Characterization of promoter elements involved in the down-regulation of topoisomerase IIalpha expression in a drug-resistant cell line.

ABSTRACT

Reduced expression of topoisomerase II is one of the mechanisms observed in cell lines and clinical samples that are resistant to topoisomerase II-targeting agents. The Chinese hamster lung cell line DC-3F/9-OH-E made resistant to 9-OH ellipticine and cross-resistant to other topoisomerase II inhibitors has previously been shown to express lower level of topoisomerase IIalpha isoform, than the parental DC-3F cell line. We have shown here that topoisomerase IIalpha promoter activity is lower in the resistant cell line. The promoter sequence responsible for the differential expression of Chinese hamster topoisomerase IIalpha gene was localized in a small promoter region, which harbors three inverted CAAT elements (ICEs) that bind transcription factor NF-Y, two GC boxes that bind Sp1 and a TATA-like element that binds unknown factors. Immunoblot analysis of cell lysates showed that the resistant line expressed reduced levels of NF-Y subunits and attenuated level of p53. Although p53 has been reported being involved in the regulation of topoisomerase II expression, it is not responsible for the reduced topoisomerase IIalpha expression in the drug resistant line. Mutational analysis of individual elements suggested that the resistant cell line has relaxed responses to ICE mutations, and the TATA-like element plays a predominant role in the regulation of topoisomerase IIalpha. Furthermore, gel mobility shift assays showed that the resistant line has a differential binding to the novel TATA-like element, which may be responsible for the down-regulation of topoisomerase IIalpha gene.