INTEGRIN ACTIVITIES DURING TUMOR PROGRESSION BY GALECTIN-3

该提案旨在确定Galectin-3在乳腺癌和前列腺癌恶性进展过程中整合素功能中的意义。肿瘤的进展通常伴随着转化细胞与细胞外基质蛋白相互作用的变化，如侵袭性和运动性的增加。这些过程受整合素的调节，而整合素又受其他细胞蛋白的调节。整合素与Galectin-3相互作用的能力促使我们质疑这种联系的生物学意义。具体地说，我们感兴趣的是检验这一假设，即Galectin-3的表达对于调节负责细胞与细胞外基质相互作用的整合素的活性是必要的。本研究的目的是：a)确定Galectin-3在细胞内对细胞外基质相互作用的重要性，b)证明Galaectin 3是整合素的关键分子伙伴，调节其功能，c)确定与Galectin-3相关的整合素异二聚体在乳腺癌和前列腺癌中的作用，以及d)分析Galectin3表达和零表达的细胞系(包括那些反义寡核苷酸)与骨和肺细胞外基质的相互作用。反义DNA技术将被用来干扰癌细胞中Galectin-3基因的表达。然后，将在体外监测Galectin-3表达减弱的细胞(反义转染体)以及正义和载体转染组与细胞外基质蛋白的细胞相互作用。预计Galectin-3的缺乏表达将导致这些细胞与细胞外基质蛋白的相互作用减弱。纯化的Galectin-3表达之间的相互作用将导致这些细胞与细胞外基质蛋白的相互作用减弱。纯化的Galectin-3与整合素，特别是α1β1之间的相互作用将通过光散射方法进行分析，以获得结合参数。利用荧光激活的细胞分选技术(FACS)，我们将寻求确定Galectin-3是否改变特定细胞整合素的表达。使用简化的Galectin-3/整合素结合实验，我们将确定Galectin-3如何调节α1β1整合素和层粘连蛋白-1之间的结合作用。我们还将确定Galectin-3生物学功能的改变如何影响其与整合素的相互作用，反之亦然。免疫沉淀方法和激光共聚焦显微镜将被用来鉴定与乳腺和前列腺上皮肿瘤细胞表面的Galectin-3相关的整合素。最后，对Galectin-3表达细胞和空表达细胞与骨细胞外基质和肺组织分泌物的黏附能力进行评价。乳腺癌细胞和前列腺癌细胞都优先转移到骨骼，其次是肺部。这些研究将为控制转移性乳腺和前列腺癌细胞的扩散和生长提供新的途径。

The proposal seeks to establish the significance of galectin-3 in integrin functions during malignant progression of breast and prostate carcinomas. The progression of tumors if normally accompanied by the alterations in the interactions of the transformed cells with extracellular matrix proteins, such as increased invasiveness and motility. These processes are regulated by integrins, which are in turn modulated by other cellular proteins. The ability of integrins to interact with galectin-3 prompted us to question the biological significance of this association. Specifically, we are interested in testing the hypothesis that the expression of galectin-3 is necessary for the modulation of the activities of integrins responsible for cell to extracellular matrix interactions. The study is designed to; a) determine the importance of galectin-3 in cell to extracellular matrix interactions, b) demonstrate that galaectin3 is a critical molecular partner of integrins which regulate their functions, c) to identify integrin heterodimers which associate with galectin-3 in the breast and prostate carcinomas and d) to analyze the interaction of galectin3- expressing and null expressing cell lines including those transfected with antisense oligos, with bone and lung extracellular matrices. Antisense DNA technologies will be employed to disrupt galectin-3 gene expression in the carcinoma cells. The interaction of cells with the diminished galectin- 3 expression (antisense transfectants) as well as sense and vector transfected controls with extracellular matrix proteins will then be monitored in vitro. It is expected that lack of galectin-3 expression will result in diminished interaction of these cells with the extracellular matrix proteins. The interactions between purified galectin-3 expression will result in diminished interaction of these cells with the extracellular matrix proteins. The interaction between purified galectin-3 and integrins, particularly alpha 1 beta1 will be analyzed by light scattering methodology to obtain binding parameters. Using fluorescence activated cell sorting techniques (FACS(, we will seek to determine whether galectin-3 alters the expression of specific cellular integrins. Using a simplified galectin-3/integrin binding assay, we will determine how galectin-3 modulates the binding interaction between alpha1 beta1 integrins and laminin-1. We will also determine how the modifications of the biological functions of galectin-3 affects its interaction with integrins and vice vera. Immunoprecipitation methodology and confocal laser microscopy will be used to identify integrins which are associated with galectin-3 on the surfaces of the breast and prostate epithelial tumor cells. Lastly, the adhesion potential of galectin-3 expressing and null expressing cells to bone extracellular matrix and to lung tissue secretions will be evaluated. Both breast and prostate carcinoma cells preferentially metastasize to the bones and to a lesser extent, the lungs. These studies will provide new avenues which may be exploited for the control of the dissemination and growth of metastatic breast and prostate tumor cells.