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English Abstract
Journal Article
Research Support, Non-U.S. Gov't
[Procaspase-3 enhances in vitro effect of cytosine deaminase-thymidine kinase fusion disuicide gene therapy system on human ovarian carcinoma].
Zhonghua Fu Chan Ke za Zhi 2005 June
OBJECTIVE: To investigate whether procaspase-3 could enhance the in vitro effect of cytosine deaminase-thymidine kinase/5-fluorocytosine + ganciclovir (CD-TK/5-FC + GCV) system in human ovarian cancer cells.
METHODS: Eukaryotic expression vectors containing procaspase-3 gene (pcDNA3-casp3) and CD-TK fusion disuicide genes regulated by human telomerase reverse transcriptase (hTERT) promoter (pBTdel-279-CD-TK) were constructed. Western blot was used to detect the expression of procaspase-3 protein in 3AO cells with transfection of pcDNA3-casp3 (3AO-pcDNA3-casp3) or pcDNA3 (3AO-pcDNA3). RT-PCR was used to detect the expression of CD and TK genes in 3AO-pcDNA3-casp3 or 3AO-pcDNA3 cells after transfection with pBTdel-279-CD-TK or pBTdel-279. Following the treatment with 5-FC + GCV, the cell survival rate and cell cycle distribution were detected by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM). Fluorogenic substrate assay and western blot were used to detect caspase-3 activity in these cells.
RESULTS: Procaspase-3 protein was expressed in 3AO-pcDNA3-casp3 cells, while not in 3AO-pcDNA3. The expressions of CD and TK genes were observed in both 3AO-pcDNA3-casp3 and 3AO-pcDNA3 after transfection with pBTdel-279-CD-TK. After treatment with 5-FC + GCV, the survival rate of 3AO-pcDNA3-casp3 + pBTdel-279-CD-TK was significantly lower than that of 3AO-pcDNA3 + pBTdel-279-CD-TK cells. After treatment with 5-FC (2 mmol/L) + GCV (10 microg/ml), there was a higher apoptotic ratio (37.98%) and S-phase block (49.67%) in 3AO-pcDNA3-casp3 + pBTdel-279-CD-TK than in 3AO-pcDNA3 + pBTdel-279-CD-TK cells (21.34% and 35.76%, respectively) (P < 0.05). Following the action of 5-FC (1 mmol/L) + GCV (1 microg/ml) for 48 h, the relative activity of caspase-3 in 3AO-pcDNA3-casp3 + pBTdel-279-CD-TK cells was 189.7, significantly higher than 44.9 in 3AO-pcDNA3 + pBTdel-279-CD-TK cells (P < 0.05).
CONCLUSION: Co-expression of procaspase-3 may lead to a significant enhancement of the efficacy of CD-TK fusion gene therapy.
METHODS: Eukaryotic expression vectors containing procaspase-3 gene (pcDNA3-casp3) and CD-TK fusion disuicide genes regulated by human telomerase reverse transcriptase (hTERT) promoter (pBTdel-279-CD-TK) were constructed. Western blot was used to detect the expression of procaspase-3 protein in 3AO cells with transfection of pcDNA3-casp3 (3AO-pcDNA3-casp3) or pcDNA3 (3AO-pcDNA3). RT-PCR was used to detect the expression of CD and TK genes in 3AO-pcDNA3-casp3 or 3AO-pcDNA3 cells after transfection with pBTdel-279-CD-TK or pBTdel-279. Following the treatment with 5-FC + GCV, the cell survival rate and cell cycle distribution were detected by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM). Fluorogenic substrate assay and western blot were used to detect caspase-3 activity in these cells.
RESULTS: Procaspase-3 protein was expressed in 3AO-pcDNA3-casp3 cells, while not in 3AO-pcDNA3. The expressions of CD and TK genes were observed in both 3AO-pcDNA3-casp3 and 3AO-pcDNA3 after transfection with pBTdel-279-CD-TK. After treatment with 5-FC + GCV, the survival rate of 3AO-pcDNA3-casp3 + pBTdel-279-CD-TK was significantly lower than that of 3AO-pcDNA3 + pBTdel-279-CD-TK cells. After treatment with 5-FC (2 mmol/L) + GCV (10 microg/ml), there was a higher apoptotic ratio (37.98%) and S-phase block (49.67%) in 3AO-pcDNA3-casp3 + pBTdel-279-CD-TK than in 3AO-pcDNA3 + pBTdel-279-CD-TK cells (21.34% and 35.76%, respectively) (P < 0.05). Following the action of 5-FC (1 mmol/L) + GCV (1 microg/ml) for 48 h, the relative activity of caspase-3 in 3AO-pcDNA3-casp3 + pBTdel-279-CD-TK cells was 189.7, significantly higher than 44.9 in 3AO-pcDNA3 + pBTdel-279-CD-TK cells (P < 0.05).
CONCLUSION: Co-expression of procaspase-3 may lead to a significant enhancement of the efficacy of CD-TK fusion gene therapy.
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