Izzo M, Antoccia A, Degrassi F, Tanzarella C
Dipartimento Biologia, Terza Università, Rome, Italy.
Mutagenesis. 1998 Sep;13(5):445-51. doi: 10.1093/mutage/13.5.445.
To study the mechanisms leading to diazepam (DZ)-induced chromosome loss, we evaluated the effect of the drug on the distribution of cytoplasmic and mitotic apparatus proteins using specific antibodies. The use of antibodies directed against dynein and kinetochores (CREST staining) suggested that chromosomes arranged in monopolar spindles were interacting with short fibers originating from the monopole. Interestingly, nearly 50% of DZ-induced monopolar mitoses showed a punctate staining of centrosomes when evaluated with an anti-gamma-tubulin antibody. The extent of phosphorylation of mitotic proteins was not affected by drug treatment, as shown by staining the cells with an antibody against mitotic phosphorylated proteins (MPM-2). After recovery of DZ, nearly 20% of anaphases were abnormal and mainly consisted of multipolar anaphases and lagging chromosomes; this was consistent with a high frequency of kinetochore-containing micronuclei as evaluated by CREST antibody staining in cells that had divided only once after drug removal, i.e. binucleate cells obtained by cytochalasin-B treatment. Our data confirmed that DZ is a powerful inducer of chromosome loss in cultured rodent cells. Moreover, our results indicate that DZ interfered with the correct assembly of centrosomes.
为研究导致地西泮（DZ）诱导染色体丢失的机制，我们使用特异性抗体评估了该药物对细胞质和有丝分裂器蛋白分布的影响。使用针对动力蛋白和动粒（着丝粒蛋白染色）的抗体表明，排列在单极纺锤体中的染色体与源自单极的短纤维相互作用。有趣的是，当用抗γ-微管蛋白抗体评估时，近50%的DZ诱导的单极有丝分裂显示中心体呈点状染色。如用抗有丝分裂磷酸化蛋白（MPM-2）抗体对细胞进行染色所示，有丝分裂蛋白的磷酸化程度不受药物处理的影响。DZ作用恢复后，近20%的后期异常，主要由多极后期和滞后染色体组成；这与在药物去除后仅分裂一次的细胞（即通过细胞松弛素B处理获得的双核细胞）中，用着丝粒蛋白染色评估的含动粒微核的高频率一致。我们的数据证实，DZ是培养的啮齿动物细胞中染色体丢失的强力诱导剂。此外，我们的结果表明，DZ干扰了中心体的正确组装。
