Chromatin texture in high grade prostatic intraepithelial neoplasia and early invasive carcinoma

OBJECTIVE: To evaluate individual nuclei from high grade prostatic intraepithelial neoplasia (PIN) lesions with early invasive carcinoma foci in the area of microinvasion and in the gland in which the microinvasion originated. STUDY DESIGN: High-resolution, digitized images of nuclei from defined locations were recorded and segmented, and karyometric variables were computed. These included a set of 93 features, which form a nuclear signature characterizing the spatial and statistical distribution of the nuclear chromatin. Nuclei in the glandular epithelium were recorded sequentially, along the basal cell layer, at increasing distances from the point of microinvasion and by random selection in the region of microinvasion. RESULTS: At a distance > 60 nuclear locations from the point of microinvasion, the nuclear signatures corresponded to those seen in high grade PIN. Between 40 and 20 nuclear locations removed from the microinvasion focus the signatures began to change gradually until at a distance of 15-5 locations they strongly resembled the signatures seen in adenocarcinoma. The total optical density decreased to values seen in adenocarcinoma, and the nuclear chromatin had finer granularity. While nuclei in high grade PIN followed a widely dispersed total optical density distribution suggestive of wide-ranging aneuploidy, the nuclei in the region of microinvasion exhibited a less dispersed and bimodal total optical density distribution. CONCLUSION: The chromatin texture signatures showed a clear trend: there was an obvious attenuation as the measured nuclei approached the microinvasion area. The decrease in total optical density at the microinvasion might suggest the emergence of one or two clones that can be responsible for the invasive phenotype.     

Change of chromatin morphology during the cell cycle detected by means of automated image analysis

Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G₁, middle S and G₂ phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G₁ through S to G₂ phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G₁, S and G₂ phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.     

Quantitative description of nuclear morphology in assessing resistance of sarcoma 180 to adriamycin

Resistance to adriamycin generally is explained through changes of cell/drug interactions that possibly reflect structural alterations of intracellular targets. One of the main targets of adriamycin is believed to be nuclear chromatin. In order to recognize chromatin alterations, we studied cell nuclei morphology and chromatin structure by means of digital image analysis. The studies were performed in both adriamycin-sensitive and -resistant Sarcoma 180 cell lines which were cultured under growth-stimulated and nonstimulated conditions. Using specially developed methods, we extracted parameters characterizing geometrical, optical, and structural properties of the cell nuclei from light microscopical images. The latter parameters concerned microscopical appearances of condensed chromatin and were described by features of high-optical-density regions. The results demonstrated that the quantitative criteria applied enabled the discrimination of sensitive and resistant cells. The most important parameters are the nuclear size, number, distribution, and optical density of condensed chromatin regions. In addition, the criteria permit recognition of changes related to differences in the growth conditions of the cells. The data of the image analysis suggest that adriamycin resistance in Sarcoma 180 cells is associated with characteristic patterns of cell nuclear morphology which can be described with a sufficient number of appropriate parameters. The advantages of image analysis are evident when these results are compared with the flow cytometric findings. The conclusion is that structural features of nuclear chromatin provide information essential for the assessment of drug resistance.     

Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells

In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.     

Phospholipids in plant and animal chromatin

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.     

Nuclei of lymphocytes of the T- and B-cell lines during normal embryogenesis and after treatment with hydrocortisone (morphometric and probabilistic-statistical parameters)

Lymphoid cells of the thymus and of the Fabricius bursa have been studied in 18-day-old chick embryos, normal and after injection of hydrocortisone on the 11th day of embryogenesis. By means of optical-structural computer analysis, the complex of morphometric and probability-statistic parameters of the nuclei in the lymphocytes are estimated: area of the nuclei, optical density of chromatin, asymmetry coefficient and variance. Normal T-lymphocytes possess less density of the nuclei, greater optical density of chromatin, greater values of negative asymmetry. The complex of these parameters can be used for identification of visually similar lymphoid cells of T- and B-lines. Under hydrocortisone effect structural changes of the nuclei in the thymus and Fabricius bursa lymphocytes of the chick embryo are uniform: increase in the area of the nuclei, decrease in optical density of chromatin, the asymmetry coefficient becomes positive.     

Electron-radioautographic analysis of 3H-thymidine distribution in the cell nuclei of the Chinese hamster

Three types of the label localization in the nuclei of Chinese hamster fibroblasts, growing for 9 and 13 hours with 3H-thymidine, were detected using electron microscopic autoradiography: 1. The label is relatively evenly distributed throughout the karyoplasm. 2. Silver grains are concentrated as stripes through the nucleus; a high label density is also found in the nuclear periphery and around the nucleolus. 3. The label is mainly concentrated over the condensed chromatin adjacent to the nuclear membrane. The cells labeled in the first half of S-phase and selected with colchicine in postsynthetic phase of the 1st and the 2nd cycles are characterized by the second and third types of label distribution. In the cell nuclei fixed in the postsynthetic period of the second cycle, the label localization in stripes is discontinuous. The results indicate that during cell transition from S to G2 the newly-synthetized DNA changes its localization in the nucleus. It is suggested that the second type of label distribution depends on the interphase chromosome concentration in definite zones of the nuclear volume after S-phase termination, and the third type label localization is connected with the formation of prophase chromosomes.     

Immunoelectron Microscopic Localization of Sperm-specific Nuclear Basic Proteins during Spermatogenesis in Anuran Amphibians

Antisera were raised in rabbits against sperm-specific nuclear basic proteins (SPs) of Bufo japonicus and Xenopus laevis . The localizations of these proteins in spermatogenic cells were then studied by electron microscopy with colloidal gold labeled antibodies as probes. The numbers of gold particles counted on ultra-thin sections of cells at various spermatogenic stages were corrected for the density per unit area, on the basis of areas determined with a digitizer. No grains were deposited during early nuclear elongation stages. Grains appeared on nuclei at the beginning of chromatin granulation, and their density increased first gradually and then sharply at the last step of spermiogenesis. Recalculation of grain counts according to the estimated nuclear volumes of Bufo spermatogenic cells also indicated a sharp increase in the amount of SPs per nucleus in the last step of spermiogenesis. No significant localization of grains in the cytoplasm was observed at any stage of spermatogenesis.     

Cytochemical and autoradiographic studies of the localization and turnover of chromatin and nucleolar associated phospholipids in plant and animal tissues

The localization of chromatin-associated phospholipids has been demonstrated on chromosomes and on chromatin of interphase nuclei by cytochemical methods either in plant and in animal tissues. Three methods of fixation are suggested which can be combined which two cytochemical methods for phospholipids detection. An additional method is represented by autoradiographic technique after incorporation of a radioactive precursor such as [3H] ethanolamine. This method has been used with good results in nuclei of lateral root apices of Vicia faba on 1 micron thick section, thus avoiding any possible contamination by nuclear membrane. In these nuclei the phospholipids appear associated with the chromatin and more intensely with the nucleolar regions. The positivity of the cytochemical reaction disappears after extraction of fixed tissue with acidified methanol chloroform and after treatment of unfixed sections with phospholipase D. The use of phospholipid precursor has allowed the study of chromatin-phospholipids synthesis in root apices of Vicia faba with respect to timings of the cell cycle. The results show that there is a strong case for a pattern of chromatin phospholipid synthesis which operates during S phase. Concerning the role of phospholipids it is suggested that they may be linked to acidic protein and may have a structural function, particularly on the nucleoli.     

Visualization of chromosome territories in interphase nuclei of ovarian nurse cells in Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Morphological changes of sperm nuclei during spermatogenesis in the brown alga Cystoseira hakodatensis (Fucales, Phaeophyceae)

Morphological changes and chromatin condensation of sperm nuclei were observed during spermatogenesis in the fucalean brown alga Cystoseira hakodatensis (Yendo) Fensholt. Ultrastructural studies have shown that the mature spermatozoid has an elongated and concave nucleus with condensed chromatin. The morphological changes and the chromatin condensation process during spermatogenesis was observed. Nuclear size decreased in two stages during spermatogenesis. During the first stage, spherical nuclei decreased in size as they were undergoing meiotic divisions and the subsequent mitoses within the antheridium. During the second stage, the morphological transformation from a spherical into an elongated nucleus occurred. Afterwards, chromatin condensed at the periphery in each nucleus, and chromatin‐free regions were observed in the center of the nucleus. These chromatin‐free regions in the center of nucleus were compressed by the peripheral chromatin‐condensed region. As the result, the elongated and concave nucleus of the mature sperm consisted of uniformly well‐condensed chromatin.     

Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study

The ultrastructural localization of a proteasomal antigen in human spermatozoa was studied by means of immunolabeling with the MPC21 monoclonal antibody and secondary gold labeled antibody with 1.4 nm gold particles in combination with silver enhancement reaction using pre-embedding technique. The labeling was found in the acrosomal and postacrosomal regions, in the connecting-piece (neck) and, in some cases, in the middle-piece and also in the residual bodies. There was no significant reaction in condensed chromatin. In some abnormal forms of spermatozoa, in which the chromatin was not well condensed, the labeling in nuclei was present. The nuclear vacuoles with looser chromatin were usually strongly labeled. The nuclei of cells representing different stages of spermatogenesis, that were present in semen samples, were also labeled.     

XCAP-C类似蛋白存在于蒜根端细胞核和染色体中

以抗XCAP-C抗体为探针,用SDS-PAGE、免疫印迹、免疫荧光和免疫电镜技术,对蒜(Allium sativa L.)根端细胞核、核骨架、染色体和染色体骨架进行研究.SDS-PAGE和免疫印迹结果表明:细胞核中的165kD多肽是XCAP-C类似蛋白,在核骨架中未检测到XCAP-C类似蛋白.免疫荧光和免疫电镜结果表明:蒜细胞核、染色体和染色体骨架中含有XCAP-C类似蛋白,该蛋白位于细胞核中的染色质区域,但核骨架不含有XCAP-C类似蛋白.     

XCAP—C类似蛋白存在于蒜根端细胞核和染色体中

以抗XCAP_C抗体为探针 ,用SDS_PAGE、免疫印迹、免疫荧光和免疫电镜技术 ,对蒜 (AlliumsativaL .)根端细胞核、核骨架、染色体和染色体骨架进行研究。SDS_PAGE和免疫印迹结果表明 :细胞核中的 16 5kD多肽是XCAP_C类似蛋白 ,在核骨架中未检测到XCAP_C类似蛋白。免疫荧光和免疫电镜结果表明 :蒜细胞核、染色体和染色体骨架中含有XCAP_C类似蛋白 ,该蛋白位于细胞核中的染色质区域 ,但核骨架不含有XCAP_C类似蛋白。     

Visualization of Chromosome Territories in Interphase Nuclei of Ovarian Nurse Cells in <Emphasis Type="Italic">Calliphora erythrocephala</Emphasis> Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei

Despite the major advancements during the last decade with respect to both knowledge of higher order chromatin organization in the cell nucleus and the elucidation of epigenetic mechanisms of gene control, the true three-dimensional (3D) chromatin structure of endogenous active and inactive gene loci is not known. The present study was initiated as an attempt to close this gap. As a model case, we compared the chromatin architecture between the genetically active and inactive domains of the imprinted Prader-Willi syndrome (PWS) locus in human fibroblast and lymphoblastoid cell nuclei by 3D fluorescence in situ hybridization and quantitative confocal laser scanning microscopy. The volumes and 3D compactions of identified maternal and paternal PWS domains were determined in stacks of light optical serial sections using a novel threshold-independent approach. Our failure to detect volume and compaction differences indicates that possible differences are below the limits of light optical resolution. To overcome this limitation, spectral precision distance microscopy, a method of localization microscopy at the nanometer scale, was used to measure 3D distances between differentially labeled probes located both within the PWS region and in its neighborhood. This approach allows the detection of intranuclear differences between 3D distances down to about 70-90 nm, but again did not reveal clearly detectable differences between active and inactive PWS domains. Despite this failure, a comparison of the experimental 3D distance measurements with computer simulations of chromatin folding strongly supports a non-random higher order chromatin configuration of the PWS locus and argues against 3D configurations based on giant chromatin loops. Our results indicate that the search for differences between endogenous active and inactive PWS domains must be continued at still smaller scales than hitherto possible with conventional light microscopic procedures. The possibilities to achieve this goal are discussed.     

DNA localization in nuclear fragments of apoptotic ameloblasts using anti-DNA immunoelectron microscopy: Programmed cell death of ameloblasts

Ameloblasts responsible for tooth enamel formation are classified into two different phases: secretion and maturation. At the transition between these secretion and maturation stages, a considerable number of cells die. In this study, we examined the morphology of degenerating ameloblasts by conventional electron microscopy, and DNA cleavage in degenerating ameloblast nuclei by the in situ terminal transferase assay. The results suggest that apoptosis (programmed cell death) in ameloblasts, including DNA ligation is induced at the transitional stage. The nuclear fragments, chromatin condensation and DNA relocation in apoptotic nuclei were examined quantitatively by post-embedding anti-DNA immunogold electron microscopy and the in situ terminal transferase assay combined with electron microscopy. Numerical analysis revealed that immunogold labeling density in the condensed chromatin of apoptotic nuclei was comparable on the average to that in the perinuclear heterochromatin of normal nuclei, and that individual apoptotic nuclear fragments exhibited highly variable gold particle density, from fragments with lower density to that of normal heterochromatin, to fragments with densities twice as high as that of normal heterochromatin. The in situ terminal transferase assay combined with electron microscopy detected DNA ends exposed by ultrathin sectioning as well as DNA cleavage by a putative endonuclease. In conclusion, the state of the DNA, including its ligation and degeneration, changes gradually during chromatin condensation and nuclear fragmentation of apoptosis.