The role of PTEN in DNA metabolism and replication

研究综述 本项目的目的是了解PTEN在DNA新陈代谢和复制中的作用。磷酸酶和 10号染色体张力蛋白同源缺失(PTEN)是最常见的缺失或突变的肿瘤之一 癌症中的抑制因子，通常在子宫内膜癌、胶质母细胞瘤、乳腺癌和前列腺癌中缺失。 PTEN是一种双特异性磷酸酶，其主要底物是脂质第二信使 磷脂酰肌醇-3，4，5-三磷酸(PIP3)和PTEN活性负调控磷脂酰肌醇-3，4，5-三磷酸。 激酶(PI3K)/AKT生长信号通路。我们实验室和其他实验室的工作表明，删除这两个 PTEN等位基因导致包括原发组织在内的各种正常组织的生长速度和增殖速度加快 小鼠胚胎成纤维细胞。此外，我们已经证明了谷氨酰胺依赖，以及 增加谷氨酰胺进入从头合成嘧啶的流量，并增加对药物的敏感性 抑制初级Pten-/-MEF从头合成嘧啶。我们认为这些结果暗示了 核苷酸代谢、细胞周期和DNA复制作为PTEN丢失的潜在途径 可能有助于放松管制的增长。我们假设使用初级Ptenflx/FLOX MEF作为一种 模型系统。 在目标1中，我们将研究PTEN表达的缺失如何使细胞对从头开始的嘧啶的抑制敏感。 通过改变细胞周期动力学进行合成。为了研究这种敏感性的分子基础，我们将 检测药物作用后DNA损伤和复制应激、细胞死亡和细胞周期检查点激活 抑制初级Pten-/-MEF从头合成嘧啶。鉴于来氟米特可能用于 PTEN缺陷型肿瘤患者，全面了解 在PTEN丢失的情况下，来氟米特的治疗将使更有效的使用。 在目标2中，我们将表征PTEN缺失对S期持续时间和DNA复制效率的影响。 为了确定初级pTen-/-MEF中S相的长度，我们将使用两种实验方法 胸腺嘧啶核苷类似物与活细胞双脉冲DNA复制标记测定S相长 结合荧光标记的细胞周期特异性蛋白进行成像。研究DNA复制 为了提高效率，我们将执行纤维梳理，以直接可视化复制DNA。对该机制的了解 在PTEN丢失的背景下改变复制动力学的背后将有助于对 癌细胞逃避生长调控的机制。

RESEARCH SUMMARY The goal of this project is to understand the role of PTEN in DNA metabolism and replication. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is one of the most frequently lost or mutated tumor suppressors in cancer, commonly lost in endometrial cancer, glioblastoma, breast cancer, and prostate cancer. PTEN is a dual-specificity phosphatase whose main substrate is the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), and PTEN activity negatively regulates the phosphoinositide 3- kinase (PI3K)/AKT growth signaling pathway. Work in our lab and others has shown that the deletion of both alleles of PTEN causes increased growth rate and proliferation in a variety of normal tissues, including in primary mouse embryonic fibroblasts (MEFs). Additionally, we have demonstrated a glutamine dependency, an increased glutamine flux into de novo pyrimidine synthesis, and an increased sensitivity to pharmacologic inhibition of de novo pyrimidine synthesis in primary Pten–/– MEFs. We propose that these results implicate the nucleotide metabolism, the cell cycle, and DNA replication as potential avenues through which the loss of PTEN could contribute to deregulated growth. We posit to explore these pathways using primary Ptenflox/flox MEFs as a model system. In Aim 1, we will investigate how the loss of PTEN expression sensitizes cells to inhibition of de novo pyrimidine synthesis through altered cell cycle dynamics. To investigate the molecular basis of this sensitivity, we will examine DNA damage and replication stress, cell death, and cell cycle checkpoint activation after pharmacologic inhibition of de novo pyrimidine synthesis in primary Pten–/– MEFs. Given the potential use of leflunomide for patients with PTEN-deficient tumors, a comprehensive understanding of the cellular consequences of leflunomide treatment in the context of PTEN loss will enable more effective use. In Aim 2, we will characterize the impact of PTEN loss on the duration of S phase and DNA replication efficiency. To determine the length of S phase in primary Pten–/– MEFs, we will use two experimental methods that allow for the determination of S phase length: dual-pulse DNA replication labeling with thymidine analogs and live cell imaging in combination with fluorescent-labeled cell cycle-specific proteins. To investigate DNA replication efficiency, we will perform fiber combing to directly visualize replicating DNA. Knowledge of the mechanism behind altered replication dynamics in the context of PTEN loss would contribute to the basic understanding of the mechanisms of evading growth regulation in cancer cells.