Molecular Studies of the p53 Pathway in Human Cancer

这些研究的目的是确定患者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，突变和表达的改变， TP 53基因或影响p53途径中涉及的某些调控基因的那些基因产生选择性的 对癌症患者的肿瘤生长和攻击行为有利。我们的具体目标如下： 目标1.对p53促凋亡反应及其与细胞凋亡的关系进行分子和功能研究， 膀胱癌的临床意义我们将确定检测TP 53的临床相关性， 突变和改变的p53表达模式。评估 我们将在表达载体中克隆p53突变体，并确定它们的表达。 活动p53通路的调控事件也将被分析，集中在遗传和表达 HDM 2研究（与Levine项目合作）。我们还将定义以下事件的发生频率和临床意义： Bax、NoxA和Noxa表达的改变，主要与治疗反应有关（与Lowe的Project合作）。 目标2.目的探讨膀胱癌p53 DNA损伤反应的临床和生物学意义 癌我们和其他人已经观察到，早期膀胱癌，而不是正常组织，表达标志物 与激活的DNA损伤反应相关，如磷酸化Chk 2。我们还确定 原发性膀胱肿瘤中的CHK 2突变（与Prives项目合作）。我们将评估 膀胱癌中的CHK 2突变及其与遗传不稳定性增加和肿瘤的相关性 进展将在膀胱癌细胞中研究Chk 2、ATM和p53的磷酸化状态。 细胞系和原发性肿瘤样品。将通过克隆Chk 2来研究CHK 2突变的后果。 表达载体中的突变体并确定它们对γ辐射的响应。目标3.为了确定 膀胱癌中Pten失活引发的p53衰老。我们最近报道了 p53参与响应Pten途径失活诱导衰老。我们还发现 p53和Pten在膀胱癌中具有协同肿瘤抑制作用，它们的伴随失活 与肿瘤进展和不良结局相关。通过使用多种技术，我们将 确定检测PTEN异常的临床相关性。此外，Akt的致癌潜力 本构活化将进行调查（与劳的项目）。机制研究将针对 进一步限定这两个路径之间的串扰（例如，沉默PTEN和/或p53表达， shRNA以表型复制衰老状态的消除，随后进行基因表达谱分析）。 鉴定的靶基因将在膀胱癌细胞系和已知肿瘤的原发性膀胱肿瘤中进行验证。 Pten和p53状态。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.