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ABSTRACT: Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types.
Journal of Biological Chemistry 03/2012; 287(17):13633-43. · 4.77 Impact Factor
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ABSTRACT: Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 degrees C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 degrees C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.
Analytical Biochemistry 07/2009; 393(1):138-40. · 3.00 Impact Factor
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ABSTRACT: Prostate cancer remains one of the most common cancers in men and treatment options for patients with advanced or metastatic disease are limited. Immunotherapeutic approaches, such as passive or active immunotherapy, have been shown to be successful in treating some cancer patients. These rely on the identification of cancer specific antigens that can be targeted by the immune system or used to diagnose as early as possible and/or to monitor the disease and its progression. Several prostate cancer antigens have already been identified; these include Prostate-Specific Membrane Antigen (PSMA), Prostate Specific Antigen (PSA), Prostatic Acid Phosphatase (PAP), PAGE-4, PSP 94, differential display 3, prostate androgen-regulated transcript 1 and six-transmembrane epithelial antigen prostate-restricted antigens and these represent potential candidate for immunotherapy against prostate cancer. The following review describes the importance of these antigens with a particular emphasis to the PAP antigen and T21, a recently identified prostate cancer associated antigen, as very promising targets for prostate therapy.
Current Cancer Therapy Reviews 04/2008; 4(2):86-95.
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ABSTRACT: There remains a need to identify novel epitopes of potential tumour target antigens for use in immunotherapy of cancer. Here, several melanoma tissues and cell lines but not normal tissues were found to overexpress the cancer-testis antigen HAGE at the mRNA and protein level. We identified a HAGE-derived 15-mer peptide containing a shorter predicted MHC class I-binding sequence within a class II-binding sequence. However, only the longer peptide was found to be both endogenously processed and immunogenic for T cells in transgenic mice in vivo, as well as for human T cells in vitro. A different class I-binding peptide, not contained within a longer class II sequence, was subsequently found to be both immunogenic and endogenously processed in transgenic mice, as was a second class II epitope. These novel HAGE-derived epitopes may contribute to the range of immunotherapeutic targets for use in cancer vaccination programs.
Cancer Immunology and Immunotherapy 01/2008; 56(12):1885-95. · 3.70 Impact Factor
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ABSTRACT: The identification of antigens that distinguish cancer cells from normal cells is of major importance for the definition of therapeutic targets in human malignancies. Using sera from cancer patients, we have previously reported on the identification of immunologically recognized proteins that belong to the family of cancer testis antigens (CTAs).
A normal testicular cDNA library was screened with pooled allogeneic sera from patients with prostate cancer using a modified SEREX approach. Subsequently we have identified and characterized a novel antigen, T21, with an expression pattern similar to that of CTAs. mRNA expression of T21 was determined using a panel of whole tissues and prostate cell lines using Q-RT-PCR. For laser microdissection, fresh prostate cancer and benign tissue was obtained using our novel validated harvesting technique. Protein expression and cellular localization of T21 were assessed in prostate cell lines using Western blotting, confocal microscopy and flow cytometry.
T21 showed tissue-restricted mRNA expression in gastric, kidney and prostate cancers, and in normal testis and prostate tissues. Following laser microdissection, T21 was significantly over-expressed in malignant compared to benign prostatic epithelium. We have demonstrated expression of T21 at the protein level and confocal microscopy on PC3 cells probed with a T21-monospecific antibody revealed cytoplasmic localization of T21 protein.
The highly restricted expression pattern of T21 makes it an attractive vaccine target for prostate cancer. Several CTAs reportedly induce cytotoxic T-lymphocyte responses, therefore it is reasonable to assume that T21 will be a valuable target for cancer immunotherapy.
The Prostate 03/2007; 67(3):274-87. · 3.48 Impact Factor
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ABSTRACT: Recent years have seen the unprecedented surge of interest in the role of CD4+ T cells and the role they play in the development of the immune response. In this symposium review, we examine the evidence for this and discuss their functions, particularly in respect to the cancer immunology, including CD4+CD25+ cells (Treg).
Cancer Immunology and Immunotherapy 02/2007; 56(1):70-80. · 3.70 Impact Factor
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ABSTRACT: Because of the central role of CD4(+) T cells in antitumour immunity, the identification of the MHC class II-restricted peptides to which CD4(+) T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR-restricted peptides using peptide immunisation of HLA-DR-transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307-319), and then extended to three candidate HLA-DR-restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DRbeta1*0101 (HLA-DR1) and HLA-DRbeta1*0401 (HLA-DR4) molecules. One of these peptides, p53(108-122), consistently induced responses in HLA-DR1- and in HLA-DR4-transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108-122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR-restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II-restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.
Cancer Immunology and Immunotherapy 04/2005; 54(3):243-53. · 3.70 Impact Factor
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ABSTRACT: During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.
Current Pharmaceutical Design 02/2005; 11(27):3501-9. · 3.87 Impact Factor
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ABSTRACT: Because of the central role of CD4+ T cells in antitumour immunity, the identification of the MHC class II–restricted peptides to which CD4+ T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR–restricted peptides using peptide immunisation of HLA-DR–transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307–319), and then extended to three candidate HLA-DR–restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DR1*0101 (HLA-DR1) and HLA-DR1*0401 (HLA-DR4) molecules. One of these peptides, p53(108–122), consistently induced responses in HLA-DR1– and in HLA-DR4–transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108–122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR–restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II–restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.
Cancer Immunology and Immunotherapy 01/2005; 54(3):243-253. · 3.70 Impact Factor
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Selman A Ali,
Murrium Ahmad,
June Lynam,
Cornelia S McLean,
Claire Entwisle,
Peter Loudon,
Esther Choolun, Stephanie E B McArdle,
Geng Li,
Shahid Mian,
Robert C Rees
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ABSTRACT: OX40 ligand (OX40L), a member of TNF superfamily, is a co-stimulatory molecule involved in T cell activation. Systemic administration of mOX40L fusion protein significantly inhibited the growth of experimental lung metastasis and subcutaneous (s.c.) established colon (CT26) and breast (4T1) carcinomas. Vaccination with OX40L was significantly enhanced by combination treatment with intra-tumour injection of a disabled infectious single cycle-herpes simplex virus (DISC-HSV) vector encoding murine granulocyte macrophage-colony stimulating factor (mGM-CSF). Tumour rejection in response to OX40L therapy required functional CD4+ and CD8+ T cells and correlated with splenocyte cytotoxic T lymphocytes (CTLs) activity against the AH-1 gp70 peptide of the tumour associated antigen expressed by CT26 cells. These results demonstrate the potential role of the OX40L in cancer immunotherapy.
Vaccine 10/2004; 22(27-28):3585-94. · 3.77 Impact Factor
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ABSTRACT: DISC-HSV is a replication incompetent herpes simplex virus that is a highly efficient vector for the transduction of genes in vivo and in vitro. We examine the ability of DISC-HSV to infect human prostate cancer cell-lines and xenograft tumor models, and induce expression of reporter and therapeutic cytokine genes.
Infection was confirmed by cellular staining for the beta-galactosidase reporter gene product, and by EM. Human GM-CSF production following DISC-hGMCSF infection was measured using ELISA. The metabolic activity of infected cells was determined by NADP/NADPH assay. Cell death was estimated by cell-cycle analysis using flow cytometry with propidium iodide staining.
Infection of DU145, PC3 and LNCaP cells with DISC-HSV was dose dependent. Cells infected with DISC-hGM-CSF released significant levels of hGM-CSF for 3 days. NADP/NADPH assay suggested that infected cells continued to be metabolically active for 3 days post-infection, which was consistent with flow cytometry findings that cell death did not occur within 7 days of infection. Tumor xenografts injected with DISC-HSV expressed beta-galactosidase, and intracellular viral particles were demonstrated using EM.
We have previously reported the rejection of established tumors following intra-tumoral injection of DISC-GMCSF. This study demonstrates the ability of DISC-HSV to infect prostate cancer and express GMCSF at significant levels. We suggest that prostate cancer is a potential target for therapy using DISC-HSV containing GM-CSF.
The Prostate 07/2003; 56(1):65-73. · 3.48 Impact Factor
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Methods in molecular medicine 02/2003; 81:359-75.
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ABSTRACT: Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-gamma, but not IL-4 cytokine production. Depletion of CD8(+) T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4(+) T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.
The Journal of Immunology 05/2002; 168(7):3512-9. · 5.79 Impact Factor
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ABSTRACT: Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 °C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 °C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.
Analytical Biochemistry 393(1):138-140. · 3.00 Impact Factor
