Influence of amsacrine (m-AMSA) on bulk and gene-specific DNA damage and c-myc expression in MCF-7 breast tumor cells.
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 alpha-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.
Bunch
R T
RT
Department of Pharmacology and Toxicology, Virginia Commonwealth, University/Medical College of Virginia, Richmond 23298.
Povirk
L F
LF
Orr
M S
MS
Randolph
J K
JK
Fornari
F A
FA
Gewirtz
D A
DA
D000677:Amsacrine / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004276:DNA, Satellite / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000187:drug effects; D015536:Down-Regulation; D015870:Gene Expression / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004276:DNA, Satellite; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D000677:Amsacrine; D004247:DNA