Development of screening systems for new anti-tumor agents using antibiotic-binding proteins and sensor-promoters

使用抗生素结合蛋白和传感器启动子开发新型抗肿瘤药物的筛选系统

基本信息

批准号：
07556093
负责人：
SUGIYAMA Masanori
金额：
$ 1.22万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Bleomycin (Bm) has been used in the combination chemotherapy based treatment of carcinoma. But its use is very much limited bccause of its untoward effects of pulmonary toxicity. So, new Bm analogues without htese side effects could be potentially indispensable in the treatment of carcinoma. We constructed a promoter-probe vector, designated pMX180, carrying a Streptomyces tyrosinase genc as a reporter gene. The present study showed that Escherichia coli harboring a plasmid pMX180tac, generated by insertion of tac promoter into pMX180, produced melanin pigment mediated by tyrosinase, when inhibitors against DNA synthesis were added to the culture medium. The Bm family of antibiotics such as pepleomycin, liblomycin and phleomycin were also effective in producing the melanin pigment. Mitomycin C was most effective in the pigment production. Nalidixic acid and azidothymidine also significantly induced the synthesis of melanin pigment. However, some inhibitors of protein and cell wall syntheses did not induce the synthesis of melanin pigment. The culture broth from Bm-producingS.verticillus induced melanin sythesis in this E.coli harboring pMX180tac, suggesting that this plasmied enables visual detection of inhibitors of nucleic acid synthesis that might be used as potent antitumor agents. The vector that we have made enables new Bm analogues to be screened directly in the culture of microorganisms that produce similar anticancer compounds, This system of screeing new Bm analogues, in a very simple, cost effective and time efficient way, would be of great importance in finding a new drug without the hazardous side effects. We call pMX180tac as an intelligent vector. The vector may have application in the efficient screening of microorganism that produce DNA synthesis inhibitors.

博来霉素（Bm）已用于基于联合化疗的癌症治疗。但由于其肺毒性的不良影响，其用途受到很大限制。因此，没有这些副作用的新型 Bm 类似物在癌症治疗中可能是不可或缺的。我们构建了一个启动子探针载体，命名为 pMX180，携带链霉菌酪氨酸酶基因作为报告基因。本研究表明，当向培养基中添加DNA合成抑制剂时，携带质粒pMX180tac（通过将tac启动子插入pMX180而产生）的大肠杆菌在酪氨酸酶介导下产生黑色素。 Bm 家族抗生素，如百霉素、利布霉素和腐草霉素，也能有效产生黑色素。丝裂霉素 C 在色素生产中最有效。萘啶酸和叠氮胸苷也显着诱导黑色素的合成。然而，一些蛋白质和细胞壁合成抑制剂并不诱导黑色素的合成。产 Bm 轮枝菌的培养液在含有 pMX180tac 的大肠杆菌中诱导黑色素合成，表明这种质粒能够目视检测可能用作有效抗肿瘤剂的核酸合成抑制剂。我们制作的载体使得能够在产生类似抗癌化合物的微生物培养物中直接筛选新的 Bm 类似物。这种以非常简单、成本有效且省时的方式筛选新 Bm 类似物的系统将非常重要寻找一种没有危险副作用的新药。我们将 pMX180tac 称为智能载体。该载体可用于有效筛选产生DNA合成抑制剂的微生物。