Apoptotic Proteins as Molecular Modulators of Stem Cell Fate

描述(由申请人提供)：尽管胚胎干细胞(ES)对于再生医学具有临床重要性，但这些细胞的许多关键生物学特征仍未被探索。我们最近发现，在ES细胞中，凋亡机制的核心成分受到不同的调控，这种独特的调控对于ES细胞对DNA损伤做出快速反应的能力至关重要。特别是，我们发现未分化的人(H)ES细胞通过维持促凋亡蛋白Bax在高尔基体的活性构象而为快速凋亡做好准备。值得注意的是，我们发现仅仅两天的分化就引起了显著的变化：Bax不再处于活跃状态，细胞对DNA损伤不再高度敏感。因此，即使在HES细胞分化的最早阶段，凋亡机制也经历了非凡的变化。更出乎意料的是，我发现这些凋亡成分的变化不仅影响了凋亡的阈值，还影响了ES细胞维持自我更新的能力。具体地说，我发现抗凋亡蛋白Mcl-1的水平随着早期分化而显著降低，仅抑制Mcl-1就足以诱导HES细胞分化。这些结果让我假设，以前被认为只参与细胞死亡过程的凋亡蛋白，是控制干细胞命运的分子开关。我的总体计划是研究分子 细胞凋亡和干细胞分化之间的交叉，目的是确定这些途径是否被癌症干细胞劫持，以达到抵抗凋亡和维持自我更新的双重目的。具体地说，在目标1中，我将研究在ES细胞分化过程中调节Bax功能的途径。我的结果还表明，将成纤维细胞重新编程为诱导的多能干细胞(IPS)与增加对凋亡的敏感性有关。我将研究多能转录因子是如何影响细胞凋亡阈值的。在目标2中，我将重点研究Mcl-1，以确定Mcl-1调节ES细胞自我更新的机制。在目标3中，我将使用胶质母细胞瘤肿瘤干细胞模型来确定抑制Mcl-1活性是否可以作为治疗工具，同时增加对凋亡的敏感性，并限制自我更新能力。这些研究无疑将揭示ES细胞中凋亡调控的关键方面，并揭示干细胞生物学的关键特征，这些特征可能对癌症治疗产生重大影响。

DESCRIPTION (provided by applicant): Despite the clinical importance of embryonic stem (ES) cells for regenerative medicine, many key biological features of these cells remain unexplored. We recently discovered that core components of the apoptotic machinery are differentially regulated in ES cells and that this unique regulation is critical for the ability ofES to respond rapidly to DNA damage. In particular, we found that undifferentiated human (h) ES cells are primed for rapid apoptosis by maintaining the pro-apoptotic protein Bax in its active conformation at the Golgi. Remarkably, we found that just two days of differentiation induced significant changes: Bax was no longer in an active state and the cells were no longer highly sensitive to DNA damage. Thus, the apoptotic machinery undergoes extraordinary changes even in the earliest stages of hES cell differentiation. Rather unexpectedly, I found that changes in these apoptotic components affected not only the thresholds of apoptosis but also the ability of ES cell to maintain self-renewal. Specifically, I found that levels of the anti- apoptotic proten Mcl-1 are markedly reduced with early differentiation and that inhibiting Mcl-1 alone was sufficient to induce differentiation of hES cells. These results have lead me to hypothesize that apoptotic proteins that were previously thought to be involved only in processes of cell death, are molecular switches controlling stem cell fate. My overall plan is to investigate the molecular intersection between apoptosis and stem cell differentiation with the goal of determining whether these pathways are hijacked by cancer stem cells for the dual purpose of resisting apoptosis and maintaining self renewal. Specifically, in Aim 1 I will examine the pathways that regulate Bax function during ES cell differentiation. My results also show that reprogramming of fibroblasts into induced pluripotent stem (iPS) cells is coupled with increased sensitivity to apoptosis. I will examine how the pluripotency transcription factors affect apoptosis thresholds. In Aim 2, I will focus my studies on Mcl-1 to determine the mechanism by which Mcl-1 regulates self-renewal in ES cells. In Aim 3, I will use a model of glioblastoma cancer stem cells to determine whether inhibition of Mcl-1 activity can be used as therapeutic tool to simultaneously increase the sensitivity to apoptosis and restrict the self-renewal capability. These studies will undoubtedly uncover critical aspects of apoptosis regulation in ES cells and reveal key features of stem cell biology that can have significant impact for cancer therapy.