Tumor Suppressor Genes in Heritable Melanoma Models

描述（由申请人提供）：几十年来，剑尾鱼的种间杂交一直用于研究黑色素瘤形成的遗传决定因素。对几个剑尾鱼回交后代中自发和紫外线诱导的黑色素瘤遗传的遗传和分子分析发现了EGFR相关的癌基因-剑尾鱼黑色素瘤受体激酶Xmrk 2-和属于细胞周期蛋白依赖性激酶抑制剂CDKN 2家族的黑色素瘤易感基因CDKN 2X。这是我们在资助这个项目的过程中发现的然而，虽然遗传证据暗示这个基因在黑色素瘤的易感性在剑尾鱼BC 1杂交从特定的十字架是强大的和令人信服的，这种假定的细胞周期调节剂在剑尾鱼肿瘤发生的具体作用仍然不确定。我们最近的研究表明，CDKN 2X在原发性黑色素瘤中过表达，并且过表达与其他细胞周期调节基因（包括细胞周期蛋白D1）的过表达以及上游酪氨酸受体激酶Xmrk 2的过表达相关。在本计画中，我们将探讨剑尾鱼生物体与细胞培养模型中的细胞周期调控基因，包括细胞周期蛋白D1、Rb与叉头转录因子。我们将开发的方法和系统，将提供一个实验框架，研究细胞的生理学和生物化学的黑色素瘤形成在剑尾鱼，从而提供更大的范围利用这种独特的实验黑色素瘤模型。我们将描述一些发生在原发性黑色素瘤发展的早期细胞和生化变化。为了实现这一目标，我们将（a）确定剑尾鱼杂交种的原发性黑素瘤中细胞周期调节组分与黑素瘤发展之间的关系，（B）在肿瘤发展的不同时间阶段，在剑尾鱼F1和BC 1杂交种产生的原发性黑素瘤中，在RNA和蛋白质水平表征特异性细胞周期调节基因的表达;这些结果将与原位肿瘤的病理学描述相关联，并且空间和时间描述将被综合，以试图鉴定原发性黑素瘤在其发展过程中如何表现出细胞周期调节的变化。我们还将（c）利用细胞培养模型研究Xmrk 2影响细胞培养模型中CDKN 2X和其他细胞周期调节因子表达的机制。

DESCRIPTION (provided by applicant): Interspecies hybrids from Xiphophorus fish have been used for decades to investigate genetic determinants of melanoma formation. Genetic and molecular analyses of inheritance of spontaneous and UV-induced melanomas in several Xiphophorus backcross hybrids have resulted in the discoveries of an EGFR-related oncogene - the Xiphophorus melanoma receptor kinase, Xmrk2 - and a melanoma susceptibility gene belonging to the CDKN2 family of cyclin-dependent kinase inhibitors, CDKN2X., which we discovered during the course of the funding of this grant. However, although the genetic evidence implicating this gene in melanoma susceptibility in Xiphophorus BC1 hybrids from particular crosses is strong and compelling, the specific role of this putative cell cycle regulator in tumorigenesis in Xiphophorus remains undefined. Our recent studies have shown that CDKN2X is overexpressed in primary melanomas, and that overexpression correlates with overexpression of other cell cycle regulating genes including cyclin D1 as well as overexpression of the upstream tyrosine receptor kinase, Xmrk2. In this project, we propose to investigate several cell cycle regulating genes in Xiphophorus organisms and cell culture models, including cyclin D1, Rb, and forkhead transcription factors. We will develop approaches and systems that will provide an experimental framework for investigating the cell physiology and biochemistry of melanoma formation in Xiphophorus, thereby providing greater scope for exploitation of this unique experimental melanoma model. We will characterize some of the early cellular and biochemical changes occurring in primary melanoma development. To accomplish this goal, we will (a) determine the relationship(s) between cell cycle regulating components and melanoma development in primary melanomas from Xiphophorus hybrids, (b) characterize the expression of specific cell cycle-regulating genes, at both the RNA and protein levels, in primary melanomas generated from Xiphophorus F1 and BC1 hybrids, at different temporal stages of tumor development; these results will be correlated with pathological descriptions of the tumors in situ, and spatial and temporal descriptions will be synthesized in an attempt to identify how primary melanomas manifest changes in cell cycle regulation as they develop. We will also (c) exploit cell culture models to investigate mechanisms by which Xmrk2 influences the expression of CDKN2X and other cell cycle regulators in cell culture models.