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PubMed1996

Interferon therapy, but not busulfan restores normal-sized megakaryopoiesis in CML--a comparative histo- and immunomorphometric study.

To assess possible alterations of megakaryocytes associated with interferon (IFN) and busulfan (BU) therapy of Ph(1+)-CML, an immunohistochemical and morphometric study was performed on trephine biopsies of the bone marrow taken before and at varying intervals during treatment. For the identification of megakaryopoiesis and its endoreduplicative activity the monoclonal antibodies CD61 (anti-platelet glycoprotein IIIa) and PC10 raised against proliferating cell nuclear antigen (PCNA) were used. We compared 60 specimens from 20 patients following IFN alpha-2b administration (in combination with IFN gamma in seven patients) with 57 specimens from 22 patients after monotherapy with BU. A close correlation with clinical follow-up studies revealed that in the IFN-treated group the prevalence of atypical micro-megakaryocytes, usually characterizing CML, was conspicuously reduced in repeatedly taken bone marrow samples. Initially, even an increase in size which was levelled to normal values during maintenance therapy was observed. These features were most prominently expressed in the 13 patients with a complete hematologic and/or partial cytogenetic response. Associated with this phenomenon was a significant enhancement of the PCNA-labelling index which indicated a stimulation of endoreduplicative (endomitotic) activity necessary for achieving normal size and ploidy. In the second group of patients treated by BU these changes were absent. For this reason, our findings are in keeping with the assumption that during IFN treatment, there is at least partial recovery and expansion of a putative normal (Ph1-) megakaryopoiesis. In conclusion, megakaryocyte morphology, i.e. normalization in size, is thought to be a useful indicator to evaluate the response to IFN in CML patients.

Adult↗
PubMed1997

Giant cell formation in cells exposed to 740 nm and 760 nm optical traps.

BACKGROUND AND OBJECTIVE: Optical trapping is becoming a useful and widespread technique for the micromanipulation of cells and organelles. Giant cell formation following optical trapping was studied to detect the potential adverse effects. STUDY DESIGN/MATERIALS AND METHODS: The nuclei of preselected single CHO cells were exposed to 740 nm and 760 nm laser microbeam generated by a titanium-sapphire tunable laser at 88 and 176 mW and different time exposures. The irradiated single cells were recorded and observed morphologically following exposure. Giant cells were tabulated and photographed. RESULTS: The irradiated cells either failed to divide, or they underwent nuclear proliferation to form giant cells through endoreduplication. CONCLUSION: Giant cells were induced by both 740 nm and 760 nm. The frequency of giant cell formation was higher for the longer time exposures and at the higher power densities. The use of an optical etalon to remove intracavity mode beating and high peak powers of the titanium-sapphire laser caused a significant reduction in the formation of giant cells.

Animals↗
PubMed1998

Teratoma with trisomy 16 in a baboon (Papio hamadryas).

A teratoma was found during a planned cesarean section in a 10-year-old primigravida baboon. This teratoma had a female sex chromosome complement and trisomy for chromosome 16. This is the first report of a teratoma in a baboon and the first report of a chromosomal abnormality in a nonhuman primate teratoma. It is also the first case in a nonhuman primate to address the mechanism of origin. Through the use of genetic markers from human chromosomes 5, 8 and 17, the origin of the teratoma was shown to be most consistent with failure of meiosis II or endoreduplication in a mature ovum, while the trisomy for chromosome 16 originated after the formation of the tumor.

Animals↗
PubMed1999

Reversal of p53-induced cell-cycle arrest.

Activation of the tumor suppressor protein p53 can lead to arrest in both G1 and G2 stages of the cell cycle and, in some cells, to apoptotic cell death. In this study, we showed that the p53 response to a chemotherapeutic drug, actinomycin D, was reversible in both normal and tumor cells, even when a substantial proportion of tumor cells were undergoing apoptosis. Despite the clear reversibility of the p53-induced cell-cycle arrest after removal of actinomycin D, a substantial proportion of the cells arrested in G2 failed to resume normal cell-cycle progression and underwent another round of DNA synthesis. This endoreduplication probably reflects a function of the cyclin-dependent kinase inhibitor p21Waf1Cip1, which is expressed in response to p53. Our observation that this abnormal re-replication of DNA occurred in both transformed and untransformed cells after reversal of a p53 response may have implications for the eventual outcome of tumor therapies in which p53 is transiently expressed in a substantial number of normal as well as tumor cells.

Apoptosis↗
PubMed1998

Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning.

Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells.

Animals↗
PubMed1976

Increased tetraploidy: cell-specific for the Gardner gene in the cultured cell.

Human hereditary tumors in vivo are known to show tissue specificity. Increased endoreduplication with tetraploidy has been shown to occur in cultures established only from tissue containing epithelium (skin and colonic polyps) from patients with the Gardner syndrome. Cultures established from blood (short-term lymphocyte and lymphoid suspension) and connective tissue (subcutaneous tissue, lipoma, mesentery, and sebaceous cyst) from the same patients did not show increased tetraploidy. These observations demonstrated that tetraploidy as an expression of the gene for the Gardner syndrome was cell-specific in vitro and added further evidence that increased tetraploidy could be used for detection of the Gardner gene in families at risk.

Cell Line↗
PubMed1984

Acute lymphoblastic leukemia supervening in a case of chronic lymphocytic leukemia after continuous 3-year treatment with chlorambucil.

A case is presented of a patient with classical chronic lymphatic leukemia (CLL) treated with continuous chlorambucil for 3 years who presented with a picture of acute leukemia. The peripheral blood still showed a prevalence of mature lymphocytes with a few blast cells, whereas the bone marrow showed a predominant population of blast cells possessing a null acute lymphoblastic leukemia phenotype. Karyotype analysis showed a prevalent hyperdiploid clone of 67 to 68 chromosomes with endoreduplication and marker chromosomes. The coexistence of a CLL-type population with the blastic, undifferentiated cell clone suggests a second malignancy superimposed on the previous leukemic process, and possibly brought about by the continuous chlorambucil treatment.

Aged↗
PubMed2001

Clone heterogeneity in diploid and aneuploid breast carcinomas as detected by FISH.

We investigated the relationship between DNA ploidy and alterations in chromosomes 1, 8, 12, 16, 17, and 18 in 63 breast carcinoma samples by static cytofluorometry and fluorescence in situ hybridization. Thirty specimens were diploid and 33 were aneuploid. In aneuploid samples, the DNA index value ranged from 1.3 to 3.1, with a main peak near tetraploid values. Diploid clones were present in 21 of 33 aneuploid specimens. Fluorescence in situ hybridization analysis showed a heterogeneous degree of alterations in diploid specimens: one sample was normal, 16 samples had one to three chromosome alterations involving mostly chromosomes 1, 16, and 17, and 13 samples an even higher degree of alterations. The 33 aneuploid specimens showed a very high number of signals (four, five, or more). All the investigated chromosomes were affected in 23 of 33 specimens. Alterations in chromosomes 1 and 17 were detected to a similar percentage in diploid and aneuploid samples, whereas chromosome 16 monosomy was more frequent in diploid samples. Overrepresentation of chromosomes 8, 12, 16, and 18 was significantly higher in aneuploid than in diploid samples. Based on these results, we suggest that diploid and aneuploid breast carcinomas are genetically related. Chromosome 1 and 17 alterations and chromosome 16 monosomy are early changes. Allelic and chromosomal accumulations occur during progression of breast carcinoma by different mechanisms. The high clone heterogeneity found in 17 of 33 aneuploid samples could not be completely explained by endoreduplication and led to the suggestion that chromosomal instability concurs with aneuploidy development. This different evolutionary pathway might be clinically relevant because clone heterogeneity might cause metastasis development and resistance to therapy.

Aneuploidy↗
PubMed1985

Severe congenital neutropenia with unique features of dysgranulopoiesis.

Congenital dysgranulopietic neutropenia (CDN) is a recently proposed entity that describes a small subgroup of children with clinically severe neutropenia. We followed and studied a 3-year-old girl with neutropenia (less than 500/mm3) and recurrent severe infections in whom repeated marrow evaluations revealed large (30-50 microns) multinucleated promyelocytes to polymorphonuclear cells with as many as 4 to 16 nuclei or nuclear lobes, respectively. In addition to the nuclear endoreduplication, ultrastructural and cytochemical evaluation of these cells demonstrated abnormalities in granule genesis and centriole structure. Concomitantly, immunoperoxidase staining indicated that many of the granules were devoid of lactoferrin but not lysozyme. In vitro proliferation studies revealed normal to increased thymidine labeling, normal numbers of colony-forming cells, and normal colony-stimulating activity from blood and marrow mononuclear cells, findings consistent with ineffective myelopoiesis. However, serum folate, B12, and lysozyme levels were normal. The nuclear and cytoplasmic abnormalities in this patient result in an extreme example of CDN, distinct from previously described cases.

Agranulocytosis↗
PubMed1990

Chromosome reproduction: units of DNA for segregation.

Evidence is summarized which indicates that the DNA loop anchoring proteins in chromosomes are effectively heterodimers that stack and are fastened into a bilaterally symmetrical array along the chromonemal axis. The evidence consists primarily of the observations made twenty five to thirty years ago on the pattern of sister chromatid exchanges and the way the DNA chains are sorted in the formation of diplochromosomes in cells that have undergone endoreduplication. The evidence indicates that each chain of DNA in the single duplex, which is assumed to run the length of a chromosome, is anchored to a bilaterally symmetrical axis of heterodimers that sort the two original chains among the four derived chromatids of each diplochromosome in a very precise way. These observations are considered in the context of investigations on the nature of scaffold proteins and the loop anchorage sequences, as well as the advances being made on the nature of DNA binding proteins and the roles of topoisomerase II.

Animals↗
PubMed1995

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.

Adenine↗
PubMed2004

Genetic evolution in colon cancer KM12 cells and metastatic derivates.

So far, CRC cell lines have contributed to descriptions of 2 patterns of genetic instability, affecting either microsatellite sequences or chromosome number and structure. Often, these patterns are mutually exclusive; while near-diploid karyotypes usually appear with MSI and chromosomal stability, near-triploid or tetraploid cells display a high degree of CIN and are stable at the microsatellite level. In the present study, we describe the genomic instability pattern of KM12 CRC cells. KM12C and derived cell lines with different metastatic properties were analyzed by conventional cytogenetics, CGH and M-FISH. Results were compared to 5 cell lines usually used as model of MSI and CIN. Concordance between our results and previously published SKY data are also reviewed. Interestingly, the poorly metastatic KM12C cell line displayed a near-diploid karyotype with high levels of structural chromosome instability and microsatellite instability. The highly metastatic KM12SM and KM12L4A cell lines showed polyploid karyotypes and maintained CIN and MSI. A comparison between karyotypes of poorly and highly metastatic KM12 cell lines allowed us to delineate a cytogenetic evolution pathway. Our results clearly demonstrated that endoreduplication was the origin of the polyploid dosages in the highly metastatic forms following the monosomic model postulated for CRC. Therefore, we demonstrate that KM12C cells and their metastatic derivates, KM12SM and KM12L4A, are a useful model of chromosomal evolution where MSI may coexist with CIN.

Cell Line, Tumor↗
PubMed2005

In vitro induction of polyploidy and chromatid exchanges by culture medium extracts of natural rubbers compounded with 2-mercaptobenzothiazole as a positive control candidate for genotoxicity tests.

We tested extracts of custom-made natural rubber samples for cytotoxicity using V79 cells and for chromosome aberration (CA) induction using CHL cells in compliance with the Japanese guidelines for basic biological tests of medical materials and devices. The samples were formulated with a high level of 2-mercaptobenzothiazole (MBT) (A); a low level of MBT (B); or zinc dibutyldithiocarbamate (ZDBC) (C). In the CA test, MBT induced mainly polyploidy, including endoreduplication, and ZDBC induced structural CAs. In the cytotoxicity test, culture medium extracts of A, B, and C suppressed colony formation to 50% of the control value at 53.1%, 94.3%, and >100%, respectively. Culture medium extracts of sample A induced polyploidy and structural CAs in the absence of an exogenous metabolic activation system (S9 mix), but at lower concentrations in its presence, indicating the existence of other leachable promutagens. The extracts of sample B induced structural CAs at the highest concentration and only with S9 mix. Sample C was negative. The facts suggest that sample A may be a candidate for a positive control for genotoxicity tests. The high frequency of polyploidy induced by sample A was not predicted by MBT, suggesting the usefulness of the test for safety evaluation of medical devices. Numerical CAs induced by MBT and sample A are discussed.

Animals↗
PubMed1988

Analysis of murine megakaryocyte colony size and ploidy: effects of interleukin-3.

Megakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in-vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin-3 (IL-3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM, a crude source of megakaryocyte colony-stimulating activity) or IL-3, the modal ploidy of day-5 colony megakaryocytes was 16N (range 2N-128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM-SCM and at all concentrations of IL-3 tested. Increasing concentrations of IL-3 in cultures of either unfractionated marrow cells or nonadherent T-lymphocyte-depleted (NATD) marrow cells stimulated similar dose-dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL-3-stimulated cultures of NATD marrow cells was significantly less (P less than 0.001) than the mean ploidy of megakaryocytes in IL-3-stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM-SCM-stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day-5 murine megakaryocyte colonies are structured as continua and that IL-3 stimulates an increase in mean colony size and numbers without affecting ploidization. T-lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM-SCM can substitute for this activity.

Animals↗
PubMed1994

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis.

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G1, most virus transformed or tumor cells are unaffected in G1 but arrest in G2 phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nuclear p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 microM SSP. MOLT-4 cells did not arrest in G1 or G2 phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G1 phase at higher DNA ploidy (tetraploidy; G1T), and then progressed through ST (rereplication) into G2T and MT. The rates of entrance to G2 and G2T were essentially identical, indicating that the rates of cell progression through S and ST as well as through G2 and G2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 microM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis.

Alkaloids↗
PubMed1994

Differential effects of transforming growth factor-beta 1 on distinct developmental stages of murine megakaryocytopoiesis.

The effect of transforming growth factor-beta 1 (TGF beta 1) on three developmental stages of megakaryocytopoiesis was investigated. Using a murine bone marrow agar culture system, titrated doses of TGF beta 1 were added to cultures assaying primitive high proliferative megakaryocyte progenitors, committed megakaryocyte precursors, and nondividing, endoreduplicating megakaryocytes. The growth of high proliferative megakaryocyte colony-forming cells (HPP-CFU-Mk) that require the growth factors interleukins-1, 3 and 6 (IL-1 + IL-3 + IL-6) for colony detection was abrogated by the addition of 1 ng TGF beta 1/ml. The sensitivity of committed megakaryocyte progenitors (colony-forming unit-megakaryocyte, CFU-Mk) to TGF beta 1 depended on the growth factor combination. TGF beta 1 (1 ng/ml) completely inhibited megakaryocyte colony formation from CFU-Mk only in cultures stimulated by low doses of IL-3. TGF beta 1 (> 10 ng/ml) could only marginally inhibit megakaryocyte colony formation generated in the presence of either high doses of IL-3 or the combination of low dose IL-3 + IL-6. TGF beta 1 inhibited both IL-3-dependent and IL-6-dependent megakaryocyte growth but tenfold higher doses of TGF beta 1 were required to inhibit growth generated by the combination of IL-3 + IL-6. The data showed that the capacity of TGF beta 1 to inhibit distinct differentiation stages of the megakaryocytopoietic lineage depended on the concentration and combination of growth factors involved.

Animals↗
PubMed2005

Genome size and endonuclear DNA replication in spiders.

Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development.

Animals↗
PubMed2005

Chromosomal instability in the cattle clones derived by somatic cell nuclear-transfer.

Cytogenetic analysis was performed on peripheral lymphocytes collected from 20 cattle clones (19 showed no overt phenotypic abnormalities except for high birth weight while 1 exhibited left forelimb contracture), the donor cell cultures from which they were derived and lymphocytes from six insemination produced control cattle. All animals and cell cultures had a modal chromosome number of 60. The frequency of abnormal cells for donor cell cultures, clones, and controls was 6.68+/-0.30%, 5.30+/-5.49%, and 5.08+/-1.04%, respectively, and did not differ significantly among the groups. There were, however, two clones derived from different donor cell cultures with high incidences of 21.29% and 20.13%, of abnormal cells consisting of pseudodiploid (near-diploid), near-triploid and near-tetraploid, and tetraploid cells. Among these two clones, one had only a few endoreduplicated nuclei although further studies are necessary to precisely define the cytological origin and nature of the abnormal cells. The clones were evaluated at multiple time points for up to 20 months of age and the incidence of abnormal lymphocytes remained stable indicating that the chromosomally abnormal nuclei found in cloned animals was not a transient event. These results show that the majority of phenotypically normal clones have normal chromosomal make up but that instability of chromosome number can occur in clones that are phenotypically normal. Therefore, cytogenetical evaluation of peripheral lymphocytes and other tissues with follow up of the phenotypical consequences of these abnormalities is warranted even in phenotypically normal clones.

Aging↗