Nutritional Cell Cycle and Differentiation Control

描述（由申请人提供）：这是一份研究化学预防剂维甲酸（RA）（维生素a的一种形式）调节细胞生长和分化的机制的提案，通过该机制将转化的表型逆转为分化的、生长受阻的表型。拟制的研究利用未成熟的、未确定的前体细胞HL-60人成髓细胞白血病细胞，当分别用RA或维生素D3 （D3）治疗时，它们经历G0阻滞和髓细胞或单核细胞分化。RA在诱导终髓细胞分化和G0阻滞的过程中引起MAPK信号的激活。ra诱导的MAPK通路激活与典型的肽激素有丝分裂RAF/MEK/ERK信号的激活不同，激活缓慢且持续时间长。增强MAPK信号可以增强ra诱导的分化，而阻断MAPK信号则可以阻止ra诱导的分化。因此，在ra诱导的导致终末分化的进行性表型变化中，MAPK信号传导是必要的，并且速率有限。RA的早期反应是BLR1膜受体的诱导表达。通过稳定转染BLR1过表达可引起RAF信号传导，加速ra诱导的髓细胞分化和G0阻滞。相比之下，双等位基因同源重组敲除的BLR1在RA反应中不能激活RAF、最终分化或G0阻滞。在被敲除的细胞中，激活RAF的异位表达挽救了它们对RA的分化和抑制能力。因此，BLR1及其伴随的RAF信号是RA诱导分化和停止的必要条件和速率限制。假设存在一个BLR1导向的分化控制点，该控制点在分化进程的某一点上起作用，通过RAF驱动的MAPK信号激活所需的转录因子并影响表型转化，从而控制超出该控制点的进程。目的1：通过确定定义BLR1的分化标记物，确定RA诱导髓细胞分化进程中的BLR1分化控制点。目的2：确定BLR1分化控制点激活导致髓细胞分化进展的转录因子集合。AIM 3：确定BLR1分化控制点用于MAPK信号并引起所需转录因子和表型变化的特定RAF磷酸化位点。HL-60的结果也将在U937和NB4中确认。这将确定ra诱导的进行性表型转化依赖于BLR1，转录因子控制在那里，以及指导这些作用的特定RAF磷酸化。这是一个范例的原型，RA通过过度活跃的RAF信号诱导分化。公共卫生相关性：通过饮食因素对癌症进行化学预防可以对减少癌症发病率的饮食建议和癌症治疗产生重大影响。视黄酸是维生素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取决于通常被认为是促进癌症的细胞信号传导过程。拟议的研究将表明，维甲酸对该信号的操纵使用了其他激素，将其从促进肿瘤转化的信号转变为逆转转化为生长受阻的正常细胞的信号，从而提供了一种增强其化学预防作用的新方法。

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.