Subcellular Localization of the Spindle Proteins Aurora A, Mad2, and BUBR1 Assessed by Immunohistochemistry

Abstract

The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.

SMAD4 Suppresses AURKA-Induced Metastatic Phenotypes via Degradation of AURKA in a TGFβ-Independent Manner

“…3E, we observed nucleic-cytoplasmic expression of SMAD4 and cytoplasmic localization of overexpressed AURKA by using immunofluorescence staining. Similar observations were made in previous studies (39,40). As shown in the PLA results, the interaction of SMAD4 and AURKA was localized to the cytosol, further supporting the findings obtained using HeLa cells (Fig.…”

“…In normal tissues, AURKA was mainly localized to centrosome, but in malignant tissues, AURKA showed additional staining in cytoplasm. Immunohistochemical analysis revealed that AURKA overexpression was frequently found in the cytoplasmic region in cancer cell (40,53). Moreover, some reports suggested that AURKA overexpression is likely to target cytoplasmic substrates related to oncogenic transformation, such as h-CPEB (54) and GSK-3b (26).…”

BUBR1 expression in benign oral lesions and squamous cell carcinomas: Correlation with human papillomavirus

“…1). In this study, we observed only immunolabeling of BUBR1 in the cell cytoplasm, as previously reported (28). Even in most cases showing high BUBR1 expression, the relationship between qualitative BUBR1 expression and clinicopathological features (location, TNM stage, treatment, recurrence and death) did not show a positive association, except regarding location when all malignant samples were analyzed together (p=0.0078±0.0003).…”

“…All the steps were performed at room temperature. A normal tonsil, which is an accessible tissue containing highly proliferative cells in its germinal centers, was chosen as a positive control sample (28). A negative control sample was performed by replacing the specific primary antibody with PBS.…”

Mitotic Checkpoints and Chromosome Instability Are Strong Predictors of Clinical Outcome in Gastrointestinal Stromal Tumors

“…Immunohistochemistry experiment was realized on tissue microarrays (TMA) containing 15 cases from the present series and carried out as previously described (20). Antigen retrieval was achieved using the Dako Target Retrieval Solution, pH 9 for 20 minutes at 98 C. Slides were incubated for 1 hour with the AURKA antibody used at a dilution…”