Differential expression of protein phosphatase type 1 isotypes and nucleolin during cell cycle arrest

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression.     

Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.     

Protein phosphatase-1 inhibitor-3 is an in vivo target of caspase-3 and participates in the apoptotic response

Inh3 (inhibitor-3) is a potent inhibitor of protein phosphatase-1 that selectively associates with PP1gamma1 and PP1alpha but not the PP1beta isoform. We demonstrate that Inh3 is a novel substrate for caspase-3 and is degraded in vivo during apoptosis induced by actinomycin D. Inh3 was not degraded in apoptotic MCF-7 cells, which lack caspase-3. These experiments establish that Inh3 is a novel physiological substrate of caspase-3. Electroporation of the caspase-3-resistant Inh3-D49A mutant into HL-60 cells resulted in a significant attenuation of apoptosis induced by actinomycin D. These results show that Inh3 degradation contributes to the apoptotic process. Immunofluorescence based examination of the subcellular localizations of Inh3 and PP1gamma1 revealed a major relocalization of the cellular pool of PP1gamma1 from the nucleolus to the nucleus and then to the cytoplasm during actinomycin D-induced apoptosis. A similar redistribution of PP1alpha from the nucleus to the cytoplasm occurred. These results are consistent with an unexpected discovery that significant fractions of the cellular pools of PP1gamma1 and PP1alpha are associated with Inh3 in HL-60 cells. Thus, Inh3 is a major factor in the cellular economy of PP1gamma1 and PP1alpha subunits. The unscheduled relocalization of this large a pool of PP1 subunits and their release from a potent inhibitor could deregulate a diverse range of essential cellular processes and signaling pathways. We discuss the significance of these findings in relation to working hypotheses whereby Inh3 destruction could contribute to the apoptotic process.     

Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.     

Double-stranded RNA mediates selective gene silencing of protein phosphatase type 1 delta isoform in HEK-293 cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1delta) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP1delta. This RNA interference did not affect the expression of lphaand gamma1 isoforms of PP1. Transfection of antisense RNA specific for PP1delta also suppressed the expression of PP1delta. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or gamma-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.     

Actinomycin D as a novel SH2 domain ligand inhibits Shc/Grb2 interaction in B104-1-1 (neu*-transformed NIH3T3) and SAA (hEGFR-overexpressed NIH3T3) cells.

Actinomycins, a family of bicyclic chromopeptide lactones with strong antineoplastic activity, were screened as inhibitors of Shc/Grb2 interaction in in vitro assay systems. To investigate the effects of actinomycin D on Shc/Grb2 interaction in cell-based experiments, we used SAA (normal hEGFR-overexpressed NIH3T3) cells and B104-1-1 (neu*-transformed NIH3T3) cells, because a large number of the Shc/Grb2 complexes were detected. Associated protein complexes containing Shc were immunoprecipitated from actinomycin D-treated cell lysates with polyclonal anti-Shc antibody. Then the association with Grb2 was assessed by immunoblotting with monoclonal anti-Grb2 antibody. The result of the immunoblotting experiment revealed that actinomycin D inhibited Shc/Grb2 interaction in a dose-dependent manner in both B104-1-1 and EGF-stimulated SAA cells. The inhibition of Shc/Grb2 interaction by actinomycin D in B104-1-1 cells also reduced tyrosine phosphorylation of MAP kinase (Erk1/Erk2), one of the major components in the Ras-MAP kinase signaling pathway. These results suggest that actinomycin D could be a non-phosphorylated natural and cellular membrane-permeable SH2 domain antagonist.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

The Protein Phosphatase Inhibitors Okadaic Acid and Calyculin A Induce Apoptosis in Human Osteoblastic Cells

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in osteoblastic cells, we examined the effects of okadaic acid (OA) and calyculin A (CA) on cultured human osteoblastic cells Saos-2 and MG63, and mouse osteoblastic MC3T3-E1 cells. After reaching confluence, these cells were exposed to varying concentrations of OA or CA. OA and CA induced cell death in all three cell lines in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were also observed in these cells by using the Hoechst 33342 stain. DNA ladder formation, a hallmark of apoptosis, was detected in Saos-2 and MG63 cells, but not in MC3T3-E1 cells by treatment of OA or CA. In the Saos-2 cells, OA- and CA-induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 10 and 2 nM,respectively, and was time-dependent from 14 to 48 h. DNA ladder formation in response to OA and CA was revealed by using conventional ethidium bromide staining of electrophoresed DNA without using autoradiography. Beyond the maximal effects at the respective concentrations, however, cell death did not indicate DNA laddering, suggesting that phosphatase activity may be required for ladder formation. Our results indicate that apoptosis in the cultured osteoblastic cells is induced by moderate inhibition of PP-1 or PP-2A based on the known selectivity of okadaic acid and of calyculin A.     

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Infection with Leishmania infantum Inhibits actinomycin D-induced apoptosis of human monocytic cell line U-937

Modulation of host cell apoptosis has been observed in many bacterial, protozoal, and viral infections. The aim of this work was to investigate the effect of viscerotropic Leishmania (L.) infantum infection on actinomycin D-induced apoptosis of the human monocytic cell line U-937. Cells were infected with L. infantum promastigotes or treated with the surface molecule lipophosphoglycan (LPG) or with parasite-free supernatant of Leishmania culture medium and submitted to action of actinomycin D as the apoptosis-inducing agent. Actinomycin D-induced apoptosis in U-937 cells was inhibited in the presence of both viable L. infantum promastigotes and soluble factors contained in Leishmania culture medium or purified LPG. Leishmania infantum affected the survival of U-937 cells via a mechanism involving inhibition of caspase-3 activation. Furthermore, protein kinase C delta (PKC delta) cleavage was increased in actinomycin D-treated U-937 cells and was inhibited by the addition of LPG. Thus, inhibition of the PKC-mediated pathways by LPG can be implicated in the enhanced survival of the parasites. These results support the claim that promastigotes of L. infantum, as well as its surface molecule, LPG, which is in part released in the culture medium, inhibit macrophage apoptosis, thus allowing intracellular parasite survival and replication.     

Cytochemical distinction of various nucleolar components in insect cells.

The fine structure of the insect Sf9 cell nucleolus has been investigated by means of different cytochemical and immunocytochemical techniques at the electron microscope level. Apart from a few perinucleolar condensed chromatin clumps, the insect cell nucleolus comprises two compartments. The first of these consists of a roundish compact zone formed of fibrillar material. The other is composed of fibrillar and granular structures organized into a network separated by interstitial spaces. But, unlike mammalian cell nucleoli, any fibrillar center has been observed in the Sf9 cell nucleolus, even after actinomycin D treatment. We also show that the compact fibrillar zone of Sf9 cell nucleoli contains silver-stainable material and DNA. In actinomycin D-treated cells, a preferential contact of this compact fibrillar zone with condensed chromatin has been visualized. Finally, silver-stainable material has been found to persist throughout the whole mitosis. These results suggest that the compact fibrillar zone at the insect Sf9 cell nucleolus should, at least partly, correspond to the fibrillar center of mammalian cell nucleoli.     

Effects of actinomycin D on ribosomal RNA and protein synthesis as revealed by high resolution autoradiography.

Incorporation of tritiated amino acids and uridine was studied in untreated and actinomycin D treated HeLa cells by high resolution autoradiography. Results showed a non-selective inhibition of protein synthesis by actinomycin, as measured by the decrease in radioactive amino acid uptake. When cells pretreated with actinomycin D were incubated with radioactive amino acids and uridine, amino acid uptake in the nucleolus still occurred, while uridine uptake was almost completely eliminated. These findings suggest that in the absence of ribosomal RNA precursor synthesis, nucleolar protein synthesis continues to some extent, and that this protein is transported to the nucleolus.     

Nucleolin interacts with US11 protein of herpes simplex virus 1 and is involved in its trafficking

Herpes simplex virus type 1 (HSV-1) infection induces profound nucleolar modifications at the functional and organizational levels, including nucleolar invasion by several viral proteins. One of these proteins is US11, which exhibits several different functions and displays both cytoplasmic localization and clear nucleolar localization very similar to that of the major multifunctional nucleolar protein nucleolin. To determine whether US11 interacts with nucleolin, we purified US11 protein partners by coimmunoprecipitations using a tagged protein, Flag-US11. From extracts of cells expressing Flag-US11 protein, we copurified a protein of about 100 kDa that was further identified as nucleolin. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. This association was confirmed in HSV-1-infected cells. We found an increase in the nucleolar accumulation of US11 in nucleolin-depleted cells, thereby revealing that nucleolin could play a role in US11 nucleocytoplasmic trafficking through one-way directional transport out of the nucleolus. Since nucleolin is required for HSV-1 nuclear egress, the interaction of US11 with nucleolin may participate in the outcome of infection.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

In vitro binding of nucleolin to double-stranded telomeric DNA

We have purified a 100 kDa protein, resolved in a Southwestern binding screen of total nuclear proteins from Hela cells with double-stranded human telomeric probe. A polyclonal antiserum raised by this protein recognizes purified nucleolin and stains nucleoli in growing Hela cells. We demonstrate that a truncated form of human nucleolin and a purified deletion derivative of mouse nucleolin bind in vitro to duplex telomeric DNA. This study suggests a new link between telomeres and the nucleolus.     

Double-stranded RNA Mediates Selective Gene Silencing of Protein Phosphatase Type 1 Delta Isoform in HEK-293 Cells

The reversible phosphorylation of proteins mediates cellular signals in eukaryotic cells. RNA interference inhibits the expression of genes and proteins in a sequence-specific manner and provides a tool to study the functions of target molecules. The effect of RNA interference on protein phosphatase isoforms in HEK-293 cells was examined. Protein phosphatase 1 delta (PP1δ) sequence-specific double-stranded RNA (dsRNA) inhibited mRNA and protein expression of the PP18. This RNA interference did not affect the expression of α and γ1 isoforms of PP1. Transfection of antisense RNA specific for PP1δ also suppressed the expression of PP1δ. It was further demonstrated by an in vitro RNA cleavage assay that extracts of HEK-293 cells catalyzed the processing of dsRNA. This cell line had much stronger mRNA expression of Dicer, an RNase III-like enzyme, than did human osteoblastic MG63 cells. The present results show that RNA interference is a useful tool to distinguish between PP1 isoforms.     

REPOPULATION OF POSTMITOTIC NUCLEOLI BY PREFORMED RNA : II. Ultrastructure

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.     

Physiology of rat-liver polysomes: Protein synthesis by stable polysomes

Certain qualitative aspects of protein synthesis in the livers of starved, starved-re-fed and actinomycin D-treated rats have been examined by polyacrylamide-gel electrophoresis. Animals were exposed to a mixture of (14)C-labelled acids for 18-20min. and killed, and an ultrasonic extract of newly formed protein in microsomal vesicles was prepared and examined by gel electrophoresis. In normal and starved-re-fed animals, 27% of the newly synthesized protein was albumin. During starvation, when RNA synthesis was decreased, the percentage of newly formed protein as albumin rose. After actinomycin D treatment of starved-re-fed rats, when only stable messenger RNA persisted in the cytoplasm, albumin synthesis increased to 63% of the total. This finding suggested that albumin was the primary protein synthesized on stable messenger RNA.