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

C-N- 和 L-MYC 对造血细胞的影响

• 批准号: 7224124
• 负责人: Edward Victor Prochownik
• 金额: $ 23.13万
• 依托单位: CHILDREN'S HOSP PITTSBURGH/UPMC HLTH SYS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7224124
• 关键词: Apoptosis; Apoptotic; Behavior; Cell Cycle Progression; Cell physiology; Cells; Cessation of life; Complement; DNA Microarray Chip; DNA Microarray format; Evolution; Family; Gene Targeting; Genes; Genomic Instability; Goals; Growth; Growth Factor; Hematopoietic; Human; IL3 Signaling Pathway; Individual; Invasive; Malignant Neoplasms; Molecular; Morphology; Myeloid Cells; Normal Cell; Normal tissue morphology; Numbers; Oncogene Proteins; Oncogenes; Pathogenesis; Pathway interactions; Phenotype; Population; Property; Proteins; Retroviral Vector; Route; Series; Staphylococcal Protein A; Transgenic Mice; Transgenic Model; Tumor Cell Invasion; Work; base; beta-Chemokines; c-myc Genes; cancer therapy; cell transformation; cellular engineering; chemokine; desire; expression vector; insight; member; novel; transcription factor; tumor; vector

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.

描述(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。C-Myc是研究最深入的成员，除了具有转化作用外，还具有促进细胞凋亡、改变细胞形态、抑制分化、加快细胞周期进程和促进基因组不稳定等作用。一个主要的目标是确定c-Myc调控的基因，因为这将为Myc表型的分子基础提供重要的洞察力。另一个目标是确定不同的Myc成员是否调控相同的靶基因。几个c-Myc靶基因可以模拟有限数量的c-Myc特性。然而，它们被证明这样做的细胞都表达内源性c-Myc，因此无法确定这些靶点是否以真正的c-Myc不依赖的方式发挥作用。我们已经鉴定出一个c-Myc靶基因MT-MC1，它具有在不表达c-Myc的细胞(c-Myc“K.O”细胞)中传递多种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被用来重建KO细胞的c-Myc表型，从而定义了一个最小的尽管不一定唯一的功能靶基因群体。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顺序转导和稳定表达。这将使我们能够在特定的目标1中，为特定的c-Myc靶基因子集构建和鉴定一系列pRetroFLOX-GFP载体。在特定的目标2中，我们将确定单个c-Myc靶基因在KO细胞中的过度表达是否可以模仿与亲本细胞相同的c-Myc表型。在特定的目标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的相反调节。与c-Myc一起，CCL6赋予依赖IL-3的髓系细胞生长因子非依赖性和转化表型，并增强已建立的肿瘤系的侵袭和转移行为。这似乎是由于CCL6的S能够诱导邻近正常细胞的凋亡性死亡，从而摧毁限制肿瘤扩散的正常组织屏障。因此，在特定的目标4中，我们将确定是否必须分泌CCL6才能给予IL-3非依赖性生长和更具侵袭性的肿瘤行为。在具体目标5中，我们将确定CCL6和c-Myc颠覆IL-3信号通路的机制(S)。在特定的目标6中，我们将确定相关的趋化因子是否具有CCL6样属性。最后，在特定的目标7中，我们将建立依赖CCL-6的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颠覆正常细胞途径的机制提供新的见解。