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1.
Article in English | IMSEAR | ID: sea-136324

ABSTRACT

Background & objectives: Dendritic cells (DCs) are potent antigen presenting cells which proceed from immature to a mature stage during their differentiation. There are several methods of obtaining long lasting mature antigen expressing DCs and different methods show different levels of antigen expressions. We investigated bone marrow derived DCs for the degree of maturation and genetically engineered antigen presentation in the presence of interleukin-4 (IL-4) as a maturity enhancer. Methods: DCs and transfected retrovirus were cultured together in the presence of granulocyte-macrophage colony stimulating factor (GMCSF)-IL4, GMCSF +IL4, lipopolysaccharide (LPS). B 7.1, B7.2 and CD11c were measured by the degree of immune fluorescence using enhanced green fluorescent protein (EGFP) shuttled retrovirus transfected antigen. Degree of MHC class I molecule with antigen presentation of antigen was also evaluated by fluorescence activated cell sorting. The antigen presenting capacity of transfected DCs was investigated. Bone marrow DCs were generated in the presence of GMCSF and IL-4 in vitro. Dividing bone marrow cells were infected with EGFP shuttled retrovirus expressing SSP2 by prolonged centrifugation for three consecutive days from day 5, 6 and 7 and continued to culture in the presence of GMSCF and IL-4 until day 8. Results: IL-4 as a cytokine increased the maturation of retrovirus transfected DCs by high expression of B 7-1 and B 7-2. Also, IL-4 induced DC enhanced by the prolonged centrifugation and it was shown by increased antigen presentation of these dendric cells as antigen presenting cell (APC). Cytolytic effects were significantly higher in cytotoxic T cell response (CTLs) mixed with transfected DCs than CTLs mixed with pulsed DCs. Interpretation & conclusions: There was an enhanced antigen presentation by prolonged expression of antigen loaded MHC class I receptors in DCs in the presence of IL-4 by prolonged centrifugation.


Subject(s)
Antigen Presentation/drug effects , Antigen Presentation/immunology , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , B7 Antigens/genetics , B7 Antigens/metabolism , Bone Marrow/immunology , Bone Marrow/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Centrifugation , Dendritic Cells/drug effects , Dendritic Cells/immunology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Genes, MHC Class I/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-4/immunology , Interleukin-4/pharmacology , Lipopolysaccharides/pharmacology , Peptides/genetics , Peptides/immunology , Retroviridae/genetics , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Transfection/methods
2.
J Biosci ; 2008 Mar; 33(1): 27-44
Article in English | IMSEAR | ID: sea-110649

ABSTRACT

Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent gene- trap lines revealed arrest-dependent induction of betagal activity, confirming the efficacy of the FACS screen.The locus of integration was identified in 15 lines. In three lines,insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY - a BRCA2--interacting protein, p8/com1 - a p300HAT -- binding protein and MLL5 - a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.


Subject(s)
Animals , Blotting, Northern , Blotting, Southern , Cell Culture Techniques , Cell Cycle , Cell Differentiation/genetics , Cell Proliferation , Cells, Cultured , Culture Media , DNA-Binding Proteins/genetics , Flow Cytometry , Gene Expression Regulation/genetics , Gene Library , Genes/physiology , Genes, Viral , Genetic Vectors , Mice , Microfilament Proteins/genetics , Models, Biological , Mutagenesis, Insertional , Myeloid-Lymphoid Leukemia Protein/genetics , Myoblasts/cytology , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Retroviridae/genetics , Transduction, Genetic , beta-Galactosidase/analysis
3.
Article in English | WPRIM | ID: wpr-20643

ABSTRACT

Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We investigated the bystander effect of retrovirusmediated gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell line (neuro-2a) in vitro and in vivo, and we examined whether the mechanism of bystander effect in neuroblastoma would also depend on connexin-dependent gap junction and/or immune response. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to ganciclovir (GCV) killing. Implanted mixtures of wildtype cells and HSV-TK transduced cells showed a potent bystander effect upon administration of GCV in A/J mice. HSV-TK/GCV system in murine neuroblastoma induced systemic immunity. Immunohistochemical staining showed many CD4+ and CD8+ cell infiltration but did not show anti-connexin 43+ cells. In conclusion, a strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma might be dependent on immune response and/or on other mechanism such as protein phosphorylation or transfer of apoptotic vesicle, rather than connexin-dependent gap junction.


Subject(s)
Animals , Apoptosis , Bystander Effect , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Connexin 43/biosynthesis , Gap Junctions , Genetic Therapy/methods , Gene Transfer Techniques , Humans , Immunohistochemistry , Mice , Neoplasm Transplantation , Neuroblastoma/therapy , Phosphorylation , Retroviridae/genetics , Simplexvirus/enzymology , Thymidine Kinase/genetics , Time Factors
4.
Article in English | WPRIM | ID: wpr-134593

ABSTRACT

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.


Subject(s)
Animals , Cell Line , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Kinetics , Peptides/genetics , Pituitary Gland, Anterior/cytology , Protein Precursors/biosynthesis , Protein Processing, Post-Translational , Protein Sorting Signals/genetics , Protein Transport , Rats , Recombinant Proteins/biosynthesis , Retroviridae/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/biosynthesis , Secretory Vesicles/metabolism , Somatostatin/biosynthesis
5.
Article in English | WPRIM | ID: wpr-134592

ABSTRACT

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.


Subject(s)
Animals , Cell Line , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Kinetics , Peptides/genetics , Pituitary Gland, Anterior/cytology , Protein Precursors/biosynthesis , Protein Processing, Post-Translational , Protein Sorting Signals/genetics , Protein Transport , Rats , Recombinant Proteins/biosynthesis , Retroviridae/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/biosynthesis , Secretory Vesicles/metabolism , Somatostatin/biosynthesis
6.
Yonsei Medical Journal ; : 76-81, 2000.
Article in English | WPRIM | ID: wpr-33454

ABSTRACT

We used retroviral-mediated gene transfer of the human interleukin (IL)-2 gene into murine neuroblastoma cells to investigate whether locally-secreted IL-2 is able to influence the generation of anti-tumor immune responses. Supernatant obtained from cultures of approximately 1 x 10(6) IL-2 gene-transduced, G-418 selected neuro-2a cells was assayed for human IL-2 production by ELISA kit. First, to estimate whether the local secretion of IL-2 from the genetically-modified tumor cells would affect their tumorigenicity in vivo, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice and tumor growth was measured weekly. And to estimate whether IL-2 transfected neuroblastoma cells protect mice from tumor development after wild-type tumor cell challenge, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice. Seven days after IL-2 gene-transfected neuroblastoma cell injection, unmodified neuro-2a cells were s.c. injected into the contralateral site of A/J mice and tumor growth was measured weekly. Finally, to estimate IL-2 effect on pre-established large tumor burdens, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice with established tumor and its growth was measured weekly. The IL-2 gene-transduced neuro-2a clones secreted 120.25-177.3 IU of IL-2 per ml per 10(6) cells during 24 hr. None of the mice injected with IL-2-secreting neuro-2a cells developed tumors within 6 weeks, while all of the mice injected with wild-type neuro-2a cells developed tumors. Immunization of mice with IL-2 gene-transfected, irradiated neuro-2a cells protected these animals against a subsequent challenge with wild-type tumor cells. Finally, the size of large neuroblastomas decreased after IL-2-secreting neuro-2a cell injection into mice. Local secretion of IL-2 gene-transduced tumor cells abrogates their tumorigenicity and induces protective immunity and may inhibit the growth of neuroblastoma.


Subject(s)
Animals , Antibody Formation , Gene Transfer Techniques , Humans , Immunization/methods , Interleukin-2/therapeutic use , Interleukin-2/genetics , Mice , Neoplasm Transplantation , Neuroblastoma/therapy , Neuroblastoma/prevention & control , Neuroblastoma/pathology , Neuroblastoma/genetics , Retroviridae/genetics , Tumor Cells, Cultured
7.
Braz. j. med. biol. res ; 32(7): 905-14, July 1999.
Article in English | LILACS | ID: lil-234898

ABSTRACT

The use of gene therapy continues to be a promising, yet elusive, alternative for the treatment of cancer. The origins of cancer must be well understood so that the therapeutic gene can be chosen with the highest chance of successful tumor regression. The gene delivery system must be tailored for optimum transfer of the therapeutic gene to the target tissue. In order to accomplish this, we study models of G1 cell-cycle control in both normal and transformed cells in order to understand the reasons for uncontrolled cellular proliferation. We then use this information to choose the gene to be delivered to the cells. We have chosen to study p16, p21, p53 and pRb gene transfer using the pCL-retrovirus. Described here are some general concepts and specific results of our work that indicate continued hope for the development of genetically based cancer treatments


Subject(s)
Rats , Mice , Animals , Humans , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors/genetics , Glioblastoma/genetics , Glioblastoma/therapy , Retroviridae/genetics , Cell Cycle/genetics , Cell Cycle/physiology , Cell Transformation, Neoplastic/genetics , Clinical Trials as Topic , Disease Models, Animal , Genes, Tumor Suppressor
8.
Indian J Exp Biol ; 1998 Jun; 36(6): 539-45
Article in English | IMSEAR | ID: sea-61328

ABSTRACT

Although there are many hurdles to overcome for successful gene therapy, there is a vast potential to permanently incorporate genes into cells to correct genetic disorders and to combat viral infections. Retroviruses, inspite of some limitations, offer the best hope in this direction and lentiviral vectors, which infect nondividing cells, may be the choice in the future, especially in gene therapy for central nervous system disorders.


Subject(s)
Animals , Genetic Therapy , Genetic Vectors , Humans , Retroviridae/genetics
10.
Medicina (B.Aires) ; 57(2): 235-44, 1997.
Article in Spanish | LILACS | ID: lil-201857

ABSTRACT

El virus del tumor mamario murino (MMTV) se considera actualmente un modelo de interés para investigar los mecanismos co-evolutivos entre los retrovirus y sus huéspedes. El MMTV es un retrovirus de tipo B que se transmite a través de la leche e induce adenocarcinomas mamarios por activación insercional de proto-oncogenes celulares. Existen também formas endógenas de estos virus integrados permanetemente en el genoma del ratón. Estos provirus se consideran el resultado de la infección de células de la línea germinal ocurridas en los últimos 4 a 5 millones de años. El marco de lectura abierto presente en el LTR 3'de los virus integrados codifica para un superantígeno (SAg) que es capaz de estimular una gran proporción de células T que comparaten la región variable de la cadena beta del TCR. La expresión de este SAg es crítica para el ciclo de vida del virus. Cuando un MMTV exógeno infecta al huésped, las células B resultan infectadas tempranamente y expresan el SAg viral. Las células T reactivas al SAg son reclutadas para responder al mismo y, como consecuencia, tanto las células T reactivas como los linfocitos B infectados se activan y comienzan a proliferar. Este hecho facilita la integración del MMTV y el incremento del número de linfocitos infectados, dando lugar a un importante aumento en la carga viral. Los linfocitos transfieren los virus a la glándula mamaria en la cual, bajo la influencia de hormonas esteroideas, se produce una gran amplificación de la carga viral. Se ha hipotetizado que la presencia de provirus Mtv endómenos conferiría una ventaja selectiva a la problación murina, ya que al inducir la deleción clonal temprana de las células T reactivas a los mismos, protegería al huésped de la infección con un virus exógeno que codifique para un SAg con reactividad cruzada. Sin embargo, resultados recientes discutidos en este trabajo sugieren que los provirus Mtv pueden resultar desventajosos para la población murina ya que son capaces de recombinar con variantes exógenas, dando lugar a partículas virales altamente tumorigénicas. Estos resultados se discuten en relación a trabajos recientes que sugieren la participación de secuencias virales altamente homólogas a los virus MMTV en la carcinogénesis mamaria humana.


Subject(s)
Mice , Animals , Biological Evolution , Disease Models, Animal , In Vitro Techniques , Mammary Tumor Virus, Mouse/genetics , Mammary Tumor Virus, Mouse/immunology , Retroviridae/genetics , Retroviridae/immunology
11.
Indian J Exp Biol ; 1989 May; 27(5): 430-6
Article in English | IMSEAR | ID: sea-63380

ABSTRACT

Phytohemagglutinin (PHA) is known to increase the synthesis of bovine leukemia virus (BLV) particles and viral antigens in short-term culture of BLV-infected neoplastic and non-neoplastic lymphocytes. This stimulation of BLV expression has been shown to be due to enhanced transcription of the viral genome by a PHA-induced protein. We have investigated the binding of 125I-labelled PHA to BLV-infected bovine B lymphocytes and subsequent events that may lead to the stimulation of BLV-p25 synthesis. We found that PHA binding to the infected cells were rapid, but only a small fraction of the bound PHA is translocated to nucleus. However, bound PHA dissociated rapidly from the cell membrane, but not from the nucleus when PHA is removed from the culture medium. Furthermore, continuous presence of PHA was not essential for optimal stimulatory activity, instead a minimum incubation with PHA for 6 hr. followed by culturing the infected cells in its absence, was sufficient to exhibit maximal stimulatory activity. Our results raise the possibility that interaction of PHA with the plasma membrane probably triggers synthesis of a protein which in turn enhances the transcription of BLV genome in vitro.


Subject(s)
Animals , B-Lymphocytes/metabolism , Cattle , Gene Expression Regulation, Viral , Leukemia Virus, Bovine/genetics , Phytohemagglutinins/metabolism , Retroviridae/genetics , Retroviridae Proteins, Oncogenic/biosynthesis
12.
Ciênc. cult. (Säo Paulo) ; 40(1): 5-14, jan. 1988. ilus
Article in Portuguese | LILACS | ID: lil-57343

ABSTRACT

O objetivo da presente revisäo é mostrar alguns aspectos relacionados com o reverso da transcriçäo, incluindo descriçäo do retrovírus, ciclo de vida do vírus, mecanismo do reverso da transcriçäo, propriedades cinéticas e imunológicas da transcritase reversa, e inibidores da enzima viral. Estäo também incluídas algumas referências sobre o retrovírus AIDS. O reverso da transcriçäo parece näo ser restrito apenas aos vírus de RNA. Alguns vírus de DNA, como o vírus da hepatite B, e o vírus do mosaico da couve-flor, e outros elementos genéticos, como pseudogenes e elementos de transposiçäo, parecem utilizar o reverso da transcriçäo para suas existências


Subject(s)
Humans , Retroviridae/enzymology , RNA-Directed DNA Polymerase/metabolism , Transcription, Genetic , DNA, Viral/genetics , Retroviridae/genetics , RNA, Viral/genetics , RNA-Directed DNA Polymerase/antagonists & inhibitors
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