A pluripotent tetraploid cell line (4nH1 cells) was established.

Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K-252a, an inhibitor of protein kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a karyotype of three homologueous chromosomes. The duration of the G1, S and G2/M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid-tetraploid-triploid transition is proposed.

To examine the alteration of cellular characteristics on ploidy transition of embryonic stem (ES) cells, octaploid cells (8H1 cells) were established from tetraploid H-1 (ES) cells, and compared with tetraploid and diploid H-1 (ES) cells (4H1 and 2H1 cells, respectively). The duration of G(1), S, and G(2)/M phases were essentially the same among 2H1, 4H1, and 8H1 cells, suggesting that cell cycle progression is conserved. The ratio of cell volume of 2H1, 4H1, and 8H1 cells was about 1 : 2 : 4, indicating that these polyploid cells were generated through cell cycle progression without cell division. The morphology of 8H1 cells was flagstone-like and flatter than that of 4H1 cells, and differed from the spindle-like shape of 2H1 cells, suggesting that transformation occurred during the ploidy transitions. Alkaline phosphatase activity was expressed equivalently in 2H1, 4H1, and 8H1 cells, and solid tumors that contained endodermal, mesodermal, and ectodermal cells were formed by 2H1, 4H1 or 8H1 cells after interperitoneal injection into the mouse abdomen, suggesting that pluripotency was preserved in the ploidy transition.

Pentaploid H1 (ES) cells (5H1 cells) were accidentally obtained through one-cell cloning of octaploid H1 (ES) cells (8H1 cells) that were established from tetraploid H1 (ES) cells (4H1 cells) polyploidized using demecolcine. The number of chromosomes of 5H1 cells was 100, unlike the 40 of diploid H1 (ES) cells (2H1 cells), 80 of 4H1, and 160 of 8H1 cells. The durations of G(1), S, and G(2)/M phases of 5H1 cells were 3, 7, and 6 h, respectively, almost the same as those of 2H1, 4H1, and 8H1 cells. The cell volume of 5H1 cells was half of that of 8H1 cells, suggesting that 5H1 cells were created through abnormal cell divisions of 8H1 cells. The morphology of growing 5H1 cells was a spherical cluster similar to that of 2H1 cells and differing from the flagstone-like shape of 4H1 and 8H1 cells. Pentaploid solid tumors were formed from 5H1 cells after interperitoneal injection into the mouse abdomen, and they contained endodermal, mesodermal, and ectodermal cells as well as undifferentiated cells, suggesting both that the DNA content of 5H1 cells was retained during tumor formation and that the 5H1 cells were pluripotent. The DNA content of 5H1 cells was stable in long-term culturing as 2H1 cells, meaning that 5H1 and 2H1 cells shared similarities in DNA structure. The excellent stability of the DNA content of 5H1 cells was explained using a hypothesis for the DNA structure of polyploid cells because the pairing of homologous chromosomes in 5H1 cells is spatially forbidden.

SummaryThe DNA content of polyploid cells sometimes decreases during subculturing. The mechanism of DNA reduction is not yet known. Precise measurements of DNA decay and related alterations in polyploid cells will be required to understand the mechanism. Diploid, tetraploid and octaploid Meth-A cells were continuously cultured for 244 d and the cellular DNA content was measured from the DNA histograms. The DNA content decays gradually with day t as expressed by f(t)ϭI p exp{ϪG(t)}, where I p is the initial ploidy, G(t)ϭat/{exp(Ϫbt)ϩct}, and a, b and c are the following parameters: aϭ0.026, bϭ0.01 and cϭ0.0175 (for tetraploid cells) or cϭ0.01 (for octaploid cells). The DNA content of diploid Meth-A cells was constant within the experimental errors. The DNA loss of polyploid cells was confirmed by a decrease in chromosome number. The cellular morphology changed in diploid and octaploid Meth-A cells, but not in tetraploid cells, suggesting that DNA loss and morphology alteration are independent. The doubling time shortened with culture time in the tetraploid and octaploid cells. The findings suggested that chromosomes are not independent in polyploid cells. The DNA content of mammalian diploid cells is well preserved during subculturing; however, that of polyploid cells is not. The DNA content of polyploid cells sometimes decreases gradually, and occasionally it decreases abruptly by half. A few studies of DNA degradation in polyploid cells have been reported. Moor, et al. (1968) concluded that near triploid is the terminal ploidy of neartetraploid Ehrlich's ascites tumor cells based on 3 years of observation. Harris (1971) showed that the chromosome number was constant in diploid cells but decreased with subculturing in tetraploid and octaploid pig kidney cells. Graves and McMillan (1984) reported that tetraploid DON and BI Chinese hamster cells were karyotypically stable but the octaploid cells were not. In spite of these long-term studies, the mechanism of DNA content reduction is not yet known.Meth-A cells always include a small population of large cells that are produced from diploid cells by spontaneous polyploidization and are eventually removed by apoptosis (Fujikawa-Yamamoto et al. 1997). Meth-A cells may be particularly susceptible to polyploid transformation. Tetraploid Meth-A cell lines have been reproducibly established from diploid Meth-A cells highly polyploidized by demecolcine (Fujikawa-Yamamoto et al. 2001). Octaploid Meth-A cells were also produced from tetraploid cells highly polyploidized by demecolcine (Fujikawa-Yamamoto et al. 2003). The diploid, tetraploid and octaploid Meth-A cells showed marked differences in the expression of cell surface hydrocarbon chains, suggesting that the ploidy transformation is accompanied by functional alterations (Fujikawa-Yamamoto and Sakuma 2003). The diploid, tetraploid and octaploid Meth-A cell lines may provide a cell system for investigating polyploid cells.