FAC IN STEM CELL FUNCTION AND OXYGEN SENSITIVITY

范可尼贫血（FA）是一种复杂的常染色体隐性遗传疾病，其特征是骨髓（BM）衰竭、恶性肿瘤发生率增加和对双功能烷化剂如丝裂霉素C（MMC）超敏。 有八种互补类型的FA具有重叠的表型，推断存在八个基因在一个共同的生化途径。 很明显，造血祖细胞区室受到范可尼贫血互补C型蛋白（FAC）丢失的影响。 然而，由于难以评估人类系统中的造血干细胞（HSC）功能，尚未进行直接研究评估造血干细胞（HSC）隔室是否受到类似影响。 在FAC的鼠同源物（Fac）中含有纯合破坏的鼠模型的开发允许对这个问题进行全面评价。 使用这种小鼠模型，我们发现Fac-/-造血祖细胞对MMC过敏，类似于FA患者。 这些数据表明，Fac-/-小鼠将是一个很好的模型系统，研究FA。 一些证据表明，FA蛋白可能在维持正常的氧化代谢中发挥作用。 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。本申请的目的是：1）确定Fac的丧失是否影响HSC功能，2）确定Fac-/-造血细胞是否对氧化应激具有增加的敏感性，和3）评估RED的过表达是否诱导Fac +/+细胞中对氧化应激的超敏反应。 这些数据将提供重要的信息，在阐明BM故障和致癌FA的发病机制，以及调查一个独特的功能Fac在正常的氧化代谢，可以提供潜在的影响，修改目前的治疗方案FA患者。

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.