Istituzione di cellule proliferativa tetraploide da non trasformate fibroblasti umani
Summary
Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.
Abstract
Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.
Introduction
Poliploidia è stata osservata non solo in tessuti specializzati di specie di mammiferi, ma anche in una varietà di condizioni patologiche, come il cancro e malattie degenerative. Poliploidi (per lo più tetraploide), le cellule sono spesso osservate nelle lesioni preneoplastiche di tessuti umani, come l'esofago 1,2 o squamose lesioni intraepiteliali di Barrett della cervice 3,4, e sono stati considerati per essere la fonte di cellule aneuploidi maligne in quei tessuti 5 , 6. Sebbene si suggerisce che la conversione di tetraploide di cellule aneuploidi potrebbe essere un evento cruciale nelle prime fasi della tumorigenesi, i meccanismi coinvolti in questo processo non sono pienamente compresi. Questo in parte perché nessun modello in vitro è stato disponibile dove non trasformate cellule umane poliploidi possono propagarsi.
Alcuni ricercatori hanno indotto tetraploidia in cellule epiteliali umane non trasformate attraverso la generazione di cellule binucleate di inhibiting cytokinesis 7-9. In questo metodo, tuttavia, le cellule diploidi inutili devono essere eliminate mediante fluorescenza-attivato cell sorting (FACS) 7,8 o la clonazione limitando la diluizione 9. Dato che queste procedure sono laboriose e non facile da eseguire, i metodi più semplici per stabilire le cellule non trasformate tetraploide si desiderano per la ricerca in questo campo.
Nella presente relazione, si descrive un protocollo per stabilire le cellule proliferative tetraploide da fibroblasti umani normali o fibroblasti umani telomerasi-immortalata da procedure relativamente semplici. Le procedure vengono utilizzate mandrino veleno demecolcine (DC) per arrestare le cellule diploidi in mitosi, e le cellule mitotiche raccolti da scrollarsi di dosso sono ulteriormente trattati con DC. cellule mitotiche diploidi trattate con corrente continua per tempo prolungato convertire in cellule G1 tetraploide, e queste cellule proliferano le cellule come tetraploide dopo l'arresto della crescita per diversi giorni dopo la rimozione di droga. Questo protocollo prevedeun metodo efficiente per la creazione di un modello utile per studiare la relazione tra il cromosoma instabilità e il potenziale oncogeno di cellule umane poliploidi.
Protocol
Representative Results
Discussion
Un grave problema nel induzione di tetraploidia da cellule diploidi di agenti chimici, sia da inibitori Citochinesi o con inibitori del fuso, è che le cellule diventano spesso un miscuglio di popolazioni diploidi e tetraploidi, e le cellule tetraploidi devono essere separati dalle cellule diploidi. approcci più comuni per l'isolamento di una popolazione tetraploide privo di cellule diploidi utilizzano FACS o clonazione, limitando la diluizione. Tuttavia, queste procedure sono laboriosa e non facile da eseguire. In…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Mrs. Matsumoto for the technical assistance.
Materials
| MEM-α | Sigma-Aldrich | M8042-500ML | |
| Trypsin-EDTA | Sigma-Aldrich | T4174 | |
| FBS | Sigma-Aldrich | 172012-500ML | |
| Demecolcine solution (10 μg/mL in HBSS) | Sigma-Aldrich | D1925-10ML | |
| BD CycleTES Plus DNA Reagent Kits | BD Biosciences | #340242 | For examination of DNA ploidy by flow cytometry |
| Human chromosome multicolor FISH probe 24XCyte | MetaSystems | #D-0125-060-DI | Specialized filter set and software for mFISH analysis are necessary |
| Isis imaging system with mFISH software | MetaSystems | Specialized probe kit is necessary |
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