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PubMed1994

[The quantitative study of the areas of active rDNA location and of the other parameters of the interphase nucleolus detectable by silver staining during the cell differentiation of the rat trophoblast].

Different quantitative parameters of nucleolar silver staining have been studied in the cambial rat trophoblast cells on the 12th, 13th and 14th days of gestation. It has been shown that the number of Ag-positive granules in the nucleoli varied from 10 to 120. The number and the total area of silver stained granules in the nuclei increased progressively in the course of polyploidization, but was not doubled passing to the next ploidy level. Nevertheless, nucleolar area increased proportionally to the ploidy degree. The mean number and the total area of Ag-stained granules as well as the nucleolar area estimated for each ploidy level did not change significantly in the course of placenta development, suggesting an unchanged level of NOR activity at the studied stages of trophoblast cell differentiation. The data obtained on the interphase nucleoli differ from the data of the analysis of the metaphase Ag-NOR at the same period of placenta development, suggesting a diversity in the interphase and metaphase NOR organization. A proportion of cells with different number of nucleoli in the cambial rat trophoblast cells was maintained unchanged in the studied period of the placenta development, the majority (80-90%) of cells contained from 1 to 3 nucleoli. Such a proportion was similar in the cells of different levels of ploidy up to 16c. In this connection the association of NORs is suggested to be in relation with switching from the polyploid mitotic cycle to the endoreduplication leading to polyteny.

Animals↗
PubMed1994

Malignant gliomas--ploidy and DNA-content before and after therapy.

DNA ploidy and S-phase percentage from nine malignant gliomas (four glioblastomas, four anaplastic astrocytomas grade 3 and one anaplastic oligoastrocytoma grade 3) have been estimated by single cell cytophotometry on biopsy and necropsy specimens. All gliomas from biopsy material showed, with the exception of two diploid tumours, a polyploid-aneuploid DNA-pattern and stem-lines of different ploidy. The most frequent stem-lines were diploid and hyperdiploid. In necropsy material, following treatment i.e. operation and combined drug and radiation therapy, the heterogeneous nature of all malignant gliomas persisted. From seven aneuploid-polyploid gliomas four showed an elevation and three of them a decrease of DI values. Diploid tumors remained diploid. Because of marked heterogeneity of ploidy patterns and the small number of tumors investigated, ploidy changes could not be used for estimation of therapy efficacy and prognosis. Further studies will be necessary to answer this question.

Adult↗
PubMed1994

Megakaryopoiesis in chronic myeloproliferative disorders: immunohistochemical evaluation of endoreduplicative activity by PCNA-staining reaction.

A morphometric analysis has been performed on bone marrow trephine biopsies following sequential double-immunostaining with monoclonal antibodies PC10 (anti-proliferating cell nuclear antigen--PCNA) and Y2/51-CD61 (anti-platelet glycoprotein IIIa) to evaluate endoreduplicative activity of megakaryopoiesis. In addition to a control group, patients included different subtypes of chronic myeloproliferative disorders (CMPDs) like chronic myeloid leukaemia (CML), polycythaemia vera (P. vera), primary thrombocythaemia (PTH) and finally primary (idiopathic) osteomyelofibrosis (OMF). In comparison with the normal bone marrow and also with P. vera and PTH a significant increase in PCNA-labelling (late G1 and S phases) of megakaryocytes was recognizable in OMF, contrasting with a striking reduction of this marker in CML. Particularly in advanced stages of OMF, secondary folate deficiency leading to a megaloblastoid appearance of erythroid precursors is a frequent finding. In pernicious anaemia previous cytokinetic studies have demonstrated an arrest in the S phase (DNA synthesis) of the cell cycle due to vitamin B12/folate (haematinic) deficiency. A similar pathomechanism may also be effective in OMF. Consequently, a block in the S phase of the cell cycle is assumed which is in keeping with the increased numbers of PC10-positive megakaryocytes. Significant correlations were calculable between megakaryocyte sizes and PCNA-staining capacity in the normal bone marrow and CMPDs. According to morphometry small-sized (hypoploid) megakaryocytes showed a prevalence of PCNA labelling. This finding is confirmative with a hypothesis on the dynamics of endoreduplicative activity of megakaryocytes, i.e. the prolongation of G1/G2 phases in larger (polyploid) elements. On the other hand, some of the giant polyploid megakaryocytes may cease endoreduplication and enter into G0 phase, which could partially explain the predominance of PCNA-negative large-sized cells of this lineage.

Aged↗
PubMed1993

[Expression of cyclin B in megakaryocytes and cells of other hematopoietic lines].

Megakaryocytes are normal bone marrow cells which have the unique ability to become polyploid. This phenomenon is termed endomitosis and its mechanism remains poorly understood at present. It is known that the cell cycle of eukaryotes, particularly at G2/M transition, is regulated by a complex with histone H1 kinase activity, the maturation- or M-phase promoting factor (MPF). We have therefore studied the expression of subunits of MPF, the p34cdc2 and cyclin B in normal bone marrow culture megakaryocytic cells, blood leukocytes and platelets as well as in human megakaryoblastic cell lines Dami, Meg-01, HEL and the promyelocytic cell line HL60. Using immunohistochemistry and electron microscopy we have observed that cyclin B was virtually undetectable in megakaryocytes and platelets, but was abundant in granulocytes, monocytes/macrophages and HL60. Studies by RT-PCR showed the presence in large quantities of mRNA of cyclin B in all cell types studied, even in megakaryocytic-like cells. These observations suggest some important implications in the understanding of the mechanisms of megakaryocyte polyploidization and related endomitosis.

Blood Platelets↗
PubMed1994

[Feasibility of DNA analysis for prognostic evaluations of colorectal cancer using touch preparations and image cytometry].

The purpose of this study is to demonstrate the applicability of DNA ploidy analysis by using touch preparations obtained from fresh and frozen tissues on the prognostic evaluations of colorectal cancer, especially the development of hepatic metastases. The specimens obtained from surgically resected colorectal carcinomas (32), and hepatic metastases (4), were lightly touched with the slide glasses. Among the 32 patients with colorectal cancers, 7 had synchronous liver metastases, and 5 metastases developed metachronously. After formalin-fixation and Feulgen staining, the DNA analysis was performed by image cytometer (ICM, CAS200R). The data were expressed as the ploidy patterns and DNA indices (p-DNAI: peak DNA index, m-DNAI: mean DNA index), and were correlated very well with those obtained from cytophotometry. The DNA ploidy pattern among the 32 colorectal cancer specimens were Aneuploid(A) in 16, Polyploid (P) in 7, Diploid (D) in 4 and D+P in 5. The cases with synchronous or metachronous liver metastases had a higher m-DNAI (1.74 +/- 0.28) and were Aneuploid in 8, P in 3, and D+P in 1 case. Aneuploidy or Polyploidy predominated among the cases with metachronous liver metastases (4 in 5 cases), with m-DNAI higher than 1.5. Thus, "m-DNA > 1.5" could be a useful prognostic indicator of the high risk group of liver metastases. The liver metastases specimens had Aneuploid and Polyploid patterns in 2 cases each, with m-DNAI ranging from 1.88 to 2.31. DNA ploidy pattern analysis using touch preparations with image cytometry may be useful in determining the prognosis for colorectal cancer.

Adult↗
PubMed1993

Evolution of DNA content in small and well-differentiated hepatocarcinoma.

DNA ploidy of 10 well-differentiated hepatocarcinomas was evaluated by densitometry on smears obtained by fine needle aspiration biopsy (FNAB) with ultrasound guidance. Because of diagnostic difficulties or an increase in tumor size, FNAB was repeated once in six cases and twice in four. All the material collected was then analyzed to study the variation of DNA content over time in these neoplasms. The study was performed on Feulgen-stained slides using an IBAS image analyzer system. DNA ploidy was evaluated by visual interpretation of the corresponding histograms and by calculating the 5c exceeding rate (5cER), 2c deviation index (2cDI) and DNA malignancy grade (DNA-MG). In the first samples seven cases were euploid (four diploid and three polyploid), with 5cER < 5; three cases were aneuploid, with 5cER > 5. In the second samples, taken after four or six weeks, eight cases became aneuploid, with 5cER > 5, and two cases were polyploid, with 5cER > 5. Four cases in which a third FNAB was performed resulted in aneuploidy, with 5cER > 5. A progressive increase in aneuploidy, 5cER and DNA-MG values was observed in six cases in the second FNAB and in one case in the third FNAB along with the increasing size of the nodules. In only one case we observed a paradoxical reduction in the 5cER in the third FNAB; the corresponding histogram remained aneuploid. Limitations could have occurred because of sampling small nodules under ultrasound guidance and because of the relatively small number of cells measured. DNA evaluation seems to be useful in the diagnosis of hepatocarcinoma and in the study of its biologic features.

Aged↗
PubMed1977

[Cytophotometric method of determination of DNA content of neoplastic cells].

The content of DNA was investigated by the method of pulsed cytophotometry in cells of the following neoplasms: pulmonary cancer, benign lesions and cancer of the stomach, melanomas of the skin, malignant lymphomas and metastases of cancer into the lymphatic node. In the normal tissues and benign tumours the number of proliferating cells (DNA value of 3p, 4p) was not significant--from 2.0 to 5.0%. Polyploid cells were absent. In tumours the number of cells varied broadly--proliferating cells from 5.0 to 27,0%, polyploid cells from 9.0 to 40.0%. The use of the method of pulsed cytophotometry in prophylactic examinations is not effective, since in many cases changes could be detected only in single cells not identifiable on DNA-cytograms. Investigation of DNA content by the method of pulsed cytophotometry in a great number of tumour cells makes it possible to determine more accurately the character of the tumoral process, the level of differentiation of the tumoral tissue.

Cell Nucleus↗
PubMed1993

[DNA-cytofluorometric, histopathological and immunohistochemical analyses of pancreatoduodenal cancers].

To study the cell characteristics of relatively small pancreatoduodenal cancers (less than 4 cm in the greatest dimension in histopathological examination), we analyzed the relation between the cell kinetics, and histopathological and immunohistochemical findings. Tumors were classified into t1 (0-2 cm in its greatest dimension) and t2 (2-4 cm). We analyzed DNA contents of five tumors of t1 and twelve of t2 by cytofluorometry. Histopathological and immunohistochemical (CA19-9 and CEA) examinations were also carried out. Nuclear DNA content histograms of the tumors in the t1 group showed all diploid pattern, while those in the t2 group exhibited 4 diploid, 7 euploid polyploid and 1 aneuploid patterns. Invasions to the lymphatic (ly), vein (v) and nerve (pn) were found in five cases of diploid tumors (3 of t1 and 2 of t2) and all (8 in t2) of non-diploid tumors. A medullary pattern of tumor growth was only noted in three tumors of t1. This pattern was not found in t2 (4 diploid, 8 non-diploid). These results suggest that the cellular DNA content increased in association with the tumor growth in size. In the histopathological findings, both the invasions (ly, v, pn) and fibrosis of the stroma more often appeared as the tumor size and polyploid and aneuploid cells increased. However, other histopathological findings and the immunohistochemical examination for CA19-9 and CEA had no relation to the tumor growth and the DNA ploidy pattern.

Aged↗
PubMed1977

[Influence of a series of substances from the group of chlorine-derivative hydrocarbons on the composition of a population of rat liver hepatocytes].

The cytophotometric and kariometric study of the liver hepatocytes in albino rats inhalationally exposed to dichlor- and trichlorpropan showed the latter to be more toxic than the former by its effect on the polyploidization of hepatocyte nuclei. The development of polyploidization was determined by the concentration level of the active agent and the length of exposure to it: the lower the concentration--the more time was required for the realization of the effect.

Animals↗
PubMed1977

[Dynamics of DNA concentration in the nuclei of rat cardiac muscle cells in experimental myocardial infarct].

The content of nuclear DNA was determined microspectrophotometrically in rat cardiac myocyte nuclei at various intervals after the onset of experimental myocardial infarction. In the intact rat heart 1.5 to 1.8% of myocyte nuclei contained tetraploid DNA. Myocardial infarction activated the process of polyploidization in the nuclei of the myocytes particularly in those surrounding the lesion. This process was most intensive during the first week. At later intervals there was an evidence of continuously increasing polyploidization.

Animals↗
PubMed1995

Morphological and cytogenetic observations of unfertilized human oocytes and abnormal embryos obtained after ovarian stimulation with pure follicle stimulating hormone following pituitary desensitization.

Morphological and cytological observations of 189 unfertilized oocytes and 40 abnormal embryos obtained from 32 patients in a routine in-vitro fertilization programme were performed. Both the oocytes and the embryos were mounted whole to preserve the original topology of all the structural elements. With the applied protocol of ovarian stimulation associating pituitary desensitization and follicle stimulating hormone stimulation, a high degree of immaturity of the unfertilized eggs was observed in comparison with previous reports. This immaturity was deduced from the higher incidence of unfertilized eggs arrested at the germinal vesicle or metaphase I stage, as well as metaphase II oocytes with multiple metaphase plates. Nine triploid and four tetraploid embryos were analysed: except for one tetraploid embryo, all the polyploid embryos cleaved. The percentages of mononucleated blastomeres in these polyploid embryos were 57 and 27% respectively. We also analysed 21 cleaving diploid embryos which exhibited a high degree of fragmentation. No more than 40% of the blastomeres contained a single nucleus. Moreover, in only one of the 21 diploid embryos could all the blastomeres be considered normal.

Blastomeres↗
PubMed1996

Genesis and evolution of high-ploidy tumour cells evaluated by means of the proliferation markers p34(cdc2), cyclin B1, PCNA and 3[H]-thymidine.

Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34(cdc2), cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal- and in high-ploidy subpopulations, almost all cells were p34(cdc2)-positive. In the modal-ploidy subpopulation only 17.1% cells were cyclin B1-positive and 85.6% PCNA-positive; in contrast, in the high-ploidy subpopulation up to 91.8% cells were cyclin B1-positive and 97.3% cells were PCNA-positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34(cdc2), both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24-h incubation with 3[H]-thymidine followed by withdrawal of the isotope, high-ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modal-ploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal-ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis.

Animals↗
PubMed1996

DNA image analysis of urinary cytology: prediction of recurrent transitional cell carcinoma.

To evaluate the utility of image analysis in monitoring patients with transitional cell carcinoma, we studied, by cytologic means and by image analysis, 78 urinary tract specimens from 66 patients, of whom 49 (74%) had a previous history of transitional cell carcinoma. The specimens consisted of 51 (65%) voided urine specimens, 12 (15%) bladder washings, 8 (10%) ureteral washings, 3 (4%) ureteral brushings, 2 (3%) renal pelvic washings, and 2 (3%) catheterized urine specimens. DNA histograms were classified into five patterns on the basis of their DNA index and the percentage of their cells with DNA content greater than 5c: diploid (single peak in the 2c region with no cells greater than 5c), intermediate (diploid with less than 10% of cells greater than 5c), aneuploid (single peak or multiple peaks between the 2c and 4c region or more than 10% of cells greater than 5c), tetraploid (at least 10% of cells in the 4c region and a corresponding peak at 8c), and polyploid (multiple peaks in the 2c, 4c, 8c, and 10c regions). Of the 78 cases, 22 were diploid, 24 were intermediate, 29 were aneuploid, one was tetraploid, and two were polyploid. Histologic confirmation or clinical follow-up was found in 29 aneuploid cases, 13 intermediate cases, and one diploid case. Most cases of carcinoma in situ (five of six) and invasive tumors (12 of 17) were aneuploid. The sensitivity was 100%, and the specificity was 73% when cytologic and image analysis results were combined. We conclude that image analysis, when combined with cytologic examination, is a reliable noninvasive diagnostic test for monitoring patients with transitional cell carcinoma; aneuploidy is specific for malignancy; and the presence of cells greater than 5c, although frequently associated with tumor recurrence, can be seen in non-neoplastic conditions.

Adult↗
PubMed1977

[Long-term reproduction of triploid and tetraploid parthenogenetic clones of silkworms during artificial thermal parthenogenesis].

The triploid and tetraploid clones of the silkworm (Bombux mori L.) are capable to be reproduced for a long time by means of thermal parthenogenesis. The activated eggs are pigmented in 80-99%. The hatching of larvae in triploid clones amounts to 20% and in tetraploid ones to 25-30%. The pigmentation of eggs and hatching of larvae greatly vary in different years: 47 to 100% and 0 to 73%, resp. In this respect, the polyploid clones are similar to those diploid ones which are characterized by the low percentage of complete parthenogenesis. The causes for the variability of parthenogenesis indices in the polyploid clones are discussed.

Bombyx↗
PubMed1977

[Pulse cytophotometric investigations concerning the biological behaviour of laryngeal carcinomas (author's transl)].

25 laryngeal carcinomas were investigated by pules cytophotometry, stained with ethidium bromide after pepsination. The karyograms were interpreted as DNA-distribution curves. In diploid populations the height of the 4c-peak was taken as a measure of proliferation rate. Aneuploidy and polyploidy were estimated by camparison with human diploid material . In our material we found 13 polyploid tumors (under them 6 aneuploid populations from 7 at all). After dividing the cases into two groups (1: with slow, 2: with rapid progression) the moiety of polyploid and aneuploid tumors and the height of the 4c-peak was significantly higher in group 2. The pulse cytophotometry gives reliable additional informations showing the great variability of biological characteristics of laryngeal carcinomas. Possibly this is a basis of a more differentiated therapy.

Aneuploidy↗
PubMed1997

Modeling, definition and applications of histogram features based on DNA values weighed by sine functions.

OBJECTIVE: To model new DNA histogram features that weigh DNA values with values of curves of a sine function and to show the definition and applications of such features. STUDY DESIGN: A simple example of a sine feature can be modeled to yield the value zero if all cells are diploid or polyploid, with values of 2c, 4c or 8c, and to yield the value 100 if all cells are aneuploid, with DNA values of 3c, 6c or 12c-e.g., cells that are probably from a malignant lesion or indicate proliferation. All other values are multiplied by the corresponding sine value. We folded the logarithmic DNA histogram with a sine curve with positive values only. RESULTS: Correlation with ploidy balance was -0.94, demonstrating the similarity of both features. The sine features, however, avoid cutpoints between diploid and aneuploid values and are therefore less influenced by minor mistakes in standardization of DNA histograms. We introduced deviation factors as variants; that led to higher sine values for higher c values. For breast carcinoma (N = 306) the sine values were spread from very low to very high values, whereas esophageal carcinomas (N = 125) were centered at a sine value of 50. In breast carcinoma the sine features also correlated with prognostic factors, including hormone receptor status. CONCLUSION: Description of DNA histogram features by graphic demonstration of their weight functions improves understanding of features. Since functions respect the cyclic events in proliferation and are not influenced by polyploidization.

Breast Neoplasms↗
PubMed1998

Effects of cytokines on platelet production from blood and marrow CD34+ cells.

The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+ CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF- or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.

Antigens, CD34↗
PubMed1998

Endomitosis of human megakaryocytes are due to abortive mitosis.

During megakaryocyte differentiation, the promegakaryoblast (immature megakaryocyte) increases its ploidy to a 2(x) DNA content by a poorly understood process called endomitosis. This leads to the formation of a giant cell, the megakaryocyte (MK), which subsequently gives rise to platelets. In this report, we show that endomitosis of human MKs is due to abortive mitosis. Human MKs were obtained by a two-step purification of CD34(+) blood or marrow precursors followed by in vitro culture in the presence of MK growth factors. Microscopic examination shows that a large number of centrosomes (up to 32) and centrioles are present in polyploid MKs. After nocodazole treatment, more than 20% of the MK are blocked in a typical pseudo-metaphase. Both spontaneous and nocodazole-induced endomitosis are associated with a breakdown of the nuclear envelope and possess a complex mitotic spindle composed of several asters. Spindle microtubules radiate from each aster, creating a spherical structure. At metaphase, expression of the kinetochore phosphoepitope recognized by the 3F3/2 antibody is lost, and the sister chromatids segregate moving toward the spindle poles. After limited segregation, the chromosomes decondense and the nuclear envelope reforms in the absence of cytokinesis, isolating all chromosomes in a single nucleus. It has been proposed that endomitosis could be due to an abnormal CDK1 activity or an absence of cyclin B1. Our results show that cyclin B1 can be detected in all MKs, including those with a ploidy of 8N or more. The cyclin B1 staining colocalizes with the mitotic spindle. Using flow cytometry, the level of cyclin B1 increased until 8N, but remained identical in 16N and 32N MKs. Cell sorting was used to separate the MKs into a 2N/4N and >4N population. Both cyclin B1 and CDK1 could be detected in the endomitotic polyploid MKs using Western blot analysis, and a histone H1 kinase activity was associated with immunoprecipitated cyclin B1. We conclude that endomitosis of human MKs is due to abortive mitosis, possibly due to alterations in the regulation of mitotic exit.

Antigens, CD34↗