Zijno A, Leopardi P, Marcon F, Crebelli R
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Rome, Italy.
Mutat Res. 1996 Dec;372(2):211-9. doi: 10.1016/s0027-5107(96)00141-8.
The application of methods based on in situ hybridization to centromeric regions to cytokinesis-blocked cells provides a convenient way for the analysis of chromosome segregation in interphase cells. In this way, the reciprocal segregation patterns in daughter nuclei can be visualized and most of the problems related to the artefactual loss or gain of chromosomes which flaw other methods are avoided. In this work, the methodology has been applied to human lymphocytes to investigate the influence of donor age on spontaneous malsegregation rates, the occurrence of multiple malsegregation events, and the effect of the cytokinesis-blocking agent cytochalasin B (Cyt B) on spontaneous and induced chromosome malsegregation. The results obtained with 14 male donors, aged 22-57 years, demonstrated a significant (p < 0.001) increase in the frequency of micronuclei and X chromosome missegregation (both non-disjunction and chromosome loss) with the increasing age of the donors. Moreover, a similar association was observed with cultures hybridized with either chromosome 8 or 18 centromere probes, suggesting that the age-related loss of fidelity in chromosome segregation in vitro may be a general trait. The investigation of the distribution of multiple malsegregation events in cultured lymphocytes of eight male and nine female donors, with the simultaneous hybridization with pairs of centromeric probes (for chromosomes X and 8 or X and 18), demonstrated a large excess of multiple events with respect to that expected by random segregation. This fact may highlight the existence of cellular subpopulation(s) prone to malsegregate, or indicate that the malsegregation of one chromosome is able to affect the fidelity of segregation of the other chromosomes. Finally, the possible influence of Cyt B on chemically induced malsegregation has been investigated with the analysis of chromosomes X and 8 signals in nuclei of lymphocyte cultures treated with vinblastine (2.5-20 ng/ml) in the presence and absence of 6 micrograms/ml Cyt B. Vinblastine induced a small increase in hyperploidy of either chromosome X or 8 at 10 ng/ml in cultures treated with Cyt B. Without Cyt B, a significant increase of hyperploidy was only observed at the highest dose assayed (20 ng/ml). This vinblastine dosage had a severe inhibitory effect on cultures treated with Cyt B, where no binucleated cells were detected. At all doses, a relatively greater mitotic index was observed in cultures with Cyt B, suggesting a synergistic effect of this drug with vinblastine. Most notably, at the two highest vinblastine dosages (10 and 20 ng/ml), a large incidence of polyploid nuclei was observed in cytokinesis-blocked cultures, whereas none or far lower increases of polyploidy were found in the absence or Cyt. B. This results provides direct evidence of the potential of Cyt B to indirectly interfere with chromosome misdistribution induced by a spindle poison, to be considered before drawing firm conclusions from kinesis-blocked systems.
将基于原位杂交的方法应用于着丝粒区域的胞质分裂阻滞细胞，为分析间期细胞中的染色体分离提供了一种便捷的方法。通过这种方式，可以观察到子核中的相互分离模式，并且避免了与其他方法中染色体人为丢失或增加相关的大多数问题。在这项工作中，该方法已应用于人类淋巴细胞，以研究供体年龄对自发错分率、多个错分事件的发生以及胞质分裂阻滞剂细胞松弛素B（Cyt B）对自发和诱导染色体错分的影响。对14名年龄在22至57岁之间的男性供体进行的研究结果表明，随着供体年龄的增加，微核和X染色体错分（包括不分离和染色体丢失）的频率显著增加（p < 0.001）。此外，在用8号或18号染色体着丝粒探针杂交的培养物中也观察到了类似的关联，这表明体外染色体分离中与年龄相关的保真度丧失可能是一个普遍特征。对8名男性和9名女性供体的培养淋巴细胞中多个错分事件的分布进行研究，同时与着丝粒探针（针对X和8号染色体或X和18号染色体）对进行杂交，结果表明多个事件的发生大大超过了随机分离预期的数量。这一事实可能突出了易发生错分的细胞亚群的存在，或者表明一条染色体的错分能够影响其他染色体分离的保真度。最后，通过分析在用长春花碱（2.5 - 20 ng/ml）处理的淋巴细胞培养物细胞核中X和8号染色体信号，研究了Cyt B对化学诱导错分的可能影响，处理时分别添加和不添加6微克/毫升的Cyt B。在用Cyt B处理的培养物中，长春花碱在10 ng/ml时导致X或8号染色体超倍体有小幅增加。在没有Cyt B的情况下，仅在最高检测剂量（20 ng/ml）时观察到超倍体有显著增加。该长春花碱剂量对用Cyt B处理的培养物有严重抑制作用，未检测到双核细胞。在所有剂量下，在用Cyt B处理的培养物中观察到有相对较高的有丝分裂指数，表明该药物与长春花碱有协同作用。最值得注意的是，在长春花碱的两个最高剂量（10和20 ng/ml）下，在胞质分裂阻滞的培养物中观察到多倍体核的发生率很高，而在没有Cyt B或添加Cyt B的情况下，多倍体的增加很少或更低。这一结果提供了直接证据，表明Cyt B有可能间接干扰纺锤体毒物诱导的染色体错分布，在从胞质分裂阻滞系统得出确凿结论之前应予以考虑。
