteins interact with pRb. PMID:20824099

  20. NF-κB signalling is attenuated by the E7 protein from cutaneous human papillomaviruses.

    PubMed

    Byg, Luise M; Vidlund, Jessica; Vasiljevic, Natasa; Clausen, Dorte; Forslund, Ola; Norrild, Bodil

    2012-10-01

    The high-risk Alpha-types of human papillomavirus (HPV) are the causative agent of cervical cancer, which is the second major cause of death among women worldwide. Recent investigations have shown that E7 from the Alpha-papillomavirus HPV-16 interacts with IKKα and IKKβ of the IKK complex in the NF-κB pathway leading to an attenuation of the activity. There is a possible link between development of non-melanoma skin cancer and cutaneous Beta-papillomavirus but if these HPV types attenuate the NF-κB pathway is unclear. Seven different E7 proteins, representing four out of the five different species of the Beta genus (HPV-20, -37, -38, -92, -93 and -96) and one from the Gamma genus (HPV-4) were investigated for potential modulation of the NF-κB pathway in U2OS cells. Our results demonstrate that E7 from all the cutaneous HPV types were capable of inhibiting the NF-κB activity as well as E7 from HPV-16. In addition, E7 proteins from the cutaneous HPV types demonstrated interaction with IKKα but not with IKKβ. The deregulation of the NF-κB pathway by cutaneous HPVs might contribute to the pathogenesis of non-melanoma skin cancers and its precursors. PMID:22776252

  1. Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein.

    PubMed Central

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

    1996-01-01

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

  2. Multifaceted Role of IRAK-M in the Promotion of Colon Carcinogenesis via Barrier Dysfunction and STAT3 Oncoprotein Stabilization in Tumors.

    PubMed

    Jenkins, Brendan J

    2016-05-01

    Dysregulated interactions between the host immune system and gut microbiota can underpin inflammation, leading to colorectal cancer (CRC). In this issue of Cancer Cell, Kesselring et al. reveal a bimodal role of the TLR/IL-1R-signaling negative regulator, IRAK-M, in promoting tumoral microbial colonization and STAT3 oncoprotein stabilization during CRC. PMID:27165738

  3. 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

    .... In the Federal Register of November 10, 2009 (74 FR 58024), FDA published a notice announcing the... Studies in Support of Special Populations; Geriatrics; Questions and Answers; Availability AGENCY: Food... announcing the availability of a guidance entitled ``E7 Studies in Support of Special Populations:...

  4. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression

    PubMed Central

    DeCaprio, James A.

    2014-01-01

    Study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. While much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al. demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F and MuvB) complex at two distinct phases of the cell cycle (1). Consistent with earlier work, HPV16 E7 can bind to the retinoblastoma tumor suppressor (RB) family member p130 (RBL2) protein and promote its proteasome-mediated destruction thereby disrupting the DREAM complex and prevent exit from the cell cycle into quiescence. In addition, they demonstrate that HPV16 E7 can bind to MuvB core complex in association with BMYB and FOXM1 and activate gene expression during the G2 and M phase of the cell cycle. Thus, HPV16 E7 acts to prevent exit from the cell cycle entry and promotes mitotic proliferation and may account for the high levels of FOXM1 often observed in poor risk cervical cancers. PMID:24166507

  5. Human papillomavirus type 16 E7 perturbs DREAM to promote cellular proliferation and mitotic gene expression.

    PubMed

    DeCaprio, J A

    2014-07-31

    The study of the small DNA tumor viruses continues to provide valuable new insights into oncogenesis and fundamental biological processes. Although much has already been revealed about how the human papillomaviruses (HPVs) can transform cells and contribute to cervical and oropharyngeal cancer, there clearly is much more to learn. In this issue of Oncogene, Pang et al., doi:10.1038/onc.2013.426, demonstrate that the high-risk HPV16 E7 oncogene can promote cellular proliferation by interacting with the DREAM (DP, RB-like, E2F and MuvB) complex at two distinct phases of the cell cycle. Consistent with earlier work, HPV16 E7 can bind to the retinoblastoma tumor suppressor (RB) family member p130 (RBL2) protein and promote its proteasome-mediated destruction thereby disrupting the DREAM complex and can prevent exit from the cell cycle into quiescence. In addition, they demonstrate that HPV16 E7 can bind to MuvB core complex in association with BMYB and FOXM1 and activate gene expression during the G2 and M phase of the cell cycle. Thus, HPV16 E7 acts to prevent exit from the cell cycle entry and promotes mitotic proliferation and may account for the high levels of FOXM1 often observed in poor-risk cervical cancers. PMID:24166507

  6. Loss of Dependence on Continued Expression of the Human Papillomavirus 16 E7 Oncogene in Cervical Cancers and Precancerous Lesions Arising in Fanconi Anemia Pathway-Deficient Mice

    PubMed Central

    Park, Soyeong; Park, Jung Wook; Pitot, Henry C.

    2016-01-01

    ABSTRACT   Fanconi anemia (FA) is a rare genetic disorder caused by defects in DNA damage repair. FA patients often develop squamous cell carcinoma (SCC) at sites where high-risk human papillomaviruses (HPVs) are known to cause cancer, including the cervix. However, SCCs found in human FA patients are often HPV negative, even though the majority of female FA patients with anogenital cancers had preexisting HPV-positive dysplasia. We hypothesize that HPVs contribute to the development of SCCs in FA patients but that the continued expression of HPV oncogenes is not required for the maintenance of the cancer state because FA deficiency leads to an accumulation of mutations in cellular genes that render the cancer no longer dependent upon viral oncogenes. We tested this hypothesis, making use of Bi-L E7 transgenic mice in which we temporally controlled expression of HPV16 E7, the dominant viral oncogene in HPV-associated cancers. As seen before, the persistence of cervical neoplastic disease was highly dependent upon the continued expression of HPV16 E7 in FA-sufficient mice. However, in mice with FA deficiency, cervical cancers persisted in a large fraction of the mice after HPV16 E7 expression was turned off, indicating that these cancers had escaped from their dependency on E7. Furthermore, the severity of precancerous lesions also failed to be reduced significantly in the mice with FA deficiency upon turning off expression of E7. These findings confirm our hypothesis and may explain the fact that, while FA patients have a high frequency of infections by HPVs and HPV-induced precancerous lesions, the cancers are frequently HPV negative. Importance   Fanconi anemia (FA) patients are at high risk for developing squamous cell carcinoma (SCC) at sites where high-risk human papillomaviruses (HPVs) frequently cause cancer. Yet these SCCs are often HPV negative. FA patients have a genetic defect in their capacity to repair damaged DNA. HPV oncogenes cause an

  7. Loss of Dependence on Continued Expression of the Human Papillomavirus 16 E7 Oncogene in Cervical Cancers and Precancerous Lesions Arising in Fanconi Anemia Pathway-Deficient Mice.

    PubMed

    Park, Soyeong; Park, Jung Wook; Pitot, Henry C; Lambert, Paul F

    2016-01-01

    Fanconi anemia (FA) is a rare genetic disorder caused by defects in DNA damage repair. FA patients often develop squamous cell carcinoma (SCC) at sites where high-risk human papillomaviruses (HPVs) are known to cause cancer, including the cervix. However, SCCs found in human FA patients are often HPV negative, even though the majority of female FA patients with anogenital cancers had preexisting HPV-positive dysplasia. We hypothesize that HPVs contribute to the development of SCCs in FA patients but that the continued expression of HPV oncogenes is not required for the maintenance of the cancer state because FA deficiency leads to an accumulation of mutations in cellular genes that render the cancer no longer dependent upon viral oncogenes. We tested this hypothesis, making use of Bi-L E7 transgenic mice in which we temporally controlled expression of HPV16 E7, the dominant viral oncogene in HPV-associated cancers. As seen before, the persistence of cervical neoplastic disease was highly dependent upon the continued expression of HPV16 E7 in FA-sufficient mice. However, in mice with FA deficiency, cervical cancers persisted in a large fraction of the mice after HPV16 E7 expression was turned off, indicating that these cancers had escaped from their dependency on E7. Furthermore, the severity of precancerous lesions also failed to be reduced significantly in the mice with FA deficiency upon turning off expression of E7. These findings confirm our hypothesis and may explain the fact that, while FA patients have a high frequency of infections by HPVs and HPV-induced precancerous lesions, the cancers are frequently HPV negative. IMPORTANCE  : Fanconi anemia (FA) patients are at high risk for developing squamous cell carcinoma (SCC) at sites where high-risk human papillomaviruses (HPVs) frequently cause cancer. Yet these SCCs are often HPV negative. FA patients have a genetic defect in their capacity to repair damaged DNA. HPV oncogenes cause an accumulation of DNA

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

    PubMed Central

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

    2015-01-01

    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

  9. 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-α. 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

  10. 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

  11. The AD1 and AD2 Transactivation Domains of E2A Are Essential for the Antiapoptotic Activity of the Chimeric Oncoprotein E2A-HLF

    PubMed Central

    Inukai, Takeshi; Inaba, Toshiya; Ikushima, Satoshi; Look, A. Thomas

    1998-01-01

    The chimeric oncoprotein E2A-HLF, generated by the t(17;19) chromosomal translocation in pro-B-cell acute lymphoblastic leukemia, incorporates the transactivation domains of E2A and the basic leucine zipper (bZIP) DNA-binding and protein dimerization domain of HLF (hepatic leukemic factor). The ability of E2A-HLF to prolong the survival of interleukin-3 (IL-3)-dependent murine pro-B cells after IL-3 withdrawal suggests that it disrupts signaling pathways normally responsible for cell suicide, allowing the cells to accumulate as transformed lymphoblasts. To determine the structural motifs that contribute to this antiapoptotic effect, we constructed a panel of E2A-HLF mutants and programmed their expression in IL-3-dependent murine pro-B cells (FL5.12 line), using a zinc-inducible vector. Neither the E12 nor the E47 product of the E2A gene nor the wild-type HLF protein was able to protect the cells from apoptosis induced by IL-3 deprivation. Surprisingly, different combinations of disabling mutations within the HLF bZIP domain had little effect on the antiapoptotic property of the chimeric protein, so long as the amino-terminal portion of E2A remained intact. In the context of a bZIP domain defective in DNA binding, mutants retaining either of the two transactivation domains of E2A were able to extend cell survival after growth factor deprivation. Thus, the block of apoptosis imposed by E2A-HLF in pro-B lymphocytes depends critically on the transactivating regions of E2A. Since neither DNA binding nor protein dimerization through the bZIP domain of HLF is required for this effect, we propose mechanisms whereby protein-protein interactions with the amino-terminal region of E2A allow the chimera to act as a transcriptional cofactor to alter the expression of genes regulating the apoptotic machinery in pro-B cells. PMID:9742120

  12. The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 Positive) and apoptosis in HeLa (HPV-18 positive).

    PubMed

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

    2013-01-01

    Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq) bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca(2+) leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer. PMID:23922932

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

    PubMed Central

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

    2013-01-01

    Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq) bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca2+ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer. PMID:23922932

  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. 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.

  16. 17 CFR 240.14e-7 - Unlawful tender offer practices in connection with roll-ups.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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 U... tenders directly from security holders in connection with a roll-up transaction as provided in...

  17. 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...

  18. 17 CFR 240.14e-7 - Unlawful tender offer practices in connection with roll-ups.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 17 Commodity and Securities Exchanges 4 2014-04-01 2014-04-01 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...

  19. 17 CFR 240.14e-7 - Unlawful tender offer practices in connection with roll-ups.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 17 Commodity and Securities Exchanges 3 2013-04-01 2013-04-01 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...

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 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. Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins

    PubMed Central

    Perepelyuk, Maryna; Maher, Christina; Lakshmikuttyamma, Ashakumary; Shoyele, Sunday A

    2016-01-01

    MicroRNAs (miRNAs) are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer. PMID:27555773

  2. Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins.

    PubMed

    Perepelyuk, Maryna; Maher, Christina; Lakshmikuttyamma, Ashakumary; Shoyele, Sunday A

    2016-01-01

    MicroRNAs (miRNAs) are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer. PMID:27555773

  3. MicroRNA-27b up-regulated by human papillomavirus 16 E7 promotes proliferation and suppresses apoptosis by targeting polo-like kinase2 in cervical cancer

    PubMed Central

    Zhao, Zhen; Mao, Xinru; Huang, Jinlan; Wu, Zixian; Zheng, Lei; Wang, Qian

    2016-01-01

    The infection with high-risk human papillomavirus is linked to cervical cancer, nevertheless, the role of miRNAs regulated by HPV oncogenes in cancer progression remain largely unknown. Here, we knocked down endogenous E6/E7 in HPV16-positive CaSki cell lines, screened differences in miRNA expression profile with control using miRNA array. 38 miRNAs were down-regulated and 6 miRNAs were up-regulated in the E6/E7 silenced CaSki cells (>2-fold changes with P <0.05). The levels of miR-27b, miR-20a, miR-24, miR-93, and miR-106b were verified by qPCR in E6/E7 silenced CaSki and SiHa cells. MiR-27b, up-regulated by E7, promoted CaSki and SiHa cell proliferation and invasion, inhibit paclitaxel-induced apoptosis. Dual-luciferase experiment confirmed miR-27b down-regulated its target gene PLK2 through the “seed regions”. The tumor suppressor PLK2 inhibited SiHa cell proliferation, reduced cell viability, and promoted paclitaxel/cisplatin -induced apoptosis. Furthermore, DGCR8 was found to mediate the up-regulation of miR-27b by HPV16 E7. Our study demonstrated that HPV16 E7 could increase DGCR8 to promote the generation of miR-27b, which accelerated cell proliferation and inhibited paclitaxel-induced cell apoptosis through down-regulating PLK2. These findings provide an insight into the interaction network of viral oncogene, miR-27b and PLK2, and support the potential strategies using antisense nucleic acid of miR-27b for therapy of cervical cancer in the future. PMID:26910911

  4. Simulation evaluation of transition and hover flying qualities of the E-7A STOVL aircraft

    NASA Technical Reports Server (NTRS)

    Franklin, James A.; Stortz, Michael W.; Gerdes, Ronald M.; Hardy, Gordon H.; Martin, James L.; Engelland, Shawn A.

    1988-01-01

    The generalized simulation model developed for the E-7A STOVL fighter-type aircraft configuration has attempted to define the limits of acceptibility for a vertical-to-horizontal-to-vertical flight transition envelope. An effort was also made to determine the control power required during hover and transition, and to evaluate whether the integration of flight and propulsion controls thus far effected achieves good flying qualities throughout the low-speed flight envelope. The results thus obtained furnish a general view of the acceptable transition corridor, expressed in terms of the minimum-climb capability.

  5. Differential Processing of let-7a Precursors Influences RRM2 Expression and Chemosensitivity in Pancreatic Cancer: Role of LIN-28 and SET Oncoprotein

    PubMed Central

    Bhutia, Yangzom Doma; Hung, Sau Wai; Krentz, Madeline; Patel, Dimal; Lovin, Dylan; Manoharan, Radhika; Thomson, J. Michael; Govindarajan, Rajgopal

    2013-01-01

    Overexpression of ribonucleotide reductase subunit M2 (RRM2), involved in deoxyribonucleotide synthesis, drives the chemoresistance of pancreatic cancer to nucleoside analogs (e.g., gemcitabine). While silencing RRM2 by synthetic means has shown promise in reducing chemoresistance, targeting endogenous molecules, especially microRNAs (miRNAs), to advance chemotherapeutic outcomes has been poorly explored. Based on computational predictions, we hypothesized that the let-7 tumor suppressor miRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in innately gemcitabine-resistant pancreatic cancer cell lines. Direct binding of let-7 miRNAs to the 3′ UTR of RRM2 transcripts identified post-transcriptional regulation of RRM2 influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and chemosensitivity responses in a poorly differentiated pancreatic cancer cell line, MIA PaCa-2. Defective processing of let-7a precursors to mature forms, in part, explained the discrepancies observed with let-7a expressional outcomes. Consistently, the ratios of mature to precursor let-7a were progressively reduced in gemcitabine-sensitive L3.6pl and Capan-1 cell lines induced to acquire gemcitabine resistance. Besides known regulators of let-7 biogenesis (e.g., LIN-28), short hairpin RNA library screening identified several novel RNA binding proteins, including the SET oncoprotein, to differentially impact let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus -resistant pancreatic cancer cells. Further, LIN-28 and SET knockdown in the cells led to profound reductions in cellular proliferation and colony-formation capacities. Finally, defective processing of let-7a precursors with a positive correlation to RRM2 overexpression was identified in patient

  6. 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

  7. Adjuvant effect of docetaxel on HPV16 L2E6E7 fusion protein vaccine in a mouse model.

    PubMed

    Su, Xiaoyan; Xu, Wei; Guan, Ran; Wang, Yunhao; Wu, Jie; Zhai, Lijuan; Chen, Gang; Hu, Songhua

    2016-09-01

    We previously demonstrated that the antineoplastic agent docetaxel enhanced the immune response to an influenza vaccine. This study evaluated the adjuvant effect of docetaxel (DOC) on the therapeutic efficacy of HPV16 L2E6E7 fusion protein (HPV-LFP) in mice inoculated with TC-1 cells. The results demonstrated that docetaxel significantly enhanced the therapeutic effect of HPV-LFP on TC-1 cell-induced tumors in mice. The injection of HPV-LFP in combination with docetaxel in TC-1 tumor-bearing mice significantly reduced tumor volume and weight, and a greater percent survival was detected than mice treated with HPV-LFP alone. The inhibition of tumors was associated with significantly increased serum antigen-specific IgG and isotypes, activated CTLs, increased IFN-γ-secreting T cells, and decreased Treg cells and IL-10-secreting cells in spleen. In addition, down-regulation of IL-10, VEGF and STAT3, up-regulation of IFN-γ and decreased Treg cells in the tumor microenvironment may also important contributing factors to the antitumor effect. It may be valuable to use a DOC-containing water to dilute HPV-LFP powder before injection in patients because of its excellent adjuvant effect on HPV-LFP and solubility in water. PMID:27233002

  8. Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

    NASA Astrophysics Data System (ADS)

    Chattopadhyay, Anasuya; O’Connor, Cornelius J.; Zhang, Fengzhi; Galvagnion, Celine; Galloway, Warren R. J. D.; Tan, Yaw Sing; Stokes, Jamie E.; Rahman, Taufiq; Verma, Chandra; Spring, David R.; Itzhaki, Laura S.

    2016-04-01

    Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a “druggable” target with small molecules.

  9. HPV oncoprotein E6 is a structure-dependent DNA-binding protein that recognizes four-way junctions.

    PubMed

    Ristriani, T; Masson, M; Nominé, Y; Laurent, C; Lefevre, J F; Weiss, E; Travé, G

    2000-03-10

    E6 is an oncoprotein implicated in cervical cancers, produced by "high-risk" human papillomaviruses. E6 is thought to promote tumorigenesis by stimulating cellular degradation of the tumour suppressor p53, but it might display other activities. Sequence similarity was recently detected between E6 and endonuclease VII, a protein of phage T4 that recognizes and cleaves four-way DNA junctions. Here, we purified recombinant E6 proteins and demonstrated that high-risk E6 s bind selectively to four-way junctions in a structure-dependent manner. Several residues in the C-terminal zinc-binding domain, the region of E6 similar to endonuclease VII, are necessary for the junction-binding activity. E6 binds to the junction as a monomer. Comparative electrophoresis shows that E6-bound junctions migrate in an extended square conformation. Magnesium inhibits the electrophoretic migration of the complexes but does not seem to influence their formation at equilibrium. This work is the first demonstration of specific binding of purified active E6 to a well-characterized DNA ligand, and suggests new modes of action of E6 in oncogenesis. PMID:10698626

  10. 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 755 bp, which contains an open reading frame (ORF) of 518 bp, encoding a 175 amino-acid polypeptide with the predicted molecular weight of 19.60 kDa 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

  11. Oncogenic nexus of cancerous inhibitor of protein phosphatase 2A (CIP2A): An oncoprotein with many hands

    PubMed Central

    De, Pradip; Carlson, Jennifer; Leyland-Jones, Brian; Dey, Nandini

    2014-01-01

    Oncoprotein CIP2A a Cancerous Inhibitor of PP2A forms an “oncogenic nexus” by virtue of its control on PP2A and MYC stabilization in cancer cells. The expression and prognostic function of CIP2A in different solid tumors including colorectal carcinoma, head & neck cancers, gastric cancers, lung carcinoma, cholangiocarcinoma, esophageal cancers, pancreatic carcinoma, brain cancers, breast carcinoma, bladder cancers, ovarian carcinoma, renal cell carcinomas, tongue cancers, cervical carcinoma, prostate cancers, and oral carcinoma as well as a number of hematological malignancies are just beginning to emerge. Herein, we reviewed the recent progress in our understanding of (1) how an “oncogenic nexus” of CIP2A participates in the tumorigenic transformation of cells and (2) how we can prospect/view the clinical relevance of CIP2A in the context of cancer therapy. The review will try to understand the role of CIP2A (a) as a biomarker in cancers and evaluate the prognostic value of CIP2A in different cancers (b) as a therapeutic target in cancers and (c) in drug response and developing chemo-resistance in cancers. PMID:25015035

  12. 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.

  13. 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.

  14. Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A.

    PubMed

    Shi, Shasha; Liu, Ke; Chen, Yanheng; Zhang, Shijun; Lin, Juanyu; Gong, Chenfang; Jin, Quanwen; Yang, Xiang-Jiao; Chen, Ruichuan; Ji, Zhiliang; Han, Aidong

    2016-07-01

    The adenovirus early region 1A (E1A) oncoprotein hijacks host cells via direct interactions with many key cellular proteins, such as KAT2B, also known as PCAF (p300/CBP associated factor). E1A binds the histone acetyltransferase (HAT) domain of KAT2B to repress its transcriptional activation. However, the molecular mechanism by which E1A inhibits the HAT activity is not known. Here we demonstrate that a short and relatively conserved N-terminal motif (cNM) in the intrinsically disordered E1A protein is crucial for KAT2B interaction, and inhibits its HAT activity through a direct competition with acetyl-CoA, but not its substrate histone H3. Molecular modeling together with a series of mutagenesis experiments suggests that the major helix of E1A cNM binds to a surface of the acetyl-CoA pocket of the KAT2B HAT domain. Moreover, transient expression of the cNM peptide is sufficient to inhibit KAT2B-specific H3 acetylation H3K14ac in vivo Together, our data define an essential motif cNM in N-terminal E1A as an acetyl-CoA entry blocker that directly associates with the entrance of acetyl-CoA binding pocket to block the HAT domain access to its cofactor. PMID:27143356

  15. Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

    PubMed Central

    Chattopadhyay, Anasuya; O’Connor, Cornelius J.; Zhang, Fengzhi; Galvagnion, Celine; Galloway, Warren R. J. D.; Tan, Yaw Sing; Stokes, Jamie E.; Rahman, Taufiq; Verma, Chandra; Spring, David R.; Itzhaki, Laura S.

    2016-01-01

    Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a “druggable” target with small molecules. PMID:27046077

  16. 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

  17. Oncogenic nexus of cancerous inhibitor of protein phosphatase 2A (CIP2A): an oncoprotein with many hands.

    PubMed

    De, Pradip; Carlson, Jennifer; Leyland-Jones, Brian; Dey, Nandini

    2014-07-15

    Oncoprotein CIP2A a Cancerous Inhibitor of PP2A forms an "oncogenic nexus" by virtue of its control on PP2A and MYC stabilization in cancer cells. The expression and prognostic function of CIP2A in different solid tumors including colorectal carcinoma, head and neck cancers, gastric cancers, lung carcinoma, cholangiocarcinoma, esophageal cancers, pancreatic carcinoma, brain cancers, breast carcinoma, bladder cancers, ovarian carcinoma, renal cell carcinomas, tongue cancers, cervical carcinoma, prostate cancers, and oral carcinoma as well as a number of hematological malignancies are just beginning to emerge. Herein, we reviewed the recent progress in our understanding of (1) how an "oncogenic nexus" of CIP2A participates in the tumorigenic transformation of cells and (2) how we can prospect/view the clinical relevance of CIP2A in the context of cancer therapy. The review will try to understand the role of CIP2A (a) as a biomarker in cancers and evaluate the prognostic value of CIP2A in different cancers (b) as a therapeutic target in cancers and (c) in drug response and developing chemo-resistance in cancers. PMID:25015035

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

    PubMed Central

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

    1995-01-01

    The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles. PMID:7707548

  19. Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis.

    PubMed Central

    Larsson, N; Marklund, U; Gradin, H M; Brattsand, G; Gullberg, M

    1997-01-01

    Oncoprotein 18 (Op18; also termed p19, 19K, metablastin, stathmin, and prosolin) is a conserved protein that regulates microtubule (MT) dynamics. Op18 is multisite phosphorylated on four Ser residues during mitosis; two of these Ser residues, Ser-25 and Ser-38, are targets for cyclin-dependent protein kinases (CDKs), and the other two Ser residues, Ser-16 and Ser-63, are targets for an unidentified protein kinase. Mutations of the two CDK sites have recently been shown to result in a mitotic block caused by destabilization of MTs. To understand the role of Op18 in regulation of MT dynamics during mitosis, in this study we dissected the functions of all four phosphorylation sites of Op18 by combining genetic, morphological, and biochemical analyses. The data show that all four phosphorylation sites are involved in switching off Op18 activity during mitosis, an event that appears to be essential for formation of the spindle during metaphase. However, the mechanisms by which specific sites down-regulate Op18 activity differ. Hence, dual phosphorylation on the CDK sites Ser-25 and Ser-38 appears to be required for phosphorylation of Ser-16 and Ser-63; however, by themselves, the CDK sites are of only minor importance in direct regulation of Op18 activity. Subsequent phosphorylation of either Ser-16, Ser-63, or both efficiently switches off Op18 activity. PMID:9271428

  20. 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

  1. 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...

  2. Activation of dendritic cells and induction of T cell responses by HPV 16 L1/E7 chimeric virus-like particles are enhanced by CpG ODN or sorbitol.

    PubMed

    Freyschmidt, Eva-Jasmin; Alonso, Angel; Hartmann, Gunther; Gissmann, Lutz

    2004-08-01

    Chimeric human papillomavirus-like particles, consisting of human papillomavirus (HPV) 16 L1-E7 fusion proteins [HPV 16 L1/E7 chimeric virus-like particles (CVLP)], are a vaccine candidate for treatment and prevention of cervical cancer. Although in preclinical studies CVLPs were shown to induce neutralizing antibodies and L1- and E7-specific T cell responses, the results of a recent clinical trial emphasized the need of improved immunogenicity of CVLPs. Here we studied the interaction of HPV 16 L1/E7 CVLPs with mouse bone marrow-derived dendritic cells (BMDCs) activated with different immune adjuvants. We found that lipopolysaccharides (LPS), unmethylated CpG motifs (CpG ODN) and sorbitol enhanced CVLP-induced stimulation of C57BL/6 mouse BMDCs as revealed by increased levels of CD40, CD80, MHC II and CD54 at the cell surface. CpG ODN and sorbitol also enhanced the presentation of Db-restricted cytotoxic T lymphocyte epitopes to HPV 16 L1- or E7-specific T lymphocytes after loading of CVLPs onto BMDCs. Treatment of BMDCs with CpG ODN in combination with CVLPs improved in vitro priming of naive T lymphocytes by CVLP-loaded BMDCs. In vivo, CVLP-loaded BMDCs were more immunogenic as compared with injection of CVLPs alone. CpG ODN and sorbitol further enhanced priming of antigen-specific T cell responses. Our data demonstrate that CpG ODN- or sorbitol-activated BMDCs substantially increase the immunogenicity of CVLPs. Implementing our results in clinical trial protocols may lead to improved activity of therapeutic HPV vaccines for the treatment of HPV-induced cancer. PMID:15456078

  3. A Synthetic E7 Gene of Human Papillomavirus Type 16 That Yields Enhanced Expression of the Protein in Mammalian Cells and Is Useful for DNA Immunization Studies

    PubMed Central

    Cid-Arregui, Angel; Juárez, Victoria; Hausen, Harald zur

    2003-01-01

    A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20- to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G + C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system. PMID:12663798

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

    SciTech Connect

    Hayakawa, Kazuo; Ikeya, Makoto; Fukuta, Makoto; Woltjen, Knut; Tamaki, Sakura; Takahara, Naoko; Kato, Tomohisa; Sato, Shingo; Otsuka, Takanobu; Toguchida, Junya

    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

  5. 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

  6. Stringy KLT relations, global symmetries, and E 7(7)-violation

    NASA Astrophysics Data System (ADS)

    Elvang, Henriette; Kiermaier, Michael

    2010-10-01

    We study consequences of the Kawai-Lewellen-Tye (KLT) relations applied to tree amplitudes in toroidal compactifications of string theory to four dimensions. The closed string tree amplitudes with massless external states respect a global SU(4) × SU(4) symmetry, which is enhanced to the SU(8) R -symmetry of mathcal{N} = 8 supergravity in the field theory limit. Our analysis focuses on two aspects: (i) We provide a detailed account of the simplest SU(8)-violating amplitudes. We classify these processes and derive explicit superamplitudes for all local 5-and 6-point operators with SU(4) × SU(4) symmetry at order α'3 . Their origin is the dilatonic operator e-6φ R 4 in the closed-string effective action. (ii) We expand the 6-point closed string tree amplitudes to order α3 and use two different methods to isolate the SU(8)-singlet contribution from e-6φ R 4. This allows us to extract the matrix elements of the unique SU(8)-invariant supersymmetrization of R 4. Their single-soft scalar limits are non-vanishing. This demonstrates that the mathcal{N} = 8 supergravity candidate counterterm R 4 is incompatible with continuous E 7(7) symmetry. From the soft scalar limits, we reconstruct to quadratic order the SU(8)-invariant function of scalars that multiplies R 4, and show that it satisfies the Laplace eigenvalue equation derived recently from supersymmetry and duality constraints.

  7. 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.

  8. 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

  9. Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16.

    PubMed Central

    Comerford, S A; McCance, D J; Dougan, G; Tite, J P

    1991-01-01

    There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant. Images PMID:1714516

  10. Frequency of antibodies against E4 and E7 from human papillomavirus type 16 in Mexican soldiers.

    PubMed

    Plett-Torres, T; Cruz-Valdez, A; Esquivel-Guadarrama, F; Hernández-Nevarez, P; Lazcano-Ponce, E; Gutiérrez-Xicotencatl, L

    2007-01-01

    The high prevalence of HPV in men's genitalia and the low frequency of virus-associated lesions gave rise to questions on the influence of infection-site on the HPV antibody profile. In a cross-sectional study, HPV infection in penis and urethra, and serum antibodies against HPV-16 E4 and E7 proteins were evaluated in 288 Mexican soldiers. The results showed that HPV prevalence was 31% (51% in penis, 11% in urethra and 38% in both sites), while 47% were multiple infections. Overall, seroprevalence was 13% for anti-E4 antibodies and 6% for anti-E7. However, the highest prevalence of anti-E4 antibodies was observed in men with HPV infection in urethra (30%), while for E7 antibodies, the highest prevalence (10%) was found in men who tested positive for HPV in penis. The prevalence of IgG and IgA anti-E4 was related to HPV-16 urethral infection, while detection of HPV-16 in penis was related to IgG anti-E7 prevalence. In conclusion, the high-risk sexual behavior observed in this population might be responsible for high HPV prevalence and multiple infections. However, the seroprevalence of E4 and E7 was similar to that observed in healthy Mexican women. These results suggest that the humoral immune response against HPV infection in men differs, depending on the site of infection. PMID:16896549

  11. 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; Rodríguez de la Fuente, Abraham Octavio; Alcocer González, Juan Manuel; Rodríguez-Flores, Laura E; Rodríguez-Padilla, Cristina; Santoyo-Stephano, Martha A; Castañeda-Garza, Esperanza; Taméz-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 μT 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

  12. 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

    2016-03-01

    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

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

    PubMed Central

    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

  14. 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.

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

    PubMed Central

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

    2012-01-01

    Background and Objectives Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. Methods We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Results Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Conclusion Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study. PMID:22655088

  16. Glioma oncoprotein Bcl2L12 inhibits the p53 tumor suppressor

    PubMed Central

    Stegh, Alexander H.; Brennan, Cameron; Mahoney, John A.; Forloney, Kristin L.; Jenq, Harry T.; Luciano, Janina P.; Protopopov, Alexei; Chin, Lynda; DePinho, Ronald A.

    2010-01-01

    Glioblastoma multiforme (GBM) is a lethal brain tumor characterized by intense apoptosis resistance and extensive necrosis. Bcl2L12 (for Bcl2-like 12) is a cytoplasmic and nuclear protein that is overexpressed in primary GBM and functions to inhibit post-mitochondrial apoptosis signaling. Here, we show that nuclear Bcl2L12 physically and functionally interacts with the p53 tumor suppressor, as evidenced by the capacity of Bcl2L12 to (1) enable bypass of replicative senescence without concomitant loss of p53 or p19Arf, (2) inhibit p53-dependent DNA damage-induced apoptosis, (3) impede the capacity of p53 to bind some of its target gene promoters, and (4) attenuate endogenous p53-directed transcriptomic changes following genotoxic stress. Correspondingly, The Cancer Genome Atlas profile and tissue protein analyses of human GBM specimens show significantly lower Bcl2L12 expression in the setting of genetic p53 pathway inactivation. Thus, Bcl2L12 is a multifunctional protein that contributes to intense therapeutic resistance of GBM through its ability to operate on two key nodes of cytoplasmic and nuclear signaling cascades. PMID:20837658

  17. Attenuated CagA oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon.

    PubMed

    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

    2011-08-26

    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

  18. 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

  19. 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.

  20. 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

  1. Expression of the MOZ-TIF2 oncoprotein in mice represses senescence

    PubMed Central

    Largeot, Anne; Perez-Campo, Flor Maria; Marinopoulou, Elli; Lie-a-Ling, Michael; Kouskoff, Valerie; Lacaud, Georges

    2016-01-01

    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, p16INK4a 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 p16INK4a 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 (p16INK4a and p19ARF), 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

  2. Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

    PubMed

    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

    2015-03-01

    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, Mann-Whitney 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

  3. 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.

  4. Bcl-2 anti-apoptotic oncoprotein suppresses angiogenesis in non-small cell lung cancer: implications in resistance to photodynamic treatment?

    NASA Astrophysics Data System (ADS)

    Koukourakis, M. I.; Giatromanolaki, A.; Skarlatos, J.; Kosma, L.; Apostolikas, N.; Beroukas, K.

    1998-07-01

    PDT cytotoxicity is likely to occur through photooxidative reactions. In that way mechanisms that define poor oxygenation should be involved in defining resistance to photo-dynamic treatment (PDT). On the other hand bcl-2 anti- apoptotic protein has been shown to delay cell death and protect cells from toxic oxidative products. We examined 134 specimens from T1,2-NO,1 staged patients treated with surgery alone. Specimens were immunohistochemically examined for vascular grade using the JC70 MoAb, and bcl-2 oncoprotein expression. Bcl-2 expression correlated with low vascular grade. Only 3/27 of bcl2+ case had high angiogenesis vs. 34/107 of cases without bcl-2 expression. In the present study we provide evidence that bcl-2 overexpression directly suppresses angiogenesis in non-small cell lung cancer, which obviously results in decreased blood supply and oxygenation. This finding implies that reduced intratumoral angiogenesis and immortalizing oncoprotein overexpression are linked to each other and may have a role in defining tumors resistant to PDT.

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

    PubMed Central

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

    1997-01-01

    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

  6. 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

  7. Recombinant adeno-associated virus expressing human papillomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer.

    PubMed

    Liu, D W; Tsao, Y P; Kung, J T; Ding, Y A; Sytwu, H K; Xiao, X; Chen, S L

    2000-03-01

    In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242-2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine. PMID:10684306

  8. 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.

  9. 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.

  10. 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.

  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 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.

  13. 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.

  14. 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.

  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

    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.

  17. 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.

  18. 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.

  19. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    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.

  20. Genetic variability in E6, E7, and L1 genes of human papillomavirus genotype 52 from Southwest China.

    PubMed

    Zhang, Yiwen; Cao, Man; Wang, Mengting; Ding, Xianping; Jing, Yaling; Chen, Zuyi; Ma, Tengjiao; Chen, Honghan

    2016-07-01

    Human papillomavirus (HPV) is the major causative agent of cervical cancer, which accounts for the second highest cancer burden in women worldwide. HPV-52, the prevalent subtype in Asia, especially in southwest China, was analyzed in this study. To analyze polymorphisms, intratypic variants, and genetic variability in the E6-E7 (n=26) and L1 (n=53) genes of HPV-52, these genes were sequenced and the sequences were submitted to GenBank. Phylogenetic trees were constructed using the neighbor-joining and Kimura 2-parameters methods, followed by analysis of the diversity of secondary structure. Finally, we estimated the selection pressures acting on the E6-E7 and L1 genes. Fifty-one novel variants of HPV-52 L1, and two novel variants of HPV-52 E6-E7 were identified in this study. Thirty single nucleotide changes were observed in HPV-52 E6-E7 sequences with 19/30 non-synonymous mutations and 11/30 synonymous mutations (five in the alpha helix and five in the beta sheet). Fifty-five single nucleotide changes were observed in HPV-52 L1 sequences with 17/55 non-synonymous mutations (seven in the alpha helix and fourteen in the beta sheet) and 38/55 synonymous mutations. Selective pressure analysis predicted that most of these mutations reflect positive selection. Identifying new variants in HPV-52 may inform the rational design of new vaccines specifically for women in southwest China. Knowledge of genetic variation in HPV may be useful as an epidemiologic correlate of cervical cancer risk, or may even provide critical information for developing diagnostic probes. PMID:26968892

  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. 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.; DiMaio, Daniel

    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.

  3. 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

  4. 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

  5. Oncoprotein HBXIP Modulates Abnormal Lipid Metabolism and Growth of Breast Cancer Cells by Activating the LXRs/SREBP-1c/FAS Signaling Cascade.

    PubMed

    Zhao, Yu; Li, Hang; Zhang, Yingyi; Li, Leilei; Fang, Runping; Li, Yinghui; Liu, Qian; Zhang, Weiying; Qiu, Liyan; Liu, Fabao; Zhang, Xiaodong; Ye, Lihong

    2016-08-15

    Abnormal lipid metabolism is a hallmark of tumorigenesis. Accumulating evidence demonstrates that fatty acid synthase (FAS, FASN) is a metabolic oncogene that supports the growth and survival of tumor cells and is highly expressed in many cancers. Here, we report that the oncoprotein, hepatitis B X-interacting protein (HBXIP, LAMTOR5) contributes to abnormal lipid metabolism. We show that high expression of HBXIP in 236 breast cancer patients was significantly associated with decreased overall survival and progression-free survival. Interestingly, the expression of HBXIP was positively related to that of FAS in clinical breast cancer tissues, and HBXIP overexpression in breast cancer cells resulted in FAS upregulation. Mechanistically, HBXIP upregulated SREBP-1c (SREBF1), which activates the transcription of FAS, by directly interacting with and coactivating nuclear receptor (NR) liver X receptors (LXR). Physiologically, LXRs are activated via a coactivator containing NR motif in a ligand-dependent manner. However, in breast cancer cells, HBXIP containing the corepressor/nuclear receptor motif with special flanking sequence could coactivate LXRs independent of ligand. Moreover, overexpressed SREBP-1c was able to activate the transcription of HBXIP, forming a positive-feedback loop. Functionally, HBXIP enhanced lipogenesis, resulting in the growth of breast cancer cells in vitro and in vivo Thus, we conclude that the oncoprotein HBXIP contributes to the abnormal lipid metabolism in breast cancer through LXRs/SREBP-1c/FAS signaling, providing new insights into the mechanisms by which cancer cells reprogram lipid metabolism in their favor. Cancer Res; 76(16); 4696-707. ©2016 AACR. PMID:26980761

  6. Systemic delivery of siRNA by actively targeted polyion complex micelles for silencing the E6 and E7 human papillomavirus oncogenes.

    PubMed

    Nishida, Haruka; Matsumoto, Yoko; Kawana, Kei; Christie, R James; Naito, Mitsuru; Kim, Beob Soo; Toh, Kazuko; Min, Hyun Su; Yi, Yu; Matsumoto, Yu; Kim, Hyun Jin; Miyata, Kanjiro; Taguchi, Ayumi; Tomio, Kensuke; Yamashita, Aki; Inoue, Tomoko; Nakamura, Hiroe; Fujimoto, Asaha; Sato, Masakazu; Yoshida, Mitsuyo; Adachi, Katsuyuki; Arimoto, Takahide; Wada-Hiraike, Osamu; Oda, Katsutoshi; Nagamatsu, Takeshi; Nishiyama, Nobuhiro; Kataoka, Kazunori; Osuga, Yutaka; Fujii, Tomoyuki

    2016-06-10

    Human papillomavirus (HPV) E6 and E7 oncogenes are essential for the immortalization and maintenance of HPV-associated cancer and are ubiquitously expressed in cervical cancer lesions. Small interfering RNA (siRNA) coding for E6 and E7 oncogenes is a promising approach for precise treatment of cervical cancer, yet a delivery system is required for systemic delivery to solid tumors. Here, an actively targeted polyion complex (PIC) micelle was applied to deliver siRNAs coding for HPV E6/E7 to HPV cervical cancer cell tumors in immune-incompetent tumor-bearing mice. A cell viability assay revealed that both HPV type 16 and 18 E6/E7 siRNAs (si16E6/E7 and si18E6/E7, respectively) interfered with proliferation of cervical cancer cell lines in an HPV type-specific manner. A fluorescence imaging biodistribution analysis further revealed that fluorescence dye-labeled siRNA-loaded PIC micelles efficiently accumulated within the tumor mass after systemic administration. Ultimately, intravenous injection of si16E6/E7 and si18E6/E7-loaded PIC micelles was found to significantly suppress the growth of subcutaneous SiHa and HeLa tumors, respectively. The specific activity of siRNA treatment was confirmed by the observation that p53 protein expression was restored in the tumors excised from the mice treated with si16E6/E7- and si18E6/E7-loaded PIC micelles for SiHa and HeLa tumors, respectively. Therefore, the actively targeted PIC micelle incorporating HPV E6/E7-coding siRNAs demonstrated its therapeutic potential against HPV-associated cancer. PMID:26979870

  7. 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

    PubMed Central

    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.

    2012-01-01

    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. PMID:22284893

  8. 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.

  9. Methylation-specific digital karyotyping of HPV16E6E7-expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis.

    PubMed

    Steenbergen, Renske D M; Ongenaert, Maté; Snellenberg, Suzanne; Trooskens, Geert; van der Meide, Wendy F; Pandey, Deeksha; Bloushtain-Qimron, Noga; Polyak, Kornelia; Meijer, Chris J L M; Snijders, Peter J F; Van Criekinge, Wim

    2013-09-01

    Transformation of epithelial cells by high-risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation profiles with genome-wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed by semi-quantitative methylation-specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV-positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high-grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16-transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may

  10. 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 α- or β-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 α- or β-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

  11. The E6AP Binding Pocket of the HPV16 E6 Oncoprotein Provides a Docking Site for a Small Inhibitory Peptide Unrelated to E6AP, Indicating Druggability of E6

    PubMed Central

    Kintscher, Susanne; Reinz, Eileen; Sehr, Peter; Bulkescher, Julia; Hoppe-Seyler, Karin; Travé, Gilles; Hoppe-Seyler, Felix

    2014-01-01

    The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design. PMID:25383876

  12. Immortalization and altered differentiation of human keratinocytes in vitro by the E6 and E7 open reading frames of human papillomavirus type 18.

    PubMed Central

    Hudson, J B; Bedell, M A; McCance, D J; Laiminis, L A

    1990-01-01

    The E6-E7 region of human papillomavirus types 16 and 18 is selectively retained and expressed in cervical carcinoma cells. In cultured human keratinocytes, expression of the E6 and E7 open reading frames of human papillomavirus type 18, under the control of its homologous promoter, resulted in high-frequency immortalization. Furthermore, by using a system that allows for stratification of keratinocytes in vitro (raft system), we observed that the morphological differentiation of these E6-E7 immortalized cells was altered such that parabasal cells extended throughout most of the epithelium, with abnormal nuclei present in the upper regions. Examination of E6-E7-expressing cell lines in the raft system at a later passage revealed that complete loss of morphological differentiation had occurred. E7 alone was a much less effective immortalizing agent than E6 and E7 together and acted only minimally to alter morphological differentiation in vitro. No such activities were found for E6 alone. High-frequency transformation of human epithelial cells thus appears to require expression of both E6 and E7 gene products. Images PMID:2153221

  13. Genetic stability of a recombinant adenovirus vaccine vector seed library expressing human papillomavirus type 16 E6 and E7 proteins

    PubMed Central

    WU, JIE; CHEN, KE-DA; GAO, MENG; CHEN, GANG; JIN, SU-FENG; ZHUANG, FANG-CHENG; WU, XIAO-HONG; JIANG, YUN-SHUI; LI, JIAN-BO

    2015-01-01

    The aim of the present study was to understand the genetic stability of a master seed bank (MSB) and a working seed bank (WSB) of an adenovirus vector vaccine expressing the human papillomavirus (HPV) type 16 E6 and E7 fusion proteins (Ad-HPV16E6E7). Microscopic examination and viral infectious efficacy were used to measure the infectious titers of the Ad-HPV16E6E7 MSB and WSB. Polymerase chain reaction was used to analyze the stability of the Ad-HPV16E6E7 target gene insertion, while western blot analysis and immunofluorescence were used to assess the expression levels of the Ad-HPV16E6E7 target protein. A C57BL/6 mouse TC-1 tumor cell growth inhibition model was used to evaluate the biological effect of Ad-HPV16E6E7 administration. The infectious titers of the Ad-HPV16E6E7 MSB and WSB were 6.31×109 IU/ml and 3.0×109 IU/ml, respectively. In addition, the expression levels of the inserted target genes and target proteins were found to be stable. In the mouse TC-1 tumor inhibition analysis, when the virus titers of the Ad-HPV16E6E7 MSB and WSB were 109 IU/ml, the tumor inhibition rate was 100%, which was significantly different when compared with the control group (χ2MSB=20.00 and χ2WSB=20.00; P<0.01). Therefore, the Ad-HPV16E6E7 vaccine seed bank is genetically stable and meets the requirements for vaccine development. PMID:25780403

  14. Regulation of ferritin mRNA translation in primary erythroblasts: exogenous c-Kit plus EpoR signaling mimics v-ErbA oncoprotein activity.

    PubMed

    Mikulits, W; Schranzhofer, M; Deiner, E M; Beug, H; Müllner, E W

    2000-08-28

    In general, translation efficiency of ferritin mRNAs is modulated by variations in iron supply. In primary avian erythroblasts undergoing short-term proliferation, however, ferritin heavy chain (ferH) mRNA is repressed at all iron levels. Yet, expression of v-ErbA oncoprotein is sufficient to reinduce ferH mRNA utilization at physiological iron concentrations. Since overexpression of the receptor tyrosine kinase c-Kit and erythropoietin receptor (EpoR) stimulates long-term proliferation of primary erythroblasts like v-ErbA, we analyzed the impact of cooperation between c-Kit and EpoR on the regulation of iron storage. Whereas endogenous c-Kit in combination with exogenous EpoR had no significant effect, ectopic overexpression of both receptors abolished translational repression of ferH mRNA upon iron administration. Thus, high-intensity signaling through c-Kit plus EpoR pathways mimics the v-ErbA-mediated regulatory phenotype. PMID:10964660

  15. 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.

  16. 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

  17. Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein.

    PubMed

    Ristriani, Tutik; Nominé, Yves; Laurent, Cécile; Weiss, Etienne; Travé, Gilles

    2002-12-01

    Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition. PMID:12460759

  18. Impact of structural polymorphism for the Helicobacter pylori CagA oncoprotein on binding to polarity-regulating kinase PAR1b.

    PubMed

    Nishikawa, Hiroko; Hayashi, Takeru; Arisaka, Fumio; Senda, Toshiya; Hatakeyama, Masanori

    2016-01-01

    Chronic infection with cagA-positive Helicobacter pylori is the strongest risk factor for atrophic gastritis, peptic ulcers, and gastric cancer. CagA, the product of the cagA gene, is a bacterial oncoprotein, which, upon delivery into gastric epithelial cells, binds to and inhibits the polarity-regulating kinase, partitioning-defective 1b (PAR1b) [also known as microtubule affinity-regulating kinase 2 (MARK2)], via its CagA multimerization (CM) motif. The inhibition of PAR1b elicits junctional and polarity defects, rendering cells susceptible to oncogenesis. Notably, the polymorphism in the CM motif has been identified among geographic variants of CagA, differing in either the copy number or the sequence composition. In this study, through quantitative analysis of the complex formation between CagA and PAR1b, we found that several CagA species have acquired elevated PAR1b-binding activity via duplication of the CM motifs, while others have lost their PAR1b-binding activity. We also found that strength of CagA-PAR1b interaction was proportional to the degrees of stress fiber formation and tight junctional disruption by CagA in gastric epithelial cells. These results indicate that the CM polymorphism is a determinant for the magnitude of CagA-mediated deregulation of the cytoskeletal system and thereby possibly affects disease outcome of cagA-positive H. pylori infection, including gastric cancer. PMID:27445265

  19. Impact of structural polymorphism for the Helicobacter pylori CagA oncoprotein on binding to polarity-regulating kinase PAR1b

    PubMed Central

    Nishikawa, Hiroko; Hayashi, Takeru; Arisaka, Fumio; Senda, Toshiya; Hatakeyama, Masanori

    2016-01-01

    Chronic infection with cagA-positive Helicobacter pylori is the strongest risk factor for atrophic gastritis, peptic ulcers, and gastric cancer. CagA, the product of the cagA gene, is a bacterial oncoprotein, which, upon delivery into gastric epithelial cells, binds to and inhibits the polarity-regulating kinase, partitioning-defective 1b (PAR1b) [also known as microtubule affinity-regulating kinase 2 (MARK2)], via its CagA multimerization (CM) motif. The inhibition of PAR1b elicits junctional and polarity defects, rendering cells susceptible to oncogenesis. Notably, the polymorphism in the CM motif has been identified among geographic variants of CagA, differing in either the copy number or the sequence composition. In this study, through quantitative analysis of the complex formation between CagA and PAR1b, we found that several CagA species have acquired elevated PAR1b-binding activity via duplication of the CM motifs, while others have lost their PAR1b-binding activity. We also found that strength of CagA-PAR1b interaction was proportional to the degrees of stress fiber formation and tight junctional disruption by CagA in gastric epithelial cells. These results indicate that the CM polymorphism is a determinant for the magnitude of CagA-mediated deregulation of the cytoskeletal system and thereby possibly affects disease outcome of cagA-positive H. pylori infection, including gastric cancer. PMID:27445265

  20. 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.

  1. E6/E7 mRNA expression analysis: a test for the objective assessment of cervical adenocarcinoma in clinical prognostic procedure.

    PubMed

    Hovland, Siri; Muller, Susanne; Skomedal, Hanne; Mints, Michael; Bergström, Jakob; Wallin, Keng-Ling; Karlsen, Frank; Johansson, Bo; Andersson, Sonia

    2010-06-01

    Detection of E6/E7 mRNA expression using the real-time nucleic acid sequence-based amplification assay (NASBA) PreTect HPV-Proofer was compared with results of human papillomavirus (HPV) DNA detection in 98 paraffin-embedded samples from patients with cervical adenocarcinoma. HR-HPV DNA was detected in 61 (62%), while HR-HPV E6/E7 mRNA was detected in 63 (64%) of the samples. Correlation between results from DNA analyses and the E6/E7 mRNA assay showed consistent results in 87% of samples (47 of 54). The results from these two methods in detecting presence of HPV infection of any type agreed in 77%. Overall agreement between the methods was seen in 82 of the 98 cases (84%). When evaluating change in sensitivity for detection of HPV positives by adding more HPV types to the HPV DNA assay, maximum sensitivity was reached by targeting four HPV types. The coverage of HPV DNA presence was 76.9%, while the E6/E7 mRNA assay achieved a maximum coverage of 80.8% using only three HPV types. Thus, E6/E7 oncogene expression analysis may provide a more objective test for assessment of neoplastic glandular cells. Further studies may reveal whether the clinical performance of the E6/E7 mRNA assay will be of prognostic value in management of cervical adenocarcinoma. PMID:20428778

  2. 1H NMR study of the dynamics of the pH modulation of axial coordination in Aplysia limacina (Val(E7)) and sperm whale double mutant His(E7)-->Val,Thr(E10)-->Arg metmyoglobin.

    PubMed

    Qin, J; Pande, U; La Mar, G N; Ascoli, F; Ascenzi, P; Cutruzzolá, F; Travaglini-Allocatelli, C; Brunori, M

    1993-11-15

    The molecular and electronic structure, thermodynamics, dynamics, and mechanism of interconversion of the pH-modulated reversible equilibria of Aplysia limacina metmyoglobin, (metMb), have been investigated by 1H NMR spectroscopy. The four identified species which interconvert slowly on the NMR time scale (lifetime > 1 ms) are metMbOH (B) at alkaline pH, five coordinate metMb (N) at acidic to neutral pH, an acidic form, A, near pH approximately 4 and an extremely low pH form, D, attributed to an equilibrium unfolded species. The presence of strong distal hydrogen bonding by Arg (E10) to bound hydroxide is detected via a significant solvent isotope effect on the metMbOH (B) hyperfine shifts. Integration of the peak intensities yields pK values of 7.7 and approximately 4 for the B<-->N and N<-->A equilibria, respectively. Saturation transfer via chemical exchange is observed for B<-->N and N<-->A, where the rates for forming metMbOH (B) and the acidic form A from N are base- and acid-catalyzed, respectively, while the reverse rates are first-order. The much slower interconversion rate for N<-->B in A. limacina metMb than His(E7) containing mammalian metMb is attributed to the fact that a ligand bond is broken rather than just proton transferred and that the equilibrium involves a major rearrangement of the orientation of Arg(E10). This conclusion is supported by 1H NMR data for the sperm whale double mutant His(E7)-->Val/Thr(E10)-->Arg metMb, which exhibits a pK approximately 8.7 for the equilibrium between five-coordinate metMb (N) and metMbOH (B) with an even slower interconversion rate than in A. limacina metMb. This double mutant metMbOH (B) exhibits hydrogen bonding by Arg (E10) with coordinated hydroxide similar to that in A. limacina metMbOH. The slow but acid-catalyzed rates of conversion of A. limacina metMb (N) to the acid species A with significantly weakened bonding of the heme iron to the axial His(F8) residue is consistent with protonation of an

  3. Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

    PubMed

    Busch, S J; Martin, G A; Barnhart, R L; Mano, M; Cardin, A D; Jackson, R L

    1992-01-01

    Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1-mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1-mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression. PMID:1730747

  4. Development of a Multiplex PCR Test with Automated Genotyping Targeting E7 for Detection of Six High-Risk Human Papillomaviruses

    PubMed Central

    de Assis, Angela Maria

    2015-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV) and viral detection tests aid in the diagnosis of precursor lesions. In the present study, a molecular test for detection of high-risk HPV DNA, called E7-HPV, was standardized and assessed in samples from women with pre-cancerous lesions. The development of the E7-HPV test for detection and genotyping of six high-risk HPV (types 16, 18, 31, 33, 45 and 52), consisted of evaluating primer quality and adjusting the multiplex PCR conditions. Primer design was based on the E7 region of each HPV, and the fluorochrome 6-FAM was added to PCR primers. Viral detection was performed by capillary electrophoresis in automated sequencer in samples obtained from 60 women (55 with ASC-H/HSIL cytology) from August to September 2013. A non-inferiority analysis was conducted with the cobas HPV test as a reference and following international guidelines for the development of new tests. The two tests had a high concordance rate in HPV16 detection (kappa=0.972), with only one discordant case (cervical intraepithelial neoplasia grade 3, negaive with cobas and positive for HPV16 by E7-HPV) and complete agreement in HPV18 detection. When comparing detection of all high-risk HPV, three cases were positive with cobas but negative with E7-HPV, and another three cases were negative with cobas but positive with E7-HPV (HPV16, 31 and 52). When we evaluate the cases initially suspected by cytology, the two tests had the same sensitivity in detection CIN2 or worse. In conclusion, the E7-HPV test has satisfactory initial results, and its development can be continued. PMID:26087285

  5. Heme oxygenase-1 inhibits phosphorylation of the Helicobacter pylori oncoprotein CagA in gastric epithelial cells

    PubMed Central

    Gobert, Alain P.; Verriere, Thomas; de Sablet, Thibaut; Peek, Richard M.; Chaturvedi, Rupesh; Wilson, Keith T.

    2012-01-01

    Summary The cytotoxin-associated gene A protein (CagA) plays a pivotal role in the etiology of Helicobacter (H.) pylori-associated gastric diseases. CagA is injected into the cytoplasm of host cells by a type IV secretion system, and is phosphorylated on tyrosine residues by the host enzyme c-Src. We previously reported that the enzyme heme oxygenase-1 (HO-1) inhibits IL-8 secretion by H. pylori-infected cells. However, the cellular mechanism by which HO-1 regulates the innate immune function of infected cells remains unknown. We now show that nitric oxide and hemin, two inducers of HO-1, decrease the level of phosphorylated CagA (p-CagA) in H. pylori-infected gastric epithelial cells and this is blocked by either pharmacologic inhibition of HO-1 or siRNA knockdown of hmox-1. Moreover, forced expression of HO-1 by transfection of a plasmid expressing hmox-1 also results in a strong attenuation of CagA phosphorylation. This occurs through the inhibition of H. pylori-induced c-Src phosphorylation/activation by HO-1. Consequently, H. pylori-induced cytoskeletal rearrangements and activation of the pro-inflammatory response mediated by p-CagA are inhibited in HO-1-expressing cells. These data highlight a mechanism by which the innate immune response of the host can restrict the pathogenicity of H. pylori by attenuating CagA phosphorylation in gastric epithelial cells. PMID:23051580

  6. Overexpression of gankyrin in mouse hepatocytes induces hemangioma by suppressing factor inhibiting hypoxia-inducible factor-1 (FIH-1) and activating hypoxia-inducible factor-1.

    PubMed

    Liu, Yu; Higashitsuji, Hiroaki; Higashitsuji, Hisako; Itoh, Katsuhiko; Sakurai, Toshiharu; Koike, Kazuhiko; Hirota, Kiichi; Fukumoto, Manabu; Fujita, Jun

    2013-03-01

    Gankyrin (also called p28 or PSMD10) is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It consists of 7 ankyrin repeats and interacts with multiple proteins including Rb, Cdk4, MDM2 and NF-κB. To assess the oncogenic activity in vivo, we produced transgenic mice that overexpress gankyrin specifically in the hepatocytes. Unexpectedly, 5 of 7 F2 transgenic mice overexpressing hepatitis B virus X protein (HBX) promoter-driven gankyrin, and one of 3 founder mice overexpressing serum amyloid P component (SAP) promoter-driven gankyrin developed hepatic vascular neoplasms (hemangioma/hemangiosarcomas) whereas none of the wild-type mice did. Endothelial overgrowth was more frequent in the livers of diethylnitrosamine-treated transgenic mice than wild-type mice. Mouse hepatoma Hepa1-6 cells overexpressing gankyrin formed tumors with more vascularity than parental Hepa1-6 cells in the transplanted mouse skin. We found that gankyrin binds to and sequester factor inhibiting hypoxia-inducible factor-1 (FIH-1), which results in decreased interaction between FIH-1 and hypoxia-inducible factor-1α (HIF-1α) and increased activity of HIF-1 to promote VEGF production. The effects of gankyrin were more prominent under 3% O2 than 1% or 20% O2 conditions. Thus, the present study clarified, at least partly, mechanisms of vascular tumorigenesis, and suggests that gankyrin might play a physiological role in hypoxic responses besides its roles as an oncoprotein. PMID:23376718

  7. Very high resolution structure of a trematode hemoglobin displaying a TyrB10-TyrE7 heme distal residue pair and high oxygen affinity.

    PubMed

    Pesce, A; Dewilde, S; Kiger, L; Milani, M; Ascenzi, P; Marden, M C; Van Hauwaert, M L; Vanfleteren, J; Moens, L; Bolognesi, M

    2001-06-22

    Monomeric hemoglobin from the trematode Paramphistomum epiclitum displays very high oxygen affinity (P(50)<0.001 mm Hg) and an unusual heme distal site containing tyrosyl residues at the B10 and E7 positions. The crystal structure of aquo-met P. epiclitum hemoglobin, solved at 1.17 A resolution via multiwavelength anomalous dispersion techniques (R-factor=0.121), shows that the heme distal site pocket residue TyrB10 is engaged in hydrogen bonding to the iron-bound ligand. By contrast, residue TyrE7 is unexpectedly locked next to the CD globin region, in a conformation unsuitable for heme-bound ligand stabilisation. Such structural organization of the E7 distal residue differs strikingly from that observed in the nematode Ascaris suum hemoglobin (bearing TyrB10 and GlnE7 residues), which also displays very high oxygen affinity. The oxygenation and carbonylation parameters of wild-type P. epiclitum Hb as well as of single- and double-site mutants, with residue substitutions at positions B10, E7 and E11, have been determined and are discussed here in the light of the protein atomic resolution crystal structure. PMID:11399085

  8. Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple in vitro stimulations of CD8+ T cells with peptide-pulsed autologous dendritic cells

    PubMed Central

    Cools, Nathalie; Ponsaerts, Peter; Lenjou, Marc; Nijs, Griet; Van Bockstaele, Dirk R; Van Tendeloo, Viggo FI; Berneman, Zwi N

    2006-01-01

    Background Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity. Results In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711–20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8+ T cells in 5/5 healthy donors. Conclusion We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro. PMID:17067378

  9. 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.

  10. The SV40 large T antigen and adenovirus E1a oncoproteins interact with distinct isoforms of the transcriptional co-activator, p300.

    PubMed Central

    Avantaggiati, M L; Carbone, M; Graessmann, A; Nakatani, Y; Howard, B; Levine, A S

    1996-01-01

    p300 is a nuclear phosphoprotein likely to be involved in the control of cell growth. Here we show that SV40 large T antigen (Tag) forms a specific complex with p300. In various Tag-expressing cell lines, the affinity of Tag for p300 was restricted to a newly identified unphosphorylated but ubiquitinated form of the protein. Further, Tag did not associate with p300 in an SV40 Tag-producing cell line (REV2) in which the original transformed phenotype (SV52) is reverted. Biochemical studies demonstrate that both the phosphorylation and the ubiquitination profile of p300 are altered in REV2 with respect to the wild-type fully transformed SV52 parental cells, wherein Tag-p300 complexes are readily detected. In contrast to Tag, the adenovirus early expression product E1a interacts with both phosphorylated and unphosphorylated forms of p300. In addition, when REV2 cells were infected with adenovirus, E1a-p300 complexes were detected, suggesting that the p300 expressed in REV2 has lost the affinity for Tag, but not for E1a. We then compared the ability of Tag and E1a to affect the transcription levels of the cAMP-responsive promoter (CRE), which is modulated in vivo by p300, in REV2 cells. We found that Tag repressed the CRE promoter in all of the cell lines in which Tag-p300 complexes were detected, but not in REV2 cells. In contrast, E1a efficiently inhibited CRE-directed transcription in this cell line. The data thus indicate that the different specificities exhibited by Tag and E1a towards the various forms of p300 are reflected in vivo as a difference in the ability of these viral oncoproteins to modulate the expression of CRE-containing genes. Images PMID:8641289

  11. Impaired ferritin mRNA translation in primary erythroid progenitors: shift to iron-dependent regulation by the v-ErbA oncoprotein.

    PubMed

    Mikulits, W; Schranzhofer, M; Bauer, A; Dolznig, H; Lobmayr, L; Infante, A A; Beug, H; Müllner, E W

    1999-12-15

    In immortalized cells of the erythroid lineage, the iron-regulatory protein (IRP) has been suggested to coregulate biosynthesis of the iron storage protein ferritin and the erythroid delta-aminolevulinate synthase (eALAS), a key enzyme in heme production. Under iron scarcity, IRP binds to an iron-responsive element (IRE) located in ferritin and eALAS mRNA leaders, causing a block of translation. In contrast, IRP-IRE interaction is reduced under high iron conditions, allowing efficient translation. We show here that primary chicken erythroblasts (ebls) proliferating or differentiating in culture use a drastically different regulation of iron metabolism. Independently of iron administration, ferritin H (ferH) chain mRNA translation was massively decreased, whereas eALAS transcripts remained constitutively associated with polyribosomes, indicating efficient translation. Variations in iron supply had minor but significant effects on eALAS mRNA polysome recruitment but failed to modulate IRP-affinity to the ferH-IRE in vitro. However, leukemic ebls transformed by the v-ErbA/v-ErbB-expressing avian erythroblastosis virus showed an iron-dependent reduction of IRP mRNA-binding activity, resulting in mobilization of ferH mRNA into polysomes. Hence, we analyzed a panel of ebls overexpressing v-ErbA and/or v-ErbB oncoproteins as well as the respective normal cellular homologues (c-ErbA/TRalpha, c-ErbB/EGFR). It turned out that v-ErbA, a mutated class II nuclear hormone receptor that arrests erythroid differentiation, caused the change in ferH mRNA translation. Accordingly, inhibition of v-ErbA function in these leukemic ebls led to a switch from iron-responsive to iron-independent ferH expression. PMID:10590077

  12. Human translocation liposarcoma-CCAAT/enhancer binding protein (C/EBP) homologous protein (TLS-CHOP) oncoprotein prevents adipocyte differentiation by directly interfering with C/EBPbeta function.

    PubMed

    Adelmant, G; Gilbert, J D; Freytag, S O

    1998-06-19

    Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBPbeta in naïve NIH3T3 cells, and their ability to inhibit C/EBPbeta-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBPbeta and inhibit C/EBPbeta DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP's putative transactivation activity. Another adipogenic transcription factor, PPARgamma2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBPbeta from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process. PMID:9624148

  13. Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms.

    PubMed Central

    Fisher, R. J.; Fivash, M.; Casas-Finet, J.; Erickson, J. W.; Kondoh, A.; Bladen, S. V.; Fisher, C.; Watson, D. K.; Papas, T.

    1994-01-01

    The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling. PMID:8003962

  14. C-terminal domain of MEIS1 converts PKNOX1 (PREP1) into a HOXA9-collaborating oncoprotein.

    PubMed

    Bisaillon, Richard; Wilhelm, Brian T; Krosl, Jana; Sauvageau, Guy

    2011-10-27

    The three-amino-acid loop extension (TALE) class homeodomain proteins MEIS1 and PKNOX1 (PREP1) share the ability to interact with PBX and HOX family members and bind similar DNA sequences but appear to play opposing roles in tumor development. Elevated levels of MEIS1 accelerate development of HOX- and MLL-induced leukemias, and this pro-tumorigenic property has been associated with transcriptional activity of MEIS1. In contrast, reduction of PKNOX1 levels has been linked with cancer development despite the absence of an identifiable transactivating domain. In this report, we show that a chimeric protein generated by fusion of the MEIS1 C-terminal region encompassing the transactivating domain with the full-length PKNOX1 (PKNOX1-MC) acquired the ability to accelerate the onset of Hoxa9-induced leukemia in the mouse bone marrow transduction/transplantation model. Gene expression profiling of primary bone marrow cells transduced with Hoxa9 plus Meis1, or Hoxa9 plus Pknox1-MC revealed perturbations in overlapping functional gene subsets implicated in DNA packaging, chromosome organization, and in cell cycle regulation. Together, results presented in this report suggest that the C-terminal domain of MEIS1 confers to PKNOX1 an ectopic transactivating function that promotes leukemogenesis by regulating expression of genes involved in chromatin accessibility and cell cycle progression. PMID:21900201

  15. Celastrol Induces Cell Apoptosis and Inhibits the Expression of the AML1-ETO/C-KIT Oncoprotein in t(8;21) Leukemia.

    PubMed

    Yu, Xianjun; Ruan, Xuzhi; Zhang, Jingxuan; Zhao, Qun

    2016-01-01

    Resistance to chemotherapy is a major challenge to improving overall survival in Acute Myeloid Leukemia (AML). Therefore, the development of innovative therapies and the identification of more novel agents for AML are urgently needed. Celastrol, a compound extracted from the Chinese herb Tripterygium wilfordii Hook, exerts anticancer activity. We investigated the effect of celastrol in the t(8;21) AML cell lines Kasumi-1 and SKNO-1. We demonstrated that inhibition of cell proliferation activated caspases and disrupted mitochondrial function. In addition, we found that celastrol downregulated the AML1-ETO fusion protein, therefore downregulating C-KIT kinases and inhibiting AKT, STAT3 and Erk1/2. These findings provide clear evidence that celastrol might provide clinical benefits to patients with t(8;21) leukemia. PMID:27144550

  16. Listeria-based HPV-16 E7 vaccines limit autochthonous tumor growth in a transgenic mouse model for HPV-16 transformed tumors

    PubMed Central

    Sewell, Duane A.; Pan, Zhen Kun; Paterson, Yvonne

    2008-01-01

    We have shown that Listeria-based cancer vaccines inhibit the growth of transplanted tumors in a transgenic mouse model of immune tolerance where HPV-16 E7 is expressed in the thyroid gland. In this study we determine whether these vaccines are able to inhibit autochthonous tumor growth in this animal model. Mice treated with Listeria vaccines expressing E7 had significantly smaller thyroid tumors than did mice treated with controls and possessed higher numbers of antigen-specific CD8+ T cells within the spleens, tumors, and peripheral blood. This study shows that Listeria-based vaccines are able to slow autochthonous tumor growth and break immunological tolerance. PMID:18680778

  17. 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; Sültmann, 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

  18. Phosphorylation by Casein Kinase I Promotes the Turnover of the Mdm2 Oncoprotein via the SCFβ-TRCP Ubiquitin Ligase

    PubMed Central

    Inuzuka, Hiroyuki; Tseng, Alan; Gao, Daming; Zhai, Bo; Zhang, Qing; Shaik, Shavali; Wan, Lixin; Ang, Xiaolu L.; Mock, Caroline; Yin, Haoqiang; Stommel, Jayne M.; Gygi, Steven; Lahav, Galit; Asara, John; Jim Xiao, Zhi-Xiong; Kaelin, William G.; Harper, J. Wade; Wei, Wenyi

    2010-01-01

    Summary Mdm2 is the major negative regulator of the p53 pathway. Here we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by Casein Kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCFβ-TRCP. Inactivation of either β-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging-agents. Moreover, SCFβ-TRCP-dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression. PMID:20708156

  19. 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.

  20. 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

  1. The PIM family of oncoproteins: Small kinases with huge implications in myeloid leukemogenesis and as therapeutic targets

    PubMed Central

    Shah, Parag P.; Mims, Alice S.; Lockwood, William W.; Kraft, Andrew S.; Beverly, Levi J.

    2014-01-01

    PIM kinases are a family of serine/threonine kinases involved in cell survival and proliferation. There is significant structural similarity between the three PIM kinases (PIM1, PIM2 and PIM3) and few amino acid differences. Although, several studies have specifically monitored the role of PIM1 in tumorigenesis, much less is known about PIM2 and PIM3. Therefore, in this study we have used in vitro cell culture models and in vivo bone marrow infection/transplantation to assess the comparative signaling and oncogenic potential of each of the three PIM kinases. All three PIM kinases were able to protect FL5.12 cells from IL-3 withdrawal induced death. Interestingly, the downstream signaling cascades were indistinguishable between the three kinases. Transplantation of murine bone marrow co-expressing MYC and PIM1, PIM2 or PIM3 caused rapid and uniformly lethal myeloid leukemia. De-induction of MYC 18 days following transplantation significantly increased the survival of mice, even with continual expression of PIM kinases. Alternatively, mice treated at the pre-leukemic stage with a PIM kinase inhibitor increased the lifespan of the mice, even with continual expression of the MYC transgene. These data demonstrate the role of PIM kinases in driving myeloid leukemia, and as candidate molecules for therapy against human malignancies. PMID:25238262

  2. 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.

  3. The BPS spectrum of the 4d {N}=2 SCFT's H 1, H 2, D 4, E 6, E 7, E 8

    NASA Astrophysics Data System (ADS)

    Cecotti, Sergio; Del Zotto, Michele

    2013-06-01

    Extending results of 1112.3984, we show that all rank 1 {N}=2 SCFT's in the sequence H 1, H 2, D 4 E 6, E 7, E 8 have canonical finite BPS chambers containing precisely 2 h(F) = 12(∆ - 1) hypermultiplets. The BPS spectrum of the canonical BPS chambers saturates the conformal central charge c, and satisfies some intriguing numerology.

  4. Transforming properties of Felis catus papillomavirus type 2 E6 and E7 putative oncogenes in vitro and their transcriptional activity in feline squamous cell carcinoma in vivo.

    PubMed

    Altamura, Gennaro; Corteggio, Annunziata; Pacini, Laura; Conte, Andrea; Pierantoni, Giovanna Maria; Tommasino, Massimo; Accardi, Rosita; Borzacchiello, Giuseppe

    2016-09-01

    Felis catus papillomavirus type 2 (FcaPV2) DNA is found in feline cutaneous squamous cell carcinomas (SCCs); however, its biological properties are still uncharacterized. In this study, we successfully expressed FcaPV2 E6 and E7 putative oncogenes in feline epithelial cells and demonstrated that FcaPV2 E6 binds to p53, impairing its protein level. In addition, E6 and E7 inhibited ultraviolet B (UVB)-triggered accumulation of p53, p21 and pro-apoptotic markers such as Cleaved Caspase3, Bax and Bak, suggesting a synergistic action of the virus with UV exposure in tumour pathogenesis. Furthermore, FcaPV2 E7 bound to feline pRb and impaired pRb levels, resulting in upregulation of the downstream pro-proliferative genes Cyclin A and Cdc2. Importantly, we demonstrated mRNA expression of FcaPV2 E2, E6 and E7 in feline SCC samples, strengthening the hypothesis of a causative role in the development of feline SCC. PMID:27236740

  5. ETV6/ARG oncoprotein confers autonomous cell growth by enhancing c-Myc expression via signal transducer and activator of transcription 5 activation in the acute promyelocytic leukemia cell line HT93A.

    PubMed

    Iriyama, Noriyoshi; Hatta, Yoshihiro; Takei, Masami

    2015-01-01

    We investigated the role of ETV6/ARG fusion gene by exposing the HT93A cell line to nilotinib. HT93A cells were cultured with or without nilotinib±50 ng/mL of granulocyte colony-stimulating factor (G-CSF). Nilotinib treatment inhibited cell growth by increasing the percentage of cells in G0/G1 phase through the decrease of phosphorylated signal transducer and activator of transcription 3 (STAT3) (Y705), STAT5 (Y694) and c-Myc expression. After stimulation with G-CSF, STAT5 but not STAT3 was significantly phosphorylated in both nilotinib-treated and untreated cells. Moreover, combination therapy with nilotinib and G-CSF returned the expression level of c-Myc, cell growth and cell cycle distribution to the control level. These findings suggest that the ETV6/ARG oncoprotein contributes to autonomous cell growth by compensating for the requirement of growth factor through activating STAT5 signaling, which leads to the up-regulation of c-Myc. Our data suggest that ETV6/ARG oncoprotein is a potential target in the treatment of leukemia. PMID:25373509

  6. Correlation of E6 and E7 levels in high-risk HPV16 type cervical lesions with CCL20 and Langerhans cells.

    PubMed

    Jiang, B; Xue, M

    2015-01-01

    The human papillomavirus (HPV)16 E6 and E7 correlation with chemokine ligand (CCL)20 expression and Langerhans cells (LCs) in cervical lesions was investigated. We enrolled 43 patients with surgically treated cervical lesions from the Department of Gynecology in our hospital, and 20 controls without cervical lesions. Subjects were divided by pathology: HPV16(-) and HPV16(+) normal cervical groups (N = 10 each), and HPV16(+) cervical intraepithelial neoplasia (CIN), cervical invasive carcinoma (N = 15 each), and in situ carcinoma (N = 13) groups. E6, E7, the LC surface marker CD1a, and CCL20 were analyzed by immunohistochemistry. E6 and E7 in HPV16-type lesions were correlated with CCL20 and LCs. The average high power field cell numbers of CD1a+ LCs in the HPV(-) and HPV(+) normal cervix groups, and the CINI-II, CINIII in situ and cervical carcinoma groups were 22.89 ± 4.84, 13.7 ± 2.26, 9.2 ± 1.68, 5.9 ± 1.59, and 5.5 ± 1.58, respectively. Significant between-group differences existed except between cervical carcinoma and CINIII groups (P < 0.05). CCL20+ rates in each group were 70, 60, 60, 15.38, and 13.33%, respectively. E6/E7-positive expression rates in each group were 20/20, 66.7/66.7, 76.9/69.2, and 86.67/73.3%, respectively. CCL20 was positively correlated with CD1a (r = 0.649), and negatively correlated with E7 (r = -0.946) and E6 (r = -0.949). CD1a was negatively correlated with E6 (r = -0.632) and E7 (r = -0.632). Downregulation of CCL20 leading to LC decline is a key factor in cervical lesions. High-risk HPV-type lesions might inhibit the chemokine CCL20 through E6 and E7 to escape the immune response. PMID:26400278

  7. 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

  8. 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

  9. 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 5–7 days. The remaining transfected hepatocytes persisted for 2–4 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

  10. Genetic variability and lineage phylogeny of human papillomavirus type-16 and -53 based on the E6, E7, and L1 genes in Southwest China.

    PubMed

    Cao, Man; Chenzhang, Yuwei; Ding, Xianping; Zhang, Yiwen; Jing, Yaling; Chen, Zuyi

    2016-10-30

    Human papillomaviruses (HPVs) are circular double-stranded DNA viruses that are highly prevalent in the general population, and account for the cervical cancer burden in women worldwide. In this study, we analyzed HPV-16, the most prevalent type worldwide, and HPV-53, a possible high-risk type from infected women in Southwest China. To characterize mutations, intratypic variants, and genetic variability in the E6, E7, and L1 genes of HPV-16 (n=97) and HPV-53 (n=15), these genes were sequenced and submitted to GenBank. Phylogenetic trees were constructed using Bayesian trees, followed by secondary structure analysis and B-cell epitope prediction. Moreover, the selection pressures of the E6, E7, and L1 genes were estimated. In total, 27 novel variants of HPV-16 and 11 novel variants of HPV-53 were identified. In the HPV-16 E6-E7-L1 sequences, 73 nucleotide changes were observed with 40/73 being non-synonymous mutations (two in the alpha helix and five in the beta sheet) and 33/73 being synonymous. In the HPV-53 E6-E7-L1 sequences, 64 nucleotide changes were observed with 26/64 being non-synonymous mutations (three in the alpha helix and one in the beta sheet) and 38/64 being synonymous. Selective pressure analysis showed that most of these mutations did not reflect positive selection. The maximal divergence between any two variants within each gene of these two HPV types ranging from 0.94%(HPV-16 L1 gene)to 2.80%(HPV-53 E6 gene). Identifying new variants of HPV-16 and -53 from women in Southwest China may be helpful to design vaccines specifically for women in Southwest China and testing methods specifically for this region. The results of our study may contribute to future researches in diagnostic probes, vaccines improvement, or screening methods for a particular population. PMID:27450917

  11. Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors.

    PubMed Central

    Auewarakul, P; Gissmann, L; Cid-Arregui, A

    1994-01-01

    The E6 and E7 early genes of human papillomavirus type 16 have been shown in vitro to play a central role in the transforming capability of this virus. To explore their effects on differentiating epithelial cells in vivo, we used a bovine cytokeratin 10 (K10) promoter to target the expression of E6 and E7 to the suprabasal layers of the epidermis of transgenic mice. In two different lines of mice efficiently expressing the transgene, animals displayed generalized epidermal hyperplasia, hyperkeratosis and parakeratosis in the skin and the forestomach, both known to be sites of K10 expression. Northern (RNA) blot analysis revealed high levels of E6 and E7 transcripts, and in situ hybridizations localized these transcripts to the suprabasal strata of epidermis. In vivo labeling of proliferating cells showed two distinct effects of E6 and E7 expression in the epidermis: (i) an increase in the number of growing cells in the undifferentiated basal layer and (ii) abnormal proliferation of differentiated cells in the suprabasal strata. The expression of c-myc in the skin of transgenics was higher than that in control animals. The induction of c-myc transcription by topical application of tetradecanoyl phorbol acetate was prevented by simultaneous treatment with transforming growth factor beta 1 in nontransgenic skin but not in transgenic skin. In addition, transforming growth factor alpha was found to be overexpressed in the suprabasal layers of the transgenic epidermis. These findings suggest that autocrine mechanisms are involved in the development and maintenance of epidermal hyperplasia. Animals of both lines developed papillomas in skin sites exposed to mechanical irritation and wounding, suggesting that secondary events are necessary for progression to neoplasia. Collectively, these results provide new insights into the tumor promoter activities of human papillomavirus type 16 in epithelial cells in vivo. Images PMID:7969162

  12. A human papillomavirus type 18 E6/E7 transgene sensitizes mouse lens cells to human wild-type p53-mediated apoptosis.

    PubMed

    Nakamura, T; Williams-Simons, L; Westphal, H

    1997-06-26

    We have studied the concerted action of factors that influence the balance between cell proliferation and cell death in the developing lens of transgenic mice. We show that a human papillomavirus type 18 (HPV18) E6/E7 transgene that predominantly expresses the viral E7 gene product triggers apoptosis in a dose dependent manner, and causes retardation of lens growth or microphakia. E7 is known to inactivate pRB, the product of the retinoblastoma gene, and to enhance the action of p53. Our earlier work had demonstrated that over-expression of p53 itself can cause apoptosis of lens cells, and that a mutant p53 allele can interfere with this process. In the present study, we examined lenses that simultaneously express different constellations of the HPV18 E6/E7, wild-type and mutant human p53, and wild-type human pRB transgenes. We observed that lens cells expressing the HPV18 transgene are more sensitive to wild-type human p53 action than normal lens cells. As a result, there is severe microphakia in lenses that express both the HPV18 and the wild-type p53 transgenes. By contrast, apoptosis was reduced in lenses that co-expressed the HPV18 and either the pRB or the mutant p53 transgene. We conclude that levels of wild-type p53 are critical, and that any excess of p53 or suppression of pRB can cause cell death. Our results encourage attempts to counteract the deleterious action of human papillomaviruses in cervical cancer by a combination of measures that decrease cell proliferation and enhance apoptosis. PMID:9223662

  13. 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

  14. SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter

    PubMed Central

    Smith, Jennifer A.; Haberstroh, Friederike S.; White, Elizabeth A.; Livingston, David M.; DeCaprio, James A.; Howley, Peter M.

    2014-01-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7. PMID:25222147

  15. SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter.

    PubMed

    Smith, Jennifer A; Haberstroh, Friederike S; White, Elizabeth A; Livingston, David M; DeCaprio, James A; Howley, Peter M

    2014-11-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7. PMID:25222147

  16. 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.

  17. Inflammatory microenvironment and human papillomavirus-induced carcinogenesis.

    PubMed

    Mangino, Giorgio; Chiantore, Maria Vincenza; Iuliano, Marco; Fiorucci, Gianna; Romeo, Giovanna

    2016-08-01

    More than 15% of the global cancer burden is attributable to infectious agents. Pathogens that cause persistent infections are strongly associated with cancer, inflammation being a major component of the chronic infections as revealed by basic, clinical and epidemiological studies. Persistent infection and viral oncoproteins induce specific cellular pathways modifications that promote tumorigenesis. Deregulated and continuous immune response leads to severe tissue and systemic damage, impaired tumor surveillance and consequent carcinogenesis promotion by selecting for metastatic and therapeutically resistant tumor phenotypes. In this review, the role of inflammatory microenvironment in the HPV-induced carcinogenesis is addressed, with a specific focus on the involvement of the immune molecules and microRNAs as well as their delivery through the microvesicle cargo. PMID:27021827

  18. Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes ▿

    PubMed Central

    Hyland, Paula L.; McDade, Simon S.; McCloskey, Rachel; Dickson, Glenda J.; Arthur, Ken; McCance, Dennis J.; Patel, Daksha

    2011-01-01

    A number of epigenetic alterations occur in both the virus and host cellular genomes during human papillomavirus (HPV)-associated carcinogenesis, and investigations of such alterations, including changes in chromatin proteins and histone modifications, have the potential to lead to therapeutic epigenetic reversion. We report here that transformed HPV16 E6/E7-expressing primary human foreskin keratinocytes (HFKs) (E6/E7 cells) demonstrate increased expression of the PRC2 methyltransferase EZH2 at both the mRNA and protein levels but do not exhibit the expected increase in trimethylated H3K27 (H3K27me3) compared to normal keratinocytes. In contrast, these cells show a reduction in global H3K27me3 levels in vitro, as well as upregulation of the KDM6A demethylase. We further show for the first time that transformation with the HPV16 E6 and E7 oncogenes also results in an increase in phosphorylated EZH2 serine 21 (P-EZH2-Ser21), mediated by active Akt, and in a downregulation of the PRC1 protein BMI1 in these cells. High-grade squamous cervical intraepithelial lesions also showed a loss of H3K27me3 in the presence of increased expression of EZH2. Correlating with the loss of H3K27me3, E6/E7 cells exhibited derepression of specific EZH2-, KMD6A-, and BMI1-targeted HOX genes. These results suggest that the observed reduction in H3K27me3 may be due to a combination of reduced activities/levels of specific polycomb proteins and increases in demethylases. The dysregulation of multiple chromatin proteins resulting in the loss of global H3K27me3 and the transcriptional reprogramming in HPV16 E6/E7-infected cells could provide an epigenetic signature associated with risk and/or progression of HPV16-associated cancers, as well as the potential for epigenetic reversion in the future. PMID:21865393

  19. Regulation of the miRNA expression by TEL/AML1, BCR/ABL, MLL/AF4 and TCF3/PBX1 oncoproteins in acute lymphoblastic leukemia (Review).

    PubMed

    Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Illades-Aguiar, Berenice; Leyva-Vázquez, Marco Antonio

    2016-09-01

    MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. miRNAs act in diverse biological processes including development, cell growth, apoptosis, and hematopoiesis. The miRNA expression is associated with specific cytogenetic changes and can also be used to discriminate between the different subtypes of leukemia in acute lymphoblastic leukemia with common translocations, it is shown that the miRNAs have the potential to be used for clinical diagnosis and prognosis. We reviewed the roles of miRNA here with emphasis on their function in human leukemia and the mechanisms of the TEL/AML1, BCR/ABL, MLL/AF4 and TCF3/PBX1 oncoproteins on miRNAs expression in acute lymphoblastic leukemia. PMID:27431573

  20. An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation.

    PubMed

    Souroullas, George P; Jeck, William R; Parker, Joel S; Simon, Jeremy M; Liu, Jie-Yu; Paulk, Joshiawa; Xiong, Jessie; Clark, Kelly S; Fedoriw, Yuri; Qi, Jun; Burd, Christin E; Bradner, James E; Sharpless, Norman E

    2016-06-01

    B cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase (EZH2), but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic Ezh2 gain-of-function mutation (EZH2(Y646F) in human; Ezh2(Y641F) in mouse) is conditionally expressed. Expression of Ezh2(Y641F) in mouse B cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Overexpression of the anti-apoptotic protein Bcl2, but not the oncoprotein Myc, or loss of the tumor suppressor protein p53 (encoded by Trp53 in mice) further accelerated lymphoma progression. Expression of the mutant Braf but not the mutant Nras oncoprotein further accelerated melanoma progression. Although expression of Ezh2(Y641F) globally increased the abundance of trimethylated Lys27 of histone H3 (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 that was associated with increased transcription at many loci. These results suggest that Ezh2(Y641F) induces lymphoma and melanoma through a vast reorganization of chromatin structure, inducing both repression and activation of polycomb-regulated loci. PMID:27135738

  1. Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2.

    PubMed Central

    Rapp, B; Pawellek, A; Kraetzer, F; Schaefer, M; May, C; Purdie, K; Grassmann, K; Iftner, T

    1997-01-01

    Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell. PMID:9261424

  2. 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

  3. Chimeric HBcAg virus-like particles presenting a HPV 16 E7 epitope significantly suppressed tumor progression through preventive or therapeutic immunization in a TC-1-grafted mouse model

    PubMed Central

    Chu, Xiaojie; Li, Yang; Long, Qiong; Xia, Ye; Yao, Yufeng; Sun, Wenjia; Huang, Weiwei; Yang, Xu; Liu, Cunbao; Ma, Yanbing

    2016-01-01

    Background Therapeutic human papillomavirus (HPV) vaccines are currently being developed. However, no therapeutic efficacy has been achieved in clinical trials for the treatment of cervical intraepithelial neoplasia or cancer. One of the important issues in increasing vaccine efficacy is determining the best way to enhance tumor antigen-specific cellular immune responses. This study aimed to explore the virus-like particles (VLPs) of hepatitis B core antigen (HBcAg) as potential therapeutic vaccine carriers and to assess its immunological characteristics. Methods Chimeric VLPs presenting a HPV 16 cytotoxic T lymphocytes epitope E749–57 (amino acid 49–57 of the E7 protein) were prepared using recombinant genes. C57BL/6 mice were immunized with VLPs and grafted with tumor cells TC-1 which is an E7-expressing tumorigenic cell line. The dynamic tumor growth was monitored and anti-tumor immune responses were investigated. Results Using a preventive strategy, immunization with VLPs resulted in nearly complete suppression of tumor growth. In treatment studies, VLP immunization significantly suppressed the tumor progression in mice carrying 2–3 mm tumors and in those bearing even larger tumors with diameters up to 8–9 mm. The VLP structure was shown to be important to induce vigorous antitumor immunity and effects. In immunized mice, enhanced E749–57-specific cellular immune responses were evidenced by increased interferon (IFN)-γ expression and decreased interleukin (IL)-4 expression in splenic lymphocytes, as well as an elevated number of effector cells expressing IFN-γ in response to the in vitro stimulation of the specific peptide E749–57. In addition, effective immune memory after VLP immunization was maintained for at least 16 weeks, preventing significant tumor growth after subsequent TC-1 challenge. Conclusion While VLPs were highly immunogenic in stimulating humoral immunity, our results strongly indicated that VLPs, such as HBcAg particles, might

  4. Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells.

    PubMed

    Bañuelos, 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

  5. Improved synthesis of (3E,7Z)-3,7-tetradecadienyl acetate, the major sex pheromone constituent of the potato pest Symmetrischema tangolias (Gyen).

    PubMed

    Ragoussis, Valentine; Perdikaris, Stamatis; Karamolegkos, Antonis; Magkiosi, Konstantina

    2008-12-24

    An efficient six-step synthesis of (3E,7Z)-3,7-tetradecadienyl acetate, the major component of the sex pheromone of the potato pest Symmetrischema tangolias (Gyen), is described, starting from the commercially available dihydropyran. The stereoselective formation of the 7Z double bond is accomplished by a Wittig reaction, while the 3E double bond is formed by a modified Knoevenagel condensation. The overall yield of the synthesis is 28%, giving the final product in high stereochemical purity (95%). The simplicity and the low cost of the herein reported synthesis suggest the potential practical use of the above pheromone in integrated management programs, for this serious insect pest. PMID:19053388

  6. Human T-cell leukemia virus type 1 oncoprotein tax represses ZNF268 expression through the cAMP-responsive element-binding protein/activating transcription factor pathway.

    PubMed

    Wang, Di; Guo, Ming-Xiong; Hu, Hai-Ming; Zhao, Zhou-Zhou; Qiu, Hong-Ling; Shao, Huan-Jie; Zhu, Chen-Gang; Xue, Lu; Shi, Yun-Bo; Li, Wen-Xin

    2008-06-13

    Expression of the human T-cell leukemia virus type 1 (HTLV-1) oncoprotein Tax is correlated with cellular transformation, contributing to the development of adult T-cell leukemia. In this study, we investigated the role of Tax in the regulation of the ZNF268 gene, which plays a role in the differentiation of blood cells and the pathogenesis of leukemia. We demonstrated that ZNF268 mRNA was repressed in HTLV-1-infected cells. We also showed that stable and transient expression of HTLV-1 Tax led to repression of ZNF268. In addition, by using reporter constructs that bear the human ZNF268 promoter and its mutants, we showed that Tax repressed ZNF268 promoter in a process dependent on a functional cAMP-responsive element. By using Tax, cAMP-responsive element-binding protein (CREB)-1, CREB-2, and their mutants, we further showed that Tax repressed ZNF268 through the CREB/activating transcription factor pathway. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated the formation of the complex of Tax.CREB-1 directly at the cAMP-responsive element both in vitro and in vivo. These findings suggest a role for ZNF268 in aberrant T-cell proliferation observed in HTLV-1-associated diseases. PMID:18375384

  7. Induced Mesophase in Mixtures of Photopolymerizable Hyperbranched Polyester and Liquid Crystal Mesogen

    NASA Astrophysics Data System (ADS)

    Kim, Namil; Kyu, Thein; Nosaka, Mami; Kudo, Hiroto; Nishikubo, Tadatomi

    2008-03-01

    Phase behavior of a mixture of eutectic liquid crystals (E7) and hyperbranched polyester (HBPEAc-COOH) has been investigated using polarized optical microscopy and differential scanning calorimetry. The observed phase diagram is an upper azeotrope, exhibiting the coexistence of nematic + isotropic phase in the vicinity of 90˜110^oC above the clearing temperature of neat E7 (60^oC). With decreasing temperature a focal-conic fan shaped texture develops in the composition range of 70˜90 wt% of E7, suggestive of induced smectic Sm-A phase in the mixture containing no known smectic phase in their neat forms. Wide angle x-ray diffraction (WAXD) technique revealed the existence of higher order mesophase(s). The phenomenon of induced mesophase in the hyperbranched polyester/E7 system will be discussed.

  8. Epstein-Barr virus oncoprotein LMP1 mediates survivin upregulation by p53 contributing to G1/S cell cycle progression in nasopharyngeal carcinoma

    PubMed Central

    GUO, LILI; TANG, MIN; YANG, LIFANG; XIAO, LANBO; BODE, ANN M.; LI, LILI; DONG, ZIGANG; CAO, YA

    2012-01-01

    Latent membrane protein 1 (LMP1) is an important oncogenic protein encoded by Epstein-Barr virus (EBV) and plays an important role in the development of nasopharyngeal carcinoma (NPC). Our previous study has shown that p53 protein was accumulated and phosphorylated in NPC, implying its transcription factor activity in NPC tumorigenesis. However, the biological function and potential downstream target of p53 mediated by LMP1 in NPC remain unknown. In this study, we found that LMP1 simultaneously induced upregulation of both p53 and survivin at the protein level, as well as their phosphorylation. Knockdown of p53 by siRNA revealed that LMP1 increased survivin expression by p53 directly. Furthermore, we found that LMP1 upregulated survivin by p53 at the transcriptional level by increasing p53-mediated survivin promoter activity and DNA binding activity. Moreover, LMP1 induced the co-localization of p53 and survivin in the nucleus, conferring to their related functions in NPC tumorigenesis. We further found that p53 promoted G1/S cell cycle progression, but did not induce apoptosis in LMP1-positive NPC cells. Collectively, these findings suggest that p53 acting as a transcription factor promotes the transcriptional activity of survivin, and further increases its protein expression and phosphorylation in the regulation of LMP1, thus, leading to G1/S cell cycle progression with no effect on apoptosis in NPC tumorigenesis. PMID:22266808

  9. 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.

  10. Performance of the HPV-16 L1 methylation assay and HPV E6/E7 mRNA test for the detection of squamous intraepithelial lesions in cervical cytological samples.

    PubMed

    Qiu, Cui; Zhi, Yanfang; Shen, Yong; Gong, Jiaomei; Li, Ya; Rong, Shouhua; Okunieff, Paul; Zhang, Lulu; Li, Xiaofu

    2015-11-01

    HPV-16 L1 methylation and E6/E7 mRNA have suggested that they had close relationship with cervical neoplastic progression. This study aimed to evaluate the clinical performance of the HPV-16 L1 methylation assay and E6/E7 mRNA test for detecting high-grade cervical lesions (CIN2+). A total of 81 women with liquid-based cytology (LBC) samples, histological results, and positive HPV-DNA test for HPV type 16 only were included in this study. HPV-16 L1 methylation and E6/E7 mRNA levels were measured using methylation-sensitive high resolution melting (MS-HRM) analysis and Quantivirus®HPV E6/E7 RNA 3.0 assay (bDNA), respectively, in the same residue of LBC samples. The current date showed a positive correlation between the HPV-16 L1 methylation and the E6/E7 mRNA levels. The L1 methylation and mRNA levels both increased with disease severity. The mRNA test method showed higher sensitivity and NPV (98.0 and 91.7% vs. 89.8 and 80.8%), while lower specificity and PPV (34.4 and 69.6% vs. 65.6 and 80.0%), than the L1 methylation assay for detecting histology-confirmed CIN2+. When using the detection method of mRNA test combined with L1 methylation assay, we obtained a sensitivity of 89.8% and a specificity of 71.9%. These findings suggest that assessment of HPV-16 L1 methylation testing combined with E6/E7 mRNA testing may be a promising method for the triage of women with HPV type 16 only. PMID:26297960

  11. 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

  12. Molecular variants of human papilloma virus 16 E2, E4, E5, E6 and E7 genes associated with cervical neoplasia in Romanian patients.

    PubMed

    Plesa, Adriana; Anton, Gabriela; Iancu, Iulia V; Diaconu, Carmen C; Huica, Irina; Stanescu, Anca D; Socolov, Demetra; Nistor, Elena; Popa, Elena; Stoian, Mihai; Botezatu, Anca

    2014-12-01

    The aim of this study was to identify and associate the sequence variations of human Papillomavirus 16 (HPV16) genes from women who live in two different areas of Romania and associate them with malignant progression. One hundred twenty-four HPV16-positive cervical isolates were collected, and the E2, E4, E5, E6 and E7 viral genes were sequenced. Two new missense mutations in the E6 gene (C279G and A305C) were found (together or alone, in association with other mutations) in 44 of 124 cases. The most frequently simultaneously mutated genes were E4/E2 hinge, E5 and E6 (p = 0.0004) in squamous cell carcinoma (SCC) samples. Also, for SCC patients, the best-correlated mutation patterns were obtained for E4/E2 hinge-E5 (r = 0.7984; p < 0.0001). No sample was found to have all of the investigated viral genes concurrently mutated. Phylogenetic analysis was performed to characterize the viral variants. Similar results were found for SCC and cervical intraepithelial neoplasia III (CINIII) cases. After all of the target gene sequences were assembled, all patients were found to be infected with viruses of the HPV16- European-German (EG) lineage, and two clusters were identified, the first (55/96 variants) from Moldavia and the second (41/96 variants) from Bucharest. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area. This study has generated a comprehensive set of sequence variation data on HPV16 circulating in Romania to join the existing data and highlight the important role of HPV16 variants during cervical carcinogenesis. PMID:25143263

  13. Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR.

    PubMed

    Clifford, Gary M; Vaccarella, Salvatore; Franceschi, Silvia; Tenet, Vanessa; Umulisa, M Chantal; Tshomo, Ugyen; Dondog, Bolormaa; Vorsters, Alex; Tommasino, Massimo; Heideman, Daniëlle A M; Snijders, Peter J F; Gheit, Tarik

    2016-08-01

    GP5+/6+-based PCR followed by reverse line blot hybridization (GP5+/6+RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5+/6+RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5+/6+RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5+/6+RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5+/6+RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5+/6+RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5+/6+RLB than for those negative for the same type by GP5+/6+RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5+/6+RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5+/6+RLB, suggesting the existence of masking effects due to competition for GP5+/6+ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. PMID:27225411

  14. 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 IκB 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

  15. MUC1-C Oncoprotein Activates ERK→C/EBPβ 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 β (C/EBPβ) transcription factor. The results further show that MUC1-C and C/EBPβ 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/EBPβ→ALDH1A1 pathway, and (ii) promotes the induction of ALDH activity in breast cancer cells. PMID:24043631

  16. Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes

    SciTech Connect

    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 IκB 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. - Highlights: • Niclosamide is a promising therapeutic candidate for adult T cell leukemia. • Niclosamide employs a novel mechanism through proteasomal degradation of Tax. • Niclosamide downregulates certain cellular pro-survival molecules.

  17. 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

  18. Late Intervention with anti-BRAFV600E Therapy Induces Tumor Regression in an Orthotopic Mouse Model of Human Anaplastic Thyroid Cancer

    PubMed Central

    Nehs, Matthew A.; Nucera, Carmelo; Nagarkatti, Sushruta S.; Sadow, Peter M.; Morales-Garcia, Dieter; Hodin, Richard A.

    2012-01-01

    Human anaplastic thyroid cancer (ATC) is a lethal disease with an advanced clinical presentation and median survival of 3 months. The BRAFV600E oncoprotein is a potent transforming factor that causes human thyroid cancer cell progression in vitro and in vivo; therefore, we sought to target this oncoprotein in a late intervention model of ATC in vivo. We used the human ATC cell line 8505c, which harbors the BRAFV600E and TP53R248G mutations. Immunocompromised mice were randomized to receive the selective anti-BRAFV600E inhibitor, PLX4720, or vehicle by oral gavage 28 d after tumor implantation, 1 wk before all animals typically die due to widespread metastatic lung disease and neck compressive symptoms in this model. Mice were euthanized weekly to evaluate tumor volume and metastases. Control mice showed progressive tumor growth and lung metastases by 35 d after tumor implantation. At that time, all control mice had large tumors, were cachectic, and were euthanized due to their tumor-related weight loss. PLX4720-treated mice, however, showed a significant decrease in tumor volume and lung metastases in addition to a reversal of tumor-related weight loss. Mouse survival was extended to 49 d in PLX4720-treated animals. PLX4720 treatment inhibited cell cycle progression from 28 d to 49 d in vivo. PLX4720 induces striking tumor regression and reversal of cachexia in an in vivo model of advanced thyroid cancer that harbors the BRAFV600E mutation. PMID:22202162

  19. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  20. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2.

    PubMed

    Glenewinkel, Florian; Cohen, Michael J; King, Cason R; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  1. The human papillomavirus type 16 E7 protein complements adenovirus type 5 E1A amino-terminus-dependent transactivation of adenovirus type 5 early genes and increases ATF and Oct-1 DNA binding activity.

    PubMed Central

    Wong, H K; Ziff, E B

    1996-01-01

    We have previously shown that conserved region 1 (CR1) of the adenovirus type 5 (Ad5) E1A protein synergizes with CR3 in the transactivation of Ad5 early genes (H.K. Wong and E. B. Ziff, J. Virol. 68:4910-4920, 1994). CR1 lies within the E1A amino terminus and binds host regulatory proteins such as the RB protein, p107, p130, and p300. Since simian virus 40 (SV40) large T antigen and human papillomavirus type 16 (HPV16) E7 protein also bind host regulatory factors, we investigated whether these viral proteins can complement E1A mutants which are defective in early gene activation. We show that the HPV16 E7 protein but not SV40 T antigen can complement mutations in the Ad5 E1A CR1 in the transactivation of viral early promoters. The inability of SV40 T antigen to complement suggests that RB binding on its own is not sufficient for early promoter transactivation by the E1A amino terminus. Nuclear runoff assays show that complementation by HPV16 E7 restores the ability of the E1A mutants to stimulate early gene expression at the level of transcription. Furthermore, nuclear extracts from the E7-transformed cells show increased binding activity of ATF and Oct-1, factors that can recognize the elements of Ad5 early genes, consistent with gene activation by E1A and E7 at the transcriptional level. PMID:8523545

  2. 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

  3. 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

  4. HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

    PubMed

    Xue, Yuezhen; Toh, Shen Yon; He, Pingping; Lim, Thimothy; Lim, Diana; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

    2015-10-27

    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

  5. 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

  6. Tobacco exposure results in increased E6 and E7 oncogene expression, DNA damage and mutation rates in cells maintaining episomal human papillomavirus 16 genomes

    PubMed Central

    Wei, Lanlan; Griego, Anastacia M.; Chu, Ming; Ozbun, Michelle A.

    2014-01-01

    High-risk human papillomavirus (HR-HPV) infections are necessary but insufficient agents of cervical and other epithelial cancers. Epidemiological studies support a causal, but ill-defined, relationship between tobacco smoking and cervical malignancies. In this study, we used mainstream tobacco smoke condensate (MSTS-C) treatments of cervical cell lines that maintain either episomal or integrated HPV16 or HPV31 genomes to model tobacco smoke exposure to the cervical epithelium of the smoker. MSTS-C exposure caused a dose-dependent increase in viral genome replication and correspondingly higher early gene transcription in cells with episomal HPV genomes. However, MSTS-C exposure in cells with integrated HR-HPV genomes had no effect on genome copy number or early gene transcription. In cells with episomal HPV genomes, the MSTS-C-induced increases in E6 oncogene transcription led to decreased p53 protein levels and activity. As expected from loss of p53 activity in tobacco-exposed cells, DNA strand breaks were significantly higher but apoptosis was minimal compared with cells containing integrated viral genomes. Furthermore, DNA mutation frequencies were higher in surviving cells with HPV episomes. These findings provide increased understanding of tobacco smoke exposure risk in HPV infection and indicate tobacco smoking acts more directly to alter HR-HPV oncogene expression in cells that maintain episomal viral genomes. This suggests a more prominent role for tobacco smoke in earlier stages of HPV-related cancer progression. PMID:25064354

  7. Environmental exposure and HPV infection may act synergistically to induce lung tumorigenesis in nonsmokers

    PubMed Central

    Cheng, Ya-Wen; Lin, Frank Cheau-Feng; Chen, Chih-Yi; Hsu, Nan-Yung

    2016-01-01

    Most studies of lung tumorigenesis have focused on smokers rather than nonsmokers. In this study, we used human papillomavirus (HPV)-positive and HPV-negative lung cancer cells to test the hypothesis that HPV infection synergistically increases DNA damage induced by exposure to the carcinogen benzo[a]pyrene (B[a]P), and contributes to lung tumorigenesis in nonsmokers. DNA adduct levels induced by B[a]P in HPV-positive cells were significantly higher than in HPV-negative cells. The DNA adduct formation was dependent on HPV E6 oncoprotein expression. Gene and protein expression of two DNA repair genes, XRCC3 and XRCC5, were lower in B[a]P-treated E6-positive cells than in E6-negative lung cancer cells. The reduced expression was also detected immunohistochemically and was caused by increased promoter hypermethylation. Moreover, mutations of p53 and epidermal growth factor receptor (EGFR) genes in lung cancer patients were associated with XRCC5 inactivation. In sum, our study indicates that HPV E6-induced promoter hypermethylation of the XRCC3 and XRCC5 DNA repair genes and the resultant decrease in their expression increases B[a]P-induced DNA adducts and contributes to lung tumorigenesis in nonsmokers. PMID:26918347

  8. Curcumin and Ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells.

    PubMed

    Kumar, Devbrat; Basu, Soumya; Parija, Lucy; Rout, Deeptimayee; Manna, Sanjeet; Dandapat, Jagneshwar; Debata, Priya Ranjan

    2016-07-01

    Cervical cancer and precancerous lesions of the cervix continue to be a global health issue, and the medication for the treatment for chronic HPV infection so far has not been effective. Potential anticancer and anti HPV activities of two known phytochemicals, Curcumin and Ellagic acid were evaluated in HeLa cervical cancer cells. Curcumin is a natural compound found in the root of Curcuma longa plant and Ellagic acid a polyphenol found in fruits of strawberries, raspberries and walnuts. The combination of Curcumin and Ellagic acid at various concentrations showed better anticancer properties than either of the drug when used alone as evidenced by MTT assay. Besides this, Curcumin and Ellagic acid also restore p53, induce ROS formation and DNA damage. Mechanistic study further indicated that Curcumin and Ellagic acid show anti-HPV activity as evidenced by decrease in the HPV E6 oncoprotein on HeLa cells. PMID:27261574

  9. Proliferative Defects in Dyskeratosis Congenita Skin Keratinocytes are Corrected by Expression of the Telomerase Reverse Transcriptase, TERT, or by Activation of Endogenous Telomerase through Expression of Papillomavirus E6/E7 or the Telomerase RNA Component, TERC

    PubMed Central

    Gourronc, Francoise A.; Robertson, Mckaylee M.; Herrig, Annie K.; Lansdorp, Peter M.; Goldman, Frederick D.; Klingelhutz, Aloysius J.

    2010-01-01

    Dyskeratosis congenita (DC) is characterized by the triad of reticulate skin pigmentation, nail dystrophy, and leukoplakia. Epidermal atrophy, hair growth defects, bone marrow failure, and increased risk of cancer are also common in DC patients. DC is caused by mutations in genes encoding for telomerase complex factors. Although there is an association of epidermal abnormalities with DC, epidermal cells from DC donors have not been previously characterized. We have isolated skin keratinocytes from affected members of a family with an autosomal dominant form of DC that is due to a mutation in the RNA component of telomerase, TERC. Here we demonstrate that, similar to DC fibroblasts from these donors, DC keratinocytes have short telomeres and a short lifespan. DC keratinocytes also exhibited impaired colony forming efficiency and migration capacity. Exogenous expression of the reverse transcriptase component of telomerase, TERT, activated telomerase levels to half that of TERT expressing normal cells and maintained telomeres at a short length with concomitant extension of lifespan. Unlike fibroblasts, transduction of human papillomavirus type 16 E6/E7 genes into DC keratinocytes activated telomerase to half that of E6/E7 expressing normal cells, and robust proliferation was observed. While expression of TERC has no measurable effect on telomerase in fibroblasts, expression of TERC in keratinocytes upregulated telomerase activity and, rarely, allowed rescue of proliferative defects. Our results point to important differences between DC fibroblasts and keratinocytes and show, for the first time, that expression of TERC can increase the lifespan of primary human epithelial cells. PMID:19558498

  10. 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

  11. SH4-domain-induced plasma membrane dynamization promotes bleb-associated cell motility.

    PubMed

    Tournaviti, Stella; Hannemann, Sebastian; Terjung, Stefan; Kitzing, Thomas M; Stegmayer, Carolin; Ritzerfeld, Julia; Walther, Paul; Grosse, Robert; Nickel, Walter; Fackler, Oliver T

    2007-11-01

    SH4 domains provide bipartite membrane-targeting signals for oncogenic Src family kinases. Here we report the induction of non-apoptotic plasma membrane (PM) blebbing as a novel and conserved activity of SH4 domains derived from the prototypic Src kinases Src, Fyn, Yes and Lck as well as the HASPB protein of Leishmania parasites. SH4-domain-induced blebbing is highly dynamic, with bleb formation and collapse displaying distinct kinetics. These reorganizations of the PM are controlled by Rho but not Rac or Cdc42 GTPase signalling pathways. SH4-induced membrane blebbing requires the membrane association of the SH4 domain, is regulated by the activities of Rock kinase and myosin II ATPase, and depends on the integrity of F-actin as well as microtubules. Endogenous Src kinase activity is crucial for PM blebbing in SH4-domain-expressing cells, active Src and Rock kinases are enriched in SH4-domain-induced PM blebs, and PM blebbing correlates with enhanced cell invasion in 3D matrices. These results establish a novel link between SH4 domains, Src activity and Rho signalling, and implicate SH4-domain-mediated PM dynamization as a mechanism that influences invasiveness of cells transformed by SH4-domain-containing oncoproteins. PMID:17959630

  12. 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.

  13. 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.

  14. 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

  15. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα protein degradation.

    PubMed

    Wang, Qian Qian; Zhou, Xin Yi; Zhang, Yan Fang; Bu, Na; Zhou, Jin; Cao, Feng Lin; Naranmandura, Hua

    2015-09-22

    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-RARα oncoprotein by As2O3, which results in initiation of PML-RARα degradation. However, after injection, As2O3 is rapidly methylated in body to different intermediate metabolites such as trivalent monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), 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 iAs(III)) and its intermediate metabolites (i.e., MMA(III)/DMA(III)) in NB4 cells. Inorganic iAs(III) predominantly showed induction of cell differentiation, while MMA(III) and DMA(III) specifically showed to induce mitochondria and endoplasmic reticulum-mediated apoptosis, respectively. On the other hand, in contrast to iAs(III), MMA(III) 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-RARα fusion protein. Moreover, methylated arsenicals can efficiently lead to cellular apoptosis, however, they are incapable of inducing NB4 cell differentiation. PMID:26213848

  16. 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

  17. Methylated arsenic metabolites bind to PML protein but do not induce cellular differentiation and PML-RARα 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-RARα 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

  18. Induced Abortion

    MedlinePlus

    ... Induced Abortion Patient Education FAQs Induced Abortion Patient Education Pamphlets - Spanish Induced Abortion FAQ043, May 2015 PDF Format Induced ... Your Practice Patient Safety & Quality Payment Reform (MACRA) Education & Events Annual ... Pamphlets Teen Health About ACOG About Us Leadership & ...

  19. The NFκB Signaling Pathway in Papillomavirus-induced Lesions: Friend or Foe?

    PubMed

    DA Costa, Rui M Gil; Bastos, Margarida M S M; Medeiros, Rui; Oliveira, Paula A

    2016-05-01

    Papillomaviruses induce a range of benign and malignant lesions in their hosts, including cervical cancer, that is associated with high-risk human papillomavirus (HPV) types. The nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) plays a pivotal role in HPV-infected cells, and its expression and activity are modulated by several viral oncoproteins. NFκB modulation seems to first facilitate viral persistence and immune evasion, and later to drive tumour progression, but the many conflicting results and the complexity of its signaling networks require great prudence while interpreting the role of NFκB in papillomaviral lesions. Accordingly, the pharmacological targeting of the NFκB pathway in HPV-induced lesions is a complex and currently unmet challenge. This review deals with recent findings concerning NFκB activation in HPV-infected cells, its role in viral persistence, cell transformation and tumour progression, and with current efforts to target this pathway for cancer prevention and therapy. PMID:27127107

  20. 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

  1. Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

    PubMed Central

    Gishizky, M L; Cortez, D; Pendergast, A M

    1995-01-01

    Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth. Images Fig. 1 Fig. 2 Fig. 3 PMID:7479904

  2. HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway

    PubMed Central

    Xue, Min; Yao, Shuihong; Hu, Minmin; Li, Wan; Hao, Tingting; Zhou, Feng; Zhu, Xiaofei; Lu, Hongmei; Qin, Di; Yan, Qin; Zhu, Jianzhong; Gao, Shou-Jiang; Lu, Chun

    2014-01-01

    Kaposi's sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV). Although the interaction between HIV-1 and KSHV plays a pivotal role in promoting the aggressive manifestations of KS, the pathogenesis underlying AIDS-KS remains largely unknown. Here we examined HIV-1 Nef protein promotion of KSHV oncoprotein K1-induced angiogenesis. We showed that both internalized and ectopic expression of Nef in endothelial cells synergized with K1 to facilitate vascular tube formation and cell proliferation, and enhance angiogenesis in a chicken CAM model. In vivo experiments further indicated that Nef accelerated K1-induced angiogenesis and tumorigenesis in athymic nu/nu mice. Mechanistic studies revealed that Nef and K1 synergistically activated PI3K/AKT/mTOR signaling by downregulating PTEN. Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3′ UTR of its mRNA. Inhibition of miR-718 expression increased PTEN synthesis and suppressed the synergistic effect of Nef- and K1-induced angiogenesis and tumorigenesis. These results indicate that, by targeting PTEN, miR-718 mediates Nef- and K1-induced angiogenesis via activation of AKT/mTOR signaling. Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target. PMID:25104021

  3. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

    PubMed Central

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  4. Translation-dependent mechanisms lead to PML upregulation and mediate oncogenic K-RAS-induced cellular senescence

    PubMed Central

    Scaglioni, Pier Paolo; Rabellino, Andrea; Yung, Thomas M; Bernardi, Rosa; Choi, Sooyeon; Konstantinidou, Georgia; Nardella, Caterina; Cheng, Ke; Pandolfi, Pier Paolo

    2012-01-01

    Expression of oncogenic K-RAS in primary cells elicits oncogene-induced cellular senescence (OIS), a form of growth arrest that potently opposes tumourigenesis. This effect has been largely attributed to transcriptional mechanisms that depend on the p53 tumour suppressor protein. The PML tumour suppressor was initially identified as a component of the PML-RARα oncoprotein of acute promyelocytic leukaemia (APL). PML, a critical OIS mediator, is upregulated by oncogenic K-RAS in vivo and in vitro. We demonstrate here that oncogenic K-RAS induces PML protein upregulation by activating the RAS/MEK1/mTOR/eIF4E pathway even in the absence of p53. Under these circumstances, PML mRNA is selectively associated to polysomes. Importantly, we find that the PML 5′ untranslated mRNA region plays a key role in mediating PML protein upregulation and that its presence is essential for an efficient OIS response. These findings demonstrate that upregulation of PML translation plays a central role in oncogenic K-RAS-induced OIS. Thus, selective translation initiation plays a critical role in tumour suppression with important therapeutic implications for the treatment of solid tumours and APL. PMID:22359342

  5. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    PubMed Central

    Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Grønn, Petter; Karlgård, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

    2012-01-01

    The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection. PMID:22235204

  6. 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

  7. 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.

  8. 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-α 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. PMID:26450919

  9. Sensitivity of APTIMA HPV E6/E7 mRNA test in comparison with hybrid capture 2 HPV DNA test for detection of high risk oncogenic human papillomavirus in 396 biopsy confirmed cervical cancers.

    PubMed

    Basu, Partha; Banerjee, Dipanwita; Mittal, Srabani; Dutta, Sankhadeep; Ghosh, Ishita; Chowdhury, Nilarun; Abraham, Priya; Chandna, Puneet; Ratnam, Sam

    2016-07-01

    The sensitivity of E6/E7 mRNA-based Aptima HPV test (AHPV; Hologic, Inc.) for detection of cervical cancer has been reported based on only a small number of cases. We determined the sensitivity of AHPV in comparison with the DNA-based Hybrid Capture 2 HPV test (HC2; Qiagen) for the detection of oncogenic HPV in a large number of cervical cancers at the time of diagnosis using cervical samples obtained in ThinPrep (Hologic). Samples yielding discordant results were genotyped using Linear Array assay (LA; Roche). Of 396 cases tested, AHPV detected 377 (sensitivity, 95.2%; 95%CI: 93.1-97.3), and HC2 376 (sensitivity, 94.9%; 95%CI: 92.7-97.1) with an agreement of 97.2% (kappa 0.7; 95%CI: 0.54-0.87). Among six AHPV+/HC2- cases, LA identified oncogenic HPV types in four including a type 73 and was negative in two. Among five AHPV-/HC2+ cases, LA detected oncogenic HPV types in two including a type 73 and was negative in three. Of 14 AHPV-/HC2- cases, 13 were genotyped. LA detected oncogenic HPV types in six, non-oncogenic types in three, and was negative in four. This is the largest study to demonstrate the sensitivity of AHPV for the detection of invasive cervical cancer and this assay showed equal sensitivity to HC2. J. Med. Virol. 88:1271-1278, 2016. © 2015 Wiley Periodicals, Inc. PMID:26693677

  10. A comparison of human papillomavirus genotype-specific DNA and E6/E7 mRNA detection to identify anal precancer among HIV-infected men who have sex with men

    PubMed Central

    Castle, Philip E.; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren M.; Lorey, Thomas S.; LaMere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Rodriguez, Ana Cecilia; Wentzensen, Nicolas

    2012-01-01

    Background Human papillomavirus (HPV) RNA detection is reportedly more specific for the detection of anogenital precancer than HPV DNA but it is unknown whether this is due to detection of RNA or due to HPV genotype restriction. Materials and Methods 363 human immunodeficiency virus (HIV)-positive men who have sex with men had two anal cytology samples taken and were evaluated using high-resolution anoscopy and biopsies of visible lesions. Anal specimens were tested for E6/E7 RNA for 5 carcinogenic HPV genotypes (HPV16, 18, 31, 33, and 45) and tested for the DNA of 13 carcinogenic HPV genotypes. Results DNA testing was more likely to be positive than RNA testing (53% vs. 48%, p = 0.02) for the same 5 HPV genotypes in aggregate. When restricted to 5 HPV genotypes targeted by the RNA test, the sensitivity to detect anal precancer was the same for DNA and RNA (81%) while RNA was more specific than DNA (65% vs. 58%, p = 0.007). By comparison, DNA detection of all 13 carcinogenic HPV genotypes was more sensitive (96% vs. 81%, p = 0.001) but much less specific (65% vs. 33%, p < 0.001) compared to RNA detection of the 5 HPV genotypes. Conclusion After controlling for HPV genotypes, RNA was only slightly more specific than DNA detection for anal precancer. Impact DNA or RNA testing for a subset of the most carcinogenic HPV genotypes may be useful for distinguishing between those HPV-positive men at higher and lower risk of anal precancer and cancer. PMID:23155136

  11. 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

  12. Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation

    PubMed Central

    Mo, Hao; Henriksson, Marie

    2006-01-01

    The Myc transcription factor plays a central role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Myc is a potent oncoprotein that is deregulated in a wide variety of human tumors and is therefore an attractive target for novel cancer therapies. Using a cellular screening approach, we have identified low-molecular-weight compounds, Myc pathway response agents (MYRAs), that induce apoptosis in a c-Myc-dependent manner and inhibit Myc-driven cellular transformation. MYRA-A inhibits Myc transactivation and interferes with the DNA-binding activity of Myc family proteins but has no effect on the E-box-binding protein USF. In contrast, MYRA-B induces Myc-dependent apoptosis without affecting Myc transactivation or Myc/Max DNA binding. Our data show that cellular screening assays can be a powerful strategy for the identification of candidate substances that modulate the Myc pathway. These compounds can be useful tools for studying Myc function and may also be of therapeutic potential as leads for drug development. PMID:16606833

  13. STAT5 is crucial to maintain leukemic stem cells in acute myelogenous leukemias induced by MOZ-TIF2.

    PubMed

    Tam, Winnie F; Hähnel, Patricia S; Schüler, 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

  14. LKB1 tumor suppressor and salt-inducible kinases negatively regulate human T-cell leukemia virus type 1 transcription

    PubMed Central

    2013-01-01

    Background Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Treatment options are limited and prophylactic agents are not available. We have previously demonstrated an essential role for CREB-regulating transcriptional coactivators (CRTCs) in HTLV-1 transcription. Results In this study we report on the negative regulatory role of LKB1 tumor suppressor and salt-inducible kinases (SIKs) in the activation of HTLV-1 long terminal repeats (LTR) by the oncoprotein Tax. Activation of LKB1 and SIKs effectively blunted Tax activity in a phosphorylation-dependent manner, whereas compromising these kinases, but not AMP-dependent protein kinases, augmented Tax function. Activated LKB1 and SIKs associated with Tax and suppressed Tax-induced LTR activation by counteracting CRTCs and CREB. Enforced expression of LKB1 or SIK1 in cells transfected with HTLV-1 molecular clone pX1MT repressed proviral transcription. On the contrary, depletion of LKB1 in pX1MT-transfected cells and in HTLV-1-transformed T cells boosted the expression of Tax. Treatment of HTLV-1 transformed cells with metformin led to LKB1/SIK1 activation, reduction in Tax expression, and inhibition of cell proliferation. Conclusions Our findings revealed a new function of LKB1 and SIKs as negative regulators of HTLV-1 transcription. Pharmaceutical activation of LKB1 and SIKs might be considered as a new strategy in anti-HTLV-1 and anti-ATL therapy. PMID:23577667

  15. Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells

    PubMed Central

    Man, Jiang-Hong; Liang, Bing; Gu, Yue-Xi; Zhou, Tao; Li, Ai-Ling; Li, Tao; Jin, Bao-Feng; Bai, Bing; Zhang, Hai-Ying; Zhang, Wei-Na; Li, Wei-Hua; Gong, Wei-Li; Li, Hui-Yan; Zhang, Xue-Min

    2010-01-01

    Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil–containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras–induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis. PMID:20628200

  16. p-Phenylenediamine induced DNA damage in SV-40 immortalized human uroepithelial cells and expression of mutant p53 and COX-2 proteins.

    PubMed

    Huang, Ya-Chun; Hung, Wen-Chun; Kang, Wan-Yi; Chen, Wan-Tzu; Chai, Chee-Yin

    2007-04-25

    p-Phenylenediamine (p-PD) is the main aromatic amine used in the formulation of hair dyes. Some epidemiologic studies have suggested that the use of p-PD-based hair dyes might be related to increased risk of human malignant tumors including bladder cancer and hematopoietic cancers. However, the toxicity and genotoxicity of p-PD on urothelial cells has not been reported yet. Therefore, we investigated the genotoxicity of p-PD on human urothelial cells and study its association with the expression of oncoproteins p53 and cyclooxygenase-2 (COX-2). Our results revealed that p-PD was able to induce DNA damage determined by Comet assay. In addition, our immunocytochemical and Western blotting results showed that p-PD induced overexpression of mutant p53 and COX-2 in a dose-dependent manner. The relationship between mutant p53 and COX-2 expression shows strong correlation. Furthermore, the accumulation of mutant p53 was linearly correlated with Comet scores. These results suggest that p-PD can induce DNA damage and accumulation of mutant p53 and COX-2 proteins; this may be one of the possible mechanisms that cause genotoxic carcinogenesis in the urothelial cells. PMID:17403587

  17. MUC1 induced by Epstein-Barr virus latent membrane protein 1 causes dissociation of the cell-matrix interaction and cellular invasiveness via STAT signaling.

    PubMed

    Kondo, Satoru; Yoshizaki, Tomokazu; Wakisaka, Naohiro; Horikawa, Toshiyuki; Murono, Shigeyuki; Jang, Kyung Lib; Joab, Irene; Furukawa, Mitsuru; Pagano, Joseph S

    2007-02-01

    Disruption of cellular adhesion is an essential pathobiologic step leading to tumor dissemination. Mucin 1 (MUC1) is a mucinous glycoprotein expressed at the surfaces of epithelial cells in many tissues and their carcinomas. MUC1 plays crucial roles in tumor invasion and metastasis, especially in opposing cell adhesion. We have shown that virus infection, specifically by the human tumor virus Epstein-Barr virus (EBV) induces a spectrum of cellular invasiveness and metastasis factors. Here we show that expression of MUC1 is increased in diverse latently EBV-infected cell lines that express latent membrane protein 1 (LMP1), the main viral oncoprotein, and that the level of MUC1 was suppressed by expression of a dominant-negative mutant of LMP1. Expression of LMP1 in EBV-negative nasopharyngeal cell lines induces expression of MUC1 through activation of the MUC1 promoter via binding of STAT1 and STAT3. Finally, LMP1 reduces cell adhesion ability, which is restored by inhibition of MUC1 expression with MUC1 small interfering RNA (siRNA). In addition, LMP1 increases cell invasiveness, which is suppressed by MUC1 siRNA. Thus, LMP1 induces MUC1, a factor important in an early step of detachment and release of tumor cells, which along with induction of other invasiveness and angiogenic factors may combine to act in a complex sequential process that culminates in metastasis of EBV-infected tumor cells. PMID:17151127

  18. 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.

  19. B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

    PubMed

    Herr, Ricarda; Köhler, Martin; Andrlová, Hana; Weinberg, Florian; Möller, 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

  20. Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells.

    PubMed

    Wesierska-Gadek, Józefa; Wandl, Stefanie; Kramer, Matthias P; Pickem, Christian; Krystof, Vladimir; Hajek, Susanne B

    2008-12-01

    Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer. PMID:18846503

  1. Silencing of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human lung cancer cells

    PubMed Central

    HU, XUANYU; GUO, WEI; CHEN, SHANSHAN; XU, YIZHUO; LI, PING; WANG, HUAQI; CHU, HEYING; LI, JUAN; DU, YUWEN; CHEN, XIAONAN; ZHANG, GUOJUN; ZHAO, GUOQIANG

    2016-01-01

    Activating enhancer-binding protein (AP)-4 is a member of the basic helix-loop-helix transcription factors, and is involved in tumor biology. However, the role of AP-4 in human lung cancer remains to be fully elucidated. In the present study, the expression of AP-4 in human lung cancer tissues and cells was investigated by reverse transcription-quantitative polymerase chain reaction, and it was observed that the level of AP-4 was increased in tumor tissues and cells compared with their normal counterparts. AP-4 expression was knocked down by transfection with a specific small interfering RNA (siRNA) in lung cancer cells, and this indicated that siRNA-mediated silencing of AP-4 inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase and induced apoptosis by modulating the expression of p21 and cyclin D1. The results of the present study suggest that AP-4 may be an oncoprotein that has a significant role in lung cancer, and that siRNA-mediated silencing of AP-4 may have therapeutic potential as a strategy for the treatment of lung cancer. PMID:27313685

  2. Roles of the PI3K/Akt pathway in Epstein-Barr virus-induced cancers and therapeutic implications.

    PubMed

    Chen, Jiezhong

    2012-12-12

    Viruses have been shown to be responsible for 10%-15% of cancer cases. Epstein-Barr virus (EBV) is the first virus to be associated with human malignancies. EBV can cause many cancers, including Burkett's lymphoma, Hodgkin's lymphoma, post-transplant lymphoproliferative disorders, nasopharyngeal carcinoma and gastric cancer. Evidence shows that phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) plays a key role in EBV-induced malignancies. The main EBV oncoproteins latent membrane proteins (LMP) 1 and LMP2A can activate the PI3K/Akt pathway, which, in turn, affects cell survival, apoptosis, proliferation and genomic instability via its downstream target proteins to cause cancer. It has also been demonstrated that the activation of the PI3K/Akt pathway can result in drug resistance to chemotherapy. Thus, the inhibition of this pathway can increase the therapeutic efficacy of EBV-associated cancers. For example, PI3K inhibitor Ly294002 has been shown to increase the effect of 5-fluorouracil in an EBV-associated gastric cancer cell line. At present, dual inhibitors of PI3K and its downstream target mammalian target of rapamycin have been used in clinical trials and may be included in treatment regimens for EBV-associated cancers. PMID:24175221

  3. Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

    SciTech Connect

    Chen, W.; Li, F.; Mead, L.; White, H.; Walker, J.; Ingram, D.A.; Roman, A.

    2007-10-10

    One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

  4. c-Abl and Arg induce cathepsin-mediated lysosomal degradation of the NM23-H1 metastasis suppressor in invasive cancer

    PubMed Central

    Sledziona, James; Cibull, Michael L.; Wang, Chi; Richards, Dana L.; Neltner, Janna M.; Beach, Carol; McCorkle, Joseph R.; Kaetzel, David M.; Plattner, Rina

    2014-01-01

    Metastasis suppressors comprise a growing class of genes whose downregulation triggers metastatic progression. In contrast to tumor suppressors, metastasis suppressors are rarely mutated or deleted, and little is known regarding the mechanisms by which their expression is downregulated. Here, we demonstrate that the metastasis suppressor, NM23-H1, is degraded by lysosomal cysteine cathepsins (L,B), which directly cleave NM23-H1. In addition, activation of c-Abl and Arg oncoproteins induces NM23-H1 degradation in invasive cancer cells by increasing cysteine cathepsin transcription and activation. Moreover, c-Abl activates cathepsins by promoting endosome maturation, which facilitates trafficking of NM23-H1 to the lysosome where it is degraded. Importantly, the invasion- and metastasis-promoting activity of c-Abl/Arg is dependent on their ability to induce NM23-H1 degradation, and the pathway is clinically relevant as c-Abl/Arg activity and NM23-H1 expression are inversely correlated in primary breast cancers and melanomas. Thus, we demonstrate a novel mechanism by which cathepsin expression is upregulated in cancer cells (via Abl kinases). We also identify a novel role for intracellular cathepsins in invasion and metastasis (degradation of a metastasis suppressor). Finally, we identify novel crosstalk between oncogenic and metastasis suppressor pathways, thereby providing mechanistic insight into the process of NM23-H1 loss, which may pave the way for new strategies to restore NM23-H1 expression and block metastatic progression. PMID:24096484

  5. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.

    PubMed Central

    Rao, L; Debbas, M; Sabbatini, P; Hockenbery, D; Korsmeyer, S; White, E

    1992-01-01

    Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis. Images PMID:1457005

  6. Characterization of p21Ras-mediated apoptosis induced by protein kinase C inhibition and application to human tumor cell lines.

    PubMed

    Liou, James S; Chen, James S; Faller, Douglas V

    2004-02-01

    Suppression of PKC activity can selectively induce apoptosis in cells expressing a constitutively activated p21Ras protein. We demonstrate that continued expression of p21Ras activity is required in PKC-mediated apoptosis because farnesyltransferase inhibitors abrogated the loss of viability in p21Ras-transformed cells occurring following PKC inhibition. Studies utilizing gene transfer or viral vectors demonstrate that transient expression of oncogenic p21Ras activity is sufficient for induction of apoptosis by PKC inhibition, whereas physiologic activation of p21Ras by growth factor is not sufficient to induce apoptosis. Mechanistically, the p21Ras-mediated apoptosis induced by PKC inhibition is dependent upon mitochondrial dysregulation, with a concurrent loss of mitochondrial membrane potential (psim). Cyclosporine A, which prevented the loss of psim, also inhibited HMG-induced DNA fragmentation in cells expressing an activated p21Ras. Induction of apoptosis by PKC inhibition in human tumors with oncogenic p21Ras mutations was demonstrated. Inhibition of PKC caused increased apoptosis in MIA-PaCa-2, a human pancreatic tumor line containing a mutated Ki-ras allele, when compared to HS766T, a human pancreatic tumor line with normal Ki-ras alleles. Furthermore, PKC inhibition induced apoptosis in HCT116, a human colorectal tumor line containing an oncogenic Ki-ras allele but not in a subline (Hke3) in which the mutated Ki-ras allele had been disrupted. The PKC inhibitor 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG), significantly reduced p21Ras-mediated tumor growth in vivo in a nude mouse MIA-PaCa-2 xenograft model. Collectively these studies suggest the therapeutic feasibility of targeting PKC activity in tumors expressing an activated p21Ras oncoprotein. PMID:14603530

  7. The roles and clinical significance of microRNAs in cervical cancer.

    PubMed

    Wang, Fenfen; Li, Baohua; Xie, Xing

    2016-02-01

    Cervical carcinogenesis induced by persistent human papillomavirus (HPV) infection represents a stepwise progression from precursors to invasive cervical cancer. Accumulated evidence has shown aberrant expression of microRNAs (miRNAs) in cervical cancer tissues and cells. Further studies reveal that miRNAs play key roles in the initiation and progression of cervical cancer, via specific signaling pathways, including E6-p53, E7-pRb, phosphoinositide-3 kinase (PI3K)-Akt, Notch, Wnt/β-catenin, and Hedgehog pathways. Some studies demonstrate that miRNAs might serve as biomarkers or therapeutic targets, presenting a potential prospect in clinical practice. All results provide new insights into the function of miRNAs and the pathogenesis of cervical cancer induced by viral oncoproteins. New approaches for miRNA-based prevention and management for cervical cancer will be developed in the future. PMID:26356641

  8. Variation in the Nucleotide Sequence of Cottontail Rabbit Papillomavirus a and b Subtypes Affects Wart Regression and Malignant Transformation and Level of Viral Replication in Domestic Rabbits

    PubMed Central

    Salmon, Jérôme; Nonnenmacher, Mathieu; Cazé, Sandrine; Flamant, Patricia; Croissant, Odile; Orth, Gérard; Breitburd, Françoise

    2000-01-01

    We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides. The most variable region (7% nucleotide divergence) included the long regulatory region (LRR) and the E6 and E7 genes. A mutation in the stop codon resulted in an 8-amino-acid-longer CRPVb E4 protein, and a nucleotide deletion reduced the coding capacity of the E5 gene from 101 to 25 amino acids. In domestic rabbits homozygous for a specific haplotype of the DRA and DQA genes of the major histocompatibility complex, warts induced by CRPVb DNA or a chimeric genome containing the CRPVb LRR/E6/E7 region showed an early regression, whereas warts induced by CRPVa4 or a chimeric genome containing the CRPVa4 LRR/E6/E7 region persisted and evolved into carcinomas. In contrast, most CRPVa, CRPVb, and chimeric CRPV DNA-induced warts showed no early regression in rabbits homozygous for another DRA-DQA haplotype. Little, if any, viral replication is usually observed in domestic rabbit warts. When warts induced by CRPVa and CRPVb virions and DNA were compared, the number of cells positive for viral DNA or capsid antigens was found to be greater by 1 order of magnitude for specimens induced by CRPVb. Thus, both sequence variation in the LRR/E6/E7 region and the genetic constitution of the host influence the expression of the oncogenic potential of CRPV. Furthermore, intratype variation may overcome to some extent the host restriction of CRPV replication in domestic rabbits. PMID:11044121

  9. Inducing labor

    MedlinePlus

    ... surrounds your baby in the womb. It contains membranes or layers of tissue. One method of inducing ... break the bag of waters" or rupture the membranes. Your health care provider will do a pelvic ...

  10. The leukemic oncoprotein NPM1-RARA inhibits TP53 activity.

    PubMed

    Swaney, Erin M; Chattopadhyay, Anuja; Abecassis, Irina; Rush, Elizabeth A; Redner, Robert L

    2016-08-01

    The variant acute promyelocytic leukemia (APL) translocation t(5;17)(q35;q21) fuses the N-terminus of nucleophosmin (NPM1) to the retinoic acid receptor alpha (RARA). We found that ectopic NPM1-RARA expression decreased TP53 protein levels in target cells. NPM1-RARA impaired TP53-dependent transcription. Cells expressing NPM1-RARA were more resistant to apoptotic stimuli. This work identifies the TP53 tumor suppressor as a novel target through which NPM1-RARA impacts leukemogenesis, and confirms the importance of impairment of TP53 in establishment of the APL phenotype. PMID:26754533

  11. The DEK oncoprotein and its emerging roles in gene regulation.

    PubMed

    Sandén, C; Gullberg, U

    2015-08-01

    The DEK oncogene is highly expressed in cells from most human tissues and overexpressed in a large and growing number of cancers. It also fuses with the NUP214 gene to form the DEK-NUP214 fusion gene in a subset of acute myeloid leukemia. Originally characterized as a member of this translocation, DEK has since been implicated in epigenetic and transcriptional regulation, but its role in these processes is still elusive and intriguingly complex. Similarly multifaceted is its contribution to cellular transformation, affecting multiple cellular processes such as self-renewal, proliferation, differentiation, senescence and apoptosis. Recently, the roles of the DEK and DEK-NUP214 proteins have been elucidated by global analysis of DNA binding and gene expression, as well as multiple functional studies. This review outlines recent advances in the understanding of the basic functions of the DEK protein and its role in leukemogenesis. PMID:25765544

  12. RNA splicing factors as oncoproteins and tumour suppressors.

    PubMed

    Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K

    2016-07-01

    The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these 'spliceosomal mutations' suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic and biological effects of spliceosomal mutations are crucial for the development of cancer therapies targeted at these mutations. PMID:27282250

  13. Fangchinoline induces G0/G1 arrest by modulating the expression of CDKN1A and CCND2 in K562 human chronic myelogenous leukemia cells

    PubMed Central

    WANG, YUPING; CHEN, JIE; WANG, LIN; HUANG, YUJI; LENG, YE; WANG, GUIYING

    2013-01-01

    Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by the oncoprotein BCR-ABL, which exhibits a constitutive tyrosine kinase activity. Imatinib mesylate (IM), an inhibitor of the tyrosine kinase activity of BCR-ABL, has been used as a first-line therapy for CML. However, IM is less effective in the accelerated phase and blastic phases of CML and certain patients develop IM resistance due to the mutation and amplification of the BCR-ABL gene. Fangchinoline, an important chemical constituent from the dried roots of Stephaniae tetrandrae S. Moore, exhibits significant antitumor activity in various types of cancers, including breast, prostate and hepatocellular carcinoma. However, the effects and the underlying mechanisms of fangchinoline in CML remain unclear. In the present study, we identified that fangchinoline inhibits cell proliferation in a dose- and time-dependent manner in K562 cells derived from the blast crisis of CML. Additional experiments revealed that fangchinoline induces cell cycle arrest at the G0/G1 phase and has no effect on apoptosis, which is mediated through the upregulation of cyclin-dependent kinase (CDK)-N1A and MCL-1 mRNA levels, as well as the downregulation of cyclin D2 (CCND2) mRNA levels. These findings suggest the potential of fangchinoline as an effective antitumor agent in CML. PMID:23596478

  14. Fangchinoline induces G0/G1 arrest by modulating the expression of CDKN1A and CCND2 in K562 human chronic myelogenous leukemia cells.

    PubMed

    Wang, Yuping; Chen, Jie; Wang, Lin; Huang, Yuji; Leng, Ye; Wang, Guiying

    2013-04-01

    Chronic myeloid leukemia (CML) is a hematopoietic stem cell disease caused by the oncoprotein BCR-ABL, which exhibits a constitutive tyrosine kinase activity. Imatinib mesylate (IM), an inhibitor of the tyrosine kinase activity of BCR-ABL, has been used as a first-line therapy for CML. However, IM is less effective in the accelerated phase and blastic phases of CML and certain patients develop IM resistance due to the mutation and amplification of the BCR-ABL gene. Fangchinoline, an important chemical constituent from the dried roots of Stephaniae tetrandrae S. Moore, exhibits significant antitumor activity in various types of cancers, including breast, prostate and hepatocellular carcinoma. However, the effects and the underlying mechanisms of fangchinoline in CML remain unclear. In the present study, we identified that fangchinoline inhibits cell proliferation in a dose- and time-dependent manner in K562 cells derived from the blast crisis of CML. Additional experiments revealed that fangchinoline induces cell cycle arrest at the G0/G1 phase and has no effect on apoptosis, which is mediated through the upregulation of cyclin-dependent kinase (CDK)-N1A and MCL-1 mRNA levels, as well as the downregulation of cyclin D2 (CCND2) mRNA levels. These findings suggest the potential of fangchinoline as an effective antitumor agent in CML. PMID:23596478

  15. Use of Human Papillomavirus DNA, E6/E7 mRNA, and p16 Immunocytochemistry to Detect and Predict anal High-Grade Squamous Intraepithelial Lesions in HIV-Positive and HIV-Negative Men Who Have Sex with Men

    PubMed Central

    Phanuphak, Nittaya; Teeratakulpisarn, Nipat; Keelawat, Somboon; Pankam, Tippawan; Barisri, Jiranuwat; Triratanachat, Surang; Deesua, Amornrat; Rodbamrung, Piyanee; Wongsabut, Jiratchaya; Tantbirojn, Patou; Numto, Saranya; Ruangvejvorachai, Preecha; Phanuphak, Praphan; Palefsky, Joel M.; Ananworanich, Jintanat; Kerr, Stephen J.

    2013-01-01

    Background Men who have sex with men (MSM) are at high risk of having anal cancer. Anal high-grade squamous intraepithelial lesion (HSIL) is the precursor of anal cancer. We explored the use of different biomarkers associated with human papillomavirus (HPV) infection and HPV-mediated cell transformation to detect and predict HSIL among HIV-positive and HIV-negative MSM. Methodology/Principal Findings A total of 123 HIV-positive and 123 HIV-negative MSM were enrolled and followed for 12 months. High-resolution anoscopy (HRA) with biopsies were performed at every visit along with anal sample collection for cytology, high-risk HPV DNA genotyping, HPV E6/E7 mRNA, and p16 immunocytochemistry. Performance characteristics and area under the receiver operator characteristics curve were calculated for these biomarkers at baseline, and Cox regression compared the usefulness of these biomarkers in predicting incident HSIL. High-risk HPV DNA, E6/E7 mRNA, and p16 immunocytochemistry each identified 43–46% of MSM whose baseline test positivity would trigger HRA referral. E6/E7 mRNA had the highest sensitivity (64.7%) and correctly classified the highest number of prevalent HSIL cases. With the exception of p16 immunochemistry, most tests showed significant increases in sensitivity but decreases specificity versus anal cytology, while the overall number of correctly classified cases was not significantly different. Baseline or persistent type 16 and/or 18 HPV DNA was the only test significantly predicting incident histologic HSIL within 12 months in models adjusted for HIV status and low-grade squamous intraepithelial lesions at baseline. Conclusions/Significance Countries with a high HIV prevalence among MSM and limited HRA resources may consider using biomarkers to identify individuals at high risk of HSIL. E6/E7 mRNA had the highest sensitivity for prevalent HSIL detection regardless of HIV status, whereas type 16 and/or 18 HPV DNA performed best in predicting development of

  16. Chronic cadmium exposure in vitro induces cancer cell characteristics in human lung cells

    PubMed Central

    Person, Rachel J.; Tokar, Erik J.; Xu, Yuanyuan; Orihuela, Ruben; Olive Ngalame, Ntube N.; Waalkes, Michael P.

    2013-01-01

    Cadmium is a known human lung carcinogen. Here, we attempt to develop an in vitro model of cadmium-induced human lung carcinogenesis by chronically exposing the peripheral lung epithelia cell line, HPL-1D, to a low level of cadmium. Cells were chronically exposed to 5 μM cadmium, a noncytotoxic level, and monitored for acquired cancer characteristics. By 20 weeks of continuous cadmium exposure, these chronic cadmium treated lung (CCT-LC) cells showed marked increases in secreted MMP-2 activity (3.5-fold), invasion (3.4-fold), and colony formation in soft agar (2-fold). CCT-LC cells were hyperproliferative, grew well in serum-free media, and overexpressed cyclin D1. The CCT-LC cells also showed decreased expression of the tumor suppressor genes p16 and SLC38A3 at the protein levels. Also consistent with an acquired cancer cell phenotype, CCT-LC cells showed increased expression of the oncoproteins K-RAS and N-RAS as well as the epithelial-to-mesenchymal transition marker protein Vimentin. Metallothionein (MT) expression is increased by cadmium, and is typically overexpressed in human lung cancers. The major MT isoforms, MT-1A and MT-2A were elevated in CCT-LC cells. Oxidant adaptive response genes HO-1 and HIF-1A were also activated in CCT-LC cells. Expression of the metal transport genes ZNT-1, ZNT-5, and ZIP-8 increased in CCT-LC cells culminating in reduced cadmium accumulation, suggesting adaptation to the metal. Overall, these data suggest that exposure of human lung epithelial cells to cadmium causes acquisition of cancer cell characteristics. Furthermore, transformation occurs despite the cell’s ability to adapt to chronic cadmium exposure. PMID:23811327

  17. Cucurbitacin IIa: a novel class of anti-cancer drug inducing non-reversible actin aggregation and inhibiting survivin independent of JAK2/STAT3 phosphorylation

    PubMed Central

    Boykin, C; Zhang, G; Chen, Y-H; Zhang, R-W; Fan, X-E; Yang, W-M; Lu, Q

    2011-01-01

    Background: Cucurbitacin (Cuc) and triterpene-derived natural products exhibit anti-cancer potential in addition to their conspicuous anti-bacterial and anti-inflammatory activity. Recently, inhibition of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling was shown to underlie the effects of Cuc family on inducing cell death in cancer. Method: We purified Cuc IIa, the active component from the medicinal plant Hemsleya amalils Diels, which shows different structural modifications from other Cuc derivatives. We investigated the mechanisms of its inhibitory effects on cancer cells in vitro and tumour growth in vivo. Results: Cuc IIa induced the irreversible clustering of filamentous actin and arrested cell cycle by the increases in G2/M populations. Cuc IIa resulted in the reduced phospho-Histone H3 and markedly increased cleavage of poly-(ADP-ribose) polymerase or PARP, immediate upstream of DNA breakdown as the result of caspase activation, consistent with mitotic blockage-induced cell death. However, unlike other Cuc members, Cuc IIa did not suppress JAK2/STAT3 phosphorylation or alter phosphorylation of mitogen-activated protein kinases. Instead, the expression of the cell cycle-regulated Inhibitor of Apoptosis Protein (IAP) survivin was reduced. Introducing oncoprotein δ-catenin, which increased survivin expression and suppressed small GTPase RhoA, reduced efficacy of Cuc IIa to induce cell death. Supporting the effects of Cuc IIa on actin cytoskeletal signaling, RhoA phosphorylation was reduced suggesting its increased activity. Conclusion: Cuc IIa is a novel class of anti-cancer drug in suppression of cancer cell expansion by disrupting the actin cytoskeleton and directing the cell to undergo PARP-mediated apoptosis through the inhibition of survivin downstream of JAK2/STAT3. PMID:21304528

  18. Transcription factor PREP1 induces EMT and metastasis by controlling the TGF-β-SMAD3 pathway in non-small cell lung adenocarcinoma.

    PubMed

    Risolino, Maurizio; Mandia, Nadia; Iavarone, Francescopaolo; Dardaei, Leila; Longobardi, Elena; Fernandez, Serena; Talotta, Francesco; Bianchi, Fabrizio; Pisati, Federica; Spaggiari, Lorenzo; Harter, Patrick N; Mittelbronn, Michel; Schulte, Dorothea; Incoronato, Mariarosaria; Di Fiore, Pier Paolo; Blasi, Francesco; Verde, Pasquale

    2014-09-01

    Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial-mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-β. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-β. PREP1 modulates the cellular sensitivity to TGF-β by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-β pathway, EMT, and metastasis in NSCLC. PMID:25157139

  19. Transcription factor PREP1 induces EMT and metastasis by controlling the TGF-β–SMAD3 pathway in non-small cell lung adenocarcinoma

    PubMed Central

    Risolino, Maurizio; Mandia, Nadia; Iavarone, Francescopaolo; Dardaei, Leila; Longobardi, Elena; Fernandez, Serena; Talotta, Francesco; Bianchi, Fabrizio; Pisati, Federica; Spaggiari, Lorenzo; Harter, Patrick N.; Mittelbronn, Michel; Schulte, Dorothea; Incoronato, Mariarosaria; Di Fiore, Pier Paolo; Blasi, Francesco; Verde, Pasquale

    2014-01-01

    Pre–B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial–mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-β. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-β. PREP1 modulates the cellular sensitivity to TGF-β by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-β pathway, EMT, and metastasis in NSCLC. PMID:25157139

  20. Rapamycin Impairs Antitumor CD8+ T-cell Responses and Vaccine-Induced Tumor Eradication.

    PubMed

    Chaoul, Nada; Fayolle, Catherine; Desrues, Belinda; Oberkampf, Marine; Tang, Alexandre; Ladant, Daniel; Leclerc, Claude

    2015-08-15

    The metabolic sensor mTOR broadly regulates cell growth and division in cancer cells, leading to a significant focus on studies of rapamycin and its analogues as candidate anticancer drugs. However, mTOR inhibitors have failed to produce useful clinical efficacy, potentially because mTOR is also critical in T cells implicated in immunosurveillance. Indeed, recent studies using rapamycin have demonstrated the important role of mTOR in differentiation and induction of the CD8+ memory in T-cell responses associated with antitumor properties. In this study, we demonstrate that rapamycin harms antitumor immune responses mediated by T cells in the setting of cancer vaccine therapy. Specifically, we analyzed how rapamycin affects the antitumor efficacy of a human papilloma virus E7 peptide vaccine (CyaA-E7) capable of eradicating tumors in the TC-1 mouse model of cervical cancer. In animals vaccinated with CyaA-E7, rapamycin administration completely abolished recruitment of CD8+ T cells into TC-1 tumors along with the ability of the vaccine to reduce infiltration of T regulatory cells and myeloid-derived suppressor cells. Moreover, rapamycin completely abolished vaccine-induced cytotoxic T-cell responses and therapeutic activity. Taken together, our results demonstrate the powerful effects of mTOR inhibition in abolishing T-cell-mediated antitumor immune responses essential for the therapeutic efficacy of cancer vaccines. PMID:26122844

  1. Tumor suppressor p16INK4A is necessary for survival of cervical carcinoma cell lines

    PubMed Central

    McLaughlin-Drubin, Margaret E.; Park, Donglim; Munger, Karl

    2013-01-01

    The tumor suppressor p16INK4A inhibits formation of enzymatically active complexes of cyclin-dependent kinases 4 and 6 (CDK4/6) with D-type cyclins. Oncogenic stress induces p16INK4A expression, which in turn triggers cellular senescence through activation of the retinoblastoma tumor suppressor. Subversion of oncogene-induced senescence is a key step during cancer development, and many tumors have lost p16INK4A activity by mutation or epigenetic silencing. Human papillomavirus (HPV)-associated tumors express high levels of p16INK4A in response to E7 oncoprotein expression. Induction of p16INK4A expression is not a consequence of retinoblastoma tumor suppressor inactivation but is triggered by a cellular senescence response and is mediated by epigenetic derepression through the H3K27-specific demethylase (KDM)6B. HPV E7 expression causes an acute dependence on KDM6B expression for cell survival. The p16INK4A tumor suppressor is a critical KDM6B downstream transcriptional target and its expression is critical for cell survival. This oncogenic p16INK4A activity depends on inhibition of CDK4/CDK6, suggesting that in cervical cancer cells where retinoblastoma tumor suppressor is inactivated, CDK4/CDK6 activity needs to be inhibited in order for cells to survive. Finally, we note that HPV E7 expression creates a unique cellular vulnerability to small-molecule KDM6A/B inhibitors. PMID:24046371

  2. Conformational Dissection of a Viral Intrinsically Disordered Domain Involved in Cellular Transformation

    PubMed Central

    Perrone, Sebastián; Salvay, Andres G.; Chemes, Lucía B.; de Prat-Gay, Gonzalo

    2013-01-01

    Intrinsic disorder is abundant in viral genomes and provides conformational plasticity to its protein products. In order to gain insight into its structure-function relationships, we carried out a comprehensive analysis of structural propensities within the intrinsically disordered N-terminal domain from the human papillomavirus type-16 E7 oncoprotein (E7N). Two E7N segments located within the conserved CR1 and CR2 regions present transient α-helix structure. The helix in the CR1 region spans residues L8 to L13 and overlaps with the E2F mimic linear motif. The second helix, located within the highly acidic CR2 region, presents a pH-dependent structural transition. At neutral pH the helix spans residues P17 to N29, which include the retinoblastoma tumor suppressor LxCxE binding motif (residues 21–29), while the acidic CKII-PEST region spanning residues E33 to I38 populates polyproline type II (PII) structure. At pH 5.0, the CR2 helix propagates up to residue I38 at the expense of loss of PII due to charge neutralization of acidic residues. Using truncated forms of HPV-16 E7, we confirmed that pH-induced changes in α-helix content are governed by the intrinsically disordered E7N domain. Interestingly, while at both pH the region encompassing the LxCxE motif adopts α-helical structure, the isolated 21–29 fragment including this stretch is unable to populate an α-helix even at high TFE concentrations. Thus, the E7N domain can populate dynamic but discrete structural ensembles by sampling α-helix-coil-PII-ß-sheet structures. This high plasticity may modulate the exposure of linear binding motifs responsible for its multi-target binding properties, leading to interference with key cell signaling pathways and eventually to cellular transformation by the virus. PMID:24086265

  3. NF-κB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral anti-sense protein HBZ.

    PubMed

    Zhi, Huijun; Yang, Liangpeng; Kuo, Yu-Liang; Ho, Yik-Khuan; Shih, Hsiu-Ming; Giam, Chou-Zen

    2011-04-01

    Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27(KIP1) protein and p21(CIP1/WAF1) mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis. PMID:21552325

  4. Fructus Gardenia Extract ameliorates oxonate-induced hyperuricemia with renal dysfunction in mice by regulating organic ion transporters and mOIT3.

    PubMed

    Hu, Qing-Hua; Zhu, Ji-Xiao; Ji, Jing; Wei, Lin-Lin; Miao, Ming-Xing; Ji, Hui

    2013-01-01

    The potent anti-hyperuricemia activities of Fructus Gardenia Extract (FGE) have been well reported. The aim of this study was to evaluate the uricosuric and nephro-protective effects of FGE and explore its possible mechanisms of action in oxonate-induced hyperuricemic mice. FGE was orally administered to hyperuricemic and normal mice for 1 week. Serum and urinary levels of uric acid, creatinine and blood urea nitrogen (BUN), and fractional excretion of uric acid (FEUA) were measured. The mRNA and protein levels of mouse urate transporter 1 (mURAT1), glucose transporter 9 (mGLUT9), ATP-binding cassette, subfamily G, 2 (mABCG2), organic anion transporter 1 (mOAT1), mOAT3, oncoprotein induced transcript 3 (mOIT3), organic cation/carnitine transporters in the kidney were analyzed. Simultaneously, Tamm-Horsfall glycoprotein (THP) levels in urine and kidney were detected. FGE significantly reduced serum urate levels and increased urinary urate levels and FEUA in hyperuricemic mice. It could also effectively reverse oxonate-induced alterations in renal mURAT1, mGLUT9, mOAT1 and mOIT3 expressions, as well as THP levels, resulting in the enhancement of renal uric acid excretion. Moreover, FGE decreased serum creatinine and BUN levels, and up-regulated expression of organic cation/carnitine transporters, improving renal dysfunction in this model. Furthermore, FGE decreased renal mABCG2 expressions in hyperuricemic mice, contributing to its beneficial actions. However, further investigation is needed in clinical trials of FGE and its bioactive components. PMID:23899832

  5. Transformation with Oncogenic Ras and the Simian Virus 40 T Antigens Induces Caspase-Dependent Sensitivity to Fatty Acid Biosynthetic Inhibition

    PubMed Central

    Xu, Shihao; Spencer, Cody M.

    2015-01-01

    ABSTRACT Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here, we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens and oncogenic Ras12V, affect fatty acid metabolism. Our results indicate that SV40/Ras12V-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (mTOR) dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras12V-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed breast epithelial cells through transduction with oncogenic Ras12V. Similar to what was observed in the transformed fibroblasts, the Ras12V-induced sensitivity to fatty acid biosynthetic inhibition was independent of the proliferative status and could be attenuated by supplementing the medium with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis. IMPORTANCE Viral oncoproteins and cellular mutations

  6. Exercise-Induced Bronchoconstriction

    MedlinePlus

    ... Conditions & Treatments ▸ Conditions Dictionary ▸ Exercise-Induced Bronchoconstriction Share | Exercise-Induced Bronchoconstriction (EIB) « Back to A to Z Listing Exercise-Induced Bronchoconstriction, (EIB), often known as exercise-induced ...

  7. Chronic cadmium exposure in vitro induces cancer cell characteristics in human lung cells

    SciTech Connect

    Person, Rachel J.; Tokar, Erik J.; Xu, Yuanyuan; Orihuela, Ruben; Ngalame, Ntube N. Olive; Waalkes, Michael P.

    2013-12-01

    Cadmium is a known human lung carcinogen. Here, we attempt to develop an in vitro model of cadmium-induced human lung carcinogenesis by chronically exposing the peripheral lung epithelia cell line, HPL-1D, to a low level of cadmium. Cells were chronically exposed to 5 μM cadmium, a noncytotoxic level, and monitored for acquired cancer characteristics. By 20 weeks of continuous cadmium exposure, these chronic cadmium treated lung (CCT-LC) cells showed marked increases in secreted MMP-2 activity (3.5-fold), invasion (3.4-fold), and colony formation in soft agar (2-fold). CCT-LC cells were hyperproliferative, grew well in serum-free media, and overexpressed cyclin D1. The CCT-LC cells also showed decreased expression of the tumor suppressor genes p16 and SLC38A3 at the protein levels. Also consistent with an acquired cancer cell phenotype, CCT-LC cells showed increased expression of the oncoproteins K-RAS and N-RAS as well as the epithelial-to-mesenchymal transition marker protein Vimentin. Metallothionein (MT) expression is increased by cadmium, and is typically overexpressed in human lung cancers. The major MT isoforms, MT-1A and MT-2A were elevated in CCT-LC cells. Oxidant adaptive response genes HO-1 and HIF-1A were also activated in CCT-LC cells. Expression of the metal transport genes ZNT-1, ZNT-5, and ZIP-8 increased in CCT-LC cells culminating in reduced cadmium accumulation, suggesting adaptation to the metal. Overall, these data suggest that exposure of human lung epithelial cells to cadmium causes acquisition of cancer cell characteristics. Furthermore, transformation occurs despite the cell's ability to adapt to chronic cadmium exposure. - Highlights: • Chronic cadmium exposure induces cancer cell characteristics in human lung cells. • This provides an in vitro model of cadmium-induced human lung cell transformation. • This occurred with general and lung specific changes typical for cancer cells. • These findings add insight to the relationship

  8. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6

    PubMed Central

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-01-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer. PMID:27364926

  9. Measurements of prompt radiation induced conductivity of alumina and sapphire.

    SciTech Connect

    Hartman, E. Frederick; Zarick, Thomas Andrew; Sheridan, Timothy J.; Preston, Eric F.

    2011-04-01

    We performed measurements of the prompt radiation induced conductivity in thin samples of Alumina and Sapphire at the Little Mountain Medusa LINAC facility in Ogden, UT. Five mil thick samples were irradiated with pulses of 20 MeV electrons, yielding dose rates of 1E7 to 1E9 rad/s. We applied variable potentials up to 1 kV across the samples and measured the prompt conduction current. Analysis rendered prompt conductivity coefficients between 1E10 and 1E9 mho/m/(rad/s), depending on the dose rate and the pulse width for Alumina and 1E7 to 6E7 mho/m/(rad/s) for Sapphire.

  10. Polycyclic Aromatic Hydrocarbons—Induced ROS Accumulation Enhances Mutagenic Potential of T-Antigen From Human Polyomavirus JC

    PubMed Central

    WILK, ANNA; RSKI, PIOTR WALIGÓ; LASSAK, ADAM; VASHISTHA, HIMANSHU; LIRETTE, DAVID; TATE, DAVID; ZEA, ARNOLD H.; KOOCHEKPOUR, SHAHRIAR; RODRIGUEZ, PAULO; MEGGS, LEONARD G.; ESTRADA, JOHN J.; OCHOA, AUGUSTO; REISS, KRZYSZTOF

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep-fried food, and in natural crude oil. Since PAH-metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T-antigen (JCV T-antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil-PAHs), and detected several carcinogenic PAHs. The oil-PAHs were tested in exponentially growing cultures of normal mouse fibroblasts (R508), and in R508 stably expressing JCV T-antigen (R508/T). The oil-PAHs were cytotoxic only at relatively high doses (1:50–1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non-toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil-PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T-antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low-fidelity repair by non-homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil-PAHs. Our results indicate for the first time carcinogenic synergy in which oil-PAHs trigger oxidative DNA damage and JCV T-antigen compromises DNA repair fidelity. PMID:23558788

  11. Human T-cell leukemia virus type 1 Tax releases cell cycle arrest induced by p16INK4a.

    PubMed Central

    Low, K G; Dorner, L F; Fernando, D B; Grossman, J; Jeang, K T; Comb, M J

    1997-01-01

    The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein causes cellular transformation by deregulating important cellular processes such as DNA repair, transcription, signal transduction, proliferation, and growth. Although it is clear that normal cell cycle control is deregulated during HTLV-1-induced cellular transformation, the effects of Tax on cell cycle control are not well understood. Flow cytometric analyses of human T cells indicate that cell cycle arrest in late G1, at or before the G1/S restriction point, by p16INK4a is relieved by Tax. Furthermore, Tax-dependent stimulation of 5-bromo-2'-deoxyuridine incorporation and transcriptional activation is inhibited by p16INK4a. This result suggests that p16INK4a is able to block Tax-dependent stimulation of DNA synthesis and cell cycle progression into S phase. In vitro binding assays with recombinant glutathione S-transferase fusion proteins and [35S]methionine-labeled proteins indicate that Tax binds specifically with p16INK4a but not with either p21cip1 or p27kip1. Furthermore, sequential immunoprecipitation assays with specific antisera and [35S]methionine-labeled cell lysates subsequent to coexpression with Tax and p16INK4a indicate that the two proteins form complexes in vivo. Immunocomplex kinase assays with cyclin-dependent kinase 4 antiserum indicate that Tax blocks the inhibition of cdk4 kinase activity by p16INK4a. This study identifies p16INK4a as a novel cellular target for Tax and suggests that the inactivation of p16INK4a function is a mechanism of cell cycle deregulation by Tax. PMID:9032327

  12. Expression profiling reveals transcriptional regulation by Fbxw7/mTOR pathway in radiation-induced mouse thymic lymphomas

    PubMed Central

    Snijders, Antoine M.; Liu, Yueyong; Su, Li; Huang, Yurong; Mao, Jian-Hua

    2015-01-01

    The tumor suppressor gene FBXW7 is deleted and mutated in many different types of human cancers. FBXW7 primarily exerts its tumor suppressor activity by ubiquitinating different oncoproteins including mTOR. Here we used gene transcript profiling to gain a deeper understanding of the role of FBXW7 in tumor development and to determine the influence of mTOR inhibition by rapamycin on tumor transcriptome and biological functions. In comparison to tumors from p53 single heterozygous (p53+/−) mice, we find that radiation-induced thymic lymphomas from Fbxw7/p53 double heterozygous (Fbxw7+/−p53+/−) mice show significant deregulation of cholesterol metabolic processes independent of rapamycin treatment, while cell cycle related genes were upregulated in tumors from placebo treated Fbxw7+/−p53+/− mice, but not in tumors from rapamycin treated Fbxw7+/−p53+/− mice. On the other hand, tumors from rapamycin treated Fbxw7+/−p53+/− mice were enriched for genes involved in the integrated stress response, an adaptive mechanism to survive in stressful environments. Finally, we demonstrated that the Fbxw7 gene signatures identified in mouse tumors significantly overlap with FBXW7 co-expressed genes in human cancers. Importantly these common FBXW7 gene signatures between mouse and human are predictive for disease-free survival in human colon, breast and lung adenocarcinoma cancer patients. These results provide novel insights into the role of FBXW7 in tumor development and have identified a number of potential targets for therapeutic intervention. PMID:26575021

  13. Integrin Engagement by the Helical RGD Motif of the Helicobacter pylori CagL Protein Is Regulated by pH-induced Displacement of a Neighboring Helix*

    PubMed Central

    Bonsor, Daniel A.; Pham, Kieu T.; Beadenkopf, Robert; Diederichs, Kay; Haas, Rainer; Beckett, Dorothy; Fischer, Wolfgang; Sundberg, Eric J.

    2015-01-01

    Arginine-aspartate-glycine (RGD) motifs are recognized by integrins to bridge cells to one another and the extracellular matrix. RGD motifs typically reside in exposed loop conformations. X-ray crystal structures of the Helicobacter pylori protein CagL revealed that RGD motifs can also exist in helical regions of proteins. Interactions between CagL and host gastric epithelial cell via integrins are required for the translocation of the bacterial oncoprotein CagA. Here, we have investigated the molecular basis of the CagL-host cell interactions using structural, biophysical, and functional analyses. We solved an x-ray crystal structure of CagL that revealed conformational changes induced by low pH not present in previous structures. Using analytical ultracentrifugation, we found that pH-induced conformational changes in CagL occur in solution and not just in the crystalline environment. By designing numerous CagL mutants based on all available crystal structures, we probed the functional roles of CagL conformational changes on cell surface integrin engagement. Together, our data indicate that the helical RGD motif in CagL is buried by a neighboring helix at low pH to inhibit CagL binding to integrin, whereas at neutral pH the neighboring helix is displaced to allow integrin access to the CagL RGD motif. This novel molecular mechanism of regulating integrin-RGD motif interactions by changes in the chemical environment provides new insight to H. pylori-mediated oncogenesis. PMID:25837254

  14. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

    PubMed

    Sheaffer, Amy K; Lee, Min S; Qi, Huilin; Chaniewski, Susan; Zheng, Xiaofan; Farr, Glen A; Esposito, Kim; Harden, David; Lei, Ming; Schweizer, Liang; Friborg, Jacques; Agler, Michele; McPhee, Fiona; Gentles, Robert; Beno, Brett R; Chupak, Lou; Mason, Stephen

    2016-01-01

    A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment. PMID:27280728

  15. A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses

    PubMed Central

    Lee, Min S.; Qi, Huilin; Chaniewski, Susan; Zheng, Xiaofan; Farr, Glen A.; Esposito, Kim; Harden, David; Lei, Ming; Schweizer, Liang; Friborg, Jacques; Agler, Michele; McPhee, Fiona; Gentles, Robert; Beno, Brett R.; Chupak, Lou; Mason, Stephen

    2016-01-01

    A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment. PMID:27280728

  16. Polymeric nanoparticles for co-delivery of synthetic long peptide antigen and poly IC as therapeutic cancer vaccine formulation.

    PubMed

    Rahimian, Sima; Fransen, Marieke F; Kleinovink, Jan Willem; Christensen, Jonatan Riis; Amidi, Maryam; Hennink, Wim E; Ossendorp, Ferry

    2015-04-10

    The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In preclinical and clinical studies adjuvants based on mineral oils (such as incomplete Freund's adjuvant (IFA) and Montanide) are used to create a sustained release depot at the injection site. While the depot effect of mineral oils is important for induction of robust immune responses, their administration is accompanied with severe adverse and long lasting side effects. In order to develop an alternative for IFA family of adjuvants, polymeric nanoparticles (NPs) based on hydrophilic polyester (poly(d,l lactic-co-hydroxymethyl glycolic acid) (pLHMGA)) were prepared. These NPs were loaded with a synthetic long peptide (SLP) derived from HPV16 E7 oncoprotein and a toll like receptor 3 (TLR3) ligand (poly IC) by double emulsion solvent evaporation technique. The therapeutic efficacy of the nanoparticulate formulations was compared to that of HPV SLP+poly IC formulated in IFA. Encapsulation of HPV SLP antigen in NPs substantially enhanced the population of HPV-specific CD8+ T cells when combined with poly IC either co-encapsulated with the antigen or in its soluble form. The therapeutic efficacy of NPs containing poly IC in tumor eradication was equivalent to that of the IFA formulation. Importantly, administration of pLHMGA nanoparticles was not associated with adverse effects and therefore these biodegradable nanoparticles are excellent substitutes for IFA in cancer vaccines. PMID:25660830

  17. Suppression of Antitumor Immune Responses by Human Papillomavirus through Epigenetic Downregulation of CXCL14

    PubMed Central

    Cicchini, Louis; Westrich, Joseph A.; Xu, Tao; Vermeer, Daniel W.; Berger, Jennifer N.; Clambey, Eric T.; Lee, Denis; Song, John I.; Lambert, Paul F.; Greer, Robert O.

    2016-01-01

    ABSTRACT High-risk human papillomaviruses (HPVs) are causally associated with multiple human cancers. Previous studies have shown that the HPV oncoprotein E7 induces immune suppression; however, the underlying mechanisms remain unknown. To understand the mechanisms by which HPV deregulates host immune responses in the tumor microenvironment, we analyzed gene expression changes of all known chemokines and their receptors using our global gene expression data sets from human HPV-positive and -negative head/neck cancer and cervical tissue specimens in different disease stages. We report that, while many proinflammatory chemokines increase expression throughout cancer progression, CXCL14 is dramatically downregulated in HPV-positive cancers. HPV suppression of CXCL14 is dependent on E7 and associated with DNA hypermethylation in the CXCL14 promoter. Using in vivo mouse models, we revealed that restoration of Cxcl14 expression in HPV-positive mouse oropharyngeal carcinoma cells clears tumors in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Further, Cxcl14 reexpression significantly increases natural killer (NK), CD4+ T, and CD8+ T cell infiltration into the tumor-draining lymph nodes in vivo. In vitro transwell migration assays show that Cxcl14 reexpression induces chemotaxis of NK, CD4+ T, and CD8+ T cells. These results suggest that CXCL14 downregulation by HPV plays an important role in suppression of antitumor immune responses. Our findings provide a new mechanistic understanding of virus-induced immune evasion that contributes to cancer progression. PMID:27143385

  18. FTY720 Induces Apoptosis of M2 Subtype Acute Myeloid Leukemia Cells by Targeting Sphingolipid Metabolism and Increasing Endogenous Ceramide Levels

    PubMed Central

    Li, Lianchun; Liu, Yuan-Fang; Wang, Jiang; Liu, Hong; Song, Heng; Jiang, Hualiang; Chen, Sai-Juan; Luo, Cheng; Li, Keqin Kathy

    2014-01-01

    The M2 subtype Acute Myeloid Leukemia (AML-M2) with t(8;21) represents an unmet challenge because of poor clinical outcomes in a sizable portion of patients. In this study,we report that FTY720 (Fingolimod), a sphingosine analogue and an FDA approved drug for treating of multiple sclerosis, shows antitumorigenic activity against the Kasumi-1 cell line, xenograft mouse models and leukemic blasts isolated from AML-M2 patients with t(8;21) translocation. Primary investigation indicated that FTY720 caused cell apoptosis through caspases and protein phosphatase 2A (PP2A) activation. Transcriptomic profiling further revealed that FTY720 treatment could upregulate AML1 target genes and interfere with genes involved in ceramide synthesis. Treatment with FTY720 led to the elimination of AML1-ETO oncoprotein and caused cell cycle arrest. More importantly, FTY720 treatment resulted in rapid and significant increase of pro-apoptotic ceramide levels, determined by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry based lipidomic approaches. Structural simulation model had also indicated that the direct binding of ceramide to inhibitor 2 of PP2A (I2PP2A) could reactivate PP2A and cause cell death. This study demonstrates, for the first time, that accumulation of ceramide plays a central role in FTY720 induced cell death of AML-M2 with t(8;21). Targeting sphingolipid metabolism by using FTY720 may provide novel insight for the drug development of treatment for AML-M2 leukemia. PMID:25050888

  19. Curcumin Nanoformulation for Cervical Cancer Treatment

    PubMed Central

    Zaman, Mohd S.; Chauhan, Neeraj; Yallapu, Murali M.; Gara, Rishi K.; Maher, Diane M.; Kumari, Sonam; Sikander, Mohammed; Khan, Sheema; Zafar, Nadeem; Jaggi, Meena; Chauhan, Subhash C.

    2016-01-01

    Cervical cancer is one of the most common cancers among women worldwide. Current standards of care for cervical cancer includes surgery, radiation, and chemotherapy. Conventional chemotherapy fails to elicit therapeutic responses and causes severe systemic toxicity. Thus, developing a natural product based, safe treatment modality would be a highly viable option. Curcumin (CUR) is a well-known natural compound, which exhibits excellent anti-cancer potential by regulating many proliferative, oncogenic, and chemo-resistance associated genes/proteins. However, due to rapid degradation and poor bioavailability, its translational and clinical use has been limited. To improve these clinically relevant parameters, we report a poly(lactic-co-glycolic acid) based curcumin nanoparticle formulation (Nano-CUR). This study demonstrates that in comparison to free CUR, Nano-CUR effectively inhibits cell growth, induces apoptosis, and arrests the cell cycle in cervical cancer cell lines. Nano-CUR treatment modulated entities such as miRNAs, transcription factors, and proteins associated with carcinogenesis. Moreover, Nano-CUR effectively reduced the tumor burden in a pre-clinical orthotopic mouse model of cervical cancer by decreasing oncogenic miRNA-21, suppressing nuclear β-catenin, and abrogating expression of E6/E7 HPV oncoproteins including smoking compound benzo[a]pyrene (BaP) induced E6/E7 and IL-6 expression. These superior pre-clinical data suggest that Nano-CUR may be an effective therapeutic modality for cervical cancer. PMID:26837852

  20. Human papillomavirus type 16 E6 activates NF-kappaB, induces cIAP-2 expression, and protects against apoptosis in a PDZ binding motif-dependent manner.

    PubMed

    James, Michael A; Lee, John H; Klingelhutz, Aloysius J

    2006-06-01

    Infection with human papillomavirus (HPV) is a critical factor in the pathogenesis of most cervical cancers and some aerodigestive cancers. The HPV E6 oncoprotein from high-risk HPV types contributes to the immortalization and transformation of cells by multiple mechanisms, including degradation of p53, transcriptional activation of human telomerase reverse transcriptase (hTERT), and degradation of several proteins containing PDZ domains. The ability of E6 to bind PDZ domain-containing proteins is independent of p53 degradation or hTERT activation but does correlate with oncogenic potential (R. A. Watson, M. Thomas, L. Banks, and S. Roberts, J. Cell Sci. 116:4925-4934, 2003) and is essential for induction of epithelial hyperplasia in vivo (M. L. Nguyen, M. M. Nguyen, D. Lee, A. E. Griep, and P. F. Lambert, J. Virol. 77:6957-6964, 2003). In this study, we found that HPV type 16 E6 was able to activate NF-kappaB in airway epithelial cells through the induction of nuclear binding activity of p52-containing NF-kappaB complexes in a PDZ binding motif-dependent manner. Transcript accumulation for the NF-kappaB-responsive antiapoptotic gene encoding cIAP-2 and binding of nuclear factors to the proximal NF-kappaB binding site of the cIAP-2 gene promoter are induced by E6 expression. Furthermore, E6 is able to protect cells from TNF-induced apoptosis. All of these E6-dependent phenotypes are dependent on the presence of the PDZ binding motif of E6. Our results imply a role for targeting of PDZ proteins by E6 in NF-kappaB activation and protection from apoptosis in airway epithelial cells. PMID:16699010

  1. EB1089, a synthetic analogue of vitamin D, induces apoptosis in breast cancer cells in vivo and in vitro

    PubMed Central

    James, Sharon Y; Mercer, Elizabeth; Brady, Matthew; Binderup, Lise; Colston, Kay W

    1998-01-01

    Effects of the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells and in nitrosomethylurea-induced rat mammary tumours in vivo were investigated.At a dose of 0.5 μg kg−1 body weight, EB1089 caused significant inhibition of tumour progression over the 28 day treatment period in the absence of a significant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 μg kg−1) produced substantial regression of the experimental tumours which was accompanied by a striking change in the histological appearance of tumours consistent with induction of tumour cell death.Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal transferase (TdT) assay, 3′ DNA breaks indicative of DNA fragmentation were detected histochemically in mammary tumour cells from animals treated with EB1089 (2.5 μg kg−1) for 14 days.Effects of the vitamin D analogue on induction of apoptosis were examined in vitro using the MCF-7 human breast cancer cell line. Using the TUNEL method, positive nuclear staining indicative of DNA fragmentation was detected in cells treated for 4 days with 10 nM EB1089. Apoptosis was also quantitated using a cell death ELISA which revealed a time and dose dependent induction of apoptosis by EB1089.The effects of EB1089 on the expression of two oncoproteins which may regulate apoptosis, bcl-2 and bax were examined by Western analysis. In MCF-7 cell cultures treated with 1,25(OH)2D3 or EB1089 (1×10−8 M), bcl-2 protein levels were decreased in a time-dependent manner relative to control levels. In contrast bax protein was not markedly regulated by these compounds. Densitometric analyses indicate that the vitamin D compounds lower the bcl-2/bax ratio favouring increased susceptibility of MCF-7 cells to undergo apoptosis.These results suggest that the synthetic vitamin D analogue EB1089 may promote tumour regression by inducing active cell death. PMID

  2. Drug-induced hypoglycemia

    MedlinePlus

    ... medlineplus.gov/ency/article/000310.htm Drug-induced hypoglycemia To use the sharing features on this page, please enable JavaScript. Drug-induced hypoglycemia is low blood sugar that results from medication. ...

  3. Flow-induced vibration

    SciTech Connect

    Blevins, R.D.

    1990-01-01

    This book reports on dimensional analysis; ideal fluid models; vortex-induced vibration; galloping and flutter; instability of tube and cylinder arrays; vibrations induced by oscillating flow; vibration induced by turbulence and sound; damping of structures; sound induced by vortex shedding; vibrations of a pipe containing a fluid flow; indices. It covers the analysis of the vibrations of structures exposed to fluid flows; explores applications for offshore platforms and piping; wind-induced vibration of buildings, bridges, and towers; and acoustic and mechanical vibration of heat exchangers, power lines, and process ducting.

  4. Cavitation-resistant inducer

    DOEpatents

    Dunn, Charlton; Subbaraman, Maria R.

    1989-01-01

    An improvement in an inducer for a pump wherein the inducer includes a hub, a plurality of radially extending substantially helical blades and a wall member extending about and encompassing an outer periphery of the blades. The improvement comprises forming adjacent pairs of blades and the hub to provide a substantially rectangular cross-sectional flow area which cross-sectional flow area decreases from the inlet end of the inducer to a discharge end of the inducer, resulting in increased inducer efficiency improved suction performance, reduced susceptibility to cavitation, reduced susceptibility to hub separation and reduced fabrication costs.

  5. Cavitation-resistant inducer

    DOEpatents

    Dunn, C.; Subbaraman, M.R.

    1989-06-13

    An improvement in an inducer for a pump is disclosed wherein the inducer includes a hub, a plurality of radially extending substantially helical blades and a wall member extending about and encompassing an outer periphery of the blades. The improvement comprises forming adjacent pairs of blades and the hub to provide a substantially rectangular cross-sectional flow area which cross-sectional flow area decreases from the inlet end of the inducer to a discharge end of the inducer, resulting in increased inducer efficiency improved suction performance, reduced susceptibility to cavitation, reduced susceptibility to hub separation and reduced fabrication costs. 11 figs.

  6. E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts.

    PubMed Central

    Fu, X; Kamps, M P

    1997-01-01

    The E2a-Pbx1 oncoprotein contains the transactivation domain of E2a joined to the DNA-binding homeodomain (HD) of Pbx1. In mice, E2a-Pbx1 transforms T lymphoblasts and fibroblasts and blocks myeloblast differentiation. Pbx1 and E2a-Pbx1 bind DNA as heterodimers with other HD proteins whose expression is tissue specific. While the transactivation domain of E2a is required for all forms of transformation, DNA binding by the Pbx1 HD is essential for blocking myeloblast differentiation but dispensable for fibroblast or T-lymphoblast transformation. These properties suggest (i) that E2a-Pbx1 causes cellular transformation by activating gene transcription, (ii) that transcription of E2a-Pbx1 target genes is normally regulated by ubiquitous Pbx proteins and tissue-specific partners, and (iii) that DNA-binding mutants of E2a-Pbx1 activate a subset of all gene targets. To test these predictions, genes induced in NIH 3T3 fibroblasts by E2a-Pbx1 were identified and examined for tissue- and stage-specific expression and their differential abilities to be upregulated by E2a-Pbx1 in NIH 3T3 fibroblasts and myeloblasts and by a DNA-binding mutant of E2a-Pbx1 in NIH 3T3 cells. Of 12 RNAs induced by E2a-Pbx1, 4 encoded known proteins (a J-C region of the immunoglobulin kappa light chain, natriuretic peptide receptor C, mitochondrial fumarase, and the 3',5'-cyclic nucleotide phosphodiesterase, PDE1A) and 5 encoded new proteins related to angiogenin, ion channels, villin, epidermal growth factor repeat proteins, and the human 2.19 gene product. Expression of many of these genes was tissue specific or developmentally regulated, and most were not expressed in fibroblasts, indicating that E2a-Pbx1 can induce ectopic expression of genes associated with lineage-specific differentiation. PMID:9032278

  7. Nuclear Factor kappa B is central to Marek’s Disease herpesvirus induced neoplastic transformation of CD30 expressing lymphocytes in-vivo

    PubMed Central

    2012-01-01

    Background Marek’s Disease (MD) is a hyperproliferative, lymphomatous, neoplastic disease of chickens caused by the oncogenic Gallid herpesvirus type 2 (GaHV-2; MDV). Like several human lymphomas the neoplastic MD lymphoma cells overexpress the CD30 antigen (CD30hi) and are in minority, while the non-neoplastic cells (CD30lo) form the majority of population. MD is a unique natural in-vivo model of human CD30hi lymphomas with both natural CD30hi lymphomagenesis and spontaneous regression. The exact mechanism of neoplastic transformation from CD30lo expressing phenotype to CD30hi expressing neoplastic phenotype is unknown. Here, using microarray, proteomics and Systems Biology modeling; we compare the global gene expression of CD30lo and CD30hi cells to identify key pathways of neoplastic transformation. We propose and test a specific mechanism of neoplastic transformation, and genetic resistance, involving the MDV oncogene Meq, host gene products of the Nuclear Factor Kappa B (NF-κB) family and CD30; we also identify a novel Meq protein interactome. Results Our results show that a) CD30lo lymphocytes are pre-neoplastic precursors and not merely reactive lymphocytes; b) multiple transformation mechanisms exist and are potentially controlled by Meq; c) Meq can drive a feed-forward cycle that induces CD30 transcription, increases CD30 signaling which activates NF-κB, and, in turn, increases Meq transcription; d) Meq transcriptional repression or activation of the CD30 promoter generally correlates with polymorphisms in the CD30 promoter distinguishing MD-lymphoma resistant and susceptible chicken genotypes e) MDV oncoprotein Meq interacts with proteins involved in physiological processes central to lymphomagenesis. Conclusions In the context of the MD lymphoma microenvironment (and potentially in other CD30hi lymphomas as well), our results show that the neoplastic transformation is a continuum and the non-neoplastic cells are actually pre-neoplastic precursor

  8. Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial

    PubMed Central

    Trimble, Cornelia L; Morrow, Matthew P; Kraynyak, Kimberly A; Shen, Xuefei; Dallas, Michael; Yan, Jian; Edwards, Lance; Parker, R Lamar; Denny, Lynette; Giffear, Mary; Brown, Ami Shah; Marcozzi-Pierce, Kathleen; Shah, Divya; Slager, Anna M; Sylvester, Albert J; Khan, Amir; Broderick, Kate E; Juba, Robert J; Herring, Timothy A; Boyer, Jean; Lee, Jessica; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L

    2016-01-01

    Summary Background Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. Methods Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). Findings Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49.5%) of 107 VGX-3100 recipients and 11 (30.6%) of 36 placebo recipients had histopathological regression (percentage point difference 19.0 [95% CI 1.4–36.6]; p=0.034). In the modified intention-to-treat analysis 55 (48.2%) of 114 VGX-3100 recipients and 12 (30.0%) of 40 placebo recipients had histopathological regression (percentage point difference 18.2 [95% CI

  9. Human papilloma virus 16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells.

    PubMed

    Tan, Shinta; Hougardy, Brigitte M T; Meersma, Gert J; Schaap, Bessel; de Vries, Elisabeth G E; van der Zee, Ate G J; de Jong, Steven

    2012-05-01

    In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells. PMID:22328720

  10. Epigenetic events during the process of cell transformation induced by carcinogens (review).

    PubMed

    Nguyen-Ba, G; Vasseur, P

    1999-01-01

    Recent studies clearly demonstrate that several environmental carcinogens lack the ability to initially induce genetic damage. In that view, multistage chemical carcinogenesis may be processed under the control of a variety of epigenetic events in addition to genotoxic impacts. The understanding of this mechanism as reviewed in this report requires knowledge of early changes induced by carcinogens in target cells, biochemical, biological and molecular reactions closely related to both sides of the growth equation: cell proliferation and programmed death. Among several cell transformation models, the most suitable for carcinogen detection and mechanistic study is the Syrian hamster embryo (SHE) cell transformation assay. This closely mimics the multistage carcinogenesis and we can examine, in a relatively short time (8 days), the mechanisms by which genotoxic and non-genotoxic agents may increase the frequency of cell transformation as a preneoplastic end-point. The mode of action of hundred of compounds, carcinogens and non-carcinogens, has been explored so far using one-stage and two-stage treatment protocols. In general, with the two-stage protocol, all carcinogens, irrespective of their genotoxic or non-genotoxic potential, give unambiguous positive results. Since perturbations of cell proliferation and death are considered essential events in the process of carcinogenesis, studies have been conducted on the dysregulation of two specific parameters, the induction of ornithine decarboxylase (ODC) an enzyme related to cell proliferation, and the apoptosis rate, when SHE cells are exposed to carcinogens. In one-stage treatment (5 h-24 h), only the promoter TPA induces ODC activity, while other carcinogens do not increase this activity. Using the two-stage exposure protocol (1 h xenobiotic/5 h TPA), all carcinogens both genotoxic and non-genotoxic, are able to stimulate ODC activity above the level obtained with TPA alone. Based on the two-stage treatment with

  11. Cell culture attenuation eliminates rMd5deltaMeq-induced bursal and thymic atrophy and renders the mutant virus as an effective and safe vaccine against Marek's disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) encodes a basic leucine zipper oncoprotein, meq, which structurally resembles jun/fos family of transcriptional activators. It has been clearly demonstrated that deletion of meq results in loss of transformation and oncogenic capacity of MDV. The rMd5'meq virus provided s...

  12. Bicistronic DNA Vaccines Simultaneously Encoding HIV, HSV and HPV Antigens Promote CD8+ T Cell Responses and Protective Immunity

    PubMed Central

    Santana, Vinicius C.; Diniz, Mariana O.; Cariri, Francisco A. M. O.; Ventura, Armando M.; Cunha-Neto, Edécio; Almeida, Rafael R.; Campos, Marco A.; Lima, Graciela K.; Ferreira, Luís C. S.

    2013-01-01

    Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8+ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8+ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses. PMID:23951135

  13. Induced pluripotency with endogenous and inducible genes

    SciTech Connect

    Duinsbergen, Dirk; Eriksson, Malin; Hoen, Peter A.C. 't; Frisen, Jonas; Mikkers, Harald

    2008-10-15

    The recent discovery that two partly overlapping sets of four genes induce nuclear reprogramming of mouse and even human cells has opened up new possibilities for cell replacement therapies. Although the combination of genes that induce pluripotency differs to some extent, Oct4 and Sox2 appear to be a prerequisite. The introduction of four genes, several of which been linked with cancer, using retroviral approaches is however unlikely to be suitable for future clinical applications. Towards developing a safer reprogramming protocol, we investigated whether cell types that express one of the most critical reprogramming genes endogenously are predisposed to reprogramming. We show here that three of the original four pluripotency transcription factors (Oct4, Klf4 and c-Myc or MYCER{sup TAM}) induced reprogramming of mouse neural stem (NS) cells exploiting endogenous SoxB1 protein levels in these cells. The reprogrammed neural stem cells differentiated into cells of each germ layer in vitro and in vivo, and contributed to mouse development in vivo. Thus a combinatorial approach taking advantage of endogenously expressed genes and inducible transgenes may contribute to the development of improved reprogramming protocols.

  14. Sensitization of cervix cancer cells to Adriamycin by Pentoxifylline induces an increase in apoptosis and decrease senescence

    PubMed Central

    2010-01-01

    Background Chemotherapeutic drugs like Adriamycin (ADR) induces apoptosis or senescence in cancer cells but these cells often develop resistance and generate responses of short duration or complete failure. The methylxantine drug Pentoxifylline (PTX) used routinely in the clinics setting for circulatory diseases has been recently described to have antitumor properties. We evaluated whether pretreatment with PTX modifies apoptosis and senescence induced by ADR in cervix cancer cells. Methods HeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, ADR or PTX + ADR. The cellular toxicity of PTX and survival fraction were determinated by WST-1 and clonogenic assay respectively. Apoptosis, caspase activation and ADR efflux rate were measured by flow cytometry, senescence by microscopy. IκBα and DNA fragmentation were determinated by ELISA. Proapoptotic, antiapoptotic and senescence genes, as well as HPV-E6/E7 mRNA expression, were detected by time real RT-PCR. p53 protein levels were assayed by Western blot. Results PTX is toxic (WST-1), affects survival (clonogenic assay) and induces apoptosis in cervix cancer cells. Additionally, the combination of this drug with ADR diminished the survival fraction and significantly increased apoptosis of HeLa and SiHa cervix cancer cells. Treatments were less effective in HaCaT cells. We found caspase participation in the induction of apoptosis by PTX, ADR or its combination. Surprisingly, in spite of the antitumor activity displayed by PTX, our results indicate that methylxantine, per se does not induce senescence; however it inhibits senescence induced by ADR and at the same time increases apoptosis. PTX elevates IκBα levels. Such sensitization is achieved through the up-regulation of proapoptotic factors such as caspase and bcl family gene expression. PTX and PTX + ADR also decrease E6 and E7 expression in SiHa cells, but not in HeLa cells. p53 was

  15. Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways.

    PubMed

    Agbottah, Emmanuel; Yeh, Wen-I; Berro, Reem; Klase, Zachary; Pedati, Caitlin; Kehn-Hall, Kyleen; Wu, Weilin; Kashanchi, Fatah

    2008-01-01

    Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-kappaB and the cell cycle pathways. The observation that NF-kappaB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-kappaB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKbeta kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target

  16. Two specific drugs, BMS-345541 and purvalanol A induce apoptosis of HTLV-1 infected cells through inhibition of the NF-kappaB and cell cycle pathways

    PubMed Central

    Agbottah, Emmanuel; Yeh, Wen-I; Berro, Reem; Klase, Zachary; Pedati, Caitlin; Kehn-Hall, Kyleen; Wu, Weilin; Kashanchi, Fatah

    2008-01-01

    Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-κB and the cell cycle pathways. The observation that NF-κB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-κB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKβ kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF

  17. Space Station Induced Monitoring

    NASA Technical Reports Server (NTRS)

    Spann, James F. (Editor); Torr, Marsha R. (Editor)

    1988-01-01

    This report contains the results of a conference convened May 10-11, 1988, to review plans for monitoring the Space Station induced environment, to recommend primary components of an induced environment monitoring package, and to make recommendations pertaining to suggested modifications of the Space Station External Contamination Control Requirements Document JSC 30426. The contents of this report are divided as Follows: Monitoring Induced Environment - Space Station Work Packages Requirements, Neutral Environment, Photon Emission Environment, Particulate Environment, Surface Deposition/Contamination; and Contamination Control Requirements.

  18. Mania Induced by Opipramol

    PubMed Central

    Firoz, Kazhungil; Khaleel, Asfia; Rajmohan, V; Kumar, Manoj; Raghuram, TM

    2015-01-01

    Antidepressants have propensity to induce manic switch in patients with bipolar disorder. Opipramol is an atypical anxiolytic and antidepressant drug which predominantly acts on sigma receptors. Although structurally resembles tricyclic antidepressant imipramine it does not have inhibitory action on the reuptake of norepinephrine/serotonin and hence it is not presumed to cause manic switch in bipolar depression. Here, we describe a case of mania induced by opipramol, in a patient with bipolar affective disorder who was treated for moderate depressive episode with lithium and opipramol and we discuss neurochemical hypothesis of opipramol-induced mania. PMID:25722522

  19. Mania induced by opipramol.

    PubMed

    Firoz, Kazhungil; Khaleel, Asfia; Rajmohan, V; Kumar, Manoj; Raghuram, Tm

    2015-01-01

    Antidepressants have propensity to induce manic switch in patients with bipolar disorder. Opipramol is an atypical anxiolytic and antidepressant drug which predominantly acts on sigma receptors. Although structurally resembles tricyclic antidepressant imipramine it does not have inhibitory action on the reuptake of norepinephrine/serotonin and hence it is not presumed to cause manic switch in bipolar depression. Here, we describe a case of mania induced by opipramol, in a patient with bipolar affective disorder who was treated for moderate depressive episode with lithium and opipramol and we discuss neurochemical hypothesis of opipramol-induced mania. PMID:25722522

  20. Noise-Induced Hearing Loss

    MedlinePlus

    ... Info » Hearing, Ear Infections, and Deafness Noise-Induced Hearing Loss On this page: What is noise-induced hearing ... additional information about NIHL? What is noise-induced hearing loss? Every day, we experience sound in our environment, ...

  1. Vitiligo, drug induced (image)

    MedlinePlus

    ... this person's face have resulted from drug-induced vitiligo. Loss of melanin, the primary skin pigment, occasionally ... is the case with this individual. The typical vitiligo lesion is flat (macular) and depigmented, but maintains ...

  2. Drug-induced hepatitis

    MedlinePlus

    ... induced hepatitis. Painkillers and fever reducers that contain acetaminophen are a common cause of liver inflammation. These ... problem. However, if you took high doses of acetaminophen , treatment should be started as soon as possible ...

  3. Gasoline-induced mucositis

    SciTech Connect

    Hoffman, D.L.; Swanson, B.Z. Jr.; Lutins, N.D.

    1980-02-01

    Gasoline-induced mucositis may become more common because of fuel shortages or increased fuel cost. Dentists should, therefore, consider this oral irritant in the differential diagnosis of oral lesions.