FUNCTIONAL ROLES OF GLUCOSE REGULATED PROTEIN GENE SYSTEM IN TARGETED TUMOR

葡萄糖调节蛋白基因系统在靶向肿瘤中的功能作用

• 批准号: 6102897
• 负责人: GUNTHER DENNERT
• 金额: $ 21.35万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6102897
• 关键词: Retroviridae; antisense nucleic acid; beta galactosidase; chloramphenicol acetyltransferase; colony stimulating factor; gene induction /repression; gene therapy; genetic promoter element; glucose; hypoxia; interleukin 2; laboratory rat; leukocytes; neoplasm /cancer therapy; neoplastic growth; nonhuman therapy evaluation; northern blottings; polymerase chain reaction; reporter genes; stress proteins; transfection; transfection /expression vector

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.

在缺乏葡萄糖和氧气的实体瘤中， 是葡萄糖调节蛋白（GRP）。特别是，GRP78升高， 大约10倍，通过转录调节。大鼠和人的启动子 GRP78已经被分离出来并显示出对葡萄糖或氧有反应 饥饿为肿瘤的靶向基因治疗开辟了新途径。 可以构建其中grp78启动子驱动表达的载体 能够诱导局部炎症和细胞死亡的基因产物。的 该提案有三个目标，第一个是审查是否grp78 在逆转录病毒载体中作为内部启动子的启动子可以 驱动肿瘤细胞中报告基因的高水平表达。录病毒 将使用grp78或SV40作为neo基因的启动子的载体， 转染B/C10 ME细胞，用秋水仙素筛选转染子。 将在体外检测葡萄糖饥饿下neo的表达。 将测定注射到小鼠中的转导细胞的报告基因 表达，并将显示是否在生长中的肿瘤在压力下，grp78 启动子在其控制下增强neo的表达。grp78启动子 将与SV40启动子进行比较，然后修饰为较低的基础水平 并优化胁迫诱导转录。这些发起人将 在培养的细胞中测试，然后在体内测定。第二个目标是 产生具有grp78启动子的载体以驱动细胞因子的表达 IL-2和GM-CSF。将在正常或应激下测定细胞因子分泌 在体外和体内诱导条件下。转导的肿瘤细胞系将 注射到小鼠中并与亲本的致瘤性比较， 非转导细胞或组成型分泌细胞因子的细胞。急性 将测定NK细胞的排斥和CTL的延迟排斥。 在 替代方法C2肌原细胞，用编码 将测试GRP78启动子控制下的细胞因子 诱导肿瘤消退的能力。 类似的尝试将由 用含有这些质粒的包装病毒感染肿瘤。的 第三个目标是检查GRP78水平对肿瘤生长的影响， 通过实验显示GRP78的诱导诱导了对CTL的抗性， 细胞毒反义载体对GRP78诱导的抑制作用 B/C10 ME消除阻力。因此，问题出现了， 应激蛋白诱导通常增强体内肿瘤生长？ 几种肿瘤将在应激后测定对CTL和TNF的敏感性 诱导 如果诱导抗性，则将它们用grp78转染 反义构建体。未能诱导GRP78应导致降低 体内肿瘤生长。