Construction of autocatalytic caspase-3 driven by amplified human telomerase reverse transcriptase promoter and its enhanced efficacy of inducing apoptosis in human ovarian carcinoma
- VernacularTitle:人端粒酶逆转录酶启动子-二步转录增强系统调控下的活性半胱氨酸天冬氨酸蛋白酶3的构建及其对卵巢上皮性癌的治疗作用
- Author:
Yue SONG
;
Keng SHEN
;
Chun-Xia HE
;
- Publication Type:Journal Article
- Keywords:
Ovarian neoplasms;
Telomerase;
Promoter regions(genetics);
Caspases
- From:
Chinese Journal of Obstetrics and Gynecology
2000;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct recombinant adenoviral vector expressing autocatalysis caspase- 3 driven by human telomerase reverse transcriptase promoter amplified by two-step transcription amplification (hTERTp-TSTA),and investigate its antitumor effect in ovarian cancer iri vitro and in vivo.Methods Recombinant adenoviruses expressing autocatalytic caspase-3(rev-caspase-3)driven by hTERTp-TSTA were prepared,which were named as AdHTVP2G5-rev-casp3.AdHT-rev-casp3,Ad-rev-casp3 and AdHTVP2G5- EGEP,which express rev-easpase-3 driven by hTERTp,cytomegalovirus promoter(CMVp)and enhanced green fluorescent protein(EGFP),respectively,were used as controls.Western blot,cell counting kit (CCK-8),flow cytometry(FCM)and TdT-mediated dUTP-biotin nick end labeling(TUNEL)were used to detect the expression of p17,active subunit of caspase-3,and p85,and to measure cell survival rates, apoptotic rates and cell cycle distribution in ovarian cell line AO and normal human umbilical vein endothelial cell line HUVEC,following treatments of AdHTVP2G5-rev-casp3.subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/e nude mice were established.Following treatments of AdHTVP2G5-rev-easp3,western blot was used to detect the expression of active caspase-3 in abdominally spread tumors and liver tissues,respectively,and the mouse survival rates and the volume of tumor nodules were measured,and the serum level of alanine transaminase (ALT)and aspartate transaminase(AST)were analyzed to monitor liver damages and HE staining was used to detect the histopathological changes of various organs.Results The levels of p17 expression in AdHTVP2G5-rev-casp3-treated AO cells were significantly higher than that in Ad-rev-casp3 or AdHT-rev- casp3 treated AO cells,while no expression was observed in AdHTVP2G5-rev-casp3-treated HUVEC.There was strong cell killing of AdHTVP2G5-rev-casp3 of hTERT positive AO cells,but not of the hTERT-negative HUVEC cells.Cell survival rate and apoptotic rate of AO cells treated with AdHTVP2G5-rev-casp3 were 17.8% and 40.2%,respectively,significantly different from that treated with AdHT-rev-casp3(75.2% and 16.1%)at the multiplicity of infection(MOI)of 70(P0.05) .Significant expressions of active caspase-3 were shown in AdHTVP2G5-rev-casp3-treated tumors,whereas no expression was shown in liver.In contrast,both tumors and liver tissues showed active caspase-3 expression following treatments of Ad-rev-casp3.AdHTVP2G5-rev-casp3 and Ad-rev-casp3 prolonged mouse survival[mean survival time of(259?14)d and(213?16)d],when compared with treatment with AdHT- rev-casp3[(177?12)d]and AdHTVP2G5-EGFP[(109?7)d;P
