TUMOR CELL ADHESIVE GLYCOPROTEINS AND METASTASIS

随着近年来在原发部位控制和复发性控制方面的进展， 疾病，转移性疾病仍然是一个主要的障碍， 改善癌症治疗。 转移级联反应是一个复杂的多步骤 一个涉及肿瘤之间同型和异型相互作用的过程 细胞和宿主细胞，除了肿瘤细胞粘附到基质 proteins. 肿瘤细胞具有多种表型特征 这使它们能够完成转移过程并形成新的病变。 然而，转移级联的一些步骤可能是速率限制性的， 可能是治疗干预的目标。 大多数，如果不是全部，步骤 转移级联反应涉及细胞表面受体， 整合素 已经发现了许多整合素粘附受体， 各种人类和啮齿动物肿瘤细胞系。 其中，玻连蛋白 受体α-v-β-3和α-II-b-β-3。α-II的表达- 最初认为b-β-3仅限于血小板， 巨核细胞谱系细胞。 最近，我们已经证明，由南方 印迹法、北方印迹法和免疫沉淀法，α- II-b-beta-3在几种人类和小鼠肿瘤系中的作用。 本研究旨在 通过以下方法测定肿瘤细胞α-II-B和β-3的cDNA序列： 通过逆转录合成第一链cDNA，并使用 聚合酶链反应技术扩增α-II-B和β-3 cDNA并对PCR产物进行测序。 接下来，α-II-b-beta-3受体 参与指示代谢潜力的表型性状，例如 粘附和铺展在内皮、血小板和肺集落上 形成将被确定，试图建立一个可能的 肿瘤细胞中α-II-β-3表达与 转移 为了进一步确定α-II-β-3的作用， 已经用抗α-II-b和/或抗α- β-3构建体。 将对α-II-b-β-3阴性细胞进行检测， 它们经历转移所必需的相互作用的能力（例如， 血小板聚集、粘附、扩散和肺定植）。 此外，细胞粘附素整联蛋白的相对贡献（即， α-II-b-β-3和α-v-β-3）与肿瘤细胞粘附， 在基质配体和内皮上铺展，肿瘤细胞诱导的血小板 将研究聚合。 表达α-II-B的肿瘤细胞系 但不是α-V（即，B 16无黑色素性黑素瘤）和另一种细胞系 同时表达α-II-B和α-v的刘易斯肺癌将 用于比较每种整合素对转移性的相对贡献 潜力 针对α-II-B和/或α-v的反义构建体 在这些比较实验中将被引入这些细胞系 然后进行粘附、血小板聚集和肺集落形成试验。 在 此外，细胞因子TGF-β、肿瘤坏死因子和白细胞介素1， 以及类二十烷酸12-羟基二十碳四烯酸，其已经被 证明调节一些整合素的表达，将进行研究 因为它们对α-半乳糖苷酶的转录和/或翻译控制的影响， II-b-beta-3和alpha-v-beta-3的表达及其功能。 细胞因子和/或类花生酸对整联蛋白功能的作用可通过以下方式调节： 磷酸化或增加它们与细胞 细胞骨架 α-II-B、α-V和β- 3，并检查受体与细胞骨架的结合。 在 这个建议，我们将使用一个全面的方法，包括分子， 生物化学和细胞生物学技术来研究相对于 细胞粘附素整联蛋白的贡献（即，alpha-II-b-beta-3和alpha-II-b-beta-3 V-β-3）对与高转移潜能相关的细胞功能的影响。

With the recent advances in primary site control and control of recurrent disease, metastatic disease remains one of the principle impediments to improved cancer treatment. The metastatic cascade is a complex multi-step process which involves homotypic and heterotypic interactions among tumor cells and host cells, in addition to tumor cell adhesion to matrix proteins. Tumor cells possess a variety of phenotypic characteristics which enable them to complete the metastatic process and form a new lesion. However, some steps of the metastatic cascade may be rate limiting and possibly targeted for therapeutic intervention. Most, if not all, steps of the metastatic cascade involve cell surface receptors such as the integrins. A number of integrin adhesion receptors have been found on various human and rodent tumor cell lines. Among them, are the vitronectin receptor alpha-v-beta-3 and alpha-II-b-beta-3. The expression of alpha-II- b-beta-3 was originally thought to be confined to platelets and megakaryocyte lineage cells. Recently, we have demonstrated, by Southern blotting, Northern blotting and immunoprecipitation, the presence of alpha- II-b-beta-3 in several human and murine tumor lines. This study is aimed to determine cDNA sequences of tumor cell alpha-II-b and beta-3 by synthesizing the first strand cDNA by reverse transcription and using the polymerase chain reaction technique to amplify the alpha-II-b and beta-3 cDNAs and sequence the PCR products. Next, the alpha-II-b-beta-3 receptor involvement in phenotypic traits indicative of metastic potential such as adhesion to and spreading on endothelium, platelets and lung colony formation will be determined, in an attempt to establish a possible correlation between alpha-II-beta-3 expression in tumor cells and metastasis. To further define the role of alpha-II-beta-3, murine tumor cell lines have been transfected with anti-alpha-II-b and/or anti-alpha- beta-3 constructs. The alpha-II-b-beta-3 negative cells will be tested for their ability to undergo interactions necessary for metastasis (e.g., platelet aggregation, adhesion, spreading and lung colonization). Moreover, the relative contribution of the cytoadhesin integrins (i.e., alpha-II-b-beta-3 and alpha-v-beta-3) to tumor cell adhesion to and spreading on matrix ligands and endothelium, tumor cell induced platelet aggregation will be studied. A tumor cell line which expresses alpha-II-b but not alpha-v (i.e., B 16 amelanotic melanoma) and another cell line (Lewis lung carcinoma) which expresses both the alpha-II-b and alpha-v will be used to compare the relative contribution of each integrin to metastatic potential. The anti-sense constructs against alpha-II-b and/or alpha-v will be introduced into these cell lines in these comparison experiments prior to adhesion, platelet aggregation and lung colony forming assays. In addition, the cytokines TGF-beta, tumor necrosis factor and interleukin 1, as well as the eicosanoid 12-hydroxyeicosatetraenoic acid, which have been demonstrated to regulate the expression of some integrins, will be studied for their effects on transcriptional and/or translational control of alpha- II-b-beta-3 and alpha-v-beta-3 expression and on their functions. Cytokines and/or eicosanoid effect on integrin function may be regulated by phosphorylation or increase in their association with the cellular cytoskeleton. The phosphorylation pattern of alpha-II-b, alpha-v and beta- 3 and the receptor association with cell skeleton will be examined. In this proposal, we will use a comprehensive approach including molecular, biochemical, and cell biological techniques to study the relative contribution of cytoadehesin integrins (i.e., alpha-II-b-beta-3 and alpha- v-beta-3 ) to cellular functions correlated with high metastatic potential.