RESUMO
To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy. In the present study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and enhanced green fluorescent protein (EGFP) separately with internal ribosome entry site (IRES). We first examined the expression and location of WEE1Hu in Chinese hamster ovary (CHO) cells and showed that WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells, which were proved by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Furthermore, inhibition of cisplatin-induced apoptosis was observed in CD34+ cells transfected with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our study suggests that the introduction of two functional genes via bicistronic vector is more powerful and efficient than single gene therapy.
Assuntos
Proteínas de Ciclo Celular/genética , Genes/genética , Proteínas Nucleares/genética , Plasmídeos/genética , Proteínas Tirosina Quinases/genética , Fator de Células-Tronco/genética , Animais , Antígenos CD34/análise , Antígenos CD34/sangue , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células , Clonagem Molecular , Cricetinae , Sangue Fetal , Imunofluorescência , Humanos , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
BACKGROUND & OBJECTIVE: Histone deacetylase (HDAC) shows a high expression in many cancer cells and the inhibitor of HDAC1, trichostatin A (TSA), can inhibit the growth of cancer cells. Hypoxia is a common feature of malignant tumors. This paper was designed to investigate the expression of HDAC1 of A549 cell strains in hypoxia condition and the effect of TSA on their proliferation and apoptosis. METHODS: The authors designed 1 normoxia group (control group) and 5 hypoxia groups (test groups): hypoxia 6h group (A), TSA + hypoxia 6h (B), hypoxia 12h group (C), hypoxia 24h group (D), TSA + hypoxia 24h (E), hypoxia 48h group (F). The expression of HDAC1 in A549 cells was examined using Western blot analysis. Proliferation, the apoptotic rates of A549 cells and the effect of TSA on them were determined using MTT method, immunohistochemistry, TUNEL method, and flow cytometry. The expression of mRNA of HDAC1 and the effect of TSA on it were determined using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The A values expressed by HDAC1 in A549 cell strains were 138+/-11 in the control group, 78+/-4, 86+/-5, 124+/-3, and 120+/-9 in test groups A, C, D, and F, respectively. The A values of HDAC1mRNA versus the A values of beta-Atin mRNA were 0.68+/-0.03 in the control group, 0.46+/-0.03, 0.45+/-0.02, 0.70+/-0.03, and 0.33+/-0.02 in test groups A, C, D, and F, respectively. The A values of the expression of PCNA in A549 cell strains were 0.13+/-0.03 in the control group, 0.10+/-0.02, 0.11+/-0.02, 0.16+/-0.02, and 0.11+/-0.03 in test groups A, B, D, and E, respectively. The A values of MTT in A549 cell strains were 0.50+/-0.06 in the control group, 0.41+/-0.04, 0.45+/-0.03, 0.59+/-0.02, and 0.45+/-0.03 in test groups A, B, D, and E, respectively. The A values of positive cells of apoptosis in A549 cell strains were 0.16+/-0.04 in the control group, 0.18+/-0.02, 0.18+/-0.05, 0.20+/-0.05, and 0.23+/-0.05 in test groups A, B, D, and E, respectively. The apoptotic rates in A549 cells were 1.11% in the control group, 18.91%,14.30%, 36.99%, and 51.92% in test groups A, B, D, and E, respectively. CONCLUSION: The expression of HDAC1 plays an important role in the proliferation and apoptosis of A549 cells, which is regulated by hypoxia. TSA may serve as a new target for therapy of lung cancer.
Assuntos
Apoptose/efeitos dos fármacos , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pulmonares/enzimologia , Divisão Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular Tumoral , Histona Desacetilase 1 , Humanos , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Antígeno Nuclear de Célula em Proliferação/análise , RNA Mensageiro/análiseRESUMO
OBJECTIVE: To explore the effects of survivin antisense RNA on taxol-induced apoptosis in leukemia cell line HL-60. METHODS: A survivin antisense eukaryotic vector pcDNA3-SVVas was transferred into HL-60 cells by electroporation. The live fraction was determined by trypan blue dye exclusion assay. Cell counting and MTT assay were performed to evaluate the sensibility of the transfected cells to taxol. Apoptosis was detected by DNA gel electrophoresis and nuclear staining. RESULTS: Two positive cell clones, HL-60 SVVas and HL-60 neo were obtained. Compared to HL-60 and HL-60 neo cells, HL-60 SVVas cells growth was significantly reduced (P < 0.05). By MTT assay, the IC(50) of taxol to HL-60 SVVas, HL-60 neo and HL-60 cells were (14.4 +/- 1.87) ng/ml, (31.9 +/- 6.38) ng/ml and (32.0 +/- 3.52) ng/ml, respectively, the difference was significant by statistic analysis (P < 0.01). Agarose gel electrophoresis of genomic DNA from HL-60 SVVas showed typical DNA ladder, but DNA from HL-60 neo and HL-60 did not. Nuclei become condense in HL-60 SVVas cells. CONCLUSION: Survivin antisense RNA could enhance taxol-induced apoptosis in leukemia cell line HL-60. This may lay an experimental foundation for further research of gene therapy in leukemia.
