CHARACTERIZATION OF L1210 CELL-GROWTH INHIBITION BY THE BACTERIAL IRON CHELATORS PARABACTIN AND COMPOUND-II

  • 1 January 1985
    • journal article
    • research article
    • Vol. 45  (10) , 4754-4759
Abstract
Microbial siderophores represent a class of iron chelators characterized by their high affinity (i.e., formation constants, > 1040 M) for ferric iron. Previously, we demonstrated that the bacterial siderophores, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2,3-dihydroxybenzamino)butryl]-2-(2-hydroxyphenyl)trans-5-methyloxazoline-4-carboxamide (Parabactin) and N1,N8-bis(2,3-dihydroxybenzoyl)spermidine (Compound II), inhibit the growth of L1210 cells and the replication of DNA (but not RNA) viruses at low micromolar concentrations. The basis for this antiproliferative effect on L1210 cells has now been investigated further. Onset of growth inhibition induced by 5 .mu.m Parabactin occurs much earlier than with an equimolar concentration of Compound II but, once established by either chelator, inhibition appears to be irreversible. Growth inhibition was fully preventable with exogenous FeCl3 when given at the same time as the chelators. Flow cytometric analysis revealed a G1-S cycle block following treatment for 4 h with either 5 .mu.M Parabactin or 30 .mu.M Compound II. The block was readily reversed with exogenous FeCl3, allowing cells to progress to mid-S phase by 3 h and to G1 again by 9 h. Thereafter, cells accumulated at a second block located at S phase. The treatment conditions required for the initial cell cycle block (at 4 h) were adapted for subsequent studies. Clonogenicity of L1210 cells in soft agar following a 4-h exposure was reduced to 22% of control by 5 .mu.M Parabactin and to 16% by 30 .mu.M Compound II. Neither growth inhibition in suspension culture nor decreased clonogenicity in soft agar could be reversed with exogenous iron, following treatment with the chelators. Both chelators caused an early and significant decrease in [14C]thymidine incorporation over the 4-h period (50% inhibitory concentration at 4 h, 0.4 .mu.M for Parabactin and 6.0 .mu.M for Compound II). [3H]Uridine incorporation was inhibited later than [14C]thymidine and to a much lesser extent, while [3H]leucine incorporation was not significantly affected. Treatment of cells with 5 .mu.M Parabactin or Compound II for 4 h decreased deoxyadenosine triphosphate pools by 38 and 70%, respectively, and increased deoxythymidine triphosphate pools by 67 and 36%, respectively, suggesting interference with ribonucleotide reductase. Indeed, extracts of cells treated for 4 h with either 5 .mu.M Parabactin or 30 .mu.M Compound II exhibit a 97 to 98% decrease in cytidine-5''-diphosphate reductase activity compared to control, whereas DNA polymerase was elevated slightly. These data suggest that interference with DNA biosynthesis by inhibition of the non-heme iron-containing enzyme, ribonucleotide reductase, represents one site of drug action involved in the early antiproliferative effects of the bacterial siderophores, Compound II and Parabactin.