Dodecaploid H1 embryonic stem cells abolished pluripotency in L15F10 medium both with and without leukemia inhibitory factor.

Two lines of dodecaploid H1 embryonic stem cells, 12H1 and 12H1(-) cells (mouse-originated cells), were established through polyploidization of two hexaploid H1 cells, 6H1 and 6H1(-) cells, which were cultured in L15F10 (7:3) medium with and without leukemia inhibitory factor (LIF), respectively. The G1, S, and G2/M phase fractions of 12H1 and 12H1(-) cells were almost the same as those of 6H1 and 6H1(-) cells, respectively, but the doubling time of cell proliferation was prolonged, suggesting that cell death occurred in 12H1 and 12H1(-)cells. The cell volumes of 12H1 and 12H1(-) cells were about double those of 6H1 and 6H1(-) cells, respectively.

Effects of etoposide on the proliferation of hexaploid H1 (ES) cells.

Etoposide is a specific inhibitor of topoisomerase II, which is an enzyme that enables double-stranded DNA to pass through another double-stranded DNA. Topoisomerase II is a major constituent of chromosome scaffold, existing at appreciable amounts in cells. To examine the effects of etoposide on the cell cycle, hexaploid H1 (ES) cells (6H1 cells) were used with diploid H1 (ES) cells (2H1 cells) as a control.

Haploid unit-ploidy transition of tetraploid and octaploid H1 (ES) cells in long-term culturing.

Haploid unit-ploidy transition in tetraploid and octaploid mouse H1 (ES) cells (4H1 and 8H1 cells, respectively) during long-term culturing was observed using flow cytometry. The DNA content of 4H1 cells was elevated from 3.5C to 4.

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

Hexaploid H1 (ES) cells (6H1 cells) were established from octaploid H1 cells (8H1 cells), as were pentaploid H1 cells (5H1 cells). 6H1 cells were compared with 5H1 cells. The number of chromosomes of 6H1 cells was 115, 20 more than the 95 of 5H1 cells.

Different responses between diploid and tetraploid H1 embryonic stem cells appeared during long-term culturing in L15F10 medium without leukemia inhibitory factor.

To examine the alteration in cellular characteristics of polyploid embryonic stem (ES) cells during long-term culturing without leukemia inhibitory factor (LIF), mouse diploid and tetraploid H-1 (ES) cells (2H1 and 4H1 cells, respectively) were cultured without LIF for approximately 5 months. 2H1 and 4H1 cells were adapted to the medium without LIF by decreasing the concentration for several passages, and they were denoted as 2H1(⁻) and 4H1(⁻) cells, respectively. DNA content of 4H1(⁻) cells decreased gradually in the early stage, increased abruptly in the second stage, and then was maintained for a long time.

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

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.

Cell cycle, morphology and pluripotency of octaploid embryonic stem cells in comparison with those of tetraploid and diploid cells.

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.

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

To examine the alteration in cellular characteristics of polyploid ES cells during long-term culturing, tetraploid H-1 (ES) cells were continuously cultured for 180 days. Cellular DNA content of the tetraploid cells decreased and reached a plateau of 3.3 C, where C represents the complement of haploid chromosomes.

Polyploidization of 2nH1 (ES) cells by K-252a and staurosporine.

Mouse 2nH1 (ES) cells were examined for polyploidization using K-252a and staurosporine. Though 2nH1 cells were polyploidized by both K-252a and staurosporine, tetraploid cells, 4nH1K cells, were obtained only from cell populations exposed to K-252a. The probability of successful establishment of tetraploid cells was 2/9, suggesting that the highly polyploidized-tetraploid transition might occur infrequently.

An hypothesis about genome structures in mammalian polyploid cells based on a new concept that genome is fractal of six hierarchies.

A new model for the spatial configurations of DNA is proposed to solve the problem of DNA loss in mammalian polyploid cells. The ordinary concept that chromosomes are situated independently in nuclei cannot account well for the DNA loss in polyploid cells. A new concept about the DNA configurations in diploid cells is constructed based on observations that have been reported.

Induction of centrosome amplification and chromosome instability in human bladder cancer cells by p53 mutation and cyclin E overexpression.

Centrosome amplification frequently occurs in human cancers and is a major cause of chromosome instability (CIN). In mouse cells, centrosome amplification can be readily induced by loss or mutational inactivation of p53. In human cells, however, silencing of endogenous p53 alone does not induce centrosome amplification or CIN, although high degrees of correlation between p53 mutation and CIN/centrosome amplification in human cancer can be detected, suggesting the presence of additional regulatory mechanism(s) in human cells that ensures the numeral integrity of centrosomes and genomic integrity.

Phorbol myristate induces apoptosis of taxol-resistant sarcoma cells in vitro.

Taxol was found to induce polyploidization and apoptosis in cultured methylcholanthrene-induced sarcoma cells (Meth-A cells), but some of the cells in G1 phase were not affected. We refer to these cells as taxol-resistant cells. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) regulator, was used to test the taxol-resistant cells.

Octaploid Meth-A cells are established from a highly polyploidized cell population.

Tetraploid Meth-A cells were polyploidized by demecolcin, an inhibitor of spindle fibre formation in M phase, and then released from the drug 1, 2, 3 and 4 days after the addition. Octaploid cells were successfully established from cell populations including hexadecaploid cells produced by 2, 3 and 4 days of exposure to demecolcin. One-day-treated cells were polyploidized octaploid cells, but they returned to tetraploid cells.

Establishment of a triploid V79 cell line from tetraploid cells obtained through polyploidization using K-252a.

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.