Single Cell Analysis of Cross Talk Among Kinase Pathways

相互作用的级联信号蛋白，特别是激酶，在癌症的发展和维持中发挥着关键作用。 尽管它们在致癌信号转导通路中很重要，但对它们的生化行为知之甚少。 )在活细胞的背景下旋转。现在很清楚，这些信号级联不只是一系列线性的酶 从离散刺激到明确的细胞反应的反应。相反，它们在复杂的网络中相互关联，以至于它们的 活细胞内的生化特性并不直观。依赖块状细胞裂解产物的传统生化研究 人口不能破译细胞信号的固有属性，这是由于不同细胞反应的平均化 人口。了解通路间的串扰、激活阈值和双稳态等生化现象 在这种情况下，必须在完整的单个细胞中进行运动分析。目前的工作是针对解码的生化行为 RAS相关通路--对癌症生物学特别重要的信号通路。这些研究将应用一种强大的新技术 单细胞对一系列独特的具有正常活性或非活性信号的肿瘤细胞株的激酶检测技术 蛋白质。这些细胞系(人HT1080/MCH603纤维肉瘤和小鼠NIH3T3)表现出一系列的致瘤特性 依赖于RAS相关信号通路中的结构性活性。在MCH603细胞的大量群体中，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 他们的致瘤表型。目前的研究将揭示信号行为是如何级联的，涉及到激酶 磷脂酰肌醇-3-激酶(P13K)、Akt、p21-激活的激酶和Erk1/2在活的单细胞中相互关联。这方面的具体目标 项目是1)确定由P13K活动产生的串扰是否需要激活P13K的阈值水平；2 确定这些激酶通路的交叉激活是协调的还是顺序的；以及3)确定MAP的交叉激活 退出P13K或Akt刺激后，激活级联反应可逆或不可逆。这些研究将提供额外的 与我们对激活激酶信号通路与恶性相关的后果的理解有关的信息 转化，侵袭性肿瘤生长和存活。

Interactingcascadesof signaling proteins, particularly kinases, play crucial roles in the development and maintenance of cancer. Despite their importance inoncogenic signal transduction pathways, little is understood about the biochemical behavior of these )roteinswithin the context of the living cell. It is now clear that these signaling cascades are not merely a linear series of enzymatic ¿eactions leadingfrom discrete stimuli to defined cellular responses. Rather, they are interrelated in complex networks such that their biochemical propertieswithin the living cell are not intuitive. Traditional biochemical studies that rely on lysates of bulk cell populationscannot decipher the inherent properties of cell signaling due to averaging of heterogeneous cellular responses across the population. To understandsuch biochemical phenomena as cross-talk between pathways, thresholds of activation and bistable states, kinaseassays must be performed in intact, single cells. The currentwork is directed at decoding the biochemical behaviors of the Ras-related pathways- signaling pathways of particular importto cancer biology. These studies will apply a powerful new technologyfor kinase assays in single cells to a unique series of tumor cell lines possessing contitutively active or inactive signaling proteins. The cell lines (human HT1080/MCH603 fibrosarcomaand mouse NIH3T3) display a range of tumorigenic characteristics dependent on the repertoire of constitutive activity in Ras-relatedSignalingpathways. 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 (P13K), Akt, p21-activated kinase and Erkl/2 interrelate in living, single cells. The specific aims for this project are to 1) determinewhether induction of crosstalk, generated by P13K activity, requires a threshold level of activated P13K; 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 P13K or Akt stimulus. These studies will provide additional information relating to our understanding of the consequencesof activationof kinase signaling pathwayswith respect to malignant transformation, aggressive tumor growth and survival.