DECREASED CP-1 (NF-Y) ACTIVITY RESULTS IN TRANSCRIPTIONAL DOWN-REGULATION OF TOPOISOMERASE II-ALPHA IN A DOXORUBICIN-RESISTANT VARIANT OF HUMAN MULTIPLE-MYELOMA RPMI-8226

Decreased topoisomerase II (Topo II) activity results in resistance to antineoplastic agents targeting this enzyme. Dox1V derived from human multiple myeloma RPMI 8226 demonstrated a 4-fold resistance to doxoru bicin in the absence of MDR1 overexpression or topo II mutations (Futs cher B.W., Foley N., Gleason-Guzman M., Meltzer P.S., Sullivan D.M., a nd Dalton W.S., Int'l. J. Cancer, 66: 520-5, 1996.). Consistent with i ts drug resistant phenotype, a 2 to 3-fold decrease in topo II express ion was identified. To investigate the molecular basis for decreased t opo II expression in Dox1V, a semi-quantitative analysis of Topo II ac tivity, protein level and mRNA transcript were performed. The results demonstrated that reduced Topo II activity is due to a decreased mRNA level, Southern blot and sequencing experiments revealed wild-type seq uence of the topo II promoter in the drug resistant cells. Transient g ene expression assays demonstrated that topo II is transcriptionally d own-regulated in Dox1V independent of the promoter sequence of the end ogenous alleles, Instead, the activity of a ubiquitous transcription f actor CP-1 (NF-Y) interacting with the topo II promoter is decreased. The decrease in CP-1/NF-Y activity in Dox1V is correlated well with th e decrease in topo II transcriptional activity, transcript level, Topo II protein and enzyme activity. Therefore, transcriptional down-regul ation resulted from a reduced CP-1/NF-Y activity is responsible for de creased topo II expression in Dox1V cells. (C) 1997 Academic Press.