Molecular Studies of the p53 Pathway in Human Cancer

拟议研究的目的是确定关于患者P53状态的数据是否有助于临床 膀胱癌的治疗。我们的工作假设是，基因突变和基因表达的改变 TP53基因，或那些影响涉及P53途径的特定调控基因的基因，产生选择性的 有利于肿瘤生长和癌症患者的攻击行为。我们的具体目标如下： 目的1.对P53促凋亡反应及其功能进行分子和功能研究。 膀胱癌的临床意义。我们将确定检测TP53的临床相关性 使用多种方法组合检测P53基因突变和表达模式的改变。要评估 对于TP53突变的后果，我们将在表达载体中克隆p53突变体，并确定其 活动。还将分析P53途径的调节事件，重点是遗传和表达 Hdm2研究(与Levine项目合作)。我们也将定义频率和临床意义。 改变了Bax、PUMA和Noxa的表达，主要与治疗反应有关(与Lowe的项目合作)。 目的2.明确膀胱癌P53 DNA损伤反应的临床和生物学意义 癌症。我们和其他人观察到早期膀胱癌，但不是正常组织，表达标志物 与激活的DNA损伤反应有关，例如磷酸化的Chk2。我们还确认了 原发膀胱肿瘤中的Chk2突变(与Prives项目合作)。我们将评估 膀胱癌中Chk2突变及其与遗传不稳定性增加和肿瘤的关系 进步。Chk2、ATM和P53在膀胱癌细胞中的磷酸化状态将被研究 系和原发肿瘤样本。Chk2突变的后果将通过克隆Chk2来研究 表达载体中的突变体，并确定它们对伽马辐射的反应。目标3.确定角色 Pten失活在膀胱癌衰老中的作用我们最近报道了 Pten途径失活导致衰老过程中P53的参与我们还发现 P53和Pten在膀胱癌中的协同抑癌作用及其伴随的失活 与肿瘤进展和不良预后有关。使用多种技术组合，我们将 确定检测PTEN异常的临床意义。此外，Akt的致癌潜能 结构激活将被调查(与Lowe的项目合作)。机械学研究的目标将是 进一步定义这两条通路之间的串扰(例如，通过以下方式抑制PTEN和/或P53的表达 ShRNA用于表观复制衰老状态的消融，随后进行基因表达谱分析)。 已确定的靶基因将在已知的膀胱癌细胞系和原发性膀胱癌中得到验证 PTEN和P53的表达情况。主要目标是将基础研究成果转化为临床应用研究。

The objective of the proposed studies is to determine whether data on a patient's p53 status aids clinical management of bladder cancer. Our working hypothesis is that mutations and altered expression of the TP53 gene, or those affecting certain regulatory genes involved in the p53 pathway, produce a selective advantage for tumor growth and aggressive behavior in cancer patients. Our specific aims are as follows: Aim 1. To conduct molecular and functional studies of the p53 pro-apoptotic response and its clinical significance in bladder cancer. We will determine the clinical relevance of detecting TP53 mutations and altered patterns of p53 expression using a combination of methods. To assess the consequences of TP53 mutations, we will clone p53 mutants in expression vectors and ascertain their activities. Regulatory events of the p53 pathway will also be analyzed, centering on genetic and expression studies of HDM2 (collaboration with Project by Levine). We will also define the frequency and clinical significance of altered Bax, PUMA and Noxa expression, mainly in relation to treatment response (working with Project by Lowe). Aim 2. To define the clinical and biological implications of p53 DNA damage response in bladder cancer. We and others have observed that early bladder cancer, but not normal tissues, express markers associated with an activated DNA damage response, such as phosphorylated Chk2. We have also identified CHK2 mutations in primary bladder tumors (collaborating with Project by Prives). We will assess the frequency of CHK2 mutations in bladder cancer, and their association with increased genetic instability and tumor progression. The phosphorylation status of Chk2, ATM, and p53 will be investigated in bladder cancer cell lines and primary tumor samples. The consequences of CHK2 mutations will be studied by cloning Chk2 mutants in expression vectors and determining their response to gamma-radiation. Aim 3. To ascertain the role of p53 in senescence triggered by Pten inactivation in bladder cancer. We have recently reported the involvement of p53 at inducing senescence in response to inactivation of the Pten pathway. We also found that p53 and Pten have cooperative tumor suppressor roles in bladder cancer, their concomitant inactivation being associated with tumor progression and poor outcome. Using a combination of techniques, we will determine the clinical relevance of detecting PTEN abnormalities. In addition, the oncogenic potential of Akt constitutive activation will be investigated (working with Project by Lowe). Mechanistic studies will be aimed at further defining the crosstalk between these two pathways (e.g., silencing PTEN and/or p53 expression by shRNA to pheno-copy the ablation of the senescent status, followed by gene expression profiling analyses). Identified target genes will be validated in bladder cancer cell lines and primary bladder tumors of known Pten and p53 status. The main goal is to translate basic research findings into clinically applied studies.