Eludicating the Molecular Mechanisms That Regulate Stem Cell Numbers in Vivo

控制干细胞自我更新与分化的分子机制为癌症生物学、衰老和再生医学的发展提供了巨大的潜力。增加干细胞库为肿瘤发生提供了条件;肿瘤具有自我更新和建立转移的“癌症干细胞”。哺乳动物中干细胞数量的关键调节因子是JAK/STAT途径。哺乳动物JAK和STAT的不适当激活会导致癌症，抑制它们的活性会阻断原代人类癌细胞的生长。这些数据表明，开发特异性抑制JAK或STAT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结果，但该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，这使得分析它们的体内功能变得困难。果蝇提供了一个解决这个问题的理想系统，因为它对干细胞数量的控制在几个组织中是保守的，包括睾丸和眼睛。果蝇只有一个jak和一个stat（称为stat 92 E），并提供简单的体内分析。本申请涉及JAK/STAT途径用于调节果蝇干细胞数量的分子机制的解剖。三个不同的过程必须以协调的方式发生，以维持干细胞群：细胞质量的增加，有丝分裂和自我更新。由于Stat 92 E是一个转录因子，离散Stat 92 E靶基因应介导其对这些过程的影响。与人类同源的三个这样的基因，细胞周期蛋白E（cycE），dmyc和chinmo，似乎直接位于Stat 92 E的下游。CycE是细胞周期的限速调节剂，其表达通过Stat 92 E以细胞自主方式增加。dMyc是果蝇中唯一的c-Myc家族成员，是细胞生长的关键调节因子。Stat 92 E增加蛋白质合成，并可能通过dMyc发挥作用。chinmo编码一种新的核蛋白，并由Stat 92 E以细胞自主的方式调控。与Stat 92 E一样，Chinmo也是睾丸干细胞自我更新所必需的。Stat 92 E调控的靶点与有丝分裂，细胞生长和自我更新的联系的鉴定是我们对JAK/STAT途径生长调节特性的理解的一个重大进展。目的1（表征JAK/STAT信号传导对增殖的影响）采用分子方法和流式细胞仪分析来测量JAK/STAT信号传导对增殖速率、细胞周期定相和关键细胞周期因子表达的影响。目的2（确定Stat 92 E是否通过dMyc调节细胞生长）使用遗传方法和FACS分析来测量Stat 92 E对细胞生长的影响，并确定其是否通过dMyc调节该过程。目的3（确定Stat 92 E是否通过chinmo调节自我更新）使用遗传和分子方法来确定Stat 92 E是否通过单独的chinmo或通过其他靶基因调节睾丸干细胞的自我更新。

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.