MAPPING GENETIC SUPPRESSORS OF EPIGENETIC CARCINOGENESIS

我们最近的发现为非致突变性产生了一个新的范例， 乳腺癌发生 我们证明了激活的Hras 1 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，NMU增强了 这些突变的表型突变，通过启动 DNA构象 我们进一步鉴定了一种细胞类型特异性DNA， 正常大鼠乳腺细胞（RMCs）Hras 1启动子内的结构 in vivo. 该结构具有H型DNA的特征，包括 一 推定的ETS转录因子结合位点。 我们的研究结果进一步 证明，根据动物的激素状态，RMCs 可以在存在结构特征的状态之间切换，或者 Hras 1启动子缺失。 因此我们称之为DNA 构造切换开关（CS）。 Fischer 344（F344） 和Sprague-Dawley（SD）菌株，其对乳腺癌敏感， 致癌作用，致癌剂量的NMU引发了这种损失， RMCs的Hras1启动子的结构。 虽然Hras 1 CS 正常RMCs在暴露后120天恢复，其损失为 在细胞中不可逆转，导致肿瘤。 相比之下， 暴露以诱导Copenhagen（Cop）大鼠CS破坏， 对乳腺癌的发生具有高度抵抗力。 NMU也未能促进 存在于乳腺癌细胞中的预先存在的Hras 1突变体的生长， 耐药Cop大鼠的上皮。此外，NMU诱导的破坏 敏感大鼠的CS被添加了 高硒大蒜的化学预防剂量。 我们的成果 表明NMU诱导的DNA构象改变促进了 通过不可逆的去调节， Hras 1和其他ets反应基因的表达，从而增加 条件性Hras 1基因突变的表型多态性。 抗性（F344 khi Cop）F1后代的表型分析进一步 表明CS破坏的抑制是由一种或多种药物引起的， Cop大鼠RMCs表达更多的显性抑制因子。我们一起 研究结果表明，这些CS破坏的遗传抑制因子 推定的肿瘤抑制基因。 本提案的总体目标是 使用遗传连锁分析来定位这种新的抑制基因， 大鼠乳腺癌发生基因组的定位研究 克隆和应用于研究人类乳腺癌的风险， 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.