反义技术沉默细胞周期蛋白依赖激酶7对体外培养人肝细胞癌HepG2细胞的抗增殖作用研究
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摘要
目的：Cdk7在细胞周期进展和RNA转录过程中具有重要的调控作用，在肿瘤样本中已经证实Cdk7有异常表达，同时，小分子非特异性Cdks抑制剂在临床前和临床试验中均显示了较强的抗肿瘤作用，因此推测在抗肿瘤药物研发中Cdk7可能是一个潜在的靶点。本研究用反义技术特异性沉默Cdk7基因，观察基因沉默对人肝细胞癌HepG2细胞增殖的影响，以确定Cdk7作为抗肿瘤药物研发靶点的可行性，同时也为开发反义药物筛选先导化合物。
方法：利用RNAstructure3.7 RNA结构模拟软件对人Cdk7mRNA进行二级结构模拟，根据模拟结构中突环、膨胀环、夹角、假结等单链结构选择反义序列，再经同源性分析，剔除其中与体内其它重要基因有同源性的片段，用体外培养HepG2细胞评估所选反义寡脱氧核苷酸片段的抗增殖活性，选择抗增殖作用最强的片段进行结构改造后再次用体外培养细胞评估反义寡脱氧核苷酸片段的抗增殖活性，作用最强的ASODN片段即为目的片段。
分别以DNA梯形条带形成实验、透射电子显微镜观察和流式细胞术考察细胞转染ASODN后DNA降解情况、凋亡细胞的超微结构特征和细胞凋亡比例以及细胞周期阻滞情况，确定细胞转染ASODN后通过何种途径引起生长抑制。以RT-PCR技术检测转染ASODN对细胞内Cdk7 mRNA水平的影响。以免疫印迹技术检测转染Cdk7 ASODN后对细胞内Cdk7蛋白水平的影响以及对细胞内视网膜纤维母细胞瘤蛋白(pRb)和Cdk2激酶蛋白磷酸化的影响。
结果：根据计算机模拟的人Cdk7 mRNA二级结构选择反义片
第一军医大学博士研究生学位论文
段，经oligowalk程序剔除可在片段内形成自身二级结构的AsoDN
片段，再经同源性分析去除其中与体内重要基因有同源性的片段，
最后获得21条符合实验要求的AsODN，用体外培养的HePGZ细胞
筛选其抗增殖作用，结果显示互补于人cdk7 mRNA第284一303位
的ASODN片段作用最强。以此序列为母体，可由其互补序列相邻
上、下游各5个碱基范围区域的互补序列再次获得10条AsoDN片
段，经全部磷酸骨架硫代修饰后，以体外抗增殖作用再次筛选，显
示互补于Cdk7 mRNA第287~306位的ASODN抗增殖活性最强，
其最大抗增殖作用为68.3士2.6%，半数抑制浓度为51.9土
8 .6nmol/L。
DNA梯形条带形成实验显示，HePGZ细胞转染50、100、200、
400nmol几ASODN 72h后，转染200和400 nmol几ASODN的细胞
中提取的DNA进行凝胶电泳可形成明显的梯形条带;用200
nlnol/L浓度ASODN转染细胞后36h提取DNA进行凝胶电泳开始
出现不明显的梯形条带，48h时梯形条带明显。透射电子显微镜对
转染ASODN细胞的超微结构进行观察显示，在转染浓度为100、
200、40Oluno讥ASODN细胞中，视野内多见凋亡细胞，主要特征
为染色质聚集，电子密度增高，细胞质内出现空泡以及脂膜包裹的
颗粒等现象。用Zoonmol几ASODN转染细胞后，48h出现较多凋亡
细胞，72h更为明显。流式细胞术分析显示转染200 Iunol几As0DN
后，细胞凋亡比例在48h突然增加，0一36h基本无凋亡，细胞周期
分析显示，S、GZ/M期细胞比例随转染时间延长逐渐下降，而GO/GI
期细胞比例逐渐增加，最高比例可达到70%以上，显示出明显的
GO/GI期阻滞。用不同浓度ASODN转染细胞72h，分析显示在浓
度>l 00 tnnoFL，凋亡细胞比例明显增加;S、GZ佩期细胞比例随浓
第一军医大学博士研究生论文
度增加逐渐下降，GO/GI期细胞所占比例则明显增加，超过80%，
显示细胞周期明显阻滞于GO/GI期。
通过RT一PCR和免疫印迹对Cdk7 mRNA和蛋白水平进行检
测，结果显示细胞用浓度为50、100、200、400 nmol几^SOnN转
染72h，其mRNA水平分别相当于转染正义ODN对照组的
48.3士6.2%、50.2士6.8%、21.3士3.3%和9.7士2.8%，蛋白水
平分别为对照组的0.64土0.16、0.43士0.08、0.18士0.03和
0.12士0.05;以浓度200 nmol几ASODN转染细胞12、24、48和72h，
Cdk7蛋白水平分别为oh对照组水平的0.48士0.06、0.33士0.04、
0 .20士0.03和0.14士0.05。
将细胞用浓度为50、100、200、400 tunol几ASODN转染72h
后，对pRb蛋白和CdkZ激酶的磷酸化水平进行分析，结果显示pRb
磷酸化水平分别为正义对照组的0.62士0.14、0.33士0.06、
0.27士0.09、0.20士0.08;CdkZ磷酸化水平为正义对照组的
0 .87士0.22、0.38士0.09、0.18士0.06和0.18士0.07;用200 nrnol几
AsoDN转染细胞12、24、45和72h后，pRb蛋白磷酸化水平分别
为对照组的0.52士0.08、0.48士0.09、0.35士0.06和0.28士0.05，
CdkZ激酶磷酸化水平为对照组的0.82士0.14、0.56士0.09、
0.32士0.06和0.37士0.08，oh处理组与正义对照组无显著差异。
结论:由于Cdk7对细胞周期进展和RNA转录两条途径均有重
要的调控作用，Cdk7在肿瘤组织中有异常高表达，利用反义技术
特异性沉默cdk7对体外培养的人肝细胞癌HePGZ细胞可产生显著
的抗增殖作用，因此认为Cdk7可作为抗肿瘤药物研发的新靶点。
研究显示ASODN沉默Cdk7基因后通过下述途径产生抗增殖作用:
第一军医大学博士研究生学位论文
cdk4、cdk6通过催化pRb蛋白磷酸化调控细胞Gl早期进展，cdkZ
催化Gl晚期进展，沉默cdk7可下调pRb蛋白和CdkZ激酶的磷酸
化
Objective: Cdk7, a key regulator in cell cycle control and RNA transcription, is overexpressed in neoplastic tissues, and low molecule weight non-target-specific Cdks inhibitors which inhibit the Cdks activity ubiquitously had shown a promising anticancer effect in preclinical and clinical trials but with a dissatisfied adverse reaction profile. Owing to all characteristics mentioned above, we hypothesized that Cdk7 was a potential target for anticancer therapeutics discovery and development. In order to validate Cdk7 as the target for anticancer agents development, we evaluated the antiproliferative effect induced by Cdk7 silencing with antisense strategy, a specific target gene knock-out tool, and tried to find the lead for further anticancer therapeutics development.
Methods: Secondary structures of homo sapiens Cdk7 mRNA were predicted by RNAstructure 3.7 software. And the antisense fragments were selected according to the partial single-strand structures such as bulges, hairpins, knots and internal loops. Fragment which should form secondary structure in itself and which be homogeneous to other important genes were out of consideration. After that, the antiproliferative effect of each fragment selected was determined by human hepatoblastoma HepG2 cells in vitro. The antiproliferative effect of the structural-modified fragments based on the most powerful one selected in the first round of screening were evaluated with the HepG2 cell culture again, then the fragment that had shown the highest potency
in a
ntiproliferation test was considered to be the hit.
DNA fragmentation was analyzed by agar electrophoresis to see if the DNA ladder was formed, infrastructure of the cells which had been transfected with ASODN was observed under the transmission electron microscope to see if the characteristics of apoptosis appeared. Indexes of apoptotic cell and cell cycle distribution in cells treated with ASODN were determined by flow cytometry. Changes of Cdk7 mRNA levels were assayed by reverse transcriptase polymerase chain reaction and changes of Cdk7 protein levels were assayed by Western blotting respectively. Phosphorylation levels of retinoblastoma and Cdk2 protein in ASODN treated cells were analyzed by Western blotting.
Results: 21 fragments were selected according to the Cdk7 mRNA structures predicted by RNAstructure software. Antiproliferative activity for each fragment was evaluated with HepG2 cell culture by MTT method after the fragments those might to form the secondary structure and fragments those had shown homogeneous to other important genes had been eliminated. The results showed that antiproliferative effect of the fragment complementary to 284-303 region was higher than that of the others. Then 10 fragments complementary to the up or down stream 1~5 bases to the complementary region were picked and antiproliferative potency for each was measured with HepG2 cell culture. The results showed that the fragment complementary to 287-306 region was capable of inhibiting cell growth by 68.3 ?2.6%, and the IC50 was l/L.
In the DNA fragmentation formation test, the DNA extracted from
-9-
cells which had been treated with 200, 400nmol/L ASODN for 72 hours showed an obvious characteristic of DNA fragmentation and formed a bright ladder with an about 200 base pairs intervals in the electrophoresis gel. And for the cell treated with constant 200nmol/L ASODN, DNA fragmentation didn't form until the cells had been treated for 36 hours, and the fragmentation was obvious at the 48h. The photographes taken by transmission electron microscope showed that the cells treated with 100, 200, 400 nmol/L ASODN were induced to programmed cell death according to the changes of the ultrastructures. The typical changes in the apoptotic cells were chromatin condensation in the nucleolus, electronic density increasing, blebbing in the cytoplasm and particle with lipid membrane forming and distracted from the cell. In the worst condition, the cell separated into a group of granule without any characteristics of a cell. The apo
方法：利用RNAstructure3.7 RNA结构模拟软件对人Cdk7mRNA进行二级结构模拟，根据模拟结构中突环、膨胀环、夹角、假结等单链结构选择反义序列，再经同源性分析，剔除其中与体内其它重要基因有同源性的片段，用体外培养HepG2细胞评估所选反义寡脱氧核苷酸片段的抗增殖活性，选择抗增殖作用最强的片段进行结构改造后再次用体外培养细胞评估反义寡脱氧核苷酸片段的抗增殖活性，作用最强的ASODN片段即为目的片段。
分别以DNA梯形条带形成实验、透射电子显微镜观察和流式细胞术考察细胞转染ASODN后DNA降解情况、凋亡细胞的超微结构特征和细胞凋亡比例以及细胞周期阻滞情况，确定细胞转染ASODN后通过何种途径引起生长抑制。以RT-PCR技术检测转染ASODN对细胞内Cdk7 mRNA水平的影响。以免疫印迹技术检测转染Cdk7 ASODN后对细胞内Cdk7蛋白水平的影响以及对细胞内视网膜纤维母细胞瘤蛋白(pRb)和Cdk2激酶蛋白磷酸化的影响。
结果：根据计算机模拟的人Cdk7 mRNA二级结构选择反义片
第一军医大学博士研究生学位论文
段，经oligowalk程序剔除可在片段内形成自身二级结构的AsoDN
片段，再经同源性分析去除其中与体内重要基因有同源性的片段，
最后获得21条符合实验要求的AsODN，用体外培养的HePGZ细胞
筛选其抗增殖作用，结果显示互补于人cdk7 mRNA第284一303位
的ASODN片段作用最强。以此序列为母体，可由其互补序列相邻
上、下游各5个碱基范围区域的互补序列再次获得10条AsoDN片
段，经全部磷酸骨架硫代修饰后，以体外抗增殖作用再次筛选，显
示互补于Cdk7 mRNA第287~306位的ASODN抗增殖活性最强，
其最大抗增殖作用为68.3士2.6%，半数抑制浓度为51.9土
8 .6nmol/L。
DNA梯形条带形成实验显示，HePGZ细胞转染50、100、200、
400nmol几ASODN 72h后，转染200和400 nmol几ASODN的细胞
中提取的DNA进行凝胶电泳可形成明显的梯形条带;用200
nlnol/L浓度ASODN转染细胞后36h提取DNA进行凝胶电泳开始
出现不明显的梯形条带，48h时梯形条带明显。透射电子显微镜对
转染ASODN细胞的超微结构进行观察显示，在转染浓度为100、
200、40Oluno讥ASODN细胞中，视野内多见凋亡细胞，主要特征
为染色质聚集，电子密度增高，细胞质内出现空泡以及脂膜包裹的
颗粒等现象。用Zoonmol几ASODN转染细胞后，48h出现较多凋亡
细胞，72h更为明显。流式细胞术分析显示转染200 Iunol几As0DN
后，细胞凋亡比例在48h突然增加，0一36h基本无凋亡，细胞周期
分析显示，S、GZ/M期细胞比例随转染时间延长逐渐下降，而GO/GI
期细胞比例逐渐增加，最高比例可达到70%以上，显示出明显的
GO/GI期阻滞。用不同浓度ASODN转染细胞72h，分析显示在浓
度>l 00 tnnoFL，凋亡细胞比例明显增加;S、GZ佩期细胞比例随浓
第一军医大学博士研究生论文
度增加逐渐下降，GO/GI期细胞所占比例则明显增加，超过80%，
显示细胞周期明显阻滞于GO/GI期。
通过RT一PCR和免疫印迹对Cdk7 mRNA和蛋白水平进行检
测，结果显示细胞用浓度为50、100、200、400 nmol几^SOnN转
染72h，其mRNA水平分别相当于转染正义ODN对照组的
48.3士6.2%、50.2士6.8%、21.3士3.3%和9.7士2.8%，蛋白水
平分别为对照组的0.64土0.16、0.43士0.08、0.18士0.03和
0.12士0.05;以浓度200 nmol几ASODN转染细胞12、24、48和72h，
Cdk7蛋白水平分别为oh对照组水平的0.48士0.06、0.33士0.04、
0 .20士0.03和0.14士0.05。
将细胞用浓度为50、100、200、400 tunol几ASODN转染72h
后，对pRb蛋白和CdkZ激酶的磷酸化水平进行分析，结果显示pRb
磷酸化水平分别为正义对照组的0.62士0.14、0.33士0.06、
0.27士0.09、0.20士0.08;CdkZ磷酸化水平为正义对照组的
0 .87士0.22、0.38士0.09、0.18士0.06和0.18士0.07;用200 nrnol几
AsoDN转染细胞12、24、45和72h后，pRb蛋白磷酸化水平分别
为对照组的0.52士0.08、0.48士0.09、0.35士0.06和0.28士0.05，
CdkZ激酶磷酸化水平为对照组的0.82士0.14、0.56士0.09、
0.32士0.06和0.37士0.08，oh处理组与正义对照组无显著差异。
结论:由于Cdk7对细胞周期进展和RNA转录两条途径均有重
要的调控作用，Cdk7在肿瘤组织中有异常高表达，利用反义技术
特异性沉默cdk7对体外培养的人肝细胞癌HePGZ细胞可产生显著
的抗增殖作用，因此认为Cdk7可作为抗肿瘤药物研发的新靶点。
研究显示ASODN沉默Cdk7基因后通过下述途径产生抗增殖作用:
第一军医大学博士研究生学位论文
cdk4、cdk6通过催化pRb蛋白磷酸化调控细胞Gl早期进展，cdkZ
催化Gl晚期进展，沉默cdk7可下调pRb蛋白和CdkZ激酶的磷酸
化
Objective: Cdk7, a key regulator in cell cycle control and RNA transcription, is overexpressed in neoplastic tissues, and low molecule weight non-target-specific Cdks inhibitors which inhibit the Cdks activity ubiquitously had shown a promising anticancer effect in preclinical and clinical trials but with a dissatisfied adverse reaction profile. Owing to all characteristics mentioned above, we hypothesized that Cdk7 was a potential target for anticancer therapeutics discovery and development. In order to validate Cdk7 as the target for anticancer agents development, we evaluated the antiproliferative effect induced by Cdk7 silencing with antisense strategy, a specific target gene knock-out tool, and tried to find the lead for further anticancer therapeutics development.
Methods: Secondary structures of homo sapiens Cdk7 mRNA were predicted by RNAstructure 3.7 software. And the antisense fragments were selected according to the partial single-strand structures such as bulges, hairpins, knots and internal loops. Fragment which should form secondary structure in itself and which be homogeneous to other important genes were out of consideration. After that, the antiproliferative effect of each fragment selected was determined by human hepatoblastoma HepG2 cells in vitro. The antiproliferative effect of the structural-modified fragments based on the most powerful one selected in the first round of screening were evaluated with the HepG2 cell culture again, then the fragment that had shown the highest potency
in a
ntiproliferation test was considered to be the hit.
DNA fragmentation was analyzed by agar electrophoresis to see if the DNA ladder was formed, infrastructure of the cells which had been transfected with ASODN was observed under the transmission electron microscope to see if the characteristics of apoptosis appeared. Indexes of apoptotic cell and cell cycle distribution in cells treated with ASODN were determined by flow cytometry. Changes of Cdk7 mRNA levels were assayed by reverse transcriptase polymerase chain reaction and changes of Cdk7 protein levels were assayed by Western blotting respectively. Phosphorylation levels of retinoblastoma and Cdk2 protein in ASODN treated cells were analyzed by Western blotting.
Results: 21 fragments were selected according to the Cdk7 mRNA structures predicted by RNAstructure software. Antiproliferative activity for each fragment was evaluated with HepG2 cell culture by MTT method after the fragments those might to form the secondary structure and fragments those had shown homogeneous to other important genes had been eliminated. The results showed that antiproliferative effect of the fragment complementary to 284-303 region was higher than that of the others. Then 10 fragments complementary to the up or down stream 1~5 bases to the complementary region were picked and antiproliferative potency for each was measured with HepG2 cell culture. The results showed that the fragment complementary to 287-306 region was capable of inhibiting cell growth by 68.3 ?2.6%, and the IC50 was l/L.
In the DNA fragmentation formation test, the DNA extracted from
-9-
cells which had been treated with 200, 400nmol/L ASODN for 72 hours showed an obvious characteristic of DNA fragmentation and formed a bright ladder with an about 200 base pairs intervals in the electrophoresis gel. And for the cell treated with constant 200nmol/L ASODN, DNA fragmentation didn't form until the cells had been treated for 36 hours, and the fragmentation was obvious at the 48h. The photographes taken by transmission electron microscope showed that the cells treated with 100, 200, 400 nmol/L ASODN were induced to programmed cell death according to the changes of the ultrastructures. The typical changes in the apoptotic cells were chromatin condensation in the nucleolus, electronic density increasing, blebbing in the cytoplasm and particle with lipid membrane forming and distracted from the cell. In the worst condition, the cell separated into a group of granule without any characteristics of a cell. The apo
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19 Stevens C, La Thangue NB.E2F and cell cycle control: a doubleedged sword.Arch Biochem Biophys. 2003; 412 (2): 157-69.
20 Knockaert M, Greengard P, Meijer L. Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol Sci, 2002, 23(9): 417-25.
21 Boulaire J, Fotedar A, Fotedar R.The functions of the Cdk-cyclin kinase inhibitor p21WAF1.Pathol Biol (Paris). 2000; 48(3): 190-202.
22 Bloom J, Pagano M.Deregulated degradation of the Cdk inhibitor p27 and malignant transformation. Semin Cancer Biol. 2003; 13(1): 41-7.
23 Sherr CJ, Roberts JM. Inhibitors of mammalian GI cyclin-dependent kinases. Genes Dev. 1995; 9(10):1149-63.
24 Ruas M, Peters G. The p 16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta. 1998; 1378(2):F115-77.
25 Nabel EG.Cdks and CKIs: molecular targets for tissue remodelling. Nat Rev Drug Discov. 2002; 1(8):587-98.
26 Chen-Kiang S. Cell-cycle control of plasma cell differentiation and tumorigenesis. Immunol Rev. 2003; 194:39-47.
27 Peter MF, Graham B, Carol M, et al. Cell cycle target validation: approaches and successes. Targets, 2003; 2(4): 154-61.
28 Laiho M, Latonen L. Cell cycle control, DNA damage checkpoints and cancer. Ann Med. 2003; 35(6):391-7.
29 Sherr CJ. Cell cycle control and cancer. Harvey Lect. 2000-2001; 96:73-92.
30 May P. Cell cycle control and cancer. Pathol Biol (Pads). 2000; 48(3): 167-73.
31 Moller MB. P27 in cell cycle control and cancer. Leuk Lymphoma. 2000; 39(1-2):19-27.
32 杨连君．细胞周期蛋白与肿瘤等疾病的关系，中国肿瘤生物治疗杂志，2003；10(3)：223—225．
33 陈汉源．细胞周期素D1与癌症．第一军医大学学报，1999；19(6)：586-591．
34 Kaldis P. The Cdk-activating kinase (CAK): from yeast to mammals. Cell Mol Life Sci. 1999; 55(2):284-96.
35 Chen J, Larochelle S, Li X, Suter B. Xpd/Ercc2 regulates CAK activity and mitotic progression. Nature. 2003; 424(6945):228-32.
36 Gu Y, Rosenblatt J, Morgan DO. Cell cycle regulation of CDK2 activity by phosphorylation of Thrl60 and Tyr15. EMBO J. 1992; 11(11):3995-4005.
37 Fleig UN, Gould KL. Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation. Semin Cell Biol. 1991; 2(4): 195-204.
38 Matsuoka M, Kato JY, Fisher RP, Morgan DO, Sherr CJ. Activation of cyclin-dependent kinase 4 (Cdk4) by mouse MO15-associated kinase. Mol Cell Biol. 1994; 14(11):7265-75.
39 Hagopian JC, Kirtley MP, Stevenson LM, Gergis RM, Russo AA, Pavletich NP, Parsons SM, Lew J. Kinetic basis for activation of CDK2/cyclin A by phosphorylation. J Biol Chem. 2001; 276 (1): 275-80.
40 Araujo SJ, Tirode F, Coin F, Pospiech H, Syvaoja JE, Stucki M, Hubscher U, Egly JM, Wood RD.Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes Dev. 2000;14(3):349-59.
41 Svejstrup, J., P. Vichi, and J.-M. Egly. 1996. The multiple roles of
transcription/repair factor TFIIH. Trends Biochem. Sci. 21:346-350
42 Rickert P, Corden JL, Lees E. Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases. Oncogene. 1999; 18(4): 1093-1102.
43 Rossignol M, Kolb-Cheynel I, Egly JM. Substrate specificity of the Cdk-activating kinase (CAK) is altered upon association with TFIIH. EMBO J, 1997; 16(7): 1628-37.
44 Wu L, Chen P, Shum CH, Chen C, Barsky LW, Weinberg KI, Jong A, Triche TJ. MAT1-modulated CAK activity regulates cell cycle G(1) exit. Mol Cell Biol. 2001; 21 (1):260-70.
45 Kaldis P, Solomon MJ. Analysis of CAK activities from human cells. Eur J Biochem. 2000; 267(13):4213-21.
46 Araujo SJ, Tirode F, Coin F, Pospiech H, Syvaoja JE, Stucki M, Hubscher U, Egly JM, Wood RD. Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes Dev. 2000; 14(3):349-59.
47 te Poele RH, Okorokov AL, Joel SP. RNA synthesis block by 5, 6-dich/oro- 1 -beta-D-ribo furanosylbenzimidazole (DRB) triggers p53-dependent apoptosis in human colon carcinoma cells. Oncogene. 1999; 18(42): 5765-72.
48 Liang YC, Tsai SH, Chen L, Lin-Shiau SY, Lin JK. Resveratrol-induced G2 arrest through the inhibition of CDK7 and p34CDC2 kinases in colon carcinoma HT29 cells. Biochem Pharmacol. 2003; 65(7): 1053-60.
49 王杉，梁斌，乔新民，彦飞飞，崔志荣．细胞周期素和细胞周期素依赖性激酶7在人乳腺癌组织中的表达及其意义，中华普通外科杂志，2002；17(10)：611-613．
50 Bartkova J, Zemanova M, Bartek J. Expression of CDK7/CAK in normal and tumor cells of diverse histogenesis, cell cycle position and differentiation. Int J Cancer, 1996; 66(6): 732-737.
51 Mouriaux F, Casagrande F, Pillaire M J, Manenti S, Malecaze F, Darbon JM. Differential expression of G1 cyclins and cyclindependent kinase inhibitors in normal and transformed melanocytes. Invest Ophthalmol Vis Sci. 1998; 39(6):876-84.
52 Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol. 2003; 4(6):457-67.
53 Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003; 349 (21) : 2042-54.
54 Kittler R, Buchholz F. RNA interference: gene silencing in the fast lane. Semin Cancer Biol. 2003; 13(4):259-65.
55 Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxy-nucleotide. Proc Natl Acad Sci U S A. 1978; 75(1): 280-4.
56 Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978; 75(1): 285-8.
57 Grillone LR, Lanz R. Fomivirsen. Drugs Today (Barc). 2001; 37(4):
245-255.
58 Geary RS, Henry SP, Grillone LR. Fomivirsen: clinical pharmacology and potential drug interactions. Clin Pharmacokinet. 2002; 41(4):255-60.
59 Jabs DA, Griffiths PD. Fomivirsen for the treatment of cytomegalovirus retinitis. Am J Ophthalmol. 2002; 133 (4): 552-6.
60 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer.Oncogene. 2003; 22(56):9087-96.
61 Biroccio A, Leonetti C, Zupi G.The future of antisense therapy: combination with anticancer treatments. Oncogene. 2003; 22(42): 6579-88.
62 Burkhard J, Uwe ZW. Antisense therapy for cancer- the time of truth, Lancet Oncol 2002; 3(11):672-83.
63 Lahn M, Sundell K, Moore S. Targeting protein kinase C-alpha (PKC-alpha) in cancer with the phosphorothioate antisense oligonucleotide aprinocarsen. Ann N Y Acad Sci. 2003; 1002: 263-70.
64 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene. 2003; 22(56):9087-96.
65 Stephens AC, Rivers RP. Antisense oligonucleotide therapy in cancer. Curr Opin Mol Ther. 2003; 5(2): 118-22.
66 Cho-Chung YS. Antisense DNAs as targeted genetic medicine to treat cancer. Arch Pharm Res. 2003; 26(3): 183-91.
67 Chen HX. Clinical development of antisense oligonucleotides as anti-cancer therapeutics. Methods Mol Med. 2003; 75:621-36.
68 Crooke ST. Progress in antisense technology. Annu Rev Med.
2004;55:61-95.
69 Holmlund JT. Applying antisense technology: Affinitak and other antisense oligonucleotides in clinical development. Ann N Y Acad Sci. 2003; 1002:244-51.
70 Lee LK, Roth CM. Antisense technology in molecular and cellular bioengineering. Curr Opin Biotechnol. 2003 ;14(5):505-11.
71 Turner DH, Sugimoto N. RNA structure prediction. Annu Rev Biophys Biophys Chem. 1988; 17:167-92.
72 Akmaev VR, Kelley ST, Stormo GD. A phylogenetic approach to RNA structure prediction. Proc Int Conf Intell Syst Mol Biol. 1999; 10-7.
73 Biroccio A, Leonetti C, Zupi G.The future of antisense therapy: combination with anticancer treatments. Oncogene. 2003; 22(42): 6579-88.
74 Bochar, D.A., Pan,Z.Q., Knights,R., Fisher, R.P., Shilatifard,A., Shiekhatta,R. Inhibition of transcription by the trimeric cyclindependent kinase 7 complex, J Biol Chem, 1999, 274(19): 13162-13166.
75 Song HF, Tang ZM, Yuan SJ, et al.. Application of secondary structure prediction in antisense drug design targeting protein kinase C-alpha mRNA and QSAR analysis. Acta Pharmacol Sin, 2000: 21(1): 80-6.
76 孔伟东，李彦豪，曾庆乐，陈勇，何晓峰．平阳霉素对血管内皮细胞的生长抑制作用和细胞周期的影响，第一军医大学学报，2003；23(8)：830-832．
77 Li X, Melamed MR, Darzynkiewicz Z. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp Cell Res. 1996; 222(1):28-37.
78 Spyratos F. DNA content and cell cycle analysis by flow cytometry in clinical samples: application in breast cancer. Biol Cell. 1993; 78(1-2):69-72.
79 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer.Oncogene. 2003; 22(56):9087-96.
80 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene. 2003; 22(56):9087-96.
81 Opalinska JB, Gewirtz AM. Therapeutic potential of antisense nucleic acid molecules. Sci STKE. 2003; 2003(206):pe47.
82 Biroccio A, Leonetti C, Zupi G. The future of antisense therapy: combination with anticancer treatments. Oncogene. 2003; 22(42): 6579-88.
83 Lavery KS, King TH. Antisense and RNAi: powerful tools in drug target discovery and validation. Curr Opin Drug Discov Devel. 2003; 6(4):561-9.
84 Stahel RA, Zangemeister-Wittke U. Antisense oligonucleotides for cancer therapy-an overview. Lung Cancer. 2003; 41 Suppl 1: S81-8.
85 Pirollo KF, Rait A, Sleer LS, Chang EH. Antisense therapeutics: from theory to clinical practice. Pharmacol Ther. 2003;99(1): 55-77.
86 Harth G, Horwitz MA, Tabatadze D, Zamecnik PC. Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl tmnsferase complex as a therapeutic strategy against tuberculosis: Proof of principle by
using antisense technology. Proc Natl Acad Sci U S A. 2002; 99(24): 15614-9.
87 Mathews DH, Sabina J, Zuker M, Turner DH. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol. 1999; 288(5):911-40.
88 Mathews DH, Burkard ME, Freier SM, Wyatt JR, Turner DH. Predicting oligonucleotide affinity to nucleic acid targets. RNA. 1999; 5(11):1458-69.
89 Schmajuk G, Sierakowska H, Kole R. Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake. J Biol Chem. 1999; 274(31):21783-9.
90 Peng Ho S, Livanov V, Zhang W, Li J, Lesher T. Modification of phosphorothioate oligonucleotides yields potent analogs with minimal toxicity for antisense experiments in the CNS. Brain Res Mol Brain Res. 1998; 62(1):1-11.
91 Lai JC, Benimetskaya L, Santella RM, Wang Q, Miller PS, Stein CA. G3139 (oblimersen) may inhibit prostate cancer cell growth in a partially bis-CpG-dependent non-antisense manner. Mol Cancer Ther. 2003; 2 (10):1031-43.
92 Wacheck V, Krepler C, Strommer S, Heere-Ress E, Klem R, Pehamberger H, Eichler HG, Jansen B. Antitumor effect of G3139 Bcl-2 antisense oligonucleotide is independent of its immune stimulation by CpG motifs in SCID mice. Antisense Nucleic Acid Drug Dev. 2002; 12(6):359-67.
93 Jahrsdorfer B, Jox R, Muhlenhoff L, Tschoep K, Krug A, Rothenfusser S, Meinhardt G, Emmerich B, Endres S, Hartmann G.
Modulation of malignant B cell activation and apoptosis by bcl-2 antisense ODN and immunostimulatory CpG ODN. J Leukoc Biol. 2002; 72(1):83-92.
94 Hacker H. Signal transduction pathways activated by CpG-DNA. Curr Top Microbiol Immunol. 2000; 247:77-92.
95 Debatin KM. Cytotoxic drugs, programmed cell death, and the immune system: defining new roles in an old play. J Natl Cancer Inst. 1997; 89(11):750-1.
96 Lenobel R, Havli L, Kryscaron PV, Otyepka M, Stmad M. Olomoucine ii, new effective cdk inhibitor with strong cytotoxic properties. ScientificWorldJoumal. 2001; 1(1 Suppl 3): 128.
97 Mihara M, Shintani S, Nakashiro K, Hamakawa H. Flavopiridol, a cyclin dependent kinase (CDK) inhibitor, induces apoptosis by regulating Bcl-x in oral cancer cells. Oral Oncol. 2003; 39(1): 49-55.
98 Edamatsu H, Gau CL, Nemoto T, Guo L, Tamanoi F. Cdk inhibitors, roscovitine and olomoucine, synergize with famesyltransferase inhibitor (FTI) to induce efficient apoptosis of human cancer cell lines.
2 Fernandes PB. Anti-cancer drug discovery and development summit. IDrugs. 2002; 5(8):757-64.
3 Clough J. New directions in cancer drug development. Drug Discov Today. 2003;8 (11):485-6.
4 Lele RD. The human genome project: its implications in clinical medicine. J Assoc Physicians India. 2003; 51:373-80.
5 Mariani SM. Functional genomics: improving cancer prognosis and drug development. MedGenMed. 2003;5(1):18.
6 Broxterman HJ, Georgopapadakou N. Cancer research 2000: drug resistance, new targets and drugs in development. Drug Resist Updat. 2000; 3(3):133-138.
7 Basik M, Mousses S, Trent J. Integration of genomic technologies for accelerated cancer drug development. Biotechniques. 2003; 35(3): 580-2, 584, 586 passim.
8 Erlich R. Cancer Drug Development: New Directions and Challenges —SMi Conference. 10-11 March 2003, London, UK. IDrugs. 2003; 6(4): 331-3.
9 Dobashi Y, Takehana T, Ooi A. Perspectives on cancer therapy: cell cycle blockers and perturbators. Curr Med Chem. 2003; 10(23): 2549
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10 王琳。正性生长因子对恶性肿瘤作用机制的研究进展，肠内与肠外营养，2003；10(3)：176—176．
11 Malumbres M, Carnero A. Cell cycle deregulation: a common motif in cancer. Prog Cell Cycle Res. 2003;5:5-18.
12 Malumbres M, Carnero A.Cell cycle deregulation: a common motif in cancer.Prog Cell Cycle Res. 2003; 5:5-18.
13 Laiho M, Latonen L.Cell cycle control, DNA damage checkpoints and cancer.Ann Med. 2003; 35(6):391-7.
14 Flemington EK, Rodriguez A. Use of gene overexpression to assess function in cell cycle control. Methods Mol Biol. 2004; 241:195-206.
15 Burtelow MA, Podust VN, Kamitz LM. Purification and identification of protein complexes that control the cell cycle. Methods Mol Biol. 2004; 241: 247-53.
16 Gitig DM, Koff A.Cdk pathway: cyclin-dependent kinases and cyclin-dependent kinase inhibitors.Methods Mol Biol. 2000; 142:109 -23.
17 Kaldis P.The Cdk-activating kinase (CAK): from yeast to mammals. Cell Mol Life Sci. 1999; 55(2):284-96.
18 Dyson N. The regulation of E2F by pRB-family proteins [J]. Genes Dev, 1998, 12(15): 2245-62.
19 Stevens C, La Thangue NB.E2F and cell cycle control: a doubleedged sword.Arch Biochem Biophys. 2003; 412 (2): 157-69.
20 Knockaert M, Greengard P, Meijer L. Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol Sci, 2002, 23(9): 417-25.
21 Boulaire J, Fotedar A, Fotedar R.The functions of the Cdk-cyclin kinase inhibitor p21WAF1.Pathol Biol (Paris). 2000; 48(3): 190-202.
22 Bloom J, Pagano M.Deregulated degradation of the Cdk inhibitor p27 and malignant transformation. Semin Cancer Biol. 2003; 13(1): 41-7.
23 Sherr CJ, Roberts JM. Inhibitors of mammalian GI cyclin-dependent kinases. Genes Dev. 1995; 9(10):1149-63.
24 Ruas M, Peters G. The p 16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta. 1998; 1378(2):F115-77.
25 Nabel EG.Cdks and CKIs: molecular targets for tissue remodelling. Nat Rev Drug Discov. 2002; 1(8):587-98.
26 Chen-Kiang S. Cell-cycle control of plasma cell differentiation and tumorigenesis. Immunol Rev. 2003; 194:39-47.
27 Peter MF, Graham B, Carol M, et al. Cell cycle target validation: approaches and successes. Targets, 2003; 2(4): 154-61.
28 Laiho M, Latonen L. Cell cycle control, DNA damage checkpoints and cancer. Ann Med. 2003; 35(6):391-7.
29 Sherr CJ. Cell cycle control and cancer. Harvey Lect. 2000-2001; 96:73-92.
30 May P. Cell cycle control and cancer. Pathol Biol (Pads). 2000; 48(3): 167-73.
31 Moller MB. P27 in cell cycle control and cancer. Leuk Lymphoma. 2000; 39(1-2):19-27.
32 杨连君．细胞周期蛋白与肿瘤等疾病的关系，中国肿瘤生物治疗杂志，2003；10(3)：223—225．
33 陈汉源．细胞周期素D1与癌症．第一军医大学学报，1999；19(6)：586-591．
34 Kaldis P. The Cdk-activating kinase (CAK): from yeast to mammals. Cell Mol Life Sci. 1999; 55(2):284-96.
35 Chen J, Larochelle S, Li X, Suter B. Xpd/Ercc2 regulates CAK activity and mitotic progression. Nature. 2003; 424(6945):228-32.
36 Gu Y, Rosenblatt J, Morgan DO. Cell cycle regulation of CDK2 activity by phosphorylation of Thrl60 and Tyr15. EMBO J. 1992; 11(11):3995-4005.
37 Fleig UN, Gould KL. Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation. Semin Cell Biol. 1991; 2(4): 195-204.
38 Matsuoka M, Kato JY, Fisher RP, Morgan DO, Sherr CJ. Activation of cyclin-dependent kinase 4 (Cdk4) by mouse MO15-associated kinase. Mol Cell Biol. 1994; 14(11):7265-75.
39 Hagopian JC, Kirtley MP, Stevenson LM, Gergis RM, Russo AA, Pavletich NP, Parsons SM, Lew J. Kinetic basis for activation of CDK2/cyclin A by phosphorylation. J Biol Chem. 2001; 276 (1): 275-80.
40 Araujo SJ, Tirode F, Coin F, Pospiech H, Syvaoja JE, Stucki M, Hubscher U, Egly JM, Wood RD.Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes Dev. 2000;14(3):349-59.
41 Svejstrup, J., P. Vichi, and J.-M. Egly. 1996. The multiple roles of
transcription/repair factor TFIIH. Trends Biochem. Sci. 21:346-350
42 Rickert P, Corden JL, Lees E. Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases. Oncogene. 1999; 18(4): 1093-1102.
43 Rossignol M, Kolb-Cheynel I, Egly JM. Substrate specificity of the Cdk-activating kinase (CAK) is altered upon association with TFIIH. EMBO J, 1997; 16(7): 1628-37.
44 Wu L, Chen P, Shum CH, Chen C, Barsky LW, Weinberg KI, Jong A, Triche TJ. MAT1-modulated CAK activity regulates cell cycle G(1) exit. Mol Cell Biol. 2001; 21 (1):260-70.
45 Kaldis P, Solomon MJ. Analysis of CAK activities from human cells. Eur J Biochem. 2000; 267(13):4213-21.
46 Araujo SJ, Tirode F, Coin F, Pospiech H, Syvaoja JE, Stucki M, Hubscher U, Egly JM, Wood RD. Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes Dev. 2000; 14(3):349-59.
47 te Poele RH, Okorokov AL, Joel SP. RNA synthesis block by 5, 6-dich/oro- 1 -beta-D-ribo furanosylbenzimidazole (DRB) triggers p53-dependent apoptosis in human colon carcinoma cells. Oncogene. 1999; 18(42): 5765-72.
48 Liang YC, Tsai SH, Chen L, Lin-Shiau SY, Lin JK. Resveratrol-induced G2 arrest through the inhibition of CDK7 and p34CDC2 kinases in colon carcinoma HT29 cells. Biochem Pharmacol. 2003; 65(7): 1053-60.
49 王杉，梁斌，乔新民，彦飞飞，崔志荣．细胞周期素和细胞周期素依赖性激酶7在人乳腺癌组织中的表达及其意义，中华普通外科杂志，2002；17(10)：611-613．
50 Bartkova J, Zemanova M, Bartek J. Expression of CDK7/CAK in normal and tumor cells of diverse histogenesis, cell cycle position and differentiation. Int J Cancer, 1996; 66(6): 732-737.
51 Mouriaux F, Casagrande F, Pillaire M J, Manenti S, Malecaze F, Darbon JM. Differential expression of G1 cyclins and cyclindependent kinase inhibitors in normal and transformed melanocytes. Invest Ophthalmol Vis Sci. 1998; 39(6):876-84.
52 Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol. 2003; 4(6):457-67.
53 Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med. 2003; 349 (21) : 2042-54.
54 Kittler R, Buchholz F. RNA interference: gene silencing in the fast lane. Semin Cancer Biol. 2003; 13(4):259-65.
55 Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxy-nucleotide. Proc Natl Acad Sci U S A. 1978; 75(1): 280-4.
56 Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978; 75(1): 285-8.
57 Grillone LR, Lanz R. Fomivirsen. Drugs Today (Barc). 2001; 37(4):
245-255.
58 Geary RS, Henry SP, Grillone LR. Fomivirsen: clinical pharmacology and potential drug interactions. Clin Pharmacokinet. 2002; 41(4):255-60.
59 Jabs DA, Griffiths PD. Fomivirsen for the treatment of cytomegalovirus retinitis. Am J Ophthalmol. 2002; 133 (4): 552-6.
60 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer.Oncogene. 2003; 22(56):9087-96.
61 Biroccio A, Leonetti C, Zupi G.The future of antisense therapy: combination with anticancer treatments. Oncogene. 2003; 22(42): 6579-88.
62 Burkhard J, Uwe ZW. Antisense therapy for cancer- the time of truth, Lancet Oncol 2002; 3(11):672-83.
63 Lahn M, Sundell K, Moore S. Targeting protein kinase C-alpha (PKC-alpha) in cancer with the phosphorothioate antisense oligonucleotide aprinocarsen. Ann N Y Acad Sci. 2003; 1002: 263-70.
64 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene. 2003; 22(56):9087-96.
65 Stephens AC, Rivers RP. Antisense oligonucleotide therapy in cancer. Curr Opin Mol Ther. 2003; 5(2): 118-22.
66 Cho-Chung YS. Antisense DNAs as targeted genetic medicine to treat cancer. Arch Pharm Res. 2003; 26(3): 183-91.
67 Chen HX. Clinical development of antisense oligonucleotides as anti-cancer therapeutics. Methods Mol Med. 2003; 75:621-36.
68 Crooke ST. Progress in antisense technology. Annu Rev Med.
2004;55:61-95.
69 Holmlund JT. Applying antisense technology: Affinitak and other antisense oligonucleotides in clinical development. Ann N Y Acad Sci. 2003; 1002:244-51.
70 Lee LK, Roth CM. Antisense technology in molecular and cellular bioengineering. Curr Opin Biotechnol. 2003 ;14(5):505-11.
71 Turner DH, Sugimoto N. RNA structure prediction. Annu Rev Biophys Biophys Chem. 1988; 17:167-92.
72 Akmaev VR, Kelley ST, Stormo GD. A phylogenetic approach to RNA structure prediction. Proc Int Conf Intell Syst Mol Biol. 1999; 10-7.
73 Biroccio A, Leonetti C, Zupi G.The future of antisense therapy: combination with anticancer treatments. Oncogene. 2003; 22(42): 6579-88.
74 Bochar, D.A., Pan,Z.Q., Knights,R., Fisher, R.P., Shilatifard,A., Shiekhatta,R. Inhibition of transcription by the trimeric cyclindependent kinase 7 complex, J Biol Chem, 1999, 274(19): 13162-13166.
75 Song HF, Tang ZM, Yuan SJ, et al.. Application of secondary structure prediction in antisense drug design targeting protein kinase C-alpha mRNA and QSAR analysis. Acta Pharmacol Sin, 2000: 21(1): 80-6.
76 孔伟东，李彦豪，曾庆乐，陈勇，何晓峰．平阳霉素对血管内皮细胞的生长抑制作用和细胞周期的影响，第一军医大学学报，2003；23(8)：830-832．
77 Li X, Melamed MR, Darzynkiewicz Z. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp Cell Res. 1996; 222(1):28-37.
78 Spyratos F. DNA content and cell cycle analysis by flow cytometry in clinical samples: application in breast cancer. Biol Cell. 1993; 78(1-2):69-72.
79 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer.Oncogene. 2003; 22(56):9087-96.
80 Dean NM, Bennett CF. Antisense oligonucleotide-based therapeutics for cancer. Oncogene. 2003; 22(56):9087-96.
81 Opalinska JB, Gewirtz AM. Therapeutic potential of antisense nucleic acid molecules. Sci STKE. 2003; 2003(206):pe47.
82 Biroccio A, Leonetti C, Zupi G. The future of antisense therapy: combination with anticancer treatments. Oncogene. 2003; 22(42): 6579-88.
83 Lavery KS, King TH. Antisense and RNAi: powerful tools in drug target discovery and validation. Curr Opin Drug Discov Devel. 2003; 6(4):561-9.
84 Stahel RA, Zangemeister-Wittke U. Antisense oligonucleotides for cancer therapy-an overview. Lung Cancer. 2003; 41 Suppl 1: S81-8.
85 Pirollo KF, Rait A, Sleer LS, Chang EH. Antisense therapeutics: from theory to clinical practice. Pharmacol Ther. 2003;99(1): 55-77.
86 Harth G, Horwitz MA, Tabatadze D, Zamecnik PC. Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl tmnsferase complex as a therapeutic strategy against tuberculosis: Proof of principle by
using antisense technology. Proc Natl Acad Sci U S A. 2002; 99(24): 15614-9.
87 Mathews DH, Sabina J, Zuker M, Turner DH. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol. 1999; 288(5):911-40.
88 Mathews DH, Burkard ME, Freier SM, Wyatt JR, Turner DH. Predicting oligonucleotide affinity to nucleic acid targets. RNA. 1999; 5(11):1458-69.
89 Schmajuk G, Sierakowska H, Kole R. Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake. J Biol Chem. 1999; 274(31):21783-9.
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