Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.     

Proliferating cell nuclear antigen (PCNA) and human malignant tumor nucleolar antigens (HMTNA) in nucleoli of human hematological malignancies

Lymphoma (Lymphocytic non-Hodgkin's malignant lymphoma) and leukemic (chronic lymphocytic, acute and chronic myeloid, myelomonocytic leukemia) cells were studied by indirect immunofluorescence to evaluate the presence of proliferating cell nuclear antigen (PCNA) and human malignant tumor nuclear antigen (HMTNA) in their nucleoli. Most cells in lymph node smears of lymphocytic non-Hodgkin's malignant lymphoma (NHML) developed a bright nucleolar fluorescence with HMTNA antibodies. PCNA was detected in nucleoli of a limited number of cells which apparently represent the proliferating cell population in these lymphomas. Similarly, in the bone marrow smears of patients with chronic lymphocytic leukemia most cells possessed a nucleolar fluorescence for HMTNA and PCNA was present in nucleoli of a limited number of cells. In the bone marrow smears of patients with myeloid or myelomonocytic leukemias most blastic or monocytoid cells also developed a bright nucleolar fluorescence with HMTNA antibodies and PCNA was present only in a small percentage of these cells. Leukemic cells with PCNA in their nucleoli like thekhuntigen might represent a proliferating cell population in late G1-early S phase.     

Studies on the human tumor nucleolar antigens

With rabbit antibodies to nuclear 0.01 M Tris-HCl, pH 8, extract or "nucleolar preparations" of human HeLa S3 cells and fluorescein-labeled goat antirabbit antibodies, bright nucleolar immunofluorescence was observed in human adenocarcinomas, squamous cell carcinomas, sarcomas, hematological neoplasms, and other malignant tumors. With these antibodies, nucleolar immunofluorescence was not found in most normal tissue specimens, benign adenomas, hyperplastic tissues, and specimens of inflammatory diseases. A study was made on the presence in benign and malignant breast tumors of a common nucleolar antigen previously found in a broad range of human malignant tumors. Bright nucleolar immunofluorescence was observed in 19/20 (95%) of known breast cancer specimens. In the group of 80 unknown samples in the "blind" study, 75 (94%) were correctly identified as malignant or benign on the basis of the presence and distribution of the nucleolar fluorescence. In a group of 67 samples in which the nucleolar fluorescence was either readily observed or virtually absent, 47/48 (98%) of the malignant tumors were correctly identified. Of the bening lesions or normal breast specimens, 18/19 (95%) were correctly identified as negative for nucleolar fluorescence. These studies extend the results previously reported for a common nucleolar antigen in a broad range of human cancers to a larger series of malignancies of a particular organ. The tumor nucleolar antigen(s) were partially characterized by isoelectric focusing on 4% polyacrylamide gels. One major band had a pI of 6.3 and a minor band had a pI of 6.1. These antigens were not found in the normal human liver nucleoli.     

Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes

Nucleoli of normal and leukemic lymphocytes were studied by cytochemical and immunofluorescence methods to provide more information on the nucleolar presence and distribution of proteins B23 and C23. Annular nucleoli of human lymphocytes represent a very convenient subject for such studies, since they consist of one centrally located large fibrillar center surrounded by RNP components. In such nucleoli, protein C23 was present mainly in the central nucleolar region and protein B23 was found mostly in the periphery. The nucleolar area immunostained for protein B23 was usually larger than that stained for protein C23. The distribution of protein C23 appeared to be similar to that of intensely stained nucleolar argyrophilic components. No substantial differences were found between the distribution of proteins B23 and C23 in nucleoli of normal and leukemic lymphocytes. In lymphocytes of patients treated with chemotherapy, the immunofluorescence was diminished for protein B23 and particularly so for protein C23.     

Nucleolar morphology and rDNA in situ hybridisation in monocytes

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

Studies on nucleoli in rosetting T and B lymphocytes of the human peripheral blood

The incidence of various nucleolar types was studied in human rosetting lymphocytes to provide an information on nucleolar types present in T and B lymphocytes of the peripheral blood. The results clearly demonstrate that both T and B lymphocytes of the peripheral blood mostly contain ring shaped nucleoli ("resting nucleoli") and less frequently other nucleolar types such as nucleoli with nucleolonemata or compact nucleoli ("active nucleoli") and micronucleoli ("inactive nucleoli"). Since all known nucleolar types and particularly micronucleoli may be observed in both T and B lymphocytes, nucleoli in these cells cannot indicate the type or origin of these cells but simply the state of the nucleolar RNA synthesis.     

Immunoelectron-microscopic localization of a proliferation-associated antigen Ki-67 in MCF-7 cells

Summary Immunocytochemistry using the monoclonal antibody Ki-67 is a commonly used method to assess proliferative activity of malignant tumours. Ki-67 reacts with proliferating cells with an antigen, whose structure, function and exact locations are unknown. We studed the subcellular location of Ki-67 in MCF-7 cells using immunoelectron microscopy. In the interphase cells, Ki-67 immunoreactivity was localized in the nucleolus, mainly in the nucleolar cortex. In particular areas of the granular component of the nucleolus were strongly stained. Weak spot-like nucleoplasmic immunostaining was also seen outside the nucleolus. During prophase Ki-67 antigen was localized on the surfaces of the condensed chromatin and during metaphase on the surface of the chromosomes. After cell division and prior to formation of new nucleoli, Ki-67 immunoreactivity was located in the nucleoplasm. Quantification of Ki-67 immunofluorescence signal by flow cytometry revealed highest Ki-67 levels in mitotic cells. The localtion of Ki-67 is very similar to certain recently described proteins of nucleolar preribosomes suggesting that Ki-67 may also be a component of the preribosomes.     

Characterization of a cell cycle-dependent nuclear autoantigen

Analysis of reactivity to nuclear antigens in autoimmune sera revealed a serum that produced a previously undescribed cell cycle-dependent immunofluorescence staining pattern. By indirect immunofluorescence using HEp-2 cells as substrate, the serum generated a speckled and nucleolar pleomorphic staining pattern. This characteristic immunofluorescence pattern was detected in different cell lines from various species indicating that the antigen was highly conserved. This serum immunoprecipitated a 85 kDa protein using an extract from [³⁵S]-labeled HeLa cells. Indirect immunofluorescence of proliferating mouse 3T3 cells displayed the characteristic pleomorphic staining which was not observed in serum-starved cells. Resting human and mouse peripheral blood lymphocytes were negative in immunofluorescence while mitogen-stimulated lymphocytes were positive. Germinal centers of mice two weeks after immunization with 2-phenyl-oxazolone showed speckled immunofluorescence staining in the dark zones whereas unimmunized mice were completely negative. Cell synchronization experiments showed a characteristic sequence of locations of the antigen during the cell cycle. In G₁, cells were completely negative. In late G₁, G₁/S and S phase, speckles were visible. In early G₂, speckles were visible, and later in G₂, the nucleoli were positive. During mitosis chromosomes were stained. Further characterization of this antibody specificity and cloning of cDNA are in progress.     

Epitope distribution and immunochemical characterization of nucleolar phosphoprotein C23 using ten monoclonal antibodies

Summary Extractable nucleolar proteins from HeLa cells were used as a source of antigen to immunize mice for monoclonal antibody (MAb) production. Ten of the resulting MAbs shown to identify nucleolar phosphoprotein (110 kD/pI 5.5) were purified and used in immunochemical studies to further characterize protein C23. All ten MAbs showed nucleolar localization by indirect immunofluorescence; one antibody (FR2) also showed some nucleoplasmic localization that was attributed to a shared epitope between protein C23 and a 72 kD nuclear/nucleolar antigen. Reciprocal antibody cross blocking studies indicated that the ten MAbs identified nine distinct epitopes on protein C23. Interestingly, seven of the nine epitopes were shown by immunofluorescence and competitive ELISA studies to be species related. Immunostained patterns of exponentially growing HeLa cells suggest that protein C23 exists in vivo solely as a 110 kD peptide. However, protein C23 was subject to rapid degradation into a number of proteolytic fragments upon extraction or storage of isolated nucleoli. The failure to find protein C23 related peptides with molecular sizes less than 110 kD in exponentially growing cells and the lack of cytoplasmic localization of any of the ten MAbs suggests that protein C23 is not a prepro-protein processed in vivo to form ribosomal proteins as previously suggested (1).     

Hepatitis-associated antigen in immunofluorescence I. Interfering factors

Synopsis By means of the indirect immunofluorescence test, Australia hepatitis-associated antigen (Au/SH) was studied in human liver cell suspensions obtained by needle biopsy. Fluorescence staining was performed with human anti-Au/SH immunoglobulins; controls with non-immune human and guinea-pig sera and with cellular substrates lacking Au/SH antigen (rat regenerating liver) were also carried out.Eight out of forty-one unselected patients showed specific fluorescence in their liver cells without any correlation with clinical diagnosis, presence of Au/SH antigen in the serum or of virus-like particles in the hepatocytes by ultrastructural examination. The positive cases were characterized by intranuclear fluorescent granules in, probably, nucleoli; this was confirmed by cytochemical investigations. However, the eight cases positive to anti-Au/SH antiserum showed a quite identical nucleolar fluorescence with non-immune human and guinea-pig sera. In addition, histochemical and ultrastructural examinations demonstrated, only in these positive cases, an increased nucleolar RNS-synthesis.The same results were obtained with cell suspensions of rat regenerating livers removed 20–24 hr after partial hepatectomy. These data strongly support that enhanced nucleolar RNA synthesis is responsible for non-specific positive fluorescence.Elution and digestion tests demonstrated that a heat-labile C'lq-like factor can interfere in the positivity of this fluorescence test.     

A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

An autoimmune antibody from scleroderma patients recognizes a component of the plant cell nucleolus

Summary An auto-antibody from human serum of patients with the autoimmune disease scleroderma was used to localize the nucleolus in meristematic cells of onion and soybean roots using indirect immunofluorescence microscopy. Similar lots of antiserum recognized a single 34 kD, nucleolar protein, fibrillarin, in a variety of animal cells (Ochs, et al. 1984, 1985). In both plants, antibody linked fluorescence is associated with the one to several nucleoli present in the interphase nucleus. The fluorescence becomes diffuse around condensing prophase chromosomes and becomes more diffused at metaphase with slightly more intense fluorescence surrounding the chromosomes. At anaphase-telophase the fluorescence is localized in dense areas within the chromosomes, presumably representing prenucleolar bodies which will form the interphase nucleoli of the daughter nuclei. This antiserum provides a new, valuable tool for the study of the nucleolus and the highly conversed nucleolar antigen(s) that it recognizes.     

Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.     

Detection of a 140 KDA nucleolar protein with an anti-PCNA autoantibody

To assess the numbers and types of PCNA (proliferating cell nuclear antigen) species, immunoprecipitation studies on HeLa cell, nuclear and nucleolar extracts were performed. A 140 KDa protein from HeLa nucleoli was immunoprecipitated by an autoantibody (E.B.) previously used to detect the proliferating cell nuclear antigens (PCNA). The 140 KDa protein was also detected in the nuclear extract of colon carcinoma cells (omega) labeled in vitro with 125I-Bolton Hunter reagent. When the growth of the colon carcinoma cells was inhibited by 1% N,N-dimethylformamide for two weeks, the 140 KDa protein was not detected which suggests this protein is associated with cell growth.     

Nucleolar immunofluorescence in human hematological malignancies.

Rabbit antibodies to the nuclear Tris extract of HeLa cells which have been shown by the indirect immunofluorescence technique to localize in nucleoli of a variety of human malignant tumors but not in a number of nontumor tissues also produced bright fluorescence in nucleoli of tumor cells in several hematological malignancies. The tumors studied included Hodgkins malignant lymphoma, non-Hodgkins malignant lymphoma, acute myeloid and acute myelomonocytic leukemia, chronic lymphatic and chronic myeloid leukemia. In contrast, none of the corresponding normal cell lines in the bone marrow exhibited bright nucleolar fluorescence. In addition, neither the cells of patients with acute infectious mononucleosis nor lymphoid hyperplasia exhibited bright nucleolar fluorescence. These studies suggest that antibodies to HeLa cell nucleolar antigens may be useful in immunodiagnosis of human malignancies.     

Immunolocalization and partial characterization of a nucleolar autoantigen (PM-Scl) associated with polymyositis/scleroderma overlap syndromes

Precipitating anti-PM-Scl antibodies are present in sera from patients with polymyositis, scleroderma, and polymyositis/scleroderma overlap syndromes. By indirect immunofluorescence microscopy, anti-PM-Scl antibodies stained the nucleolus in cells of different tissues and species, suggesting that the antigen is highly conserved. By electron microscopy, anti-PM-Scl antibodies reacted primarily with the granular component of the nucleolus. Drugs that inhibit rRNA synthesis had a marked effect on the expression of PM-Scl antigen. In actinomycin D-treated cells, immunofluorescence staining by anti-PM-Scl was significantly reduced with residual staining restricted to the granular regions of nucleoli. Treatment with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole (DRB) also selectively reduced nucleolar staining. On a molecular level, anti-PM-Scl antibodies precipitated 11 polypeptides with molecular weights (Mr) ranging from 110,000 to 20,000. The Mr 80,000 and 20,000 polypeptides were phosphorylated. Evidence suggests that the PM-Scl antigen complex may be related to a preribosomal particle.     

S-phase patterns of cyclin (PCNA) antigen staining resemble topographical patterns of DNA synthesis. A role for cyclin in DNA replication?

The sequence of cyclin (proliferating cell nuclear antigen, PCNA), antigen staining throughout the cell cycle of African green monkey kidney cells (BS-C-1) has been determined by indirect immunofluorescence using PCNA autoantibodies specific for this protein. Patterns of cyclin staining observed between the beginning of S-phase and maximum DNA synthesis are similar to those reported in human AMA cells [(1985) Proc. Natl. Acad. Sci. USA 82, 3262-3266], while those detected thereafter are significantly different; the most striking feature being the continuous staining of the nucleoli up to or very near the S/G2 border of the cell cycle. Using [3H]thymidine autoradiography and indirect immunofluorescence of the same cells we show a remarkable correlation between cyclin antigen distribution and topographical patterns of DNA synthesis. In addition, we present evidence showing that DNase I treatment of Triton-extracted monolayers abolishes cyclin antigen staining but does not result in a substantial release of this protein. Taken together the above observations argue for a role of cyclin in some aspect of DNA replication.