DISRUPTION OF CELL CYCLE GENES IN BREAST CANCER

描述：信使核糖核酸帽结合蛋白eif4E起着重要的作用。 通过调节G1-S期事件控制细胞增殖 过渡，并在多个层面上进行监管。不正常的高水平 EIF4E是维持乳腺癌细胞表型所必需的。这个 乳腺癌及乳腺癌细胞株活检组织中eIF4E的表达 相对于良性纤维腺瘤和对照细胞是增加的。一位直接的 乳腺癌研究证实eIF4E在乳腺癌中的作用 细胞(MDA-435)eIF4E表达下降50%，原因是 一种反义结构，其产生能力显著降低 裸鼠体内的肿瘤。申请者将使用显性负性突变体 EIF4E验证过度表达eIF4E导致乳房的假说 癌症。此外，他们还将使用基于结构的站点定向 诱变和随机诱变及筛选鉴定eIF4E突变体 对信使核糖核酸有很高的亲和力，以了解更多关于信使核糖核酸结合的知识。 突变体可用于癌症(显性阴性)、核的基因治疗 酸基疫苗，或在生物技术中。具体目标是：(A) 确定(1)表达eIF4E的显性否定形式在 乳腺癌细胞和(2)过表达野生型和高表达的作用 EIF4E在非恶性乳腺上皮细胞中的亲和突变体。效应 关于eIF4F复合体的形成，细胞周期扰动与 在软琼脂中促进增殖、集落生长和肿瘤形成 裸鼠将被确定。(B)确定具体的 EIF4E的mRNA结合位点附近的环可以改变其亲和力 MRNA.(C)使用随机诱变、细菌表面展示系统和 荧光m7G探针用于鉴定结合信使核糖核酸帽子的eIF4E突变 有很高的亲和力。(D)开展共晶生长研究， M7GpppG和m7G封端的寡核苷酸，几个结合的突变体 与野生型eIF4E有较高的亲和力。

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.