Active centromere and chromosome identification in fixed cell lines
Active centromere and chromosome identification in fixed cell lines
复制标题
固定细胞系中的活性着丝粒和染色体鉴定
作者：
T. T. Beh;R. MacKinnon;P. Kalitsis
在细胞分裂过程中，着丝粒在确保染色体分离的保真度方面起着至关重要的作用。然而，在癌症和体质性疾病中，染色体上存在一个以上的活性着丝粒可能是染色体不稳定性的一个促成因素，也可能对疾病进展具有预测价值，因此检测功能正常的着丝粒非常重要。到目前为止，广泛用于功能性着丝粒检测的抗体主要作用于新鲜收获的细胞，而大多数细胞遗传学样品长期储存在甲醇-乙酸固定剂中。因此，我们的目的是确定抗体，将识别活性着丝粒抗原甲醇-乙酸固定的细胞。测试了一组活性着丝粒蛋白抗体，我们发现兔抗人CENP-C单克隆抗体识别甲醇-乙酸中固定的细胞的活性着丝粒。然后，我们测试并比较了已建立的方法即着丝粒荧光原位杂交（cenFISH）、着丝粒蛋白免疫荧光（CENP-IF）和多色FISH（mFISH）的组合，并显示了CENP-IF与cenFISH一起，然后用前述抗CENP-C抗体进行mFISH（CENP-IF-cenFISH-mFISH）的有用性。我们进一步证明了我们的方法在两种具有高比例着丝粒缺陷的癌细胞系中的实用性，即新着丝粒和功能性双着丝粒。我们建议将使用市售兔单克隆抗CENP-C的CENP-IF-cenFISH-mFISH方法纳入已建立的方法中，例如双着丝粒染色体测定（DCA）、产前核型筛查以及体质和癌症核型分析。这种方法将提供一个更准确的评估着丝粒异常状态的染色体不稳定性疾病。
The centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. However, in cancer and constitutional disorders, the presence of more than one active centromere on a chromosome may be a contributing factor to chromosome instability and could also have predictive value in disease progression, making the detection of properly functioning centromeres important. Thus far, antibodies that are widely used for functional centromere detection mainly work on freshly harvested cells whereas most cytogenetic samples are stored long-term in methanol-acetic acid fixative. Hence, we aimed to identify antibodies that would recognise active centromere antigens on methanol-acetic acid fixed cells. A panel of active centromere protein antibodies was tested and we found that a rabbit monoclonal antibody against human CENP-C recognises the active centromeres of cells fixed in methanol-acetic acid. We then tested and compared combinations of established methods namely centromere fluorescence in situ hybridisation (cenFISH), centromere protein immunofluorescence (CENP-IF) and multicolour FISH (mFISH), and showed the usefulness of CENP-IF together with cenFISH followed by mFISH (CENP-IF-cenFISH-mFISH) with the aforementioned anti-CENP-C antibody. We further demonstrated the utility of our method in two cancer cell lines with high proportion of centromere defects namely neocentromere and functional dicentric. We propose the incorporation of the CENP-IF-cenFISH-mFISH method using a commercially available rabbit monoclonal anti-CENP-C into established methods such as dicentric chromosome assay (DCA), prenatal karyotype screening in addition to constitutional and cancer karyotyping. This method will provide a more accurate assessment of centromere abnormality status in chromosome instability disorders.
影响因子：
11.2
作者：
A. Roschke;G. Tonon;Kristen S Gehlhaus;Nicolas McTyre;K. Bussey;S. Lababidi;D. Scudiero;J. Weinstein;I. Kirsch
通讯作者：
A. Roschke;G. Tonon;Kristen S Gehlhaus;Nicolas McTyre;K. Bussey;S. Lababidi;D. Scudiero;J. Weinstein;I. Kirsch