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Characterization of L1210 cell growth inhibition by the bacterial iron chelators parabactin and compound II
Abstract:
Microbial siderophores represent a class of iron chelators characterized by their high affinity (i.e., formation constants, greater than 10(40) 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 (Biochem. Biophys. Res. Commun., 121: 848-854, 1984). The basis for this antiproliferative effect on L1210 cells has now been investigated further. Onset of growth inhibition induced by 5 microM 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 microM Parabactin or 30 microM 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 microM Parabactin and to 16% by 30 microM 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 microM for Parabactin and 6.0 microM 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 microM Parabactin or Compound II for 4 h decreased deoxy-adenosine 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 microM Parabactin or 30 microM Compound II exhibit a 97 to 98% decrease in cytidine-5'-diphosphate reductase activity compared to control, whereas DNA polymerase was elevated slightly.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Microbial siderophores like Parabactin and Compound II inhibit L1210 cell growth by blocking DNA synthesis. Iron can reverse early cell cycle arrest, but later effects and reduced clonogenicity are irreversible, indicating complex antiproliferative mechanisms.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Microbial siderophores are high-affinity iron chelators.
- Parabactin and Compound II inhibit L1210 cell growth and viral replication.
- The antiproliferative mechanism of these siderophores requires further investigation.
Purpose of the Study:
- To elucidate the mechanism of antiproliferative effects of Parabactin and Compound II on L1210 cells.
- To investigate the role of iron in mediating the effects of these siderophores.
- To assess the impact of siderophore treatment on cell cycle progression and DNA synthesis.
Main Methods:
- Cell culture (L1210 cells)
- Flow cytometry for cell cycle analysis
- Soft agar assays for clonogenicity
- Measurement of nucleotide pool incorporation ([14C]thymidine, [3H]uridine, [3H]leucine)
- Enzyme activity assays (ribonucleotide reductase, DNA polymerase)
Main Results:
- Parabactin and Compound II induce G1-S and S phase cell cycle blocks, reversible by iron initially.
- Siderophore treatment significantly reduces L1210 cell clonogenicity.
- Both siderophores inhibit [14C]thymidine incorporation and ribonucleotide reductase activity.
- Iron supplementation prevents early cell cycle arrest but not irreversible growth inhibition or reduced clonogenicity.
Conclusions:
- Parabactin and Compound II exhibit potent antiproliferative effects on L1210 cells.
- The mechanism involves interference with DNA synthesis, particularly ribonucleotide reductase activity.
- While iron can modulate early responses, sustained treatment leads to irreversible effects on cell proliferation and clonogenicity.