Polyploid Metaphases Research Articles

Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

Новые горизонты цитогенетики при первичном миелофиброзе

ABSTRACT Aim. To evaluate the potential of a new cytogenetic technique in patients with primary myelofibrosis (PMF). Materials and methods. 48-hour blood cell cultures (accord-ing to Singh et al., 2013) were used for cytogenetic study in 11 PMF patients (5 female, 6 men, aged 32–60 years; median 48.6 years). GTG-banding and different types of fluorescence in situ hybridization (FISH) techniques were used for identifica-tion of chromosomal aberrations. Results. The incidence of abnormal karyotypes in blood cul-tures was significantly higher than that in standard bone mar-row cultures (82 vs 27 %; p < 0.01). The polyploid clones were found in blood cultures of 45 % of patients. Structural chro-mosomal aberrations were found in chromosomes 6, 1, 3, as well as 16 and 17 (in 2 and 1 patients with each aberration, respectively). In all but one patients these abnormalities in dip-loid and polyploid metaphases were identical. Partial 1q tri-somy resulted from adding of additional (1q21–1q44) material translocated to the short arm of chromosome 5 to the material of 2 normal homologue of chromosome 1. It seems that 1q+, i(17q) and some others chromosomal abnormalities were sec-ondary, whereas 6p21 locus involvement may be a primary defect in PMF. The t(3;6)(q25;p21) translocation described for the first time and confirmed by FISH should be considered a variant of well-known translocation t(1;6). Allo-HSCT in 2 pa-tients with 1q+ was successful, whereas there were problems with engraftment in a female patient with prognostically unfa-vorable t(3;3)(q21;q26) translocation associated with the

Polyploidy in myelofibrosis: analysis by cytogenetic and SNP array indicates association with advancing disease

BackgroundMyelofibrosis occurs as primary myelofibrosis or as a late occurrence in the evolution of essential thrombocythaemia and polycythaemia vera. It is the rarest of the three classic myeloproliferative neoplasms (MPN). Polyploidy has only rarely been reported in MPN despite the prominent involvement of abnormal megakaryocytes. The use of peripheral blood samples containing increased numbers of haematopoietic progenitors has improved the output from cytogenetic studies in myelofibrosis and together with the use of single nucleotide polymorphism arrays (SNPa) has contributed to an improved knowledge regarding the diverse genetic landscape of this rare disease.ResultsCytogenetic studies performed on a consecutive cohort of 42 patients with primary or post ET/PV myelofibrosis showed an abnormal karyotype in 24 cases and of these, nine showed a polyploid clone. Six of the nine cases showed a tetraploid (4n) subclone, whereas three showed mixed polyploid subclones with both tetraploid and octoploid (4n/8n) cell lines. The abnormal clone evolved from a near diploid karyotype at the initial investigation to a tetraploid karyotype in follow-up cytogenetic analysis in four cases. In total, six of the nine polyploid cases showed gain of 1q material. The remaining three cases showed polyploid metaphases, but with no detectable structural karyotypic rearrangements. Three of the nine cases showed chromosome abnormalities of 6p, either at diagnosis or later acquired. SNPa analysis on eight polyploid cases showed additional changes not previously recognised by karyotype analysis alone, including recurring changes involving 9p, 14q, 17q and 22q. Except for gain of 1q, SNPa findings from the polyploid group compared to eight non-polyploid cases with myelofibrosis found no significant differences in the type of abnormality detected.ConclusionsThe study showed the use of peripheral blood samples to be suitable for standard karyotyping evaluation and DNA based studies. The overall profile of abnormalities found were comparable with that of post-MPN acute myeloid leukaemia or secondary myelodysplastic syndrome and cases in the polyploidy group were associated with features of high risk disease. The above represents the first documented series of polyploid karyotypes in myelofibrosis and shows a high representation of gain of 1q.

Primary mantle tissue culture from the bivalve mollusc Mytilus galloprovincialis: Investigations on the growth promoting activity of the serum used for medium supplementation

The main objective of the present work was to evaluate with a quantitative approach the effects of different chicken serum (CS) concentrations in the medium used for mussel mantle tissue culturing. Our results showed that a CS level of 20% was optimal. Under these conditions, the cultures reached a maximum mean number of mitotic figures per slide exceeding widely 100. Cultures were also achieved to test whether CS must be heat treated to inactivate complement components before use for medium enrichment. We demonstrated that heat inactivation did not significantly change the promoting activity of the CS. Finally, the growth-stimulatory properties of CS were compared to those of fetal calf serum (FCS). The best results were obtained with 30% FCS. The difference with 20% CS was not statistically significant, but the FCS yielded a much higher level of polyploid metaphases than the CS. Since it had no adverse effect such as polyploid metaphases induction, was readily commercially available and relatively less expensive than other additives, CS at the concentration of 20%, without heat-decomplementation, is routinely used as growth medium supplement for mussel mantle cell culturing in our laboratory. Even though our primary cultures do not have the potency of continuous cell lines, it is possible to use such cultures for in vitro experiments.

Parthenin, a sesquiterpene lactone of Parthenium hysterophorus L. is a high toxicity clastogen

The in vitro and in vivo genotoxicity of parthenin, a sesquiterpene lactone from Parthenium hysterophorus L. with allergenic and irritant action, was assessed in three short-term tests: bacterial reversion in Salmonella typhimurium and Escherichia coli, in vitro chromosomal aberrations in peripheral blood lymphocytes and micronuclei in mouse peripheral blood. Parthenin was not mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 but a weak response was observed in TA 102 (+S9) from 0.19 to 1.22 μmole per plate. Concentrations of 7.62 μmole per plate or higher were toxic, but the effect was reduced when S9 was present. Screening of oxidative mutagenesis with E. coli strains IC 188 and IC 203 gave negative results. Parthenin induced chromosomal aberrations, mainly chromatid breaks, in blood lymphocytes exposed to 10–60 μM during 20 h. An association was found with cytotoxicity, since concomitant nuclear alterations such as pycnosis, micronuclei and karyorrhexis were observed. Sister chromatid exchanges (SCEs) in lymphocytes were not influenced by exposure to parthenin; rather a decrease was observed at 60 μM. On the other hand, a minor increment in polyploid metaphases was found at 40 μM. When a single intraperitoneal (i.p.) dose of 4–31 mg/kg of parthenin was administered to mice, a positive increase in the micronucleated reticulocyte (RET) frequency was observed at 48 h for both sexes at the highest dose.

Induction of polyploidy and apoptosis after exposure to high concentrations of the spindle poison nocodazole.

The proportions of aneuploid/polyploid versus euploid cells formed after treatment with spindle poisons like nocodazole are of course dependent on the relative survival of cells with numerical chromosome aberrations. This work aimed at studying the survival of polyploid cells formed after treatment with a nocodazole concentration sufficient to significantly decrease tubulin polymerization (0.1 microg/ml). First, normal primary lymphocytes were analysed and the following complementary chromosomal parameters were quantified: mitotic index, frequency of abnormal mitoses, polyploid metaphases and apoptotic cells. The results clearly indicate a positive correlation between abnormal mitotic figures, apoptosis and the induction of polyploidy. They therefore led to a single cell approach in which both apoptosis and polyploidy induction could be scored in the same cell. For this purpose, actively proliferating cells are required and two human leukaemic cell lines were used, KS (p53-positive) and K562 (p53-negative), which have a near-triploid karyotype. Cells were separated into an apoptotic and a viable fraction by means of annexin-V staining and flow cytometry. In KS, treatment with nocodazole induced a similar fraction of hexaploid cells in both the viable and apoptotic fraction, but no dodecaploid cells were ever observed. In contrast, a population of dodecaploid cells (essentially viable) was clearly observed in the K562 cell line. The results in KS, as compared with K562, confirm that wild-type p53 can prevent further cycling of polyploid cells by blocking rereplication. The most probable explanation for these data is that not only the mitotic spindle but also interphase microtubules are sensitive to nocodazole treatment. Our data thus strongly suggest that besides the G(1)/S checkpoint under the control of p53, the G(2)/M transition may be sensitive to depolymerization of microtubules, possibly under the control of Cdc2, Bcl-2, Raf-1 and/or Rho.

Aberrations of cell cycle and cell death in normal development of the chick embryo growth plate

The epiphyses of femurs from 7.5–15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, ‘dark chondrocytes’ representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours. This connection is briefly analysed in view of some current concepts of carcinogenesis.

Hyperdiploid acute myeloid leukemia Relationship between blast size and karyotype demonstrated by fluorescence in situ hybridization

Hyperdiploidy is a rare finding in acute myeloid leukemia (AML), and the cases described so far showed characteristic large and bizarre blasts. We further demonstrate this morphologic/karyotypic relationship in a case of hyperdiploid AML by fluorescence in situ hybridization. Together with previous observations, we propose that in myeloid leukemias, increase in DNA content is accompanied by a corresponding increase in blast size. If large and bizarre blasts are shown on morphologic analysis, polyploid metaphases on cytogenetic preparations should also be analyzed, as they may be derived from these cells.

Induction of polyploidy in human lymphocytes in vitro by excess adenine, but not by adenosine.

It is known that high levels of DNA precursors can be both clastogenic and mutagenic in cultured cell lines and in vivo. The purpose of the present study was to examine at an observational level the cytogenetic effects of adenine and adenosine in primary human cell cultures. Human peripheral blood lymphocytes from four donors were cultured and treated with a range of concentrations of adenine and adenosine. Although no increase in sister chromatid exchange (SCE) frequency was observed with either compound, there was a statistically significant, dose-related increase in the proportion of polyploid cells in cultures treated with adenine, but not in those treated with adenosine. Some of the polyploid metaphases found after adenine treatment contained diplochromosomes, suggesting that endoreduplication might have been involved in polyploid formation in these cells. It is concluded that a high level of adenine can cause genetic changes in human lymphocytes by interfering with mitosis, perhaps by disturbing the balance of DNA precursor pools.

Cytogenetic biomonitoring of an Italian population exposed to pesticides: chromosome aberration and sister-chromatid exchange analysis in peripheral blood lymphocytes

Chromosome aberrations (CA) and sister-chromatid exchanges (SCE) were measured in lymphocytes of (A) 32 healthy individuals working in the flower industry and exposed to pesticides, (B) 32 individuals exposed as above and hospitalized for bladder cancer, and (C) 31 controls. Compounds to which floriculturists were exposed included 18 nitro-organic herbicides and fungicides, 9 nitro-organic fungicides, 12 organophosphate and organothiophosphate insecticides, 4 hydrocarbon derivative herbicides and 5 inorganic fungicides and insecticides. 150 and 70 metaphases per individual were scored for CA and SCE, respectively. A significant increase in the incidence of CA and SCE was observed in both exposed groups. Cancer patients showed the presence of rare rearrangements (dicentrics, rings and quadriradials) that were not observed in controls and were present at a lower frequency in healthy exposed people. Hyperdiploid and polyploid metaphases were also significantly increased in the 2 exposed groups compared to controls. Stratifying for age or smoking habits, although affecting the significance of individual data, did not change the substance of the results.

Chromosome abnormalities in tuberous sclerosis

In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.

The induction of numerical chromosome aberrations in human lymphocyte cultures and V79 Chinese hamster cells by diethyl sulfate

Human lymphocyte cultures were treated with increasing concentrations of diethyl sulfate (DES) at different times after stimulation with phytohemagglutinin (0, 24 and 45 h) and were scored for numerical chromosomal aberrations at different culture times (52, 72 and 96 h). A total of 3500 metaphases were analyzed. A statistically significant ( p < 0.01) increase in hypodiploid and hyperdiploid metaphases was found throughout the tested dose range (0.1–3 mM DES); the increase in polyploid metaphases was statistically significant at 1 mM ( p < 0.05) and 3 mM ( p < 0.01) DES. In human lymphocytes treated in G 0, DES also induced chromatid breaks as well as micronuclei. In V79 Chinese hamster cells, DES induced micronuclei and polyploidy.

Chromosome abnormalities in myelofibrosis.

Cytogenetic analysis was performed on megakaryocyte-enriched cell fractions obtained by separation on discontinuous Percoll gradients from blood, bone marrow, and ascitic fluid of 12 patients with myelofibrosis with myeloid metaplasia (MMM). The most common abnormalities appeared to involve chromosomes 1, 3, 7, 8, 11, 13, 21 and 22. Although no specific chromosome rearrangements were observed in megakaryocytic fractions from different tissues of the patients with MMM, the presence in one case of a 3q-abnormality restricted to polyploid metaphases only, and of a 3q paracentric inversion in another patient, seems to confirm a close relationship between the involvement of chromosome 3 and dysplastic thrombopoiesis.

Herpes simplex virus gene products involved in the induction of chromosomal aberrations

The effect of short-term herpes simplex virus type 1 (HSV-1) infection on chromosomes of human diploid fibroblasts was examined. In addition to chromosomal breaks, gaps and pulverization, three kinds of cytogenetic damage (double minutes, polyploidy and endoreduplication) not yet reported following productive infection with HSV or other animal viruses were frequently observed. Consistent with previous studies suggesting that the expression of immediate-early and/or early viral gene products is required for the induction of chromosomal damage, was the observation that cells infected at the nonpermissive temperature with HSV-1 temperature-sensitive mutants defective in the gene for the immediate-early transcriptional regulatory protein, ICP4, and three early viral gene products — DNA polymerase ( pol), the major HSV DNA-binding protein (ICP8) and an HSV-2 mutant defective in alkaline nuclease — exhibited altered patterns of chromosomal damage relative to the effects of wild-type virus on infected cells. These findings suggest a direct or indirect role for all four gene products in the induction of chromosomal damage. In cells infected with wild-type virus for 4 h or longer, HSV proved to be a more potent mitotic arresting agent than colcemid. Moreover, studies with selected mutants indicate that HSV pol specifically may be involved in mitotic arrest. Additionally, in cells infected at the non-permissive temperature with a pol mutant, the number of polyploid metaphases was reduced 4-fold relative to that seen in wild-type virus-infected cells suggesting a role for HSV pol in the amplification of cellular DNA.

Chromosomal changes in a documented case of malignant histiocytosis: Significance of polyploidy

A case of malignant histiocytosis was studied by cytology, cytochemistry, electron microscopy, and cytogenetics. It was shown that the malignant cells expressed a fully differentiated histiocytic pattern with high macrophagic activity. This correlated with the presence of polyploid metaphases. The significance of polyploid cells in the definition of malignant histiocytosis is discussed.

A cytogenetic study of subcutaneous murine macrophages and multinucleate giant cells.

Macrophages and multinucleate giant cells (MGC), collected by subcutaneous implantation of melinex discs into the dorsum of mice for 7 days, were examined cytogenetically. Two per cent of the metaphases seen were polyploid and were considered to represent dividing MGC. Twenty-two per cent of the diploid metaphases showed chromosomal damage. Seventy per cent of damage consisted of chromosomal gaps, but the remaining 30% consisted of breaks and chromosomal fragments which would lead to unbalanced karyotypes in the next generation. More than 50% of the polyploid metaphases analysed had chromosomal damage, including 16.6% which displayed premature chromatin condensation, the damage being more severe in polyploid than in diploid metaphases. The chromosomal damage parallels that previously reported in resident peritoneal macrophages and in cultured exudate macrophages. The cause of such damage is unclear, but it was concluded that, because of the extent of damage, mitosis is unlikely to play a major part in the maintenance of macrophages or MGC at inflammatory sites.

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced chromosome aberrations in mouse keratinocyte cell lines: a possible genetic mechanism of tumor promotion.

The effect of the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the induction of numerical and structural chromosomal aberrations, double minute chromosomes (DM) and homogeneously staining regions (HSR) was investigated in two mouse keratinocyte cell lines (HEL-30 and HEL-37) using G- and C-banded chromosomal preparations. The frequency of polyploid metaphases and of structural aberrations increased several fold during the first two cell cycles. The increase of gaps, chromatid and isochromatid breaks was accompanied by inter- and intrachromosomal exchanges, which were virtually absent in control cultures. The induction of chromosomal aberrations was similarly expressed in both cell lines at different passage levels and was largely independent of the TPA dose applied (10(-9) - 10(-6) M). The non-promoting phorbolester 4-O-methyl-TPA (10(-6) M) did not produce any chromosomal aberrations above the control level. Effects were already visible within one cell cycle (24 h) after treatment and increased with longer and multiple exposure. Moreover, the TPA-induced chromosomal aberrations persisted for several days after elimination of the promoter and further cultivation of cells in fresh medium. Structural alterations were not randomly distributed but chromosomes 1 and 2 were preferentially involved in breaks, while chromosomes 1-3 and 9-13 were mainly involved in chromosomal exchanges. Chromosomes 7 and 14, which are numerically underrepresented in HEL cells, were least involved in breaks. Most interesting, the frequency of metaphases carrying DMs increased 2- to 3-fold after TPA treatment, and the alterations were partially reversible after removal of TPA. These early effects of TPA at the chromosomal level could play an essential role in the mechanism of tumor promotion and may be responsible for the observed persistence of alterations induced during the first stage of tumor promotion.

Early chromosome changes in diploid Chinese hamster cells after infection with Simian virus 40.

AbstractPrimary and secondary Chinese hamster (ChH) embryo cells infected with SV40 exhibited chromosomal changes within one cell generation (24 h). The initial change seen is an increase in the number of polyploid metaphases (&gt;10%). The majority of the polyploid cells are tetraploid (8×), however, higher ploidy values were also observed (16 ×, 32 × and 64 ×). In approximately 20–30% of the polyploid metaphases there were chromosome changes such as breaks, abnormal chromosomes and loss and addition of chromosomes. The remaining polyploids were normal in regard to chromosome number and chromosome morphology. A primary mouse embryo cell system also responded to SV40 infection with an increase in the number of polyploid cells within 48 h.

CHANGES IN MITOTIC INDICES IN ROOTS OF VICIA FABA L.

SummaryRoots of Vicia faba were treated with IAA (6.26 × 10−4 m) for 3 hours and fixed immediately or 5, 11 or 23 hours later. Mitotic indices (MI) fell in these treated roots; the decrease was apparent at 5 hours and was significant at 11 and 23 hours, indicating that IAA was inhibiting some interphase processes.Other V. faba roots were treated with colchicine (0.025%) or IAA (4.7 × 10−4 m) or they were given successive treatments with colchicine or IAA and fixed up to 201 hours later. (a) In roots treated with colchicine, the MI rose for the first 24 hours and then fell to values below 1, where it remained for several days. (b) In roots treated with IAA, a fall in MI was observed within the first 2 days but it eventually returned to normal. This recovery of normal levels of mitotic activity was dependent upon the age of the roots at the time of treatment, the youngest roots died after treatment with IAA while older roots recovered completely. (c) Roots treated with IAA 24 hours after treatment with colchicine showed a decrease in MI in the following 24 hours similar to that found in roots treated only with colchicine. However, the MI of these roots subsequently rose to values that were not significantly different from those of the controls, i.e. there is an IAA induced stimulation of mitotic activity.Polyploid cells are found in division following treatment with colchicine. Changes in the frequencies of polyploid metaphases occur if roots are also treated with IAA, suggesting, as we have seen above, that IAA affects the duration of mitotic cycles.These results provide further evidence that a decrease in the endogenous levels of auxins occurs in roots treated with colchicine and this decrease can be partially restored by exogenous auxin. It is suggested that the changes in levels of mitotic activity in treated meristems is due, at least in part, to the colchicine induced fall in endogenous auxins.

Abnormalities of human chromosomes following therapeutic irradiation.

Blood leucocytes were cultured from six men before and at intervals after therapeutic irradiation of the abdomen and pelvis. There was no evidence of increased aneuploidy after exposure. By the end of radiotherapy, 32 % of metaphases showed chromosome structural aberrations (control 2 %); these declined to 8 % by the end of the follow-up (17–19 months after treatment). Cells with ‘unstable’ aberrations declined faster than ‘stable’ cells. No clones of cells were observed. Many polyploid metaphases were seen in the cultures after irradiation. There was no evidence that they were derived from circulating tumour cells or other large cells. The irradiated bone marrow was aplastic. Cells from unirradiated bone marrow had normal chromosomes. Thus, there was no evidence for the propagation of small lymphocytes (or other cells) with radiation-induced chromosome ‘markers’ in the bone marrow.