MORPHOLOGICAL MECHANISMS OF ORGANELLE FUNCTION AND TRANSFORMATION IN CULTURE CELL

培养细胞中细胞器功能和转化的形态机制

• 批准号: 3916303
• 负责人: M C WILLINGHAM
• 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/3916303
• 关键词: antigen antibody reaction; antitumor antibody; cellular oncology; clathrin; daunorubicin; drug resistance; electron microscopy; exocytosis; gel electrophoresis; histochemistry /cytochemistry; hormone binding protein; human subject; hybridomas; immunochemistry; immunoconjugates; immunocytochemistry; immunofluorescence technique; immunoperoxidase; membrane proteins; microinjections; microscopy; monoclonal antibody; morphology; neoplasm /cancer immunotherapy; neoplastic transformation; oncoproteins; organelles; ovary neoplasms; phosphatidylcholines; protein biosynthesis; protein kinase C; tissue /cell culture; transposon /insertion element; tumor antigens

Neoplastic transformation produces many changes in cell physiology, some of which can be studied using morphologic techniques. We have used morphologic methods to study three general areas: 1) We have used light microscopic cytochemical methods to isolate and characterize monoclonal antibodies to antigens present on ovarian cancer cells for use in diagnosis and in the construction of immunotoxins for therapy of human cancer. We had previously isolated OVB3, an antibody currently being evaluated in phase I clinical trials as an immunotoxin; the isolation of similar tumor- specific antibodies is an on-going project. Technical improvements have streamlined the morphologic hybridoma screening processing time to less than two days, allowing primary screening on cultured cells and secondary screening on tissue samples with the same initial 100 mu L supernatant sample. 2) We have studied aspects of multidrug-resistance of human tumors in cultured cells and in tissues. We have localized P170, the product of the human MDR1 gene, in normal tissues using monoclonal antibody MRK16, and in addition to its location in the GI tract, adrenal, kidney and liver, we have also found it in capillaries in the central nervous system. We have studied the reactivity of monoclonal antibody C219 that reacts with an epitope of P170 present on the inner surface of the plasma membrane, and found that this antibody cross-reacts with other proteins in some tissues, such as the heavy chain of cardiac and slow-twitch muscle myosin, in human, monkey, and rat tissues. We have also examined the expression of human P170 introduced through a viral vector in MDCK cells and found that P170 is expressed in a polarized fashion on the apical surface of these cells, similar to its distribution in normal kidney proximal tubules. 3) In other studies, we have examined the effects of microinjected protein kinase C on cell morphology; we have localized the E5 oncogene product from papilloma virus in cultured cells; we have also localized monoclonal antibodies of nuclear pore carbohydrate epitopes using immunocytochemistry and microinjected lectins; and we have localized P58, a major thyroid hormone binding protein, to the cytosol of rapidly growing cells.

肿瘤转化会导致细胞生理上的许多变化， 其中一些可以用形态学技术来研究。我们有 使用形态学方法研究了三个一般领域：1)我们有 用光镜细胞化学方法分离和鉴定 抗卵巢抗原的单抗的特性 用于诊断和构建的癌细胞 用于治疗人类癌症的免疫毒素。我们之前已经 分离的OVB3，一种目前处于I期评估的抗体 临床试验作为一种免疫毒素；类似肿瘤的分离- 特定的抗体是一个正在进行的项目。技术改进 简化了形态杂交瘤的筛选过程 时间不到两天，允许对培养物进行初步筛查 细胞及其在组织样本上的二次筛选 最初的百亩L上清液样本。2)我们研究了几个方面 体外培养的人肿瘤多药耐药的研究 纸巾。我们已经本地化了P170，这是人类MDR1的产物 基因，在正常组织中使用单抗MRK16，并在 除了它在胃肠道、肾上腺、肾脏和 肝脏，我们也在中枢神经的毛细血管中发现了它 系统。我们研究了单抗C219的反应性。 它与存在于内表面的P170表位发生反应 发现这种抗体会发生交叉反应 与某些组织中的其他蛋白质结合，如重链 人、猴和大鼠的心肌和慢收缩肌球蛋白 纸巾。我们还检测了人p170的表达。 通过病毒载体导入MDCK细胞，发现p170 在这些细胞的顶端表面以极化的方式表达 细胞，与其在正常肾脏近端的分布相似 小管。3)在其他研究中，我们检查了 显微注射蛋白激酶C对细胞形态的影响；我们有 人乳头瘤病毒E5癌基因产物在培养细胞中的定位 细胞；我们还定位了核孔的单抗 应用免疫细胞化学和显微注射技术研究碳水化合物表位 凝集素；我们已经定位了P58，一种主要的甲状腺激素结合 蛋白质，进入快速生长的细胞的胞浆。