RB4 Intravesical Gene Therapy: Mechanisms of Cell Death

描述(申请人提供)：一种改良的视网膜母细胞瘤基因结构利用Rb基因的第二起始密码子，编码一个94kD的蛋白质(pRB94。与野生型Rb蛋白相比，它是一种明显更有效的肿瘤抑制和细胞毒剂，迄今对所有测试的肿瘤类型都有效，无论组织类型、Rb或除端粒酶以外的其他基因状态。该项目的一个长期目标是了解导致这种强大影响的细胞和分子pRB94相互作用。初步结果表明，pRB94特异性诱导肿瘤细胞死亡的一个关键机制可能涉及产生快速的端粒磨损和染色体危机。这些结果使得RB94细胞杀伤和肿瘤抑制的机制(S)潜在地独一无二于所有其他被研究的药物或方式，并且已经在所有端粒酶阳性肿瘤或永生化细胞中发生，但在肿瘤或含有ALT途径的永生化细胞中不发生，即端粒酶阴性细胞。RB94也被发现对正常的人细胞，包括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，一种方法将是确定对正常端粒复合体的干扰是否在RB94引起的端粒磨损、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被深入研究。技术将包括使用微阵列、激光共聚焦扫描、分析染色体和端粒状态、通过Western blotting和免疫化学染色检查RB94特定蛋白质的相互作用，以及免疫沉淀和假定的RB94特定相关蛋白质的测序。研究将扩大到检测额外的端粒酶阳性或阴性肿瘤细胞和基因改变的非致癌永生化细胞对RB94细胞的杀伤作用。这些变化是否依赖于caspase也将被研究。另一个具体目标是优化膀胱内基因治疗，并确定AdRB94对浅表性膀胱癌的疗效。我们使用GFP表达细胞建立的人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被发现可以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，可能会对开发一种新的治疗复发性浅表性膀胱癌的方法以及潜在的其他肿瘤类型产生重大影响，并为RB94的独特性质提供分子基础。

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.