Nucleolar Size Research Articles

What the nucleolus says to a tumour pathologist

The importance of nucleolar changes in cancer cells is underestimated in tumour pathology. There is evidence that the nucleolus is the mirror of a series of metabolic changes that characterize cancer cells. Cell entry into the cell cycle is always associated with up-regulation of the nucleolar function and increased nucleolar size, which are also directly dependent on the rapidity of cell cycle progression. Furthermore, alterations of the major tumour suppressor retinoblastoma (Rb) and p53 pathways also contribute to the stimulation of nucleolar function and to nucleolar enlargement. High cell growth fraction, high cell growth rate and disruption of the Rb and p53 pathways are responsible for greater aggressiveness of cancer tissues. Therefore, the evaluation of nucleolar size allows one to obtain reliable information on the clinical outcome of the cancer: the greater the nucleolar size, the worse the tumour prognosis. Indeed, a series of studies carried out on numerous human tumours has shown that nucleolar hypertrophy (prominent nucleolus) was an independent predictive and prognostic parameter of a fatal clinical outcome.

A short cytochemical note on the nucleolar and cytoplasmic RNA concentration in differentiating cells represented by human erythroblasts

The present study was undertaken to provide more information on the nucleolar and cytoplasmic RNA concentration in differentiating cells of the erythroid lineage. These cells represent a convenient model to study cell differentiation since all stages are morphologically well characterised. The bone marrow of patients suffering from the chronic phase of chronic myeloid leukaemia without a large increase in the granulocyte to erythroid ratio provided erythroblasts for computer-assisted image density measurements of RNA in nucleoli and cytoplasm at the single cell level. The measurements indicated a significant decrease of the nucleolar and cytoplasmic RNA concentration only in advanced stages of erythroblast differentiation (polychromatic and orthochromatic erythroblasts). The ratio of the nucleolar to cytoplasmic RNA concentration was otherwise very stable and did not change during differentiation, being similar in the early and advanced stages of erythroblastic development. In contrast, the nucleolar size significantly decreased even during the early stages of erythroid development (basophilic erythroblasts). This marked decrease in the nucleolar diameter in differentiating erythroblasts and the less marked decrease in the nucleolar RNA concentration suggest that the amount of RNA in the nucleolus is closely associated with nucleolar size rather than on its concentration within the nucleolar body.

Silver staining and nucleolar patterns in human heteroploid and measles-carrier cells.

Silver staining revealed 10, 11 and 7 active rDNA sites (Ag-NORs) in acrocentrics of human diploid fibroblasts, near-triploid Lu106 cells (a HeLa-derivative) and measles-carrier Lu106 cells, respectively. Thus, the heteroploid cell lines showed no significant increase in Ag-NORs. They also had consistent Ag-staining patterns, in accordance with their karyotypic uniformity. The numbers of Ag-NORs agreed with the numbers of acrocentrics present. The fewer Ag-NORs in the measles-carrier cells showed increase of the Ag-stained material indicating regulated ribosomal gene activity. Similarly, in interphase cells stained with toluidine blue, the nucleoli were fewer but larger in the measles-carrier cells. By combining Ag-staining and G-banding in control and measles-carrier Lu106 cells, it was possible to identify the Ag-positive acrocentrics. These included normal and marker chromosomes. In both cell lines. Ag-positive chromosomes No. 15 were frequent. Numbers and sizes of nucleoli at anaphase, telophase and early interphase in monolayers corresponded to the Ag-staining patterns in air-dried metaphases. Thus, silver-stained regions in the metaphase chromosomes develop and grow into larger nucleoli during interphase. Ag-staining of some interphase nuclei in the measles-carrier cells comprised materials other than those found at the primary NORs.

On the nucleolar and cytoplasmic RNA density during "cell dedifferentiation" represented by blastic transformation of human mature T lymphocytes - a cytochemical study.

The present study was undertaken to provide information on the nucleolar and cytoplasmic density in specimens stained for RNA during "cell dedifferentiation" represented by blastic transformation of mature T lymphocytes. Nucleolar and cytoplasmic RNA's were visualized using a simple cytochemical method followed by computer assisted densitometry and size measurements of digitised images. An increased nucleolar and cytoplasmic RNA density accompanying the blastic transformation was significant after 48 hours of cultivation with phytohemaglutinin (PHA) when stimulated cells were characterized the largest nucleolar size reflecting S or G2 phase of the cell cycle. On the other hand, significantly larger ratio of the nucleolar to cytoplasmic density was noted only after a shorter cultivation when stimulated cells were presumably in the G1 phase. Thus the increased nucleolar and cytoplasmic RNA density together represented an accompanying phenomenon of the cell proliferation and cycling state. From the methodical point of view, the nucleolar and cytoplasmic RNA densitometry appeared as a simple as well as useful additional method to study "dedifferentiation" or various cell states at the single cell level. In addition, it was also interesting that the increase of the nucleolar diameter in stimulated cells was much larger than that of the nucleolar density. Such difference suggested that the RNA content in nucleoli was related mainly to their size.

The Target of Rapamycin Pathway Antagonizes pha-4/FoxA to Control Development and Aging

FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. We previously identified the AAA+ ATPase homolog ruvb-1 as a potent suppressor of pha-4 mutations. Here we show that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity. We find that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan, but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations. The data suggest that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. We suggest a model in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease.

Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage

Drosophila neuroblasts and ovarian stem cells are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim-NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.

Cytologic findings of acquired cystic disease‐associated renal cell carcinoma: A report of two cases

Renal tumors may arise in the setting of end-stage renal cell disease. The risk is 100 times that of the normal population with an incidence ranging from 3-7%. The most common malignant tumor to arise in the setting of acquired cystic disease of the kidney is the acquired cystic disease-associated renal cell carcinoma (ACD-associated RCC). The cytomorphologic features of ACD-associated RCC, which has not been described previously, show moderately cellular specimens composed of clusters of cells with papillary configuration. The cells ranged from polygonal to columnar and contained abundant eosinophilic granular cytoplasm. The nuclei were round and centrally located, and the chromatin was finely granular with prominent central nucleoli that corresponded to Fuhrman's grade 3 nucleolar size. The main differential diagnosis is type 2 papillary renal cell carcinoma, from which it can be distinguished based on clinical findings only at this moment.

The RNA Content of Nucleolar Bodies Is Related to Their Size – a Cytochemical Study on Human Monocytes and Lymphocytes in Blood Smears and Blood Cytospins

The present study was undertaken to provide more information on the relationship between the nucleolar size and RNA density. Mature monocytes circulating in human peripheral blood appeared to be very convenient for such study because they contain multiple nucleoli of various sizes in one and the same nucleus. In addition, nucleoli without perinucleolar chromatin represented by nucleolar bodies are easy to be visualized by a simple cytochemical procedure for RNA demonstration. The diameter and density of NoBs in specimens stained for RNA were determined by computer-assisted measurements of individual cells. According to the results, the nucleolar RNA content was apparently related to the nucleolar size because the RNA density of small and large NoBs was practically the same. In addition, the diameter measurements also indicated that one or two of several NoBs in one nucleus were dominant – larger – than the others. It should also be mentioned that the diameter and RNA density of NoBs were studied in monocytes in peripheral blood smears regardless of their limited number. Additional study on lymphocytes indicated that the preparation procedure of smears modified both diameter and density of the measured parameters less than the preparation of cytospins as demonstrated by lower variability of the resulting values. Thus, the observed differences between smears and cytospins also indicated that the specimen preparation procedures should always be considered during evaluation of the nucleolar size or staining intensity of nucleoli or cytoplasm due to the presence of RNA.

Relationships between transcription, silver staining, and chromatin organization of nucleolar organizers in Secale cereale

The nucleolar organizer regions (NORs) are composed of hundreds of rRNA genes, typically spanning several megabases. Cytologically, NORs include regions that are highly condensed and regions that are decondensed, the latter corresponding to regions at which associated proteins stain intensively with silver (Ag-NORs) and where active rRNA gene transcription is thought to occur. To test the relationship between rRNA gene activity, NOR silver staining, and rDNA (genes coding for rRNA) chromatin condensation, we used the DNA methyl-transferase inhibitor 5-azacytidine to evaluate the correlation between the epigenetic regulation of rRNA genes and NOR silver staining in the plant Secale cereale. Following 5-azacytidine treatment, we observed an increase in rRNA gene transcription as well as a reduction in the number of cells showing a significant difference in the size of the silver-stained domains in the two NORs. These transcriptional changes occurred concomitantly with an increase in nuclear and nucleolar size and were associated with the reallocation of most of the rDNA from perinucleolar heterochromatin into the nucleolus. Collectively, these results suggest that rRNA gene transcription, silver staining, and NOR decondensation are interrelated in S. cereale.

Outcome prediction for renal cell carcinoma: evaluation of prognostic factors for tumours divided according to histological subtype

A wide variety of parameters have been investigated for their prognostic significance in mixed series of renal cell carcinoma (RCC). The classification of RCC into separate types with differing morphology, genotype and probable clinical outcome has led to a re-evaluation of many prognostic parameters with studies confined to a single RCC morphotype. Tumour stage remains the most important predictor of RCC outcome and recent investigations have focused upon tumour diameter and the prognostic significance of stromal, vascular and lymphatic invasion within the renal sinus. In large tumour series, morphotype has been correlated with patient survival, with clear cell RCC being associated with a less favourable outcome than chromophobe RCC and to a lesser extent papillary RCC, for organ confined tumours. The prognostic significance of nuclear grading remains controversial. Fuhrman grading has been shown to have prognostic utility for clear cell RCC in some series. Recent studies have shown that for papillary RCC, grading should be based upon nucleolar size and that Fuhrman grading is inappropriate for chromophobe RCC. Proliferative indices based upon a variety of markers have been correlated with outcome for clear cell RCC (Ki-67, AgNORs, p21(waf1/cip1) and p27(Kip1)) and papillary RCC (Ki-67, AgNORs), although in some series prognostic significance was lost on multivariate analysis. The presence of tumour necrosis has been shown to predict survival for clear cell and chromophobe RCC, and in clear cell RCC quantification of tumour vascular density has been correlated with outcome. Several molecular markers have been investigated for prognostic significance, mostly in clear cell RCC. Although some of these markers have been shown to be significantly associated with survival, these findings remain to be confirmed in large scale follow-up studies.

Mean diameter of nucleolar bodies in cultured human leukemic myeloblasts is mainly related to the S and G2 phase of the cell cycle.

Mean diameter of nucleolar bodies (nucleoli without the perinucleolar chromatin) per cell was studied in human leukemic myeloblasts represented by K 562 and Kasumi 1 cell lines which originated from chronic and acute myeloid leukaemia. The measurement of mean diameter of nucleolar bodies in specimens stained for RNA was very simple. Such approach eliminated the variability of the perinucleolar chromatin discontinuous shell which might influence the measured nucleolar size as suggested by earlier studies. Ageing of K 562 myeloblasts produced a significant decrease of cells in S+G2 phase of the cell cycle accompanied by a significant reduction of mean diameter of nucleolar bodies (MDNoBs) per cell. In contrast, treatment of Kasumi 1 myeloblasts with histone deacetylase inhibitor - Trichostatin A - produced a large incidence of resistant cells in S+G2 phase which were characterised by a large increase of MDNoBs. Thus, MDNoBs in leukemic myeloblasts might be a helpful tool to estimate the incidence of cells in the S+G2 phase at the single cell level in smear preparations when the number of cells is very small.

The “root” experiment of the “cervantes” spanish soyuz mission: Cell proliferation and nucleolar activity alterations in arabidopsis roots germinated in real or simulated microgravity

In an experiment conducted in the “Cervantes” Spanish Soyuz Mission, a 10-day flight to the International Space Station, Arabidopsis seeds were germinated, seedlings grew for 4 days at 22ºC, and they were fixed in flight with paraformaldehyde. A ground 1 g control experiment was replicated, and an additional experiment in simulated microgravity, using a Random Positioning Machine, was performed in the same conditions. Structural, morphometric and immunocytochemical data were compared. Glutaraldehyde-fixed seedlings similarly grown in the Space Shuttle (STS-84 Mission) were also used for ultrastructural and morphometric studies. Seedlings grown for 4 days in real or simulated weightlessness showed a longer size than the ground 1 g control. Root meristematic cells showed an enhanced proliferating rate, but ribosome biogenesis was reduced, as inferred from the nucleolar size and from the levels of the nucleolar protein nucleolin. This could be the consequence of the acceleration of the cell cycle, with shortening of its phases. Weightlessness induces stress in the plant, influencing cellular processes decisive for development and morphogenesis. This stress may endanger the plant and would require the action of compensating specialized defence mechanisms.

Nucleolus size variation during meiosis and NOR activity of a B chromosome in the grasshopper Eyprepocnemis plorans

The number of nucleoli and nucleolar area were measured in meiotic cells from males of the grasshopper Eyprepocnemis plorans collected in three natural populations. Number of nucleoli per cell showed no significant correlation among cells in different meiotic stages, but there was strong positive correlation for nucleolar area between leptotene and interkinesis cells in individuals from distant populations (Salobreña in Spain, and Smir in Morocco). No correlation was, however, observed for both parameters between the meiotic stages analysed in individuals from the population of Torrox (Spain). The number of nucleoli at leptotene was about double the number at interkinesis, as expected from the double ploidy level at leptotene and the corresponding double number of rDNA clusters. Leptotene nucleolar area, however, was about fourfold that in interkinesis, presumably due to higher requirements for ribosome biogenesis in meiosis I than meiosis II. In Torrox, diplotene cells showed a lower number of nucleoli but larger nucleolar area than in leptotene cells, suggesting an increase in nucleolus size during prophase I. Significant differences were found among populations for nucleolar area but not for number of nucleoli, the smallest nucleolar area being observed in Torrox, which is the population harbouring the most parasitic B chromosome variant. No clear effects on nucleolar area or number of nucleoli were associated with the B-chromosome number. However, B-chromosome effects on the nucleolar area were apparent in the Torrox population when data were analysed with respect to a B-chromosome odd-even pattern in leptotene and interkinesis cells. However, in diplotene cells no odd-even pattern was observed for both nucleolar parameters, suggesting that the increase in nucleolar size from leptotene to diplotene dilutes the leptotene odd-even pattern. The rDNA distally located in the B chromosome was associated with a nucleolus in 6.5% out of the 247 diplotene cells analysed. The implications of these findings are discussed in the context of B chromosomes as stress-causing genome parasites and the nucleolus as a sensor of stress.

Nucleolar enlargement, nuclear eccentricity and altered cell body immunostaining characteristics of large-sized sensory neurons following treatment of rats with paclitaxel

Paclitaxel-induced sensory neuropathy is a problematic side-effect of cancer chemotherapy. Previous studies in rodents have shown paclitaxel treatment to have many effects on different parts of the peripheral nervous system, but those responsible for its bothersome clinical side-effects are still unclear. In the current study, we sought to obtain information about the involvement of sensory neurons in paclitaxel neurotoxicity at the level of the dorsal root ganglion. Rats were treated with a clinically relevant dose of paclitaxel (87.5 mg/m 2 weekly for a total of nine doses) to induce a sensory neuropathy; then their L5 dorsal root ganglia were studied by morphometry and immunohistochemistry. Paclitaxel treatment was generally well tolerated, and slowed conduction velocity and prolonged conduction latencies in the peripheral sensory nerves without altering conduction in the central or motor pathways of the H-reflex arc. In the L5 dorsal root ganglion, nucleolus size and the number of neurons with eccentric nuclei were increased only in a subpopulation of dorsal root ganglion neurons with cell body cross-sectional areas greater than 1750 μm 2, which made up less than 10% of the total population. Paclitaxel treatment increased immunohistochemical staining for activating transcription factor-3 (ATF-3), c-Jun and neuropeptide Y (NPY) but only in a small percentage of neuronal cell bodies and mainly in those with large cell bodies. In conclusion, we have demonstrated that nucleolar enlargement, nuclear eccentricity, ATF-3, c-Jun and NPY are neuronal markers of paclitaxel-induced sensory neuropathy, however, these axotomy-like cell body reactions are infrequent and occur in mainly large-sized sensory neurons.

Nucleolar variation in response to nutritional condition in juvenile pejerrey Odontesthes bonariensis (Valenciennes)

The variations of the nucleolar characteristics size and number were studied in the gill and liver of Odontesthes bonariensis under different feeding regimes in order to test the hypothesis that functional genomes at the nucleolus, which determine variation of size (heterogeneity) in juvenile fishes, are modulated by environmental factors such as food availability. The programme that controls genetic changes of the nucleolar characteristics of the diploid cells of O. bonariensis was different from that described in the diploid cells of cyprinid fishes. No variations of the mean nucleolar number per nucleus were observed in liver or gill cells in relation to the feeding regime. Conversely, significant reduction of the mean nucleolar volume per nucleus (VMN) and change in the shape of the frequency distribution of the total nucleolar volume per cell was evident in both cell types in response to starvation or food restriction. Tissue specificity was observed in relation to the absolute values of the nucleolar characteristics; however, the relative behaviour was comparable in digestive and non‐digestive tissues. The time‐course reduction of VMN in gill and liver cells of restrictively fed fish followed a negative exponential. A good correlation between VMN and the condition index of the fish (K) was observed after long‐term food restriction; however, a faster response of VMN was observed after short‐term fasting events. Results supported the stated hypothesis and showed nucleolar activity as a direct, rapid and sensitive indicator of the nutritional status of the fish.

An ultrastructural study of reproduction in the sporocysts of Prosorhynchoides gracilescens (Digenea: Bucephalidae)

The germinal development in Prosorhynchoides gracilescens sporocysts was studied using electron microscopy. The germinal cells proliferated and developed within multiple floating germinal masses located in the terminal portions of sporocyst branches. The germinal masses were composed of supporting and germinal cells. Supporting cells possessed numerous flattened extensions that spread around and between developing germinal cells to form three-dimensional mesh network, which maintained the integrity of the germinal mass. Morphological evidences of close interactions between supporting and germinal cells were numerous gap junctions between the two cell types and phagocytosis of small fragments of germinal cells cytoplasm by supporting cells. The germinal cells displayed structural differences that seemed to reflect their sequential developmental changes. These changes included (1) cell growth and increase of organelles number, (2) dispersion of nuclear chromatin and increase of nucleolus size, (3) polarization of the cell, (4) appearance of specific structures such as nuage and laminated inclusions. The germinal cells left the germinal masses to finish their differentiation in the body cavity and then cleaved to give rise to cercarial embryos. Ultrastructural features of the germinal elements of P. gracilescens sporocysts are discussed in the light of existing controversy concerning the nature of the germinal sacs reproduction.

Cajal body number and nucleolar size correlate with the cell body mass in human sensory ganglia neurons

This paper studies the cell size-dependent organization of the nucleolus and Cajal bodies (CBs) in dissociated human dorsal root ganglia (DRG) neurons from autopsy tissue samples of patients without neurological disease. The quantitative analysis of nucleoli with an anti-fibrillarin antibody showed that all neurons have only one nucleolus. However, the nucleolar volume and the number of fibrillar centers per nucleolus significantly increase as a function of cell body size. Immunostaining for coilin demonstrated the presence of numerous CBs in DRG neurons (up to 20 in large size neurons). The number of CBs per neuron correlated positively with the cell body volume. Light and electron microscopy immunocytochemical analysis revealed the concentration of coilin, snRNPs, SMN and fibrillarin in CBs of DRG neurons. CBs were frequently associated with the nucleolus, active chromatin domains and PML bodies, but not with telomeres. Our results support the view that the nucleolar volume and number of both fibrillar centers and CBs depend on the cell body mass, a parameter closely related to transcriptional and synaptic activity in mammalian neurons. Moreover, the unusual large number of CBs could facilitate the transfer of RNA processing components from CBs to nucleolar and nucleoplasmic sites of RNA processing.

Age-related changes in histogram pattern of anterior horn cells in human cervical spinal cord

The purpose of the present study was to clarify age-related changes in histograms of spinal anterior horn cells. The study examined Rexed lamina IX of the C7 spinal cord segment in 22 men who had died of non-spinal disease (age range, 0-85 years). First, we confirmed that the size of nucleoli exhibited a linear relationship to the diameter of spinal anterior horn cells by preparing histograms of nucleoli. Second, this formula was used to create histograms of cervical anterior horn cells. Results were as follows: (i) diameter of nucleoli ranged from 2.0 microm to 6.0 microm; (ii) in each subject, no changes were seen in histogram patterns among ventral, intermediate, dorsal and overall sections; (iii) at < or =20 years of age, histograms displayed a single peak for the diameter of nucleoli at about 4.0-4.5 microm; (iv) at 21-60 years of age, histograms displayed two peaks, at about 3.5-4.0 microm and 5.0-5.5 microm; and (v) at 61-85 years of age, histograms displayed a single peak at about 5.0-5.5 microm.

The RNA Methyltransferase Misu (NSun2) Mediates Myc-Induced Proliferation and Is Upregulated in Tumors

Myc is a well-known proto-oncogene, but its functions in normal tissue remain enigmatic. In adult epidermis, Myc stimulates exit from the stem cell compartment, decreasing cell adhesion and, by an unknown mechanism, triggering proliferation of transit-amplifying cells. We describe a novel direct target gene of Myc, Misu, that is expressed at low levels in normal epidermis but is upregulated on Myc activation. Misu encodes a previously uncharacterized RNA methyltransferase with high sequence homology to NSun2 and defines a new family of mammalian SUN-domain-containing proteins. The nucleolar localization of Misu is dependent on RNA polymerase III transcripts, and knockdown of Misu decreases nucleolar size. In G2 phase of the cell cycle, Misu is found in cytoplasmic vesicles, and it decorates the spindle in mitosis. Misu expression is highest in S phase, and RNAi constructs block Myc-induced keratinocyte proliferation and cell-cycle progression. Misu is expressed at low levels in normal tissues, but is highly induced in a range of tumors. Growth of human squamous-cell-carcinoma xenografts is decreased by Misu RNAi. Misu is a novel downstream Myc target that methylates RNA polymerase III transcripts. Misu mediates Myc-induced cell proliferation and growth and is a potential target for cancer therapies.

On the nucleolar size and density in human early granulocytic progenitors, myeloblasts.

Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing. The results clearly demonstrated that values of the nucleolar diameter depended on the procedures used for visualising nucleoli. It seems to be also clear that a close relationship exists between the diameter of nucleoli and their number since the larger the number of nucleoli per cell the smaller their mean size. However, one of multiple nucleoli present in the nucleus is usually significantly larger. Moreover, the possibility exists that the variability of nucleolar diameter of leukemic myeloblasts and thus the heterogeneity of these cells might depend on various stages of the cell cycle as supported by nucleolar measurements on aging leukemic myeloblasts (K 562 cells) in vitro. Since the staining density of small and large nucleoli did not differ substantially after staining for RNA, it seems to be likely that the nucleolar size is directly related to the total RNA content in myeloblasts. In addition, karyometry combined with RNA cytochemistry still appears to be an useful tool to study nucleoli at the single cell level.