Characterization of P-selectin Ligands on Carcinomas

描述（由申请人提供）： P-选择素表达于活化的内皮细胞和血小板上，是一种细胞表面糖蛋白，介导宿主细胞-肿瘤细胞相互作用，与血液转移过程相关。P-选择素在血行播散中的直接作用的最有说服力的证据是与野生型对照相比，P-选择素缺陷小鼠中的转移的显著抑制。因此，鉴定人肿瘤细胞上负责P-选择素介导的宿主-肿瘤细胞粘附的“功能性”配体对于理解血行转移将是重要的，并可能为其控制提供新的途径。虽然P-选择素配体已被确定在肿瘤细胞系，其功能和生物学意义尚未被证明在一个动态（非静态）设置。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和在体内流动条件下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尚未被探索。在本申请中，人结肠癌细胞模型（LS 174 T）用于P-选择素配体的鉴定、分离和生物化学和生物物理表征，因为结肠癌在美国是普遍的，并且也是具有血行扩散倾向的那些肿瘤之一。目的1使用基于流动的粘附测定和高度特异性的酶、抗体和糖缀合物生物合成抑制剂来检验完整LS 174 T细胞上的P-选择素配体是新型蛋白酶敏感的O-连接唾液酸化糖缀合物的假设。此外，将使用分子力探针测定P-选择素和完整LS 174 T细胞之间的P-选择素-配体键的解离速率常数和强度。在目标2中，我们将使用亲和层析结合标准分子和细胞生物学技术从LS 174 T细胞中分离推定的P-选择素配体。目的3将表征分离的配体的生物化学和生物物理性质，并测试其在流动下支持P-选择素介导的结合的能力，以及其与L-和E-选择素的交叉反应性。从这些研究中获得的知识将促进新型抗转移剂的设计，以及我们对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.