【作者】 梁越欣；

【导师】 李电东； 柳惠图；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2005， 博士

【摘要】 力达霉素(Lidamycin，LDM)又称C1027，是一种烯二炔类(enediyne)抗生素。它以其强烈的杀伤肿瘤细胞的活性和独特的切割DNA方式而备受重视。LDM分子由一个发色团和一个酸性的辅基蛋白非共价结合而组成。发色团是LDM的活性部分，能够引起DNA分子断裂，该作用具有核苷酸和序列的特异性；辅基蛋白起保护作用。以往的研究表明，LDM既能在体内外强烈的抑制肿瘤生长、抗肿瘤转移，又具有一定的抗病毒作用。LDM因其突出的临床应用前景而值得深入研究。 细胞裂亡即有丝分裂性细胞死亡(mitotic cell death)，在肿瘤治疗中已渐受关注，其精确机制尚不清楚。我们曾经报道过LDM在低浓度下可诱导人肝癌BEL-7402细胞和人乳腺癌MCF-7细胞裂亡。本文的研究目的在于探索LDM诱导细胞裂亡的分子机制及其可能的信号转导通路。 我们用低浓度LDM(0.1 nmol／L和0.5 nmol／L)分别处理人肝癌BEL-7402细胞和正常人肝L-02细胞2 h，发现LDM能够明显的诱导BEL-7402细胞和L-02细胞发生裂亡，其主要特征为：细胞体积增大、发生G2-M期阻断、出现多核化等。用MTT法测定生长曲线，发现与不加药的对照组细胞相比，加药组细胞生长速率均明显降低，细胞倍增时间延长，增殖受到抑制。低浓度LDM处理细胞后72 h，用Hoechst33342和PI联染观察到一种与典型凋亡不同的染色质凝集方式：核膜一直保持完整，细胞仍贴壁，染色质呈散点状凝集，不形成凋亡小体。琼脂糖凝胶电泳检测不到DNA梯带。流式细胞术也检测不到凋亡相关的“亚G1峰”。用Giemsa染色发现，加入低浓度LDM培养2 h，BEL-7402细胞于撤药后12 h出现多核化细胞，而L-02细胞出现多核化现象要比其滞后大约36 h。多核化细胞的形态各异，LDM作用下多核化的BEL-7402细胞微核数可达12-15个；而LDM引起的L-02细胞多核化一般不超过10个微核。0.5 nmol／L LDM作用后72 h，BEL-7402细胞和L-02细胞的多核化比例分别达到35.7％和14.1％，G2-M期阻滞分别达23.7％和60.9％。有丝分裂指数检测表明，在低浓度LDM作用下两种细胞的M期细胞百分数均明显增加。线粒体膜电位的检测结果显示，经低浓度LDM作用后，发生裂亡的细胞线粒体膜电位并未下降。细胞间接免疫荧光实验首次发现，低浓度LDM作用后细胞的中心体过复制，多极有丝分裂纺锤体形成。线粒体信号转导通路是凋亡的主要通路之一，Western blotting结果显示其在裂亡过程中未被激活。这些结果提示，经力达霉素诱导所致的BEL-7402细胞和L-02细胞裂亡与中心体过复制有关，并且不依赖于线粒体通路。异常的中心体导致多极有丝分裂纺锤体形成，进而引发染色体的不均等分配和以多核化或微核化大细胞为特更多还原

【Abstract】 Lidamycin (also designated C1027) is a member of the enediyne antibiotic family, has highly potent cytotoxicities toward tumor cells. It consists of a chromophore and an apoprotein, and the former has the ability to attack DNA, whereas the latter plays the role as a protecting protein. It has been observed that lidamycin can directly cleave DNA strands with preference of nucleotide and sequence. In addition to antitumor activities, lidamycin can inhibit the replication of viruses. The accumulation evidences indicate that lidamycin is a promising drug for clinical usage and valuable to be further studied.Mitotic cell death has been focused on in tumor therapy. However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the molecular mechanism and signal transduction of lidamycin-induced mitotic cell death.With 0.1 nmol/L or 0.5 nmol/L lidamycin for 2 h, followed by a 72-hour incubation in drug-free medium, the treated human hepatoma BEL-7402 cells and normal human liver L-02 cells both displayed mitotic cell death characterized by enlargement of cell volume, appearance of multinucleation, and arrest in G2-M phase of cell cycle. We measured the cells growth curves by the MTT assay. Compared with the control cells, the treated cells showed the marked growth rate decrease and the delay in double time of proliferation. Subconfluent cells were continuously incubated for 72 h after exposure to low concentration of lidamycin for 2 h, and then were co-stained with the DNA-specific fluorescent dyes Hoechst 33342 and propidium iodide. A unique and atypical chromatin condensation occurred. The cells with this kind of condensation are characterized by integrated karyotheca, adhering on the bottom, and appearance of small "dots" representing segregated condensed chromatin without apoptotic bodies. The genomic DNA samples were extracted from the BEL-7402 cells and L-02 cells incubated continuously for 72 h after 2-hour treatments of lidamycin. The DNA ladder was never obtained in each sample whatever the exposure to 0.1 nmol/L or 0.5 nmol/L lidamycin. Neither BEL-7402 cells nor L-02 cells displayed apoptotic peaks after treatment of lidamycin at a low concentration. With Giemsa staining, the multinucleation was observed in BEL-7402 cells at the 12th hour after lidamycin treatment, whereas the appearance of更多还原