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: 6721446
• 负责人: WILLIAM Francis BENEDICT
• 金额: $ 33.6万
• 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/6721446
• 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 基因的第二个起始密码子，编码 94 KD 蛋白 (pRB94)。它是比野生型 RB 蛋白明显更有效的肿瘤抑制剂和细胞毒剂，并且迄今为止，无论组织类型、RB 或其他基因状态如何，对所有测试的肿瘤类型均有效，但端粒酶除外。该项目的长期目标是了解端粒酶的情况。引起如此有效作用的细胞和分子 pRB94 相互作用 初步结果表明 pRB94 特异性诱导肿瘤细胞死亡的关键机制可能涉及端粒快速磨损和染色体危机的产生。肿瘤抑制可能与迄今为止检查的所有其他药物或方式不同，并且发生在所有端粒酶阳性肿瘤或永生化细胞中，但不存在于含有 ALT 的肿瘤或永生化细胞中途径，即端粒酶阴性细胞。 RB94 还被发现对正常人类细胞没有细胞毒性或生长抑制作用，包括尿路上皮细胞，这些细胞也是端粒酶阴性的。因此，一种方法是确定对正常端粒复合体的干扰是否在 RB94 产生的端粒磨损以及随后的染色体不稳定和细胞死亡中发挥关键作用。中心体的作用和 STK15 激酶活性的变化也将得到深入研究。技术包括使用微阵列、共焦激光扫描、染色体和端粒状态分析、通过蛋白质印迹和免疫化学染色检查 RB94 特异性蛋白质相互作用，以及通过对假定的 RB94 特异性相关蛋白质进行测序进行免疫沉淀。研究将扩大到检查 RB94 对其他端粒酶阳性或阴性肿瘤细胞以及基因改变的非致瘤永生化细胞的杀伤作用。还将研究这些变化是否依赖于半胱天冬酶。另一个具体目标是优化膀胱内基因治疗并确定 AdRB94 对浅表性膀胱癌的效果。我们使用 GFP 表达细胞开发的膀胱内人类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。 Syn3 已被发现能够显着增加腺病毒介导的基因转移，而本身却没有毒性。如果这些研究成功，可能会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 的独特特性提供分子基础。