Apoptotic Proteins as Molecular Modulators of Stem Cell Fate

描述（由申请人提供）：尽管胚胎干（ES）细胞在再生医学中具有临床重要性，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，凋亡机制的核心成分在ES细胞中受到不同的调节，这种独特的调节对于ES对DNA损伤迅速反应的能力至关重要。特别是，我们发现，未分化的人（h）ES细胞的快速凋亡，通过维持促凋亡蛋白Bax在其活性构象在高尔基体。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不再处于活性状态，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。出乎意料的是，我发现这些凋亡成分的变化不仅影响凋亡的阈值，而且影响ES细胞维持自我更新的能力。具体地，我发现抗凋亡蛋白Mcl-1的水平在早期分化时显著降低，并且单独抑制Mcl-1足以诱导hES细胞的分化。这些结果使我假设，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 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，以达到抵抗细胞凋亡和维持自我更新的双重目的。具体来说，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。我的研究结果还表明，成纤维细胞重编程为诱导多能干细胞（iPS）与细胞凋亡的敏感性增加。我将研究多能性转录因子如何影响凋亡阈值。在目标2中，我将把我的研究重点放在Mcl-1上，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癌症干细胞模型来确定Mcl-1活性的抑制是否可以用作治疗工具，以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细胞凋亡调控的关键方面，并揭示干细胞生物学的关键特征，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.