Deregulated expression of Nucleophosmin 1 in gastric cancer and its clinicopathological implications.

BACKGROUND: The process of gastric carcinogenesis still remains to be elucidated. The identification of genes related to this process may help to reduce mortality rates through early diagnosis and the development of new anticancer therapies. Nucleophosmin 1 (NPM1) acts in ribosome biogenesis, centrosome duplication, maintenance of genomic stability, and embryonic development. Recently, NPM1 has been implicated in the tumorigenesis processes. Here, we evaluated NPM1 gene and protein expression in gastric tumors and in corresponding non-neoplastic gastric samples. METHODS: NPM1 protein expression was determined by Western blot in 17 pairs of gastric tumors and corresponding non-neoplastic gastric tissue. The protein immunoreactivity was observed in 12 tumor samples. mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in 22 pairs of gastric tumors and in matched non-neoplastic gastric tissue. RESULTS: NPM1 protein expression was significantly reduced in gastric cancer samples compared to matched non-neoplastic gastric samples (P = 0.019). The protein level of NPM1 was reduced at least 1.5-fold in 35% of tumors compared to paired non-neoplastic gastric tissue. However, NPM1 immunoreactivity was detected in neoplastic and non-neoplastic cells, including in intestinal metaplastic, gastritis and inflammatory cells. NPM1 was mainly expressed in nucleus and nucleolus subcellular compartments. The staining intensity and the percentage of immunoreactive cells varied among the studied cases. The NPM1 mRNA level was reduced at least 1.5-fold in 45.5% of samples and increased in 27.3% of samples. An inverse correlation between protein and mRNA expression was detected (r = -0.509, P = 0.037). Intestinal-type gastric cancer presented higher mRNA levels than diffuse-type (P = 0.026). However, reduced NPM1 protein expression was associated with intestinal-type gastric cancer compared to matched non-neoplastic gastric samples (P = 0.018). In addition, tumors from patients with known distant metastasis presented reduced NPM1 protein levels compared to tumors from patients without distant metastasis (P < 0.001). CONCLUSION: Although the expression of NPM1 is heterogeneous in gastric tumors, our results suggest that NPM1 down-regulation may have a role in gastric carcinogenesis and may help in the selection of anticancer treatment strategies.

Redefining the Scl-70 indirect immunofluorescence pattern: autoantibodies to DNA topoisomerase I yield a specific compound immunofluorescence pattern.

OBJECTIVES: To report on a novel IIF pattern specifically associated with antibodies to DNA topo I. METHODS: A novel compound IF pattern, designated Scl-70 pattern, was characterized in routine ANA-HEp-2 IIF screening. Within the last 3 years, all serum samples presenting the Scl-70 pattern at the ANA-HEp2 IIF screening were tested for anti-topo I reactivity. Conversely, 16 serum samples with known anti-topo I reactivity and affinity-purified anti-topo I antibody preparations were tested for the Scl-70 pattern. RESULTS: The Scl-70 pattern comprised the staining of five cellular regions: nucleus, nucleolus and cytoplasm in interphase cells; nucleolar organizing region (NOR) and chromosomes in mitotic cells. All 81 serum samples selected as Scl-70 pattern reacted with topo I. All 16 anti-topo I samples and antibody preparations reproduced the Scl-70 pattern. This compound IF pattern was consistently observed in different commercial HEp-2 cell slides and in home-made slides with HEp-2 cells and human fibroblasts fixed with alternative protocols. Double IIF experiments demonstrated the co-localization of topo I and human upstream binding factor at the NOR. CONCLUSIONS: The Scl-70 pattern belongs to the group of compound IF patterns that hold strong association with the respective autoantibody specificities, such as that observed with centromere protein F (CENP-F) and nuclear mitotic apparatus-1 (NuMA-1) protein. The identification of the Scl-70 pattern at routine ANA-HEp-2 IIF screening may lead to implementation of specific tests for the identification of anti-topo I antibodies. In addition, the Scl-70 pattern outlines cellular domains other than those previously reported for topo I, which is of interest for further understanding the roles of this enzyme in cell biology.

The nucleoprotein and the viral RNA of infectious salmon anemia virus (ISAV) are localized in the nucleolus of infected cells.

The infectious salmon anemia virus (ISAV), which belongs to the new genus Isavirus of the Orthomyxoviridae family, is an important pathogen of the salmon farming industry. Indirect immunofluorescence assays carried out with monoclonal antibodies specific for the nucleoprotein (NP) reveal differential staining of sub-cellular compartments in infected cells. Particularly interesting was the staining of the nucleolus, which showed co-localization with nucleolin in CHSE-214, EPC and SHK-1 cells infected with ISAV. These results were confirmed by co-immunoprecipitation studies showing an interaction between NP and nucleolin. In addition, in situ hybridization carried out with probes specific for each of the 8 RNA segments of ISAV showed that the genomic as well as the anti-genomic strands were also localized in the nucleolus. These results suggest a role of the nucleolus in the replication and/or in the packaging of the ISAV genome.

Nop53p interacts with 5.8S rRNA co-transcriptionally, and regulates processing of pre-rRNA by the exosome.

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. One of the early stages of pre-rRNA processing includes formation of the two intermediate complexes pre-40S and pre-60S, which then form the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, ribosomal proteins and processing factors. The yeast nucleolar protein Nop53p has previously been identified in the pre-60S complex and shown to affect pre-rRNA processing by directly binding to 5.8S rRNA, and to interact with Nop17p and Nip7p, which are also involved in this process. Here we show that Nop53p binds 5.8S rRNA co-transcriptionally through its N-terminal region, and that this protein portion can also partially complement growth of the conditional mutant strain Deltanop53/GAL::NOP53. Nop53p interacts with Rrp6p and activates the exosome in vitro. These results indicate that Nop53p may recruit the exosome to 7S pre-rRNA for processing. Consistent with this observation and similar to the observed in exosome mutants, depletion of Nop53p leads to accumulation of polyadenylated pre-rRNAs.

Histone H1 of Trypanosoma cruzi is concentrated in the nucleolus region and disperses upon phosphorylation during progression to mitosis.

Phosphorylation of histone H1 is intimately related to the cell cycle progression in higher eukaryotes, reaching maximum levels during mitosis. We have previously shown that in the flagellated protozoan Trypanosoma cruzi, which does not condense chromatin during mitosis, histone H1 is phosphorylated at a single cyclin-dependent kinase site. By using an antibody that recognizes specifically the phosphorylated T. cruzi histone H1 site, we have now confirmed that T. cruzi histone H1 is also phosphorylated in a cell cycle-dependent manner. Differently from core histones, the bulk of nonphosphorylated histone H1 in G(1) and S phases of the cell cycle is concentrated in the central regions of the nucleus, which contains the nucleolus and less densely packed chromatin. When cells pass G(2), histone H1 becomes phosphorylated and starts to diffuse. At the onset of mitosis, histone H1 phosphorylation is maximal and found in the entire nuclear space. As permeabilized parasites preferentially lose phosphorylated histone H1, we conclude that this modification promotes its release from less condensed and nucleolar chromatin after G(2).

CGI-55 interacts with nuclear proteins and co-localizes to p80-coilin positive-coiled bodies in the nucleus.

The human protein CGI-55 has been described as a chromo-helicase-DNA-binding domain protein (CHD)-3 interacting protein and was also found to interact with the 3'-region of the plasminogen activator inhibitor (PAI)-1 mRNA. Here, we used CGI-55 as a "bait" in a yeast two-hybrid screen and identified eight interacting proteins: Daxx, Topoisomerase I binding RS (Topors), HPC2, UBA2, TDG, and protein inhibitor of activated STAT (signal transducer and activator of transcription) (PIAS)-1, -3, and -y. These proteins are either structurally or functionally associated with promyelocytic leukemia nuclear bodies (PML-NBs), protein sumoylation, or the regulation of transcription. The interactions of CGI-55 with Daxx, Topors, PIASy, and UBA2 were confirmed by in vivo colocalization experiments in HeLa cells, by using green (GFP) and red fluorescence fusion proteins. A mapping study of the CGI-55 binding site for these proteins revealed three distinct patterns of interaction. The fact that CGI-55-GFP has been localized in cytoplasm and nucleus in a dotted manner, and its interaction with proteins associated with PML-NBs, suggested that CGI-55 might be associated with nuclear bodies. Although Daxx and Topors co-localized with promyelocytic leukemia protein (PML), CGI-55 itself as well as PIASy and UBA2 showed only little co-localization with PML. However, we observed that CGI-55 localizes to the nucleolus and co-localizes with p80-coilin positive nuclear-coiled bodies.

Ultrastructural changes of the carp (Cyprinus carpio) hepatocyte nucleolus during seasonal acclimatization.

BACKGROUND INFORMATION: The eurythermal fish carp (Cyprinus carpio) adjusts to the seasonal changes in the temperature and photoperiod of its habitat through diverse cellular and molecular mechanisms. We have observed that ribosomal biogenesis is modulated during the acclimatization process and correlates with profound phenotypic changes, reflecting a seasonal-dependent ultrastructural appearance of the nucleolar components. Previous studies using classical techniques showed that in winter-adapted carp the nucleolus appears to be segregated. In the present work, we have reassessed the nucleolar ultrastructural organization of the carp in summer- and winter-adapted fish by using more specific cytochemical and immunocytological techniques. RESULTS: The acetylation method provided evidence that the nucleolar organization is different between winter- and summer-adapted carp. In winter-adapted fish the fibrillar component appears as a unique mass surrounded by several granular caps, whereas in summer-adapted carp the fibrillar component forms few cordons surrounded by granular masses. The nucleolar structure and distribution of the condensed chromatin observed varies upon seasonal acclimatization. In winter the nucleolar chromatin is densely packed in masses that surround the nucleolus, whereas during summer it displays a rather looser organization formed by filaments that not only surround the nucleolus, but also go through the nucleolar body. Using the TdT (terminal deoxynucleotidyl transferase)-immunogold labelling technique, we detected condensed and decondensed nucleolar chromatin, and found some labelling of fibrillar components in both seasons. When liver tissue from summer-adapted carp was treated with AMD (actinomycin D), we observed that the rearrangement of the nucleolar components and condensed chromatin were similar to that found in winter-adapted fish, with differences in the distribution of the perinucleolar chromatin. CONCLUSIONS: The acetylation and TdT-immunogold labelling experiments indicated that the rearrangement of the nucleolar components of winter-adapted carp is very similar to the AMD-treated summer-adapted carp nucleolus, with the latter representing the repression of the ribosomal biogenesis that occurs during the cold season. Nevertheless, the distribution of the condensed perinucleolar chromatin in winter-adapted carp compared with AMD-treated cells suggests that the transcription of rRNA genes in winter-adapted fish is less strongly inhibited and does not lead to the classical segregation of the nucleolus of that described after AMD treatment. In addition, we have confirmed that carp hepatocyte nucleoli comprise only two main structural compartments: a fibrillar component and a granular component. Fibrillar centres were not observed.

Interference image analysis of heat-shocked HeLa cells.

HeLa cells were studied with the interference microscope 1 h after heat shock at temperatures of 40 degrees C and 43 degrees C and also under conditions of recovery from the shock. The aim was to investigate changes in patterns of cellular dry mass distribution with the heat shock, based on variation of interference colours in interphase cells. A change in concentration and distribution of a partly high-salt-resistant material in the nuclear and perinuclear regions of the cells was found to be induced by the heat shock at 43 degrees C, a situation which reverted to control under recovery conditions. A similar interference image response was obtained for the heat shock assay at 40 degrees C, but it was detected only during the 4 h recovery period, suggesting that it could have been elicited later. The material induced by the heat shock and visualized by the analysis of interference images is assumed to be a part of the nuclear matrix-intermediate filament cell fraction.