FUNCTIONAL ROLES OF GLUCOSE REGULATED PROTEIN GENE SYSTEM IN TARGETED TUMOR

在缺乏葡萄糖和氧气的实体肿瘤中，高表达的蛋白质 是葡萄糖调节蛋白(GRPs)。特别是，GRP78被提升 大约10倍，通过转录调控。大鼠和人的启动子 GRP78已被分离出来，并被证明对葡萄糖或氧气有反应 饥饿为肿瘤的靶向基因治疗开辟了新的途径。 可以构建GRP78启动子驱动表达的载体 能够引起局部炎症和细胞死亡的基因产物。这个 提案有三个目标，第一个是审查GRP78 启动子在逆转录病毒载体中作为内部启动子，可以 推动报告基因在肿瘤细胞中的高水平表达。逆转录病毒 使用GRP78或SV40作为neo基因启动子的载体将是 转导入B/C10ME细胞和秋水仙碱筛选的转染体。 将在体外检测葡萄糖饥饿条件下neo的表达情况。 注射到小鼠体内的转导细胞将被检测报告基因 并将显示在生长中的肿瘤是否在应激状态下，GRP78 启动子在其调控下增强neo的表达。GRP78启动子 将与SV40启动子进行比较，然后修改为更低的基础水平 并优化应激诱导转录。这些推动者将是 在培养细胞中进行测试，然后在体内进行测试。第二个目标是 构建带有GRP78启动子的载体以驱动细胞因子的表达 IL-2和GM-CSF。细胞因子的分泌将在正常或应激状态下进行检测 体外和体内诱导条件。转导的肿瘤细胞系将 注射入小鼠体内并与亲本相比致瘤性， 非转导细胞或结构性分泌细胞因子的细胞。急性 将检测NK细胞的排斥反应和CTL的迟发性排斥反应。在……里面 另一种方法C2肌源性细胞，用编码载体转染 在GRP78启动子控制下的细胞因子将被测试 诱导肿瘤消退的能力。类似的尝试将通过以下方式进行 用含有这些质粒的包装病毒感染肿瘤。这个 第三个目标是检查GRP78水平对肿瘤生长的影响 通过实验证明GRP78的诱导可以诱导对CTL的抗性 细胞毒性。反义载体抑制GRP78的诱导作用 B/C10ME消除了阻力。因此出现了这样一个问题： 应激蛋白的诱导普遍增强体内肿瘤的生长？ 应激后将检测几种肿瘤对CTL和肿瘤坏死因子的敏感性 归纳法。如果诱导产生抗性，就会将GRP78基因导入它们 反义结构。未能诱导GRP78应导致下降 肿瘤在体内生长。

In solid tumors deprived of glucose and oxygen, highly expressed proteins are glucose regulated proteins (GRPs). In particular, GRP78 is elevated about 10 fold, by transcriptional regulation. Promoters of rat and human grp78 have been isolated and shown to respond to glucose or oxygen starvation opening new approaches to targeted gene therapy of tumors. Vectors can be constructed in which the grp78 promoter drives expression of gene products able to induce local inflammation and cell death. The proposal has three goals, the first is to examine whether the grp78 promoter, serving as an internal promoter in a retroviral vector, can drive high level expression of a reporter gene in tumor cells. Retroviral vectors using either grp78 or SV40 as promoters, for the neo gene, will be transduced into B/C10ME cells and tranfectants selected by colchicine. Expression of neo under glucose starvation will be examined in vitro. Transduced cells injected into mice will be assayed for reporter gene expression and will show whether in growing tumors under stress, the grp78 promoter enhances expression of neo under its control. The grp78 promoter will be compared to the SV40 promoter, then modified to lower basal level and to optimize stress inducible transcription. These promoters will be tested in cultured cells, then assayed in vivo. The second goal is to generate vectors with the grp78 promoter to drive expression of cytokines IL-2 and GM-CSF. Cytokine secretion will be assayed under normal or stress induced conditions in vitro and in vivo. Transduced tumor cell lines will be injected into mice and tumorigenicity compared to that of parental, non-transduced cells or cells secreting cytokines constitutively. Acute rejection by NK cells and delayed rejection by CTL will be assayed. In alternative approaches C2 myogenic cells, transfected with vectors coding for cytokines under control of the grp78 promoter will be tested for ability to induce tumor regression. Similar attempts will be made by infection of tumors with packaged virus containing these plasmids. The third goal is to examine effects of GRP78 levels on tumor growth prompted by experiments showing that induction of GRP78 induces resistance to CTL cytotoxicity. Suppression of GRP78 induction by antisense vectors in B/C10ME eliminates resistance. Therefore the question arises does induction of stress protein in general enhance in vivo tumor growth? Several tumors will be assayed for sensitivity to CTL and TNF after stress induction. If resistance is induced they will be transfected with grp78 antisense constructs. Failure to induce GRP78 should lead to decreased tumor growth in vivo.