Fornari F A, Jarvis W D, Grant S, Orr M S, Randolph J K, White F K, Mumaw V R, Lovings E T, Freeman R H, Gewirtz D A
Department of Medicine, Medical College of Virginia, Richmond 23298.
Cell Growth Differ. 1994 Jul;5(7):723-33.
The effects on DNA integrity of continuous (72-h) exposure of human MCF-7 breast adenocarcinoma cells to 50 nM doxorubicin (a concentration which can be maintained in the plasma by continuous infusion) were characterized by bisbenzimide spectrofluorophotometry, cell flow cytometry, agarose gel electrophoresis, and neutral elution. Spectrofluorophotometry and cell flow cytometry indicated the presence of DNA fragmentation, which was maximal at 24 h. Resolution of these fragments on agarose gels failed to demonstrate "laddered" oligosomal profiles. Neutral elution analysis at 24 h indicated that doxorubicin induced fragmentation of nascent, but not mature, double-stranded DNA. Drug-treated cells exhibited endoreduplication and significant shifts in cell cycle distribution, (i.e., increased G0/G1 and G2/M fractions and a markedly reduced S-phase fraction). These alterations occurred without inhibiting the incorporation of [3H]dThd into cellular DNA; in fact, both the rate and magnitude of [3H]dThd incorporation increased progressively. Doxorubicin also produced a sustained decline in c-myc mRNA levels that paralleled both growth arrest and induction of DNA fragmentation. Ultrastructural examination revealed morphological alterations consistent 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 autocatalysis (e.g., lipofuschin-containing vacuoles). A gradual decline in cell number was observed, with loss of approximately 35% of the cell population after 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)
通过双苯甲酰亚胺荧光分光光度法、细胞流式细胞术、琼脂糖凝胶电泳和中性洗脱法，对人MCF - 7乳腺腺癌细胞连续（72小时）暴露于50 nM阿霉素（通过连续输注可在血浆中维持的浓度）对DNA完整性的影响进行了表征。荧光分光光度法和细胞流式细胞术表明存在DNA片段化，在24小时时达到最大值。这些片段在琼脂糖凝胶上的分离未能显示出“阶梯状”寡聚体图谱。24小时的中性洗脱分析表明，阿霉素诱导新生双链DNA而非成熟双链DNA发生片段化。药物处理的细胞表现出核内复制和细胞周期分布的显著变化（即G0/G1和G2/M期分数增加，S期分数显著降低）。这些改变在不抑制[3H]胸苷掺入细胞DNA的情况下发生；事实上，[3H]胸苷掺入的速率和幅度都逐渐增加。阿霉素还使c - myc mRNA水平持续下降，这与生长停滞和DNA片段化的诱导平行。超微结构检查揭示了与分化诱导一致的形态学改变（例如，脂质含量和线粒体密度增加、紧密连接和分泌导管的出现），并进一步提示了自催化的可能性（例如，含脂褐素的液泡）。观察到细胞数量逐渐下降，72小时后约35%的细胞群体丧失。（摘要截短至250字）
