DISRUPTION OF CELL CYCLE GENES IN BREAST CANCER

乳腺癌细胞周期基因的破坏

• 批准号: 6439261
• 负责人: CURT H. HAGEDORN
• 金额: $ 3.59万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6439261
• 关键词: X ray crystallography; antineoplastics; athymic mouse; binding proteins; binding sites; breast neoplasms; cell cycle; cell growth regulation; cell proliferation; epithelioma; epithelium; fluorescent dye /probe; gene expression; guanosine triphosphate; messenger RNA; mutant; neoplasm /cancer genetics; neoplastic cell; oligonucleotides; protein binding; protein structure function; site directed mutagenesis; tissue /cell culture

DESCRIPTION: The mRNA cap binding protein, eIF4E, plays an essential role in the control of cell proliferation by regulating events at the G1-S phase transition and is regulated at multiple levels. Abnormally high levels of eIF4E are required to maintain the phenotype of breast cancer cells. The levels of eIF4E in biopsies of breast cancer and breast cancer cell lines are increased relative to benign fibroadenomas and control cells. A direct role for eIF4E in breast cancer is evidenced by studies of mammary carcinoma cells (MDA-435) exhibiting a 50 percent decrease in eIF4E expression, due to an antisense construct, that have a markedly reduced ability to produce tumors in nude mice. The applicants will use dominant negative mutants of eIF4E to test the hypothesis that overexpression of eIF4E causes breast cancer. In addition, they will use structure based site directed mutagenesis and random mutagenesis with screening to identify eIF4E mutants that have a high affinity for mRNA to learn more about mRNA binding. Mutants may be used in gene therapy of cancer (dominant negatives), nucleic acid based vaccines, or in biotechnology. Specific objectives are: (A) To determine (1) the effect of expressing dominant negative forms of eIF4E in breast cancer cells and (2) the effect of overexpressing wild-type and high affinity mutants of eIF4E in nonmalignant breast epithelial cells. Effects on eIF4F complex formation, cell cycle perturbations associated with increased proliferation, colony growth in soft agar, and tumor formation in nude mice will be determined. (B) To determine what residues in specific loops adjacent to the mRNA binding site of eIF4E can alter the affinity for mRNA. (C) To use random mutagenesis, a bacterial surface display system and fluorescent m7G probes to identify mutations of eIF4E that bind mRNA caps with high affinities. (D) to initiate cocrystal growth studies, with m7GpppG and m7G capped oligoribonucleotides, of several mutants that bind mRNA with a higher affinity than wild-type eIF4E.

描述：mRNA 帽结合蛋白 eIF4E 发挥着重要作用 通过调节 G1-S 期事件来控制细胞增殖 转型并受到多个层面的监管。 异常高水平 eIF4E 是维持乳腺癌细胞表型所必需的。 这 乳腺癌和乳腺癌细胞系活检中 eIF4E 的水平 相对于良性纤维腺瘤和对照细胞增加。 直接一个 乳腺癌研究证明了 eIF4E 在乳腺癌中的作用 细胞 (MDA-435) 的 eIF4E 表达下降 50%，这是由于 反义结构，其产生的能力显着降低 裸鼠体内的肿瘤。 申请人将使用显性失活突变体 eIF4E 检验 eIF4E 过度表达导致乳腺癌的假设 癌症。 此外，他们将使用基于结构的站点定向 诱变和随机诱变以及筛选以确定 eIF4E 突变体 对 mRNA 具有高亲和力，以了解有关 mRNA 结合的更多信息。 突变体可用于癌症（显性阴性）、核酸的基因治疗 酸基疫苗或生物技术。 具体目标是： (A) 确定 (1) 在中表达 eIF4E 显性失活形式的效果 乳腺癌细胞和（2）过表达野生型和高表达的效果 非恶性乳腺上皮细胞中 eIF4E 的亲和力突变体。 效果 eIF4F 复合物形成、细胞周期扰动与 增加软琼脂中的增殖、集落生长以及肿瘤形成 裸鼠将被确定。 (B) 确定特定的残基 与 eIF4E mRNA 结合位点相邻的环可以改变对 mRNA。 (C) 使用随机诱变、细菌表面展示系统和 荧光 m7G 探针可识别结合 mRNA 帽的 eIF4E 突变 具有很高的亲和力。 (D) 启动共晶生长研究， m7GpppG 和 m7G 加帽寡核糖核苷酸，几种结合的突变体 与野生型 eIF4E 相比，mRNA 具有更高的亲和力。