RB4 Intravesical Gene Therapy: Mechanisms of Cell Death

RB4膀胱内基因治疗：细胞死亡机制

• 批准号: 7020047
• 负责人: WILLIAM Francis BENEDICT
• 金额: $ 32.81万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020047
• 关键词: Adenoviridae; athymic mouse; bladder neoplasm; cell death; cell line; centrosome; combination cancer therapy; confocal scanning microscopy; cytotoxicity; disease /disorder model; enzyme activity; gene therapy; immunocytochemistry; immunoprecipitation; microarray technology; neoplasm /cancer chemotherapy; neoplasm /cancer therapy; neoplastic cell; nonhuman therapy evaluation; protein protein interaction; protein sequence; retinoblastoma protein; telomerase; telomere; transfection /expression vector; tumor suppressor genes; western blottings

DESCRIPTION (provided by applicant): A modified retinoblastoma gene construct utilizes the second start codon of the RB gene and encodes for a 94 KD protein (pRB94. It is a markedly more potent tumor suppressor and cytotoxic agent than the wild-type RB protein and has been effective against all tumor types tested to date irrespective of tissue type, RB or other gene status, except for that of telomerase. A long-term objective of this project is to understand the cellular and molecular pRB94 interactions that cause such potent effects. Preliminary results suggest that a key mechanism of pRB94 specific induced tumor cell death may involve the production of rapid telomere attrition and chromosomal crisis. These results make the mechanism(s) of RB94 cell kill and tumor suppression potentially unique from all other agents or modalities examined to date and has occurred in all telomerase positive tumors or immortalized cells but not in tumor or immortalized cells containing an ALT pathway, i.e. telomerase negative cells. RB94 also has been found not to be cytotoxic or growth inhibitory to normal human cells, including urothelial cells, which are also telomerase negative. One approach will therefore be to determine if interference with the normal telomere complex plays a key role in RB94 produced telomere attrition, with subsequent chromosomal instability and cell death. The role of centrosomes and changes in STK15 kinase activity will also be studied in depth. Techniques will be include the use of microarrays, confocal laser scanning, analysis of chromosomal and telomere status, examination of RB94 specific protein interactions by Western blotting and immunochemical staining as well as immunoprecipitation with sequencing of putative RB94-specific related proteins. Studies will be expanded to examine RB94 cell kill in additional telomerase positive or negative tumor cells and genetically altered, non-tumorigenic immortalized cells. Whether or not these changes are caspase dependent will also be studied. Another specific aim is to optimize intravesical gene therapy and determine the effect of AdRB94 on superficial bladder cancer. An intravesical human bladder cancer model developed by us using GFP expressing cells will be utilized for this purpose. To increase adenovirus-mediated transfer the reagent, Syn3, will be used. Syn3 has been found to markedly increase adenoviralmediated gene transfer without being toxic itself. If these studies are successful, it could have a significant influence in developing a new modality of treatment for recurrent superficial bladder cancer and potentially for other tumor types as well as provide the molecular basis for the unique properties of RB94.

描述（由申请人提供）：修饰的视网膜母细胞瘤基因构建体利用RB基因的第二起始密码子并编码94KD蛋白（pRB94.它是一种比野生型RB蛋白明显更有效的肿瘤抑制剂和细胞毒性剂，并且迄今为止对所有测试的肿瘤类型都有效，而不管组织类型、RB或其他基因状态，除了端粒酶。该项目的一个长期目标是了解导致这种有效作用的细胞和分子pRB94相互作用。初步结果表明，pRB 94特异性诱导肿瘤细胞死亡的关键机制可能涉及快速端粒磨损和染色体危象的产生。这些结果使得RB94细胞杀伤和肿瘤抑制的机制可能与迄今为止检查的所有其他试剂或模式不同，并且已经发生在所有端粒酶阳性肿瘤或永生化细胞中，但不发生在含有ALT途径的肿瘤或永生化细胞中，即端粒酶阴性细胞。还发现RB94对正常人细胞（包括也是端粒酶阴性的尿路上皮细胞）没有细胞毒性或生长抑制作用。因此，一种方法是确定对正常端粒复合物的干扰是否在RB 94产生的端粒磨损中起关键作用，随后是染色体不稳定和细胞死亡。中心体的作用和STK15激酶活性的变化也将深入研究。技术将包括使用微阵列，共聚焦激光扫描，染色体和端粒状态分析，检查RB94特异性蛋白质的相互作用，通过Western印迹和免疫化学染色以及免疫沉淀与推定的RB94特异性相关蛋白质的测序。研究将扩大到检查RB94细胞在其他端粒酶阳性或阴性肿瘤细胞和遗传改变的非致瘤性永生化细胞中的杀伤。还将研究这些变化是否是半胱天冬酶依赖性的。另一个具体目标是优化膀胱内基因治疗，并确定AdRB 94对浅表性膀胱癌的影响。我们使用GFP表达细胞开发的膀胱内人膀胱癌模型将用于此目的。为了增加腺病毒介导的转移，将使用试剂Syn3。已发现Syn3显著增加腺病毒介导的基因转移，而本身没有毒性。如果这些研究成功，它可能对开发复发性浅表性膀胱癌和其他肿瘤类型的新治疗方式产生重大影响，并为RB94的独特性质提供分子基础。