INDUCTION OF DIFFERENTIATION AND GROWTH ARREST ASSOCIATED WITH NASCENT (NONOLIGOSOMAL) DNA FRAGMENTATION AND REDUCED C-MYC EXPRESSION IN MCF-7 HUMAN BREAST-TUMOR CELLS AFTER CONTINUOUS EXPOSURE TO A SUBLETHAL CONCENTRATION OF DOXORUBICIN

The effects on DNA integrity of continuous (72-h) exposure of human MC F-7 breast adenocarcinoma cells to 50 nM doxorubicin (a concentration which can be maintained in the plasma by continuous infusion) were cha racterized by bisbenzimide spectrofluorophotometry, cell flow cytometr y, agarose gel electrophoresis, and neutral elution. Spectrofluorophot ometry and cell flow cytometry indicated the presence of DNA fragmenta tion, which was maximal at 24 h. Resolution of these fragments on agar ose gels failed to demonstrate ''laddered'' oligosomal profiles. Neutr al elution analysis at 24 h indicated that doxorubicin induced fragmen tation of nascent, but not mature, double-stranded DNA. Drug-treated c ells exhibited endoreduplication and significant shifts in cell cycle distribution, (i.e., increased C-0/G(1) and C-2/M fractions and a mark edly reduced S-phase fraction). These alterations occurred without inh ibiting the incorporation of [H-3]dThd into cellular DNA; in fact, bot h the rate and magnitude of [H-3]dThd incorporation increased progress ively. Doxorubicin also produced a sustained decline in c-myc mRNA lev els that paralleled both growth arrest and induction of DNA fragmentat ion. Ultrastructural examination revealed morphological alterations co nsistent with the induction of differentiation (e.g., increased lipid content and mitochondrial density, appearance of tight junctions, and secretory ducts) and further suggested the possibility of autocatalysi s (e.g, lipofuschin-containing vacuoles). A gradual decline in cell nu mber was observed, with loss of approximately 35% of the cell populati on after 72 h. These findings suggest that growth arrest (and possibly cell death) following chronic exposure of MCF-7 breast tumor cells to a low (generally sublethal) concentration of doxorubicin may be a con sequence of damage to nascent DNA leading to down-regulation of c-myc expression and the induction of differentiation.