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

Abstract

Using a DNA microarray, we analyzed about 16,600 genes for changes in expression associated with the differentiation of human promyelocytic leukemia HL-60 cells induced by 1α,25-dihydroxyvitamin D 3 (DVD). Many of the up-regulated genes could be correlated to differentiation-associated changes toward a monocyte/macrophage lineage, and many down-regulated genes could be correlated to repressed cell growth. The present study revealed the down-regulated gene expression of importins and exportins 1, 5, 7, and exportin-tRNA. Thus, the present results confirmed our previous findings of down-regulation of exportin 1 and exportin-tRNA by DVD. Gene expression of exportin 6 is suggested to be regulated differently from that of exportins 1, 5, 7, and exportin-tRNA. The down-regulation of nuclear transport factors may be deeply associated with the differentiation of HL-60 cells induced by DVD.The active form of vitamin D 3 , 1α, 25-dihydroxyvitamin D 3 (DVD), results from sequential hydroxylation of vitamin D 3 in the liver and kidney and is responsible for calcium homeostasis in the body. Vitamin D 3 metabolites have an important role in the proliferation of hematopoietic cells, lymphocytes, and keratinocytes, as well as other cell types (17). DVD is a potent inducer of cell differentiation in human leukemia HL-60 cells (6,10,18,24). The HL-60 cell line, derived from a single individual with acute promyelocytic leukemia, provides a unique in vitro system for studying the cellular and molecular events involved in the proliferation and differentiation of normal and leukemic cells of the granulocyte/monocyte/macrophage lineage (5).In spite of extensive studies (20,25), it remains unclear how DVD exhibits biological activities including induction of differentiation, growth inhibition, and apoptosis. In our previous study, we examined changes in gene expression during the DVD-induced differentiation of HL-60 cells employing a DNA microarray technique and found the gene expression of the β subunit of eukaryotic translation initiation factor 2 to be suppressed, suggesting a linkage to differentiation-associated growth inhibition of these cells (7,22). Since the microarray used covered only 425 genes, the study provided us with limited information.In the present study, we analyzed about 16,600 genes for changes in expression associated with the DVD-induced differentiation of HL-60 cells. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to examine the expression of some selected genes of our interest to evaluate the validity of cDNA microarray analysis.

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

“…Our previous cDNA microarray analysis (11) revealed the down-regulated expression of most importins and exportins in DVD-treated HL-60 cells ( Table 2). The changes in the expression of exportins have been confirmed by Q-PCR (9, 11).…”

“…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-expression of importins in HL-60 cells before and after DVD treatment for 72 h (Fig.…”

The anti-fibrotic effect of green tea with a high catechin content in the galactosamine-injured rat liver

“…XM_213440), 5'-CAATTCCTGGC GTTACCTTG-3' and 5'CAGTGAGCACTGAAG CGAAA-3' for TGF-β1 (NM_021578), 5'-ACAT CATCCCTGCATCCACT-3' and 5'-TCTGGG ATGGAATTGTGAGG-3' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (XM_573896), and 5'-ACCGTGAAAAGATGACCCA-3' and 5'-AGGAAGGAAGGCTGGAAGA-3' for β-actin (NM_031144). Amplified DNA was subjected to electrophoresis in 2% agarose, stained with SYBR Green I (Molecular Probes Ltd., Eugene, Oregon, USA), and imaged and calculated using a FluorImager (Molecular Dynamics Tokyo Ltd., Tokyo, Japan) as described previously (1,19). Expected sizes of amplified DNA were 278 bp, 143 bp, 522 bp, and 457 bp for collagen α1 (I), TGF-β1, GAPDH, and β-actin, respectively.…”

“…To prevent possible contamination, samples were treated with deoxyribonuclease (RT-grade, Wako Pure Chemical Industries Ltd.) as recommended by the manufacturer (1,19). RT-PCR was performed as described previously (1,19).…”

Deciphering the calcitriol-induced transcriptomic response in keratinocytes: presentation of novel target genes

“…6). The most important examples encompass primary, immortalized, or malignant prostate , Kovalenko et al 2010, Wang et al 2011, colon , squamous cell carcinoma (Akutsu et al 2001, Lin et al 2002, myelocytic/monocytic leukemia (Savli et al 2002, Suzuki et al 2006, Heikkinen et al 2011, ovarian , coronary artery smooth muscle (Wu-Wong et al 2006), (pre-) adipocytic (Sun et al 2008), lymphoblastoid (Ramagopalan et al 2010), or breast cancer (Swami et al 2003, Kriebitzsch et al In silico prediction of putative VDR-RXR heterodimer binding sites within the promoter regions of RTq-PCR-validated calcitriol target genes. Columns specify applied matrix concepts, relative position/orientation with respect to the relevant TSS, calculated statistics, and matching consensus motifs.…”