DNA DAMAGE AND GENE EXPRESSION IN BREAST CANCER

乳腺癌中的 DNA 损伤和基因表达

• 批准号: 2096925
• 负责人: David A. Gewirtz
• 金额: $ 9.31万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-16 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2096925
• 关键词: DNA binding protein; DNA damage; DNA topoisomerases; MCF7 cell; antineoplastics; breast neoplasms; chloramphenicol acetyltransferase; drug screening /evaluation; enzyme activity; enzyme inhibitors; gene expression; genetic promoter element; genetic regulation; genetic transcription; human therapy evaluation; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; neoplasm /cancer pharmacology; nuclear runoff assay; oncoproteins; phosphorylation; posttranscriptional RNA processing; protooncogene; transfection /expression vector; western blottings

The biochemical and molecular perturbations which mediate the cytostatic and cytotoxic effects of antineoplastic drugs which damage DNA are not understood. The studies proposed in this application are predicated upon the hypothesis that down regulation of the expression of the c-myc oncogene and alterations in the levels and activity of the myc oncoprotein may be critical components of one pathway of growth arrest in response to DNA damage& in MCF-7 breast tumor cells. In order to test this hypothesis, we propose to demonstrate that transfection of MCF-7 cells with a c-myc construct driven by a constitutive promoter reduces or abrogate sensitivity to the topoisomerase II inhibitors, VM-26 and m-AMSA; in contrast, a similar transfection of K562 human leukemic cells (where c-myc appears to be uninvolved in growth regulation) with constitutively expressed c-myc should fail to alter cell sensitivity to these drugs. The nature of c-myc down-regulation will be defined by discriminating between effects of VM-26 and m-AMSA at the level of transcription (transcript initiation and elongation) and transcript stability; the involvement of the c-myc promoter region in the cellular response to VM-26 and m-AMSA will be established by monitoring drug effects on CAT activity using a myc promoter-CAT construct transfected into MCF-7 cells. The association of the myc oncoprotein with growth arrest 'will be defined by determining the influence of VM-26 and m-AMSA on oncoprotein levels, the phosphorylation state of the oncoprotein, and binding of the oncoprotein to its consensus sequence. Determination of the capacity of various DNA damaging drugs (and ionizing radiation) to produce collateral modulation of c-myc expression and growth arrest will serve to establish whether c-myc is uniformly involved in the cellular response to DNA damage in MCF-7 cells. Finally, the paradigm relating down-regulation of c-myc expression to growth arrest in response to DNA damage will be evaluated in other experimental models of breast cancer.

介导细胞抑制的生化和分子扰动 抗肿瘤药物的细胞毒作用不会损伤DNA 明白了。本申请中提出的研究基于 假设 c-myc 表达下调 癌基因以及 myc 癌蛋白水平和活性的变化 可能是响应生长停滞的一种途径的关键组成部分 MCF-7 乳腺肿瘤细胞中的 DNA 损伤。为了检验这个假设， 我们建议证明用 c-myc 转染 MCF-7 细胞 由组成型启动子驱动的构建体减少或消除 对拓扑异构酶 II 抑制剂 VM-26 和 m-AMSA 的敏感性；在 相比之下，K562人类白血病细胞的类似转染（其中c-myc 似乎不参与生长调节） 表达的 c-myc 应该不会改变细胞对这些药物的敏感性。这 c-myc 下调的性质将通过区分来定义 VM-26 和 m-AMSA 在转录水平上的影响（转录本 起始和延伸）和转录稳定性；的参与 VM-26 和 m-AMSA 细胞反应中的 c-myc 启动子区域 将通过使用 myc 监测药物对 CAT 活性的影响来确定 启动子-CAT构建体转染至MCF-7细胞中。该协会的 具有生长停滞的 myc 癌蛋白将通过确定 VM-26 和 m-AMSA 对癌蛋白水平、磷酸化的影响 癌蛋白的状态，以及癌蛋白与其共有序列的结合 顺序。测定各种 DNA 损伤药物（和 电离辐射）产生 c-myc 表达的附带调节 生长停滞将有助于确定 c-myc 是否一致 参与 MCF-7 细胞中 DNA 损伤的细胞反应。最后， c-myc表达下调与生长停滞相关的范式 对 DNA 损伤的反应将在其他实验模型中进行评估 乳腺癌。