Murine & Human In Vivo Models of Melanoma Formation

项目3利用人类和小鼠模型系统来解决标志性遗传事件之间的联系 黑色素瘤进展(p16INK4a缺失和N-RAS/B-RAF突变)与DMADAGE反应 黑素细胞。该项目结合了对新的黑色素瘤小鼠模型的分析和全面的 一个大型临床注释的人类黑色素瘤数据库的分子和免疫组织化学研究。在……里面 具体目的1，我们结合了两个不同的RAS等位基因，条件失活的p53和p16INK4a等位基因， 以及一种可诱导的、黑素细胞特异性的Cre等位基因，以产生新的黑色素瘤小鼠模型 高度忠于这种肿瘤的人类遗传学。在这些模型中，所有致癌事件都被限制在 黑素细胞室，包括体细胞p16/p53失活或K-RAS激活，两个 的等位基因(Tyr-Cre-ER-T和条件性p16INK4a)是新发现的，未发表。在……里面 特定目标2，我们将这些新的黑色素瘤小鼠模型与新生儿UV-B暴露相结合，以 促进体内黑色素瘤的发生。特殊目标2还包括详细的免疫组织化学分析 紫外线照射前后DNA损伤反应和衰老标志物表达的动力学研究 B在RAS激活、p16INK4a缺失和/或p53缺失的情况下进行治疗。这一特定目标采用了 用类似的方法分析免疫缺陷小鼠在紫外线照射和不照射情况下的真皮重建 使用项目1和项目2提供的试剂进行暴露。在特定目标3中，我们扩展了对人类的分析 福尔马林固定和石蜡包埋的原发黑素细胞病变之间的关系 Ras/RAF/p16INK4a突变、ERK丝裂原活化、衰老与DNA损伤反应 从痣到转移性肿瘤的进展。这一具体目标包括全面的 DNA损伤反应和衰老的几个标志物的免疫组织化学分析 N-RAS、B-RAF和p16INK4a基因突变分析这个特定的目标是有动力的(250个黑素细胞 从痣到转移性黑色素瘤)来解释原发肿瘤的分子异质性，但 仍然发现了这些标志性基因事件之间具有生物学意义的关系，DNA损伤 反应和黑色素瘤进展。我们希望这项工作将进一步加深我们对人类的基本理解 黑色素瘤的进展以及确定疾病进展和结果的新的临床预测因子。

Project 3 utilizes human and murine model systems to address the links between signature genetic events in melanoma progression (p16INK4a loss and N-RAS/B-RAF mutation) and the DMAdamage response in melanocytes. This project combines analyses of novel murine models of melanoma with a comprehensive molecular and immunohistochemical study of a large, clinically annotated human melanoma database. In specific aim 1, we combine two different RAS alleles, conditionally inactivatable p53 and p16INK4a alleles, and an inducible, melanocyte-specific CRE allele to produce new murine models of melanoma that are highly faithful to the human genetics of this tumor. In these models, all oncogenic events are restricted to the melanocytic compartment, and include somatic inactivation of p16/p53 or somatic activation of K-RAS, Two of the alleles (Tyr-CRE-ER-T and conditional p16INK4a) are newly characterized and unpublished. In specific aim 2, we combine these novel murine models of melanoma with neonatal UV-B exposure to facilitate in vivo melanomagenesis. Specific aim 2 also includes a detailed immunohistochemical analysis of the kinetics of the expression of markers of the DNA damage response and senescence with or without UV- B treatment in the setting of RAS activation, p16INK4a loss and/or p53 loss. This specific aim employs a similar approach to analyze human dermal reconstructs in immunodeficient mice with and without UV exposure using reagents supplied from projects 1 and 2. In specific aim 3, we extend our analysis of human primary formalin-fixed and paraffin-embedded melanocytic lesions to identify the relationship among RAS/RAF/p16INK4a mutation, ERK MAP kinase activation, senescence and the DNA damage response in the progression from nevus to metastatic tumor. This specific aim includes a comprehensive immunohistochemical analysis of several markers of the DNA damage response and senescence, as well as a mutational analysis of N-RAS, B-RAF and p16INK4a. This specific aim is powered (250 melanocytic lesions from nevus to metaststic melanoma) to account for molecular heterogeneity in primary tumors but still uncover biologically significant relationships among these signature genetic events, the DNA damage response and melanoma progression. We expect this work will further our basic understanding of human melanoma progression as well as identify new clinjcal predictors of disease progression and outcome.