RESUMEN
We characterized new transgenic varieties of poplar with multiple insect-resistant and salt stress tolerant genes. Two insect-resistant Bacillus thuringiensis (Bt) genes, Cry1Ac and Cry3A, and a salt-tolerant gene, Betaine aldehyde dehydrogenase (BADH) were inserted into a vector, p209-Cry1Ac-Cry3A-BADH. The clone of Populus x euramericana was transformed by the vector using the Agrobacterium-mediated method. Three transgenic lines were assessed using genetic detection and resistance expression analysis. PCR revealed that exogenous genes Cry1Ac, Cry3A, BADH and selective marker gene NPTII were present in three transgenic lines. Quantitative real-time PCR (qPCR) showed significant differences in the transcriptional abundance of three exogenous genes in different lines. Results of assays for Bt toxic proteins showed that the Cry1Ac and Cry3A toxic protein content of each line was 12.83-26.32 and 2108.91-2724.79 ng/g, respectively. The Cry1Ac toxic protein content of different lines was significantly different; the Cry3A toxic protein content was about 100 times higher than that of the Cry1Ac toxic protein. The insect-resistance test revealed the mortality rate of transgenic lines to Hyphantria cunea L1 larvae varied by 42.2-66.7%, which was significantly higher than non-transgenic lines. The mortality rate of L1 and L2 Plagiodera versicolora larvae was 100%. The insecticidal effect of transgenic lines to P. versicolora larvae was higher than that to H. cunea larvae. NaCl stress tolerance of three transgenic lines under 3-6% NaCl concentration was significantly higher than that of non-transgenic lines.
Asunto(s)
Control Biológico de Vectores/métodos , Populus/genética , Tolerancia a la Sal/genética , Transgenes , Bacillus thuringiensis/genética , Toxinas Bacterianas/genética , Populus/parasitología , Populus/fisiologíaRESUMEN
MP [4-(3',3'-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27(KIP1) protein and p21(CIP1) mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21(CIP1), p16(INK4a) and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzofuranos/administración & dosificación , Endófitos/química , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Xylariales/química , Proteínas Reguladoras de la Apoptosis/genética , Benzofuranos/aislamiento & purificación , Proteínas de Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cycadopsida , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Citometría de Flujo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/efectos de los fármacos , Genes p16/efectos de los fármacos , Células HeLa , Humanos , Proteínas Nucleares/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/efectos de los fármacos , Transcripción Genética , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/efectos de los fármacosRESUMEN
MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1 , p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.
Asunto(s)
Humanos , Apoptosis/efectos de los fármacos , Benzofuranos/administración & dosificación , Endófitos/química , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Xylariales/química , Proteínas Reguladoras de la Apoptosis/genética , Benzofuranos/aislamiento & purificación , Proteínas de Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , /efectos de los fármacos , /efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Citometría de Flujo , Factores de Transcripción Forkhead/efectos de los fármacos , Cycadopsida , /efectos de los fármacos , Células HeLa , Proteínas Nucleares/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Genética , Factores de Transcripción/efectos de los fármacos , Proteínas Supresoras de Tumor/efectos de los fármacosRESUMEN
The tissue inhibitor of metalloproteinases (TIMP)-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs) but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line), L-02 (a normal liver cell line) and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma) using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60 percent and reduced the migration to about 30 percent. Meanwhile, our data showed that interleukin-6 (IL-6) (100 ng/mL) could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.
Asunto(s)
Humanos , Carcinoma Hepatocelular/genética , /genética , Neoplasias Hepáticas/genética , Metaloproteinasa 9 de la Matriz/genética , /genética , Inhibidor Tisular de Metaloproteinasa-1/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , /metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , /metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Regulación hacia ArribaRESUMEN
The tissue inhibitor of metalloproteinases (TIMP)-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs) but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line), L-02 (a normal liver cell line) and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma) using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60% and reduced the migration to about 30%. Meanwhile, our data showed that interleukin-6 (IL-6) (100 ng/mL) could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.