Mechanisms of thymidine phosphorylase angiogenesis

申请者提供的描述)：对 血管生成在实体瘤生长和转移中的作用 提示抗血管生成治疗可能是一种可行的治疗方法。 晚期疾病。胸苷磷酸化酶(TP)最近被发现是 一种血管生成因子，这是一种基于其与 先前描述的血管生成因子，血小板衍生的内皮细胞 生长因子(PD-ECGF)。TP在许多人体固体中高水平表达 肿瘤，包括结直肠癌，其表达水平一直以来 在许多临床研究中，包括我们进行的一项研究中，与 新生血管、肿瘤侵袭性和患者预后不良。 我们等人对TP的免疫组织化学评估表明 TP在许多(但不是所有)结肠肿瘤中的表达升高主要发生在 肿瘤相关巨噬细胞(TAM)，而不是在肿瘤细胞中。我们有 发现人类单核细胞系(THP1)表达更高水平的TP 比人结肠癌细胞系更强。我们还发现，肿瘤坏死因子-α刺激了 TP在THP1细胞和WiDR结肠癌细胞中的表达，我们将使用 这两个细胞系以及TAMs的原代培养以确定 调控TP表达的分子基础(即转录， 转录后)。茶多酚的血管生成活性似乎依赖于其 以2-D-脱氧核糖(2dR)为介体的催化活性 血管内皮细胞的激活。我们建立了一个体外模型，在这个模型中 人类癌细胞或单核细胞可以与正常人共培养 内皮细胞(HUVEC)，并可诱导其在TP依赖的情况下迁移 举止。我们将使用这个模型来开始确定TP 刺激HUVEC和人微血管内皮细胞(HMEC)迁移， 使用胸腺嘧啶核苷和2dR的类似物，我们将合成来验证这一假设 这种迁移只与2dR的形成有关，并通过使用TP抑制剂 我们已经制作了一种中和TP抗体来检验TP的假设 细胞内的作用足以刺激HUVEC迁移。一点儿 进一步了解茶多酚的作用机制。我们的研究表明 介导TP和VEGF诱导的人脐静脉内皮细胞的特异性整合素的差异 迁移是第一个探索TP对内皮细胞作用的人 细胞。我们还发现TP诱导FAK的酪氨酸磷酸化 (粘着斑激酶)，一种在细胞中起中心作用的蛋白质 连接、迁移和信令。我们将继续研究，以确定 茶多酚激活的整合素相关信号转导通路 在HUVEC和HMEC中，检验这些最初不同于 血管生成因子如血管内皮生长因子和碱性成纤维细胞生长因子的作用。 有明确定义的细胞表面受体。除了提供基本的 对TP结肠癌血管生成作用机制的了解 研究可以为优化利用当前和未来的 以整合素为靶点的抗血管生成药物。

DESCRIPTION provided by applicant): There is an increasing appreciation of the role of angiogenesis in the growth and metastasis of solid tumors, and data suggest that antiangiogenesis therapy could be a viable approach to treatment of advanced disease. Thymidine phosphorylase (TP) has been recently found to be an angiogenic factor, an observation that was based on its identity to a previously described angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF). TP is expressed at high levels in many human solid tumors including colorectal cancers, and the level of its expression has been correlated in numerous clinical studies, including one we conducted, with neovascularization, tumor aggressiveness, and poor patient prognosis. Immunohistochemical evaluation of TP by us and others has demonstrated that elevated TP expression in many, but not all, colon tumors occurs mainly in tumor-associated macrophages (TAMs), rather than in the tumor cells. We have found that a human monocytic cell line (THP1) expressed a higher level of TP than human colon carcinoma cell lines. We also found that TNF-alpha stimulated TP expression in THP1 cells and in WiDr colon carcinoma cells, and we will use these two cell lines as well as primary cultures of TAMs to determine the molecular basis for the regulation of TP expression (i.e. transcriptional, post-transcriptional). TP's angiogenic activity appears to be dependent on its catalytic activity, with 2-D-deoxyribose (2dR) serving as the putative mediator of endothelial cell activation. We have established an in vitro model in which human cancer cells or monocytes can be co-cultured with normal human endothelial cells (HUVEC), and can induce their migration in a TP-dependent manner. We will use this model to begin to determine the mechanisms by which TP stimulates HUVEC and human microvascular endothelial cell (HMEC) migration, using analogs of thymidine and 2dR we will synthesize to test the hypothesis that migration is solely related to 2dR formation, and by using a TP inhibitor we have made and a neutralizing TP antibody to test the hypothesis that TP's intracellular actions are sufficient to stimulate HUVEC migration. Little further is known of the mechanism of action of TP. Our studies showing differences in the specific integrins which mediate TP vs. VEGF-induced HUVEC migration are the first to explore the cellular actions of TP on endothelial cells. We have also found that TP induces tyrosine phosphorylation of FAK (focal adhesion kinase), a protein that plays a central role in cell attachment, migration, and signaling. We will continue our studies to define the integrin-associated signal transduction pathways that are activated by TP in HUVEC and HMEC, testing the hypothesis that these differ initially from those occurring in response to angiogenic factors such as VEGF and bFGF, which have clearly defined cell-surface receptors. In addition to providing a basic understanding of the mechanisms of angiogenic action of TP colon cancers, these studies could provide insights to optimize the use of current and future anti-angiogenic agents which target integrins.