Nutritional Cell Cycle and Differentiation Control

描述(由申请人提供)：这是一项研究化学预防药物维甲酸(RA)调节细胞生长和分化以将转化表型逆转为分化、生长受阻表型的机制的提案。这项研究利用了一种未成熟的、未固定的前体细胞，HL-60人髓母细胞白血病细胞，当用RA或维生素D3(D3)处理时，它分别经历G0期停滞和髓系或单核细胞分化。RA在诱导髓系终末分化和G0停滞的过程中导致MAPK信号的激活。RA诱导的MAPK通路的激活不同于典型的促有丝分裂激素RAF/MEK/ERK信号通路的激活，因为激活是缓慢和持久的。增强MAPK信号转导促进RA诱导分化，阻断MAPK信号转导抑制RA诱导分化。因此，在RA诱导的导致终末分化的进行性表型变化中，MAPK信号是必要的和速率限制的。对RA的早期反应是诱导BLR1膜受体的表达。稳定表达BLR1可诱导RAF信号转导，加速RA诱导的髓系分化和G0期停滞。与之相反，BLR1基因被双列同源重组敲除后，不能激活RAF、终末分化或对RA的G0阻断。在敲除细胞中异位表达激活的RAF挽救了它们分化和阻止RA反应的能力。因此，BLR1及其伴随的RAF信号对RA诱导分化和停滞是必要的和限速的。假设存在一个BLR1定向的分化控制点，它作用于分化过程中的某个点，通过RAF驱动的MAPK信号来控制超过它的过程，激活所需的转录因子，影响表型转换。其具体目的是：目的1：通过确定决定BLR1分化的分化标志物，确定BLR1在RA诱导的髓系分化过程中的分化控制点。目的2：确定BLR1分化控制点激活的导致髓系分化进展的转录因子集合。目的：确定BLR1分化控制点对MAPK信号的特异性RAF磷酸化位点，并引起所需的转录因子和表型变化。HL-60的结果也将在U937和NB4中得到证实。这将决定RA诱导的进行性表型转换在哪里依赖于BLR1，那里控制的转录因子，以及指导这些效应的特定RAF磷酸化。这是RA通过过度活跃的RAF信号诱导分化的范例的原型。公共卫生相关性：饮食因素对癌症的化学预防可以对降低癌症发病率的饮食建议以及癌症治疗产生重大影响。维甲酸是维生素A的一种活性形式，具有化学预防作用，但其作用机制现在似乎依赖于通常被归因于癌症促进的细胞信号过程。这项拟议中的研究将表明，维甲酸对该信号的操纵使用其他激素，将其从促进肿瘤转化为逆转转化为生长受阻的正常细胞，从而提供了一种新的方法来增强其化学预防作用。

DESCRIPTION (provided by applicant): This is a proposal to study the mechanism by which the chemopreventive agent, retinoic acid (RA) which is a form of vitamin A, regulates cell growth and differentiation to reverse a transformed phenotype to a differentiated, growth arrested phenotype. The proposed studies utilize an immature, uncommitted precursor cell, HL-60 human myeloblastic leukemia cells, which undergoes G0 arrest and either myeloid or monocytic differentiation when treated with RA or vitamin D3 (D3) respectively. RA causes activation of MAPK signaling in the process of inducing terminal myeloid differentiation and G0 arrest. The RA-induced MAPK pathway activation diverges from that of typical peptide hormone mitogenic RAF/MEK/ERK signaling in that activation is slow and long lived. Enhancing MAPK signaling enhances RA-induced differentiation while blocking it prevents RA-induced differentiation. MAPK signaling is thus necessary and rate limiting in RA-induced progressive phenotypic changes leading to terminal differentiation. An early response to RA is the induced expression of the BLR1 membrane receptor. BLR1 over expression by stable transfection causes RAF signaling and accelerates RA-induced myeloid differentiation and G0 arrest. In contrast, the BLR1 knock out by bialleilic homologous recombination fails to activate RAF, terminally differentiate or G0 arrest in response to RA. Ectopic expression of activated RAF in the knock out cells rescues their ability to differentiate and arrest in response to RA. BLR1 and its attendant RAF signaling are thus necessary and rate limiting for RA to induce differentiation and arrest. The hypothesis is that there is a BLR1 directed differentiation control point that acts at a certain point during progression of differentiation to control progress beyond it through RAF driven MAPK signaling to activate needed transcription factors and effect phenotypic conversion. The specific aims are: AIM 1: Identify the BLR1 differentiation control point during progression of myeloid differentiation induced by RA by determining the differentiation markers that define it. AIM 2: Determine the ensemble of transcription factors that the BLR1 differentiation control point activates to cause progression of myeloid differentiation. AIM 3: Determine the specific RAF phosphorylation sites that the BLR1 differentiation control point uses to MAPK signal and cause the needed transcription factor and phenotypic changes. The results for HL-60 will be confirmed in U937 and NB4 as well. This will determine where RA-induced progressive phenotypic conversion depends on BLR1, the transcription factors controlled there, and the specific RAF phosphorylations directing these effects. This is the prototype of a paradigm where RA induces differentiation via hyperactive RAF signaling. PUBLIC HEALTH RELEVANCE: Chemoprevention of cancer by dietary factors can have a significant effect on dietary recommendations to reduce cancer incidence and also on cancer treatment. Retinoic acid is an active form of vitamin A that has chemopreventive effects, but its mechanism of action now appears to depend on cellular signaling processes customarily attributed to cancer promotion. The proposed research will show that manipulation of that signal by retinoic acid uses other hormones to change it from one that promotes neoplastic transformation to one that reverses transformation to a growth arrested, normal like cell, thereby providing a new way to enhance its chemopreventive effects.