Determinants of Melanocyte Transformation and Melanoma P

大多数人类浅表播散型和结节型临床亚型的黑色素瘤都含有NRAS小GTP酶或BRAF蛋白激酶的激活突变。同样的突变在黑色素细胞痣中以大致相同的频率发生，这可能是黑色素瘤的先兆。为了进一步了解激活的BRAF在人类黑色素瘤中的作用，我们正在进行一项观察，即我们在人类黑色素瘤细胞中的BRAF与分子伴侣热休克蛋白(HSP)-90相互作用。HSP-90此前已被证明与另一种形式的皇家空军-CRAF相互作用。CRAF和HSP-90之间的相互作用与天然产物格尔丹霉素(一种HSP90 ATPase活性的抑制剂)或其半合成类似物17-AAG的干扰会破坏CRAF的稳定，并抑制其直接激活下游MAP激酶信号级联的能力。我们已经分析了17-AAG对人黑色素瘤细胞系BRAF稳定性和下游MAPK信号的影响。在所研究的2/6人黑色素瘤细胞系中，17-AAG在1微摩尔的浓度下似乎使BRAF不稳定，但在其中4/6的细胞系中使CRAF不稳定。尽管BRAF在用17-AAG处理的大多数细胞系中持续存在，但在每个表现出MEK和ERK磷酸化的细胞系和另外两个被检查的人黑色素瘤细胞系中，MAP激酶级联的下游激活受到抑制，表现为缺乏MEK和ERK的磷酸化。我们对17-AAG在不引起BRAF降解的情况下抑制依赖于BRAF的MEK和ERK的激活的机制很感兴趣，并主要使用生化方法来验证关于这一机制的假说。为了分析在黑素细胞中表达NRAS和BRAF中黑色素瘤特异性突变的效果，我们正在开发一种四环素诱导的转基因小鼠系统，该系统使用多巴铬互变酶(DCT)启动子，允许在体内控制诱导小鼠黑素细胞中的基因表达。使用DCT启动子可以在胚胎发育的早期诱导这些基因的表达，也可以在假定的黑素细胞干细胞中诱导表达，这已经在小鼠毛囊的隆起区被描述过。其中一些工作是与NCI的格伦·梅利诺博士合作完成的。初步的鉴定实验表明，转基因的表达是在小鼠卵泡的预期位置诱导的。需要对转基因品系进行更广泛的表征，以描述转基因表达模式和表达的诱导性。诱导系对于研究BRAF和NRAS的激活突变体在分离的小鼠黑素细胞中表达的影响，以及在适当的遗传背景下建立这些突变导致黑色素瘤形成的倾向是非常有用的。

Most human melanomas of the superficial spreading and nodular clinical subtypes contain activating mutations in either the NRAS small GTPase or the BRAF protein kinase. The same mutations occur at approximately the same frequency in melanocytic nevi, which can be precursors to melanoma. To understand further the role of activated BRAF in human melanoma, we are pursuing an observation we made that BRAF in human melanoma cells interacts with the molecular chaperone heat shock protein (HSP)-90. HSP-90 has previously been demonstrated to interact with another form of RAF, CRAF. Disruption of the interaction between CRAF and HSP-90 with the natural product geldanamycin, an inhibitor of HSP90 ATPase activity, or its semi-synthetic analog, 17-AAG, destabilizes CRAF and inhibits its ability to direct activation of the downstream MAP kinase signaling cascade. We have analyzed the effect of 17-AAG upon BRAF stability and downstream MAP kinase signaling in human melanoma cell lines. 17-AAG at concentrations up to 1 micromolar appeared to destabilize BRAF in 2/6 human melanoma cell lines examined but destabilized CRAF in 4/6 of these cell lines. Despite the persistence of BRAF in the majority of cell lines treated with 17-AAG, downstream activation of the MAP kinase cascade was inhibited, shown by absence of MEK and ERK phosphorylation, in each of the cell lines exhibiting and in 2 additional human melanoma cell lines examined. We are interested in the mechanism by which 17-AAG inhibits BRAF-dependent activation of MEK and ERK without inducing BRAF degradation and are testing hypotheses about this mechanism primarily using a biochemical approach. To analyze the effects of expressing melanoma-specific mutations in NRAS and BRAF in melanocytes, we are developing an tetracycline-inducible transgenic mouse system using the dopachrome tautomerase (Dct) promoter to permit the controlled induction of gene expression in vivo in murine melanocytes. Using the Dct promoter may render it possible to induce expression of these genes early in embryogenesis as well as in the putative melanocyte stem cell that has been described in the bulge region of the murine hair follicle. Some of this work is being done in collaboration with Dr. Glenn Merlino of NCI. Initial characterization experiments suggest that expression of transgenes is induced in the expected location of the murine follicle. More extensive characterization of transgenic lines will be required to describe the transgene expression pattern and inducibility of expression. Inducible lines should be quite useful for studying the effects of expression of activated mutants of BRAF and NRAS in isolated murine melanocytes, and establishing the propensity of these mutations to cause melanoma formation in appropriate genetic backgrounds.