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Two cis-acting elements in the p120 gene play important roles in transcription; the region from -537 to -278 is necessary for initiation of transcription, and the region from -1426 to -1223 is necessary for efficient transcription. The distal element(s) which lies upstream of -278 is required for initiation of transcription.
A gene for human proliferating cell nucleolar protein p120 has been isolated from a human genomic library using p120 cDNA as a probe. The gene spanned 12 kilobase pairs and was composed of 15 exons and 14 introns. Unusual splice junction sequences, (AT) and (AC), instead of (GT) and (AG), respectively, were located at the splice sites for intron F. The 5' flanking region was analyzed for putative cis-acting factor binding sites. The region contained a TATA-like sequence and the CCAAT box. CAT assays indicated that the region -2532 /+ 102 was necessary for transcription. Two specific areas at -1444/-1224 and -537/-278 were shown to be important for transcription. Two "GC" boxes and two "GC"-rich sequences were observed Other sites upstream of these sites were analyzed for homology to other gene control regions.
Blastic cells of acute lymphoid, acute myeloid and acute myelomonocytic leukaemias were studied by means of indirect immunofluorencence to provide more information on the presence of satellite nucleoli in blood cells. According to results, satellite nucleoli were found in a small but constant number of blastic cells disregarding their type and type of acute leukaemia. Satellite nucleoli exhibited a positive immunoreaction for fibrillarin and protein B23 which are characteristic for main nucleolar components. These findings suggest that satellite nucleoli contain fibrillar centers as well as dense fibrillar and granular components or at least proteins characteristic for these nucleolar components. Similarly as in normal and pathological cells of completely different origin, in blastic cells of acute leukaemias the number of satellite nucleoli per cells ranged between 1 and 2.
The electrostimulation of the cicatricial healing in the abdominal wall were tested in 119 Wistar rats by means of bipolar rectangularly pulsed current (0.87 Hz, +/- 25 mu A) and direct current (1 mu A). Doubling of the fascia of an abdominal wall hernia was carried out in three groups of rats. In the first group the operational region was stimulated by rectangularly pulsed current, strong proliferated cicatrices were formed with premature production of fibroblasts and collagenous fibres, fast maturation, and high strength. In the second group it was stimulated by direct current, a less exact longitudinal orientation of the collagenous fibres and a slower cicatricial maturation were shown. The third group applied as a control showed a cicatricial distension up to 5 mm. The electrostimulation of the proliferation of connective tissue and cicatricial healing could be pointed out between different tissues (peritoneum and fascia).
The amount of human nucleolar protein p120 is small or nondetectable in most normal resting cells but increases greatly in some actively proliferating cells. The time course was compared for accumulation of mRNAs for p120, c-myc, and histones in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The mRNAs for p120 and c-myc were undetectable in unstimulated peripheral blood mononuclear cells; phytohemagglutinin stimulation increased the p120 mRNA level within 2 h, and it reached a maximum within 24-48 h. The c-myc mRNA level increased within 4 h and had a biphasic maximum at 4 h and 24 h. Indirect immunofluorescence staining and Western blot analysis showed an increase in the level of p120 protein in the nucleolus following the increase in its mRNA level, which suggests that p120 expression is transcriptionally controlled. Both p120 mRNA and c-myc mRNA levels were significantly decreased in 12-O-tetradecanoyl-phorbol-13-acetate-differentiated HL-60 leukemic cells and in confluent normal immortalized human fibroblasts (WSI). These data indicate that p120 is expressed very early in the G1 phase of the cell cycle and suggest that it has an important function in the cell cycle.
Proliferating cell nucleolar antigen P40 is a late G1-specific protein, which was found in a variety of human tumors (A. Chatterjee, J. W. Freeman, and H. Busch. Cancer Res., 47: 1123-1129, 1987). Two overlapping complementary DNA clones for antigen P40 were isolated by immunoscreening a lambda gt11 human expression library. The complete nucleotide sequence of the clones was determined. The complementary DNAs encode the Mr 30,000 portion of the COOH-terminal portion of the protein. The mRNA for P40 was 2.8 kilobases long and was expressed maximally in G1 cells in cell cycle. A series of deletion mutants of the expressed peptide was constructed and the deletion mutants were expressed in Escherichia coli. Using these mutants, the epitope region of P40 recognized by a P40-specific monoclonal antibody was identified. The hydropathy plot based on the protein sequence revealed that this region of the protein is largely hydrophilic. This protein is unique and differs in sequence from other proliferating cell nuclear/nucleolar antigen proteins of similar molecular weight such as protein B23 and cyclin.
A cDNA encoding human nucleophosmin (protein B23) was obtained by screening a human placental cDNA library in lambda gtll first with monoclonal antibody to rat nucleophosmin and then with confirmed partial cDNA of human nucleophosmin as probes. The cDNA had 1311 bp with a coding sequence encoding a protein of 294 amino acids. The identity of the cDNA was confirmed by the presence of encoded amino acid sequences identical with those determined by sequencing pure rat nucleophosmin (a total of 138 amino acids). The most striking feature of the sequence is an acidic cluster located in the middle of the molecule. The cluster consists of 26 Asp/Glu and 1 Phe and Ala. Comparison of human nucleophosmin and Xenopus nucleolar protein NO38 shows 64.3% sequence identity. The N-terminal 130 amino acids of human nucleophosmin also bear 50% identity with that of Xenopus nucleoplasmin. Northern blot analysis of rat liver total RNA with a partial nucleophosmin cDNA as probe demonstrated a homogeneous mRNA band of about 1.6 kb. Similar observations were made in hypertrophic rat liver and Novikoff hepatoma. However, the quantity of nucleophosmin mRNA is 50- and 5-fold higher in Novikoff hepatoma and hypertrophic rat liver, respectively, when compared with normal rat liver. Dot blot analysis also showed a nucleophosmin mRNA ratio of 64:5:1 in the three types of rat liver. When the protein levels were compared with Western blot immunoassays, Novikoff hepatoma showed 20 times more nucleophosmin, while only about 5 times more nucleophosmin was observed in hypertrophic rat liver than in unstimulated normal liver.
Ultrastructurally, metachromatic leukodystrophy (MLD) is marked by characteristic features such as herringbone, prismatic and tufaceous patterns which are typically encountered within oligodendrocytes of the central nervous system (CNS) and in Schwann cells (PNS). These patterns can be documented in late infantile, juvenile, and adult forms. In the latter, aging of the ailing individual adds another component, the accumulation of lipopigments which are marked by an opaque supposedly lipid droplet and a granular component. While MLD-specific lysosomal residual bodies occur in myelinforming cells, lipopigments accrue in neurons and to a lesser degree in astrocytes. MLD represents a unique example in which these two separate lysosomal storage processes combine to form a wide spectrum of ultrastructurally divergent MLD-lipopigments affecting several cell type in the CNS and PNS. Lipopigments and MLD-specific lysosomal inclusions also assemble in sweat gland epithelial cells again combined within the same residual body and in Schwann cells of non-myelinated axons which may also regularly display lipopigments, contrary to Schwann cells of myelinated axons. Comparative studies on sweat glands in childhood and adult forms confirm the earlier observations that composite MLD-lipopigments are frequent in adult MLD. Thus, although lipopigment formation and MLD are different processes, their common occurrence in MLD provides evidence of the mutual morphogenic influence which results in a diversified population of MLD-lipopigments. Whether there is absolute increase of lipopigment formation beyond the age-related level remains to be clarified by quantitative and morphometric data. The presence of lipopigments in childhood MLD in sweat gland epithelial cells favors the concept of accelerated lipopigment formation while the lysosomal compartment is already stimulated by an inborn error of lysosomal metabolism. Whether this principle also prevails in adult forms of other non-MLD lysosomal disorders remains to be elucidated by respective ultrastructural investigations.
Biopsy specimens of 66 medulloblastomas were investigated by means of S-antigen and rod-opsin immunocytochemistry. The patients were operated between 1969 and 1988 and the medical records were retrospectively evaluated to correlate the immunocytochemical features of the tumors to the course of the disease. S-antigen- and rod-opsin-immunoreactive tumor cells were found in 19 out of 66 cases. Since in the normal non-neoplastic state immunoreactive S-antigen and rod-opsin are restricted to retinal photoreceptors and a class of pinealocytes derived from photoreceptor cells, the occurrence of these proteins in certain tumor cells of medulloblastomas suggests a differentiation of these cells along the photoreceptor cell lineage and allows the identification of a special subtype of medulloblastoma displaying photoreceptor-specific characteristics. This subtype appears to be closely related to retinoblastomas and pineal cell tumors. The incidence of this subtype corresponds to approximately 30% of all medulloblastomas. Correlation between the demonstration of immunoreactive S-antigen and rod-opsin and the course of the disease revealed a 10-year survival rate of 50.6% for patients with medulloblastomas displaying photoreceptor-specific characteristics and maximally 11% for patients suffering from medulloblastomas devoid of these markers. Although the statistical evaluation does not provide a significant result, the estimated P-value of 0.085 indicates a distinct trend toward a better prognosis for patients suffering from medulloblastomas with photoreceptor-specific features. The validity of this trend needs to be proven in further studies with a greater number of patients.
Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag-staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase-specific domains which contain both rDNA and RNA polymerase I.
The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.
Nucleolar antigens p145 and p120 are associated with proliferating cells (Freeman, J.W.; McRorie, D.K.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Spohn, W.H.; Busch, H. Cancer Res. 46:3593; 1986 and Freeman, J.W.; Busch, R.K.; Gyorkey, P.; Gyorkey, F.; Ross, B.E.; Busch, H. Cancer Res. 48:1244; 1988) and are not detectable in normal resting cells. Recent immunoelectron microscopic studies (Ochs, R.L.; Reilly, M.T.; Freeman, J.W.; Busch, H. Cancer Res. 48:6523; 1988) suggest that the two antigens have overlapping nucleolar localizations. In this study the nucleolus was physicochemically and biochemically studied to determine whether p145 and p120 were associated with a common nucleolar component. Antigen p145 was associated with 40-80 S ribonucleoprotein particles (RNPs), and the p145 antigen was not detected in HeLa cells following in situ RNAse digestion. P120 was found in a 40-80 S, RNAse resistant complex. Sequential extraction of HeLa nucleoli showed that most of antigen p145 was extractable in 10 mM Tris with 0.2% deoxycholate, whereas p120 was found in a nucleolar residue fraction requiring DNAse and high salt treatment for optimal extraction. Neither antigen p145 nor p120 was detectable in normal resting tissues. Antigen p145 was detected in all proliferating tissues examined, including a variety of malignant tumors (ten of ten), benign tissues including adenomas and hyperplasias (eight of eight), and in normal proliferating cells such as colonic epithelium and spermatogonia of the testes. Antigen p120 was not detected in all tumors, being absent in three of seven lymphomas and in one melanoma examined.(ABSTRACT TRUNCATED AT 250 WORDS)
A monoclonal antibody to a human tumor nucleolar 120 kD protein was developed by Freeman et al. (Cancer Res. 48: 1244-1251, 1988). Its complementary DNA (cDNA) has been isolated and sequenced (Fonagy et al., submitted). To determine the relative messenger RNA (mRNA) level for protein p120, cellular mRNA was extracted, slot-blotted onto nitrocellulose filters, and hybridized to radioactive p120 cDNA fragments. Human tumor cells contained 15-60 times more p120 mRNA than human term placenta. The rat Novikoff hepatoma ascites cell mRNA hybridized to the p120 cDNA probes, but the p120 monoclonal antibody did not react with the Novikoff hepatoma proteins. Novikoff hepatoma mRNA contained 8 times as much p120 mRNA as normal rat liver. As a control, a cDNA was used for protein B23, an abundant nucleolar protein; there were 3.5, 29, and 14 times more B23 mRNA than p120 mRNA in normal rat liver, Novikoff hepatoma ascites cells, and HeLa cells, respectively. Whereas the increased levels of the mRNA and protein B23 reflect increased activity of the nucleolus for any increment of nucleolar function, the increased levels of p120 mRNA and the p120 protein reflect the activity of the G1 phase of the cell cycle. The elevated level of p120 mRNA in tumors may reflect the heightened G1 cascade in transformed cells.
The 120 kDa proliferating-cell nucleolar antigen described by Freeman et al. (Cancer Res. 48:1244; 1988) is the most cancer specific of the proliferation-associated nucleolar proteins identified thus far. It is localized in a novel nucleolar microfibrillar structure recently described by Ochs et al. (Cancer Res. 48:6523; 1988). The amino acid sequence has been determined by a combination of cDNA and genomic DNA sequences. This molecule contains, consecutively, four major domains: a basic domain, an acidic domain, a hydrophobic and methionine-rich domain, and a domain rich in cysteine and proline residues. The isolated cDNA was shown to code for the HeLa P120 protein as shown by a similarity in immunoreactivity, mobility on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and patterns of partial digestion of the Escherichia coli-expressed P120 and the HeLa nucleolar P120 protein. This protein is of special interest because it is expressed in early G1 and, in studies to date, it has not been detected in benign tumors and most normal resting tissues.
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In the course of development of a "library" of monoclonal antibodies to nucleolar proteins, a monoclonal antibody to a nuclear antigen with a molecular weight of 150,000 was obtained. Using this monoclonal antibody as an immunocytochemical probe, low immunofluorescence was demonstrated in human peripheral blood lymphocytes or HL-60 cells treated with retinoic acid. In contrast, a high degree of immunofluorescence was detected in rapidly proliferating human cells (HeLa, Hep-2, HL-60), in Novikoff hepatoma cells, and in phytohemagglutinin-activated human blood lymphocytes. Immunodot blot analysis indicated there were low levels of Mr 150,000 protein in quiescent lymphocytes and increasing amounts after stimulation with phytohemagglutinin. The increased level of Mr 150,000 protein thus is associated with cell proliferation.
The human proliferation-associated nucleolar antigen p120 was localized to substructures within HeLa cell nucleoli by immunofluorescence and immunoelectron microscopy of cells whose nucleoli were segregated by drug treatment or extracted with nucleases. By indirect immunofluorescence, protein p120 was localized diffusely throughout all interphase nucleoli. However, high resolution immunoelectron microscopy demonstrated that protein p120 staining delineated a network of 20-30-nm diameter beaded fibrils distributed throughout the nucleolus. This distribution was unique compared to that of the nucleolar proteins p145, RNA polymerase I, or B23 which were examined simultaneously. Drug-induced segregation of nucleoli by actinomycin D or dichlorobenzimidazole riboside, followed by immunoelectron microscopy, indicated that protein p120 was concentrated at the periphery of the granular region in segregated nucleoli. In situ nuclease digestion of cells with DNase I and/or RNase A did not release p120 from the nucleolus. Instead, p120 immunoreactivity was retained within phase-dense residual nucleoli. These results provide evidence that protein p120 is associated with, and in fact delineates, a network of fibrils which is retained in the nucleolar residue fraction of proliferating cells.
Our laboratory has reported (J. W. Freeman et al., Cancer Res., 48:1244-1251, 1988) a proliferation-associated Mr 120,000 nucleolar antigen (p120), that was found in human tumors but was not detectable in most normal resting tissues and in benign tumors. To study further the function and the localization of this protein, we have investigated various methods of microinjecting p120 monoclonal antibodies into cells. For comparison, we have used a monoclonal antibody to protein C23, a nucleolar protein found in high levels in most cells. To determine optimal conditions for loading of antibodies to nucleolar antigens into mechanically disrupted HeLa cells, we studied the effects of ionic strengths of loading buffers, various antibody concentrations, and optimal time for loading and antibody localization. With ascites fluids in isotonic buffer containing antibodies to nucleolar proteins p120 and C23, a maximum number of cells, 86 and 84%, respectively, were loaded following a 20-min incubation. Hypotonic buffers decreased the percentage of cells loaded (22%); hypertonic buffers reduced cell viability. The optimal concentration of purified antibody yielding a maximum number of loaded cells (81%) was 2.5 mg/ml. Higher concentrations of antibody resulted in residual cytoplasmic staining without increasing the percentage of loaded cells. With antibody concentrations less than 2.5 mg/ml, a linear decrease was noted in the percentage of cells loaded with a decrease in intensity of fluorescence. Following antibody loading, nucleolar fluorescence was observed by 12 h and the intensity increased at 24 h. Localization of the p120 antibody was followed through mitosis where it was perichromosomal and equally divided between the chromosomes at metaphase. A decrease of nucleolar immunofluorescence intensity and percentage of cells labeled were observed in successive cell generations.