Selection of chemical inhibitors of oncoproteins

癌蛋白化学抑制剂的选择

基本信息

批准号：
6515050
负责人：
MAURIZIO BOCCHETTA
金额：
$ 14.8万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-05-25 至 2004-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6515050
关键词：
enzyme linked immunosorbent assay high throughput technology host organism interaction human papillomavirus inhibitor /antagonist mesothelioma microorganism culture oncoproteins p53 gene /protein protein protein interaction recombinant proteins retinoblastoma protein simian virus 40 transfection /expression vector tumor antigens viral carcinogenesis virus antigen virus protein

项目摘要

DESCRIPTION: (provided by applicant) Human Papilloma Viruses (HPVs) have been conclusively proven as causative agents of ano-genital tumors, and some tumors of the head and neck. A growing body of evidence relates Simian Virus 40 (SV40) with tumors of the mesothelium, brain, and bone. Both HPV and SV40 deregulate the p53 and pRb tumor suppressors pathways through binding of virus-encoded oncoproteins to the cellular p53 and pRb. Antisense technology targeting the HPV and SV40 oncoproteins leads to growth inhibition and apoptosis in cell lines derived from HPV-positive cervical cancers, and from SV40-positive malignant mesotheliomas, respectively. This evidence suggest that the HPV and SV40 oncoproteins represent valuable targets for the treatment of specific types of human cancer. Accordingly, both immuno-therapy and gene-therapy approaches to target HPV E6 and E7 are subjects of pre-clinical or clinical trials for the treatment of cervical cancer, and similar strategies have been proposed for the treatment of SV40-positive mesotheliomas. So far, immuno-therapy approaches have failed to provide a sufficient response in vivo, and genetic approaches are hampered by the lack of an efficient delivery system. We propose an alternative approach: the screening of chemical libraries to identify molecules capable of interfering with the binding of SV 40 and HPV oncoproteins to cellular p53 and pRb in vitro. These strategies require the analysis of a large panel of chemicals, a task feasible only if high-throughput assays to study the interactions of the viral oncoproteins with their cellular targets are available. These assays would require relatively high amounts of viral oncoproteins and tumor suppressors with proper post-translational modifications to ensure biological activity. Such requirement can be fulfilled if the protein substrates are expressed in human cells. However, human cell systems for protein over-expression are presently unavailable. We discovered that SV40-transformed human mesothelial cells (HM) can be used to obtain mg amounts of the SV40 large tumor antigen (Tag) in complex with cellular p53 and pRb. We propose to take advantage of this cell system to identify chemical inhibitors of the SV40 Tag-cellular tumor suppressors interactions. Moreover SV40-transformed mesothelial clones can be used as a basis to propagate "high copy number", episomal expression vectors in actively replicating human mesothelial ce!ls. Such vectors may allow over-expression of proteins requiring post-translational modifications for proper biological activity in human cells. We propose to use this experimental system to overproduce and purify carrier-conjugable HPVl6 E6 and E7. Recombinant E6 and E7 will be subsequently used to develop ELISA-based in vitro assays to study the HPVl6 E6 and E7 binding to p53 and pRb, respectively. Finally, we propose to employ the latter assays for the screening of chemical libraries in order to find inhibitors of the HPV E6 and E7. The identification of putative inhibitors of the SV 40 and HPV oncoproteins may lead to the development of novel anticancer drugs. Furthermore, the experiments proposed may contribute novel technology for the over-expression and purification of potentially any protein in actively replicating human mesothelial cells.

描述：（申请人提供）人类乳头状瘤病毒（HPV）已被最终证明是病毒肛门生成肿瘤的药物和头颈部的一些肿瘤。成长证据主体将猿猴病毒40（SV40）与肿瘤有关间皮，大脑和骨骼。 HPV和SV40都放弃了P53和PRB 肿瘤抑制途径通过病毒编码的癌蛋白与细胞p53和PRB。针对HPV和SV40的反义技术癌蛋白会导致细胞系的生长抑制和凋亡来自HPV阳性宫颈癌，以及从SV40阳性恶性肿瘤间皮瘤分别。这些证据表明HPV和SV40 癌蛋白代表了处理特定类型的宝贵目标人类癌。因此，免疫疗法和基因疗法的方法目标HPV E6和E7是临床前或临床试验的受试者已经提出了宫颈癌的治疗和类似的策略 SV40阳性间皮瘤的治疗。到目前为止，免疫治疗方法未能在体内提供足够的反应和遗传缺乏有效的交付系统会阻碍方法。我们提出另一种方法：化学文库筛选到识别能够干扰SV 40和HPV结合的分子在体外对细胞p53和PRB的癌细胞蛋白。这些策略需要分析一大批化学物质，仅当任务是可行的高通量测定法研究病毒癌蛋白的相互作用有其细胞靶标。这些测定需要具有较高量的病毒癌蛋白和肿瘤抑制剂适当的翻译后修饰以确保生物活性。这样的如果蛋白质底物在人类中表达，则可以满足需求细胞。但是，目前的蛋白质过表达的人类细胞系统是不可用。我们发现SV40转化的人间皮细胞（HM）可用于获得Mg量的SV40大型肿瘤抗原（TAG）与细胞p53和PRB复合物。我们建议利用这个单元鉴定SV40标记细胞肿瘤的化学抑制剂的系统抑制器相互作用。此外，SV40转换的介间克隆可以是用作传播“高拷贝数”的基础，偶发表达矢量在积极复制人间皮ce！时。这样的向量可能允许蛋白质的过度表达，需要翻译后修饰的蛋白质人类细胞中适当的生物学活性。我们建议使用此实验过量生产和净化载体可结合的HPVL6 E6和E7的系统。重组E6和E7随后将用于开发基于ELISA的体外测定研究HPVL6 E6和E7与p53和PRB的结合，分别。最后，我们建议使用后者的测定法筛选化学文库，以查找HPV E6的抑制剂和 E7。 SV 40和HPV的推定抑制剂的鉴定癌蛋白可能导致新型抗癌药的发展。此外，提出的实验可能为在主动中的过度表达和纯化潜在的任何蛋白质复制人间皮细胞。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Different susceptibility of human mesothelial cells to polyomavirus infection and malignant transformation.

人间皮细胞对多瘤病毒感染和恶变的易感性不同。

DOI：
发表时间：
2003
期刊：
Cancer research
影响因子：
11.2
作者：
Carbone,Michele;Burck,Carol;Rdzanek,Monica;Rudzinski,Jennifer;Cutrone,Rochelle;Bocchetta,Maurizio
通讯作者：
Bocchetta,Maurizio

SV40 and Notch-I: multi-functionality meets pleiotropy.

SV40 和 Notch-I：多功能性与多效性的结合。