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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33 degrees C and arrest growth when transferred to the nonpermissive temperature of 39 degrees C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined.
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PMID:Characterization of murine thymic stromal-cell lines immortalized by temperature-sensitive simian virus 40 large T or adenovirus 5 E1a. 166 72

Cells of the monocyte lineage act as a major reservoir for HIV, and ways of enhancing the resistance of mononuclear phagocytes to HIV replication would be useful for delaying the onset of AIDS in infected individuals. Seif et al. (J. Virol. 65:664, 1991) have recently shown the possibility of obtaining stable antiviral expression (SAVE), directed against three nonretroviral RNA viruses, and normal cell viability in a significant percentage of murine BALB/c 3T3 cells transformed with an IFN-beta expression plasmid under the control of the 0.6-kb XhoII-NruI promoter region of the murine H-2Kb MHC gene. In the present paper, we show that it is possible to establish SAVE in human promonocytic cells. Cells of the human promonocytic U937 line were stably transfected with a human IFN-beta expression plasmid carrying the neo- and human IFN-beta-coding sequences under the control of the H-2Kb promoter fragment previously used in murine cells. After selection with G418, two transformed clones were isolated that released small amounts of human IFN-beta into the culture medium, without affecting the expression of CD4 and leucocyte function-associated Ag-1 differentiation Ag. The presence of construct-derived IFN-beta mRNA was demonstrated by polymerase chain reaction amplification of cDNA, and the level of 2-5A synthetase, one of the major IFN-induced antiviral proteins, was shown to be constitutively increased. These clones were less permissive for HIV-1 than control clones transformed with the neo gene only. The antiviral state could be modulated by anti-IFN-beta antibodies, in that the continuous presence of antibodies in the culture medium abolished the enhanced resistance to HIV-1 replication, whereas the withdrawal of the antiserum restored the antiviral state, indicating that it did indeed result from the constitutive synthesis of human IFN-beta. These results demonstrate the possibility of restricting HIV-1 replication in human promonocytic cells by establishing SAVE. Further exploration of this method as a possible approach to somatic cell gene therapy of HIV infection appears worthwhile.
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PMID:Enhanced resistance to HIV-1 replication in U937 cells stably transfected with the human IFN-beta gene behind an MHC promoter fragment. 194 Mar 55

A "hybrid gene" (MTKb) comprised of the human metallothionein IIA promoter ligated to the genomic sequence of the major histocompatibility complex class I (H-2Kb) gene was subcloned into the expression vector pSV2neo and transfected into the natural killer (NK) cell-sensitive YAC-1 lymphoma. The Kb gene product was readily detectable on the cell surface of G418-resistant transfectants using both Kb-specific monoclonal antibodies and H-2b-specific cytolytic T cells. Unlike control pSV2neo transfectants, MTKb-pSV2neo transfectants were relatively resistant to lysis by NK cells from H-2a, H-2b, H-2k or H-2 (a x b)F1 haplotype mice. These data strongly suggest that the effects of MHC expression on susceptibility to NK cells can be mediated by a single and well-defined class I molecule, Kb.
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PMID:Class I (H-2Kb) gene transfection reduces susceptibility of YAC-1 lymphoma targets to natural killer cells. 234 64

In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by > 95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by > 95%. Western analysis revealed a 30% decrease in the level of perforin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.
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PMID:Stably transfected antisense granzyme B and perforin constructs inhibit human granule-mediated lytic ability. 765 32

Three commonly used transfection techniques (electroporation, calcium phosphate precipitation and scrape loading) and a novel procedure combining the latter two methods were evaluated and conditions optimised for successful transfection of human HepG2 cells with plasmid DNA incorporating a mouse MHC Class I gene and a selectable marker (neomycin transferase gene) conferring resistance to G418. While transfection with linear DNA by scrape-loading gave satisfactory results, transfer of cloned circular DNA by electroporation, calcium phosphate precipitation or a combined use of scrape-loading and calcium phosphate gave best results for HepG2 cells.
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PMID:A comparative evaluation of three transfection procedures as assessed by resistance to G418 conferred to HEPG2 cells. 806 22

Experimental models of vaccination with tumor cells engineered to produce interleukin-4 (IL-4) have shown that the local release of this cytokine is associated with the development of antitumor immunity that may induce regression of established cancer. The aim of this study was to transduce a human melanoma cell line with the gene coding for human IL-4, and to analyze cytokine production, phenotypic characteristics, and antigen expression after transduction. A retroviral vector, constructed by inserting IL-4 cDNA into the LXSN vector, was used to infect the human melanoma cell line Me14932, known to express the MHC class I HLA-A2 and the melanoma-associated antigen Melan-A/MART-1, recognized by HLA-A2-restricted T-cells. The confluence of all G418-resistant cells (Me14932/IL-4) was then analyzed for proviral integration and IL-4 mRNA expression. Substantially stable IL-4 release was detected by ELISA in the supernatant of transduced cells, ranging from 1.6 to 4.6 ng/ml per 10(5) cells per 24 hr; such a cytokine displayed a specific biologic activity, as revealed by the stimulation of blast cell proliferation and the inhibition of lymphokine activated killer cell (LAK) induction by IL-2. After 200 Gy irradiation, IL-4 release remained detectable for 5 weeks, whereas cell proliferation ceased within 7 days. Morphology and immunophenotypic characteristics of the parental cell line (expression of MHC classes I and II, ICAM-1, LFA 3, melanoma-associated antigens, etc.) were retained by the IL-4 gene-transduced melanoma as assayed by microscopy and immunofluorescence; likewise, susceptibility to lysis by LAK cells as well as a T-cell clone recognizing the Melan-A/MART-1 antigen did not change. These results, together with the lack of replication-competent retrovirus, suggest that the Me14932/IL-4 cell line displays suitable characteristics for its use in the treatment of HLA-matched melanoma patients.
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PMID:A human melanoma cell line transduced with an interleukin-4 gene by a retroviral vector releases biologically active IL-4 and maintains the original tumor antigenic phenotype. 857 15

Abstract Interferon gamma (IFNgamma) is an important cytokine with immunomodulatory properties that include activation of immune cells and induction of class I and class II major histocompatibility complex antigens. In this study a retroviral vector was used to introduce the IFNgamma gene into EMT6 tumor cells to assess the effect of IFNgamma gene expression on tumor immunogenicity. Transfectants were selected in G418-containing tissue-culture medium and were determined to express the inserted IFNgamma gene by reverse transcriptase/polymerase chain reaction. Flow-cytometric analysis revealed that parental unmodified EMT6 cells constitutively expressed only class I MHC and were poorly responsive to exogenous IFNgamma stimulation, whereas class II MHC was induced in IFNgamma-transfected cells. The induction of class II MHC in IFNgamma-transfected cells correlated with the expression of a mouse class II transactivator gene that was dormant in unmodified or mock-transfected cells. In addition, IFNgamma-gene-transfected tumor cells were found to secrete up to 17 ng IFN (equivalent to 75 units/10(6) cells) by enzyme-linked immunosorbent assay (ELISA). Whereas parental EMT6 cells grew unchecked, the growth of genetically modified tumor cells was significantly inhibited in immunocompetent mice. Rechallenge of animals that rejected an IFNgamma-transfected EMT6 clone (EMT6-B17) with parental EMT6 cells resulted in tumor rejection, suggesting that IFNgamma-transfected EMT6 cells were able to induce long-term immunity. Mixing experiments using gene-transfected and unmodified tumor cells demonstrated that 10% of IFNgamma-transfected cells in the population was sufficient to protect mice against subsequent challenge with tumorigenic EMT6 cells. These studies demonstrate that the immunogenicity of tumor cells that are poorly responsive to exogenous IFNgamma can be enhanced by inserting and expressing the IFNgamma transgene. These findings also suggest a role for class II MHC in reducing tumorigenicity of the EMT6 tumor and inducing long-term tumor immunity.
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PMID:Interferon gamma (IFNgamma) gene transfer of an EMT6 tumor that is poorly responsive to IFNgamma stimulation: increase in tumor immunogenicity is accompanied by induction of a mouse class II transactivator and class II MHC. 862 May 27

LB leukemia is a nonimmunogenic T cell tumor which spontaneously arose in a BALB/c mouse; efforts to induce immunological rejection of the leukemic cells have always failed. The leukemic cells grow rapidly and progressively in the syngeneic host invading spleen, lymph nodes and liver. A cell line (LBC) was developed from the original tumor. Both the original tumor and the cell line have been characterized as expressing the Thy 1+, CD3-, CD25+, MHC class I+, class II-, CD4- (original tumor), CD4+ (cell line), CD8+, gp70-, J11d.2+ phenotypes. Immunization of syngeneic mice with irradiated LBC cells induced cytotoxic T lymphocytes as well as anti-LBC antibodies which reacted with components of 14, 16 and 27 kDa present on LB tumor cells, LBC cell line and normal thymocytes but not on normal lymph node cells. Immunization of syngeneic mice with LBC cells partially protected them against subsequent challenge with the original tumor cells. The effect of sera from tumor-bearing mice and the super-natants from short term cultures were studied on cell proliferation. An inhibitory activity was demonstrated in these fluids, which was abrogated by addition of exogenous IL-2. ELISA showed the presence of soluble IL-2R alpha chain both in the conditioned medium as well as in the serum, which was demonstrated to be responsible for the inhibitory activity. The soluble IL-2R was produced by LB leukemic cells and exerted the inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL-2. Using reverse-transcription PCR, mRNA for IL-2 was found to be present in tumor cells. Our findings indicate that LB cell proliferation is mediated by an autocrine pathway involving endogenous IL-2 generation, despite the fact that these cells are not dependent on exogenous IL-2 to grow in culture. The relationship between tumorigenicity and expression of MHC class II was also investigated. In vitro treatment with IFN-gamma failed to induce the expression of class II antigens in LBC cell line. Therefore these cells were tri-transfected by a liposome-mediated protocol with 1-A alpha d, I-A beta d genes and pSV2neo. Cells were selected to grow in medium containing Genetecin (G418) and surviving transfectants were cloned. Three I-A+ clones were obtained (LBCT) and were used to induce a specific CTL response against tumor cells. Syngeneic mice inoculated with 10(3) LBCT cells failed to develop a tumor while the DT50 of mice injected with 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). It is suggested that neoexpression of MHC class II molecules enhances anti-tumor response by transforming tumor cells into professional antigen-presenting cells, which may be used to improve tumor-specific immunity in the autologous host.
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PMID:[Immunobiological characterization of murine LB leukemia and the LBC cell line]. 922 74

To generate non-small cell lung cancer (NSCLC)-reactive lymphocytes, we transfected an HLA-A2-expressing human NSCLC line (1355) with the cDNA encoding the lymphocyte co-stimulatory molecule CD80. Following selection in G418, 1355.7 demonstrated stable cell-surface expression of CD80. Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established in 600 IU/ml IL-2 using HLA-A2+ normal donor peripheral blood mononuclear cells (PBMCs) stimulated with 1355-P (parental), 1355.7 or IL-2 alone. In 7 of 9 MLTCs, those stimulated with 1355.7 demonstrated enhanced growth after 30 to 45 days of culture. The predominant lymphocyte to grow in all MLTCs was a CD3+alphabeta+CD4+ T cell. In one case, lymphocytes stimulated with 1355.7 (MLTC 2389.7) exhibited preferential lysis of 1355. MLTC 2389.7 was cloned by limiting dilution, and 2 resultant cloids were shown to be NSCLC-reactive and dependent on both MHC class 1 and CD3 in their recognition of tumor cells. Additionally, allogeneic MLTCs were established using three HLA-A2+ NSCLC patients' PBMC. The predominant lymphocyte to grow in these MLTCs was a CD3+ alphabeta+CD8+ T cell. In cytotoxicity studies, MLTC-UKY25.7 demonstrated preferential lysis of 1355-P, 1355.7 and an HLA-A2+ NSCLC cell line, 1650. Lymphocytes from this MLTC did not lyse K562, Daudi or an HLA-A2- NSCLC cell line, 647. Our data suggest that CD80-expressing NSCLC tumor cells may enhance the generation of specific CTLs in vitro. These CTLs could be important reagents for use in cellular immunotherapy and/or in isolating tumor antigens for potential tumor vaccine development.
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PMID:CD80 expression in an HLA-A2-positive human non-small cell lung cancer cell line enhances tumor-specific cytotoxicity of HLA-A2-positive T cells derived from a normal donor and a patient with non-small cell lung cancer. 983 60

Staphylococcus enterotoxin A (SEA) stimulates T cells bearing certain TCR beta-chain variable regions, when bound to MHC-II molecules, and is a potent inducer of CTL activity and cytokines production. To decrease toxicity of SEA to the normal MHC-II(+) cells and to localize the immune response induced by SEA to the tumor site, my colleague previously genetically fused SEA with B7.1 transmembrane region (named as SEAtm) to make SEA express on the surface of tumor cells and tumor cells modified with SEAtm could induce efficient antitumor immunity in vitro. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger antitumor immune responses than single-modified vaccines. In this study, we modified the tumor cell vaccine with B7.1 and SEAtm to improve efficiency in the application of SEA. First, SEAtm gene was subcloned from recombinant plasmid pLXSNSEP by PCR and murine B7.1 gene was cloned from splenocytes derived from C57BL/6 mice by RT-PCR. Then, the eukaryotic co-expression vector of SEA and murine B7.1 gene was constructed and named as pcDNA-BIS. B16 cell lines stably expressing SEA and/or B7.1 were established by screening with G418 after transfection and inactivated for the preparation of tumor cell vaccines to treat mice bearing established B16 tumors. The results indicated that the dual-modified tumor cell vaccine B16/B7.1+SEAtm (B16-BIS) elicited significantly stronger antitumor immune responses in vivo when compared with the single-modified tumor cell vaccines B16/B7.1 (B16-B7.1) and B16/SEAtm (B16-SEAtm), and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine with superantigen and co-stimulatory molecule.
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PMID:Tumor cells with B7.1 and transmembrane anchored staphylococcal enterotoxin A generate effective antitumor immunity. 1681 54


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