Subject(s)
Humans , Anticholesteremic Agents/therapeutic use , Apolipoproteins B/genetics , Muscular Atrophy, Spinal/drug therapy , Oligonucleotides, Antisense/therapeutic use , RNA Splicing/genetics , SMN Complex Proteins/genetics , Exons/genetics , Models, Biological , Muscular Atrophy, Spinal/genetics , Oligonucleotides, Antisense/geneticsABSTRACT
Suvivin is a novel member of the inhibitor of apoptosis protein (IAP) family, which is known to be over-expressed in various carcinomas and associated with their biologically aggressive characteristics. The aim of this study was to investigate survivin expression in human medullary thyroid carcinoma (MTC) and a MTC cell line TT, correlate suvivin expression with clinicopathologic features of MTC, and test effects of antisurvivin oligonucleotides (ASODNs) on growth and apoptosis of TT cells. Survivin expression was immunohistochemically determined in formalin-fixed and paraffin-embedded specimens obtained from 10 cases of normal thyroid (NT) and 10 cases of MTC, and in TT cells. In TT cells, we confirmed survivin expression and its down-regulation by ASODNs using RT-PCR and Western blot analyses, and investigated effects of ASODNs on viability and growth by MTT assay and apoptosis by apoptotic analyses including DNA laddering assay, acridine orange/ethidium bromide staining and flow cytometric cell cycle analysis. Immunohistochemical analysis showed high survivin expression in MTC and TT cells, whereas no immunoreactivity was detectable in NT. Statistical analyses revealed no significant correlation of survivin expression with the clinicopathologic features of MTC. In TT cells, survivin expression at both mRNA and protein levels was confirmed and could be down-regulated by ASODNs concomitant with decrease in viability and growth, and increase in apoptosis. Our results suggest that survivin plays an important role in MTC independent of the conventional clinicopathologic factors, and ASODNs is a promising survivin-targeted gene therapy for MTC.
Subject(s)
Male , Humans , Female , Adult , Time Factors , Thyroid Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Oligonucleotides, Antisense/genetics , Neoplasm Proteins/genetics , Microtubule-Associated Proteins/genetics , Gene Expression Regulation, Neoplastic/drug effects , Down-Regulation/drug effects , Dose-Response Relationship, Drug , Cell Survival/drug effects , Cell Proliferation/drug effects , Cell Line, Tumor , Carcinoma, Medullary/metabolism , Apoptosis/drug effectsABSTRACT
The HL-60 cells were transfected with chk1 antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31%, significantly higher than that by the sense blocking (10.34%, 0.025 < P < 0.05). In HL-60 cells transfected with chk1 antisense chain, the G2/M phase arrest was attenuated and the cells in G2/M phase were accounted for 38.42%, significantly lower than those of the cells transfected with chkl sense chain (54.64%, 0.005 < P < 0.01). It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.
Subject(s)
Apoptosis/radiation effects , Cell Cycle/radiation effects , HL-60 Cells , Oligonucleotides, Antisense/genetics , Protein Kinases/genetics , Protein Kinases/metabolism , Radiation Tolerance/genetics , TransfectionABSTRACT
Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912 +/- 0.022 to 0.215 +/- 0. 017, and the protein level of MBD1 gene also decreased from (80.19 +/- 5.05) % to (35.11 +/- 4.05) %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group (P < 0.01). It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.
Subject(s)
Biliary Tract Neoplasms/metabolism , Biliary Tract Neoplasms/pathology , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Eukaryotic Cells/metabolism , Gene Expression Regulation, Neoplastic , Genetic Vectors , Oligonucleotides, Antisense/genetics , Plasmids/genetics , Transcription Factors/biosynthesis , Transcription Factors/genetics , TransfectionABSTRACT
Interleukin (IL)-4 inhibits proliferation of several human cancer cell lines in vitro. Although IL-4 is known to regulate proliferation of lymphocytes by modulating p27KIP1 expression, the mechanism involved in the IL-4-induced growth inhibition of nonhematopoietic cancer cells has not been fully elucidated. Previously, we reported that IL-4 suppressed proliferation of human renal cell carcinoma (RCC) cell lines in vitro. Here, we show that IL-4 inhibits cell cycle progression at the G1 phase in Caki-1 cells by increasing the expression of p21WAF1 and interferon regulatory factor (IRF)-1, and decreasing the cyclin dependent kinase (CDK) 2 activity. Up-regulation of p21WAF1 and IRF-1 expression is transcriptional, but independent of p53. The levels of p21WAF1 and IRF-1 proteins were enhanced as early as 1 h after IL-4 treatment. CDK2 activity started to decline at 4 h after IL-4 treatment, and by 24 h, was ~50% of the control. Neither the protein expressions of p27KIP1 and p16INK4a, nor the phosphorylation level of pRb was changed. The importance of p21WAF1 and IRF-1 in the growth inhibition induced by IL-4 was confirmed by antisense oligonucleotide transfection. Both of p21WAF1 and IRF-1 antisense oligonucleotides prevented IL-4-mediated growth inhibition by ~30% compared to the respective sense oligonucleotides. In summary, our study indicated that p21WAF1 and IRF-1 mediate the growth inhibitory effect of IL-4 in human RCC cells.
Subject(s)
Humans , CDC2-CDC28 Kinases/metabolism , Carcinoma, Renal Cell/genetics , Cell Cycle/drug effects , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , DNA-Binding Proteins/genetics , Gene Expression/drug effects , Interleukin-4/pharmacology , Kidney Neoplasms/genetics , Oligonucleotides, Antisense/genetics , Phosphoproteins/geneticsABSTRACT
La aplicación de la genética molecular a la biología de la enfermedad humana tiene un profundo efecto en la comprensión de los mecanismos moleculares de la patogénesis de la enfermedad. En esta revisión se proporciona una introducción y bosquejo de la terapia génetica, y se presentan y discuten las técnicas más actualizadas para hacer la entrega de genes asi como las hazañas clínicas y experimentales realizadas hasta la fecha. El desarrollo de métodos de entrega de genes a células de mamífero lleva a la posibilidad de tratar la enfermedad humana a través de terapias basadas en genes. Como resultado, conceptos y métodos que podían considerarse ciencia ficción hace 50 años se aplican ahora en el tratamiento de diversas enfermedades. La aplicación de la terapia génica está rompiendo las fronteras tradicionales en la cual la medicina moderna ha estado sumergida. Sin embargo, no obstante los progresos, un gran número de dificultades técnicas necesitan ser resueltas antes de que la terapia génica pueda ser segura y eficientemente aplicada en la clínica. Los desarrollos tecnológicos, particularmente la entrega de genes y el trasplante celular, son críticos para la práctica exitosa de la terapia génica