Characterization of P-selectin Ligands on Carcinomas

描述(由申请人提供)： P-选择素是一种细胞表面糖蛋白，表达于活化的内皮细胞和血小板上，介导宿主细胞与肿瘤细胞的相互作用，与血行转移相关。P-选择素在血源性扩散中直接作用的最令人信服的证据是，与野生型对照相比，P-选择素缺陷小鼠的转移受到显著抑制。因此，识别肿瘤细胞上与P-选择素介导的宿主-肿瘤细胞黏附有关的“功能性”配体，对于了解血源性转移机制具有重要意义，并可能为血源性转移的控制提供新的途径。虽然P-选择素配体已经在肿瘤细胞系上被发现，但它们的功能和生物学意义还没有在动态(非静态)环境中得到证实。正如文献中所讨论的，必须区分在体外静态条件下可以与选择素结合的结构，以及在体内流动条件下确实与选择素相互作用的“功能性”配体。此外，P-选择素与肿瘤细胞上的P-选择素配体结合的详细生物物理特征以前还没有被探索过。在这项应用中，由于结肠癌在美国很常见，也是有血源性扩散倾向的肿瘤之一，因此使用人类结肠癌细胞模型(LS174T)来鉴定、分离P-选择素配体(S)，并对其进行生化和生物物理表征。目的1利用基于流动的黏附实验和高度特异性的酶、抗体和糖偶联物生物合成抑制剂，验证完整的LS174T细胞上的P-选择素配体是一种新的蛋白酶敏感的O-连接唾液酸糖结合物的假说。此外，还将使用分子力探针测定P-选择素与完整LS174T细胞之间的解离速率常数和P-选择素配体键的强度。在目标2中，我们将利用亲和层析结合标准的分子和细胞生物学技术从LS174T细胞中分离出可能的P-选择素配体(S)。目的3鉴定分离配体(S)的生化和生物物理性质，并测试其在流动状态下支持P-选择素介导的结合的能力，以及它与L-和E-选择素的交叉反应。从这些研究中获得的知识将促进新型抗转移药物的设计，并促进我们对P-选择素/配体相互作用在转移中的作用的理解。

DESCRIPTION (provided by applicant): P-selectin, expressed on activated endothelial cells and platelets, is a cell surface glycoprotein which mediates host cell-tumor cell interactions relevant to the process of blood-borne metastasis. The most convincing evidence for a direct role of P-selectin in hematogenous spread is the marked inhibition of metastasis in P-selectin-deficient mice compared to wild-type controls. Thus, identification of "functional" ligands on human tumor cells responsible for P-selectin-mediated host-tumor cell adhesion will be important to the understading of hematogenous metastasis and may offer novel avenues for its control. Although P-selectin ligands have been identified on tumor cell lines, their function and biologic significance have yet to be demonstrated in a dynamic (non-static) setting. As has been argued in the literature, distinctions must be drawn between structures that can bind to selectins under static conditions in vitro, and "functional" ligands that do interact with selectins under flow conditions in vivo. Moreover, the detailed biophysical characteristics of P-selectin binding to P-selectin ligands on tumor cells have not been previously explored. In this application, a human colon carcinoma cell model (LS174T) is used for the identification, isolation, and biochemical and biophysical characterization of the P-selectin ligand(s), since colon cancer is prevalent in the United States and is also among those tumors with a propensity for hematogenous spread. Aim 1 tests the hypothesis that the P-selectin ligand on intact LS174T cells is a novel protease-sensitive, O-linked sialylated glycoconjugate, using flow-based adhesion assays and highly specific enzymes, antibodies and glycoconjugate biosynthesis inhibitors. Moreover, the dissociation rate constant and strength of P-selectin- ligand bonds between P-selectin and intact LS174T cells will be determined using a molecular force probe. In Aim 2, we will isolate the putative P-selectin ligand(s) from LS174T cells using affinity chromatography coupled with standard molecular and cell biology techniques. Aim 3 will characterize the biochemical and biophysical properties of the isolated ligand(s), and test its ability to support P-selectin-mediated binding under flow, as well as its cross-reactivity with L- and E- selectin. Knowledge gained from these studies will advance the design of novel anti-metastatic agents, and our understanding of the roles of P-selectin/ligand interactions in metastasis.