Experimental Transformation of Human Melanocytes in Vivo

描述（由申请人提供）：最近的研究已经在大多数人类黑色素瘤中发现了B-RAF基因的一种新突变，但对导致黑色素瘤的遗传和生物学事件的清晰理解仍然难以捉摸。对黑色素瘤发展的实验研究受到两个主要困难的阻碍：i）小鼠不是用于黑色素瘤相关研究的理想实验动物，因为正常黑色素细胞在小鼠与人皮肤中的位置不同; ii）生物学上的早期病变不能从病理学家获得，因为它们的尺寸小并且需要对整个病变进行组织学扫描。我们的实验室已经开发了一种人黑色素瘤模型，其中将来自健康供体的人皮肤移植到免疫缺陷小鼠中，随后皮内腺病毒载体介导的三种生长因子bFGF（FGF 2）、SCF（c-kit配体）和ET-3（内皮素-3）的表达，并伴随UVB（B范围的紫外线）照射。在仅仅三到四周的治疗期后，在人类皮肤移植物中出现了高度侵袭性的病变，在组织学上类似于患者的原发性黑色素瘤。然而，在缺乏生长因子的情况下，病变消退，并且从病变培养的细胞显示出有限的寿命。第一个目的提出使用新的皮肤重建模型在黑素细胞和基质成纤维细胞中转导基因来诱导致瘤性黑色素瘤。我们将测试当黑色素细胞被基质和环境刺激激活时是否会发生黑色素瘤，以及当引入突变基因如突变的B-RAF时它们是否会变得永生和不依赖生长因子。第二个目的是研究黑色素细胞转化为黑色素瘤的机制，测试生长因子bFGF，SCF和ET-3，当由基质成纤维细胞产生时，与UVB合作激活生长，存活和抗凋亡的关键途径的假设。我们将把我们的调查重点放在MAPK，AKT，PKC和NF κ B信号通路，因为我们预计，它们的激活，以及在定义的基因畸变，导致生长自主性和无限的增殖潜力。我们独特的模型允许系统解剖导致人类黑色素瘤形成的生物和分子事件。

DESCRIPTION (provided by applicant): Recent investigations have identified a novel mutation in the B-RAF gene in the majority of human melanomas but a clear understanding of genetic and biologic events leading to melanoma remains elusive. Experimental investigations on melanoma development have been hampered by two major difficulties: i) the mouse is not an ideal experimental animal for melanoma-related studies because normal melanocytes are differently located in mouse versus human skin; ii) biologically early lesions cannot be obtained from pathologists because of their small size and the necessity for histologic scanning of the entire lesion. Our laboratory has developed a model of human melanoma in which human skin from healthy donors is grafted to immunodeficient mice, followed by intradermal adenovirus vector-mediated expression of three growth factors, bFGF (FGF 2), SCF (c-kit ligand) and ET-3 (endothelin-3) and concomitant irradiation with UVB (ultraviolet light in the B range). After a treatment period of only three to four weeks, highly invasive lesions developed in the human skin grafts that histologically resembled primary melanomas in patients. However, the lesions regressed in the absence of growth factors and cells cultured from the lesions showed limited life span. The first aim proposes to induce tumorigenic melanomas using a novel skin reconstruction model for transduction of genes in melanocytes and stromal fibroblasts. We will test whether melanomas develop when melanocytes are activated by stromal and environmental stimuli, and whether they become immortal and growth factor independent when mutated genes such as mutated B-RAF are introduced. The second aim investigates the mechanisms of transformation of melanocytes to melanoma, testing the hypothesis that the growth factors bFGF, SCF, and ET-3, when produced by stromal fibroblasts, cooperate with UVB to activate critical pathways for growth, survival, and anti-apoptosis. We will focus our investigations on the MAPK, AKT, PKC, and NFKappaB signaling pathways, because we expect that their activation, together with aberrations in defined genes, leads to growth autonomy and limitless proliferative potential. Our unique model allows a systematic dissection of the biological and molecular events leading to human melanoma formation.