eriments were per formed by quantitative, Aurora A specific, indirect immunofluorescence with asynchronized cells, to detect occurrence of mitoses and multipolar mitoses in view of their own cell cycle dynamics. Parallel hematoxylin and eosin staining Paclitaxel human endothelial cells con firmed the data on mitotic cells morphologically and pericentrin specific indirect immunofluorescence Inhibitors,Modulators,Libraries confirmed the presence of Aurora A associated supernu merary centrosomes. To specify the previous flow cytometric analyses, which only provided data on the total number of G2 M phase cells, the mitotic index was evaluated in indirect immunofluorescence analysis of Aurora A and nuclear staining. For each cell line at least 100 cells were counted in three independent experiments. This revealed the highest mitotic index in OE21, followed by OE33, OE19, Kyse 410 and EPC hTERT cells.
Similarly, the occurrence of multipolar mitoses was assessed by quantifying Inhibitors,Modulators,Libraries indirect immunofluorescence analysis of Aurora A and nuclear stainings. For this, in each cell line at least 80 mitoses were counted in three independent experiments. Aurora A positive multipolar mitoses were most frequent in OE33 followed by OE21 and Kyse 410 cells. OE19 cells as well as EPC hTERT cells, if any, only had single Aur ora A positive multipolar mitoses. Presence of supernu merary centrosomes in these multipolar mitoses was confirmed by pericentrin staining. These data suggest that similarly high Inhibitors,Modulators,Libraries Aurora A expression alone is insufficient to induce prominent multipolar mitoses in aneuploid esophageal cancer cells.
Distinct p53 mutations contribute to multipolar mitoses in esophageal cancer cells In view of the role of p53 in post mitotic cell cycle control, centrosome duplication and Aurora A interac tion as well as its frequent mutation in eso phageal carcinogenesis, we next determined p53 mutation status, p53 protein Inhibitors,Modulators,Libraries expression and intracellular localization in the control EPC hTERT cell line and in the four esophageal cancer cell lines. The control EPC hTERT cells exhibited a wild type p53 sequence and showed weak p53 protein expression in immunoblot and indirect immunofluores cence analysis. This wild type p53 protein was located in the cytoplasm of EPC hTERT cells. In contrast, all ESCC and BAC cell lines displayed p53 mutations, OE21 cells exhibited p53 muta tions in exon 4, which introduce a stop codon at the N terminus of the p53 core domain.
The p53 protein of OE21 cells lacks almost the entire DNA binding domain, the tetrameriza tion domain and the extreme C terminus. This protein, if Cilengitide at all being expressed, is most likely non functional since almost all domains are missing, including the Aurora A interaction sites Serine 215 and 315. Indeed, immuno blot analysis did not detect this largely truncated p53 protein and immunofluorescence showed only weak and rather diffusely localized p53 staining directly in OE21 cells. Kyse 410 cells displayed a point mutation in exon 10 of the tetramerization domain. This mutation was neither found i