DNA Loss and Related Alterations in Long-term Cultures of Diploid, Tetraploid and Octaploid Meth-A Cells

Abstract

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.

DNA stable pentaploid H1 (ES) cells obtained from an octaploid cell induced from tetraploid cells polyploidized using demecolcine

“…Harris (1971) showed that the chromosome number was constant in diploid cells but decreased with subculturing in tetraploid and octaploid pig kidney cells. While tetraploid and octaploid Meth-A cells lost the DNA content in long-term culturing (Fujikawa-Yamamoto et al, 2005), triploid V79 Chinese hamster cells were stable, as were the parent diploid cells (Fujikawa-Yamamoto et al, 2002).…”

“…The DNA content of tetraploid and octaploid Meth-A cells decayed gradually with culturing and reached a plateau phase (Fujikawa-Yamamoto et al, 2005). While several studies reported DNA loss in polyploid cells, the DNA content of triploid V79 cells was stable (Fujikawa-Yamamoto et al, 2002), except in a special case where the cells were suspension-cultured .…”

Alteration and preservation of cellular characteristics in long-term culture of tetraploid H-1 (ES) cells

“…The equation also indicates that the rate of reduction of DNA is 0.04 C/day in the initial stage, –df(0)/dt = 0.04, and that the DNA content at the final stage is 3.3 C, f(∞) = 3.3. We have reported that the DNA decay of tetraploid Meth‐A cells can be described using the above equation with a = 0.0026, b = 0.01 and c = 0.0175 8 . The values of these parameters might be different depending on the type of cells.…”

“…Whether the DNA content in polyploid cells is stable is still unknown. The DNA content of octaploid and tetraploid Meth‐A cells (a methylcholanthrene‐induced mouse abdominal dropsy sarcoma cell line) gradually decreased with culturing, and eventually reached a plateau phase 8 . In contrast, the DNA content of triploid V79 Chinese hamster lung cells that were established from the diploid cells was stable during long‐term culturing 9 .…”

Hexaploid H1 (ES) cells established from octaploid H1 cells are as DNA stable as pentaploid H1 cells

“…Harris [6] showed that the chromosome number was constant in diploid cells, but decreased with subculturing in tetraploid and octaploid pig kidney cells. Tetraploid [7] and octaploid [8] Meth-A cells exhibited gradual decay of DNA with culturing and reached a plateau phase [9]. Triploid V79 cells, however, were DNA-stable [10], except in a special case where the cells were suspension-cultured [11].…”