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: 6881558
• 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/6881558
• 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）：一种修饰的视网膜母细胞瘤基因构建物利用RB基因的第二个起始密码子编码94kd蛋白（pRB94）。与野生型RB蛋白相比，它是一种明显更有效的肿瘤抑制剂和细胞毒性剂，迄今为止，它对所有类型的肿瘤都有效，与组织类型、RB或其他基因状态无关，但端粒酶除外。该项目的长期目标是了解细胞和分子pRB94的相互作用，导致这种强有力的影响。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。这些结果使得RB94细胞杀伤和肿瘤抑制的机制可能是迄今为止研究过的所有其他药物或模式中唯一的，并且发生在所有端粒酶阳性的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。RB94也被发现对正常人类细胞没有细胞毒性或生长抑制作用，包括端粒酶阴性的尿路上皮细胞。因此，一种方法将是确定对正常端粒复合物的干扰是否在RB94产生的端粒磨损、随后的染色体不稳定和细胞死亡中起关键作用。中心体的作用和STK15激酶活性的变化也将被深入研究。技术将包括使用微阵列，共聚焦激光扫描，分析染色体和端粒状态，通过Western blotting和免疫化学染色检查RB94特异性蛋白相互作用，以及免疫沉淀与推定的RB94特异性相关蛋白测序。研究将扩大到检测RB94细胞在其他端粒酶阳性或阴性肿瘤细胞和基因改变的非致瘤性永生化细胞中的杀伤作用。这些变化是否依赖于半胱天冬酶也将被研究。另一个具体的目的是优化膀胱内基因治疗，并确定AdRB94对浅表性膀胱癌的影响。我们利用表达GFP的细胞建立的膀胱内人类膀胱癌模型将用于这一目的。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。如果这些研究取得成功，将对开发一种治疗复发性浅表性膀胱癌和其他类型肿瘤的新模式产生重大影响，并为RB94的独特性质提供分子基础。