Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Introduction of the interleukin-4 (IL-4) gene into cells derived from human tumor tissue provides a means for generating a specific tumor vaccine. Such a vaccine could be produced by either transducing tumor-derived stromal cells with the IL-4 vector and coinjecting tumor cells, or by transducing the tumor cells themselves. We have developed a protocol for culturing cells from non-small cell lung tumors and routinely produce tumor cultures from 25% of tumors, and stromal cultures from > 80% of specimens. Several of these cultures were transduced with the incompetent retroviral vector G1NaSvi4.25, which encodes the human IL-4 cDNA and the G418-resistance gene. Infection of cells by viral titers of 2-5 x 10(4) plaque-forming units/ml, and a multiplicity of infection of 0.1:1 to 1:1 yielded transfer efficiencies of 3.3-32.0 transfectants per 10(4) cells in six of eight attempts. Following selection with the neomycin analog G418, IL-4-producing cells were isolated. IL-4 titers ranged from 142 to 593 U/ml/10(6) in a 24-h collection. Successful transfer of the IL-4 gene was demonstrated by polymerase chain reaction amplification of cDNA derived from reverse-transcribed total RNA, by immunohistochemistry, and by enzyme-linked immunosorbent assay. The IL-4-producing cells were shown to stimulate the proliferation of autologous peripheral blood lymphocytes in one individual by 7.5-fold over control and by 4.1-fold over non-IL-4 producing tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transfer and expression of the human interleukin-4 gene in carcinoma and stromal cell lines derived from lung cancer patients. 828 Jul 14

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

Human autologous dermal fibroblasts have been cultured, transduced with the interleukin-4 (IL-4) gene and used as a vaccine together with irradiated autologous tumor cells in patients with cancer participating in a phase I/II clinical trial at the University of Pittsburgh Cancer Institute. In support of this clinical trial, methods have been devised to facilitate isolation of fibroblasts from freshly harvested skin specimens, to enhance their outgrowth in large-scale cultures, and to assay cytokine (IL-4) production following transduction with the cytokine gene +/- irradiation. Fibroblasts were isolated from skin specimens by enzymatic digestion, grown in primary cultures, and transduced with a retroviral vector containing the gene for human IL-4 and the NeoR gene as a selectable marker. Following selection in G418, the irradiated, IL-4-producing fibroblasts were administered to patients in a vaccine containing irradiated autologous tumor cells. Seventy-eight specimens of human skin were processed to obtain fibroblast suspensions. Cultures of fibroblasts were established from 68 of the 78 specimens (87%). Of 33 transduced and selected fibroblast cultures, 21 produced at least 1,000 units of IL-4/24 hours per 10(6) cells, as determined by ELISA, and 17/33 or 51% were used for therapy. The primary cultures were typically maintained for up to seven or eight passages. The mean +/- SD overall time for obtaining a required number of transduced, selected cells was 53 +/- 4 days. The fibroblasts continued to produce IL-4 in culture for 3 weeks even after irradiation. Similar results have been obtained with a retroviral vector encoding IL-12. This study shows that human dermal fibroblasts can be consistently and reproducibly expanded and genetically modified to serve as a source of cytokines or other gene products for gene therapy trials.
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PMID:Successful culture and selection of cytokine gene-modified human dermal fibroblasts for the biologic therapy of patients with cancer. 880 Jul 42

The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.
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PMID:Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus. 1067 Jun 49