FAC IN STEM CELL FUNCTION AND OXYGEN SENSITIVITY

Fanconi贫血(FA)是一种复杂的常染色体隐性遗传病，其特征是骨髓(BM)衰竭、恶性肿瘤发生率增加以及对双功能烷化剂如丝裂霉素C(MMC)超敏。FA有八种互补类型，具有重叠的表型，推测在一个共同的生化途径中存在八个基因。很明显，造血祖细胞受到Fanconi贫血补体C型蛋白(FAC)缺失的影响。然而，由于难以评估造血干细胞(HSC)在人体系统中的功能，目前还没有进行直接研究来评估HSC是否受到类似的影响。小鼠模型的发展包含在FAC的小鼠同源基因(FAC)中存在纯合子中断，这使得对这一问题的全面评估成为可能。利用这个小鼠模型，我们发现FAC-/-造血祖细胞对MMC超敏，类似于FA患者。这些数据表明，FAC-/-小鼠将是研究FA的良好模型系统。一些证据表明，FA蛋白可能在维持正常氧化代谢方面发挥作用。此外，最近在COS细胞中观察到FAC调节NADPH：细胞色素P450还原酶(RED)活性，这是一种产生氧自由基并激活几种药物(如MMC)的重要酶，支持这一假设。这项应用的目的是：1)确定FAC缺失是否影响HSC功能，2)确定FAC-/-造血细胞是否对氧化应激更敏感，以及3)评估RED过表达是否会诱导FAC+/+细胞对氧化应激的超敏反应。这些数据将为阐明骨髓衰竭和FA的致癌机制提供重要信息，并研究FAC在正常氧化代谢中的独特作用，可能为修改FA患者的现有治疗方案提供潜在的启示。

Fanconi anemia (FA) is a complex, autosomal recessive disorder characterized by bone marrow (BM) failure, increased incidence of malignancies, and hypersensitivity to bifunctional alkylating agents such as mitomycin C (MMC). There are eight complementation types of FA that have overlapping phenotypes inferring the existence of eight genes in a common biochemical pathway. It is clear that the hematopoietic progenitor compartment is affected by loss of Fanconi anemia complementation type C protein (FAC). However, no direct studies have been conducted evaluate whether the hematopoietic stem cell (HSC) compartment is similarly affected due to difficulties in assessing HSC function in the human system. The development of a murine model containing a homozygous disruption in the murine homologue (Fac) of FAC allows comprehensive evaluation of this question. Using this murine model, we showed that Fac -/- hematopoietic progenitors were hypersensitive to MMC, similar to FA patients. These data suggest that Fac -/- mice will be a good model system to study FA. Several lines of evidence suggest that FA proteins may have a role in maintaining normal oxidative metabolism. In addition, the recent observation in COS cells that FAC modulates NADPH:cytochrome p450 reductase (RED) activity, an important enzyme that generates oxygen radicals and activates several drugs such as MMC supports this hypothesis. The goals of this application are: 1) to determine whether loss of Fac affects HSC function, 2) to determine if Fac -/- hematopoietic cells have an increased sensitivity to oxidative stress, and 3) to evaluate whether overexpression of RED induces a hypersensitivity to oxidative stresss in Fac +/+ cells. These data will provide important information in elucidating the pathogenesis of BM failure and carcinogenesis in FA as well as investigate a distinct function of Fac in normal oxidative metabolism that could provide potential implications for modifying current treatment protocols for FA patients.