Influence of amsacrine (m-AMSA) on bulk and gene-specific DNA damage and c- myc expression in MCF-7 breast tumor cells

• Published in: Biochemical pharmacology Vol. 47; no. 2; pp. 317 - 329
• Main Authors: Bunch, Roderick T., Povirk, Lawrence F., Orr, Michael S., Randolph, Joyce K., Fornari, Frank A., Gewirtz, David A.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 20.01.1994; Elsevier Science
• Subjects: Amsacrine - pharmacology; Antineoplastic agents; Biological and medical sciences; c-myc; Cell Division - drug effects; Chemotherapy; DNA - drug effects; DNA Damage; DNA, Satellite - drug effects; DNA, Single-Stranded - drug effects; Down-Regulation; Gene Expression - drug effects; Genes, myc - drug effects; Humans; m-AMSA; Medical sciences; Pharmacology. Drug treatments; RNA, Messenger - analysis; Tumor Cells, Cultured - drug effects; dsDNA, per cent of total DNA remaining double stranded (acid-precipitable DNA/total DNA × 100); m-AMSA; F DS, fraction of hybridization sequence remaining double-stranded compared with control (density of hybridization signal treated sample/control); c-myc; DNA damage; SSB, single-strand break; GAPD, glyceraldehyde phosphate dehydrogenase; SDS, sodium dodecyl sulfate; DSB, double-strand break; AU/SB, alkaline unwinding/Southern blot; ssDNA, single-stranded DNA; m-AMSA, amsacrine, 4′-(9-acridinylamino)methanesulfon- m-anisidide; TCA, trichloroacetic acid; Antineoplastic agent; DNA topoisomerase (ATP-hydrolysing); Enzyme; Enzyme inhibitor; Gene expression; In vitro; Biological activity; Chromosome break; Isomerases; DNA; C-Onc gene; Mechanism of action; Tumor cell
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/0006-2952(94)90023-X

In the MCF-7 human breast tumor cell line, the aminoacridine, m-AMSA, induces protein-associated DNA strand breaks consistent with inhibition of topoisomerase II. However, neither single-strand nor double-strand breaks in DNA, determined using conventional assays, show a consistent relationship with m-AMSA-induced inhibition of growth. In contrast, when DNA strand breaks are determined by alkaline unwinding under the high salt conditions of the alkaline unwinding/Southern blotting (AU/SB) assay, developed by our laboratories, damage to DNA corresponds closely with growth inhibition. The AU/SB assay, which is capable of assessing breaks within large-scale domains (upwards of 1 megabase) surrounding genes of interest, was further utilized to explore the capacity of m-AMSA to induce damage within specific genomic regions that may regulate cell growth. Regions encompassing the transcriptionally active oncogenes, c- myc and c- fos, were found to be more susceptible to m-AMSA-induced strand breaks than the region encompassing the non-transcribed α-satellite DNA or the genome as a whole (bulk DNA). These findings demonstrate that m-AMSA may produce more pronounced damage within specific genomic regions than in bulk DNA. m-AMSA also preferentially altered expression of the c- myc oncogene; at an m-AMSA concentration where growth was inhibited by between 70 and 80%, steady-state c- myc mRNA levels declined to approximately 10–15% of control levels within 2–3 hr; furthermore, concentration-dependent reductions in c- myc expression appeared to coincide with growth inhibition. In addition, inhibition of [ 3H]thymidine incorporation after 2 hr directly paralleled inhibition of growth, suggesting an early effect at the level of DNA biosynthesis, possibly related to the down-regulation of c- myc expression. It is proposed that specific lesions, e.g., in regions surrounding the c- myc gene, as well as generalized lesions in DNA may lead to growth inhibition mediated by down-regulation of the expression of select growth regulatory genes, such as c- myc.