Doxorubicin is a widely used anticancer drug due to its broad spectrum of antitumor activity. Various mechanisms have been proposed for its cytostatic activity, including DNA intercalation, topoisomerase II inhibition, generation of free radicals and apoptosis. The present study aims to further clarify the cytostatic activity of doxorubicin by its specific effect on (a) DNA damage, (b) micronucleation and (c) apoptosis, using a combination of different methods and cell systems such as human lymphocytes and HL-60 human leukemic cells. DNA lesions were analyzed by the alkaline comet assay in combination with formamidopyrimidine (Fpg) and human 8-oxoguanine (hOGG1) repair enzymes. Micronucleation was investigated by the Cytokinesis-Block Micronucleus assay (CBMN) in combination with Fluorescence In Situ Hybridization analysis. Impairment on mitotic apparatus was investigated by double immunofluorescence of β- and γ-tubulin. Apoptotic cell frequency was determined by the CBMN cytome assay. Complementary to the above, caspase-3 level was investigated by Western blot. It was found that doxorubicin generates DNA breakage induced by oxidative damage in DNA bases, which can be repaired by the Fpg and hOGG1 enzymes. Increased micronucleus frequency was identified mainly through chromosome breakage and, at a lesser extent, through chromosome delay. Analysis of mitotic spindle showed disturbance of chromosome orientation and centrosome duplication and/or separation, leading to aneuploidy. Enhanced frequency of apoptotic leukemic cells was also observed. Caspase-3 seems to be involved in the generation of apoptosis. The aforementioned findings derived from different treatment schedules, doses and time of exposure on primary versus transformed cells extend our knowledge about doxorubicin genotoxicity and contribute to the better understanding of the mechanisms by which doxorubicin induces genotoxic effects on human cells.

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阿霉素在人细胞中的裂解，促细胞生成和凋亡特性的联合研究

阿霉素由于其广泛的抗肿瘤活性而被广泛使用。已经提出了用于其细胞抑制活性的各种机制，包括DNA嵌入，拓扑异构酶II抑制，自由基的产生和细胞凋亡。本研究旨在通过结合使用多种方法和细胞系统（例如人淋巴细胞和HL-60），通过对阿霉素（a）DNA损伤，（b）微核化和（c）凋亡的特异性作用来进一步阐明阿霉素的细胞抑制活性。人类白血病细胞。通过与甲酰胺基嘧啶（Fpg）和人8-氧代鸟嘌呤（hOGG1）修复酶结合的碱性彗星试验分析DNA损伤。通过胞质分裂-块微核试验（CBMN）结合荧光原位杂交分析研究了微核化。通过β-和γ-微管蛋白的双重免疫荧光研究了有丝分裂装置的损伤。通过CBMN细胞因子测定法确定凋亡细胞频率。与上述互补的是，通过Western印迹研究了caspase-3水平。已发现阿霉素会由于DNA碱基的氧化损伤而导致DNA断裂，该断裂可被Fpg和hOGG1酶修复。增加的微核频率主要是通过染色体断裂鉴定的，而在较小程度上是通过染色体延迟鉴定的。有丝分裂纺锤体的分析显示染色体方向的干扰和中心体复制和/或分离，导致非整倍性。还观察到凋亡性白血病细胞的频率增加。Caspase-3似乎与细胞凋亡有关。