Control of DNA synthesis in polyploid mammalian cells

To test the hypothesis that the duration of DNA synthesis is an inverse function of nuclear size or DNA content, the S phase was calculated from PLM analysis for pseudodiploid, tetraploid, and octaploid lines of Chinese hamster cells growing as a monolayer or in suspension. S phase times were found not to be significantly different between polyploid lines and the diploid lines from which they were derived, regardless of the conformation of the nucleus. There is no evidence, therefore, that would implicate the nuclear membrane, or nuclear surface area/volume relationships, in the control of DNA synthesis.     

Intranuclear and cytoplasmic annulate lamellae in the polyploid giant cells of the trophoblast

Intranuclear and cytoplasmic annulate lamellae in polyploid giant cells of the trophoblast have been studied in rat placenta on days 12--17 of development. The annulate lamellae are present in the cytoplasm within a limited time, being visible on day 12 only. These are arranged in bundles near the nucleus to be moving then to the cytoplasm. The end parts of annulate lamellae are broadened to make cisterns of rough endoplasmic reticulum. Unlike the cytoplasmic annulate lamellae, those found within the nucleus are seen in part of the nuclei investigated throughout the whole period examined to look as single structures (not gathered in bundles), they can be branching, separating closed spaces within the nucleus (making local swellings in the loci of branching; the latter having electron dense or transparent vesicles). Association with nuclear chromatin in some regions is a peculiar feature of the intranuclear annulate lamellae. This association is especially obvious at endoprophase in the cycle ofthe polytene nucleus during the somatic conjugation--chromonemes unite in a bundle and condense. Ultrastructural changes of the annulate lamellae is noted throughout the polytene nucleus cycle and during the cell differentiation. It is supposed that in the case of temporary labile chromosome polyteny in the nuclear cycle, which is characteristic of mammalian trophoblasts, annulate lamellae can well compare, in their function, with the synaptonemal complex--these prevent from too tight associations of homologues in the course of somatic conjugation of chromosomes.     

Accumulation of polyploid cells and G2-phase cells during ascites tumor growth

Ehrlich ascites tumor cells from the plateau phase of growth were transplanted into new hosts, pulse-labeled with tritiated thymidine and blocked with repeated injections of vinblastine. When unlabeled cells were analyzed for their cellular DNA content utilizing a cytophotometric technique it was found that in relation to the total number of cells (labeled plus unlabeled), 13% had a 2C DNA content, 36% a 4C DNA content and 5% an 8C DNA content at 0.5 hours after transplantation. By 24 hours the distributions changed dramatically: the initially unlabeled 2C cells were now 4C, the 36% of the cells that were initially 4C partitioned into 24% that were still 4C and 12% that progressed to 8C, and the initial 8C cells remained 8C. These studies indicate that the accumulation of 4C cells during the plateau phase of growth is due to a combination of G2 diploid and G1 tetraploid cells.     

Limiting dilution analysis of the specificity of influenza-immune cytotoxic T cells

The frequency of memory T cells in the spleens of mice primed with the A/Puerto Rico/8/34/1 (H1N1) (PR8) influenza A virus was determined using limiting dilution protocols. The mean frequency of memory cytotoxic T lymphocytes (CTL) in spleen populations from mice primed with PR8 and restimulated in vitro with the same virus ranged, in six experiments, from 1 in 1600 to 1 in 4800. In the same experiments, the frequencies of CTL capable of lysing targets infected with the heterologous A/Hong Kong/×31/68 (H3N2) (HK) virus ranged from 1 in 1700 to 1 in 4700 nucleated spleen cells. Thus, at least 80% of PR8 (H1N1) influenza-specific cytotoxic T cells are lytic for both HK (H3N2)- and PR8-infected target cells. Further analysis of the specificity of a series of monoclonal influenza-specific CTL was achieved by expanding limit dilution cultures and then testing lytic capacity for targets infected with a range of influenza A viruses. This approach confirmed that the great majority of PR8-primed influenza-specific CTL are cross-reactive for a variety of influenza A subtypes. These experiments demonstrate the feasibility of quantitating different influenza-immune CTL specificities at a stage very close to removal of cells from the animal.     

Limiting dilution analysis of the stem cells for T cell lineage

Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.     

Underreplication of repetitive DNA in polyploid cells of Pisum sativum.

Earlier reported results of a difference in the amount of DNA between 2C meristematic plumula cells and 2C cortex cells of the epicotyls are confirmed by fluorometric Feulgen-DNA determinations. Renaturation experiments of isolated DNA suggest that this difference in DNA is probably correlated with an underreplication of some highly repetitive DNA sequences in cells preparing endomitosis.     

The breeding of two polyploid rice lines with the characteristic of polyploid meiosis stability

Polyploidization is a basic feature of plant evolution. Nearly all of the main food, cotton and oil crops are polyploid. When ploidy levels increase, yields double; this phenomenon suggested a new strategy of rice breeding that utilizes wide crosses and polyploidization dual advantages to breed super rice.Because low seed set rates in polyploid rice usually makes it difficult to breed, the selection of Ph-liked gene lines was emphasized. After progenies of indica-japonica were identified and selected, two polyploid lines, PMeS-1 and PMeS-2 with Polyploid Meiosis Stability (PMeS) genes were bred. The procedure included seven steps: selecting parents, crossing or multiple crossing, back-crossing, doubling chromosomes, identifying the polyploid, and choosing plants with high seed set rates that can breed themselves into stable lines. The characteristics of PMeS were determined by observing meiotic behaviors and by cross-identification of seed sets. PMeS-1 and PMeS-2, (japonica rice), have several characteristics different from other polyploid rice lines, including a higher rate of seed set (more than 65%, increasing to more than 70% in their F1 offspring); and stable meiotic behaviors (pairing with bivalents and quarivalents nearly without over-quarivalent in prophase, nearly without lagging chromosomes in metaphase and without micronuclei in anaphase and telophase). The latter was obviously different from control polyploid line Dure-4X, which displayed abnormal meiotic behaviors including a higher rate of multivalents, univalents and trivalents in prophase, lagging chromosomes in metaphase and micronuclei in anaphase and telophase. There were also three differences of the breeding method between PMeS lines and normal diploid lines: chromosomes doubling, polyploidism identifying and higher seed set testing. The selection of PMeS lines is the first step in polyploid rice breeding; their use will advance the progress of polyploid rice breeding, which will in turn offer a new way to breed super rice.     

Progressive telomere dysfunction causes cytokinesis failure and leads to the accumulation of polyploid cells

Most cancer cells accumulate genomic abnormalities at a remarkably rapid rate, as they are unable to maintain their chromosome structure and number. Excessively short telomeres, a known source of chromosome instability, are observed in early human-cancer lesions. Besides telomere dysfunction, it has been suggested that a transient phase of polyploidization, in most cases tetraploidization, has a causative role in cancer. Proliferation of tetraploids can gradually generate subtetraploid lineages of unstable cells that might fire the carcinogenic process by promoting further aneuploidy and genomic instability. Given the significance of telomere dysfunction and tetraploidy in the early stages of carcinogenesis, we investigated whether there is a connection between these two important promoters of chromosomal instability. We report that human mammary epithelial cells exhibiting progressive telomere dysfunction, in a pRb deficient and wild-type p53 background, fail to complete the cytoplasmatic cell division due to the persistence of chromatin bridges in the midzone. Flow cytometry together with fluorescence in situ hybridization demonstrated an accumulation of binucleated polyploid cells upon serial passaging cells. Restoration of telomere function through hTERT transduction, which lessens the formation of anaphase bridges by recapping the chromosome ends, rescued the polyploid phenotype. Live-cell imaging revealed that these polyploid cells emerged after abortive cytokinesis due to the persistence of anaphase bridges with large intervening chromatin in the cleavage plane. In agreement with a primary role of anaphase bridge intermediates in the polyploidization process, treatment of HMEC-hTERT cells with bleomycin, which produces chromatin bridges through illegimitate repair, resulted in tetraploid binucleated cells. Taken together, we demonstrate that human epithelial cells exhibiting physiological telomere dysfunction engender tetraploid cells through interference of anaphase bridges with the completion of cytokinesis. These observations shed light on the mechanisms operating during the initial stages of human carcinogenesis, as they provide a link between progressive telomere dysfunction and tetraploidy.     

Spontaneous mutation rate to thioguanine resistance is decreased in polyploid hamster cells

The mutation rate to thioguanine resistance was 3.11 X 10(-6) in a near diploid V79 hamster cell line and 7.58 X 10(-8) in a near tetraploid derivative produced with colchicine. The specific activities of glucose-6-phosphate dehydrogenase and phosphoglycerate kinase of the tetraploid line were greater than that of the diploid which suggests that twice the number of active X chromosomes were present in the tetraploid. These results are compatible with the hypothesis that spontaneous variants resistant to thioguanine arise through mutation and chromosomal segregation, as has been suggested for induced mutations in tetraploid hamster cells.     

Assessing the monophyly of polyploid Gossypium species

The origin and monophyly of the polyploid cotton (Gossypium) species has been largely accepted, despite the lack of explicit phylogenetic evidence. Recent studies in other polyploid systems have demonstrated that multiple origins for polyploid species are much more common than once thought, raising the possibility that Gossypium polyploids also had multiple origins, as postulated by some authors. To test the monophyly of polyploid cotton, we sequenced a 2.8-kb intergenic region from all diploid species belonging to the genome groups from which the polyploid originates. The resulting phylogenetic analyses strongly support a single origin of polyploid cotton involving a D-genome ancestor related to Gossypium raimondii and an A-genome ancestor that was sister to both extant A-genome species.     

Expression characterization of testicular DMRT1 in both Sertoli cells and spermatogenic cells of polyploid gibel carp

Dmrt1 has been suggested to play significant roles in sex determination and differentiation, but various expression patterns and cell types have been observed in the testis of vertebrates. Polyploid gibel carp, because of the multiple modes of unisexual gynogenesis and sexual reproduction, has become a unique case to explore the evolution of sex determination and differentiation. However, the sex-determination related genes in gibel carp have remained unknown. In this study, we identified and characterized 4 cDNAs of Dmrt1 genes. Subsequently, a polyclonal antibody specific to CagDMRT1 was prepared to examine its expression and distribution patterns at protein level. Significantly, both relative real-time PCR and Western blot detection confirmed predominant expression of CagDmrt1 in the adult testis of gibel carp. Moreover, the intensive expression of CagDMRT1 around spermatogenic cysts was revealed during spermatogenesis. And, following immunofluorescence co-localization of CagDMRT1 and CagVASA, a prominent CagDMRT1 expression in Sertoli cells and a mild CagDMRT1 expression in spermatogenic cells including spermatogonia and primary spermatocytes were clearly characterized. The CagDMRT1 signal in Sertoli cells is extensively distributed in both nuclei and cytoplasm, while the CagDMRT1 in spermatogonia and primary spermatocytes is mainly expressed in nuclei, and there is only the remained CagDMRT1 signal in the cytoplasm of secondary spermatocytes. These findings suggest that DMRT1 should be related to testis differentiation and spermatogenesis in gibel carp.     

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.