Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli.

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively. Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.

Establishment and characterization of a mouse FM3A cell mutant resistant to topoisomerase II-inhibitor NC-190.

We established an NC-190-resistant cell line, FM/NC-R, from the murine mammary carcinoma cell line FM3A and examined some of its characteristics. FM/NC-R cells were prepared by mutagen treatment followed by exposure to NC-190 in the culture medium. FM/NC-R cells were 76.5 times more resistant against NC-190 than FM3A cells as measured by their growth in vitro. FM/NC-R cells also showed cross-resistance to etoposide with NC-190. Neither NC-190 nor etoposide increased the lifespan of FM/NC-R-bearing mice at doses that prolonged the lifespan of FM3A-bearing mice more than four times. This resistance was not due to the change in the concentration of NC-190 in the cells, and there was no change in the expression of P-glycoprotein, a drug efflux pump in the cells. NC-190 and etoposide are inhibitors of DNA topoisomerase II, but there was no difference in cellular content of DNA topoisomerase II between the two cell lines as determined by Western blot analysis. The stabilization of DNA-DNA topoisomerase II cleavable complexes induced by NC-190 was lost in FM/NC-R cells. It was found that Gly881, which is located in the ATP binding site, was replaced by Arg in topoisomerase IIalpha of FM/NC-R cells. These results indicate that the NC-190-resistant cell line FM/NC-R contains a mutated DNA topoisomerase IIalpha.

The topoisomerase II-inhibitor NC-190 reduces the level of thymidine kinase mRNA in murine tumor cells.

The novel antitumor compound NC-190 strongly inhibited the growth of FM3A cells with an IC50 of 0.019 microg/ml (0.042 microM) when cultured with NC-190 for 48 h. NC-190 potently suppressed DNA synthesis, with 90% inhibition observed at 0.1 microg/ml of NC-190. RNA and protein syntheses were also suppressed under the same conditions, but to a lesser extent. We then measured the cellular enzymatic activities of DNA polymerase alpha, RNA polymerase, thymidine kinase, thymidylate synthase and Leu-tRNA synthetase of FM3A cells cultured with or without NC-190. Of these 5 enzymes, the activity of thymidine kinase was most strongly suppressed by NC-190, by 77%. Although NC-190 did not directly inhibit the activitiy of thymidine kinase in a cell-free system, expression of mRNA of thymidine kinase was suppressed by 75% in NC-190-treated cells. These results indicate that NC-190 can suppress the expression of the gene for thymidine kinase and the inhibition of thymidine kinase contributes to the inhibition of cell growth by NC-190 together with the inhibition of topoisomerase II.