Proteomic Study of Novel Platinum Compounds in Glioma

描述（由申请人提供）：胶质瘤仍然是一个严重的公共卫生问题，没有有效的治疗方法，因此确定新的化疗药物是一个优先事项。同样重要的是，及时开发分子工具可以将患者分为可能的应答者和无应答者，从而有效地靶向治疗。由三核BBR3464发现的一个新的铂化合物家族，在体外杀死胶质瘤细胞方面比现有的铂制剂更有效。最有效的化合物BBR3610在胶质瘤细胞克隆实验中具有8 nM的IC90，而顺铂的IC90为2 mM。我们正在尝试通过各种手段建立反应性肿瘤的分子图谱，并研究这些有前景的药物的作用机制，并在本提案中寻求支持基于二维液相色谱显示蛋白质组的最新方法。蛋白质在第一个维度上通过色谱聚焦柱分离，在第二个维度上通过反相HPLC柱分离。然后使用专门的软件显示214 nM处的吸光度数据，以便快速识别表达差异。除了交钥匙和适合高通量之外，这种方法使蛋白质完全处于液相中，使下游分析（如质谱分析）的回收变得容易。我们建议将这项技术应用于两个模型系统，目的是测试铂化合物反应的细胞死亡与蛋白质组的特征性变化有关的假设，并且所涉及的蛋白质可用于分析患者。在第一个模型中，我们将在体外和体内用等剂量的顺铂和BBR化合物治疗胶质瘤细胞系，并评估对其蛋白质组的影响。将比较同一细胞系的敏感株和耐药株。在第二个模型中，我们将比较p53突变在蛋白质水平上对细胞对铂化合物的反应的影响，p53突变可以使细胞敏感或保护细胞，通过改造p53-null星形胶质细胞来表达人类p53突变体。我们希望确定蛋白质组的变化，这些变化是铂化合物有效杀死胶质瘤细胞的特征，因为这些信息可能有助于识别可能对给定药物有反应的患者，并有助于理解其作用机制。

DESCRIPTION (provided by applicant): Gliomas remain a serious public health problem with no effective treatment, making the identification of new chemotherapeutics a priority. Equally important is the timely development of molecular tools allow the stratification of patients into likely responders and non-responders, so that therapies can be targeted effectively. A new family of platinum compounds, founded by the trinuclear BBR3464, is much more potent than established platinum agents in the killing of glioma cells in vitro. The most potent compound, BBR3610 has an IC90 in a glioma cell clonogenic assay of 8 nM, as compared to 2 mM for cisplatin. We are attempting to establish molecular profiles of responding tumors, and investigating the mechanism of action of these promising drugs by various means, and in this proposal seek support for the newest approach, which is based on displaying the proteome using two-dimensional liquid chromatography. Proteins are separated in the first dimension by a chromatofocusing column and in the second dimension on a reverse phase HPLC column. Absorbance data at 214 nM is then displayed using specialized software allowing the rapid identification of expression differences. In addition to being turnkey and suited to high throughput, this approach leaves proteins entirely in the liquid phase making recovery for downstream analysis, such as mass spectrometry, easy. We propose to apply this technology to two model systems with the aim of testing the hypothesis that cell death in response to platinum compounds is associated with characteristic changes in the proteome and that the proteins involved can be used to profile patients. In the first model, we will treat glioma cell lines with isodoses of cisplatin and BBR compounds in vitro and in vivo and assess the impact on their proteomes. Sensitive and resistant isolates of the same cell lines will be compared. In the second model, we will compare the impact of p53 mutation, which can sensitize or protect cells, on the response of cells to platinum compounds at the protein level, by using transformed p53-null astrocytes engineered to express human p53 mutants. We hope to identify changes in the proteome that are characteristic of effective glioma cell killing by platinum compounds, as such information may facilitate identifying patients likely to respond to a given drug, as well as aid in understanding their mechanism of action.