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Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells.

Fichter CD, Herz C, Münch C, Opitz OG, Werner M, Lassmann S - BMC Cell Biol. (2011)

Bottom Line: Single multipolar mitoses occurred in OE19 (1.0 ± 1.0%) cells.Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only.Additional p53 loss of function mutations are necessary for this to occur, at least for invasive esophageal cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, University Medical Center, Freiburg, Germany.

ABSTRACT

Background: Aurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC) and Barrett's adenocarcinoma (BAC) remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines.

Results: A control esophageal epithelial cell line (EPC-hTERT) had normal Aurora-A and -B gene copy numbers and expression, was p53 wild type and displayed bipolar mitoses. In contrast, both ESCC (OE21, Kyse-410) and BAC (OE33, OE19) cell lines were aneuploid and displayed elevated gene copy numbers of Aurora-A (chromosome 20 polysomy: OE21, OE33, OE19; gene amplification: Kyse-410) and Aurora-B (chromosome 17 polysomy: OE21, Kyse-410). Aurora-B gene copy numbers were not elevated in OE19 and OE33 cells despite chromosome 17 polysomy. Aurora-A expression and activity (Aurora-A/phosphoT288) was not directly linked to gene copy numbers and was highest in Kyse-410 and OE33 cells. Aurora-B expression and activity (Aurora-B/phosphoT232) was higher in OE21 and Kyse-410 than in OE33 and OE19 cells. The mitotic index was highest in OE21, followed by OE33 > OE19 > Kyse-410 and EPC-hTERT cells. Multipolar mitoses occurred with high frequency in OE33 (13.8 ± 4.2%), followed by OE21 (7.7 ± 5.0%) and Kyse-410 (6.3 ± 2.0%) cells. Single multipolar mitoses occurred in OE19 (1.0 ± 1.0%) cells. Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only.

Conclusions: High Aurora-A expression alone is not associated with overt multipolar mitoses in aneuploid ESCC and BAC cancer cells, as specifically shown here for OE21 and OE33 cells, respectively. Additional p53 loss of function mutations are necessary for this to occur, at least for invasive esophageal cancer cells. Further assessment of Aurora kinases and p53 interactions in cells or tissue specimens derived from non-invasive dysplasia (ESCC) or intestinal metaplasia (BAC) are necessary to disclose a potential causative role of Aurora kinases and p53 for development of aneuploid, invasive esophageal cancers.

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p53 in normal esophageal epithelial cells and esophageal cancer cells. A. The p53 protein and its domain structure (modified from [39]). The normal esophageal epithelium cell line EPC-hTERT carries no p53 mutation whereas all esophageal cancer cell lines exhibit mutations of p53, but each in a different functional domain. Mutations are indicated by arrows (↓) and possible protein truncations by crosses (×). B. Photograph of p53 protein expression by immunoblot analysis, representative for 3 independent experiments. Due to mutations, the p53 protein of OE21 and OE19 is truncated, resulting in protein masses of about 14 kDa and 40 kDa, respectively. For OE19 this shift in protein mass was seen at the immunoblot (asterisks), the 14 kDa protein of OE21 cells was below the size of detectable proteins in these 10% SDS gels. β-Actin served as laoding control. C. p53 indirect immunofluorescence (green) and DNA counterstaining by DAPI (blue). Mutated p53 accumulates in the nucleus of Kyse-410, OE33 and OE19 cells. The truncated p53 protein of OE21 cells lacks almost all protein domains and is only weakly expressed. Refer to text for discussion of p53 mutations.
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Figure 5: p53 in normal esophageal epithelial cells and esophageal cancer cells. A. The p53 protein and its domain structure (modified from [39]). The normal esophageal epithelium cell line EPC-hTERT carries no p53 mutation whereas all esophageal cancer cell lines exhibit mutations of p53, but each in a different functional domain. Mutations are indicated by arrows (↓) and possible protein truncations by crosses (×). B. Photograph of p53 protein expression by immunoblot analysis, representative for 3 independent experiments. Due to mutations, the p53 protein of OE21 and OE19 is truncated, resulting in protein masses of about 14 kDa and 40 kDa, respectively. For OE19 this shift in protein mass was seen at the immunoblot (asterisks), the 14 kDa protein of OE21 cells was below the size of detectable proteins in these 10% SDS gels. β-Actin served as laoding control. C. p53 indirect immunofluorescence (green) and DNA counterstaining by DAPI (blue). Mutated p53 accumulates in the nucleus of Kyse-410, OE33 and OE19 cells. The truncated p53 protein of OE21 cells lacks almost all protein domains and is only weakly expressed. Refer to text for discussion of p53 mutations.

Mentions: In view of the role of p53 in post-mitotic cell cycle control, centrosome duplication and Aurora-A interaction [41-45,53] as well as its frequent mutation in esophageal carcinogenesis [4,10,11], we next determined p53 mutation status [54,55], p53 protein expression and intracellular localization [39] in the control EPC-hTERT cell line and in the four esophageal cancer cell lines (Figure 5).


Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells.

Fichter CD, Herz C, Münch C, Opitz OG, Werner M, Lassmann S - BMC Cell Biol. (2011)

p53 in normal esophageal epithelial cells and esophageal cancer cells. A. The p53 protein and its domain structure (modified from [39]). The normal esophageal epithelium cell line EPC-hTERT carries no p53 mutation whereas all esophageal cancer cell lines exhibit mutations of p53, but each in a different functional domain. Mutations are indicated by arrows (↓) and possible protein truncations by crosses (×). B. Photograph of p53 protein expression by immunoblot analysis, representative for 3 independent experiments. Due to mutations, the p53 protein of OE21 and OE19 is truncated, resulting in protein masses of about 14 kDa and 40 kDa, respectively. For OE19 this shift in protein mass was seen at the immunoblot (asterisks), the 14 kDa protein of OE21 cells was below the size of detectable proteins in these 10% SDS gels. β-Actin served as laoding control. C. p53 indirect immunofluorescence (green) and DNA counterstaining by DAPI (blue). Mutated p53 accumulates in the nucleus of Kyse-410, OE33 and OE19 cells. The truncated p53 protein of OE21 cells lacks almost all protein domains and is only weakly expressed. Refer to text for discussion of p53 mutations.
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Figure 5: p53 in normal esophageal epithelial cells and esophageal cancer cells. A. The p53 protein and its domain structure (modified from [39]). The normal esophageal epithelium cell line EPC-hTERT carries no p53 mutation whereas all esophageal cancer cell lines exhibit mutations of p53, but each in a different functional domain. Mutations are indicated by arrows (↓) and possible protein truncations by crosses (×). B. Photograph of p53 protein expression by immunoblot analysis, representative for 3 independent experiments. Due to mutations, the p53 protein of OE21 and OE19 is truncated, resulting in protein masses of about 14 kDa and 40 kDa, respectively. For OE19 this shift in protein mass was seen at the immunoblot (asterisks), the 14 kDa protein of OE21 cells was below the size of detectable proteins in these 10% SDS gels. β-Actin served as laoding control. C. p53 indirect immunofluorescence (green) and DNA counterstaining by DAPI (blue). Mutated p53 accumulates in the nucleus of Kyse-410, OE33 and OE19 cells. The truncated p53 protein of OE21 cells lacks almost all protein domains and is only weakly expressed. Refer to text for discussion of p53 mutations.
Mentions: In view of the role of p53 in post-mitotic cell cycle control, centrosome duplication and Aurora-A interaction [41-45,53] as well as its frequent mutation in esophageal carcinogenesis [4,10,11], we next determined p53 mutation status [54,55], p53 protein expression and intracellular localization [39] in the control EPC-hTERT cell line and in the four esophageal cancer cell lines (Figure 5).

Bottom Line: Single multipolar mitoses occurred in OE19 (1.0 ± 1.0%) cells.Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only.Additional p53 loss of function mutations are necessary for this to occur, at least for invasive esophageal cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Pathology, University Medical Center, Freiburg, Germany.

ABSTRACT

Background: Aurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC) and Barrett's adenocarcinoma (BAC) remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines.

Results: A control esophageal epithelial cell line (EPC-hTERT) had normal Aurora-A and -B gene copy numbers and expression, was p53 wild type and displayed bipolar mitoses. In contrast, both ESCC (OE21, Kyse-410) and BAC (OE33, OE19) cell lines were aneuploid and displayed elevated gene copy numbers of Aurora-A (chromosome 20 polysomy: OE21, OE33, OE19; gene amplification: Kyse-410) and Aurora-B (chromosome 17 polysomy: OE21, Kyse-410). Aurora-B gene copy numbers were not elevated in OE19 and OE33 cells despite chromosome 17 polysomy. Aurora-A expression and activity (Aurora-A/phosphoT288) was not directly linked to gene copy numbers and was highest in Kyse-410 and OE33 cells. Aurora-B expression and activity (Aurora-B/phosphoT232) was higher in OE21 and Kyse-410 than in OE33 and OE19 cells. The mitotic index was highest in OE21, followed by OE33 > OE19 > Kyse-410 and EPC-hTERT cells. Multipolar mitoses occurred with high frequency in OE33 (13.8 ± 4.2%), followed by OE21 (7.7 ± 5.0%) and Kyse-410 (6.3 ± 2.0%) cells. Single multipolar mitoses occurred in OE19 (1.0 ± 1.0%) cells. Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only.

Conclusions: High Aurora-A expression alone is not associated with overt multipolar mitoses in aneuploid ESCC and BAC cancer cells, as specifically shown here for OE21 and OE33 cells, respectively. Additional p53 loss of function mutations are necessary for this to occur, at least for invasive esophageal cancer cells. Further assessment of Aurora kinases and p53 interactions in cells or tissue specimens derived from non-invasive dysplasia (ESCC) or intestinal metaplasia (BAC) are necessary to disclose a potential causative role of Aurora kinases and p53 for development of aneuploid, invasive esophageal cancers.

Show MeSH
Related in: MedlinePlus