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: 6966572
• 负责人: Edward Victor Prochownik
• 金额: $ 24.4万
• 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/6966572
• 关键词: DNA damage; apoptosis; athymic mouse; cell cycle proteins; cell growth regulation; cell proliferation; cell transformation; chimeric proteins; chromosome aberrations; cysteine endopeptidases; flow cytometry; gene expression; genetically modified animals; green fluorescent proteins; molecular oncology; neoplasm /cancer genetics; oncoproteins; p53 gene /protein; phosphoprotein phosphatase; protein structure function; protooncogene; transcription factor; western blottings

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（由申请人提供）：MYC癌蛋白（C-，N-和L-MYC）是转录因子，与许多人类癌的发病机理有关。除了转化外，最深入研究的成员还促进凋亡，改变形态，抑制分化，加速细胞周期进程并促进基因组不稳定性。一个主要目标是识别由C-MYC调节的基因，因为这将为MYC表型的分子基础提供重大见解。另一个目标是确定不同的MYC成员是否调节相同的靶基因。几个C-MYC靶基因可以模仿有限数量的C-MYC特性。但是，已证明它们这样做的细胞都表达了内源性C-MYC，因此无法确定这些靶标是否以真正与C-Myc无关的方式行为。我们已经确定了C-MYC靶基因MT-MC1，该基因具有独特的特性，即在工程为不表达C-MYC的细胞中赋予多种C-MYC表型（C-MYC“ K.O”细胞）。这表明MT-MC1以及精心选择的其他补体基因可能用于重建KO细胞中的C-MYC表型，从而定义了最小，尽管不一定是独特的功能性靶基因群体。因此，我们设计了一种新型的逆转录病毒载体（预氟霉素GFP），该载体允许无限数量的基因被依次转导且稳定地表达。这将使我们在特定的目标1中构建和表征一系列为C-MYC靶基因的定义子集的前氟-GFP载体。在特定目标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中相同的C-MYC表型。在特定的目标3中，我们将连续表达KO细胞或转基因小鼠中的C-MYC靶基因，以概括“完整”的C-MYC表型。在相关研究中，我们表明CCL6趋化因子受C-MYC和L-MYC的相反调节。 CCL6与C-MYC一起赋予生长因子独立性和转化为IL-3依赖性髓样细胞，并增强已建立肿瘤系的侵入性和转移行为。这似乎是CCL6在相邻正常细胞中诱导凋亡死亡的能力，从而破坏了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，以赋予IL-3独立的生长和更具侵略性的肿瘤行为。在特定目标5中，我们将确定CCL6和C-MYC颠覆IL-3信号通路的机制。在特定的目标6中，我们将确定相关趋化因子是否具有类似CCL6的特性。最后，在特定的目标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，这些研究将提供有关C-MYC及其转录靶标的机制的新见解。