IC Communication in Breast Cancer Metastasis to Bone

描述(来自申请人的逐字描述)：支持 乳腺癌向骨的优先转移目前还知之甚少。我们 提出缝隙连接细胞间通讯(GJIC)有助于 促进异型细胞-细胞向骨转移的乳腺癌 乳腺癌细胞与成骨细胞的相互作用。这些 相互作用有助于乳腺癌细胞跨成骨细胞迁移， 这是骨转移的关键一步。这样做的长期目标是 项目是描述缝隙连接在乳腺中的表达和功能 癌细胞(转移、转移抑制和非转移)及其方式 这通过激活胞浆内钙动员和肌球蛋白起作用 轻链激酶(MLCK)活性对乳腺癌细胞转骨母细胞的影响 迁移和骨转移。这些目标将通过 完成五个具体目标：(1)检测乳房GJIC同型 乳腺癌细胞株与乳腺癌细胞间的异型GJIC 成骨细胞；(2)量化乳腺癌细胞接触的影响，在 GJTC和细胞内钙动员抑制剂的存在和不存在， 成骨细胞hFOB 1.9细胞内钙离子浓度([Cai])的研究 乳房接触后成骨细胞MLCK活性的定量研究 癌细胞；(4)量化乳腺癌细胞的跨细胞迁移 通过成骨细胞单层；和(5)基因改变表达 乳腺癌细胞中的连接蛋白与体内转移潜能的评估 骨转移模型。作为乳腺癌细胞株，我们将使用MDA-MB-23I 和MDA-MB-435，这些细胞表达最近发现的转移 抑制基因BRMS1，这些细胞表达绿色荧光蛋白(GFP) 和适当的媒介控制。我们还将检查非转移性MDA-MB-468 和Neol 1/435乳腺癌细胞。GJIC将通过染料转移进行量化 技术，微量荧光分光光度法测定[Ca~(2+)]，体外测定MLCK活性 磷酸化分析和细胞数量测定的跨细胞迁移 通过Matrigel的迁移，利用共聚焦显微镜。骨转移会 利用绿色荧光蛋白标记的乳腺癌细胞进行荧光法评估。这个 该项目的结果将为深入了解 乳腺癌细胞优先转移到骨骼，这是 开发抑制骨转移的方法。

DESCRIPTION (Verbatim from the Applicant): The mechanism underlying the preferential metastasis of breast cancer to bone is poorly understood. We propose that gap junctional intercellular communication (GJIC) contributes to breast cancer metastasis to bone by facilitating heterotypic cell-cell interactions between breast cancer cells and osteoblastic cells. These interactions contribute to breast cancer cell transosteoblastic migration, which is a critical step in bone metastasis. The long-term goals of this project are to characterize gap junction expression and function in breast cancer cells (metastasizing, metastasis suppressed and non-metastatic) and how this contributes, through activation of cytosolic Ca2+ mobilization and myosin light chain kinase (MLCK) activity, to breast cancer cell transosteoblastic migration and bone metastasis. These goals will be accomplished through the completion of five Specific Aims: (1) examine homotypic GJIC among breast cancer cell lines and heterotypic GJIC between breast cancer cells and osteoblastic cells; (2) quantify the effect of breast cancer cell contact, in the presence and absence of inhibitors of GJTC and cytosolic Ca2+ mobilization, on cytosolic Ca2+ concentration ([Ca +i]) in osteoblastic hFOB 1.9 cells; (3) quantify MLCK activity in osteoblastic cells following contact with breast cancer cells; (4) quantify transcellular migration of breast cancer cells through osteoblastic monolayers; and (5) genetically alter expression of connexins in breast cancer cells and assess metastatic potential in an in vivo model of bone metastasis. As breast cancer cell lines, we will use MDA-MB-23 I and MDA-MB-435, these cells expressing the recently identified metastasis suppressing gene BRMS1, these cells expressing green fluorescent protein (GFP) and appropriate vector controls. We will also examine non-metastasic MDA-MB-468 and neol 1/435 breast cancer cells. GJIC will be quantified by dye transfer techniques, [Ca2+], by microspectrofluorometry, MLCK activity by in vitro phosphorylation assays, and transcellular migration by quantification of cell migration through Matrigel, utilizing confocal microscopy. Bone metastasis will be assessed fluorometrically utilizing GFP tagged breast cancer cells. The results of this project will provide insights into the mechanism by which breast cancer cells preferentially metastasize to bone, an important step in developing approaches to inhibit bone metastasis.