MAPPING GENETIC SUPPRESSORS OF EPIGENETIC CARCINOGENESIS

我们最近的发现已经产生了一种新的非诱变的范例 乳腺癌的发生。我们证明了激活的Hras1 在NMU诱导的肿瘤中发现的突变作为背景突变出现 在发育中的腺体细胞内，该NMU增强了 这些突变的表型外显性通过启动基因的改变 DNA构象。我们进一步鉴定了一种特定于细胞类型的DNA 正常大鼠乳腺细胞Hras1启动子的结构 在活体内。该结构具有H型DNA的特征，包括 一个 推测为ETS转录因子结合位点。我们的结果进一步 证明根据动物的荷尔蒙状态，RMCs 可以在结构特征存在的状态之间切换，或者 在Hras1启动子中缺失。出于这个原因，我们称之为DNA 构造一个构型拨动开关(CS)。在费舍尔344(F344) 和对乳腺敏感的Spraogue-Dawley(SD)菌株 致癌，致癌剂量的NMU启动了这种丢失 RMCs的Hras1启动子的结构。而Hras1 CS是 在暴露后120天恢复到正常的RMC，其损失是 在导致肿瘤的细胞中是不可逆转的。相比之下，同样的 暴露诱导哥本哈根(Cop)大鼠CS中断 高度抵抗乳腺癌的发生。NMU也未能推广 乳腺中已存在的Hras1突变体的生长 耐药Cop大鼠上皮细胞。此外，NMU诱导的 敏感大鼠的CS在饲料中添加一种 化学预防剂量的高硒大蒜。把我们的结果加在一起 提示NMU诱导的DNA构象变化促进了 通过不可逆转地放松管制来发展先前存在的突变体 Hras1和其他ETS反应基因的表达，从而增加 条件性基因hras1的表型外显。 抗病(F344 Khi Cop)F1子代的进一步表型分析 表明抑制CS干扰的药物是一种或一种 COP大鼠肾小球系膜细胞表达更多优势抑制子。在一起我们的 研究结果表明，CS中断的这些基因抑制因子是 可能的肿瘤抑制基因。这项提案的总体目标是 利用遗传连锁分析定位这一新的抑制基因(S) 乳腺癌变对大鼠基因组定位的准备 早期克隆及其在乳腺癌风险研究中的应用 检测和预防。

Our recent findings have generated a new paradigm for nonmutagenic mammary carcinogenesis. We demonstrated that the activating Hras1 mutations found in NMU-induced tumors arise as background mutations within cells of the developing gland, and that NMU enhanced the phenotypic penetrance of these mutations by initiating alterations in DNA conformation. We further identified a cell-type-specific, DNA structure within the Hras1 promoter of normal rat mammary cells(RMCs) in vivo. The structure has characteristics of H-form DNA and includes a putative ets transcription factor binding site. Our results further demonstrated that depending upon hormonal status of the animals, RMCs can switch between states where the structural feature is present or absent from the Hras1 promoter. For this reason, we call this DNA structure a Conformation Toggle Switch(CS). In the Fischer 344(F344) and Sprague-Dawley(SD) strains, which are sensitive to mammary carcinogenesis, a carcinogenic dose of NMU initiated the loss of this structure from the Hras1 promoter of RMCs. While the Hras1 CS was restored in normal RMCs by 120 days after exposure, its loss was irreversible in cells that gave rise to tumors. By contrast, the same exposure to induce CS disruption in Copenhagen(Cop) rats, which are highly resistant to mammary carcinogenesis. NMU also failed to promote the outgrowth of pre-existing Hras1 mutants present in the mammary epithelium of resistant Cop rats. Moreover, NMU-induced disruption of the CS in sensitive rats was inhibited by a diet supplemented with a chemopreventive dose of high selenium garlic. Together our results suggest that NMU-induced alterations in DNA conformation promote the outgrowth of pre-existing mutants by irreversibly deregulating expression of Hras1 and other ets-responsive genes, thereby increasing the phenotypic penetrance of the conditional Hras1 oncomutations. Phenotypic analysis of resistant(F344 khi Cop)F1 progeny further indicated that the suppression of CS disruption was medicated by one or more dominant suppressors expressed in RMCs of Cop rats. Together our findings indicated that these genetic suppressors of CS disruption are putative tumor suppressor genes. The overall goal of this proposal is to use genetic linkage analysis to map this novel suppressor(s) of mammary carcinogenesis on the rat genome in preparation for positional cloning and application to studies of human breast cancer risk, early detection and prevention.