Re-evaluation of autoantibodies and clinical overview of silicone-related disorders.
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Adult↗
Biomedical subjects
R L Ochs
Publications and source records attributed to R L Ochs.
Quantitative changes of the two major AgNOR proteins, nucleolin and protein B23, related to stimulation of rDNA transcription.
The relationship between the amount of the two major AgNOR proteins, nucleolin and protein B23, and the rate of ribosomal RNA (rRNA) synthesis was studied in cortisol-treated and regenerating rat hepatocytes after partial hepatectomy. In both experimental models the synthesis of rRNA was greatly stimulated, but only in regenerating hepatocytes was the increased synthesis associated with cells entering the mitotic cycle. Nucleolin and protein B23 were identified on SDS-polyacrylamide gels of nuclear proteins transferred to nitrocellulose and detected (1) by immunoreaction with specific monoclonal antibodies revealed by second antibodies coupled to peroxidase followed by chemiluminescence or (2) by the silver-staining procedure for AgNOR proteins. Nucleolin and protein B23 were then quantified by computerized densitometric analysis of the immunolabeling signals or the silver-stained bands at 105 kDa (nucleolin) and 39 kDa (protein B23). The synthesis of rRNA was measured by evaluating the amount of radioactivity incorporated into pre-rRNA after [3H]orotic acid injection. Densitometric analysis of silver-stained bands and immunolabeling signals showed no change in nucleolin and protein B23 amounts in cortisol-stimulated hepatocytes, whereas a moderate increase was found in regenerating hepatocytes at 12 h after partial hepatectomy. In both cortisol-stimulated and regenerating hepatocytes the synthesis of rRNA was highly increased (2.6-fold and 4.3-fold above the control level, respectively). To ascertain the relationship between quantitative changes in nucleolin and protein B23 and stimulation of rRNA transcriptional activity in regenerating hepatocytes, the quantitative distribution of these proteins was also investigated in the early times of regeneration using silver-stained nitrocellulose-transblotted nuclear proteins. The quantity of protein B23 was unchanged until 12 h after partial hepatectomy, whereas nucleolin appeared to be slightly increased at 9 h (1.15-fold above the control value) after partial hepatectomy. On the other hand, at just 6 h after partial hepatectomy, a significant increase of rRNA synthesis occurred in regenerating rat hepatocytes (1.8-fold above the control value). These data demonstrated that stimulation of rRNA transcriptional activity occurring in rat hepatocytes after cortisol treatment and in the early times after partial hepatectomy was not associated with quantitative changes in the amounts of nucleolin and protein B23.
Animals↗
Nuclear remodeling in response to steroid hormone action.
Steroid and similar hormones comprise the broadest class of gene regulatory agents known, spanning vertebrates through the lower animals, and even fungi. Not unexpectedly, therefore, steroid receptors belong to an evolutionarily highly conserved family of proteins. After complexing with their cognate ligands, receptors interact with hormone response elements on target genes and modulate transcription. These actions are multifaceted and only partly understood, and include large-scale changes in the structure and molecular composition of the affected cell nuclei. This chapter examines steroid hormone action and the resultant nuclear remodeling from the following perspectives: (1) Where are the receptors located? (2) Which nuclear domains are most affected? (3) Are there extended or permanent nuclear changes? (4) What is the role of coiled bodies and similar structures in this regard? To address these and related questions, information is drawn from several sources, including vertebrates, insects, and malignant tissues. Entirely new data are presented as well as a review of the literature.
Animals↗
Formation of nuclear bodies in hepatocytes of estrogen-treated roosters.
As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antibody to the coiled body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 coiled body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.
Animals↗
The quantity of nucleolar proteins nucleolin and protein B23 is related to cell doubling time in human cancer cells.
BACKGROUND: The quantity of the silver-stained nucleolar proteins (AgNOR proteins) measured in situ in cytohistologic preparations is related to the rapidity of cell proliferation. The term "AgNOR proteins" comprises several proteins. The relationship between the individual AgNOR protein amount and cell proliferating activity is not yet known. We studied the quantitative distribution of the individual AgNOR proteins, with specific attention to the two major AgNOR proteins, nucleolin and protein B23, in seven human cancer cell lines characterized by different cell doubling times. DESIGN: The doubling time of cancer cells was measured by counting the asynchronously growing cells at regular time intervals. The AgNOR proteins were quantified in situ, after a specific one-step staining procedure, by computerized image analysis. For the quantitative evaluation of nucleolin and protein B23, two methods were followed. Nuclear proteins after separation by SDS-PAGE were transferred onto nitrocellulose membranes and were either: 1) stained by the silver staining procedure for AgNOR proteins or 2) treated with anti-nucleolin and anti-protein B23 mAb followed by reaction with secondary Ab linked to peroxidase and revealed by chemiluminescence and autoradiography. In both cases, measurement of individual AgNOR protein and nucleolin and protein B23 amount was carried out using computerized densitometric analysis. RESULTS: Integrated density values of the silver-stained bands at 105 kDa (nucleolin) and 38 to 39 kDa (protein B23) represented, in all cell lines, more than 60% of the total silver-stained band value. A relationship was found between the densitometric values of silver-stained nucleolin and protein B23 and rapidity of cell proliferation (r = 0.85 and r = 0.86, respectively, p < 0.05). The values of nucleolin and protein B23 obtained using the Western blots were strictly related to the rapidity of cell proliferation (r = 0.93 and 0.96, respectively, p < 0.001). Finally, a good correlation was observed between the mean AgNOR protein area value, as defined in cytologic preparations in situ, and nucleolin and protein B23 amounts as evaluated in silver-stained nitrocellulose membranes (r = 0.92 and r = 0.90, respectively, p < 0.01) and in Western blots (r = 0.95 and r = 0.94, respectively, p < 0.001). CONCLUSIONS: These data indicate that the quantitative changes of AgNOR proteins observed in cytohistologic preparations in situ mainly reflect the quantitative changes of nucleolin and protein B23 and demonstrate that nucleolin and protein B23 amounts are inversely related to cell doubling time in human cancer cells.
Adenocarcinoma↗
Chondrocyte apoptosis induced by nitric oxide.
Chondrocytes stimulated with IL-1 produce high levels of nitric oxide (NO), which inhibits proliferation induced by transforming growth factor-beta or serum. This study analyzes the role of NO and IL-1 in the induction of chondrocyte cell death. NO generated from sodium nitroprusside induced apoptosis in cultured chondrocytes as demonstrated by electron microscopy, 4',6-dianidino-2-phenylindole dihydrochloride staining, FACS analysis, and histochemical detection of DNA fragmentation. Similar results were obtained with two other NO donors, 3-morpholinosynonimide-hydrochloride and s-nitroso-N-acetyl-D-L-penicillamine. In contrast, oxygen radicals generated by hypoxanthine/xanthine oxidase caused necrosis but did not induce chondrocyte apoptosis. To analyze whether endogenously generated NO induces apoptosis, chondrocytes were stimulated with IL-1, but there was no evidence for apoptotic changes. Combinations of NO inducers such as IL-1, lipopolysaccharide, tumor necrosis factor, and interferon-gamma also failed to trigger apoptosis. IL-1-stimulated chondrocytes are known to produce oxygen radicals that react with NO to form products that can induce cell death in other systems. We thus tested IL-1 in combination with the oxygen radical scavengers N-acetyl cysteine, dimethyl sulfoxide, or 5,5'-dimetylpyrroline 1-oxide. Under these conditions IL-1 was able to induce apoptosis, which was inhibited in a dose-dependent manner by the NO synthase inhibitor N-monomethyl L-arginine. Conversely, endogenous oxygen radicals induced by inflammatory mediators caused necrosis under conditions in which the simultaneous production of NO was reduced. These results suggest that NO, but not oxygen radicals, is the primary inducer of apoptosis in human articular chondrocytes.
Adult↗
Autoantibodies in interstitial cystitis.
We have identified and partially characterized autoantibodies from the sera of patients with interstitial cystitis. Our characterization included initial screening by antinuclear antibody testing on human HEp-2 cell substrate and mouse kidney/stomach tissue substrate, titering and subtyping of positive sera, and Western blotting to identify target autoantigens. Of 96 interstitial cystitis patients 35 (36%) were positive for antinuclear antibodies at titers of 1/40 or greater. Among the antinuclear antibody patterns observed 24 were dense fine nuclear speckles, 7 were nucleolar, 3 were mitochondrial and 1 was coarse nuclear speckles. All but 4 of the antinuclear antibody positive sera were exclusively of the IgG class. As determined by unique antinuclear antibody staining patterns and by specificities on Western blots, interstitial cystitis autoantibodies appear to recognize novel autoantigens not previously described in patients with systemic autoimmune diseases, such as lupus, scleroderma and Sjögren's syndrome.
Adult↗
Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis.
We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, containing factors essential for pre-rRNA processing, are recruited to these sites of rRNA transcription and processing.
Animals↗
Coiled bodies in the nucleolus of breast cancer cells.
Coiled bodies are a special type of small round nuclear body, composed of coiled fibers and granules, especially prominent in the nucleoplasm of highly active cells (Brasch and Ochs (1992) Exp. Cell Res. 202, 211-223). Although no specific function has been assigned to coiled bodies, they contain spliceosome snRNAs and proteins, as well as the nucleolar U3 RNA-associated protein fibrillarin. In the present study, we have used antibodies to the coiled body-specific protein p80-coilin, together with double-label immunofluorescence, confocal microscopy and immunoelectron microscopy, to examine the distribution of coiled bodies in a number of different breast cancer cell lines. By immunofluorescence, all cell lines had prominent coiled bodies in the nucleoplasm and several cell lines appeared to have coiled bodies within the nucleolus itself. Double-label immunofluorescence and confocal laser scanning microscopy confirmed the nucleolar localization of coiled bodies. Besides containing p80-coilin, nucleoplasmic and nucleolar coiled bodies contained fibrillarin and Sm proteins. By conventional and immunoelectron microscopy, nucleolar coiled bodies appeared as discrete structures within the nucleolus in a number of different morphotypes, distinct from the normal nucleolar domains of granular component, dense fibrillar component, and fibrillar centers. While the significance of finding coiled bodies in the nucleolus of certain breast cancer cell lines is at present unknown, this represents the first report of coiled bodies and Sm staining in the nucleolus of mammalian cells.
Autoantigens↗
Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.
Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.
Amino Acid Sequence↗
Electron microscopic localization of ribosomal DNA in rat liver nucleoli by nonisotopic in situ hybridization.
We have used postembedding nonisotopic in situ hybridization, with biotinylated rat ribosomal DNA (rDNA) as a probe and streptavidin coupled to 10-nm colloidal gold particles as the detection system, to localize rDNA sequences in rat liver nucleoli at the electron microscopic level. For comparison purposes, immunoelectron microscopy was performed for the detection of DNA. Our results indicate that ribosomal DNA sequences are enriched in the dense fibrillar component of the rat liver nucleolus. These data are discussed in relation to the putative site(s) for transcription of ribosomal genes.
Animals↗
Autoantibodies to a novel cell cycle-regulated protein that accumulates in the nuclear matrix during S phase and is localized in the kinetochores and spindle midzone during mitosis.
We have employed human autoantibodies to characterize a novel cell cycle-regulated nuclear protein, provisionally designated p330d (doublet polypeptide of 330 kDa). The expression and intracellular distribution of this protein was followed throughout the cell cycle using immunofluorescence microscopy, laser confocal microscopy, immunoelectron microscopy and flow cytometry. p330d was expressed only in proliferating cells and began accumulating in the nucleus during early S phase. The protein reached maximum expression levels during G2/M. In situ extractions with detergent, salt and nucleases failed to abolish the nuclear staining of interphase cells, suggesting a tight binding of p330d to the nuclear matrix during interphase. p330d was concentrated in the kinetochores during prophase but was relocated to the spindle midzone at the onset of anaphase. By late telophase, it was localized predominantly in the intercellular bridge regions flanking the midbody and disappeared gradually as the daughter cells separated. Immunoblotting analysis showed that the autoimmune sera recognized a doublet of 330 kDa, and affinity-purified antibodies from this doublet reproduced the fluorescence staining pattern of the whole serum. We propose that p330d is a novel member of the class of 'chromosomal passenger' proteins, which are associated transiently with centromeres during early mitosis and are then redistributed to other sites of the mitotic apparatus after the metaphase/anaphase transition. Possible in vivo functions for p330d and related proteins might include roles in centromere/kinetochore maturation and assembly, chromosome segregation, central spindle stabilization and cytokinesis.
Autoantibodies↗
Antinuclear autoantibodies in women with silicone breast implants.
Clinical syndromes resembling autoimmune diseases have been reported in women who have had breast augmentation procedures. To see whether there is a humoral immune response in these diseases that is similar to the immune response in their idiopathic counterparts, we assessed the immunological specificity of antinuclear antibodies (ANAs) and certain epidemiological features in 24 patients, all of whom (with 1 exception) had received silicone gel breast implants. ANA specificities were identified by indirect immunofluorescence, immunodiffusion, western blot analysis, and immunoprecipitation of radiolabelled intracellular proteins. Of 11 patients who had symptoms and signs that met criteria for defined autoimmune diseases, 7 had scleroderma or subsets of this disorder and the others had systemic lupus erythematosus, rheumatoid arthritis, or overlapping autoimmune diseases. High ANA titres were present in 10 of these 11 patients and the ANA specificities were similar to those found in the idiopathic forms of the corresponding autoimmune diseases. Trauma, with resultant rupture of implants, accelerated onset of symptoms. 13 other patients had autoimmune disorders of a less clearly defined nature and low titres of ANAs whose specificities could not be identified. ANAs are associated with the development of autoimmune complications in women with silicone breast implants. Further studies are needed to see whether this relation is one of cause and effect and whether ANAs might be early serological markers preceding development of autoimmune symptoms.
Academies and Institutes↗
Nuclear bodies (NBs): a newly "rediscovered" organelle.
Nuclear bodies (NBs) were first described in detail some 30 years ago, by conventional electron microscopy, as prominent interchromatin structures found primarily in the nuclei of malignant or hyperstimulated animal cells. Subsequent studies have shown that NBs are ubiquitous organelles, but they are numerically and morphologically quite varied. With the recent discovery of human autoantibodies against several key nuclear antigens present in some NBs, these structures are once again the subject of much attention. At least one class of NBs, coiled bodies, has been shown to be nucleolus-derived and to contain not only nucleolus-associated antigens, but also many of the snRNP components involved in pre-mRNA splicing. These data suggest that coiled bodies, and perhaps other NBs as well, are multifunctional and may be involved in the processing or transport of both pre-mRNA and pre-rRNA. Further evidence is provided showing that NBs constitute distinct nuclear domains whose functional significance is just now emerging.
Animals↗
Centromere autoantigens are associated with the nucleolus.
Because of their importance as target antigens in scleroderma and since all other major autoantigens in scleroderma can be localized to the interphase nucleolus, we were interested in a further investigation of the potential relationship between interphase centromeres and the nucleolus. Using human anticentromere autoantibodies (ACA) from patients with the CREST form of scleroderma as probes in indirect immunofluorescence microscopy, we observed nonrandom interphase "clumping" of centromeres in a distribution suggestive of nucleoli. By double-label immunofluorescence comparing the localization of centromeres to nucleolar proteins Ki-67, fibrillarin, or protein B23 (nucleophosmin), interphase centromeres appeared to be localized around and within nucleoli. A number of different ACA sera were tested on HEp-2, HeLa, PtK2, Indian muntjac, 3T3, and NRK cells, all with identical results indicating colocalization between centromeres and nucleoli. Immunoelectron microscopy revealed that interphase centromeres were distributed free in the nucleoplasm, in contact with the nuclear envelope, in contact with and on the periphery of nucleoli, and totally embedded within the confines of the nucleolus itself. Interestingly, actinomycin D treatment dissociated centromeres from localization within the segregated nucleolus. To determine if interphase centromeres were integral components of nucleoli, nucleoli were isolated according to classical methods. By double-label immunofluorescence, immunoelectron microscopy, and Western blotting, it was demonstrated that centromere autoantigens copurified with isolated nucleoli. These studies offer proof that some interphase centromeres can be associated with, and may even be considered part of, the interphase nucleolus. Furthermore, all of the major autoantigens in scleroderma can now be localized to the nucleolus.
Autoantigens↗
Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies.
Patients with hepatocellular carcinoma (HCC), gastrointestinal, lung, and ovarian cancers were shown to have autoantibodies to nuclear and nucleolar antigens as detected by immunofluorescence on cell substrates. The frequency of antinuclear antibodies (ANAs) was significantly higher (P less than 0.001) in patients with HCC (57/184 = 31%) than in patients with chronic hepatitis or liver cirrhosis (25/187 = 13%). Although a range of fluorescence patterns was observed, a higher percentage of nucleolar fluorescence was detected in HCC, and three of these nucleolar antigens were identified. They were NOR-90, nucleolus organizer region doublet polypeptides of 93 and 89 kDa involved in RNA polymerase I transcription; fibrillarin, a 34 kDa protein of the nucleolar U3 ribonucleoprotein particle which is engaged in preribosomal RNA processing; and nucleophosmin/protein B23, a 37 kDa polypeptide which is associated with ribosome maturation and cellular proliferation. All these antigens are nucleolar components that are engaged in some aspect of ribosome biosynthesis. Since autoantibodies to these nucleolar antigens have also been found in systemic autoimmune diseases, they do not represent autoimmune reactions unique to cancer but might reflect reaction pathways related to immune responses that are antigen-driven. The ANA response in HCC appears to be dynamic reactions to this antigen-drive since some patients with chronic liver disease showed seroconversion to ANA positivity, marked increase in titer and/or change in antibody specificity preceding or coincident with clinical detection of HCC. These changes in ANA showed a close temporal relationship with transformation from long-established chronic liver disease to HCC.
Antibodies, Neoplasm↗
Detection of fibrillarin in nucleolar remnants and the nucleolar matrix.
In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.
Animals↗
Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies.
Studies with human autoimmune sera identified auto-antibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the coiled body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the coiled body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.
Animals↗