CYTOTOXICITY AND DNA-DAMAGE ASSOCIATED WITH PYRAZOLOACRIDINE IN MCF-7BREAST-CANCER CELLS

We examined the effects of pyrazoloacridine (PZA), an investigational anticancer agent in clinical trials, on cytotoxicity, DNA synthesis, a nd DNA damage in MCF-7 human breast carcinoma cells. With PZA concentr ations ranging from 0.5 to 50 mu M for durations of 3-72 hr, cytotoxic ity increased in proportion to the total PZA exposure (concentration x time). Inhibition of DNA and RNA syntheses increased with increasing PZA concentration x time (mu M . hr). A 24-hr exposure to 1 and 10 mu M PZA reduced DNA synthesis to 62 and 5% of control, respectively, dec reased the proportion of cells in S phase with accumulation of cells i n G(2) + M phase, and inhibited cell growth at 72 hr by 68 and 100%. N ewly synthesized DNA was more susceptible to damage during PZA exposur e, with subsequent induction of parental DNA damage. Significant damag e to newly synthesized DNA as monitored by alkaline elution was eviden t after a 3-hr exposure to greater than or equal to 5 mu M PZA. Longer PZA exposures (greater than or equal to 10 mu M for 16 hr) were requi red to elicit damage to parental DNA. Induction of single-strand break s in parental DNA correlated closely with induction of double-strand b reaks and detachment of cells from the monolayer. PZA-mediated DNA fra gmentation was not accompanied by the generation of oligonucleosomal l addering in MCF-7 cells, but induction of very high molecular weight D NA fragmentation (0.5 to 1 Mb) was detected by pulsed-field gel electr ophoresis. In vitro binding of PZA to linear duplex DNA (1 kb DNA ladd er) and closed, circular plasmid DNA was demonstrated by a shift in mi gration during agarose electophoresis. PZA interfered with topoisomera se I- and II-mediated relaxation of plasmid DNA in a cell-fret system, but the cytotoxic effects of PZA did not appear to involve a direct i nteraction with topoisomerase I or II (stabilization of the topoisomer ase I- or II-DNA cleavable complex). PZA-mediated cytotoxicity correla ted strongly with inhibition of DNA and RNA syntheses, and damage to b oth nascent and parental DNA. Neither the cytotoxicity of PZA nor indu ction of double-stranded DNA fragmentation was prevented by aphidicoli n, indicating that PZA-mediated lethality occurred in the absence of D NA replication. Since free radical formation was not detected, inducti on of nascent and parental DNA damage appeared to be a consequence of the avid binding of PZA to DNA, presumably by interfering with the acc ess of replication, repair, and transcription enzyme complexes.