Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe here a simple and easily manipulatable Escherichia coli-based genetic system that permits us to identify bacterial gene products that modulate the sensitivity of bacteria to tumoricidal agents, such as DMP 840, a bisnaphthalimide drug. To the extent that the action of these agents is conserved, these studies may expand our understanding agents is conserved, these studies may expand our understanding of how the agents work in mammalian cells. The approach briefly is to use a library of E. coli genes that are overexpressed in a high copy number vector to select bacterial clones that are resistant to the cytotoxic effects of drugs. AtolC bacterial mutant is used to maximize permeability of cells to hydrophobic organic molecules. By using DMP 840 to model the system, we have identified two genes, designated mdaA and mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located between the parE and parC genes, and encodes a protein of 22 kDa. mdaA maps to E. coli map coordinate 18, is located adjacent to the glutaredoxin (grx) gene, and encodes a protein of 24 kDa. Specific and regulatable overproduction of both of these proteins correlates with DMP 840 resistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. In contrast, overproduction of the MdaA protein produces resistance only to Adriamycin. Based on its drug-resistance properties and its location between genes that encode the two subunits of the bacterial topoisomerase IV, we suggest that mdaB acts by modulating topoisomerase IV activity. The location of the mdaA gene adjacent to grx suggests it acts by a drug detoxification mechanism.
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PMID:A general genetic approach in Escherichia coli for determining the mechanism(s) of action of tumoricidal agents: application to DMP 840, a tumoricidal agent. 756 50

DMP 840 ((R,R)-2,2'-[1,2-ethanediylbis[imino(1-methyl-2, 1-ethanediyl)]-bis(5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione] dimethanesulfonate) is a novel bis(naphthalimide) that has shown promising antitumor activity in a variety of preclinical model systems. The compound binds to DNA with high affinity and intercalates, but the mechanism of cell killing has not been elucidated. We have used yeast strains to test whether DMP-840 is active against either topoisomerase I or II. We found that temperature-sensitive top2 mutants resistant to etoposide or amsacrine also confer resistance to DMP-840. In addition, cells overexpressing yeast topoisomerase II were hypersensitive to the drug. By contrast, top1 deletions rendered cells hypersensitive to the drug. These results strongly suggest that DMP-840 acts against eukaryotic topoisomerase II and kills cells by converting the enzyme into a cellular poison. We verified that DMP-840 is active against eukaryotic topoisomerase II by demonstrating that the drug stimulates formation of a cleavage complex with purified yeast topoisomerase II in vitro. We also demonstrated that the drug is active against human topoisomerase II by showing that expression of human topoisomerase II restored sensitivity of resistant yeast cells to DMP-840. We have also directly demonstrated that DMP-840 acts as a poison against purified human topoisomerase II alpha. Taken together, these results indicate that DMP-840 acts like other intercalating topoisomerase II poisons; it kills eukaryotic cells by stabilizing the cleavage complex of topoisomerase II with DNA.
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PMID:The bis(naphthalimide) DMP-840 causes cytotoxicity by its action against eukaryotic topoisomerase II. 948 61