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

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Hsing L. Wu, Chen Y. Hsu, Wen H. Liu, Benjamin Y.M. Yung. International Journal of Cancer. Volume 81, Issue 6, pages 923–929, 11 June 1999

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.

Apoptosis, the physiological mode of cell death, is representative of an endogenous mechanism which can be selectively triggered by cells in response to largely unknown stimuli. During apoptosis, a series of well-defined degenerative changes occur within the cell which ultimately result in degradation of the nuclear DNA into oligonucleosome chains (Wyllie, 1980) and fragmentation of the cell into neat “bite-size” pieces for efficient disposal by neighboring cells or marauding macrophages (Savill et al.,1989). According to Wyllie et al. (1980), at the morphological level, necrosis is associated with cell swelling, rupture of membranes and dissolution of an organized structure while apoptosis is characterized by cell shrinkage and chromatin condensation. Apoptosis has been described as programmed, as opposed to accidental, cell death (Wyllie, 1980).

Identification of the genes and their products that are involved in responses to growth stimuli is essential for understanding normal cell growth and death. During the past decade, numerous regulatory factors which control the balance between a cycling and quiescent state have been identified. These factors include proto-oncogenes and negative and positive regulatory growth factors. Other proliferation-associated molecules are being studied to determine their potential role in cell-growth regulation (Valdez et al.,1994; Chou and Yung, 1995). Nucleophosmin/B23, also called protein B23, NO38 or numatrin (Schmidt-Zachmann et al.,1987; Yung et al.,1985), is a major nucleolar phosphoprotein that displays a number of activities. These include a potential role as a positive regulator of cell proliferation. Nucleophosmin/B23 is significantly more abundant in tumor and proliferating cells than in normal resting cells (Chan et al.,1989). Nucleophosmin/B23 mRNA is 50- and 5-fold higher in Novikoff hepatoma and hypertrophic rat liver, respectively, compared to normal rat liver (Chan et al.,1989). Nucleophosmin/B23 is localized in granular regions of the nucleolus (Peculis and Gall, 1992), associated with pre-ribosomal particles (Yung et al.,1985), and forms hexamers (Yung and Chan, 1987) which may be essential for the assembly of ribosomes. Our previous results have shown that nucleophosmin/B23 translocates from nucleoli to nucleoplasm during the stationary phase of growth (Yung et al.,1990b) or during treatment with certain anti-tumor drugs, particularly DNA intercalators (Yung et al.,1985, 1990a; Wu et al.,1995). Valdez et al. (1994) have demonstrated that nucleophosmin/B23 binds to amino acid sequence 24–56 of protein p120, a cell cycle–related protein. In light of many potential roles, it is conceivable that nucleophosmin/B23 is involved in the regulation of cell proliferation.

Normal diploid somatic cells lose approximately 50 to 200 bp of telomeric DNA/mean population doubling due to the inability of DNA polymerase to completely replicate the end parts of linear chromosomes (Harley et al.,1990). In contrast, germ cell lines and most immortal cell lines maintain their telomeres at a constant length, irrespective of the number of divisions they undergo, due to the activity of telomerase, a ribonucleoprotein that synthesizes and adds telomeric repeats onto chromosomal termini. Since telomerase activity is absent in most normal human somatic cells and tissues that have limited replicative spans (Kim et al.,1994) but is activated during cellular immortalization (Counter et al.,1992), a telomere hypothesis of cell aging and immortalization has been proposed, in which the attrition of telomeric sequences ultimately interferes with the expression of genes required for continued cell growth (Allsopp et al.,1992). Strong correlative support for this hypothesis was also provided by the observation of telomerase activity in 98% of established immortal cell lines and 90% of tumors tested and the absence of telomerase activity in over 50 normal human somatic tissues (Kim et al.,1994). Telomerase is down-regulated during terminal differentiation and in quiescent cells (Sharma et al.,1995; Holt et al.,1996). Further, there is evidence that telomere length controls the life span of cells and, hence, cellular senescence (Harley et al.,1990; Allsopp et al.,1992; Bodnar et al.,1998).

Berberine, an alkaloid, originated from Chinese herbal medicine, where it is used as an antibiotic; its anti-bacterial activity has been demonstrated against many species (Ghosh et al.1985). The drug was subsequently screened for anti-cancer activity following evidence of anti-neoplastic properties (Zhang, 1990). We have shown that berberine induces apoptosis in human leukemia HL-60 cells (Kuo et al.1995) and have observed differential dose-dependent effects on the cell cycle and induction of apoptosis in BALB/c3T3 cells (Yang et al.,1996). To better understand the induction of apoptosis by berberine, attempts have been made to determine the effects of berberine on nucleophosmin/B23 mRNA expression and telomerase activities. Our results show that nucleophosmin/B23 mRNA and telomerase activity are down-regulated during berberine-induced apoptosis of HL-60 cells. Whether there is a link between down-regulation of nucleophosmin/B23 and berberine-induced apoptosis thus becomes an important question. For this reason, we have investigated the influence of nucleophosmin/B23 over-expression on berberine-induced apoptosis. We report here that over-expression of nucleophosmin/B23 decreases the responsiveness of HL-60 cells to berberine-induced apoptosis.

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