RESUMO
The expression of the interleukin 4 (IL-4) receptor (IL-4R) and effects of human recombinant IL-4 on human gastric carcinoma cell lines were studied. We demonstrated that IL-4 inhibited the growth of gastric carcinoma cells in a dose dependent manner (0.1-100 units/ml) in a [3H]thymidine incorporation proliferation assay. The gastric carcinoma cells varied in sensitivity to treatment with low dose IL-4. Treatment of cells with IL-4 altered the morphology of the cells to a "flattened" morphological shape resembling differentiation. The IL-4-mediated growth inhibition was significantly abrogated by neutralization of IL-4 with specific anti-IL-4 antibody. IL-4R expression on the cell surface was determined by assessing biotin-labeled IL-4 binding to cells using flow cytometry. IL-4R expression ranged from 5 to 85% of total cell population in the gastric carcinoma cell lines assessed. There was a positive correlation between the sensitivity to IL-4-mediated growth inhibition and IL-4R expression. By Northern blot analysis, we demonstrated that mRNA of IL-4R was expressed in the gastric carcinoma cells. Using in situ hybridization, we confirmed that IL-4R mRNA was expressed in the gastric carcinoma cell at the single cell level. By using a sensitive polymerase chain reaction technique, we demonstrated that gastric carcinoma cells expressed IL-4 mRNA, suggesting a possible autocrine loop. These studies indicate that IL-4 can significantly modulate gastric carcinoma cells that possess IL-4R. IL-4R on gastric carcinoma cells may be a potential therapeutic target site for IL-4-directed therapy.
Assuntos
Interleucina-4/farmacologia , Receptores Mitogênicos/análise , Neoplasias Gástricas/patologia , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Membrana Celular/química , Relação Dose-Resposta a Droga , Humanos , Interleucina-4/metabolismo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores de Interleucina-4 , Receptores Mitogênicos/metabolismo , Neoplasias Gástricas/química , Neoplasias Gástricas/metabolismo , Células Tumorais CultivadasRESUMO
We have previously reported that interleukin 4 (IL-4) inhibits the growth of human gastric carcinoma cells. To investigate the mechanism for this inhibition we analyzed the effect of IL-4 on cell cycle progression of the IL-4-sensitive gastric carcinoma cell line, HTB-135. IL-4 significantly inhibited cell cycle G1-S-phase progression. To assess the postreceptor molecular events that transduced the negative-growth signals by IL-4, we analyzed the expression of cell cycle nuclear-regulating factors such as retinoblastoma gene product (Rbp), c-myc, c-myc protein (c-mycp), and cyclin D1 expression which are known to be regulators of G1-S-phase transition. IL-4 was found to induce an unphosphorylated form of Rbp within 24 h and significantly reduce the phosphorylated form at 48 h. The transition of Rbp to a hypophosphorylated form concurs with the decrease in c-myc gene expression and c-mycp. In addition, we demonstrated that IL-4 down-regulated p34cdc2, a kinase associated with Rbp phosphorylation and cyclin D1. Cyclin D1, considered as a critical nuclear regulatory factor of G0-G1 to S-phase transition was down-regulated 24 and 48 h post-IL-4 treatment as well. These studies suggest that IL-4 inhibits gastric cell proliferation by blocking cell cycle progression by down-regulating several key G0-G1 cell cycle nuclear-regulating factors.
Assuntos
Adenocarcinoma/patologia , Fase G1/efeitos dos fármacos , Interleucina-4/farmacologia , Neoplasias Gástricas/patologia , Proteína Quinase CDC2/análise , Ciclinas/análise , Genes myc , Humanos , Proteína do Retinoblastoma/análise , Células Tumorais CultivadasRESUMO
A preclinical model was used to determine if transfection of the interleukin 2 (IL-2) gene into human melanoma cells would augment the response of autologous and allogeneic peripheral blood lymphocytes (PBLs) from melanoma patients. IL-2 gene was transfected into three human melanoma cell lines; secretion of IL-2 from stable transfected cells was confirmed by enzyme-linked immunosorbent assay. The PBL response to these melanoma cells was then examined in a mixed-lymphocyte tumor reaction using PBLs from eight melanoma patients. The PBL response to autologous (P < 0.01) or human leukocyte antigen A cross-reactive (P < 0.05) transfected melanoma cells was significantly higher than it was to nontransfected melanoma cells. These data suggest that IL-2 gene transfection may be an important strategic approach to enhancing specific immune responses induced by a polyvalent melanoma cell vaccine.
Assuntos
Interleucina-2/genética , Melanoma/imunologia , Transfecção , Sequência de Bases , Terapia Genética , Antígenos HLA-A/imunologia , Humanos , Imunidade Celular , Imunoterapia , Melanoma/terapia , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Cytotoxic T cells have been implicated in the control of the progression of human melanoma. Most studies on human tumor T cell immunity have focused on the CD3+CD8+ cytotoxic T lymphocyte (CTL) phenotype; however, CD3+CD4+ CTL are important effector cells in other diseases and may also contribute to antimelanoma immunity. In this study we compared the functional activity of CD3+CD4+ and CD3+CD8+ CTL lines generated against autologous melanoma cells. CD8+ CTL had twofold higher cytotoxicity and serine esterase activity than CD4+ CTL. CD8+ CTL also were better binders to autologous melanoma cells. Binding of both CD4+ and CD8+ CTL to melanoma cells was significantly inhibited by ICAM-1 mAb. Interleukin-2 (IL-2) and IL-4 secretion was induced in both CD4+ and CD8+ CTL after stimulation by melanoma cells. A reverse transcriptase polymerase chain reaction performed on specific messenger RNA showed that both CD4+ and CD8+ CTL expressed IL-1, IL-2 and IL-4; CD4+ CTL also expressed interferon gamma (IFN). Both CTL phenotypes expressed receptors for IL-2 and IFN but only CD4+ CTL expressed the receptor for IL-4. Methods to augment CD4+ CTL growth were assessed using different combinations of cytokines. The combination of IL-2, IL-4 and IFN provided the optimal stimulation. Treatment of melanoma target cells with IL-4 and IFN enhanced CD4+ CTL recognition activity. CD4+ T cells are associated with antigen memory response and helper function, therefore activation of CD4+ CTL may be more beneficial with respect to long-term protective antimelanoma immunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunoterapia , Melanoma/imunologia , Melanoma/terapia , Sequência de Bases , Relação CD4-CD8 , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Adesão Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Citocinas/farmacologia , Citocinas/fisiologia , Citotoxicidade Imunológica/imunologia , Esterases/sangue , Humanos , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Melanoma/patologia , Dados de Sequência Molecular , Fenótipo , Receptores de Citocinas/fisiologia , Células Tumorais CultivadasRESUMO
The MAGE-1 gene was recently characterized to encode an immunogenic tumor Ag on several types of human tumors, including melanoma. This Ag is expressed in a wide variety of human tumors and not in normal cells, except testicular tissue, as assessed through specific mRNA analysis. In this study we cloned the MAGE-1 gene exon 3 region from a colon carcinoma cell line and expressed it in Escherichia coli. The recombinant MAGE-1 protein was affinity purified. By using Western blot analysis, IgG and IgM anti-MAGE-1 Abs were detected in the sera of melanoma patients. Fifty-three patients immunized with a melanoma cell vaccine (MCV) were assessed for anti-MAGE-1 IgG responses by using a MAGE-1 Ag-specific ELISA. The MCV consisted of three melanoma cell lines that expressed MAGE-1. Comparisons of anti-MAGE-1 IgG response pre-MCV treatment with 12- to 16-wk post-MCV treatment were made. Fifty-seven percent of the patients immunized with the MCV showed significant enhancement of IgG response to recombinant MAGE-1 protein. Patients who responded had no particular HLA-A or -B allele expression pattern. Melanoma patients immunized with whole cell MCV containing MAGE-1 can enhance anti-MAGE-1 IgG Abs. Recombinant MAGE-1 protein can be used to assess patient response to MAGE-1 and will be investigated as a potential cancer vaccine against a wide variety of human tumors that express MAGE-1.
Assuntos
Anticorpos Antineoplásicos/biossíntese , Antígenos de Neoplasias/imunologia , Melanoma/imunologia , Proteínas de Neoplasias , Vacinas/imunologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Ensaio de Imunoadsorção Enzimática , Antígenos HLA-A/imunologia , Humanos , Imunoglobulina G/biossíntese , Antígenos Específicos de Melanoma , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/imunologiaRESUMO
A 2.1-kilobase cDNA coding for a surface protein of mammary epithelial cells has been isolated from a mouse mammary gland lambda gt11 cDNA library. Sequence analysis of this cDNA reveals an open reading frame of 1389 base pairs that defines a protein with a molecular mass of 51.5 kDa. Structural analysis of the predicted sequence identifies two putative functional domains of the protein: (i) an N-terminal cysteine-rich region that is similar to epidermal growth factor-like domains of Drosophila Notch-1 protein and (ii) a large segment of the sequence that exhibited 54.5% identity with C-terminal domains of human coagulation factors VIII and V. These similarities in structure are used to predict the possible functions of the protein and its means of interaction with the cell surface. mRNA expression was detectable in mammary tissue from nonpregnant animals but was maximal in the lactating gland. In cultured cells, mRNA levels also correlated with the degree of cellular differentiation.
Assuntos
Fator de Crescimento Epidérmico/genética , Fator VIII/genética , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica , Biblioteca Gênica , Glândulas Mamárias Animais/metabolismo , Camundongos , Dados de Sequência Molecular , Mucina-1 , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Development of effective cancer vaccines would help prevent and control tumor progression. A novel approach of immunizing against tumor antigens is in vivo gene vaccination. We have developed a fusigenic viral liposome vector using HVJ (hemagglutinating virus of Japan) and liposome to deliver human tumor antigen genes effectively to cells in vivo. Plasmids containing the human tumor antigen genes MAGE-1 and MAGE-3 were encapsulated in fusigenic viral liposomes and injected into mice intramuscularly. MAGE-1 and -3 recombinant proteins were used in Western blotting and affinity ELISA for assessment of antibody responses. Mice immunized with MAGE-1 and -3 gene vaccine individually were shown to produce anti-MAGE-1 and -3 IgG antibody responses respectively. Animals immunized with plasmid alone did not induce anti-MAGE-1 or -3 IgG responses. Antibody responses could be enhanced on reimmunization with the gene vaccines. Muscle biopsies taken after vaccine injection were verified to express gene-specific mRNA transcripts. Mice immunized with MAGE-1 or -3 gene vaccines were shown to induce antibodies that could cross-react with the respective recombinant proteins. This study demonstrates that in vivo immunization using HVJ-liposome containing human tumor antigen genes can effectively deliver and induce immune responses to the respective whole proteins.