Population polymorphism of silver-stained nucleolar organizer chromosome regions in man

Activity of nucleolar organizer regions (NORs) of acrocentric chromosomes determined by Ag-staining was comparatively studied in 40 individuals of Bulgarian and 40 individuals of Russian populations. Chromosome 21 was found to be significantly more often stained in both populations. The other NORs did not differ significantly in staining from the means. No differences were noted between individual NORs, in respect of the intensity of Ag-staining in both populations, except chromosome 15 which showed markedly decreased staining capacity in Russians. The data obtained are compared with those published in literature concerning four other populations.     

Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears.

Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).     

Digital image analysis of silver-stained nucleolar organizer region--associated proteins in endometrial cytologic samples

OBJECTIVE: Digital image analysis was applied to determine the number, area and size of silver-stained nucleolar organizer regions (AgNORs) in cytologic samples from curettage in normal, hyperplastic and malignant endometrium. STUDY DESIGN: Thirty-two archival cytologic smears from curettage (previously stained by the Papanicolaou method) with the histologic diagnosis (4 inactive endometrium, 5 secretion, 5 proliferation, 5 simple hyperplasia, 5 complex hyperplasia, 3 atypical hyperplasia, 5 adenocarcinoma, grade 1) were analyzed with the AgNOR technique. Count, area and size of AgNORs were analyzed in 50 cells per sample using a magnification of 1,000x. Quantitative analysis was performed on an SFORM digital imaging system. Data were analyzed with the SPSS/PC+ program. Mann-Whitney and chi 2 tests were performed. RESULTS: The average value of AgNOR count increased from normal to hyperplastic endometrium and well-differentiated adenocarcinoma. Differences were significant except between atypical hyperplasia and adenocarcinoma. Four, five and more AgNORs in 40% or more of the nuclei were found in complex and atypical hyperplasia and adenocarcinoma. Proliferation, and simple and atypical hyperplasia had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic had similar mean values of AgNOR area. The mean total AgNOR area value increased from normal to hyperplastic and well-differentiated adenocarcinoma. Differences were statistically significant. AgNOR size in well-differentiated adenocarcinoma was significantly different from that in normal endometrium and different grades of hyperplasia. CONCLUSION: Digital image analysis of AgNOR count, area and size enabled a distinction to be made between normal, hyperplastic and malignant endometrium.     

Number and distribution of silver-stained nucleolar organizer regions and evolutionary relationships in domestic pigs

Variation in the numbers of silver-stained nucleolar organizer regions (Ag-NORS) were examined in 36 breeds of domestic pig of different geographic origins and five subspecies of wild boar. The relationship between Ag-NORs and evolution of domestic pigs was investigated. In all pigs observed, Ag-NORs were localized on the secondary constriction of chromosomes 10 and 8. The mean Ag-NOR numbers varied from 2.0–4.0, and decreased gradually with the different geographical distribution from south to north in China and from east to west in Europe. This regular change was caused mainly by the differences of frequency in chromosome 8 Ag-NOR type and was closely related to the evolution of domestic pig breeds.     

Evidence for the inheritance of silver-stained nucleolus organizer regions

Summary The inheritance of nucleolus organizer regions (NORs) was investigated by examining the degree of silver-staining in individual acrocentric chromosomes in two successive generations. The study was undertaken in six Down's syndrome children and their respective parents. Quinacrine fluorescent polymorphisms were used to identify individual acrocentrics and to determine which of the child's acrocentrics were informative as to parental homologue of origin. Of the 66 acrocentrics in the six children, 31 were informative. The correlation between the degree of silver-staining in the child's chromosomes and the respective parental chromosomes of origin was highly significant (P<0.001), with a correlation coefficient of 0.90. The results suggest that the degree of Ag-AS staining is characteristic for a particular chromosome and that this characteristic is an inherited property.     

Distribution of silver-stained interphase nucleolar organizer regions as a parameter to distinguish neoplastic from nonneoplastic reactive cells in human effusions

The distribution of interphasic nucleolar organizer regions (NORs) was studied in cytologic preparations of human serous effusions in order to differentiate malignant cells from nonmalignant reactive cells. The study was carried out on 80 cases of metastatic adenocarcinoma, 10 cases of mesothelioma, 10 reactive pleural effusions and 5 peritoneal washings. Visualization of NORs at the light microscopic level was obtained using a silver-staining technique for acidic proteins selectively associated with NORs. The morphologic data were also statistically evaluated by means of an automated image analyzer. The quantity of silver-stained NORs was higher in cancer cells (both mesothelioma and adenocarcinoma) than in reactive mesothelial cells. Moreover, NORs were more irregularly distributed within the nucleoli and were more variably sized in cancer cells than in reactive mesothelial cells.     

Silver-stained nucleolar organizer proteins in chondrosarcoma.

Silver-stained nucleolar proteins (AgNORs) were counted in primary chondrosarcomas of three histologic grades and in metastatic chondrosarcomatous lesions in the lung. The AgNOR numbers of neoplastic cells in primary tumors increased stepwise from grade 1 (4.42 +/- 1.11) through grade 2 (4.94 +/- 1.31) to grade 3 (6.97 +/- 1.10). There was a significant difference in AgNOR numbers between grade 3 and both grades 1 and 2 (p less than 0.001). Furthermore, the mean number of AgNORs in metastatic lesions (9.75 +/- 0.83) was significantly higher than that in primary sites (p less than 0.001). The number of AgNORs therefore reflects the grade of the chondrosarcoma. The results in the present study indicate that silver colloid staining is a useful technique for determining the histologic grade and evaluating the proliferative activity of chondrosarcomas.     

Dimorphic nucleolar organizer regions in the frog Rana blairi.

The nucleolar organizer-specific staining procedure, ammoniacal silver (Ag-AS), has been used to study the distribution and size of the nucleolar organizer regions (NORs) in chromosomes of the frog Rana blairi (Mecham, Littlejohn, Oldham, Brown and Brown). The somatic metaphase karyotype of this frog is similar to that of other frogs of the Rana pipiens species complex, numerically (2n=26) and morphologically. Secondary constrictions are detectable in untreated Giemsa-stained metaphase preparations as achromatic gaps in the long arms of a pair of submetacentric chromosomes (no. 10). These constrictions are the only regions which are deeply stained with the Ag-AS method and are thus identified as the nucleolar organizer regions (Ag-NORs). In each of the three individuals, the Ag-NORs as visualized on the homologues are of unequal length.     

Importance of interphase nucleolar organizer regions in tumor pathology.

The importance of the distribution of silver-stained nucleolar organizer regions (Ag-NORs) in interphase nuclei for diagnostic and prognostic purposes in tumor pathology has been reviewed. The available data demonstrated that interphase Ag-NOR evaluation may be of help in distinguishing malignant from hyperplastic or normal cells. On the other hand, there is increasing evidence that a relationship exists between the quantity of interphase Ag-NORs and the prognosis of malignant tumors: the greater the number of interphase Ag-NORs, the worse is the prognosis. This can be explained by the observation that the interphase Ag-NOR quantity is strictly related to the cell proliferation rate. The procedures used for the measurement of the interphase Ag-NOR quantity are also critically discussed.     

Polymorphisms of Ag-stained nucleolar organizer regions in man

Summary Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population.The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation.In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.     

Compensatory changes in silver-stainability of nucleolar organizer regions in mice.

Silver-stainability of nucleolar organizer regions (NORs) that contain genes for ribosomal RNA (rDNA) was investigated using two mouse strains, BALB/cCrSlc and MOA, and their hybrid progeny. The patterns of segregation of the rDNA clusters were analyzed in terms of chromosomal C-banding and by use of a polymorphic probe for the variable region in backcrossed N2 and N3 individuals. The results indicate that the intensity of Ag-NOR staining is stably inherited in most of the rDNA clusters, irrespective of different genetic backgrounds. In some clusters, such as those on chromosome 12 of BALB/cCrSlc, a modulation of the intensity is observed. This modulation seems to be due to compensatory activation via a change in the number of actively transcribed genes. The change from silver-negative to silver-positive staining of the NOR of chromosome 12 of BALB/cCrSlc was correlated with demethylation of the genes.     

Evaluation of nucleolar organizer region-associated proteins in endometrial pathology.

In the current study the argyrophil staining technique for NOR proteins (Ag-NORs) has been performed on cases of different endometrial lesions, trying to find an aid in differentiating atypical hyperplasia from well differentiated carcinoma in biopsy specimens. We conclude that the Ag-NOR count, even though in endometrial carcinoma is significantly exceeding that of atypical hyperplastic endometrium, could be a misleading discriminator, because of a wide overlap of values in individual cases.     

Importance of interphase nucleolar organizer regions in tumor pathology

The importance of the distribution of silverstained nucleolar organizer regions (Ag-NORs) in interphase nuclei for diagnostic and prognostic purposes in tumor pathology has been reviewed. The available data demonstrated that interphase Ag-NOR evaluation may be of help in distinguishing malignant from hyperplastic or normal cells. On the other hand, there is increasing evidence that a relationship exists between the quantity of interphase Ag-NORs and the prognosis of malignant tumors: the greater the number of interphase Ag-NORs, the worse is the prognosis. This can be explained by the observation that the interphase Ag-NOR quantity is strictly related to the cell proliferation rate. The procedures used for the measurement of the interphase Ag-NOR quantity are also critically discussed.     

Procedures for specific detection of silver-stained nucleolar proteins on western blots.

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.     

Polymorphism of nucleolar organizer regions of chromosomes in human embryos

NOR activity in metaphase chromosomes from extraembryonic and embryonic tissues of 9-12 week human fetuses was studied after standard silver staining. Significant interidividual variations in the average cummulative NOR activity was assessed by means of one-factor dispersion analysis. No significant intertissue fluctuation of NOR activity was found. Total number of NOR+ chromosomes demonstrated no correlation with the embryonic age. Steady growth of an average cummulative NOR activity respective of progressive embryonic age was proven by correlation analysis method. Unequal participation of NOR-bearing chromosomes of D- and G-groups during early embryonic development in human was shown.     

Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.     

Ribosomal RNA gene loci and silver-stained nucleolar organizer regions associated with heterochromatin in Alaskan char Salvelinus malma and chum salmon Oncorhynchus keta

Nucleolus-forming 5.8S, 18S and 28S ribosomal RNA gene (rDNA) loci were assigned by fluorescence in situ hybridization (FISH) to the distal half of the short arms of a large-sized submetacentric pair in the Alaskan char (Salvelinus malma) and to the distal region of the long arms of a medium-sized submetacentric pair in the chum salmon (Oncorhynchus keta), respectively. In each species, heteromorphic FISH signals, spanning whole satellite region and secondary constriction, imply an intraspecific variation in the size of rDNA loci. Size variation of silver-stained nucleolar organizer regions (AgNORs) was also apparent between or within the assigned rDNA loci in each species, suggesting a possible inter- or intralocus inactivation of rDNAs. C-band positivity of assigned rDNA loci and AgNORs unequivocally showed their association with heterochromatin in these species.