demecolcine and Chromosomal-Instability

Recent studies indicate that mammalian chromosomes contain discrete cis-acting loci that control replication timing, mitotic condensation, and stability of entire chromosomes. Disruption of the large non-coding RNA gene ASAR6 results in late replication, an under-condensed appearance during mitosis, and structural instability of human chromosome 6. Similarly, disruption of the mouse Xist gene in adult somatic cells results in a late replication and instability phenotype on the X chromosome. ASAR6 shares many characteristics with Xist, including random mono-allelic expression and asynchronous replication timing. Additional "chromosome engineering" studies indicate that certain chromosome rearrangements affecting many different chromosomes display this abnormal replication and instability phenotype. These observations suggest that all mammalian chromosomes contain "inactivation/stability centers" that control proper replication, condensation, and stability of individual chromosomes. Therefore, mammalian chromosomes contain four types of cis-acting elements, origins, telomeres, centromeres, and "inactivation/stability centers", all functioning to ensure proper replication, condensation, segregation, and stability of individual chromosomes.

Topics: Animals; Aurora Kinases; Chromosomal Instability; Chromosomes; Demecolcine; DNA Damage; DNA Replication Timing; Humans; Mammals; Mitosis; Phenotype; Protein Serine-Threonine Kinases; Regulatory Sequences, Nucleic Acid; Replication Origin; X Chromosome Inactivation

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.

Topics: Cell Line; Cell Proliferation; Chromosomal Instability; Demecolcine; DNA; Female; Fibroblasts; Flow Cytometry; Fluorescent Dyes; Humans; Karyotyping; Mitosis; Tetraploidy

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4 days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2 weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3 days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.

Topics: Aneuploidy; Cell Culture Techniques; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Centrosome; Chromosomal Instability; Demecolcine; DNA; Fibroblasts; Humans; Spindle Apparatus; Tetraploidy

DNA content of diploid H1 (ES) cells (2H1 cells) has been shown to be stable in long-term culture; however, tetraploid and octaploid H1 (ES) cells (4H1 and 8H1 cells, respectively) were DNA-unstable. Pentaploid H1 (ES) cells (5H1 cells) established recently have been found to be DNA-stable; how, then is cell DNA stability determined? To discuss ploidy stability, decaploid H1 (ES) cells (10H1 cells) were established from 5H1 cells and examined for DNA stability.. 5H1 cells were polyploidized using demecolcine (DC) and 10H1 cells were obtained by one-cell cloning.. Number of chromosomes of 10H1 cells was 180 and durations of their G(1), S, and G(2)/M phases were 3, 7 and 6 h respectively. Volume of 10H1 cells was double that of 5H1 cells and morphology of 10H1 cells was flagstone-like in shape. 10H1 cells exhibited alkaline phosphatase activity and their DNA content decayed in 91 days of culture. 10H1 cells injected into mouse abdomen formed solid tumours that contained several kinds of differentiated cells with lower DNA content, suggesting that 10H1 cells were pluripotent and DNA-unstable. Loss of DNA stability was explained using a hypothesis concerning DNA structure of polyploid cells as DNA reconstructed through ploidy doubling was arranged in mirror symmetry in a new configuration.. In the pentaploid-decaploid transition of H1 cells, cell cycle parameters and pluripotency were retained, but morphology and DNA stability were altered.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Division; Chromosomal Instability; Demecolcine; DNA; Embryonic Stem Cells; G1 Phase; G2 Phase; Mice; Polyploidy; S Phase

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.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Lineage; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomal Instability; Chromosome Aberrations; Demecolcine; DNA; Gene Expression Regulation, Neoplastic; Mice; Neoplasms; Pluripotent Stem Cells; Polyploidy; Stem Cell Transplantation

The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and mutations in three of these, BLM, WRN, and RECQL4, are associated with human diseases and a cellular phenotype that includes genomic instability. To date, no human disease has been associated with mutations in RECQL and no cellular phenotype has been associated with its deficiency. To gain insight into the physiological function of RECQL, we disrupted Recql in mice. RECQL-deficient mice did not exhibit any apparent phenotypic differences compared to wild-type mice. Cytogenetic analyses of embryonic fibroblasts from the RECQL-deficient mice revealed aneuploidy, spontaneous chromosomal breakage, and frequent translocation events. In addition, the RECQL-deficient cells were hypersensitive to ionizing radiation, exhibited an increased load of DNA damage, and displayed elevated spontaneous sister chromatid exchanges. These results provide evidence that RECQL has a unique cellular role in the DNA repair processes required for genomic integrity. Genetic background, functional redundancy, and perhaps other factors may protect the unstressed mouse from the types of abnormalities that might be expected from the severe chromosomal aberrations detected at the cellular level.

Topics: Alleles; Animals; Cells, Cultured; Chromosomal Instability; Demecolcine; DNA Damage; Electroporation; Embryonic Stem Cells; Fibroblasts; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Radiation, Ionizing; RecQ Helicases; RNA, Messenger; Sister Chromatid Exchange; Tissue Distribution