Elucidating the molecular mechanisms that regulate stem cell numbers in vivo

描述(申请人提供)：控制干细胞自我更新与分化的分子机制在癌症生物学、衰老和再生医学方面具有巨大的发展潜力。增加干细胞库为肿瘤的形成提供了条件；肿瘤具有自我更新和建立转移的“癌症干细胞”。哺乳动物干细胞数量的一个关键调节因素是JAK/STAT通路。哺乳动物JAK和STATs的不适当激活会导致癌症，抑制它们的活性会阻止原代人类癌细胞的生长。这些数据表明，开发专门抑制JAK或STATs的药物可以为癌症患者提供新的治疗方法。尽管有这些令人信服的观察，但这一途径用于调节哺乳动物干细胞数量的机制尚未阐明。哺乳动物有多个JAK和STAT基因，这使得分析它们在体内的功能变得困难。果蝇提供了一个理想的系统来解决这个问题，因为它对干细胞数量的控制在几个组织中都是保守的，包括睾丸和眼睛。果蝇只有一个JAK和一个STAT(称为stat92E)，可以方便地进行活体分析。这一应用涉及对JAK/STAT通路用于调节果蝇干细胞数量的分子机制的剖析。为了维持干细胞种群，必须以一种协调的方式发生三个不同的过程：细胞质量增加、有丝分裂和自我更新。由于Stat92E是一个转录因子，离散的Stat92E靶基因应该介导其在这些过程中的作用。有三个这样的基因与人类同源基因Cyclin E(CycE)、dmyc和chinmo似乎直接位于Stat92E的下游。CycE是细胞周期的限速调节因子，其表达被Stat92E以细胞自主的方式上调。DMyc是果蝇中唯一的c-Myc家族成员，是细胞生长的重要调节因子。Stat92E促进蛋白质合成，并可能通过dMyc起作用。Chinmo编码一种新的核蛋白，并受Stat92E以细胞自主的方式调节。和Stat92E一样，睾丸干细胞也需要Chinmo来自我更新。鉴定与有丝分裂、细胞生长和自我更新有关的Stat92E调控靶标是我们理解JAK/STAT途径生长调控特性的重大进展。目的1(表征JAK/STAT信号对细胞增殖的影响)利用分子方法和流式细胞仪分析JAK/STAT信号对细胞增殖率、细胞周期周期和关键细胞周期因子表达的影响。目的2(确定Stat92E是否通过dMyc调控细胞生长)利用遗传学方法和流式细胞仪分析来检测Stat92E对细胞生长的影响，并确定它是否通过dmyc调控这一过程。目的3(确定Stat92E是否通过chinmo调节睾丸干细胞的自我更新)利用遗传学和分子生物学方法确定Stat92E是否通过chinmo单独或通过额外的靶基因来调节睾丸干细胞的自我更新。由于进化与公共卫生相关，JAK和STAT基因在其他生物中以非常相似的形式存在，例如果蝇。果蝇是一种优秀的遗传有机体，多年来一直在研究实验室中使用，并在识别和表征对基本生物过程至关重要的基因方面取得了巨大成功。我们的研究旨在了解JAK/STAT通路如何调节果蝇的干细胞数量。

DESCRIPTION (provided by applicant): The molecular mechanisms controlling stem cell self-renewal versus differentiation hold great potential for advances in cancer biology, aging and regenerative medicine. Increasing the pool of stem cells provides a condition for oncogenesis; tumors have "cancer stem cells" that self-renew and establish metastases. A critical regulator of stem cell numbers in mammals is the JAK/STAT pathway. Inappropriate activation of mammalian JAKs and STATs cause cancer and inhibiting their activity blocks the growth of primary human cancer cells. These data suggests that developing drugs which specifically inhibit JAKs or STATs, could offer novel treatments for cancer patients. Despite these compelling observations, the mechanisms utilized by this pathway to regulate stem cell numbers in mammals have not yet been elucidated. Mammals have multiple jak and stat genes, making the analysis of their in vivo function difficult. Drosophila offers an ideal system to address this issue, as its control of stem cell numbers is conserved in several tissues, including the testis and eye. Drosophila has only one jak and one stat (called stat92E) and offers facile in vivo analysis. This application concerns the dissection of molecular mechanisms used by the JAK/STAT pathway to regulate stem cell number in Drosophila. Three distinct processes must occur in a coordinated manner for stem cell populations to be maintained: increase in cellular mass, mitosis and self-renewal. Since Stat92E is a transcription factor, discrete Stat92E target genes should mediate its effects on these processes. Three such genes with human homologs, cyclin E (cycE), dmyc and chinmo, appear to reside directly downstream of Stat92E. CycE is the rate-limiting regulator of the cell cycle and its expression is increased by Stat92E in a cell- autonomous manner. dMyc is the sole c-Myc family member in Drosophila and is a critical regulator of cellular growth. Stat92E increases protein synthesis and may act through dMyc to do so. chinmo encodes a novel nuclear protein and is regulated by Stat92E in a cell-autonomous manner. Like Stat92E, Chinmo is required within testis stem cells for their self-renewal. The identification of Stat92E-regulated targets with links to mitosis, cellular growth and self-renewal is a major advance in our understanding of the growth-regulatory properties of the JAK/STAT pathway. Aim 1 (Characterize the effects of JAK/STAT signaling on proliferation) employs molecular methods and FACS analysis to measure the effects of JAK/STAT signaling on proliferation rates, cell cycle phasing and expression of critical cell cycle factors. Aim 2 (Determine if Stat92E regulates cellular growth through dMyc) uses genetic approaches and FACS analysis to measure the effects of Stat92E on cellular growth and to determine if it regulates this process through dmyc. Aim 3 (Determine if Stat92E regulates self-renewal in through chinmo) uses genetic and molecular approaches to determine if Stat92E regulates self-renewal in testis stem cells through chinmo alone or through additional target genes. PUBLIC HEALTH RELEVANCE Due to evolution, JAK and STAT genes exist in very similar forms in other organisms, such as the fruit fly Drosophila. The fruit fly is an excellent genetic organism that has been used for many years in research laboratories and with great success to identify and characterize genes critical for basic biological processes. Our study is designed to eludicate how the JAK/STAT pathway regulates stem cell numbers in Drosophila.