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PubMed1992

Effect of anticancer agents on cell cycle of regenerating hepatocytes in rats.

The effect of three anticancer agents, cisplatin, doxorubicin, and mitomycin, on liver regeneration after 70% partial hepatectomy in rats was investigated by total DNA content of the liver and flow cytometric analysis of hepatocyte nuclei using two-color staining of anti-bromodeoxyuridine monoclonal antibody and propidium iodide. Total DNA content of regenerating liver 7 days after hepatectomy showed significant suppression of regeneration by these agents (P less than 0.01). The inhibitory effects of the agents on the cell cycle of hepatocytes by flow cytometric analysis were (1) a delay of the peak or a decrease in the proportion of S phase nuclei and/or (2) polyploidization of the nuclei, demonstrated by accumulation of 8c and occasionally 16c nuclei, of which the DNA contents were four and eight times as much as that of diploid (2c) cell nuclei, respectively. The former (1) suggests G0 or G1 phase block, and the latter (2) G2 phase block. In terms of total DNA content of regenerating liver, the inhibitory effect was most prominent in the cisplatin-administered groups. The polyploidization of nuclei was most remarkable in the mitomycin-administered groups. Although the total DNA content recovered to the level of control at 6 weeks after hepatectomy, the polyploidization effect persisted in the drug-administered groups. These agents had no cytocidal action on proliferating hepatocytes as can be seen from the aspartate aminotransferase and alanine aminotransferase serum levels. We conclude that in a short-term observation, the anti-cancer agents significantly inhibit liver regeneration, although the inhibitory effect on DNA synthesis does not last long.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Puromycin resistance in Chinese hamster cells: genetic and biochemical studies of partially resistant, unstable clones.

Resistance to 10 microgram/ml of puromycin has been analyzed in V79 Chinese hamster cells. Clones that were isolated in 10 microgram/ml of puromycin and subsequently cultivated in its absence consistently lost their resistance. One clone was analyzed in detail by recloning in the presence and absence of puromycin, and it was found that non-puromycin cultivated subclones also lost their resistance and regained inhibition profiles similar to the V79 parent. Reconstruction experiments between sensitive and resistant cells demonstrated that the yield of mutants was not affected by metabolic cooperation. The mutation rate was calculated to be 1 x 10(-7) per cell generation, and was the same within the limits of statistical error in a colchicine-produced polyploid derivative of the V79 line. Although a number of resistant clones were found to have polyploid karyotypes, the polyploid V79 lines was not more resistant to puromycin, nor did it possess a higher frequency of puromycin resistant cells. Studies employing radiolabeled puromycin established that resistance was due to a lowered uptake of puromycin and that an inverse relationship existed between resistance level and uptake rate.

Animals↗
PubMed1994

Determination of DNA ploidy, nuclear size, and proliferative activity by means of the computer-assisted image analysis of Feulgen-stained nuclei in 68 soft tissue tumors of adults.

The diagnostic values of the ploidy level, the proliferative activity, and the nuclear size in a series of 68 soft tissue tumors of adults were determined by digital cell image analysis of Feulgen-stained nuclei from formalin-fixed, paraffin-embedded tissues. The DNA ploidy level was characterized by calculating the DNA index (DI) and the percentage of the diploid and polyploid cells, and by typing the DNA histogram. Proliferative activity assessments were a function of the determination of the proliferation index (PI), ie, the percentage of cells engaged in the S phase of the cell cycle (SPF value). The present series included 19 benign and 49 malignant soft tissue tumors. The results show that DNA aneuploidy, as assessed by both the DI and the DNA histogram type, cannot be used as a discriminatory parameter for distinguishing between benign and malignant soft tissue tumors. Indeed, some benign cases may be highly aneuploid, whereas some highly malignant soft tissue tumors may be definitely diploid. In contrast, the determination of the percentage of polyploid cell nuclei seems to be a useful parameter in distinguishing between benign and malignant cases. In fact, the benign soft tissue tumors showed a very significantly lower mean percentage value of polyploid cell nuclei than the malignant cases. The determination of the proliferative activity also discriminated significantly between the benign and the malignant cases, the former proliferating more slowly than the latter. Lastly, the determination of nuclear size made it possible to differentiate the primary malignant soft tissue tumors, whether recurrent or not, that were associated with metastasis from those free of metastasis.

Adult↗
PubMed1986

Enhanced polyploidy by glutaraldehyde in cultured HL60 leukemic cells.

Glutaraldehyde 10(-4) M weakened cell proliferation of HL60 cultured cells and enhanced the appearance of giant polyploid cells, up to 32.5% after 6 days. The size and structure of these cells, the quantitative changes in their DNA content with respect to diploid ones demonstrate their polyploid nature, which may be corrected by the occurrence of pluripolar mitoses. However the slowing down of cell proliferation is not enough to orient the cells towards differentiation. Maturation of polyploid cells may be stimulated by retinoic acid and dexamethasone as for diploid ones. Several possible mechanisms of polyploidy are discussed. Except the possibility that the cells may directly fuse, the mechanisms which are considered, may involve a preprophase inhibition, a mitotic arrest at metaphase or a reduction of asters which may result in a defect in cytokinesis, the latter followed by secondary fusion of nuclei.

Aldehydes↗
PubMed1992

The effect of gamma-irradiation on the toxicity of malathion in V79 Chinese hamster cells and Molt-4 human lymphocytes.

There is growing interest in the irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Extensive research has been done on this process, and irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. The question has been raised, however, whether irradiation of pesticide residues might produce radiation products that were more toxic or less toxic than the original pesticide. To address this question, we observed the effects of 10 kGy of gamma-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. We found no significant differences between the effects of irradiated and unirradiated malathion on any of these end-points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte cells, micronuclei formation was not affected by irradiated or unirradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in the cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems.

Animals↗
PubMed1987

Flow cytometric and karyotypic analyses of androgen-responsive and androgen-independent Shionogi mouse mammary tumours.

The androgen-responsive (AR) Shionogi mouse mammary carcinoma is a heterogeneous tumour composed of AR and androgen-independent (AI) cells. We characterized the cells of the AR tumour and those of its AI derivative by flow cytometric analysis of DNA content and karyotypic analysis of metaphase spreads. Both tumours had diploid and near tetraploid populations of cells. However, the AR and AI malignant cells of these tumours both appeared to be polyploid. A decrease in the polyploid population of the AR tumour accompanied tumour regression following castration, but this population was restored when tumour growth resumed. Although karyotypic analysis of metaphase spreads showed wide variations in chromosome numbers among the polyploid population, the range, 55-88 chromosomes, was found in both AR and AI tumours. In addition, the same chromosome anomalies, including a marker chromosome, were identified in both tumours. Since the AR and AI malignant cells could not be distinguished on the basis of their DNA content or karyotype, the cell types may not represent genetically distinct populations of cells. The AR cells may undergo alterations in gene expression in adapting to their androgen-free environment.

Androgens↗
PubMed1983

Liver polyploidy: influence of age and of dietary restriction.

Polyploidy increases with age in mammalian liver. Since dietary restriction increases lifespan, the question posed in this study was whether liver polyploidy would be influenced by dietary protein restriction. Restricted mice were fed a 4% protein diet, while control mice received a 26% protein diet. Polyploidy was determined from measurements of nuclear diameter on liver of 3 week, 1 1/2, 3, 10, 20 and 24-1/2 month old Swiss albino mice. The percentage of polyploid cells increased steadily with age. The rate of increase, expressed as the polyploidization index, was greater in the fully fed control animals as compared to the animals on protein restriction. Protein restriction thus retards the age associated process of polyploidization of liver nuclei. Nuclear diameter increased slightly with age, but was not influenced by protein restriction.

Aging↗
PubMed2005

Budded karyoplasts from multinucleated fibroblast cells contain centrosomes and change their morphology to mitotic cells.

Cell changes to accelerated rates of growth are determined by random, new gene mutations that have their origin in vitro in a morphologically visible process of cell alterations. It is a continuous process that, step by step, transforms the normal cell into extended life (EL) cells. These latter cells with limited life spans can further transform to immortalized cells (i.e., cell lines) by the same sequence of morphological cell changes. In contrast to human epithelial cells, fibroblasts in culture have not given rise spontaneously to EL cells. Therefore, it was assumed that some of the cell changes (i.e., cell indicators of the process of transformation) might not be present in near senescent fibroblast cell cultures. As a positive control to normal fibroblast expansion to senescence, the same cells were stressed by inadequate nutrition and confluence. Another positive control was cell indicators induced by SV40 infections. The consecutive sequence of the cell indicators in the transformation process reported previously were: (1) large polyploid cells with nuclei that contained more than two genomes, (2) fragmentation/amitosis of the polyploid nuclei to bi- and multinucleated cells (MNCs), and (3) nuclear budding (i.e., karyoplasts) from MNCs that gave rise to EL cell colonies with various longevities beyond the senescent phase. The present study shows that MNCs and karyoplasts were present in the near-senescent fibroblast cell cultures. Furthermore, new data on the following aspect of the cell transformation process are presented: (1) association of the nuclear fragmentation process with death of cells, (2) cytological markers that distinguish between fragmentation-MNCs versus MNCs from cell fusion, (3) cytological changes of karyoplasts that go through mitotic division to produce daughter cells, and (4) presence of two centrosomes (spindle polar bodies) in the budded karyoplasts. These new findings are discussed in regard to the nuclear fragmentation process in polyploid cells which gives rise to smaller, viable nuclei in MNCs with reduced numbers of whole genomes.

Biomarkers↗
PubMed2006

Regulation of growth by ploidy in Caenorhabditis elegans.

Some animals, such as the larvae of Drosophila melanogaster, the larvae of the Appendicularian chordate Oikopleura, and the adults of the nematode Caenorhabditis elegans, are unusual in that they grow largely by increases in cell size. The giant cells of such species are highly polyploid, having undergone repeated rounds of endoreduplication. Since germline polyploid strains tend to have large cells, it is often assumed that endoreduplication drives cell growth, but this remains controversial. We have previously shown that adult growth in C. elegans is associated with the endoreduplication of nuclei in the epidermal syncitium, hyp 7. We show here that this relationship is causal. Manipulation of somatic ploidy both upwards and downwards increases and decreases, respectively, adult body size. We also establish a quantitative relationship between ploidy and body size. Finally, we find that TGF-beta (DBL-1) and cyclin E (CYE-1) regulate body size via endoreduplication. To our knowledge, this is the first experimental evidence establishing a cause-and-effect relationship between somatic polyploidization and body size in a metazoan.

Animals↗
PubMed2005

Polyploidy and genome evolution in plants.

Genome doubling (polyploidy) has been and continues to be a pervasive force in plant evolution. Modern plant genomes harbor evidence of multiple rounds of past polyploidization events, often followed by massive silencing and elimination of duplicated genes. Recent studies have refined our inferences of the number and timing of polyploidy events and the impact of these events on genome structure. Many polyploids experience extensive and rapid genomic alterations, some arising with the onset of polyploidy. Survivorship of duplicated genes are differential across gene classes, with some duplicate genes more prone to retention than others. Recent theory is now supported by evidence showing that genes that are retained in duplicate typically diversify in function or undergo subfunctionalization. Polyploidy has extensive effects on gene expression, with gene silencing accompanying polyploid formation and continuing over evolutionary time.

Evolution, Molecular↗
PubMed2004

Pattern of divergence of amino acid sequences encoded by paralogous genes in human and pufferfish.

We used phylogenetic analyses of protein families containing two or more pairs of orthologues in the genomes of human and pufferfish (Takifugu rubripes) to test the hypothesis that these sequences show a strong signal of polyploidization events hypothesized to have occurred early in vertebrate history. In order to test for evidence of two distinct rounds of polyploidization (the 2R hypothesis), we compared the pattern of amino acid sequence divergence of proteins encoded by genes duplicated just prior to the most recent common ancestor of human and pufferfish with that of proteins encoded genes duplicated earlier. These sequence divergences were statistically indistinguishable, contrary to the prediction of the 2R hypothesis. The variance of amino acid sequence divergences between paralogues was significantly greater than expected from that of orthologues in the same families. Estimation of gene duplication times assuming a molecular clock provided earlier estimates than expected, suggesting that it may not be appropriate to time the duplication of paralogues using rate estimates derived from orthologous comparisons. Overall, the results indicate that amino acid sequences do not provide a strong signal supporting the hypothesis that gene duplications early in vertebrate history occurred by polyploidization. On the other hand, the data are easily explained under an alternative model that gene duplications occurred at different times in different vertebrate gene families.

Animals↗
PubMed2004

Phylogenetic analysis of Hordeum (Poaceae) as inferred by nuclear rDNA ITS sequences.

Hordeum (Poaceae, Triticeae) occurs with 31 species worldwide in temperate regions, with the exception of Australasia. About 50% of the species are polyploids (4x, 6x) or occur as di- and polyploid forms. To analyze the phylogenetic relationships among diploid and polyploid taxa of the genus the nuclear rDNA internal transcribed spacer region (ITS) was analyzed for 91 accessions, representing all Hordeum species, together with 10 outgroup species. PCR products were either directly sequenced (outgroups) or cloned and eight clones per individual were analyzed. Phylogenetic analysis revealed four major clades that concur with the four genome groups in Hordeum (H, I, Xa, and Xu). Allopolyploids, putative autopolyploids, and species groups within the closely related H-genome clade could be identified. The ITS data indicate times of independent evolution of paralogous rDNA clusters on different chromosomes intermitted by sweeps of homogenization among these clusters and bi-directional homogenization of the clusters in diploids. Penalized likelihood analysis revealed an age of about 12 million years (my) for the genus and indicated the start of a rapid radiation in the H-genome group about 2.5 my ago in South America and Asia.

Cell Nucleus↗
PubMed1975

Heteropyknosis of the chromosomes in liver cells of different ploidy: a nuclear image study.

The chromatin densities of Feulgen-stained diploid, tetraploid, octoploid and binucleate cells of smear preparations of the liver of female field voles, Microtus agrestis, were examined by means of image analysis. The ratio of the areas of flattened nuclei of 2n, 4n and 8n ploidy was about 1 : 1.62 :2.60 and that of the relative DNA content 1 : 2 : 4. In flattened polyploid nuclei, the chromosomes are more densely arranged than in diploid. In diploid nuclei, absolutely and percentually smaller areas of dark chromatin particles were found than in polyploid nuclei. After correction of the grey values with a density factor, the frequency distribution curves of all ploidy classes were found to be nearly identical. The results show that the percentage of heteropyknotic chromosome material is the same in diploid, polyploid and binucleate liver cells.

Animals↗
PubMed1986

Age-dependent changes in nuclear DNA content and cell size of presumably normal human corneal endothelium.

In order to investigate a relationship between cellular DNA-content and enlargement in cell size of human corneal endothelium in healthy population, both nuclear DNA-cytofluorometry and cell morphometry were performed on individual cells of presumably normal corneas taken from six autopsy cases. The cornea was treated in 0.2 M phosphate buffer containing 60 mM Na2EDTA and fixed with absolute methanol. The endothelial-cell layer was separated from Descemet's membrane by placing the tissue in distilled water. The specimen was marked with rhodamine-labelled wheatgerm agglutinin (WGA)-lectin for identifying cell boundaries and subsequently stained with DAPI for measurement of DNA contents. The area of each cell was measured by means of a color image analyser and compared with its cytofluorometric nuclear DNA content. The enlarged endothelial cells which appeared with age contained diploid, polyploid or multiple nuclei. The polyploid and multinucleated cells had larger mean and maximum cell sizes and more irregular and polymorphous shapes than diploid cells. There were neither small polyploid nor small multinucleated cells. It was suggested that the loss of human corneal endothelium was compensated in vivo by cell size enlargement with or without doubling of nuclear DNA.

Adolescent↗
PubMed1996

Alteration of DNA ploidy and cell nuclearity in human hepatocellular carcinoma associated with HBV infection.

BACKGROUND/AIMS: Hepatocellular carcinoma usually arises in cirrhotic livers as a complication of chronic liver disease, and may show a variable trend towards increasing ploidy. The aim of this study was to investigate possible associations between different etiological factors, particularly hepatitis B virus and hepatitis C virus infection, and alteration of DNA-ploidy and nuclearity of neoplastic hepatocytes. METHODS: DNA-ploidy, the percentage of binucleated cells in the total cell population and the fraction of mononucleated hepatocytes in the polyploid compartment were assessed by image cytometry on cellular suspensions obtained by fine-needle biopsy from 60 hepatocellular carcinomas in patients whose viral status had previously been assessed. RESULTS: Significantly higher DNA-ploidy values (p = 0.005), with a reduction in the percentage of binucleated hepatocytes (p = 0.003) and an increase in the fraction of mononucleated hepatocytes in the polyploid compartment (p < 0.0001), were found in hepatocellular carcinoma with actual or previous hepatitis B virus infection (including also HCV+ve patients) in comparison to those not associated with hepatitis B virus infection, but not when HCV+ve hepatocellular carcinomas were compared to HCV-ve ones. Statistically significant differences for ploidy values (p < 0.05), percentage of binucleated hepatocytes (p < 0.05) and fraction of mononucleated hepatocytes in the polyploid compartment (p = 0.003) were also found between hepatocellular carcinoma associated only to hepatitis B virus infection ("pure" hepatitis B virus cases) and those associated only to hepatitis C virus infection ("pure" hepatitis C virus cases). CONCLUSIONS: Hepatocellular carcinoma associated with a previous or actual hepatitis B virus infection shows a peculiar phenotypical appearance, characterized by a trend towards increasing ploidy and reduction of binuclearity.

Adult↗
PubMed2001

Mechanism of the radiosensitization induced by vinorelbine in human non-small cell lung cancer cells.

Vinorelbine (Navelbine, KW-2307), a semisynthetic vinca alkaloid, is a potent inhibitor of mitotic microtubule polymerization. The aims of this study were to demonstrate radiosensitization produced by vinorelbine in human non-small cell lung cancer (NSCLC) PC-9 cells and to elucidate the cellular mechanism of radiosensitization. A clonogenic assay demonstrated that PC-9 cells were sensitized to radiation by vinorelbine with a maximal sensitizer enhancement ratio at a 10% cell survival level of 1.35 after 24-h exposure to vinorelbine at 20 nM. After 24-h exposure to vinorelbine at 20 nM, the approximately 67% of the cells that had accumulated in the G2/M-phase were cultured in the absence of vinorelbine and then irradiated at a dose of 8 Gy. Flow cytometric analyses showed prolonged G2/M accumulation concomitant with continuous polyploidization, and induction of apoptosis was observed in the cells subjected to the combination of vinorelbine-pretreatment and radiation. Polyploidization and induction of apoptosis were confirmed by morphological examination and a DNA fragmentation assay, respectively. We concluded that vinorelbine at a minimally toxic concentration moderately sensitizes human NSCLC cells to radiation by causing accumulation of cells in the G2/M-phase of the cell cycle. Prolonged G2/M accumulation concomitant with continuous polyploidization and increased susceptibility to induction of apoptosis may be associated with the cellular mechanism of radiosensitization produced by vinorelbine.

Carcinoma, Non-Small-Cell Lung↗
PubMed1975

Investigation of DNA-content of leukoplakia cells or oral mucosa.

DNA-histograms of smears of 11 plain leukoplakias, one of a precancerous leukoplakia, three of leukoplakia carcinoma in early stage, and one of a keratotic seqamous cell carcinoma of the floor of the mouth were prepared. The cell nuclei of the plain leukoplakia are mostly diploid, a small proportion has a DNA-content of 4 C. Intermediate values or polyploid nuclei are absent. In precancerous leukoplakia, intermediate values were noticed; in the cases of carcinoma in early stage, intermediate and polyploid nuclei were present, and in one case only polyploid nuclei. Only 2C and 4C nuclei occurred in a case of keratotic squamous cell carcinoma, which in comparison to earlier examined cases must be termed DNA negative.

Adult↗
PubMed2003

Stathmin expression and megakaryocyte differentiation: a potential role in polyploidy.

OBJECTIVE: Megakaryopoiesis is characterized by two major processes, acquisition of lineage-specific markers and polyploidization. Polyploidy is a result of endomitosis, a process that is characterized by continued DNA replication in the presence of abortive mitosis. Stathmin is a major microtubule-regulatory protein that plays an important role in the regulation of the mitotic spindle. Our previous studies had shown that inhibition of stathmin expression in human leukemia cells results in the assembly of atypical mitotic spindles and abnormal exit from mitosis. We hypothesized that the absence of stathmin expression in megakaryocytes might be important for their abortive mitosis. MATERIALS AND METHODS: The experimental models that we used were human K562 and HEL cell lines that can be induced to undergo megakaryocytic differentiation and primary murine megakaryocytes generated by in vitro culture of bone marrow cells. The megakaryocytic phenotype was evaluated by flow cytometry and light microscopy. The DNA content (ploidy) was analyzed by flow cytometry. Stathmin expression was analyzed by Western and Northern blotting and by RT-PCR. RESULTS: Our studies showed an inverse correlation between the level of ploidy and the level of stathmin expression in megakaryocytic cell lines and in primary cells. More importantly, inhibition of stathmin expression in K562 cells enhanced the propensity of these cells to undergo endomitosis and to become polyploid upon induction of megakaryocytic differentiation. In contrast, inhibition of stathmin expression interfered with the ability of the cells to acquire megakaryocyte-specific markers of differentiation. CONCLUSION: Based on these observations, we propose a model of megakaryopoiesis in which stathmin expression is necessary for the proliferation and differentiation of early megakaryoblasts and its suppression in the later stages of megakaryocytic maturation is necessary for polyploidization.

Animals↗