Adriamycin-induced inhibition of mitochondrial-encoded polypeptides as a model system for the identification of hotspots for DNA-damaging agents.
It has recently been shown that the anti-cancer drug Adriamycin forms drug-DNA adducts which function as 'virtual' interstrand cross-links in cells, and these cross-links are specific for GpC sequences. The objective of this work was to determine whether all GpC sites are equally susceptible to the formation of Adriamycin-DNA adducts in the mitochondrial genome or whether any 'hotspots' exist whereby lesions are formed preferentially at particular GpC-containing sequences. The mitochondrial genome was used as a model system as it provides a series of contiguous genes, all of which lack introns and in which transcription is driven from a single promoter. With the absence of nucleotide excision repair, this provides an excellent system with which to observe Adriamycin-induced DNA damage since such lesions are reflected as an inhibition of mitochondrial protein synthesis. HeLa cells were treated with Adriamycin and the extent to which synthesis of individual mitochondrial-encoded proteins was inhibited was quantitated. Mitochondrial protein synthesis was found to be inhibited in a discontinuous manner, corresponding to regions rich in 5'-GpC sequences. These results therefore indicate that Adriamycin-DNA adducts do not form randomly with GpC sites throughout the mitochondrial genome, but instead appear to form preferentially at regions of high GpC content. This selective inhibition of mitochondrial-encoded proteins demonstrates the potential of this method for the in situ detection of localized regions of binding by DNA-acting drugs.
Sharples
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Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Cullinane
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Phillips
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D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001483:Base Sequence; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D004640:Emetine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011506:Proteins / Q000235:genetics; D013379:Substrate Specificity
D000903:Antibiotics, Antineoplastic; D003432:Cross-Linking Reagents; D004272:DNA, Mitochondrial; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D004317:Doxorubicin; D004640:Emetine