INTEGRIN ACTIVITIES DURING TUMOR PROGRESSION BY GALECTIN-3

该提案旨在确定在乳腺癌和前列腺癌恶性进展过程中半乳糖凝集素-3在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改变，如侵袭性和运动性增加。这些过程由整合素调节，而整合素又由其他细胞蛋白调节。整合素与半乳糖凝集素-3相互作用的能力促使我们质疑这种关联的生物学意义。具体来说，我们感兴趣的是验证一种假设，即半乳糖凝集素-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。这项研究的目的是；A)确定细胞中半乳糖凝集素-3对细胞外基质相互作用的重要性，b)证明半乳糖凝集素-3是调节其功能的整合素的关键分子伙伴，c)鉴定在乳腺癌和前列腺癌中与半乳糖凝集素-3相关的整合素异源二聚体，d)分析半乳糖凝集素-3表达和不表达的细胞系（包括转染反义寡核苷酸的细胞系）与骨和肺细胞外基质的相互作用。反义DNA技术将被用于破坏癌细胞中半乳糖凝集素-3基因的表达。然后在体外监测细胞与凝集素- 3表达减少（反义转染）以及细胞外基质蛋白转染的意义和载体对照的相互作用。预计缺乏半乳糖凝集素-3的表达将导致这些细胞与细胞外基质蛋白的相互作用减少。纯化的半乳糖凝集素-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少。纯化的半乳糖凝集素-3和整合素之间的相互作用，特别是α 1 β a1将通过光散射方法分析，以获得结合参数。利用荧光活化细胞分选技术（FACS），我们将试图确定半凝集素-3是否会改变特定细胞整合素的表达。使用简化的半乳糖凝集素-3/整合素结合试验，我们将确定半乳糖凝集素-3如何调节α 1 β 1整合素和层粘连蛋白-1之间的结合相互作用。我们还将确定半乳糖凝集素-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。免疫沉淀法和共聚焦激光显微镜将用于鉴定与乳腺和前列腺上皮肿瘤细胞表面的半凝集素-3相关的整合素。最后，我们将评估半乳糖凝集素-3表达细胞和无表达细胞对骨细胞外基质和肺组织分泌物的粘附潜力。乳腺癌和前列腺癌细胞都倾向于转移到骨骼，在较小程度上转移到肺部。这些研究将为控制转移性乳腺和前列腺肿瘤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.