Differentiation-associated alteration in gene expression of importins and exportins in human leukemia HL-60 cells

Abstract

Employing the DNA microarray technique, we previously reported the alteration in gene expression of nucleocytoplasmic transport factors, importins and exportins, induced by 1,25-dihydroxyvitamin D3 (DVD) in human leukemia HL-60 cells. Here, we used the quantitative reverse transcription-polymerase chain reaction method to confirm such previous findings, and compared them with those from the cells treated with all-trans-retinoic acid (ATRA). The results indicated that the gene expression of the transport factors examined was mostly down-regulated following differentiation induced by DVD and ATRA, but importin α5 gene expression was up-regulated in either case. The differences were found in the gene expression of importin α3 and exportin 6 between the cells after treatments with DVD and ATRA. These variations may be related to the difference between HL-60 cell lineages differentiating into monocytes/macrophages and granulocytes. The present findings provide further evidence to support the important roles of importins and exportins in cell differentiation.In eukaryotic cells, the nucleus is separated from the cytoplasm by the nuclear envelope. Macromolecules such as RNA transcripts generated in the nucleus are exported from the nucleus to the ribosomes in the cytoplasm, and proteins synthesized in the cytoplasm, such as histones, polymerases, and transcription factors, are imported into the nucleus. The importin/exportin transport system is involved in the nucleocytoplasmic transport of such cargo molecules (1-4, 6-8). In this system, proteins to be imported or exported have generally a nuclear localization signal (NLS) sequence or a nuclear export signal (NES) sequence. Importin α binds to NLS within protein cargoes and links them to importin β to enter into the nucleus. In some cases, the cargo molecule with NLS directly binds to β and is transported into the nucleus.In an analogous fashion, exportin recognizes NES in the cargo protein and the complex is exported from the nucleus by binding to the GTP-bound form of guanine nucleotide binding protein Ran. There are at least 18 importin and 6 exportin genes in humans and 15 importin and 6 exportin genes in mice. Alteration of the expression level of importins and exportins would affect the transport efficiency, and play a crucial role in development, differentiation, and transformation.It is well-known that human promyelocytic leukemia HL-60 cells can be induced to differentiate into monocytes/macrophages and granulocytes by 1,25-dihydroxyvitamin D3 (DVD) and all-trans-retinoic acid (ATRA), respectively (5). Employing the DNA microarray technique, we previously reported the alteration in gene expression of importins and exportins induced by DVD in HL-60 cells (11). In the present study, we used the quantitative real-time polymerase chain reaction (Q-PCR) method to con-

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

“…When HL-60 cells were treated with 0.1 nM TPA for 1 day or 100 nM DVD for 3 days, a number of the cells became attached to the culture flask (data not shown). We reported that DVD suppressed cell growth (21) and that DVD and ATRA caused the change in gene expression of proteins involved in nucleocytoplasmic transportation (22). Thus, the present conditions appear to be sufficient to induce differentiation in HL-60 cells.…”

“…To prevent possible contamination, samples were treated with deoxyribonuclease (RT-grade, Wako Pure Chemical Industries Ltd.) as recommended by the manufacturer. Q-PCR was performed using the Thermal Cycler Dice (TaKaRa Bio., Tokyo, Japan) as described previously (22). Primers for 67LR designed using a software OLIGO4.0-s were 5'-GC CATTGAAAACCCTGCTG-3' and 5'-GCTGCCTG GATCTGGTTAGTG-3' (GenBank accession no.…”

“…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.…”

Effects of chronic ingestion of catechin-rich green tea on hepatic gene expression of gluconeogenic enzymes in rats

“…The remaining portions of the liver samples were kept at −85°C in RNAlater (Applied Biosystems Japan Ltd., Tokyo, Japan) for Q-PCR. Q-PCR was performed using the Thermal Cycler Dice (TaKaRa Bio., Tokyo, Japan) as described previously (19). Primers were purchased from TaKaRa Bio., and are listed in Table 1.…”

Effects of catechin-rich green tea on gene expression of gluconeogenic enzymes in rat hepatoma H4IIE cells

“…To prevent possible contamination, samples were treated with deoxyribonuclease (RT-grade, Wako Pure Chemical Industries Ltd.) as recommended by the manufacturer (1). Q-PCR was performed using the Thermal Cycler Dice (TaKaRa Bio., Tokyo, Japan) as described previously (1,15). Primers were purchased from TaKaRa Bio.…”