Human promyelocytic leukemia HL-60 cells were induced to undergo granulocytic di erentiation by treatment with retinoic acid (RA, 10 mM, 1 ± 5 days). The steady-state level of nucleophosmin/B23 mRNA decreased during the RA-induced di erentiation. There was also decrease in the level of total cellular nucleophosmin/B23 protein during the RA-induced di erentiation. Stabilization and nuclear run-on assays indicate that the decrease in nucleophosmin/B23 mRNA in RA-treated HL-60 cells was transcriptionally regulated. Unlike c-myc mRNA, there was virtually no decline of nucleophosmin/B23 mRNA during the growth arrest by serum-starvation. The decrease in nucleophosmin/B23 mRNA expression in HL-60 cells subsequent to retinoic acid treatment can thus be attributed to cellular di erentiation rather than the growth arrest induced by RA. Nucleophosmin/B23 antisense oligomer treatment signi®cantly potentiated RA-induced cellular di erentiation. Results of this study suggest that nucleophosmin/ B23 is one of the key elements in the down-regulation of nucleolar function for cellular di erentiation.

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. © 2000 Wiley‐Liss, Inc.

The steady-state level of nucleophosmin/B23 mRNA decreased during berberine-induced (25g/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 overexpressed 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 15g/ml berberine for 48 to 96 hr. DNA extracted from pCR3 cells contained more fragmented DNA than pCR3-B23 cells during treatment with 15g/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. Cancer 81:923-929, 1999.1999 Wiley-Liss, Inc.

Human myelogenous leukaemia K562 cells were induced to undergo megakaryocytic differentiation by treatment with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (20 nM, 24 -72 h). The steady-state level of nucleophosmin/B23 mRNA decreased during the TPA-induced differentiation. There was also decrease in the level of cellular nucleophosmin/B23 protein and appearance of its degraded product (25 kDa) during the TPA-induced differentiation. Furthermore, K562/B23 (wild type), K562/D1 (D280 -294) and K562/D2 (D263 -294) cells were less, while K562/D3 (D244 -294) cells were more responsive to TPA-induced differentiation as compared to K562/vector or parental K562 cells. Activation of the ERK/MAPK was observed in parental K562 cells upon TPA treatment (5 nM, 5 -30 min). As compared to K562/vector cells, less activation of ERK/MAPK was observed in K562/D2 cells, while ERK/MAPK was highly activated in K562/D3 cells upon TPA treatment. Our results indicate that nucleophosmin/B23 plays an important role in TPA-induced differentiation of K562 cells and the amino acids 244 -294 at C-terminal of nucleophosmin/B23 could be an important site for regulation of cellular response to differentiation.

No abstract