Tumor Suppressor Genes in Heritable Melanoma Models

描述(由申请人提供)：剑尾鱼的物种间杂交已被用于研究黑色素瘤形成的遗传决定因素已有数十年。对剑尾鱼自发性黑色素瘤和紫外线诱导黑色素瘤遗传的遗传学和分子分析导致了与EGFR相关的癌基因-剑尾鱼黑色素瘤受体激酶Xmrk2-和黑色素瘤易感基因CDKN2X的发现，该基因属于细胞周期蛋白依赖性激酶抑制剂CDKN2家族，我们在这笔资金的过程中发现了这两个基因。然而，尽管该基因与剑尾鱼Bc1杂交后代黑色素瘤易感性有关的遗传证据是强有力的和令人信服的，但这种假定的细胞周期调节因子在剑尾蛇肿瘤发生中的具体作用仍未确定。我们最近的研究表明，CDKN2X在原发黑色素瘤中过表达，并且这种过表达与其他细胞周期调节基因的过表达有关，包括细胞周期蛋白D1和上游酪氨酸受体激酶Xmrk2的过表达。在这个项目中，我们计划研究剑鱼生物和细胞培养模型中的几个细胞周期调控基因，包括细胞周期蛋白D1、Rb和叉头转录因子。我们将开发方法和系统，为研究剑鱼黑色素瘤形成的细胞生理和生物化学提供实验框架，从而为开发这一独特的实验性黑色素瘤模型提供更大的空间。我们将描述一些发生在原发黑色素瘤发展过程中的早期细胞和生化变化。为了实现这一目标，我们将(A)确定细胞周期调节成分与黑色素瘤发展之间的关系(S)，(B)确定特定的细胞周期调节基因在RNA和蛋白质水平上的表达，在肿瘤发展的不同时间阶段，从剑尾鱼F1和Bc1杂交种产生的初级黑色素瘤中；这些结果将与原位肿瘤的病理描述相关联，并将综合空间和时间描述，试图确定原发黑色素瘤在发展过程中如何在细胞周期调节中明显变化。我们还将(C)利用细胞培养模型来研究Xmrk2在细胞培养模型中影响CDKN2X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.