Benjamin Yat-Ming Yung

Berberine complexes with DNA in the berberine-induced apoptosis in human leukemic HL-60 cells

Berberine, an alkaloid initially isolated from Chinese herbal medicine exhibited the ability to induce morphological changes and internucleosomal DNA fragmentation, characteristic of apoptosis in promyelocytic leukemia HL-60 cells. Cell cycle studies showed that only about 20% of the cells underwent apoptosis at the early time (6 h) of berberine (25 micrograms/ml) treatment; these appeared to be cells in S phase at the time of berberine treatment. At extended time (6-48 h), cells were cell cycle arrested, the number of cells of each phase, particularly the cells of S phase decreased and much more (> 50%) of the cells appeared with DNA content less than G1. Attempts were also made to isolate possible berberine-DNA complexes from cell cultures treated with berberine (25 micrograms/ml; 2-24 h). Shifts of absorption maxima of berberine in the direction of longer wavelengths were observed in the isolated berberine-DNA complexes. Palmatine, an analog of berberine, which was not able to induce apoptosis, also complexed with DNA in cells treated with palmatine (25 micrograms/ml; 2-24 h). Our results suggest that some important cellular processes other than the intracellular DNA-interacting action of berberine may be involved in the berberine-induced apoptosis in HL-60 cells.

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 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.

Identification and characterization of a hexameric form of nucleolar phosphoprotein B23

Under native purification conditions, an oligomeric form (Mr = 230,000) and monomeric form (37,000) of protein B23 were purified by affinity chromatography. Both forms were identified by Western blot immunoassay and ELISA. The molecular weight of the oligomeric form of protein B23 was estimated to be 230,000 with a Stokes radius and a sedimentation coefficient of 51 A and 10 S, respectively. The oligomer (230 kDa) of protein B23 was dissociated into monomers (37 kDa) by treatment with 7 M urea. Quantitation of the monomer by gel scanning densitometry indicated that the oligomeric form of protein B23 is a hexamer containing four alpha and two beta monomers (37 kDa). A trace amount of nucleic acids (amounting to less than 3% of the total mass) was detected in the affinity-purified oligomers of protein B23. Protein B23 may be a structural element which is involved in ribosome transport or assembly in the nucleus.

Berberine-induced apoptosis of human leukemia HL-60 cells is associated with down-regulation of nucleophosmin/B23 and telomerase activity

The steady‐state level of nucleophosmin/B23 mRNA decreased during berberine‐induced (25μg/ml, 24 to 96 hr) apoptosis of human leukemia HL‐60 cells. A decline in telomerase activity was also observed in HL‐60 cells treated with berberine. A stable clone of nucleophosmin/B23 over‐expressed in HL‐60 cells was selected and found to be less responsive to berberine‐induced apoptosis. About 35% to 63% of control vector–transfected cells (pCR3) exhibited morphological characteristics of apoptosis, while about 8% to 45% of nucleophosmin/B23‐over‐expressed cells (pCR3‐B23) became apoptotic after incubation with 15μg/ml berberine for 48 to 96 hr. DNA extracted from pCR3 cells contained more fragmented DNA than pCR3‐B23 cells during treatment with 15μg/ml berberine for 24 to 48 hr. Our results indicate that berberine‐induced apoptosis is associated with down‐regulation of nucleophosmin/B23 and telomerase activity. We also suggest that nucleophosmin/B23 may play an important role in the control of the cellular response to apoptosis induction. Int. J. Cancer81:923–929, 1999.

The Nucleolar Phosphoprotein B23 Interacts with Hepatitis Delta Antigens and Modulates the Hepatitis Delta Virus RNA Replication

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH2-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.

Nucleophosmin acts as a novel AP2α‐binding transcriptional corepressor during cell differentiation

Nucleophosmin (NPM) is an important nucleolar phosphoprotein with pleiotropic functions in various cellular processes. In this study, we have further examined the largely uncharacterized role of NPM in transcriptional regulation by uncovering novel NPM‐binding transcriptional factors. Among potential interactors, we found that activating protein transcription factor 2 (AP2)α forms a complex with NPM during retinoic‐acid‐induced cell differentiation. We show that this complex is recruited to the promoters of certain retinoic‐acid‐responsive genes, including NPM itself. Such binding of AP2α, and consequent recruitment of NPM, is selective and dependent on a consensus AP2α‐binding sequence. Remarkably, suppression of NPM by RNA interference alleviates the repression of gene expression mediated by retinoic acid and AP2α. Our findings further show that, on promoter binding, NPM probably exerts its repressive effect by inducing a change in local chromatin structure that also engages histone deacetylases. This study unveils a hitherto unrecognized transcriptional corepressor function of the NPM protein, and highlights a novel mechanism by which NPM regulates cell growth and differentiation.

Identification of differentially expressed genes in oral squamous cell carcinoma (OSCC) : Overexpression of NPM, CDK1 and NDRG1 and underexpression of CHES1

To identify cellular genes that could potentially serve as predictive molecular markers for human oral cancer, we employed differential display analysis to compare the gene expression profiles between oral squamous cell carcinoma (OSCC) and histopathologically normal epithelium tissues. Comparative real‐time RT‐PCR was used to confirm the gene expression in 52 OSCC patients, and a 2‐fold difference was defined as over‐ or underexpression. A total of 7 genes were identified: NPM, CDK1, NDRG1, HMGCR, EF1A, NAC and CHES1. In the cancer tissues, NPM, CDK1 and NDRG1 were significantly overexpressed (an average of 7.6‐, 17.2‐ and 12.9‐fold, respectively), and CHES1 was underexpressed (15‐fold). The frequencies of the differential expression were 40, 56, 67 and 46%, respectively in NPM, CDK1, NDRG1 and CHES1. In Western blot analysis, the protein expressions of NPM, CDK1 and NDRG1 were also increased in the cancer tissues, consistent with the mRNA expression results. To further evaluate clinicopathological associations in these genes, Pearson chi‐square analysis was employed. Levels of CDK1 and NDRG1 were associated with poorly differentiated tumors (p = 0.043 and 0.023), suggesting that these genes participate in the mechanism of tumor transformation. Expressions of CDK1 and NDRG1, and CDK1 and CHES1 were mutually statistically correlated (p = 0.001 and 0.014), indicating that these genes share a very close regulatory relationship or interact synergistically in oncogenesis. In conclusion, we identified 7 genes that are differentially expressed in OSCC, and we provide the first evidence that NPM, CDK1 and NDRG1 are overexpressed and CHES1 is underexpressed in oral cancer. These results serve as a fundamental base for employing these genes in future clinical applications.

Over-expression of nucleophosmin/B23 decreases the susceptibility of human leukemia HL-60 cells to retinoic acid-induced differentiation and apoptosis.

Stable clones of HL‐60 cells in which nucleophosmin/B23 was over‐expressed were established. Less percentages (4–20%) of nucleophosmin/B23 over‐expressed (pCR3‐B23) cells exhibited the morphological characteristic of apoptosis as compared with control vector‐transfected (pCR3) cells (6–53%) during the 10 μM RA treatment for 1–4 days. In flow cytometry analysis, a block in the G1 phase was noted in all the pCR3‐B23 and pCR3 cells after 2 days of 10 μM RA treatment and continued to be observed at all times measured up to 6 days. Smaller peaks of apoptotic cells with less than G1 DNA content were observed in pCR3‐B23 as compared with pCR3 cells after 4–6 days of 10 μM RA treatment. As measured by expressions of differentiation markers and the functional assessment of the ability to reduce nitrobluetetrazolium, our results further showed that over‐expression of nucleophosmin/B23 decreased the response of the cells to RA‐induced differentiation. Less cleavage of PARP and in vitro caspase‐3 activity were observed in PCR3‐B23 cells as compared with pCR3 cells treated with 10 μM RA for 3–4 days. IRF‐1 was induced after 6 hr of 10 μM RA treatment in the pCR3‐B23 and pCR3 cells. Significantly more nucleophosmin/B23 was co‐immunoprecipitated with IRF‐1 from pCR3‐B23 cells than from pCR3 cells during RA treatment (10 μM; 24 hr, 96 hr). The IRF‐1 transcriptional activity was found to be attenuated in pCR3‐B23 cells as compared with pCR3 cells during the treatment of cells with RA. Nucleophosmin/B23, through interacting with IRF‐1, plays an important role in the control of the susceptibility of cells to RA‐induced differentiation and apoptosis. Int. J. Cancer 88:392–400, 2000.

Involvement of nucleophosmin/B23 in the response of HeLa cells to UV irradiation

The steady‐state mRNA level of nucleophosmin/B23 in HeLa cells increased after UV irradiation. Nucleophosmin/B23 antisense transfection potentiated ultraviolet (UV)‐induced cell killing. A block in G2/M phase, larger peak of apoptotic cells and higher caspase‐3 in vitro activity were noted in nucleophosmin/B23 antisense‐transfected cells compared with vector‐transfected cells after UV treatment. Irradiated cells that received vector plasmid exhibited increased levels of [3H]thymidine incorporation due to DNA repair synthesis. In contrast, irradiated cells that received nucleophosmin/B23 antisense plasmid exhibited no such increase of [3H]thymidine incorporation, indicating inhibition of DNA repair. Cotransfection of cells with vector allowed repair of the damaged chloramphenicol acetyl transferase (CAT) reporter and rescue of CAT activity by host repair machinery. CAT activity in cells cotransfected with nucleophosmin/B23 antisense was less (<50%) than that of vector‐transfected cells, indicating reduction of host nucleotide excision repair activity. Lower protein expressions of nucleophosmin/B23 and proliferating cell nuclear antigen (PCNA) were observed in nucleophosmin/B23 antisense‐transfected cells compared with vector‐transfected cells with or without UV treatment. Cotransfection of nucleophosmin/B23 antisense‐transfected HeLa cells with PCNA construct made the cells less susceptible to UV‐induced cell killing. Our results indicate that nucleophosmin/B23 correlates with PCNA and DNA repair capacity in cellular sensitivity to UV.

In vivo interaction of nucleophosmin/B23 and protein C23 during cell cycle progression in HeLa cells

By using the cross-linking reagent, DSP, efforts were made to identify the protein(s) that interact with nucleophosmin/B23. A cross-linked protein complex at molecular weight of about 140 kDa was recognized by both nucleophosmin/B23 and protein C23 MAbs. Both C23 and nucleophosmin/B23 could be detected from the cross-linked complex immunoprecipitated by C23 MAb. The association between nucleophosmin/B23 and protein C23 while being observed at interphase and cytokinesis, was not detected in prometaphase and metaphase cells. Interactions of nucleophosmin/B23 with C23 not only could be found in cells in which nucleophosmin/B23 and C23 were both mainly localized to the nucleolus, but also in cells in which nucleophosmin/B23 and C23 had translocated from the nucleolus to the nucleoplasm during actinomycin D-induced cell growth inhibition. The purified recombinant GST-B23 being phosphorylated by prometaphase cell extracts (nocodazole-arrested cells) or cdc2 kinase could still be co-immunoprecipitated with C23. Consequently, the fact that nucleophosmin/B23 did not interact with C23 during mitosis could not be explained simply by mitotic phosphorylation of nucleophosmin/B23. Our findings suggest some possibilities for further elucidation of the actions of nucleophosmin/B23 and protein C23 in cell cycle progression and cell growth.