Etablering av proliferativ tetraploid celler från icke-transforme humanfibroblaster
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
Polyploidi har observerats inte bara i specialiserade vävnader hos däggdjursarter, utan också i en mängd olika patologiska tillstånd, såsom cancer och degenerativa sjukdomar. Polyploida (mestadels tetraploida celler) observeras ofta i preneoplastiska lesioner av mänskliga vävnader, såsom Barretts esofagus 1,2 eller skivepitelcancer intraepitelial lesioner i livmoderhalsen 3,4, och har ansetts vara en källa till maligna aneuploida celler i dessa vävnader 5 , 6. Även om det föreslås att omvandlingen av tetraploid till aneuploida celler kan vara en avgörande händelse i de tidiga stadierna av tumörbildning, är de mekanismer som är involverade i denna process inte helt klarlagt. Detta beror delvis på att ingen in vitro-modell har funnits där icke-transforme polyploida humana celler kan föröka sig.
Vissa forskare har inducerat tetraploidy i icke-transforme humana epitelceller genom generering av binucleated celler genom inhibiting cytokines 7-9. I denna metod är emellertid onödiga diploida celler måste elimineras genom fluorescensaktiverad cellsortering (FACS) 7,8 eller kloning genom begränsande spädning 9. Eftersom dessa förfaranden är arbetskrävande och inte lätt att utföra, för att enklare metoder fastställa icke-transforme tetraploida celler önskas för forskning på detta område.
I denna rapport beskriver vi ett protokoll för att etablera proliferativa tetraploida celler från normala humana fibroblaster eller telomeras-förevigat humana fibroblaster genom relativt enkla förfaranden. Procedurerna använder spindelgift demekolcin (DC) för att stoppa diploida celler i mitos och mitotiska celler som samlats in genom att skaka av ytterligare behandlas med DC. Diploida mitotiska celler som behandlats med DC för längre tid konvertera till tetraploida G1 celler, och dessa celler förökar sig som tetraploida celler efter tillväxtstopp under flera dagar efter borttagning läkemedel. Detta protokoll geren effektiv metod för att skapa en användbar modell för att studera sambandet mellan kromosom instabilitet och den onkogena potentialen hos polyploida humana celler.
Protocol
Representative Results
Discussion
Ett stort problem i induktion av tetraploidy från diploida celler genom kemiska medel, antingen genom cytokines hämmare eller spindel hämmare, är att cellerna blir ofta en blandning av diploida och tetraploida populationer, och tetraploida celler måste separeras från diploida celler. Vanligaste metoder för isolering av en tetraploid befolkning utan diploida celler använder FACS eller kloning genom begränsande utspädning. Emellertid är dessa förfaranden är arbetskrävande och inte lätt att utföra. I denna …
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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