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Discovery of TP53 splice variants in two novel papillary urothelial cancer cell lines

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Abstract

Background

Using a novel cell culture technique, we established two new cell lines, BC44 and BC61, from papillary urothelial carcinoma and analyzed them for genetic changes typical of this tumor type.

Methods and results

Karyotyping revealed aneuploid karyotypes with loss of chromosome 9 and rearranged chromosome 5p. Molecular analysis showed CDKN2A deletions but wild-type PIK3CA. BC61 contained a G372C FGFR3 mutation. TP53 was not mutated in either cell line and BC61 expressed normal full-length protein. In contrast, BC44 exclusively expressed cytoplasmic and nuclear p53Δ40 and 133 isoforms from the alternative promoter P2 as revealed by Western blotting, immunocytochemistry and PCR. The only discernible difference in TP53 in BC44 was homozygosity for the deletion allele of the rs17878362 polymorphism in the P2 promoter. Expression of p53 isoforms was also detected in a few other urothelial carcinoma cell lines and tumor cultures and in 4 out of 28 carcinoma tissues.

Conclusion

In urothelial cancers, TP53 is typically inactivated by mutations in one allele and loss of the wildtype allele and more frequently in invasive compared to papillary carcinomas. We show that some urothelial carcinomas may predominantly or exclusively express isoforms which are not detected by commonly used antibodies to epitopes located in the p53 TA amino-terminal region. Expression of these isoforms may constitute a further mode of p53 inactivation in urothelial carcinoma. Our findings raise the question to which extent this mechanism may compromise wildtype p53 function in papillary tumors in particular, where point mutations in the gene are rare.

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Acknowledgments

We are grateful to Ms. Andrea Meyer for initial establishment of the cell lines and to Prof. Rainer Jaenicke and Dr Günter Niegisch for helpful discussions. We are indebted to Dr. Bořivoj Vojtĕšek (Masaryk Memorial Cancer Institute, Brno, Czech Republic) for a generous gift of the DO12 antibody. We also thank Ms. Ina Bachmann for performing the comparable genomic hybridization analysis.

The study was supported by a start-up grant of the Forschungskommission der Medizinischen Fakultät of the Heinrich Heine University (H.H.S.), by a guest professorship of the German Academic Exchange Service (J.H.) and by the grant no. MSM 0021620819 ‘Replacement of and support to some vital organs of the Ministry of Education of the Czech Republic’ to J.H. A.K. is supported by a PhD fellowship from the Jürgen-Manchot foundation.

Conflict of interest

There are no conflicts of interest by any of the authors

Author information

Authors and Affiliations

  1. Urologische Klinik, Heinrich-Heine-Universität, Moorenstr. 5, 40225, Düsseldorf, Germany

    Annemarie Koch, Jiri Hatina, Hans-Helge Seifert, Andrea R. Florl & Wolfgang A. Schulz

  2. Department of Biology, Charles University Faculty of Medicine, Plzen, Czech Republic

    Jiri Hatina

  3. Institut für Humangenetik und Anthropologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225, Düsseldorf, Germany

    Harald Rieder

  4. Klinik für Urologie und Kinderurologie, Hegau-Bodensee-Klinikum, Virchowstr. 10, 78224, Singen, Germany

    Hans-Helge Seifert

  5. Institut für Rechtsmedizin, Heinrich-Heine-Universität, Moorenstr. 5, 40225, Düsseldorf, Germany

    Wolfgang Huckenbeck

  6. Institut für Pathologie, Heinrich-Heine-Universität, Moorenstr. 5, 40225, Düsseldorf, Germany

    Frank Jankowiak

  7. Institute of Pathology, University Hospital Erlangen, Erlangen, Germany

    Robert Stoehr

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  1. Annemarie Koch
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Correspondence to Wolfgang A. Schulz.

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Supplementary figure S1

p53 methylation analysis. Methylation-specific PCR with primers specific for methylated CpGs (M) or primers for unmethylated CpGs (U) following bisulfite treatment. Primers are located in the 5′-UTR in a sequence methylated in certain leukemia samples. The urothelial cancer cell lines BC44, BC61, 647v, 639v, 5637, VmCub1, HT1376, SW1710 and RT4 are all completely unmethylated in the analysed region. (GIF 464 kb)

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Koch, A., Hatina, J., Rieder, H. et al. Discovery of TP53 splice variants in two novel papillary urothelial cancer cell lines. Cell Oncol. 35, 243–257 (2012). https://doi.org/10.1007/s13402-012-0082-8

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