A PTEN-Dependent Size Checkpoint in Human Cancer Cells

描述（由申请人提供）：在广泛的常见人类癌症中发现通过失活PTEN肿瘤抑制基因中的突变或激活PIK 3CA癌基因中的突变来激活PIP 3信号传导。虽然PIP 3信号转导的生物化学细节正在迅速出现，但人类癌细胞中PIP 3途径激活的表型后果仍然不太清楚。因此，该项目的长期目标是确定PIP 3激活在人类癌细胞中的作用。在我们最初的初步研究中，我们采用人体细胞基因靶向来创建PTEN+/+和PTEN-/-人癌细胞系的等基因组。使用这些和相关的模型系统，我们发现，PTEN控制一种新的辐射诱导的大小检查点在人类细胞中，这是不同的和遗传上可分离的辐射诱导的，p53依赖的G1和G2检查点。据我们所知，这种DNA损伤诱导的大小停滞的存在既没有在任何生物体中被假设或证明。自首次提交本申请以来，我们已采用人体细胞基因靶向来创建PIK 3CA基因靶向的人癌细胞的等基因组，并且还创建了表达野生型、致癌或失活形式的PIK 3CA的NIH 3 T3细胞系。使用这些系统，我们已经获得了初步的数据，表明PIK 3CA癌基因，如PTEN，可能能够调节大小检查点功能以及。在本申请中，我们提出了三个相关的具体目标，以进一步追求和扩展这些观察。目的#1 -鉴定PTEN依赖性大小检查点的其他诱导物。目的#2 -证明在未转化的人细胞中存在PTEN依赖性大小检查点。目的#3 -表征辐射诱导的p53依赖性G1/G2检查点和辐射诱导的PTEN依赖性细胞大小检查点之间的串扰。追求这些目标将使我们能够进一步表征肿瘤抑制基因调控的辐射诱导的细胞大小检查点的特性和机制。

DESCRIPTION (provided by applicant): Activation of PIP3 signaling by inactivating mutations in the PTEN tumor suppressor or activating mutations in the PIK3CA oncogene is found in a wide range of common human cancers. Though the biochemical details of PIP3 signal transduction are rapidly emerging, the phenotypic consequences of PIP3 pathway activation in human cancer cells remain less well defined. As such, the long-term goal of this project is to define the effects of PIP3 activation in human cancer cells. In our initial preliminary studies, we employed human somatic cell gene targeting to create isogenic sets of PTEN+/+ and PTEN-/- human cancer cell lines. Using these and related model systems, we found that PTEN controls a novel radiation-induced size checkpoint in human cells that is distinct and genetically separable from the radiation-induced, p53-dependent G1 and G2 checkpoints. To our knowledge, the existence of such a DNA damage-inducible size arrest has neither been postulated or demonstrated in any organism. Since the initial submission of this application we have employed human somatic cell gene targeting to create an isogenic set of PIK3CA gene targeted human cancer cells, and also created NIH3T3 cell lines that express wild-type, oncogenic, or inactive forms of PIK3CA. Using these systems we have obtained preliminary data suggesting that, like PTEN, the PIK3CA oncogene may be able to regulate size checkpoint function as well. In this application we propose three related specific aims to further pursue and extend these observations. Aim #1 - Identify additional inducers of the PTEN-dependent size checkpoint. Aim #2 - Demonstrate the presence of the PTEN-dependent size checkpoint in untransformed human cells. Aim #3 - Characterize crosstalk between the radiation-induced, p53-dependent G1/G2 checkpoints and the radiation-induced, PTEN-dependent cell size checkpoint. Pursuit of these aims will enable us to further characterize the properties and mechanism(s) of a tumor suppressor gene-regulated, radiation-induced cell size checkpoint.