DISRUPTION OF CELL CYCLE GENES IN BREAST CANCER

描述：mRNA帽结合蛋白eIF 4 E在细胞凋亡中起重要作用。 通过调节G1-S期事件控制细胞增殖 过渡，并在多个层面进行监管。 异常高水平的 eIF 4 E是维持乳腺癌细胞表型所必需的。 的 乳腺癌和乳腺癌细胞系活组织检查中eIF 4 E的水平 相对于良性纤维腺瘤和对照细胞增加。 直接 乳腺癌研究证实了eIF 4 E在乳腺癌中的作用 细胞（MDA-435）表现出50%的eIF 4 E表达下降，由于 反义构建体，其具有显著降低的产生 裸鼠肿瘤。 申请人将使用显性阴性突变体， eIF 4 E以检验eIF 4 E过表达导致乳腺癌的假设。 癌 此外，他们将使用基于结构的网站定向 诱变和随机诱变并筛选以鉴定eIF 4 E突变体 对mRNA有高亲和力的细胞来了解更多关于mRNA结合的信息。 突变体可用于癌症的基因治疗（显性阴性），核酸治疗， 酸基疫苗或生物技术。 具体目标是：（A） 确定（1）在大肠杆菌中表达eIF 4 E的显性阴性形式的影响， 乳腺癌细胞和（2）过表达野生型和高表达的影响 非恶性乳腺上皮细胞中eIF 4 E的亲和突变体。 影响 对eIF 4F复合物形成的影响，与 增加的增殖，软琼脂中的集落生长， 将确定裸鼠。 (B)为了确定在特定的 与eIF 4 E的mRNA结合位点相邻的环可以改变eIF 4 E与mRNA结合位点的亲和力。 mRNA。 (C)为了使用随机诱变，细菌表面展示系统和 荧光m7 G探针，用于鉴定结合mRNA帽的eIF 4 E突变 高亲和力。 (D)启动共晶生长研究， m7 GpppG和m7 G加帽的寡核糖核苷酸，结合 与野生型eIF 4 E相比，mRNA具有更高的亲和力。

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.