C-N-, and L-MYC Effects on Hematopoietic Cells

描述（由申请人提供）：Myc癌蛋白（c-， N-和L-Myc）是转录因子，参与许多人类癌症的发病机制。除了转化外，研究最深入的成员c-Myc还促进细胞凋亡，改变形态，抑制分化，加速细胞周期进程，促进基因组不稳定性。一个主要的目标是确定由c-Myc调节的基因，因为这将为了解Myc表型的分子基础提供重要的见解。另一个目标是确定不同的Myc成员是否调节相同的靶基因。一些c-Myc靶基因可以模拟有限数量的c-Myc特性。然而，它们所处的细胞都表达内源性c-Myc，因此不可能确定这些靶标是否以真正的c-Myc独立的方式表现。我们已经确定了一个c-Myc靶基因MT-MC1，它具有在不表达c-Myc的工程细胞（c-Myc“K.O”细胞）中赋予多种c-Myc表型的独特特性。这表明MT-MC1和一组精心挑选的其他互补基因可能用于重建KO细胞中的c-Myc表型，从而定义一个最小的（尽管不一定是唯一的）功能靶基因群。因此，我们设计了一种新的逆转录病毒载体（pRetroFLOX-GFP），它允许无限数量的基因被顺序转导并稳定表达。这将使我们能够在Specific Aim 1中构建和表征一系列针对c-Myc靶基因子集的pRetroFLOX-GFP载体。在Specific Aim 2中，我们将确定KO细胞中单个c-Myc靶基因的过表达是否可以模仿与亲本细胞相同的c-Myc表型。在Specific Aim 3中，我们将在KO细胞或转基因小鼠中连续表达c-Myc靶基因，以概括“完整”的c-Myc表型。在相关研究中，我们发现CCL6趋化因子受c-Myc和L-Myc的相反调控。与c-Myc一起，CCL6赋予il -3依赖性骨髓细胞生长因子独立性和转化表型，并增强已建立肿瘤系的侵袭和转移行为。这似乎是由于CCL6能够诱导邻近正常细胞的凋亡性死亡，从而破坏限制肿瘤扩散的正常组织屏障。因此，在Specific Aim 4中，我们将确定是否必须分泌CCL6才能促进il -3独立生长和更具侵袭性的肿瘤行为。在Specific Aim 5中，我们将确定CCL6和c-Myc破坏IL-3信号通路的机制。在Specific Aim 6中，我们将确定相关趋化因子是否赋予ccl6样特性。最后，在Specific Aim 7中，我们将开发一种ccl -6依赖性肿瘤侵袭的转基因模型。总之，这些研究将为c-Myc及其转录靶点在建立转化细胞的过程中破坏正常细胞通路的机制提供新的见解。

DESCRIPTION (provided by applicant): Myc oncoproteins (c-, N-, and L-Myc) are transcription factors, which are involved in the pathogenesis of many human cancers. In addition to being transforming, c-Myc, the most intensely studied member also promotes apoptosis, alters morphology, inhibits differentiation, accelerates cell cycle progression, and promotes genomic instability. A major goal is to identify the genes regulated by c-Myc, since this would provide significant insight into the molecular basis of Myc's phenotypes. Another goal is to determine whether different Myc members regulate the same target genes. Several c-Myc target genes can mimic a limited number of c-Myc properties. However, the cells in which they have been shown to do so all express endogenous c-Myc, thus making it impossible to determine whether these targets behave in a truly c-Myc-independent manner. We have identified a c-Myc target gene, MT-MC1, which has the unique property of imparting multiple c-Myc phenotypes in cells engineered to not express c-Myc (c-Myc "K.O" cells). This suggests that MT-MC1, and a carefully selected group of other complementing genes, might be used to reconstruct the c-Myc phenotype in KO cells, thus defining a minimum, although not necessarily unique, functional target gene population. We have therefore devised a novel retroviral vector (pRetroFLOX-GFP) that permits an unlimited number of genes to be sequentially transduced and stably expressed. This will allow us in Specific Aim 1, to construct and characterize a series of pRetroFLOX-GFP vectors for a defined subset of c-Myc target genes. In Specific Aim 2, we will determine whether over-expression of individual c-Myc target genes in KO cells can mimic the same c-Myc phenotypes as they do in parental cells. In Specific Aim 3, we will consecutively express c-Myc target genes in KO cells or transgenic mice in order to recapitulate the "complete" c-Myc phenotype. In related studies, we have shown that the CCL6 chemokine is regulated oppositely by c-Myc and L-Myc. Together with c-Myc, CCL6 imparts growth factor independence and a transformed phenotype to IL-3-dependent myeloid cells and enhances the invasive and metastatic behavior of established tumor lines. This appears to be a result of CCL6's ability to induce apoptotic death in adjacent normal cells, thus destroying the normal tissue barriers that limit tumor spread. Therefore, in Specific Aim 4, we will determine whether CCL6 must be secreted in order to impart IL-3-independent growth and more aggressive tumor behavior. In Specific Aim 5, we will determine the mechanism(s) by which CCL6 and c-Myc subvert the IL-3 signaling pathway. In Specific Aim 6, we will determine whether related chemokines impart CCL6-like properties. Finally, in Specific Aim 7, we will develop a transgenic model of CCL-6-dependent tumor invasion. Together, these studies will provide new insights into the mechanisms by which c-Myc and its transcriptional targets subvert normal cellular pathways en route to establishing a transformed cell.