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

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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.