RESUMO
Newcastle disease virus (NDV) is an avian paramyxovirus with replication competence in human tumor cells and interesting anti-neoplastic and immune stimulatory properties. In order to increase tumor selectivity of replication, we prepared mutants from the avirulent strain Ulster with monocyclic replication cycle and adapted them for multicyclic replication in human melanoma cells. Two mutants (M1 and M2) showed interesting functional differences: while M2 showed T cell co-stimulatory effects in a tumor-specific cytotoxic T lymphocyte (CTL) assay, M1 did not. A distinct difference of these 2 virus mutants appeared also when testing their capacity to induce interferon-alpha and -beta as well as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules in human monocytes. Sequence analysis of the hemagglutinin-neuraminidase (HN) molecules of the 2 virus mutants showed 7 non-silent mutational differences. Upon cloning of the HN mutant genes into an expression vector and transfection of cells, only HN derived from M2 (HN-M2) was detected at the cell surface by immunostaining with specific antibodies and showed hemadsorption and neuraminidase activity. In order to define which amino acid was responsible for the loss of functional activity of HN derived from M1 (HN-M1), distinct HN mutants were generated via site-directed mutagenesis and tested. Substitution of serine 200 by a proline abrogated HN expression and its hemadsorption and neuraminidase activities. Molecular modeling revealed that proline 200 in HN influences flexibility of a loop near the entrance to the neuraminidase active site, a function that may be crucial for the functions of this viral protein.
Assuntos
Proteína HN/química , Vírus da Doença de Newcastle/metabolismo , Serina/química , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Clonagem Molecular , Cricetinae , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Vetores Genéticos , Proteína HN/metabolismo , Humanos , Interferon Tipo I/metabolismo , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica , RNA/metabolismo , Vírus da Floresta de Semliki/genética , Linfócitos T Citotóxicos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
T cell immunity in breast cancer is suggested to play a role in tumor dormancy, a period of stability which can correspond to the time interval between primary treatment and tumor recurrence. Bone marrow in breast cancer patients seems to be particularly important because it is highly enriched with cancer specific memory T cells. Similar cells can be found in peripheral blood, but these appear to be functionally anergic. The immune system of primary operated breast cancer patients does not seem to be completely anergized. Bone marrow derived memory T cells can be reactivated ex vivo and show functional reactivity, including tumor rejection in NOD/SCID mice. Promising results were obtained from a postoperative phase-II active specific immunotherapy study. In this study, 32 patients treated with an optimal formulation of a virus-modified autologous tumor vaccine (ATV-NDV) appeared to have a significant 5-year survival benefit. Our results suggest that cancer reactive memory T cells which are enriched in the bone marrow of breast cancer patients, can be activated ex vivo via autologous dendritic cells pulsed with breast cancer tumor antigens, or they can be activated in situ via a tumor vaccine, which combines tumor antigens with virus infection. The findings should encourage further studies in breast cancer on active specific immunotherapy with tumor vaccines or adoptive immunotherapy with activated memory T cells.
