权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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) 人乳头瘤病毒(HPV)已被确凿地证明是由无生殖器肿瘤的代理人，以及一些头部和颈部的肿瘤。一个不断增长的大量证据表明猿猴病毒40(SV40)与人的肿瘤有关间皮、脑和骨。HPV和SV40均使P53和pRb基因失控肿瘤抑制因子与病毒编码的癌蛋白结合的途径细胞内的P53和pRb。针对HPV和SV40的反义技术癌蛋白导致细胞生长抑制和细胞凋亡来自HPV阳性的宫颈癌和SV40阳性的恶性肿瘤分别为间皮瘤。这一证据表明，HPV和SV40 癌蛋白是治疗特定类型肿瘤的有价值的靶点人类癌症。因此，免疫治疗和基因治疗都是治疗目标人乳头瘤病毒E6和E7是对于宫颈癌的治疗，已经提出了类似的策略 SV40阳性间皮瘤的治疗。到目前为止，免疫疗法这些方法未能在体内提供足够的反应，而且基因由于缺乏有效的交付系统，这些方法受到阻碍。我们提出一种替代方法：筛选化学文库以鉴定能够干扰SV 40与HPV结合的分子癌蛋白对细胞P53和pRb的体外作用。这些策略需要分析一大批化学品，只有在以下情况下才是可行的研究病毒癌蛋白相互作用的高通量方法他们的细胞目标是可用的。这些化验需要相对较高的病毒癌蛋白和肿瘤抑制因子适当的翻译后修饰以确保生物活性。是这样的如果蛋白质底物在人体内表达，就可以满足要求细胞。然而，目前人类细胞系统中蛋白质的过度表达不可用。我们发现SV40转化的人间皮细胞(HM) 可用于获得毫克量的SV40大肿瘤抗原(TAG) 与细胞内P53和pRb的复合体。我们打算利用这间牢房用于鉴定SV40标记细胞肿瘤化学抑制物的系统抑制者相互作用。此外，SV40转化的间皮克隆可以作为传播高拷贝数、异构体表达载体的基础在积极复制人间皮细胞中。这样的载体可以允许需要翻译后修饰的蛋白质的过度表达人体细胞中的适当生物活性。我们打算利用这项实验过量生产和纯化载体结合型HPV16E6和E7的系统。重组E6和E7将随后用于开发基于EL ISA的体外实验研究HPV16 E6和E7与P53和pRb的结合，分别进行了分析。最后，我们建议采用后一种分析方法来分析筛选化学文库以寻找HPVE6和HPVE6的抑制剂 E7。人乳头瘤病毒40和人乳头瘤病毒可能抑制物的鉴定癌蛋白可能导致新的抗癌药物的开发。此外，所提出的实验可能会为未来的研究提供新的技术。活性蛋白的高效表达和纯化复制人类间皮细胞。

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

High throughput testing of the SV40 Large T antigen binding to cellular p53 identifies putative drugs for the treatment of SV40-related cancers.

对 SV40 大 T 抗原与细胞 p53 结合的高通量测试确定了用于治疗 SV40 相关癌症的推定药物。