Single Cell Analysis of Cross Talk Among Kinase Pathways

描述（由申请人提供）：信号蛋白（特别是激酶）的相互作用级联在癌症的发展和维持中起关键作用。尽管它们在致癌信号转导途径中的重要性，但对这些蛋白质在活细胞中的生物化学行为知之甚少。现在清楚的是，这些信号级联不仅仅是从离散刺激到确定的细胞反应的线性系列酶促反应。相反，它们在复杂的网络中相互关联，使得它们在活细胞内的生化特性不是直观的。传统的生物化学研究依赖于大量细胞群的裂解物，由于整个群体的异质细胞反应的平均化，因此无法破译细胞信号传导的固有特性。为了理解这样的生化现象，如通路之间的串扰、激活阈值和激活状态，激酶测定必须在完整的单细胞中进行。目前的工作是针对解码Ras相关途径的生化行为-对癌症生物学特别重要的信号传导途径。这些研究将为单细胞激酶测定提供一种强大的新技术，用于一系列独特的肿瘤细胞系，这些肿瘤细胞系具有组成性活性或非活性信号蛋白。细胞系（人HT 1080/MCH 603纤维肉瘤和小鼠NIH 3 T3）显示出一系列依赖于Ras相关信号通路组成性活性的致瘤特征。在大量的MCH 603细胞群体中，我们的数据支持这样的观点，即这些通路之间的阈值行为和串扰与其致瘤表型的相对侵袭性密切相关。目前的研究将揭示涉及激酶磷酸肌醇-3-激酶（PI 3 K），Akt，p21激活激酶和Erk 1/2的信号级联的行为如何在活的单细胞中相互关联。该项目的具体目的是：1）确定P13 K活性产生的串扰诱导是否需要活化PI 3 K的阈值水平; 2）确定这些激酶途径的交叉活化是协调的还是顺序的; 3）确定在PI 3 K或Akt刺激撤销后MAP激酶级联的串扰活化是可逆的还是不可逆的。这些研究将为我们理解激酶信号通路激活对恶性转化、侵袭性肿瘤生长和生存的影响提供更多信息。

DESCRIPTION (provided by applicant): Interacting cascades of signaling proteins, particularly kinases, play crucial roles in the development and maintenance of cancer. Despite their importance in oncogenic signal transduction pathways, little is understood about the biochemical behavior of these proteins within the context of the living cell. It is now clear that these signaling cascades are not merely a linear series of enzymatic reactions leading from discrete stimuli to defined cellular responses. Rather, they are interrelated in complex networks such that their biochemical properties within the living cell are not intuitive. Traditional biochemical studies that rely on lysates of bulk cell populations cannot decipher the inherent properties of cell signaling due to averaging of heterogeneous cellular responses across the population. To understand such biochemical phenomena as cross-talk between pathways, thresholds of activation and bistable states, kinase assays must be performed in intact, single cells. The current work is directed at decoding the biochemical behaviors of the Ras-related pathways- signaling pathways of particular import to cancer biology. These studies will apply a powerful new technology for kinase assays in single cells to a unique series of tumor cell lines possessing constitutively active or inactive signaling proteins. The cell lines (human HT1080/MCH603 fibrosarcoma and mouse NIH3T3) display a range of tumorigenic characteristics dependent on the repertoire of constitutive activity in Ras-related signaling pathways. In mass populations of MCH603 cells our data support the notion that the threshold behavior and crosstalk among these pathways closely relate to the relative aggressiveness of their tumorigenic phenotype. The current studies will reveal how the behavior of signaling cascades involving the kinases phosphoinositide-3-kinase (PI3K), Akt, p21-activated kinase and Erk1/2 interrelate in living, single cells. The specific aims for this project are to 1) determine whether induction of crosstalk, generated by P13K activity, requires a threshold level of activated PI3K; 2) determine if cross-activation of these kinase pathways is coordinate or sequential; and 3) determine if cross-talk activation of MAP kinase cascades is reversible or irreversible after withdrawal of the PI3K or Akt stimulus. These studies will provide additional information relating to our understanding of the consequences of activation of kinase signaling pathways with respect to malignant transformation, aggressive tumor growth and survival.