CHARACTERIZATION OF HUMAN CARCINOMA T & TN AUTOANTIGENS

人类癌症的特征

基本信息

批准号：
3165842
负责人：
GEORG F SPRINGER
金额：
$ 13.27万
依托单位：
ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-06-01 至 1993-03-31
项目状态：
已结题

项目摘要

Immunoreactive Thomsen-Friedenreich (T) antigen and its precursor, Tn, occur in approximately 90% of all human (adeno) carcinomas (CAs) and in their metastases. In nonCA tissues, these antigens are occluded and inaccessible to the immune system. T & Tn are the immediate precursors of blood type M,N antigens. T & Tn are the first universal CA-diagnostic markers; they are adhesion molecules and persistent autoantigens, from incipience onward; T & Tn's surface densities are often prognostic. Our proposed structures of the T & Tn epitopes based on stepwise degradation and biosynthesis were later confirmed by organic synthesis. The focus will be on Stage I ductal breast CAs. We plan: Study of fresh surgical specimens of primary, preferably Stage I, CAs and tissue-cultured CAs plus control tissues. Full characterization of CA-T & -Tn's structure, location and integration in carrier molecules, macromolecular conformation and distribution in CA. To define extent of T & Tn epitope-clustering, and to determine I & Tn epitopes' interrelationship in CA using metabolically labeled breast CA cells. Immunohistochemical evaluation (peroxidase, gold- silver) with light (ultra)microscopy of spatial distribution of T & Tn epitopes with our anti-T & -Tn rodent monoclonal (Mo) and human monospecific, polyclonal antibodies (Abs). Quantitation of T & Tn epitopes on breast CA cells and on control tissues by RIA and by computerized image analysis, relating T & Tn densities of CA to their ploidy, and correlation of T & Tn densities to invasiveness and autoimmunogenicity of different breast CAs. Lectin and Ab affinity chromatography (C) to separate CA glycoproteins based on their predominant Tn, T and N & M epitopes. Purification of T- & Tn-active glycopeptides obtained by pronase digestion and their reductively beta-eliminated oligosaccharides (OS) by gel filtration, ion-exchange & high-performance liquid C. To determine OS composition by hydrolysis, enzymatically, colorimetrically and by gas C (GC) of alditol acetates, and structures using glycosidases, permethylation, GC-mass spectrometry (GC/MS) & NMR. Amino acid sequencing will be by subtractive Edman degradation. To isolate guinea pig L-lO hepatoCA Tn-, T- and N- & M-active glycolipids (GLs) on s large scale, and define the carbohydrate structures of the T- & Tn-specific GLs. Study of human CAs and lymphomas for expression and nature of T- & Tn-active GLs; relate findings to degree of malignancy. Immunostaining with our MoAbs to locate and identify active GLs. To analyze OS of active L-lO GLs by GC/MS after trifluoroacetolysis, reduction and permethylation.

免疫反应性 Thomsen-Friedenreich (T) 抗原及其前体， Tn，出现在大约 90% 的人类（腺）癌中（CA）及其转移。在非 CA 组织中，这些抗原被封闭并且免疫系统无法进入。 T 和 Tn 是 M、N 型血抗原的直接前体。 T 和 Tn 是第一个通用 CA 诊断标记；它们是粘附分子以及从一开始就持续存在的自身抗原； T & Tn 的表面密度通常具有预测意义。我们建议的结构基于逐步降解和生物合成的 T 和 Tn 表位后来通过有机合成得到证实。重点将放在 I 期导管乳腺 CA。我们计划：研究新鲜手术原代标本，最好是 I 期 CA 标本和组织培养标本 CA 加上对照组织。 CA-T 和 -Tn 的完整表征载体分子的结构、位置和整合， CA 中的大分子构象和分布。定义 T 和 Tn 表位聚类的程度，并确定 I 和 Tn 使用代谢标记的 CA 中表位的相互关系乳腺 CA 细胞。免疫组织化学评估（过氧化物酶、金- 银）用光（超）显微镜观察 T 的空间分布 & Tn 表位与我们的抗 T & -Tn 啮齿动物单克隆 (Mo) 和人类单特异性多克隆抗体 (Abs)。定量通过 RIA 检测乳腺 CA 细胞和对照组织上的 T 和 Tn 表位并通过计算机图像分析，将 T 和 Tn 密度关联起来 CA 与其倍性，以及 T 和 Tn 密度与不同乳腺CA的侵袭性和自身免疫原性。凝集素和抗体亲和层析 (C) 分离 CA 基于其主要 Tn、T 和 N & M 表位的糖蛋白。通过链霉蛋白酶纯化 T 和 Tn 活性糖肽消化及其还原性β-消除寡糖 (OS) 通过凝胶过滤、离子交换和高性能液体 C. 通过水解、酶法测定 OS 组成，糖醇乙酸酯的比色法和气体 C (GC)，以及使用糖苷酶、全甲基化、GC-质谱分析结构 (GC/MS) 和核磁共振。氨基酸测序将通过消减埃德曼进行降解。分离豚鼠L-10肝CA Tn-、T-和N-& 大规模的 M-活性糖脂 (GL)，并定义 T 和 Tn 特异性 GL 的碳水化合物结构。研究人类 CA 和淋巴瘤中 T 和 Tn 活性的表达和性质 GL；将发现结果与恶性程度联系起来。免疫染色用我们的 MoAb 可以定位和识别活性 GL。分析操作系统三氟乙酰分解、还原和后通过 GC/MS 检测活性 L-10 GL 全甲基化。