A GENERAL GENETIC APPROACH IN ESCHERICHIA-COLI FOR DETERMINING THE MECHANISM(S) OF ACTION OF TUMORICIDAL AGENTS - APPLICATION TO DMP-840, ATUMORICIDAL AGENT

We describe here a simple and easily manipulatable Escherichia coli-ba sed 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 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 cyt otoxic effects of drugs. A tolC bacterial mutant is used to maximize p ermeability of cells to hydrophobic organic molecules. By using DMP 84 0 to model the system, we have identified two genes, designated mdaA a nd mdaB, that impart resistance to DMP 840 when they are expressed at elevated levels. mdaB maps to E. coli map coordinate 66, is located be tween the parE and parC genes, and encodes a protein of 22 kDa. mdaA m aps to E. coli map coordinate 18, is located adjacent to the glutaredo xin (grx) gene, and encodes a protein of 24 kDa. Specific and regulata ble overproduction of both of these proteins correlates with DMP 840 r esistance. Overproduction of the MdaB protein also imparts resistance to two mammalian topoisomerase inhibitors, Adriamycin and etoposide. I n contrast, overproduction of the MdaA protein produces resistance onl y to Adriamycin. Based on its drug-resistance properties and its locat ion between genes that encode the two subunits of the bacterial topois omerase IV, we suggest that mdaB acts by modulating topoisomerase IV a ctivity. The location of the mdaA gene adjacent to grx suggests it act s by a drug detoxification mechanism.