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Mithramycin selectively inhibits transcription of G-C containing DNA
D M Miller1, D A Polansky, S D Thomas
1Comprehensive Cancer Center, University of Alabama, Birmingham 35294.
Abstract:
Mithramycin induces a reversible inhibition of cellular RNA synthesis without affecting DNA synthesis. The authors have shown this drug induces myeloid differentiation of HL-60 promyelocytic leukemia cells and is an effective agent in certain patients with chronic granulocytic leukemia. In order to investigate the mechanism by which this drug inhibits RNA synthesis we have compared the effect of mithramycin on RNA synthesis by whole cells, isolated nuclei, and RNA synthesis by isolated E. coli RNA polymerase and eukaryotic RNA polymerase II. Exposure of HL-60 cells to mithramycin at concentrations of 4.6 X 10(-7) m or higher for 48 hours causes an almost immediate inhibition of RNA synthesis (up to 85% at 4 hours) with only modest cytotoxicity at these concentrations. Endogenous RNA synthesis by isolated nuclei can be inhibited by mithramycin only at high concentrations (greater than 10(-5) m), suggesting that mithramycin primarily may inhibit initiation, rather than elongation. Mithramycin inhibits in vitro transcription of salmon sperm DNA by E. coli RNA polymerase at DNA:drug ratios similar to those required for RNA synthesis inhibition in whole cells. Similar DNA binding studies with synthetic oligonucleotides demonstrate that mithramycin is a potent inhibitor of transcription of Poly dG.dC by E. coli RNA polymerase but has no effect on transcription of Poly dA.dT. The rapid inhibition of whole cell and isolated RNA polymerase transcription, and the relative insensitivity of isolated nuclei, suggest mithramycin may interact with specific DNA sequences in order to inhibit the initiation of RNA synthesis in intact cells.
Insights
Mithramycin rapidly inhibits cellular RNA synthesis by targeting DNA, potentially at specific sequences, without affecting DNA synthesis. This mechanism explains its efficacy in leukemia and myeloid differentiation.
Area of Science:
- Molecular Biology
- Biochemistry
- Pharmacology
Background:
- Mithramycin is known to inhibit RNA synthesis and induce myeloid differentiation in HL-60 leukemia cells.
- Its precise mechanism of action on RNA synthesis, particularly the distinction between initiation and elongation, requires further investigation.
Purpose of the Study:
- To elucidate the mechanism by which mithramycin inhibits cellular RNA synthesis.
- To compare the effects of mithramycin on RNA synthesis in whole cells, isolated nuclei, and purified RNA polymerases.
Main Methods:
- Comparing mithramycin's effect on RNA synthesis in whole HL-60 cells, isolated nuclei, and in vitro transcription assays using E. coli and eukaryotic RNA polymerase II.
- DNA binding studies with synthetic oligonucleotides (Poly dG.dC and Poly dA.dT) to assess sequence specificity.
Main Results:
- Mithramycin rapidly inhibits RNA synthesis in whole HL-60 cells (up to 85% inhibition within 4 hours) at concentrations of 4.6 X 10(-7) m or higher, with minimal cytotoxicity.
- Inhibition of endogenous RNA synthesis in isolated nuclei required significantly higher mithramycin concentrations (greater than 10(-5) m), suggesting a primary effect on initiation rather than elongation.
- Mithramycin potently inhibited in vitro transcription of Poly dG.dC by E. coli RNA polymerase, but not Poly dA.dT, indicating sequence-specific DNA interaction.
Conclusions:
- Mithramycin's rapid inhibition of RNA synthesis in intact cells, coupled with its relative insensitivity in isolated nuclei, suggests it primarily inhibits the initiation of RNA synthesis.
- The drug likely interacts with specific DNA sequences, as evidenced by its differential effects on synthetic DNA polymers.
- These findings provide a mechanistic basis for mithramycin's observed biological effects, including its therapeutic potential in certain leukemias.