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Abstract
BackgroundThe 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.ResultsA 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.ConclusionsWe 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.
Highlights
The centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions
We report on and discuss (1) the screening outcome of several kinetochore antibodies for fixed cells, (2) the difference between the proposed method involving immunocytochemistry, fluorescence in situ hybridisation (FISH) and multicolour FISH (mFISH), and the combination of other methods, and (3) the potential utility of the proposed method with the positive antibody, rabbit monoclonal anti-CENP-C, in identifying chromosomes with structural centromere defects in clinical samples of patients with congenital diseases or cancer as exemplified using T-47D, a breast cancer cell line, and SN12C, a renal cancer cell line, from the NCI60 cancer panel
Centromere antibody screening With the awareness that most samples in the cytogenetic laboratories are stored long-term in methanol-acetic acid fixative and with the expectation that centromere status screening will provide useful information for these laboratories, we decided to screen several commercially available antibodies that target components of an active centromere using fixed HCT-15, a near diploid and lowly rearranged human colon cancer cell line [4]
Summary
The centromere plays a crucial role in ensuring the fidelity of chromosome segregation during cell divisions. One of the hallmarks of cancer is genome instability, often characterised by the presence of aneuploidy and genetic heterogeneity resulting from chromosome missegregation or defective DNA repair followed by the failure to enter cellular arrest or death [1, 2]. Such genetic heterogeneity ranges from the extent seen in leukaemias, generally presented with simple chromosomal rearrangements, to carcinomas that are often complex [3, 4]. A properly functioning centromere is essential for correct chromosome segregation during cell divisions
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