Silver-stained Nucleolar Organizer Regions Research Articles

Quantity of AgNORs in gastric endocrine carcinoid tumours as a potential prognostic tool.

In order to assess if the quantity of silver-stained nucleolar organizer region (AgNOR) proteins represents a prognostic tool in gastric carcinoids, a standardised AgNOR analysis was performed on 24 samples collected from the pathology archives of the Universities of Messina and Parma; the samples were taken at surgery from 11 males and 13 females (mean age 55 yrs, age range 28-77 yrs); 13 cases were defined as Type I, 1 case as Type II and 10 cases as Type III; 16 cases showed a diameter <1 cm, 8 >1 cm. Only 6 tumours were deeply invasive, breaking through the muscularis propria or the subserosa. The proliferative status of carcinoids performed by Ki67 protein antibodies was available in 20/24 cases. The quantification of AgNORs was performed according to the guidelines of the Committee on AgNOR Quantification and the mean area (microm2) of AgNORs per nucleus (NORA) was determined by means of image analyser and specific software programs. The relationship between NORA values and Ki67 data was investigated by Spearman correlation test. The mean NORA value of all 24 gastric carcinoids was 1.279 microm2 (SD 0.404); values ranged from 0.734 to 2.142 microm2. A significantly higher (p < 0.001) mean NORA value (1.736 microm2; SD 0.283) was found in tumours larger than 1 cm, in comparison to the smaller neoplasms (1.051 microm2; SD 0.214); moreover, cases showing deep wall invasion exhibited a mean NORA value of 1.765 microm2 (SD 0.276), significantly higher (p < 0.001) than those with superficial growth (1.118 microm2; SD 0.296). Finally, a similar highly significant difference was seen between type III carcinoids (1.615 microm2; SD 0.375) and type I-II (1.040 microm2; SD 0.208). A linear relationship between Ki67 and corresponding NORA values was obtained by the Spearman correlation test (p = 0.001). No other significant correlations were found between mean NORA values and other clinico-pathological parameters. The AgNOR method seems to be an additional tool potentially able to predict the prognosis of this kind of endocrine tumour, facilitating the identification of fast-growing tumours and being able to directly correlate with the size, deep invasion of gastric wall and tumour type, generally considered as the best prognostic indicators.

To the intranucleolar translocation of AgNORs in leukemic early granulocytic and plasmacytic precursors

Early leukemic granulocytic and plasmacytic precursors were studied in vitro and in vivo to provide an information on the intranucleolar distribution of AgNORs (silver stained nucleolus organizer regions). In most of these cells AgNORs appeared as clusters of silver stained particles distributed in the whole nucleolar body. On the other hand, in some leukemic early granulocytic precursors, i.e., in myeloblasts and promyelocytes enlarged AgNORs were translocated in the nucleolar peripheral part. In addition, the number of translocated AgNORs at the nucleolar periphery was significantly smaller. Such translocation of a reduced number of AgNORs was easily produced by experimental aging, i.e., starving of cultured leukemic early granulocytic precursors (HL-60 and K562 cells) in vitro and seems to be reversible. Similar translocation of a reduced number of AgNORs was also produced by aging of leukemic plasmacytic precursors. Thus, the translocation of the reduced number of AgNORs to the nucleolar periphery in some blastic leukemic hematopoietic cells might be an useful marker of their aging at the single cell level. However, more studies in this direction are required in the future.

Assessment of Proliferative Activity by AgNOR and PCNA in Prostatic Tissues of Ram Lambs Implanted with Zeranol

The aim of this study was to assess cellular proliferation using silver-stained nucleolar organizer regions (AgNOR) and the proliferating cell nuclear antigen (PCNA) in various tissues in the prostate of ram lambs implanted with increasing zeranol doses and to compare the sensitivity of different tissues of lamb prostate to zeranol. Twenty-four Akkaraman lambs were implanted with increasing zeranol doses, including 12 mg (n = 8), 24 mg (n = 8) and 96 mg (n = 8), with eight lambs serving as controls. After 33 days, the prostate tissues of the lambs were stained using AgNOR and PCNA techniques. The prostate tissues were divided into two compartments--the epithelial tissues, including glandular acinus, collecting duct and penile urethra, and the non-epithelial tissues, including interstitial tissue and striated muscle. AgNOR dots and PCNA index on each prostatic tissue were counted under a light microscope and were evaluated statistically. AgNOR staining in the treatment groups showed a higher score in the non-epithelial tissues than the epithelial components, whereas the PCNA index was significant in the epithelial tissues and non-epithelial tissues had very low PCNA immunostaining. According to the PCNA index, collecting duct epithelium showed more sensitivity to increasing zeranol doses and according to AgNOR counts, there was no difference of sensitivity to zeranol among tissues of the same origin. Both AgNOR counts and PCNA indexes seem to be valuable proliferating markers for the epithelial components of ram prostate, but PCNA index had no significance in relation to the non-epithelial components in contrast to AgNOR counts. Therefore, the controversial results arising from the combined use of both techniques as proliferating markers for the ram prostate should be considered in further studies.

A morphological and cytochemical note to the reversible intranucleolar translocation of AgNORs (silver stained nucleolus organizer regions) in early leukemic granulocytic progenitors represented by cultured K 562 cells

A morphological and cytochemical note to the reversible intranucleolar translocation of AgNORs (silver stained nucleolus organizer regions) in early leukemic granulocytic progenitors represented by cultured K 562 cells

Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the Acipenseriformes

The shortnose sturgeon Acipenser brevirostrum was revealed to have a larger number of chromosomes than previously reported for other sturgeon species. Its chromosome number ranged from 362 to 372 (of ten specimens examined), showing intraindividual variation. The karyotype of metaphase with the highest chromosome number (372) consisted of 89 pairs of macrochromosomes and 97 pairs of microchromosomes (fundamental number; NF = 550). Although the microchromosomes were relatively shorter than the macrochromosomes, most of them had discernible arms and centromeres. Silver-stained nucleolar organizer regions (Ag-NORs) were localized on the telomeric regions of 5 pairs of chromosomes (Ag-NORs = 10): 4 were made up of small meta/submetacentrics and 1 of acrocentrics. Polyploidy of A. brevirostrum should be hexaploid based on the karyotype, numerous chromosomes, Ag-NORs, and previously reported large genome size (ca. 13 pg DNA/cell).

Comparative study of DNA synthesis and nucleolar organizer regions of sinusoid littoral cells in mouse regenerating liver

Variations in DNA synthesis (DNAs) and Nucleolar Organizer Regions (NORs) were studied in the littoral cell population from regenerating liver of C3HS inbred mice standardized for periodicity analysis. Immunohistochemical detection of Bromodeoxyuridine (BrdU) with a monoclonal antibody and silver staining of NORs (AgNORs) were assessed by means of a digital image analysis system in histological sections. Tissue samples were obtained every four hours from the 30th to the 54th hours after a partial hepatectomy. The results showed, in both parameters, a gradual increment of the values during the period studied, with highest values (DNAs 107.1 +/- 16.1 SE; AgNORs 77.3 +/- 3.4 SE) located at 16:00/54 Time of Day / Hours Post-Hepatectomy (TD/HPH), which were significantly different (p <0.001) from the values of the first sample (DNAs 38.1 +/- 9.5 SE; AgNORs 27.3 +/- 1.0 SE) taken at 16:00/30 TD/HPH. The results of our experiment demonstrate the existence of a strong correlation of DNA synthesis measured by BrdU immunohistochemistry and AgNORs numbers in sinusoid littoral cells from mouse regenerating liver.

Quantification of silver-staining nucleolar organizer region in epithelial cells of tongue of mice after exposure to, or intake of, alcohol

The purpose of the current study was to evaluate the effect of alcohol on the proliferative activity of epithelial cells in the lingual mucosa of mice by means of silver-staining nucleolar organizer region (AgNOR) count and area measurements. Forty-eight CF1 mice were separated into three groups. The test groups were submitted to topical exposure to, or intake of, 40% (volume/volume) ethyl alcohol. Biopsy specimens were collected from the middle third of the dorsal tongue at 0, 6, and 12 months, and samples were stained according to the AgNOR technique. Mean number and mean area of AgNOR per nucleus were calculated for 50 basal layer cells and 50 intermediate layer cells. Increases in mean number and mean area of AgNOR per nucleus in intermediate cells were observed at 12 months in the alcohol intake group ( P < .05). Results showed that intake of 40% alcohol increased epithelial cell proliferation in the dorsal surface of lingual mucosa.

Nucleolar Organizer Regions as a Prognostic Indicator in Epithelial Cancers of the Ovary

Recent studies have shown that quantification of silver-stained nucleolar organizer region associated proteins (AgNORs) is correlated with ploidy and proliferative activity in several neoplasms. The prognostic value of this marker, however, has not been well defined in epithelial ovarian carcinomas (EOCs). We studied 84 cases of EOCs in an attempt to assess the potential prognostic significance of AgNORs and to compare the results with other prognostic factors in EOCs. Clinical, operative and treatment details, and follow-up data were recorded for each case. In univariate analysis, a highly significant correlation of AgNOR count with clinical stage (p < 0.01), histologic grade (p < 0.05), response to chemotherapy (p < 0.05), and biologic behavior (p < 0.01) was found. High AgNOR counts were observed in late stage (III and IV) tumors, grade 3 carcinomas, tumors in patients with partial response to chemotherapy, progressive disease, recurrence, or death from tumor. In multivariate analysis, AgNOR count at cut off 3.5 was found to be the third prognostic variable after response to therapy and stage. These observations suggest that AgNOR count holds promise for prediction of tumor aggressiveness in EOCs. However, its role as an independent prognostic variable of patient survival remains questionable.

Local invasiveness of ameloblastoma. Role played by matrix metalloproteinases and proliferative activity.

Ameloblastoma is an odontogenic neoplasm characterized by local invasiveness and recurrence. In this study we analysed the role played by matrix metalloproteinases (MMPs) in the local invasiveness of ameloblastoma. We also attempted to establish a relationship between the presence of MMPs and the proliferative activity of ameloblastoma cells. Immunohistochemistry was carried out to detect different MMPs in formalin-fixed paraffin-embedded samples of human ameloblastoma. Immunohistochemistry, however, does not establish whether a given MMP is latent or active. To address this point, we carried out biochemical methods, namely zymography and Western blotting. Our results showed expression of latent and active forms of MMPs 1, 2 and 9 in ameloblastoma. These enzymes may digest bone matrix and release mitogenic factors, which would increase tumour proliferation. This possibility prompted us to study the proliferation of ameloblastoma cells located in close proximity to bone. Silver-stained nucleolar organizer region morphometry revealed that ameloblastoma cells in the vicinity of bone show increased proliferation, when compared with controls. Our results suggest an interdependent mechanism involving MMPs and proliferation of ameloblastoma cells, which may contribute to the local invasiveness of this tumour.

Satellited Chromosomes, Nucleolus Organizer Regions and Nucleoli of Brassica campestris L., B. nigra(L.) Koch, and Sinapis arvensis L.

Giemsa staining was used to characterize the satellited chromosomes, and silver staining, to detect nucleolus organizer regions and nucleoli in Brassica campestris (genome: AA, 2n = 20), B. nigra (BB, 2n = 16) and Sinapis arvensis (SS, 2n = 18). B. campestris had one pair of satellited chromosomes, while B. nigra had three pairs. S. arvensis had five distinct satellited chromosomes and a sixth chromosome with a faint satellite. Silver-stained nucleolus organizer regions (Ag-NORs) were localized at the secondary constrictions of the satellited chromosomes. Two Ag-NORs and a maximum number of two nucleoli were revealed in B. campestris, Heteromorphism for size of Ag-NORs and nucleoli was observed in one accession of B. campestris. In B. nigra, six Ag-NORs and six nucleoli were observed at early and late telophase, respectively. In S. arvensis, six Ag-NORs and six nucleoli were revealed. Size differences were noticed within and between pairs of Ag-NORs. The presence of three pairs of active NORs in B. nigra and S. arvensis is an additional support for the hypothesis of a basic number of ×= 3 in the Brassiceae.

Ribosomal RNA Gene Loci and Their Nucleolar Activity in Brassica alboglabra Bailey

Fluorescence in situ hybridization (FISH) and silver staining techniques were used to reveal the number, chromosomal sites and nucleolar activity of ribosomal RNA (rRNA) gene loci in Brassica alboglabra Bailey (genome: CC, 2n = 18). Two major and two minor rRNA gene loci were disclosed using FISH. The two major rRNA gene loci were located on the secondary constrictions of the satellited chromosome pair 9, the two minor rRNA gene loci, on the terminal region of the short arms of the non-satellited chromosome pair 8. Two silver-stained nucleolus organizer regions (Ag-NORs) were detected on the secondary constrictions of chromosome pair 9. Two major nucleoli were recorded in telophase nuclei. In interphase nuclei, two micronucleoli were observed in addition to the two major nuceoli. Thus, it is suggested that the satellited chromosome pair 9 with Ag-NORs contains major active rRNA gene loci leading to the formation of major nucleoli, while the non-satellited chromosome pair 8 without detectable Ag-NORs carries minor active rRNA gene loci responsible for the formation of micronucleoli.

Variation in size of Ag-NORs and fluorescent rDNA in situ hybridization signals in six breeds of domestic pig.

Variation of the size of silver-stained nucleolar organizer regions (NORs) of chromosomes 10 and 8 was studied in pigs of six breeds (Sus scrofa L.). The silver deposits were quantified by image analysis and the results were normalized for each Ag-NOR chromosome. In general, normalized values for chromosomes 10 were higher than those for chromosomes 8, suggesting that the NOR activity of chromosomes 10 is dominant as compared to that of chromosomes 8. However, high values for chromosomes 8 were found in the Meishan breed and in some Piétrain pigs, indicating a high transcriptional activity of the rRNA genes on these chromosomes. In some pigs, the relative quantities of rDNA in chromosomes 10 and 8 were investigated by fluorescent in situ hybridization and the results were compared with those of the silver staining procedure. It is concluded that Ag-NOR sizes on chromosomes 10 are relatively well correlated to the number of rRNA genes, whereas the absence or the small size of Ag-NORs on chromosomes 8, often observed in pigs, is the result of low NOR activity rather than of absence of rDNA.

Karyotype description of five species of Trichomycterus (Teleostei: Siluriformes: Trichomycteridae)

Trichomycteridae is a family of small catfish which are widely distributed throughout Southern Central America and South America. The present study showed that the cis-Andean species Trichomycterus florensis, Trichomycterus sp. aff. Trichomycterus itatiyae, Trichomycterus reinhardti, Trichomycterus davisi and Trichomycterus auroguttatus had 2n = 54 chromosomes (42 metacentric, 10 submetacentric and 2 subtelocentric), with T. reinhardti, T. auroguttatus and T. sp. aff. T. itatiyae exhibiting only one chromosome pair with silver-stained nucleolus organizer regions (NORs). The cytogenetic data suggest the existence of at least two groups of species in the cis-Andean representatives of the genus Trichomycterus. In the first group the first metacentric pair is considerably larger than the second metacentric pair and the NORs occur in the pericentromeric position of the short arm of a large submetacentric pair while in the second group the first and second metacentric pairs are about the same size and larger than the other metacentric pairs and the NORs are located in the pericentromeric position of the long arm of a large metacentric pair. The relative conservatism of the karyotype of the cis-Andean Trichomycterus species contrasts with the wide diversification observed in the trans-Andean species, reinforcing the hypothesis that the genus is not monophyletic.

Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent

In this study, cyto-genotoxic effects of a textile mill effluent on fish Oreochromis niloticus were investigated using the micronucleus (MN) test and methods to analyze interphase silver-stained nucleolar organizer regions (AgNORs). Fishes were exposed to three different concentrations of textile mill effluent (5, 10 and 20% (v/v)) for 3, 6 and 9 days. Cyclophosphamide (2 mg/l) was used as a positive control. Micronucleus frequencies were examined in peripheral blood erythrocytes and gill cells. Nuclear abnormalities (NA) other than micronuclei such as binuclei, lobed nuclei, blebbed nuclei and notched nuclei were also evaluated in peripheral erythrocytes. Interphase AgNOR parameters were examined in epithelial cells obtained from the edge of caudal fins after 90 and 180 min of exposure. As a result, dose-dependent increases in the frequencies of micronuclei and other NA in erythrocytes were observed. MN frequencies in gill cells also significantly increased, while the interphase AgNOR parameters in fin cells decreased, as a result of textile effluent and cylophosphamide treatments.

Argyrophilic nuclear organizer regions in nasopharyngeal carcinoma and paraneoplastic epithelia.

Nucleolar organizer regions (NORs), which are loops of DNA containing ribosomal RNA genes, have been shown to correlate with cell proliferation and malignant transformation. The diagnostic value of silver staining NORs (AgNORs) in preneoplastic epithelia and nasopharyngeal carcinoma (NPC) by the quantitative assessment of AgNORs proteins is evaluated. Silver-staining of NORs was applied to 70 paraffin sections of NPC biopsy specimens, including 29 samples of the adjacent normal glandular epithelia (GE), 19 hyperplastic/dysplastic columnar epithelia (CE) in the adjacent mucosa, 10 hyperplastic/dysplastic squamous epithelia (SE) in the adjacent mucosa, 54 differentiated nonkeratinizing carcinoma (DC), and 16 undifferentiated carcinoma (UC). The morphometric features of AgNORs in preneoplastic epithelia and cancer cells were analyzed by image cytometric analysis and then compared. The AgNOR count, mean AgNOR area, and AgNOR area-count ratio increased significantly from GE to CE to DC or UC. The mean nuclear area, AgNOR area, and AgNOR area-count ratio increased significantly from GE to SE to UC, whereas overlapping AgNOR values were observed between SE and DC. UC had significantly higher values than DC in mean nuclear area, AgNOR area, and AgNOR area-count ratio. The morphometric analysis of AgNORs reflected cell proliferative activity and histologic differentiation of tumor rather than malignant transformation in different nasopharyngeal epithelia and NPC. AgNOR area and AgNOR area-count ratio are the most valuable features for differential diagnosis of normal, preneoplastic, and cancer cells.

In vivo response to the different LET regions of a 55 MeV helium ion beam.

We evaluated the in vivo response to heavy particle irradiation in rat tail epidermis using silver-stained nucleolar organizer regions (AgNOR) as the end-point. The energy degradation of the beam across the circular section of the tail allowed us to study the damage elicited by two different LET regions of a helium beam, i.e. non-Bragg peak (NBP) and Bragg peak (BP), at different sites on the same sample. The tails were locally irradiated with a helium ion beam at different fluences. AgNOR exhibited marked variations between tissue areas only a few micrometers apart within each tail exposed to a given beam fluence. An analysis of the AgNOR variations in NBP and BP areas of tails exposed to different beam fluences revealed a dose-dependent effect. The AgNOR provide quantitative evidence of differential damage in neighboring tissue areas exposed to different LET regions of a helium-ion beam.

AgNOR count as objective marker for dysplastic features in oral leukoplakia.

Dysplasia is an important feature of leukoplakia. Because agreement among oral pathologists is poor regarding lesional diagnosis, silver stainable nucleolar organizer regions (AgNORs) as replicatory markers may have a place in objectively characterizing dysplasia in tissue specimens. We studied 41 normal oral epithelia, 51 oral leukoplakia (26 dysplastic, 25 non-dysplastic), and 51 cases of squamous cell carcinoma specimens for their mean AgNOR counts. Mean AgNOR counts increased gradually from normal epithelium to non-dysplastic to dysplastic leukoplakia to squamous cell carcinoma. Using ROC analysis, we determined a mean AgNOR count cut-point (2.37) that can be used to distinguish between dysplastic and non-dysplastic leukoplakia. The test had a sensitivity of 75% and specificity of 83% with area under the curve being 88%. Mean AgNOR count could be a valuable criterion for defining objective parameters for diagnosis/determination of dysplasia distinguishing between dysplastic and non-dysplastic leukoplakia.

AgNORs as an Early Marker of Sensitivity to Radiotherapy in Gynecologic Cancer

To evaluate the changes induced in silver-stained nucleolar organizer regions (AgNORs) by the first fraction of a radiotherapy protocol for gynecologic cancer on exfoliated cytologic samples to predict the therapeutic success of the full protocol. Thirteen gynecologic cancer patients who were scheduled for radiotherapy were included in the study. Cell smears were taken from the affected area before and after the first fraction of a radiotherapy protocol and silver stained for AgNORs. AgNORs per nucleus were counted under a light microscope. Local disease control by the full radiotherapy protocol was assessed at one year by the Papanicolaou technique. Local success of radiotherapy was greater for lesions with higher pretreatment AgNOR counts and for lesions that underwent a greater percentage reduction in AgNOR counts after the first fraction. We correlated local success of the full radiotherapy protocol with a predictive index based on AgNOR counts obtained before and after the first fraction. A predictive index based on AgNOR counts can predict, as early as after the first fraction, the local control of disease by a full radiotherapy protocol. Knowledge of the probability of success long before the protocol is completed would allow reevaluation of therapeutic options.

Standardized AgNOR analysis in actinic keratosis.

To assess if the quantity of silver-stained nucleolar organizer region (AgNOR) proteins predicts the behavior of actinic keratosis (AK), we performed a standardized AgNOR analysis on 51 cases of AK; in addition, 10 cases of squamous cell (SCC) and 10 cases of basal cell (BCC) carcinomas and 10 normal skin samples were also studied. AgNOR analysis was performed on formalin-fixed and paraffin-embedded sections according to the guidelines of the Committee on AgNOR Quantification (1995), evaluating the mean area (microm(2)) of AgNORs per nucleus (NORA). A highly significant P value (< 0.001) was found in the comparison among NORA values of normal skin (1.869 microm(2); SD + 0.332), AK (3.988 microm(2); SD + 0.914), BCC (3.044 microm(2); SD + 0.254), and SCC (5.286 microm(2); SD + 0.920). In AK, a progressive increase of mean NORA values was observed moving from Stage I (3.161 microm(2); SD + 0.600) to Stage II (3.455 microm(2); SD + 0.562), Stage III (4.360 microm(2); SD + 0.295), and Stage IV (5.168 microm(2); SD + 0.694); highly significant differences (P < 0.001) were noted when Stages I or II were compared with Stage III or Stage IV or between these latter stages. The AgNOR quantity may identify AKs with high proliferative activity and increased tendency to develop into invasive SCC.

Chemoprevention of azoxymethane-induced rat aberrant crypt foci by dietary zerumbone isolated from Zingiber zerumbet

The modifying effects of dietary feeding of zerumbone isolated from Zingiber zerumbet on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Expression of cyclooxygenase (COX)-2 in colonic mucosa exposed to AOM and/or zerumbone was also assayed. In addition, we assessed the effects of zerumbone on cell proliferation activity of crypts by counting silver-stained nucleolar organizer regions protein (AgNORs) in colonic cryptal cell nuclei. To induce ACF rats were given three weekly subcutaneous injections of AOM (15 mg/kg body weight). They were also fed the experimental diet containing 0.01% or 0.05% zerumbone for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced 84 ± 13 ACF/rat at the end of the study (week 5). Dietary administration of zerumbone caused reduction in the frequency of ACF: 72 ± 17 (14% reduction) at a dose of 0.01% and 45 ± 18 (46% reduction, p<0.001) at a dose of 0.05%. Feeding of zerumbone significantly reduced expression of COX-2 and prostaglandins in colonic mucosa. Zerumbone feeding significantly lowered the number of AgNORs in colonic crypt cell nuclei. These findings might suggest possible chemopreventive ability of zerumbone, through suppression of COX-2 expression, cell proliferating activity of colonic mucosa, and induction of phase II detoxification enzymes in the development of carcinogen-induced ACF.