MOLECULAR TARGETS OF PERINATAL ENDOCRINE DISRUPTION

根据我们的数据显示，使用经典内分泌干扰剂己烯雌酚（DES）治疗新生儿可通过直接机制改变成年仓鼠子宫雌激素反应性，我们提出了两个相关的工作假设：1)成年仓鼠子宫雌激素反应性的改变是新生儿在发育早期DES诱导的关键调控基因表达失衡的结果；2)基质-上皮相互作用使得DES改变的早期基因表达模式可能在两个组织间室之间有所不同。基于这些假设，我们将追求具体目标：确定在成年仓鼠子宫中引起不适当雌激素反应的新生儿DES损伤的直接细胞和分子靶点。幸运的是，强大的新基因筛选和克隆策略的改进版本现在以试剂盒形式可用，并且需要很少的细胞总RNA作为起始材料。此外，我们能够将新生儿子宫中存在的相对未分化的间质室与简单的管腔上皮清晰地分离开来，这将有助于确定des引起的直接改变的细胞位置。这种筛选工作应该1)产生一系列新发现的遗传元件，其中一些必须涉及重要的生物学功能（雌激素反应性增殖/凋亡/肿瘤形成），2)随后是在基因组，RNA和蛋白质水平上有序确定每个元件的分子结构和功能动力学。每一个这样的过程所定义的研究机会的范围将包括许多层次的努力，包括适合我们的一些独立教师，博士后研究助理，或我们的研究生或本科生水平的学生。因此，该项目与AREA奖励计划的既定目的特别相关。除了提供围产期内分泌干扰这一有争议的话题的重要机制信息外，该项目必将有利于我们更大的长期目标：1)描绘雌激素调节正常子宫生长和形态发生的基本机制；2)确定导致正常生长过程变性从而导致不受调节的肿瘤状态的机制改变。这些在生物医学上是重要的，因为：1)成功的受孕和妊娠需要正常的子宫形态和功能，2)雌激素依赖性子宫肿瘤是当代美国社会相当高的发病率和死亡率的原因。

From our data showing that neonatal treatment with the classic endocrine-disrupting agent diethylstilbestrol (DES) acts to alter estrogen responsiveness in the adult hamster uterus by a direct mechanism, we propose two related working hypotheses are: 1) The altered estrogen responsiveness that occurs in the adult hamster uterus is the result of neonatal DES-induced imbalances in the expression of key regulatory genes during early development; and 2) Stromal-epithelial interactions make it likely that DES- altered patterns of early gene expression will differ between the two tissue compartments. Based on these hypotheses, we will pursue the Specific Aim to: Identify the immediate cellular and molecular targets of the neonatal DES insult that cause inappropriate estrogen responses in the adult hamster uterus. Fortunately, improved versions of powerful new genetic screening and cloning strategies are now available in kit form and require very little total cellular RNA as starting material. Also, determining the cellular site of the immediate DES-induced alterations will be facilitated by our ability to cleanly separate the simple luminal epithelium from the relatively undifferentiated stromal compartment that is present in neonatal uteri. This screening effort should 1) generate a succession of newly identified genetic elements, several of which must be involved in important biological functions (estrogen-responsive proliferation/apoptosis/neoplasia), and 2) be followed by an ordered determination of each element s molecular structure and functional dynamics at the genomic, RNA, and protein level. The spectrum of research opportunities defined by each such process would encompass efforts at many levels including those appropriate for several of our independent faculty members, for post-doctoral research associates, or for our students at either the graduate or undergraduate level. Thus this project is particularly relevant to the stated purpose of the AREA Award Program. In addition to providing important mechanistic information about the controversial topic of perinatal endocrine disruption, this project will surely benefit our larger long-term objectives to: 1) Delineate the basic mechanisms whereby estrogen regulates normal uterine growth and morphogenesis, and 2) Identify mechanistic alterations that cause degeneration of the normal growth process and thereby leads to the unregulated neoplastic state. These are biomedically important because: 1) Successful conception and gestation demands normal uterine form and function, and 2) Estrogen-dependent uterine neoplasms are responsible for considerable morbidity and mortality in contemporary American society.