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Effects of adriamycin on supercoiled DNA and calf thymus nucleosomes studied with fluorescent probes
Abstract:
The interaction of the antitumor drug Adriamycin with nucleotides, polynucleotides, RNA, calf thymus nucleosomes, and DNA (including pBR322 supercoiled DNA) has been studied using fluorescent probes. The lanthanide terbium is known to interact with guanine and xanthosine to produce high fluorescence enhancement. The nature of the interaction of the lanthanide with the heterocyclic ring in guanine appears to involve both the C-2 and N-7 groups. A striking decrease in fluorescence enhancement was observed with all of the polynucleotides, RNA, DNA, and nucleosomes after treatment with Adriamycin at molar ratios of 1:200 or less. It appears that Adriamycin interacts with the guanine ring, displacing or preventing terbium access to its second site of binding. However, with supercoiled DNA and nucleosomes, the displacement followed a destabilization of the helix at very low drug concentrations. The binding affinities of calf thymus DNA, pBR322 DNA, and calf thymus nucleosomes at 37 degrees for Adriamycin were of the same order of magnitude. Reaction with N-pyrene maleimide, a fluorescent probe which binds to histone H3, showed that Adriamycin interacted with the nucleosome to increase the binding of the probe (only, however, at drug ratios far greater than those required to produce effects with DNA). No compositional changes of supercoiled or nucleosomal DNA or nucleosomal histones were observed by agarose gel or sodium dodecyl sulfate:polyacrylamide gel electrophoresis, respectively. The classic intercalating agent, ethidium bromide, produced minimal displacement of the lanthanide from DNA, although an effect with RNA at high drug concentrations was observed.
Insights
Adriamycin, an antitumor drug, interferes with terbium binding to guanine in DNA and RNA. It also destabilizes DNA and nucleosomes at low concentrations, impacting their structure.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Adriamycin is an antitumor drug with known DNA-binding properties.
- Terbium ions (Tb3+) exhibit fluorescence enhancement upon binding to guanine bases in nucleic acids.
- Understanding drug-nucleic acid interactions is crucial for drug development.
Purpose of the Study:
- To investigate the interaction of Adriamycin with various nucleic acid structures using fluorescent probes.
- To elucidate the mechanism by which Adriamycin affects terbium binding to guanine.
- To assess the impact of Adriamycin on DNA and nucleosome stability.
Main Methods:
- Fluorescence spectroscopy using terbium as a probe for guanine binding.
- Utilizing N-pyrene maleimide to probe histone H3 interactions within nucleosomes.
- Agarose gel electrophoresis and SDS-PAGE to assess DNA and histone integrity.
Main Results:
- Adriamycin significantly reduced terbium fluorescence enhancement in DNA, RNA, and nucleosomes, indicating interference with terbium-guanine binding.
- Low concentrations of Adriamycin destabilized supercoiled DNA and nucleosomes.
- Adriamycin increased N-pyrene maleimide binding to histone H3 at high concentrations, suggesting nucleosome interaction.
- No compositional changes in DNA or histones were detected via electrophoresis.
Conclusions:
- Adriamycin interacts with the guanine ring, potentially displacing terbium binding sites.
- The drug can destabilize DNA and nucleosome structures at low concentrations.
- Adriamycin's effects on nucleic acids are concentration-dependent and distinct from intercalating agents like ethidium bromide.