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

博来霉素（Bm）已被用于以联合化疗为基础的肿瘤治疗。但由于其肺毒性的不良反应，其应用受到很大限制。因此，寻找新的无毒副作用的Bm类似物在肿瘤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pMX 180，携带链霉菌酪氨酸酶基因作为报告基因。本研究表明，大肠杆菌携带质粒pMX180 tac，通过插入tac启动子到pMX180产生的，产生黑色素的酪氨酸酶介导的，当对DNA合成的抑制剂添加到培养基中。Bm家族的抗生素如培洛霉素、利布霉素和腐草霉素也有效地产生黑色素。丝裂霉素C在色素产生中最有效。萘啶酸和叠氮胸苷也显著诱导黑色素的合成。然而，一些蛋白质和细胞壁合成的抑制剂不诱导黑色素的合成。产Bm的轮状芽孢杆菌的培养液在携带pMX180 tac的大肠杆菌中诱导黑色素合成，这表明该质粒能够视觉检测可能用作有效抗肿瘤剂的核酸合成抑制剂。我们所做的载体使新的Bm类似物能够直接在产生类似抗癌化合物的微生物培养物中筛选。这种筛选新Bm类似物的系统，以一种非常简单、成本有效和时间有效的方式，对于寻找没有危险副作用的新药具有重要意义。我们称pMX180tac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。

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.