CYTOTOXICITY AND BYSTANDER KILLING FOR HSV-TK SUBSTRATES

说明：（申请人摘要）疱疹病毒cDNA的转移 单纯病毒胸苷激酶HSV-TK）进入肿瘤细胞， 更昔洛韦（GCV）活化为细胞毒性核苷酸。 这 这种方法的新颖之处在于，不仅表达HSV-TK的细胞被杀死， 通过用GCV治疗，但邻近的非HSV-TK表达细胞被 也被杀了。 这种现象被称为旁观者效应， 一些动物肿瘤的完全消退，现在正在临床上 审判 申请人的实验室最近的研究表明， GCV在胶质母细胞瘤和结肠中诱导大于4-log的细胞杀伤 癌细胞系相比，只有1 - 2 log细胞杀伤诱导 其他HSV-TK底物，如胸腺嘧啶阿拉伯糖苷（araT）。 的 申请人最近观察到另一种HSV-TK底物，D- 碳环-2 '-脱氧鸟苷（CdG），诱导类似于 与GCV的观察结果一致。 数据表明，GCV是独特的，因为它 弱抑制DNA合成，使细胞通过 细胞周期时，GCV核苷酸水平高，导致大量 细胞凋亡 此外，我们证明， 在HSV-TK细胞中与GCV相加增强细胞毒性， 在旁观者细胞中协同作用。 尽管其他研究人员 细胞间隙连接通讯（GJIC） 介导GCV的旁观者杀伤，我们的研究表明， GJIC缺陷也表现出显著的旁观者杀伤作用， HSV-TK细胞和GCV的存在。 因为这个项目的潜力 显著的抗肿瘤活性的方法，这里提出的研究 将阐明多对数细胞毒性和旁观者的机制 为GCV杀人 在具体目标1中，细胞毒性机制将 测定GCV和CdG，并与毒性较小的araT进行比较， 所有表达HSV-TK的细胞。 这些实验将集中在 GCV的摄取和代谢，并表征其对DNA的影响 合成和细胞周期进程。 具体目标2将评估 在旁观者细胞的细胞毒性机制，并阐明新的 GCV核苷酸在GJIC缺陷细胞中转移的机制。具体 目的3阐明羟基脲增强GCV的机制 细胞毒性并评价其在裸鼠中的治疗潜力 人肿瘤模型。 特异性目的4将评估延长的凋亡 用GCV观察到的反应以及bcl-2或bcl-xl对其的抑制。 这些 研究将确定细胞毒性和 GCV的旁观者效应，结果将形成基础， 这种治疗癌症的新方法的改进临床实施 化疗

DESCRIPTION: (Applicant's Abstract) Transfer of cDNA for the herpes simplex virus thymidine kinase HSV-TK) into tumor cells allows activation of ganciclovir (GCV) to a cytotoxic nucleotide. This approach is novel in that, not only are cells that express HSV-TK killed by treatment with GCV, but neighboring, non-HSV-TK-expressing cells are also killed. This phenomenon, called the bystander effect, has produced some complete regressions of tumors in animals and is now in clinical trials. Recent studies in the applicant's laboratory have shown that GCV induced a greater than 4-log cell kill in glioblastoma and colon carcinoma cell lines compared to only a 1 - 2 log cell kill induced by other HSV-TK substrates such as thymine arabinoside (araT). The applicant has recently observed that another HSV-TK substrate, D- carbocyclic-2'-deoxyguanosine (CdG), induces multi-log kill similar to that observed with GCV. The data suggest that GCV is unique in that it weakly inhibits DNA synthesis, allowing cells to progress through the cell cycle when GCV nucleotide levels are high, resulting in massive apoptotic cell death. Furthermore, we demonstrate that hydroxyurea enhances cytotoxicity with GCV additively in HSV-TK cells and synergistically in bystander cells. Although other investigators have implicated gap junctional intercellular communication (GJIC) as mediating bystander killing with GCV, our studies show that cells deficient in GJIC also exhibit significant bystander killing in the presence of HSV-TK cells and GCV. Because of the potential of this approach for significant antitumor activity, the studies proposed here will elucidate the mechanism of multi-log cytotoxicity and bystander killing for GCV. In Specific Aim 1, the mechanism of cytotoxicity will be determined for GCV and CdG and compared to the less toxic araT using cells which all express HSV-TK. These experiments will focus on the uptake and metabolism of GCV, and characterize its effects on DNA synthesis and cell cycle progression. Specific Aim 2 will evaluate the mechanism of cytotoxicity in bystander cells and elucidate the novel mechanism of GCV nucleotide transfer in GJIC-deficient cells. Specific Aim 3 will elucidate the mechanism by which hydroxyurea enhances GCV cytotoxicity and evaluate its therapeutic potential in a nude mouse human tumor model. Specific Aim 4 will evaluate the prolonged apoptotic response observed with GCV and its inhibition by bcl-2 or bcl-xl. These studies will define the critical determinants of the cytotoxic and bystander effects with GCV, and the results will form the basis for improved clinical implementation of this novel approach to cancer chemotherapy.