Abstract
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.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Austin E. A., Graves J. F., Hite L. A., Parker C. T., Schnaitman C. A. Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus. J Bacteriol. 1990 Sep;172(9):5312–5325. doi: 10.1128/jb.172.9.5312-5325.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bugg B. Y., Danks M. K., Beck W. T., Suttle D. P. Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7654–7658. doi: 10.1073/pnas.88.17.7654. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chatterjee P. K., Sternberg N. L. Using cell-fractionation and photochemical crosslinking methods to determine the cellular binding site(s) of the antitumor drug DMP 840. Photochem Photobiol. 1995 Apr;61(4):360–366. doi: 10.1111/j.1751-1097.1995.tb08623.x. [DOI] [PubMed] [Google Scholar]
- Chu G. Cellular responses to cisplatin. The roles of DNA-binding proteins and DNA repair. J Biol Chem. 1994 Jan 14;269(2):787–790. [PubMed] [Google Scholar]
- Cohen G., Sternberg N. Genetic analysis of the lytic replicon of bacteriophage P1. I. Isolation and partial characterization. J Mol Biol. 1989 May 5;207(1):99–109. doi: 10.1016/0022-2836(89)90443-9. [DOI] [PubMed] [Google Scholar]
- Gottesman M. M., Pastan I. The multidrug transporter, a double-edged sword. J Biol Chem. 1988 Sep 5;263(25):12163–12166. [PubMed] [Google Scholar]
- Gupta R. S., Gupta R., Eng B., Lock R. B., Ross W. E., Hertzberg R. P., Caranfa M. J., Johnson R. K. Camptothecin-resistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase I. Cancer Res. 1988 Nov 15;48(22):6404–6410. [PubMed] [Google Scholar]
- Guzman L. M., Belin D., Carson M. J., Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121–4130. doi: 10.1128/jb.177.14.4121-4130.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hosking L. K., Whelan R. D., Shellard S. A., Bedford P., Hill B. T. An evaluation of the role of glutathione and its associated enzymes in the expression of differential sensitivities to antitumour agents shown by a range of human tumour cell lines. Biochem Pharmacol. 1990 Oct 15;40(8):1833–1842. doi: 10.1016/0006-2952(90)90364-q. [DOI] [PubMed] [Google Scholar]
- Kapp L. N., Painter R. B. Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells. Int J Radiat Biol. 1989 Nov;56(5):667–675. doi: 10.1080/09553008914551891. [DOI] [PubMed] [Google Scholar]
- Kato J., Nishimura Y., Imamura R., Niki H., Hiraga S., Suzuki H. New topoisomerase essential for chromosome segregation in E. coli. Cell. 1990 Oct 19;63(2):393–404. doi: 10.1016/0092-8674(90)90172-b. [DOI] [PubMed] [Google Scholar]
- Kirshenbaum M. R., Chen S. F., Behrens C. H., Papp L. M., Stafford M. M., Sun J. H., Behrens D. L., Fredericks J. R., Polkus S. T., Sipple P. (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 (DMP 840), a novel bis-naphthalimide with potent nonselective tumoricidal activity in vitro. Cancer Res. 1994 Apr 15;54(8):2199–2206. [PubMed] [Google Scholar]
- Kröger M., Wahl R., Rice P. Compilation of DNA sequences of Escherichia coli (update 1993). Nucleic Acids Res. 1993 Jul 1;21(13):2973–3000. doi: 10.1093/nar/21.13.2973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lambert B., Segal-Bendirdjian E., Esnault C., Le Pecq J. B., Roques B. P., Jones B., Yeung A. T. Recognition by the DNA repair system of DNA structural alterations induced by reversible drug-DNA interactions. Anticancer Drug Des. 1990 Feb;5(1):43–53. [PubMed] [Google Scholar]
- Lee M. S., Wang J. C., Beran M. Two independent amsacrine-resistant human myeloid leukemia cell lines share an identical point mutation in the 170 kDa form of human topoisomerase II. J Mol Biol. 1992 Feb 20;223(4):837–843. doi: 10.1016/0022-2836(92)90245-f. [DOI] [PubMed] [Google Scholar]
- Liu Y. X., Hsiung Y., Jannatipour M., Yeh Y., Nitiss J. L. Yeast topoisomerase II mutants resistant to anti-topoisomerase agents: identification and characterization of new yeast topoisomerase II mutants selected for resistance to etoposide. Cancer Res. 1994 Jun 1;54(11):2943–2951. [PubMed] [Google Scholar]
- Lomovskaya O., Lewis K. Emr, an Escherichia coli locus for multidrug resistance. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8938–8942. doi: 10.1073/pnas.89.19.8938. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McRipley R. J., Burns-Horwitz P. E., Czerniak P. M., Diamond R. J., Diamond M. A., Miller J. L., Page R. J., Dexter D. L., Chen S. F., Sun J. H. Efficacy of DMP 840: a novel bis-naphthalimide cytotoxic agent with human solid tumor xenograft selectivity. Cancer Res. 1994 Jan 1;54(1):159–164. [PubMed] [Google Scholar]
- Pierce J. C., Sternberg N., Sauer B. A mouse genomic library in the bacteriophage P1 cloning system: organization and characterization. Mamm Genome. 1992;3(10):550–558. doi: 10.1007/BF00350620. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Van Dyk T. K., Majarian W. R., Konstantinov K. B., Young R. M., Dhurjati P. S., LaRossa R. A. Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions. Appl Environ Microbiol. 1994 May;60(5):1414–1420. doi: 10.1128/aem.60.5.1414-1420.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber C. A., Salazar E. P., Stewart S. A., Thompson L. H. Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells. Mol Cell Biol. 1988 Mar;8(3):1137–1146. doi: 10.1128/mcb.8.3.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Webster R. E. The tol gene products and the import of macromolecules into Escherichia coli. Mol Microbiol. 1991 May;5(5):1005–1011. doi: 10.1111/j.1365-2958.1991.tb01873.x. [DOI] [PubMed] [Google Scholar]
- Zwelling L. A., Hinds M., Chan D., Mayes J., Sie K. L., Parker E., Silberman L., Radcliffe A., Beran M., Blick M. Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II. J Biol Chem. 1989 Oct 5;264(28):16411–16420. [PubMed] [Google Scholar]