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：（由申请人提供）人类乳头瘤病毒 (HPV) 已被最终证明是致病因素肛门生殖器肿瘤和某些头颈部肿瘤的病原体。一个不断成长的大量证据表明猿猴病毒 40 (SV40) 与人类肿瘤有关间皮、脑和骨。 HPV 和 SV40 都会解除 p53 和 pRb 的管制通过病毒编码的癌蛋白与肿瘤抑制途径结合细胞 p53 和 pRb。针对 HPV 和 SV40 的反义技术癌蛋白导致细胞系生长抑制和凋亡 HPV 阳性宫颈癌和 SV40 阳性恶性间皮瘤，分别。该证据表明 HPV 和 SV40 癌蛋白代表了治疗特定类型癌症的有价值的靶点人类癌症。因此，免疫治疗和基因治疗方法目标HPV E6和E7是临床前或临床试验的对象宫颈癌的治疗，并且已经提出了类似的策略 SV40阳性间皮瘤的治疗。迄今为止，免疫治疗方法未能在体内提供足够的反应，并且遗传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：筛选化学库鉴定能够干扰 SV 40 和 HPV 结合的分子体外将癌蛋白转化为细胞 p53 和 pRb。这些策略需要分析一大组化学品，这项任务只有在以下情况下才可行研究病毒癌蛋白相互作用的高通量测定其细胞目标是可用的。这些测定需要相对大量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。这样的如果蛋白质底物在人体内表达，则可以满足要求细胞。然而，用于蛋白质过度表达的人类细胞系统目前尚不成熟。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（标签）与细胞 p53 和 pRb 形成复合物。我们建议利用这个细胞识别 SV40 标签细胞肿瘤化学抑制剂的系统抑制剂相互作用。此外，SV40 转化的间皮克隆可以用作传播“高拷贝数”附加型表达载体的基础积极复制人类间皮细胞。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适当的生物活性。我们建议使用这个实验系统过量产生并纯化可缀合载体的 HPV16 E6 和 E7。重组 E6 和 E7 随后将用于开发基于 ELISA 的研究 HPVl6 E6 和 E7 与 p53 和 pRb 结合的体外测定，分别。最后，我们建议采用后一种分析方法筛选化学文库以寻找 HPV E6 抑制剂和 E7。 SV 40 和 HPV 推定抑制剂的鉴定癌蛋白可能会导致新型抗癌药物的开发。此外，所提出的实验可能会为以下方面提供新技术：积极地过度表达和纯化潜在的任何蛋白质复制人类间皮细胞。

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

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

SV40 和 Notch-I：多功能性与多效性的结合。