Zusammenfassung
Hintergrund
Tumorzelllinien stellen wertvolle präklinische Modelle sowohl zur Aufklärung tumorbiologischer Mechanismen als auch zur Identifizierung therapeutischer Zielstrukturen und Prüfung von pharmakologisch aktiven Wirkstoffen dar. Aus menschlichen Harnblasenkarzinomen wurden bisher etwa 50 Tumorzelllinien etabliert, vorwiegend aus invasiven und metastatischen Tumoren. Zwei davon (T24 und 253J) wurden experimentell zu mehreren Progressionsserien weiterentwickelt. Diese Modelle haben wichtige neue Erkenntnisse zu späteren Phasen der Tumorprogression und metastatischen Disseminierung erbracht. Nur wenige Zelllinien stehen allerdings als Modelle der papillären Harnblasenkarzinogenese zur Verfügung, zudem keine Progressionsserie aus nichtinvasiven, papillären Harnblasentumorzelllinien.
Material und Methode
Während der Etablierung einer Doxorubicin-resistenten Variante der papillären Harnblasenkarzinomzelllinie BFTC-905 haben wir eine Zellkolonie identifiziert, die offenbar Zellen mit unterschiedlichem Wachstumsmuster beinhaltete. Die anschließende Subkultivierung ergab 3 Tochterzelllinien: BFTC-905-kompakt, BFTC-905-diffus und BFTC-905-diffus M. Zu ihrer grundlegenden Charakterisierung wurde die Zellmorphologie, die membranständige Expression von E-Cadherin, der Karyotyp sowie Invasivität und Klonogenität im Agar untersucht. Die klonale Herkunft der BFTC-905-Tochterzelllinien wurde mittels molekulargenetischer Methoden analysiert.
Ergebnisse
Die Zelllinien der BFTC-905-Progressionsserie weisen Unterschiede in mehreren transformationsrelevanten phänotypischen Charakteristika auf. Die beiden diffusen Zelllinien (BFTC-905-diffus und BFTC-905-diffus M) unterschieden sich von der BFTC-905-kompakt-Zelllinie durch ihr deutlich weniger organisiertes Wachstumsmuster, das neben aneinander adhärierenden Zellen auch isoliert wachsende Zellen beinhaltete – dieser relative Verlust der Zell-Zell-Adhäsion ging mit entsprechendem Verlust der membranständigen Expression von E-Cadherin einher. Bei der BFTC-905-diffus-M-Tochterzelllinie wurde eine dramatische Zunahme der Chromosomenanzahl zu einem hypertetraploiden Karyotyp gefunden. Gleichzeitig kam es bei dieser Tochterzelllinie zur Entwicklung der Fähigkeit zum verankerungsunabhängigen Wachstum im Weichagar. Alle 3 Zelllinien der BFTC-905-Progressionsserie blieben nichtinvasiv. Die molekulargenetische Herkunftsanalyse bestätigte den klonalen Ursprung der Zelllinien.
Schlussfolgerungen
Die neu etablierte BFTC-905-Progressionsserie weist gegenüber anderen publizierten Progressionsserien 2 Charakteristika der frühen Progression des Harnblasenkarzinoms auf, nämlich eine dynamische Veränderung der Expression von E-Cadherin und eine komplexe karyotypische Entwicklung. Daher könnte sie zum weiteren Verständnis der Pathobiologie des Harnblasenkarzinoms wesentlich beitragen.
Abstract
Background
Tumour cell lines represent valuable preclinical models to decipher underlying biology and identify potential therapy targets and pharmacologically useful compounds. Approximately 50 human bladder cancer cell lines have been established to date, mainly from invasive and metastatic tumours. Two of these, namely T24 and 253J, were experimentally further developed into progression series. These models have provided important insights into later tumour progression events and metastatic dissemination. Only a few cell lines are available as models of non-invasive papillary bladder cancer and no progression series have yet been established.
Material and methods
During the course of establishing a doxorubicin-resistant variant cell line of the human papillary bladder carcinoma cell line BFTC-905, a unique cell colony was identified, apparently involving cells with divergent growth patterns. Subsequent subculturing yielded three daughter cell lines, BFTC-905-compact, BFTC-905-diffuse und BFTC-905-diffuse M. Their fundamental characterization included basic cell morphology, cell membrane expression of E-Cadherin, karyotype analysis, invasion and colony forming capacity in soft agar. The clonal origin of the newly established daughter cell lines was assessed by means of molecular genetic methods.
Results
We could identify important differences in multiple transformation related traits among the cell lines of the BFTC-905 progression series. Both diffuse cell lines (BFTC-905-diffuse und BFTC-905-diffuse M) differed from the BFTC-905-compact cell line by growing in a less organized,“diffuse” manner, which involved colonies of cells exhibiting apparently normal cell-to-cell adhesion as well as individual cells outside of them. This diminution of the cell-to-cell adhesion was accompanied by a corresponding decrease of membranous E-Cadherin. The BFTC-905-diffuse M cell line displayed a dramatic increase in the overall chromosome number, resulting in a hypertetraploid karyotype. At the same time, this cell line, as the only one in the progression series, acquired the ability to grow independent of anchorage in soft agar. All three cell lines remained noninvasive. Allelic distribution of highly polymorphic DNA-markers in the cell lines of the BFTC-905 progression series provided unequivocal evidence of their common origin.
Conclusion
The newly established BFTC-905 progression series manifests two aspects of the early progression of non-invasive bladder carcinoma, not exhibited by any other progression series published so far, namely dynamic changes in the expression of E-Cadherin and a complex karyotypic evolution. It may thus contribute important insights into further understanding of the pathobiology of bladder cancer.
Abbreviations
- BBN:
-
N-butyl-N-(4-hydroxybutyl)-nitrosamine
- CGH:
-
comparative genomic hybridization
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- EDTA:
-
ethylenediamine tetraacetic acid
- EMT:
-
epithelial-to-mesenchymal transition
- FANF:
-
N-[4-(5-nitro-2-furyl)-2-thiasolyl]-formamide
- FITC:
-
fluorescein isothiocyanate
- MMP:
-
matrix metalloproteinase
- MNU:
-
N-methyl-N-nitrosourea
- PBS:
-
phospate-buffered saline
- RT:
-
Raumtemperatur
- SV40:
-
Simian virus 40
- SCID:
-
severe combined immunodeficiency
- TIMP:
-
tissue inhibitor of metalloproteinases
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Danksagung
Die Arbeit entstand im Rahmen einer DAAD-Gastdozentur von PD Jiri Hatina an der Heinrich-Heine-Universität Düsseldorf. Die Arbeit im Forschungslaboratorium der Urologischen Klinik der Heinrich-Heine-Universität Düsseldorf wurde teilweise durch die Christiane und Claudia-Hempel-Stiftung unterstützt. J.H. ist durch das Forschungsprojekt MSM 0021620819 des Ministeriums für Schulwesen der Tschechischen Republik gefördert. Wir danken Frau Dr. Blanka Šedivá für Hilfe bei der statistischen Analyse der Daten.
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Hatina, J., Huckenbeck, W., Rieder, H. et al. Harnblasenkarzinomzelllinien als Modellsysteme zur Pathobiologie des Harnblasenkarzinoms. Urologe 47, 724–734 (2008). https://doi.org/10.1007/s00120-008-1687-4
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DOI: https://doi.org/10.1007/s00120-008-1687-4
Schlüsselwörter
- Harnblasenkarzinomzelllinien
- Progressionsserien
- Metastatische Disseminierung
- BFTC-905-Progressionsserie
- Pathobiologie des Harnblasenkarzinoms