ROLE OF TOPOISOMERASE II IN NBQ MEDIATED TUMOR CELL DIFFERENTIATION

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3-nitrobenzothiazolo[3,2-a] quinolinium (NBQ) intercalates in DNA and shows a strong potential as antitumor agent. Studies in our laboratory on its mode of action revealed that the cytotoxicity of NBQ is associated with topoisomerase II- mediated DNA strand breaks. Moreover, NBQ significantly enhances in vivo lens regeneration in the adult newt Notophtalmus viridescens, and induces in vitro the differentiation of HL-60 and DFaDu tumor cell lines. NBQ, at non-lethal doses that stimulate differentiation, binds in vitro topoisomerase II to purified DNA. The involvement of topoisomerase II in the cytotoxic and differentiation activities of NBQ, brings into question what is the role of topoisomerase II in the NBQ-induced differentiation. Knowledge of the molecular mechanism(s) through which NBQ induces differentiation, in contrast to its cytotoxic effect, will further a potentially new mode of cancer chemotherapy. To explore this question, we will study the effects of NBQ and eight analogs using a combination of molecular and cellular approaches. Because the DNA sequence localization of the cleavable complex, formed by NBQ and the various analogs, with topoisomerase II should be identical, the comparison of drug effects is more amenable to interpretation. We propose to characterize the cytotoxic activity of the eight NBQ analogues and evaluate athe inhibition f topoisomerase activity. The differentiation ability of each analog will compared to NBQ (80 - 90% HL-60 cells differentiated into the granulocytic series). To understand the mechanism of action of NBQ and analogs, we will evaluate; the formation of protein linked strand breaks in intact cells, the inhibition of topoisomerase activity i vitro, and promotion of chromosomal abnormalities. The effects of NBQ analogs on the cell cycle will be detected at concentration that induce differentiation compared with cytotoxic concentrations. The pattern of topoisomerase II-mediated DNA cleavage stimulated by NBQ will be compared to the cleavage pattern stimulated by NBQ analogs and the topoisomerase II poisons doxorubicin, VP-16 and m-AMSA. Finally, the effects of NBQ analogs will be studied in the resistant HL-60 and m-AMSA. Finally, the effects of NBQ analogs will be studied in the resistant HL-60 cell line (HL-60/NBA), which allows us to study the basis for differences in activity, specially, the induction of differentiation.