1,25-Dihydroxyvitamin D3 suppresses exportin expression in human promyelocytic leukemia HL-60 cells

Abstract

The active form of vitamin D 3 , 1α,25-dihydroxyvitamin D 3 (DVD), is a potent inducer of cell differentiation and inhibits proliferation of cells such as human promyelocytic leukemia HL-60 cells.In the present study, we examined the effects of DVD on the expression of exportin-1 and exportin-t, which play essential roles in the transport of mRNA and tRNA, respectively. The results of reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction indicated that DVD down-regulated the gene expression of these exportins. Western blotting revealed the decreased production of these proteins in DVD-treated cells. Thus, the present findings of decreased expression of exportin-1 and exportin-t induced by DVD can be correlated to inhibition of the proliferation of HL-60 cells.The physiologically active vitamin D 3 metabolite, 1α,25-dihydroxyvitamin D 3 (DVD), not only contributes to calcium homeostasis but also regulates cell proliferation and differentiation (4,10,13,16). It is a potent inducer of the differentiation of human promyelocytic leukemia HL-60 cells into the monocyte/macrophage lineage (4, 10, 16). In our previous study, we demonstrated that DVD suppressed the gene expression of eukaryotic translation initiation factor 2 (eIF-2) in these cells (15). Since eIF-2 is one of the key initiation factors controlling translation for the synthesis of new proteins, the down-regulated expression could be correlated to growth inhibition of these cells by DVD.It is known that nuclear transport factors play important roles in cellular proliferation and differentiation (6). These shuttling factors include importins and exportins. Importins play pivotal roles in transport into the nucleus of specific substrates such as NF-kappaB, ribosomal protein L12, and the signal recognition particle protein (3,5,6,12). On the other hand, nuclear export of specific macromolecules is mediated by exportins. RNAs are synthesized in the nucleus and after several steps in the maturation process are exported to the cytoplasm. Exportin-1 (XPO1), also called CRM1, is essential for the transport of mRNA (14) and exportin-t (XPOt) is a specific mediator of the export of tRNA (8).Previously it was shown that 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of HL-60 cells resulted in the down-regulation of importin α1 and α4 expression (6). Since DVD is similar to the phorbol ester in that it can lead these cells toward a macrophage phenotype (10), DVD is expected to down-regulate the expression of importins. However, there has been little information on the effects of these drugs on the expression of exportins, which might be correlated to their growth inhibitory activity. Therefore, in the present study, we examined the effects of DVD on expression of XPO1 (14, 17) and XPOt (8) which play essential roles in the transport

DNA microarray analysis of changes in gene expression induced by 1,25-dihydroxyvitamin D3 in human promyelocytic leukemia HL-60 cells

“…The results of the present microarray analysis (Table 4) confirmed our previous findings that the expression of exportin-tRNA and exportin 1/CRM1 was down-regulated at both the gene and protein levels in DVD-treated HL-60 cells (23). In addition, the present study examined the gene expression of exportin 5,6, and 7 by RT-PCR and quantitative real-time PCR to validate the results of the microarray analysis.…”

“…Previously, we found that exportin-tRNA and exportin 1 were down-regulated at both the gene and protein expression levels (23). The present microar- The data from three experimetns were processed as described in Fig.…”

Differentiation-associated alteration in sensitivity to apoptosis induced by (-)-epigallocatechin-3-O-gallate in HL-60 cells

“…The lysates were heated at 100°C for 3 min and sonicated for 30 s. After SDS-polyacrylamide gel electrophoresis, blotted proteins on nitrocellulose membrane were probed with goat anti-67LR antibody (F18) (24) which recognizes the 37 kDa precursor protein of 67LR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-β-actin IgG (Affinity BioReagents, CO, USA), horseradish peroxidase-conjugated rabbit anti-goat IgG (Santa Cruz Biotechnology), and swine horseradish peroxidase-conjugated anti-rabbit IgG (DAKO A/S, Denmark). Chemiluminescence was detected using ECL plus Western Blotting Detection System (GE Healthcare Biosciences, Tokyo) according to the directions provided by the manufacturer and visualized using as described previously (20).…”

“…HL-60 cells were cultured in 10% fetal bovine serum in RPMI 1640 medium containing 50 U/mL penicillin, 50 μg/mL streptomycin, and 2.5 μg/mL amphotericin B at 37°C under a 5% CO 2 atmosphere. HL-60 cells were treated with 1 μM ATRA (Wako Pure Chemical Industries, Ltd. Osaka, Japan) for 3 days, 0.5 nM TPA (Wako Pure Chemical Industries) for 1 day, or 100 nM DVD for 3 days (20)(21)(22) to the treatment with EGCG (Funakoshi Co. Ltd., Tokyo). The differentiated cells were then incubated with EGCG at various concentrations in the cell culture medium at 37°C for 24 h in the presence of each differentiation inducer, and compared with the results from undifferentiated cells.…”

Perspectives of Differentiation Therapies of Acute Myeloid Leukemia: The Search for the Molecular Basis of Patients’ Variable Responses to 1,25-Dihydroxyvitamin D and Vitamin D Analogs

“…This was in common with U937 cells which after initial proliferative burst, later showed elevated levels of cyclin-dependent kinase inhibitors, p21 Waf1/Cip1 and p27 Kip1 (105). Other mechanisms include a decrease in the level of gene expression of exportin and importins (which are involved in regulating the traffic of substrates for protein production) (106) and elf2 (a translation initiation factor) (107) in HL60 cells suggesting decreased protein synthesis and suppression of translation.…”