Peculiarities of mitosis and nucleolar characteristics of the birch plantlets under antropogenous pollution

A study was made of some cytogenetic characteristics (mitotic activity, the level and spectrum of pathological mitosis, nucleolar features in root tip cells) in birch plantlets. The seeds were collected in four districts of Voronezh and in the ecologically clean territory. The index of mitotic activity has a considerable resistance to anthropogenous pollution. In the experimental areas, the level and spectrum of pathological mitosis increase. In contaminated areas we observed changes of nucleolar characteristics (the increased surface area of nucleoli and their higher number in cells, the increased number of cells with highly active types of nucleoli, the appearance of residual nucleoli). These changes can be considered as possible mechanisms of adaptation to stress due to antropogenous pollution. It is suggested that the use of such indices as single nucleolar surface area or the level of pathological mitosis may be perspective for cytogenetic monitoring of the environment, and for prognostification of environmental conditions suitable or unsuitable for the human health.     

Studies on satellite nucleoli in human blastic cells of acute leukaemias.

Blastic cells of acute lymphoid, acute myeloid and acute myelomonocytic leukaemias were studied by means of indirect immunofluorencence to provide more information on the presence of satellite nucleoli in blood cells. According to results, satellite nucleoli were found in a small but constant number of blastic cells disregarding their type and type of acute leukaemia. Satellite nucleoli exhibited a positive immunoreaction for fibrillarin and protein B23 which are characteristic for main nucleolar components. These findings suggest that satellite nucleoli contain fibrillar centers as well as dense fibrillar and granular components or at least proteins characteristic for these nucleolar components. Similarly as in normal and pathological cells of completely different origin, in blastic cells of acute leukaemias the number of satellite nucleoli per cells ranged between 1 and 2.     

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.     

A quantitative study of Ag-positive nucleolar segments revealed by silver staining in the interphase nuclei of trophoblast cells from the rat placental connective zone

A cytomorphological study was made of silver stained nucleoli in interphasic nuclei of trophoblast cells from the rat placenta connective zone, in addition to calculation of Ag-positive spherules in the nucleoli. The prevalent number of Ag-positive nucleolar spherules in the nuclei was 6, corresponding to the number of nucleolar organizers (NOR's) in the diploid chromosome complement of the rat. The mean number of Ag-positive spherules in the nucleoli progressively increase in the course of polyploidization from 2c to 32c; variability of the spherule number also increasing. The mean area of nucleoli is found to increase in proportion to the ploidy degree. A high correlation is found between the number of Ag-positive spherules and the area of nucleoli in the nucleus (r = 0.78). This appropriateness is exhibited at all the ploidy levels. The number of Ag-spherules and the area of nucleoli are found to depend slightly on the number of nucleoli. The possibility to use the number of Ag-positive spherules as a criterion of the activity of the NOR in interphasic nuclei is discussed.     

Comparison of impact of EMCV replication on the nuclear apparatus of NIH 3T3 and HEp-2 cells

We have investigated differences between the actions of encephalomyocarditis virus (EMCV) on cytometric indices in cultured NIH 3T3 and HEp-2 cells, which are characterized by different levels of transformation. HEp-2 cells surviving 48 h after EMCV infection showed lower nuclear ploidy, reduced nuclear area, fewer nucleoli and a higher percentage of euploid cells. There was a significant increase of nucleolar/nuclear DNA 6-24 h after EMCV infection. However, EMCV had markedly different effects on NIH 3T3 cells: there was a consistent increase in population ploidy, but the average number of nucleoli and the number of euploid cells in the population remained constant. The nucleolar/nuclear DNA ratio was almost unchanged. These different viral effects might be explained by the contrasting levels of differentiation of the cultured cell lines. The number of nucleoli does not depend on the amount of nuclear DNA in either viral-infected or intact cells but on the euploidy-to-aneuploidy ratio. The ratio of the sums of the nucleolar perimeters to the nuclear perimeter increases linearly with the number of nucleoli per nucleus in both intact and virus-infected cells. In both cell lines, the amount of DNA per nucleolus decreases as the number of nucleoli increases.     

Ultrastructural changes in nucleoli and fibrillar centers under the effect of local ultraviolet microbeam irradiation of interphase culture cells

As shown previously, ultraviolet (uv) microbeam irradiation of one of the two mature nucleoli within an interphase cell nucleus causes significant diminution and inactivation of the irradiated nucleolus and compensatory growth and activation of the nonirradiated one. In the present work we describe the results of an ultrastructural study of this phenomenon. The changes in the nucleoli were examined by means of complete series of ultrathin sections obtained from seven irradiated pig kidney cells. The compensatory hypertrophy of the nonirradiated nucleoli is shown to be accompanied by a nearly twofold increase in the number of fibrillar centers (FCs) and by a decrease in their linear dimensions compared with the control cells of the same ploidy. In the degraded nucleoli the number of FCs decreases, but their dimensions increase. Ultraviolet microbeam irradiation causes dramatic diminution of the dense fibrillar component within the irradiated nucleoli as well. The nucleolar capacity for compensatory hypertrophy indicates that in addition to active ribosomal genes, mature nucleoli also contain "silent" genes capable of being activated under extreme conditions to sustain the required level of rRNA synthesis. It is assumed that activation of latent ribosomal genes is accompanied by FC "fragmentation" without a considerable increase in their total volume per cell.     

Human large intestine adenocarcinoma cells (CaCo-2) in the process of cultivation

Using cytomorphometry and cytophotometry cells of human large intestine adenocarcinoma (CaCo-2) were studied under condition of a 10 day cultivation. A reverse dependence was established between proliferative activity and monolayer density. The increase of the latter inhibits proliferation and promotes the formation of islets of polymorph cells. 2c-cells could be seen only at the beginning of culture growth; a larger part of cells polyploidized by cell blocking in G2-phase. These cells do not divide, which is testified by the absence of 2c-cells, but some part of 4c-cells start the next cycle, accumulates 8c-DNA and then divides, replenishing the 4c-cells population. In the process of cultivation, we observed an increase in the number and total volume of nucleoli in the nuclei, and a rise in DNA amount in the peri-nucleolar chromatin. The formation of numerous 4c-cells with multi-nucleolar nuclei may define an increase of functional activity of CaCo-2 culture as the whole, whereas the formation of separated groups of such cells in the monolayer may denote a possible initiation of their differentiation.     

Simultaneous quantification of nucleoproteins and comparison of methyl green-pyronin Y and Feulgen staining in sections of oral squamous cell carcinoma,dysplastic lesions and normal mucosa

A fundamental difference between normal cells and tumor cells is the proliferative activity of the nucleus and nucleolus, which increases progressively from normal to oral dysplastic mucosa to oral squamous cell carcinoma (OSCC). This activity is evaluated routinely using hematoxylin and eosin (H & E) staining, but in some cases, inter-observer variability occurs among pathologists. We evaluated cellular proliferation by staining sections with the methyl green-pyronin Y procedure and the Feulgen reaction. We also compared the efficacy of methyl green-pyronin Y and Feulgen staining for studying nuclear and nucleolar features in oral dysplastic mucosa and in different grades of OSCC. Sections cut from formalin fixed, paraffin embedded blocks of five normal mucosa, 15 dysplastic mucosa, 10 well-differentiated OSCC, 10 moderately differentiated OSCC and five poorly differentiated OSCC cases were stained with Hematoxylin and Eosin, methyl green-pyronin Y and the Feulgen reaction. The mean diameters of the nuclei and number of nucleoli showed significant differences. A progressive increase in diameter of the nucleus and number of nucleoli was observed from normal mucosa through poorly differentiated OSCC. We observed that methyl green-pyronin Y stain is more useful than Feulgen and hematoxylin and eosin for simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine examination.     

New indices in morphometry of nuclear structure in the NIH 3T3 cell line

We analyzed the connection of changes in nucleus ploidy with changes in nucleolar apparatus of NIH 3T3 cells. The quantity of nucleoli does not depend on the quantity of nucleolar DNA, but instead depends on euploidy: the majority of euploid cells have 1-3 nucleoli. The quantity of DNA in the nucleolus is correlated with the quantity of nucleolar DNA, and does not depend on ploidy changes. The nucleolar area has a tendency to increase in line with an increase in their numbers in the nucleus. The relationship of the quantity of DNA in the nucleolus with that of the nucleus is stable. During the process of increase in the number of nucleoli in a nucleus, there is a corresponding decrease in the quantity of DNA in each nucleolus, and there is likewise no increase in the sum of nucleolar DNA. The ratio of sums of the nucleolar perimeters to nuclear perimeter is a significant factor, which increases linearly along with an increase in the number of nucleoli in a nucleus.     

Tissue and sex differences in the expression of nucleoli in mouse somatic cells.

In Mus musculus, the nucleolus organizer regions (NORs), or sites of ribosomal RNA-encoding genes, map at three chromosomal pairs. A silver procedure was modified to stain nucleoli in interphasic somatic cells of mice. The number of nucleoli per cell nucleus was determined in squashed cells of kidney, liver and pancreas obtained from male and female mice. In liver and pancreas cells the average number of nucleoli per cell was 4.84 and 4.66, respectively, and only 2.83 in kidney cells (p < 0.001). Less than 8% of pancreas cells and about 15% of liver cells contained more than 6 nucleoli per cell, which was the maximum expected number. In addition, the number of nucleoli per cell was significatively different (p < 0.01) when male and female liver or pancreas cells (not kidney cells) were compared. In both cases, female cells presented more nucleoli than the respective male cells. Assuming that the available NORs are the same, the variable number of nucleoli in the examined cell types would be the consequence of a tissue specific NOR regulation. The apparent influence of sex on this regulation is noted.     

Nucleolin and Nucleoli in Ependymocytes and Tanycytes of the Third Ventricle of the Rat Brain

The aim of the present study was to compare structure of the nucleoli of ependymocytes, tanycytes, and secretory cells of the subcommissural organ using immunohistochemical staining for nucleolin and confocal laser microscopy. The study was performed in samples from the diencephalon of adult male Wistar rats (n = 6). The samples were fixed in zinc–ethanol–formaldehyde, a fixative providing a high level of preservation of antigen determinants. In the present study, we estimated diameters of nucleoli and their number in various types of cells lining the third ventricle. We compared for the first time the nucleoli of different subpopulations of tanycytes and report data on the distribution of nucleolin protein in the cells lining the ventricles. The content and location of nucleolin reflect the functional state of the cell. Our data will promote understanding of the interrelationships between the indices of the nucleolar apparatus and the functional state of the cell under various conditions, including stress, neoplastic transformation, and other pathological conditions.     

RNA synthesis in the circular nucleoli of hepatocytes]

By analysis of serial sections it has been revealed that the so-called ring-like nucleoli of hepatocytes consist of a cavity with an amorphous contents surrounded by fibrillar and granular material. Such nucleoli are sometimes encountered in normal animals; the number of ring-like nucleoli increases considerably in chronic pathological process caused by repeated CCl4 injections. The capacity of RNA synthesis in the ring-like nucleoli was revealed by means of electron-microscopic autoradiography.     

Properties and Functions of a New Nucleolar Protein,Surf-6, in 3T3 Mouse Cells

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle.     

Qualitative and structural peculiarities of manifestation of nucleoli-forming chromosome regions in breast cancer cells

Qualitative differentiated study and calculation of 44404 main morphofunctional types of nucleoli has been perfomed on Ag-positive punction specimens from 50 patients with glandular breast cancer. Accordance of different for their activity morhofunctional types of nucleoli and extranucleolar argentofil granules to proliferative activity rates, malignant transformation and dystrophic changes of tumor cells has been demonstrated.     

Properties and functions of a new nucleolar protein, Surf-6, in 3T3 mouse cells

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. II. Mechanisms of polyploidization

Peculiarities of the structure of interphase nuclei, mitotic activity, and Ki-67 protein intranuclear immunolocalization were studied to elucidate mechanisms of genome multiplication in proliferative and differentiating invasive extravillous trophoblast cells in the human placenta. The presence of numerous chromocenters was shown to be a characteristic feature of proliferative cell nuclei of both villous and extravillous trophoblast. At the beginning of extravillous trophoblast cell differentiation, i.e. in the proximal part of cell columns, some amount of cells with large nuclei containing enlarged chromocenters were found. DNA content was measured simultaneously with counting the number of chromocenters in similarly looking nuclei of squash preparations of placental villi. The increase in the ploidy level up to 4c-8c, accompanied by a slight increase in the number of chromocenters being not proportional to the ploidy level and not exceeding the diploid number of chromosomes of the human genome, was demonstrated. This suggests that genome multiplication of extravillous trophoblast cells may be accomplished by endoreduplication. In addition, pictures of endomitosis were seen at early steps of differentiation of EVT cells. The lack of polyploid mitotic figures or any obvious polyploidizing or restitutional mitoses suggests that these are not of considerable importance in genome multiplication of human EVT cells. However, the prevalence of metaphases at the boundary of the distal part of cell columns suggests that restitutional mitoses may be involved, even partly, in human trophoblast cell polyploidization. At later steps of differentiation, i.e. in the distal part of cell columns, the nuclear structure obviously changes, with a uniform "network" chromatin arrangement prevailing, whereas numerous chromocenters and features of endomitosis are no longer seen. The pattern of Ki-67 protein immunolocalization is also changing along the invasive pathway. In the proliferating stem cells and trophoblast cells of the proximal part of cell columns, Ki-67 was localized in the karyoplasm, chromocenters and numerous small nucleoli, whereas in the distal part of cell columns this protein was detected predominantly in 1-2 large nucleoli. The comparative analysis of the literature data on Ki-67 localization at different stages of cell cycle provided another evidence that EVT cells in the course of invasion may switch to the endoreduplication cycle. In agreement with the relevant report on rodent placentation, our present data suggest that acquirement of an invasive phenotype of EVT cells is accompanied by switching from mitotic division to endoreduplication cycle.     

Behavior of HEp-2 cell line cells, their nuclei and nucleoli during cultivation and under the effect of Na-ds-RNA

The behavior of human larynx cancer cells (HEp-2) and of their nuclei and nucleoli during the cultivation without the influence of Na-ds-RNA and after its introduction into the medium was investigated by methods of cytomorphometry and cytophotometry. The density of monolayer (the number of cells on the area unit), percentage of two-nuclear cells, the number of nucleoli in the nuclei, mitotic coefficient, volume and total surface of nuclei and nucleoli have been measured. In addition, the mass of DNA in the nuclei and that of the total RNA and DNA in the nuclei and in each nucleolus was measured. Cells in the culture, not subjected to the influence of Na-ds-RNA, were weakly differentiated, kept active proliferation, and their population contained a small number of two-nuclear elements and a high share of multi-nuclear cell. During cultivation, these indices became even more pronounced, which is typical for the increase in cell malignancy. Under the influence of Na-ds-RNA, the proliferate activity decreases, the number of double-nuclear cells increases, while that of multi-nucleolar cell decreases; also, the share of cells with one- and two-nucleolar nuclei increases. The authors conclude that Na-ds-RNA may have antineoplastic activities, clearly evidenced from its influence on the culture of transformed HEp-2 cells.     

Nucleolus and large nucleolar aggregates of condensed chromatin in interphase nuclei of L 929 cells

Summary Three-dimensional reconstructions show that the nucleoli from L 929 cells are associated with one or several large aggregates of chromatin displaying a honeycomb-like structure. The form and the number of both nucleoli and honeycomb structures vary as the cells emerge from the resting state and enter exponential growth. Quantitative data show that the number of honeycomb structures decreases as the number of nucleoli diminishes; both numerical regressions are significant. In addition, the nucleoli and the honeycomb structures enlarge when the cells enter the exponential growth phase.In resting cells the number of honeycomb structures is correlated to the number of nucleoli. Therefore we conclude that the large nucleolar mass of condensed chromatin, which in L 929 cells displays a honeycomb structure, contains a portion of the nucleolar organizing region.     

Nucleolar morphology and rDNA in situ hybridisation in monocytes

Summary The aim of this study was to correlate morphological changes of nucleoli of non-proliferating monocytes to their functional activity, since nucleolar morphology is currently considered as a diagnostic marker for cell proliferation. Monocytes from healthy donors were fractionated by current counterflow centrifugation and kept in culture for 6 days. Cells were stimulated by the addition of 200 units/ml interferon (IFN). Under this stimulus the monocytes show no proliferation but a strongly augmented expression of type I Fc IgG receptor, human leucocyte antigen DR, human leucocyte antigen DP and human leucocyte antigen DQ. Morphological changes after stimulation included the appearance of multinucleated cells, typical signs of the activation of rRNA synthesis indicated by an increase in nucleolar size, and changes in nucleolar structure such as the appearance of reticulate and compact nucleoli. The number of nucleolus organiser regions (NORs) visualised by in situ hybridisation was compared with the position and number of nucleoli visualised by silverstaining in interphase cells. In comparison with control cultures, activated monocytes show a distinct increase in the number of those NORs that take part in the formation of nucleoli. Our results show that, in non-proliferating activated monocytes, the morphology of nucleoli and the increase of NOR activity are similar to those in proliferating cells. NOR activation is therefore an indicator for cellular activity, but is not necessarily correlated with proliferation.