Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of...Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of 13%. The CD had pH optimum of 7.2, a temperature optimum of 40℃ - 45℃, activation of energy (Ea) of 8.4 KJ/mol and a t1/2 of 1.10 hr. A. parasiticus CD stoichiometrically deaminated Cyto-sine and 5-fluorocytosine (5-FC) with the KM values of 0.19mM and 0.30 mM respectively. Studies on the effect of pH on KM and Vmax gave pKa1 of 5.8 and pKa2 of 6.3 with enthalpy of ionization of 43.01 KJ/mole suggesting histidine in the active site. The enzyme was strongly inhibited by Ca2+, Co2+, Zn2+ and Hg2+ and completely inhibited by Cu2+ and Fe2+ at 50 mM. Therefore, A. parasiticus CD can be compared with CDs from other sources that are used in suicide gene therapy.展开更多
Background：Human sulfatase-1 （Hsulf-l） is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans （HSPGs）,altering the binding of several growth factors and cytokines to HSPG t...Background：Human sulfatase-1 （Hsulf-l） is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans （HSPGs）,altering the binding of several growth factors and cytokines to HSPG to regulate cell proliferation,cell motility,and apoptosis.We investigated the role of combined cancer gene therapy with Hsulf-l and cytosine deaminase/5-fluorocytosine （CD/5-FC） suicide gene on a hepatocellular carcinoma （HCC） cell line,HepG2,in vitro and in vivo.Methods：Reverse transcription polymerase chain reaction and immunohistochemistry were used to determine the expression of Hsulf-1 in HCC.Cell apoptosis was observed through flow cytometry instrument and mechanism of Hsulf-1 to enhance the cytotoxicity of 5-FC against HCC was analyzed in HCC by confocal microscopy.We also establish a nude mice model of HCC to address the effect of Hsulf-1 expression on the CD/5-FC suicide gene therapy in vivo.Results：A significant decrease in HepG2 cell proliferation and an increase in HepG2 cell apoptosis were observed when Hsulf-1 expression was combined with the CD/5-FC gene suicide system.A noticeable bystander effect was observed when the Hsulf-1 and CD genes were co-expressed.Intracellular calcium was also increased after HepG2 cells were infected with the Hsulf-1 gene.In vivo studies showed that the suppression of tumor growth was more pronounced in animals treated with the Hsulf-1 plus CD than those treated with either gene therapy alone,and the combined treatment resulted in a significant increase in survival.Conclusions：Hsulf-1 expression combined with the CD/5-FC gene suicide system could be an effective treatment approach for HCC.展开更多
Objective To investigate the efficiency of the cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines.Methods We used cell culture, infectivity and sensitivity tests, to observe bystande...Objective To investigate the efficiency of the cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines.Methods We used cell culture, infectivity and sensitivity tests, to observe bystander effect by animal tests.Results Established prostate cancer cell lines are eventually infectible by adenoviral vector. The ratio of vector/cell at which infection occurs depends on the specific cell line. The peak of expression of the transferred cytosine deaminase gene occurred in cells at different time, but persisted beyond 11 days. These prostate cell lines are sensitized to 5-fluorocytosine by infection with adenoviral vector carrying the cytosine deaminase gene. Only 5% of the LNCap and 10% of the RM-1 cells were infected and produced 100% cell death. In the animal test, there was significant inhibition of tumor growth at a ratio of 400 vector particles/cell with the systematic treatment of 5-fluorocytosine.Conclusions Adenoviral vector carrying a cytosine deaminase transcription unit can sensitize prostate cancer cell lines to 5-fluorocytosine. The system can significantly inhibit the growth of prostatic tumors in mice.展开更多
目的探讨Survivin启动子介导的CD/5-FC自杀系统的抗肿瘤效果。方法利用Survivin启动子替换p EGFP-N1载体上的巨细胞病毒(CMV)启动子,构建重组表达载体p Sur P-EGFP,通过报告基因绿色荧光蛋白(GFP)的表达确定Survivin启动子的特异性;将...目的探讨Survivin启动子介导的CD/5-FC自杀系统的抗肿瘤效果。方法利用Survivin启动子替换p EGFP-N1载体上的巨细胞病毒(CMV)启动子,构建重组表达载体p Sur P-EGFP,通过报告基因绿色荧光蛋白(GFP)的表达确定Survivin启动子的特异性;将酿酒酵母的胞嘧啶脱氨酶(CD)基因克隆在p Sur P-EGFP中,构建成重组载体p Sur P-CD,转染肿瘤细胞后利用毛细管电泳仪检测前药5-氟胞嘧啶(5-FC)的转化效率,同时分析肿瘤细胞的凋亡。结果 GFP的表达表明Survivin启动子具有较好的肿瘤靶向性;毛细管电泳检测表明CD能够有效地将5-FC转化为5-FU,显微镜观察和流式分析表明了Survivin启动子介导的CD的抗肿瘤效果。结论 Survivin启动子能有效地介导CD自杀基因的表达,具有较好的靶向性抗肿瘤效果。展开更多
目的以含胞嘧啶脱氨酶(CD)基因的pCMVCD重组表达质粒转染SHG-44胶质瘤细胞,体外观察5氟-胞嘧啶(5-FC)对转染CD基因的胶质瘤细胞的凋亡诱导作用。方法体外扩增、酶切鉴定pCMVCD质粒并采用DNA序列测定pCMVCD质粒中的CD基因;脂质体L ipofec...目的以含胞嘧啶脱氨酶(CD)基因的pCMVCD重组表达质粒转染SHG-44胶质瘤细胞,体外观察5氟-胞嘧啶(5-FC)对转染CD基因的胶质瘤细胞的凋亡诱导作用。方法体外扩增、酶切鉴定pCMVCD质粒并采用DNA序列测定pCMVCD质粒中的CD基因;脂质体L ipofectam ine 2000介导pCMVCD质粒转染SHG-44细胞,G418筛选培养获取抗性细胞克隆(即SHG-44/CD细胞);免疫细胞化学检测SHG-44/CD细胞的CD基因蛋白水平表达;流式细胞仪、TUNEL实验及透射电子显微镜观察5-FC对表达CD基因的SHG-44/CD细胞凋亡的诱导作用。结果含CD基因的pCMVCD质粒成功转染进入SHG-44细胞,获取了含CD基因的SHG-44/CD细胞,免疫细胞化学染色显示SHG-44/CD细胞成功地表达了CD。在含5-FC的培养液中培养,SHG-44/CD细胞出现典型的凋亡形态,TUNEL显示凋亡细胞比例极高;透射电镜可见凋亡改变;流式细胞术检测凋亡率达18.6%,凋亡率呈剂量依赖性。结论建立了SHG-44恶性人脑胶质瘤细胞的CD/5-FC自杀基因系统。诱导SHG-44/CD胶质瘤细胞产生凋亡可能是脑胶质瘤CD基因疗法的重要机制。展开更多
文摘Cytosine Deaminase (CD) from Aspergillus parasiticus was purified and characterized. Time course for maximal CD production (50 μmol/min/mg) was at 72 hrs. The enzyme was purified 387.73 folds with an overall yield of 13%. The CD had pH optimum of 7.2, a temperature optimum of 40℃ - 45℃, activation of energy (Ea) of 8.4 KJ/mol and a t1/2 of 1.10 hr. A. parasiticus CD stoichiometrically deaminated Cyto-sine and 5-fluorocytosine (5-FC) with the KM values of 0.19mM and 0.30 mM respectively. Studies on the effect of pH on KM and Vmax gave pKa1 of 5.8 and pKa2 of 6.3 with enthalpy of ionization of 43.01 KJ/mole suggesting histidine in the active site. The enzyme was strongly inhibited by Ca2+, Co2+, Zn2+ and Hg2+ and completely inhibited by Cu2+ and Fe2+ at 50 mM. Therefore, A. parasiticus CD can be compared with CDs from other sources that are used in suicide gene therapy.
文摘Background：Human sulfatase-1 （Hsulf-l） is an endosulfatase that selectively removes sulfate groups from heparan sulfate proteoglycans （HSPGs）,altering the binding of several growth factors and cytokines to HSPG to regulate cell proliferation,cell motility,and apoptosis.We investigated the role of combined cancer gene therapy with Hsulf-l and cytosine deaminase/5-fluorocytosine （CD/5-FC） suicide gene on a hepatocellular carcinoma （HCC） cell line,HepG2,in vitro and in vivo.Methods：Reverse transcription polymerase chain reaction and immunohistochemistry were used to determine the expression of Hsulf-1 in HCC.Cell apoptosis was observed through flow cytometry instrument and mechanism of Hsulf-1 to enhance the cytotoxicity of 5-FC against HCC was analyzed in HCC by confocal microscopy.We also establish a nude mice model of HCC to address the effect of Hsulf-1 expression on the CD/5-FC suicide gene therapy in vivo.Results：A significant decrease in HepG2 cell proliferation and an increase in HepG2 cell apoptosis were observed when Hsulf-1 expression was combined with the CD/5-FC gene suicide system.A noticeable bystander effect was observed when the Hsulf-1 and CD genes were co-expressed.Intracellular calcium was also increased after HepG2 cells were infected with the Hsulf-1 gene.In vivo studies showed that the suppression of tumor growth was more pronounced in animals treated with the Hsulf-1 plus CD than those treated with either gene therapy alone,and the combined treatment resulted in a significant increase in survival.Conclusions：Hsulf-1 expression combined with the CD/5-FC gene suicide system could be an effective treatment approach for HCC.
文摘Objective To investigate the efficiency of the cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines.Methods We used cell culture, infectivity and sensitivity tests, to observe bystander effect by animal tests.Results Established prostate cancer cell lines are eventually infectible by adenoviral vector. The ratio of vector/cell at which infection occurs depends on the specific cell line. The peak of expression of the transferred cytosine deaminase gene occurred in cells at different time, but persisted beyond 11 days. These prostate cell lines are sensitized to 5-fluorocytosine by infection with adenoviral vector carrying the cytosine deaminase gene. Only 5% of the LNCap and 10% of the RM-1 cells were infected and produced 100% cell death. In the animal test, there was significant inhibition of tumor growth at a ratio of 400 vector particles/cell with the systematic treatment of 5-fluorocytosine.Conclusions Adenoviral vector carrying a cytosine deaminase transcription unit can sensitize prostate cancer cell lines to 5-fluorocytosine. The system can significantly inhibit the growth of prostatic tumors in mice.
文摘目的探讨Survivin启动子介导的CD/5-FC自杀系统的抗肿瘤效果。方法利用Survivin启动子替换p EGFP-N1载体上的巨细胞病毒(CMV)启动子,构建重组表达载体p Sur P-EGFP,通过报告基因绿色荧光蛋白(GFP)的表达确定Survivin启动子的特异性;将酿酒酵母的胞嘧啶脱氨酶(CD)基因克隆在p Sur P-EGFP中,构建成重组载体p Sur P-CD,转染肿瘤细胞后利用毛细管电泳仪检测前药5-氟胞嘧啶(5-FC)的转化效率,同时分析肿瘤细胞的凋亡。结果 GFP的表达表明Survivin启动子具有较好的肿瘤靶向性;毛细管电泳检测表明CD能够有效地将5-FC转化为5-FU,显微镜观察和流式分析表明了Survivin启动子介导的CD的抗肿瘤效果。结论 Survivin启动子能有效地介导CD自杀基因的表达,具有较好的靶向性抗肿瘤效果。
文摘目的以含胞嘧啶脱氨酶(CD)基因的pCMVCD重组表达质粒转染SHG-44胶质瘤细胞,体外观察5氟-胞嘧啶(5-FC)对转染CD基因的胶质瘤细胞的凋亡诱导作用。方法体外扩增、酶切鉴定pCMVCD质粒并采用DNA序列测定pCMVCD质粒中的CD基因;脂质体L ipofectam ine 2000介导pCMVCD质粒转染SHG-44细胞,G418筛选培养获取抗性细胞克隆(即SHG-44/CD细胞);免疫细胞化学检测SHG-44/CD细胞的CD基因蛋白水平表达;流式细胞仪、TUNEL实验及透射电子显微镜观察5-FC对表达CD基因的SHG-44/CD细胞凋亡的诱导作用。结果含CD基因的pCMVCD质粒成功转染进入SHG-44细胞,获取了含CD基因的SHG-44/CD细胞,免疫细胞化学染色显示SHG-44/CD细胞成功地表达了CD。在含5-FC的培养液中培养,SHG-44/CD细胞出现典型的凋亡形态,TUNEL显示凋亡细胞比例极高;透射电镜可见凋亡改变;流式细胞术检测凋亡率达18.6%,凋亡率呈剂量依赖性。结论建立了SHG-44恶性人脑胶质瘤细胞的CD/5-FC自杀基因系统。诱导SHG-44/CD胶质瘤细胞产生凋亡可能是脑胶质瘤CD基因疗法的重要机制。
