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Interstrand cross-linking by adriamycin in nuclear and mitochondrial DNA of MCF-7 cells
C Cullinane1, S M Cutts, C Panousis
1Department of Biochemistry, La Trobe University, Bundoora, Victoria 3083, Australia.
Abstract:
Activation of Adriamycin by formaldehyde leads to the formation of drug-DNA adducts in vitro and these adducts stabilise the DNA to such a degree that they function as virtual interstrand cross-links. The formation of these virtual interstrand cross-links by Adriamycin was investigated in MCF-7 cells using a gene-specific interstrand cross-linking assay. Cross-linking was measured in both the nuclear-encoded DHFR gene and in mitochondrial DNA (mtDNA). Cross-link formation increased linearly with Adriamycin concentration following a 4 h exposure to the drug. The rate of formation of Adriamycin cross-links in each of the genomes was similar, reaching maximal levels of 0.55 and 0.4 cross-links/10 kb in the DHFR gene and mtDNA respectively, following exposure to 20 micro M Adriamycin for 8 h. The interstrand cross-link was short lived in both DNA compartments, with a half-life of 4.5 and 3.3 h in the DHFR gene and mtDNA respectively. The kinetics of total Adriamycin adduct formation, detected using [(14)C]Adriamycin, was similar to that of cross-link formation. Maximal adduct levels (30 lesions/10 kb) were observed following incubation at 20 micro M drug for 8 h. The formation of such high levels of adducts and cross-links could therefore be expected to contribute to the mechanism of action of Adriamycin.
Insights
Adriamycin forms DNA adducts that act as virtual interstrand cross-links in cancer cells. These cross-links, found in both nuclear and mitochondrial DNA, are transient and contribute to Adriamycin
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Background:
- Adriamycin (doxorubicin) is a widely used chemotherapy agent.
- Drug-induced DNA damage, including adducts and cross-links, is a key mechanism of action for many chemotherapeutics.
- Understanding the specific types and kinetics of DNA damage induced by Adriamycin is crucial for optimizing its therapeutic efficacy and managing side effects.
Purpose of the Study:
- To investigate the formation and characteristics of Adriamycin-induced virtual interstrand cross-links in MCF-7 cells.
- To compare Adriamycin cross-linking in nuclear DNA (DHFR gene) and mitochondrial DNA (mtDNA).
- To correlate the formation of drug-DNA adducts with cross-link formation and explore their contribution to Adriamycin's mechanism of action.
Main Methods:
- Utilized a gene-specific interstrand cross-linking assay to quantify Adriamycin-induced cross-links.
- Measured cross-linking in both the nuclear-encoded dihydrofolate reductase (DHFR) gene and mitochondrial DNA (mtDNA).
- Quantified total Adriamycin adducts using [(14)C]Adriamycin and determined the kinetics of adduct and cross-link formation and removal.
Main Results:
- Adriamycin exposure led to the formation of virtual interstrand cross-links in both nuclear and mitochondrial DNA.
- Cross-link formation increased linearly with Adriamycin concentration and exposure time.
- Maximal cross-link levels were observed at 20 micro M Adriamycin after 8 hours, with similar rates in DHFR and mtDNA.
- The induced interstrand cross-links were transient, with half-lives of 4.5 hours in the DHFR gene and 3.3 hours in mtDNA.
- Total Adriamycin adduct formation kinetics mirrored cross-link formation, reaching maximal levels of 30 lesions/10 kb.
Conclusions:
- Adriamycin effectively forms virtual interstrand cross-links in both nuclear and mitochondrial DNA within cancer cells.
- The transient nature of these cross-links suggests a dynamic interaction between Adriamycin, DNA, and cellular repair mechanisms.
- The substantial levels of both adducts and cross-links formed support their significant role in Adriamycin's cytotoxic mechanism of action.