ISOLATION AND CHARACTERIZATION OF GENES CODING FOR CARCINOMA-ASSOCIATED ANTIGENS

癌相关抗原基因编码的分离和表征

• 批准号: 3813405
• 负责人: J KANTOR
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3813405
• 关键词: DNA; DNA methylation; antitumor antibody; breast neoplasms; carcinoembryonal antigen; carcinoma; colon neoplasms; complementary DNA; electrophoresis; gene expression; genetic library; genetic transcription; glycoproteins; immunoglobulin genes; interferons; molecular cloning; monoclonal antibody; mucins; neoplasm /cancer immunodiagnosis; neoplasm /cancer immunology; neoplastic transformation; nucleic acid hybridization; nucleic acid probes; proline; serine; threonine; tissue /cell culture; transfection; tumor antigens

Monoclonal antibodies (MAbs) have defined several antigens associated with human carcinomas. Two of the most widely studied antigens are carcinoembryonic antigen (CEA) and TAG-72. CEA is a 180,000-kd glycoprotein and TAG-72 is a high- molecular-weight mucin. Recent studies have demonstrated that CEA belongs to a multigene family related to the immunoglobulin supergene family. These include CEA, normal cross-reacting antigen (NCA), biliary glycoprotein (BGP), and human pregnancy-specific beta 1-glycoprotein (SP1). A 550-base pair CEA probe derived from cloned complementary DNA was used to carry out Southern analysis of the DNA isolated from normal and colon tumor cell lines. At high stringency, the CEA probe detected seven BamHI fragments in all DNAs analyzed. Results of the Southern analysis demonstrate no amplification or rearrangement of the CEA genes in tumor cells. Methylation-sensitive restriction endonucleases, Hpa(II) and Hha(I), were used to compare the degrees of methylation of the CEA family of genes in normal and colon tumor cell lines. The results demonstrate that the CEA family of genes exists in a state of hypermethylation in normal cells. In contrast, the CEA gene(s) are relatively hypomethylated in the tumor cell lines, suggesting a correlation between the state of methylation and degree of expression of the CEA gene(s). Using a CEA cDNA probe, we have investigated the phenomenon of up-regulation of CEA expression in human tumor cell lines by recombinant human gamma-interferon (Hu-IFN-gamma). An increase in MAb binding was shown to be accompanied by a 6- to 11-fold increase in the steady-state levels of three CEA transcripts with sizes of 4.2, 3.5, and 2.8 kb. Studies are also in progress to identify and characterize the gene(s) encoding the TAG-72 mucin antigen. A number of mucin genes representing several tissue types have been cloned. These include the genes encoding the epitopes for breast cancer, HMFG2, DF3 and H23 and the pancreatic antigen DuPAN-2. Nucleic acid sequence analysis of these isolated genes has shown them to be identical and that the difference resides in the sugar epitopes detected by the MAb. The dominant feature of the breast and pancreatic mucin clones was an extremely GC-rich, 60-bp tandem repeat sequence that encoded a repeating peptide unit rich in serine, threonine, and proline.

单克隆抗体（MAb）已经定义了几种与 人类癌研究最广泛的两种抗原是 癌胚抗原（CEA）和TAG-72。CEA是一种180，000 kd的糖蛋白 TAG-72是高分子量粘蛋白。最近的研究 表明CEA属于一个多基因家族， 免疫球蛋白超基因家族这些包括CEA，正常的交叉反应 抗原（NCA）、胆汁糖蛋白（BGP）和人妊娠特异性 β 1-糖蛋白（SP1）。一个550碱基对的CEA探针， 使用互补DNA进行DNA的Southern分析 分离自正常和结肠肿瘤细胞系。在高严格性下， CEA探针在分析的所有DNA中检测到7个BamHI片段。结果 Southern分析表明，没有扩增或重排的 肿瘤细胞中的CEA基因。甲基化敏感性限制性内切核酸酶， Hpa（II）和Hha（I）用于比较甲基化的程度。 正常和结肠肿瘤细胞系中的CEA基因家族。结果 证明CEA家族基因存在于 在正常细胞中的甲基化。相反，CEA基因是 在肿瘤细胞系中相对低甲基化，这表明 甲基化状态和癌胚抗原表达程度之间 基因。利用CEA cDNA探针，我们研究了 重组人肝癌细胞系中癌胚抗原表达的上调 人γ-干扰素（Hu-IFN-γ）。显示MAb结合增加 伴随着稳态水平的6至11倍增加， 三种CEA转录物，大小分别为4.2、3.5和2.8 kb。研究也 正在鉴定和表征编码TAG-72的基因 粘蛋白抗原一些代表几种组织类型的粘蛋白基因 被克隆了。这些包括编码乳腺癌抗原决定簇的基因， 癌症、HMFG 2、DF 3和H23以及胰腺抗原DuPAN-2。核酸 对这些分离基因的序列分析表明它们是相同的 差异在于单克隆抗体检测到的糖表位。 乳腺和胰腺粘蛋白克隆的主要特征是 一个富含GC的60 bp串联重复序列，编码一个重复的 富含丝氨酸、苏氨酸和脯氨酸的肽单位。