Dutra A, Pak E, Wincovitch S, John K, Poirier M C, Olivero O A
Cytogenetics and Microscopy Core, National Human Genome Research Institute, National Cancer Institute, NIH, Bethesda, MD 20892-4255, USA.
Cytogenet Genome Res. 2010;128(1-3):105-10. doi: 10.1159/000298794. Epub 2010 Apr 20.
Normal diploid somatic mammalian cell division generates 2 daughter cells as a result of a strict and well-controlled mitotic process. However, some defects during the progression of that process could generate an unbalanced distribution of chromosomes, aneuploidy and eventually, a malignant phenotype. Previous observations using a transgenic mouse model with diminished DNA repair capacity revealed the presence of nuclear buds (NBs) induced in vitro by the nucleoside analog zidovudine (Retrovir(R), 3'-azido-3'-deoxythymidine, AZT). Here we used bone marrow mesenchymal cells, taken from mice with the Xpa(-/-)Trp53(+/-) genotype, that were cultured and exposed to 0 and 100 muM AZT for 24 hours. Fixed and denatured cells were processed by fluorescence in situ hybridization (FISH) with whole chromosome painting probes used to identify chromosomes in cells growing on glass chamber slides (2 probes/slide). A variety of sizes and shapes of NBs were observed. Some NBs had a large connection with the main nucleus (>(1/4) of the NB diameter), others hada smaller connection (<(1/4) of the NB diameter), some were circular and positioned close to the nucleus, while some resided in the cytoplasm separated from the nucleus or connected by a thin chromatin strand. We had hypothesized that NBs would progress in the process of budding until separation occurred, but this was not proven by time-lapse photography studies performed for 20 hours. From 1,126 cells scored in the unexposed cultures, 10.39 % of cells carried NBs, while from 1,108 cells scored in the AZT-exposed cultures 29.16% of cells carried NBs (p = 0.001). In AZT-exposed cells there were a total of 322 NBs scored; 46.6% or 150 NBs contained positive signals for one or both probes used, while 53% or 172 NBs had no probe signal. In addition, FISH analysis showed no preferential localization of any chromosome within the NBs. Among the NBs that carried no probe signal, the presence of positive signals with inversion of DAPI imaging demonstrated centromeric content. It has been hypothesized that NBs occur as a result of expulsion of amplified DNA from the main nucleus; however, this data demonstrates that NBs may contain any chromosome, suggesting that NBs do not consist of just amplified DNA.
正常的二倍体哺乳动物体细胞通过严格且受良好控制的有丝分裂过程产生2个 daughter 细胞。然而，该过程进展期间的一些缺陷可能导致染色体分布不均衡、非整倍体，最终产生恶性表型。先前使用DNA修复能力降低的转基因小鼠模型进行的观察显示，核苷类似物齐多夫定（叠氮胸苷，AZT）在体外诱导产生了核芽（NBs）。在此，我们使用来自Xpa(-/-)Trp53(+/-)基因型小鼠的骨髓间充质细胞，将其培养并暴露于0和100μM的AZT中24小时。固定并变性的细胞通过荧光原位杂交（FISH）处理，使用全染色体涂染探针来识别在玻璃腔室载玻片上生长的细胞中的染色体（每张载玻片2个探针）。观察到了各种大小和形状的NBs。一些NBs与主核有较大连接（>NB直径的1/4），其他的连接较小（<NB直径的1/4），一些呈圆形且靠近核定位，而一些位于与核分离的细胞质中或由细染色质链连接。我们曾假设NBs会在出芽过程中进展直至发生分离，但对其进行20小时的延时摄影研究并未证实这一点。在未暴露培养物中计数的1126个细胞中，10.39%的细胞带有NBs，而在暴露于AZT的培养物中计数的1108个细胞中，29.16%的细胞带有NBs（p = 0.001）。在暴露于AZT的细胞中，总共计数到322个NBs；46.6%或150个NBs对所用的一种或两种探针含有阳性信号，而53%或172个NBs没有探针信号。此外，FISH分析显示任何染色体在NBs内均无优先定位。在没有探针信号的NBs中，DAPI成像反转时阳性信号的存在表明有着丝粒成分。有人曾假设NBs是由于扩增的DNA从主核中排出而产生的；然而，这些数据表明NBs可能包含任何染色体，这表明NBs并非仅由扩增的DNA组成。
