AIM: To evaluate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynudeotide (As-ODN) on telomerase activity and cell apoptosis in colon cancer cell line SW480.METHODS: As-ODN was t...AIM: To evaluate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynudeotide (As-ODN) on telomerase activity and cell apoptosis in colon cancer cell line SW480.METHODS: As-ODN was transfected into cells SW480 by liposomal transfection. Cultured cells were divided into three groups: ASODN (5′GGAGCGCGCGGCATCGCGGG-3), sense oligodeoxynucleotide (5′-CCCGCGATGCCGCGCGCTCC-3; SODN) and control. The concentration of oligodeoxynucleotide and lipsome was 10 μmol/L and 16 mg/L, respectively. The activity of telomerase was examined by telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (ELISA), and cell apoptosis was observed by morphology and flow cytometry in each group.RESULTS: Telomerase activity began to be down-regulated or inhibited when cells SW480 were treated with As-ODN for 72 h, and cell apoptosis was induced.CONCLUSION: It is suggested that hTERT As-ODN might specially inhibit the activity of telomerase in colon cancer cells and it is further proved that the hTERT gene has a significant correlation with telomerase activity. Further evidence is needed to prove whether hTERT As-ODN is a potential tool for the treatment of colon cancer.展开更多
AIM: To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treatedwith cant...AIM: To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treatedwith cantide mixed with lipofectin, or lipofectin alone. Afterinducted for 6 h at 37℃, 10% FCS in DMEM was replacedin each well. After the treatment repeated twice to threetimes in each concentration of cantide, hTERT mRNA andprotein expression were measured by RT-PCR and Westernblot analysis, respectively. Telomerase aclivity was determinedby TRAP-ELISA assay. CPP32- and ICE-like activity was alsoinvestigated using CasPACE assay system at 48 h aftercantide treatment, and apoptosis was evaluated using theDeadEnd assay at 24, 48 and 72 h after cantide treatment.RESULTS: Compared to the control cells, the cells treated with cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively.The telomerase activity was decreased as the concentration of cantide increased at 48 h. At the concentration of 800 nM,the telomerase activity in the treated HepG2 and SMMC7721 cells was only 17.1% (P＜0.01) and 20.3% (P＜0.01)of that in untreated cells. The levels of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8- and 3.0-fold (P＜0.05) at 48 h, and the levels of ICE-like protease activity also increased by 2.6- and 3.2-fold (P＜0.05)respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63% and 52% (P＜0.01), respectively. By contrast, 8%and 9% of the cells were apoptosis after 72 h treatment with lipofectin alone.CONCLUSION: Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.展开更多
根据 Ty3-gypsy 反转录转座子反转录酶的保守序列设计简并引物,从中原牡丹(Paeonia suffruticosa Andrews)品种‘洛阳红’和野生种卵叶牡丹(Paeonia qiui Y. L. Pei et D. Y. Hong)中扩增出430 bp 左右的目标片段。目的条带经回收、克...根据 Ty3-gypsy 反转录转座子反转录酶的保守序列设计简并引物,从中原牡丹(Paeonia suffruticosa Andrews)品种‘洛阳红’和野生种卵叶牡丹(Paeonia qiui Y. L. Pei et D. Y. Hong)中扩增出430 bp 左右的目标片段。目的条带经回收、克隆、测序及相关生物信息学软件进行序列分析后,获得了13 条来自牡丹的 Ty3-gypsy 反转录转座子反转录酶序列。这些核苷酸序列具有较高的异质性,主要表现为缺失突变,序列长度变化范围为 412 ～ 446 bp,同源性范围为 71.5% ～ 94.8%。翻译成氨基酸后,有 12 条序列出现 1 ～ 9 个不同程度的终止密码子突变,3 条序列出现移框突变。其核苷酸序列经过系统聚类后可分为 6 个家族。将其氨基酸序列与已登录的不同物种 Ty3-gypsy 反转录转座子反转录酶的氨基酸序列进行聚类分析,结果表明与其他植物具有较高的同源性,表明它们间可能存在着 Ty3-gypsy 反转录转座子的横向传递。展开更多
Influence of fullerene (C60) derivatives on M-MuLV reverse transcriptase activity is investi- gated. Two water-soluble fullerene derivatives, fullerol (C60(OH)23-24) and trimalonic acid C60 (TMA C60, C63(COOH)6) are u...Influence of fullerene (C60) derivatives on M-MuLV reverse transcriptase activity is investi- gated. Two water-soluble fullerene derivatives, fullerol (C60(OH)23-24) and trimalonic acid C60 (TMA C60, C63(COOH)6) are used in the experiments and their effects on in vitro reverse transcription-polymerase chain reaction (RT-PCR) of β-actin mRNA in HeLa cells are determined. PCR products are detected by agarose gel electrophoresis. It is found that the amounts of PCR products decrease with addition of either of two fullerene derivatives to RT reaction mixture. The inhibition of fullerene derivatives is dose-dependent, and IC50 values of fullerol and TMA C60 are 0.089 and 0.039 mmol/L, respectively. The amount of PCR products obtained by direct addition of fullerol or TMA C60 to PCR are greater than those obtained by addition of the equivalent amount of fullerol or TMA C60 to RT, indicating an inhibitory ef- fect of fullerol or TMA C60 on RT. The compensative experiment of M-MuLV reverse transcriptase shows that increasing enzyme amounts can antagonize the activity of fullerol or TMA C60. These results imply that fullerenes can inhibit M-MuLV reverse transcriptase activity, with the inhibition of TMA C60 slightly stronger than fullerol, and that their potential in treatment of diseases induced by RNA viruses such as leukemia virus needs further investigation.展开更多
基金the Science and Technology Research Project of Hubei Province,No.2002AA301C72
文摘AIM: To evaluate the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynudeotide (As-ODN) on telomerase activity and cell apoptosis in colon cancer cell line SW480.METHODS: As-ODN was transfected into cells SW480 by liposomal transfection. Cultured cells were divided into three groups: ASODN (5′GGAGCGCGCGGCATCGCGGG-3), sense oligodeoxynucleotide (5′-CCCGCGATGCCGCGCGCTCC-3; SODN) and control. The concentration of oligodeoxynucleotide and lipsome was 10 μmol/L and 16 mg/L, respectively. The activity of telomerase was examined by telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (ELISA), and cell apoptosis was observed by morphology and flow cytometry in each group.RESULTS: Telomerase activity began to be down-regulated or inhibited when cells SW480 were treated with As-ODN for 72 h, and cell apoptosis was induced.CONCLUSION: It is suggested that hTERT As-ODN might specially inhibit the activity of telomerase in colon cancer cells and it is further proved that the hTERT gene has a significant correlation with telomerase activity. Further evidence is needed to prove whether hTERT As-ODN is a potential tool for the treatment of colon cancer.
基金the National Natural Science Foundation of China, No.39870879the Special Funds for Major State Basic Research of China,No.G 1998051103
文摘AIM: To investigate the anti-tumor mechanism of antisenseoligodeoxynucleotide cantide against hTERT.METHODS: Tumor cells were cultured overnight and grownto 50-60% confluence. HepG2 and SMMC-7721 were treatedwith cantide mixed with lipofectin, or lipofectin alone. Afterinducted for 6 h at 37℃, 10% FCS in DMEM was replacedin each well. After the treatment repeated twice to threetimes in each concentration of cantide, hTERT mRNA andprotein expression were measured by RT-PCR and Westernblot analysis, respectively. Telomerase aclivity was determinedby TRAP-ELISA assay. CPP32- and ICE-like activity was alsoinvestigated using CasPACE assay system at 48 h aftercantide treatment, and apoptosis was evaluated using theDeadEnd assay at 24, 48 and 72 h after cantide treatment.RESULTS: Compared to the control cells, the cells treated with cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively.The telomerase activity was decreased as the concentration of cantide increased at 48 h. At the concentration of 800 nM,the telomerase activity in the treated HepG2 and SMMC7721 cells was only 17.1% (P＜0.01) and 20.3% (P＜0.01)of that in untreated cells. The levels of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8- and 3.0-fold (P＜0.05) at 48 h, and the levels of ICE-like protease activity also increased by 2.6- and 3.2-fold (P＜0.05)respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63% and 52% (P＜0.01), respectively. By contrast, 8%and 9% of the cells were apoptosis after 72 h treatment with lipofectin alone.CONCLUSION: Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.
文摘Influence of fullerene (C60) derivatives on M-MuLV reverse transcriptase activity is investi- gated. Two water-soluble fullerene derivatives, fullerol (C60(OH)23-24) and trimalonic acid C60 (TMA C60, C63(COOH)6) are used in the experiments and their effects on in vitro reverse transcription-polymerase chain reaction (RT-PCR) of β-actin mRNA in HeLa cells are determined. PCR products are detected by agarose gel electrophoresis. It is found that the amounts of PCR products decrease with addition of either of two fullerene derivatives to RT reaction mixture. The inhibition of fullerene derivatives is dose-dependent, and IC50 values of fullerol and TMA C60 are 0.089 and 0.039 mmol/L, respectively. The amount of PCR products obtained by direct addition of fullerol or TMA C60 to PCR are greater than those obtained by addition of the equivalent amount of fullerol or TMA C60 to RT, indicating an inhibitory ef- fect of fullerol or TMA C60 on RT. The compensative experiment of M-MuLV reverse transcriptase shows that increasing enzyme amounts can antagonize the activity of fullerol or TMA C60. These results imply that fullerenes can inhibit M-MuLV reverse transcriptase activity, with the inhibition of TMA C60 slightly stronger than fullerol, and that their potential in treatment of diseases induced by RNA viruses such as leukemia virus needs further investigation.
