Establishment of Proliferative Tetraploid Cells from Normal Human Fibroblasts

Abstract

The chromosomal instability of polyploid cells, which leads to the formation of aneuploid cells, is causally related to carcinogenesis in human tissues. However, the precise link between the chromosomal instability of polyploid cells and oncogenic transformation of them remains elusive. This is partly because we lack an experimental model in which non-transformed polyploid human cells can propagate in vitro. In a previous report, we demonstrated that proliferative tetraploid cells can be established from TIG-1 human fibroblasts by treatment with the spindle poison demecolcine (DC, colcemid) for 4 days. However, this procedure could not be applied to other human fibroblast strains because the resulting cells proliferated as a mixture of diploid and tetraploid populations. Here, we report a modified procedure to establish proliferative tetraploid cells from human fibroblasts of the BJ strain with minimum contamination by diploid cells. In the modified procedure, DC-arrested mitotic cells were collected by mitotic shake-off and treated with DC for an additional 3 days. DC-treated cells restarted proliferation as tetraploid cells after several days of growth arrest and showed similar growth to that of untreated diploid cells. The MDM2 antagonist Nutlin-3a activated p53 in established tetraploid cells and suppressed their growth, indicating that these cells have functional p53. These results contradicted the hypothesis that p53 functions as the tetraploidy checkpoint and prevents proliferation of tetraploid cells. Tetraploid cells established by our method could be a valuable model for the study of chromosomal instability and the oncogenic potential of polyploid cells.

Establishment of proliferative tetraploid cells from telomerase‐immortalized normal human fibroblasts

“…When establishing tetraploid cells for carcinogenesis studies, the TP53 status must be investigated because of the hypothesis that TP53 functions as the tetraploidy checkpoint and prevents proliferation of tetraploid cells ( Di Leonardo et al, ; Khan and Wahl, ; Andreassen et al, ; Vogel et al, ; Margolis, ; Aylon et al, ) . However, our previous studies found that proliferation of tetraploid cells established by our method is similar to the original diploid cells despite having functional TP53 ( Ohshima and Seyama, ) . In this study, tetraploid cells established from immortalized TIG‐1 cells had functional TP53 because treatment with the MDM2 antagonist Nutlin‐3a or a DNA‐damaging agent etoposide activated TP53 and CDKN1A, and suppressed cell growth.…”

“…When establishing these cells, continuous treatment of immortalized TIG‐1 cells with DC for 4 days resulted in a mixture of diploid and tetraploid populations; the diploid population could not be eliminated. Therefore, we employed a mitotic shake‐off method, which was applied to establish tetraploid cells from BJ human fibroblasts in our previous report ( Ohshima and Seyama, ) . Treatment with DC for 1 day to arrest cells at mitosis and an additional 3 days after mitotic shake‐off resulted in proliferation of cells with tetraploid DNA content after growth arrest for about 1 week.…”

“…We showed that tetraploid cells capable of propagating indefinitely can be established from immortalized human fibroblasts by the method we reported previously (Ohshima and Seyama, 2013). In this procedure, we employed a combination of a shake-off method and DC treatment to establish tetraploid cells.…”

“…When establishing these cells, continuous treatment of immortalized TIG-1 cells with DC for 4 days resulted in a mixture of diploid and tetraploid populations; the diploid population could not be eliminated. Therefore, we employed a mitotic shakeoff method, which was applied to establish tetraploid cells from BJ human fibroblasts in our previous report (Ohshima and Seyama, 2013).…”

“…In previous studies, we showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment for several days with the spindle poison demecolcine (DC) ( Ohshima and Seyama, ) . Established tetraploid cells formed bipolar spindles with two centrosomes in mitosis and proliferate similarly to the original diploid cells.…”