DISRUPTION OF CELL CYCLE GENES IN BREAST CANCER

乳腺癌细胞周期基因的破坏

• 批准号: 2692054
• 负责人: CURT H. HAGEDORN
• 金额: $ 18.23万
• 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 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2692054
• 关键词: X ray crystallography; antineoplastics; athymic mouse; binding proteins; breast neoplasms; cell cycle; cell growth regulation; cell proliferation; epithelioma; epithelium; fluorescent dye /probe; gene expression; guanosine triphosphate; messenger RNA; molecular site; 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起着至关重要的作用 通过调节G1-S相的事件来控制细胞增殖 过渡，并在多个层次上进行调节。 异常高 需要EIF4E维持乳腺癌细胞的表型。 这 乳腺癌和乳腺癌细胞活检活检中的EIF4E水平 相对于良性纤维瘤和对照细胞增加。 直接 乳腺癌的研究证明了EIF4E在乳腺癌中的作用 细胞（MDA-435）由于EIF4E表达降低50％，因此 一种反义结构，其产生能力明显降低 裸鼠的肿瘤。 申请人将使用主要的负面突变体 EIF4E检验以下假设：EIF4E的过表达引起乳房 癌症。 此外，他们将使用基于结构的网站定向 诱变和随机诱变与筛选以鉴定EIF4E突变体 对mRNA具有很高的亲和力，以了解有关mRNA结合的更多信息。 突变体可用于癌症的基因治疗（主要阴性），核酸 基于酸的疫苗或生物技术。 具体目标是：（a） 确定（1）表达EIF4E的主要负面形式的效果 乳腺癌细胞和（2）过表达野生型和高高的影响 eIF4E的亲和力突变体在非恶性乳房上皮细胞中。 效果 在EIF4F复合物形成上，细胞周期扰动与 增加的增殖，软琼脂的菌落生长和肿瘤形成 将确定裸鼠。 （b）确定特定的残留物 与EIF4E的mRNA结合位点相邻的环可以改变对的亲和力 mRNA。 （c）使用随机诱变，细菌表面显示系统和 荧光M7G探针以鉴定结合mRNA帽的EIF4E突变 具有高亲和力。 （d）启动共晶增长研究， M7GPPPG和M7G限制了几种结合的突变体的寡核苷酸 与野生型EIF4E相比，亲和力更高的mRNA。