Objective： To investigate the specific blockage effect of individual antisense RNA on mutant p53 gene in vitro. Methods： The single strand antisense transcription system containing mt-p53 exon 8 sequence （pGEM3zf（...Objective： To investigate the specific blockage effect of individual antisense RNA on mutant p53 gene in vitro. Methods： The single strand antisense transcription system containing mt-p53 exon 8 sequence （pGEM3zf（＋/-）p53exon8） was constructed. The ligation of antisense RNAwith mt-p53 gene was confirmed by in situ hybridization; MDA-MB-231 human breast cancer cells were transfected with ASp53exon8＇RNA cotionic liposome-mediated. Expression of mt-p53 protein was examined by immunocytochemical staining and Western blot. Cell proliferation was evaluated by MTT assay; Cell cycle distribution was determined by flow cytometry （FCM）; Apoptosis was observed by TUNEL. Results： In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. ASp53exon8＇RNA transfection induced inhibition of cell proliferation, G2/M phase arrest and increasing apoptotic rates. In addition, expression of p53 protein was down-regulated. Conclusion： pGEM3zf（＋/-）p53exon8 was well constructed and ASp53exon8＇RNA can block mt-p53 gene expression specifically and then inhibit MDA-MB-231 cell proliferation in vitro, which may serve as therapeutic means for human malignancy.展开更多
Objective： To investigate the relationship between the objective response to combination chemotherapy of taxanes plus cisplatin in non-small cell lung cancer （NSCLC） and docetaxel plus cisplatin （DC regime） induc...Objective： To investigate the relationship between the objective response to combination chemotherapy of taxanes plus cisplatin in non-small cell lung cancer （NSCLC） and docetaxel plus cisplatin （DC regime） induced senescence of tumor cells in vitro. And its relation to mutant P53 protein （m-P53） was also to be evaluated. Methods： Sixty-seven specimen obtained from NSCLC patients from January 1, 2003 to June 30, 2006. The patients consisted of 48 males and 19 females, ranging in age from 54 to 82 years （mean, 67.5 years）, 41 cases were diagnosed as pathological stage Ⅲb, 26 cases were diagnosed as stage Ⅳ. Thirty-nine tumors were confirmed to be adenocarcinomas, 28 were confirmed to be squamous cell carcinomas. All patients accepted 2-6 cycles combination chemotherapy of Taxanes （docetaxel 40 mg/m^2, d1; d8, or paclitaxel 175 mg/m^2, d1） plus cisplatin （CDDP, 25 mg/m^2, d2-4）. Patients were divided into chemoresponsive （CR ＋ PR） and chemoresistant （SD ＋ PD） groups according to objective response status which was evaluated by RECIST system. Tumor cells from specimens of bronchoscopic, surgical biopsy and pleural effusion cell collection had been cultured and treated with DC in vitro. The m-P53 of culture supernatant was measured by ABC-ELISA kit before DC treatment. The telomerase activity was determined by the telomeric repeat amplification protocol （TRAP） based PCR-ELISA kit and apoptosis was determined by TdT-mediated d-UTP-X nick-end labeling （TUNEL） assay. Data represent as both actual detected and positive value. The senescence of tumor cells defined as that, apoptosis rate increased more than 50% to control, and telomerase activity decreased less than 50% to control. Results： There was no significant difference between clinical treatment response and sex, pathological type, specimen origin, or m-P53 status in cultured cell supernatant. Telomerase activity and apoptosis rate was positive in 61.1% （41/67） and 25.4%（17/67） of all samples respectively. A significant difference of senescence of tumor cells treated by DC, was existed between chemoresponsive and chemoresistant patients groups （P 〈 0.05）. Multinomial logistic regression analyses shew that telomerase activity decreased less than 50% in vitro may be an indicator of clinical response for taxanes plus cisplatin chemotherapy. Odds ratio was 4.226, P 〈 0.05. Conclusion： For NSCLC, DC induce lung cancer tumor cells senesce in vitro may be a promising predicator for clinical response, but the relationship between objective response by chemotherapy and detectable m-P53 or DC induced apoptosis is still obscure.展开更多
基金Key Project Fund of Basic Research, Tianjin Municipal Science and Technology Commission (No. 033801511), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2004–2005).
文摘Objective： To investigate the specific blockage effect of individual antisense RNA on mutant p53 gene in vitro. Methods： The single strand antisense transcription system containing mt-p53 exon 8 sequence （pGEM3zf（＋/-）p53exon8） was constructed. The ligation of antisense RNAwith mt-p53 gene was confirmed by in situ hybridization; MDA-MB-231 human breast cancer cells were transfected with ASp53exon8＇RNA cotionic liposome-mediated. Expression of mt-p53 protein was examined by immunocytochemical staining and Western blot. Cell proliferation was evaluated by MTT assay; Cell cycle distribution was determined by flow cytometry （FCM）; Apoptosis was observed by TUNEL. Results： In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. ASp53exon8＇RNA transfection induced inhibition of cell proliferation, G2/M phase arrest and increasing apoptotic rates. In addition, expression of p53 protein was down-regulated. Conclusion： pGEM3zf（＋/-）p53exon8 was well constructed and ASp53exon8＇RNA can block mt-p53 gene expression specifically and then inhibit MDA-MB-231 cell proliferation in vitro, which may serve as therapeutic means for human malignancy.
文摘Objective： To investigate the relationship between the objective response to combination chemotherapy of taxanes plus cisplatin in non-small cell lung cancer （NSCLC） and docetaxel plus cisplatin （DC regime） induced senescence of tumor cells in vitro. And its relation to mutant P53 protein （m-P53） was also to be evaluated. Methods： Sixty-seven specimen obtained from NSCLC patients from January 1, 2003 to June 30, 2006. The patients consisted of 48 males and 19 females, ranging in age from 54 to 82 years （mean, 67.5 years）, 41 cases were diagnosed as pathological stage Ⅲb, 26 cases were diagnosed as stage Ⅳ. Thirty-nine tumors were confirmed to be adenocarcinomas, 28 were confirmed to be squamous cell carcinomas. All patients accepted 2-6 cycles combination chemotherapy of Taxanes （docetaxel 40 mg/m^2, d1; d8, or paclitaxel 175 mg/m^2, d1） plus cisplatin （CDDP, 25 mg/m^2, d2-4）. Patients were divided into chemoresponsive （CR ＋ PR） and chemoresistant （SD ＋ PD） groups according to objective response status which was evaluated by RECIST system. Tumor cells from specimens of bronchoscopic, surgical biopsy and pleural effusion cell collection had been cultured and treated with DC in vitro. The m-P53 of culture supernatant was measured by ABC-ELISA kit before DC treatment. The telomerase activity was determined by the telomeric repeat amplification protocol （TRAP） based PCR-ELISA kit and apoptosis was determined by TdT-mediated d-UTP-X nick-end labeling （TUNEL） assay. Data represent as both actual detected and positive value. The senescence of tumor cells defined as that, apoptosis rate increased more than 50% to control, and telomerase activity decreased less than 50% to control. Results： There was no significant difference between clinical treatment response and sex, pathological type, specimen origin, or m-P53 status in cultured cell supernatant. Telomerase activity and apoptosis rate was positive in 61.1% （41/67） and 25.4%（17/67） of all samples respectively. A significant difference of senescence of tumor cells treated by DC, was existed between chemoresponsive and chemoresistant patients groups （P 〈 0.05）. Multinomial logistic regression analyses shew that telomerase activity decreased less than 50% in vitro may be an indicator of clinical response for taxanes plus cisplatin chemotherapy. Odds ratio was 4.226, P 〈 0.05. Conclusion： For NSCLC, DC induce lung cancer tumor cells senesce in vitro may be a promising predicator for clinical response, but the relationship between objective response by chemotherapy and detectable m-P53 or DC induced apoptosis is still obscure.
