Apoptotic Proteins as Molecular Modulators of Stem Cell Fate

描述（由申请人提供）：尽管胚胎干细胞在再生医学中具有重要的临床意义，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胚胎干细胞中受到不同的调控，这种独特的调控对于胚胎干细胞快速响应DNA损伤的能力至关重要。特别是，我们发现未分化的人胚胎干细胞通过维持高尔基体上促凋亡蛋白Bax的活性构象而引发快速凋亡。值得注意的是，我们发现仅仅两天的分化就诱导了显著的变化：Bax不再处于活跃状态，细胞不再对DNA损伤高度敏感。因此，即使在he细胞分化的早期阶段，凋亡机制也经历了非同寻常的变化。出乎意料的是，我发现这些凋亡成分的变化不仅影响凋亡阈值，还影响ES细胞维持自我更新的能力。具体来说，我发现抗凋亡蛋白Mcl-1的水平随着早期分化而显著降低，并且仅抑制Mcl-1就足以诱导hES细胞分化。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分子

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.