Analysis of topoisomerase II‐mediated DNA cleavage of the c‐myc gene during HL60 differentiation

Abstract

We have investigated the effect of mAMSA, a potent topoisomerase II inhibitor, on the c-myc proto-oncogene of the acute promyelocytic leukemia HL60 cell line during its differentiation. When HL60 cells were induced by dimethylsulfoxide (DMSO) to terminally differentiate, a rapid drop m the level of c-myc mRNA was observed, followed by an arrest of cell proliferation. In contrast, the level of topoisomerase II mRNA was transiently Increased with a maximum at 6 h after DMSO addition and was then completely abolished after 48 h, indicating that topoisomerase II is activated during the onset of HL60 differentiation. In exponentially growing cells, treatment by mAMSA results in the formation of topoisomerase II-mediated double strand DNA breaks in the c-myc gene at positions where topoisomerase II would normally nick and reseal the two strands. In HL60 cells treated with both mAMSA and DMSO, the sites in the c-myc gene at which mAMSA had induced cleavage were not altered. However, a DNA cleavage site located at the end of the first c-myc exon (position +3100), was strongly stimulated by mAMSA and DMSO treatment. This site fell within a DNase I hypersensitive region encompassing the MYC intron factor 1 (MIFl) binding site, where transcription elongation is normally blocked during differentiation. These data indicate that a change of topoisomerase II binding to critical regulatory region of the c-myc gene is associated with the downregulation of this gene during differentiation.

Differential regulation of p21waf-1/cip-1 and Mdm2 by etoposide: etoposide inhibits the p53-Mdm2 autoregulatory feedback loop

“…In addition Mdm2 is a short-half life protein which is very actively transcribed so will be the site of enhanced topoisomerase activity. Similar ®ndings of etoposide hypersensitivity were previously described for DNAse 1 sensitive region in the c-myc gene (Riou et al, 1993). It is of note that c-myc and mdm2 both form episomes and are frequently ampli®ed in human tumour's suggesting their chromatin conformation may be unusual.…”

“…Etoposide is a topoisomerase II (topo II) poison which inhibits the resealing activity of topo II, resulting in a bulky DNA protein complex. The presence of etoposide and UV-induced lesions in DNA can directly impair transcription (Ljungman and Zhang, 1996;Riou et al, 1993). If the Mdm2 transcription is inhibited by etoposide damage then removal of etoposide, which results in reversal of etoposide topo II complex, should allow transcription to proceed.…”

Transcriptional Consequences of Topoisomerase Inhibition

“…Translocations, regional amplifications, and viral insertions and mutations, sometimes at vast distances either 5Ј or 3Ј from the c-myc promoters, all deregulate c-myc transcription (28,36,50). Although topoisomerase inhibitors influence c-myc expression (2,6,16,19,43,48,56,57,58), it is unknown whether this results from perturbation of c-myc DNA and chromatin structure driven by changes in the localization and levels of torsional strain or whether this results from indirect effects. If c-myc transcription is sensitive to torsional strain, then changes in c-myc mRNA levels, promoter structure, and the distribution of RNA polymerase II molecules along the gene should all respond to topoisomerase inhibitors.…”

Nuclear factor‐κB, induced in human carcinoma cell line A2780 by the new anthracycline men 10755, is devoid of transcriptional activity

“…37 The observed downmodulation of c-myc expression could be directly linked to topo II poisoning since gene-specific cleavage sites in the c-myc promoter and first exon have been described. 44,45 Therefore, we found in MEN 10755-treated A2780 cells a pattern of gene modifications that can lead to cell death, with no evidence of prosurvival gene expression ascribable to NF-B activity. Indeed, since p53 has also been described to mediate the transcriptional repression of the antiapoptotic genes included in our analysis, 11,46 most of the gene modifications we observed in A2780 cells following MEN 10755 treatment could be ascribed to p53.…”