Leukemia (1997) 11, 1453–1458  1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Mechanical agitation induces gene expression of NOR-1 and its closely related orphan nuclear receptors in leukemic cell lines S Bandoh 1 , T Tsukada 1 , K Maruyama 1 , N Ohkura 2 and K Yamaguchi 1 1 Growth Factor Division, National Cancer Center Research Institute, Tokyo; and 2 Terumo R&D Center, Kanagawa, Japan NOR-1, NGFI-B and Nurr1 are closely related transcription fac- inducible in vivo under various conditions including organ tors that constitute a distinct subfamily within the nuclear regeneration, 6,11 seizure 22 and inflammation. 23 receptor superfamily. Genes for these proteins are immediate– Several leukemic cell lines serve as good models of hemato- early genes, and are inducible in diverse cell types by various poietic cell differentiation. These include HL-60, KG-1, U937, stimuli. In the present study, we investigated the effect of K562 and THP-1 cells. During experiments in which these mechanical agitation on the gene expression of these tran- scription factors in cultured suspension cells by the quantitat- cells were treated with various reagents, we found that mech- ive reverse transcription-polymerase chain reaction. We found anical agitation induced NOR-1 gene expression in some of that mechanical agitation transiently induced NOR-1, NGFI-B them. In the present study, we investigate the effect of agi- and Nurr1 mRNAs in several leukemic cell lines in a dose- tation on the gene expression of NOR-1, NGFI-B and Nurr1 dependent manner. This induction was most pronounced in the in various cell lines by quantitative reverse transcription- HL-60 promyelocytic leukemia cell line, but also occurred to a polymerase chain reaction (RT-PCR). 20,21 The agitation- lesser extent in other cell lines including KG-1, THP-1 and U937 cells. The induction was attenuated by serum or albumin, which induced gene expression was dose dependent and most pro- are shear stress protectants for suspension culture cells. nounced in HL-60 cells, one of the most widely used cell lines These reagents did not suppress forskolin-induced NOR-1 for studying differentiation and immediate–early gene gene expression. These findings suggest the involvement of expression. The effect was attenuated by adding serum or fluid shear stress in agitation-induced immediate–early gene albumin to the culture medium, suggesting that the induction expression. Since even moderate agitation could cause the may be caused by fluid shear stress 24–26 evoked by the mech- induction, investigators should be cautious when evaluating the expression of immediate–early genes in some leukemic anical agitation. Although it remains to be clarified whether cell lines. mechanical agitation results in significant biological conse- Keywords: agitation; immediate–early gene; HL-60; NOR-1; NGFI- quences, investigators should be cautious when evaluating the B; Nurr1 expression of immediate–early genes in certain cell lines. Introduction Materials and methods NOR-1 is a putative transcription factor originally identified Cell cultures in the fetal rat brain, 1 that has close structural homology to NGFI-B 2 (also called Nur77 3 and TR3 4 ) and Nurr1 5 (also HL-60 (promyelocytic leukemia), THP-1 (monocytic leu- called RNR-1 6 and NOT 7 ), which constitute a distinct subfam- kemia), U937 (histiocytic lymphoma), K562 (chronic mye- ily within the nuclear receptor superfamily. 8 Since specific logenous leukemia), Raji (Burkitt lymphoma), KG-1 (acute ligands for these molecules have not yet been identified, they myelogenous leukemia) and KATO III cells (gastric carcinoma) are often called orphan nuclear receptors. NOR-1, NGFI-B were obtained from Human Science Research Resources and Nurr1 have been implicated in neuroendocrine regu- Bank, Tokyo, Japan. All cell lines were cultured in RPMI-1640 lation, 5,9 neuronal differentiation, 2,10 liver regeneration 6,11 and medium containing 10% fetal bovine serum (FBS) (Mitsubishi T cell apoptosis. 12,13 However, little is known about the mol- Kasei, Tokyo, Japan) and maintained at 37°C in 5% CO 2 . For ecular mechanisms by which these transcription factors are agitation studies, 5 × 10 5 (HL-60, THP-1, U937, K562, Raji involved in such diverse biological processes. NGFI-B and and KG-1) or 3 × 10 5 (KATO III) cells were plated in 10 ml of Nurr1 form heterodimers with RXR, a receptor for 9-cis reti- growth medium in 75 cm 2 tissue culture flasks (Iwaki, Tokyo, noic acid. 14,15 NOR-1 fuses to the truncated EWS gene pro- Japan) 1 day before study. duct to form an anomalous chimeric protein in human chond- rosarcomas. 16,17 These findings suggest that these orphan receptors are involved in the control of cell growth and differ- Agitation of cells and total RNA isolation entiation by modulating the retinoic acid signaling pathway. We have shown that NOR-1, NGFI-B and Nurr1 genes are Cells were agitated by manually inclining the culture flask. immediate–early genes that are transiently induced in diverse One cycle of agitation consisted of continuously moving the cell types by a variety of stimuli such as forskolin, 12-O-tetra- flask from an upright, to a horizontal position and back again. decanoylphorbol-13 acetate and growth factors. 18,19 These After defined repetitions of this procedure (1 cycle/s), cells immediate–early genes are ubiquitously expressed in vivo, but were cultured, then total RNA was isolated using a kit predominantly in the nervous, endocrine and immune systems (RNeasy; Qiagen, Hilden, Germany). To study the inhibiting under basal conditions. 20,21 Expression of these genes is also effect of albumin, cells were cultured in growth medium con- taining 10% FBS plus 0, 0.5 or 1% (w/v) of bovine albumin (Fraction V: Daiichi Pure Chemicals, Tokyo, Japan). Cells Correspondence: Shuji Bandoh, Growth Factor Division, National were agitated or stimulated with forskolin (Calbiochem, La Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo Jolla, CA, USA) at a final concentration of 1 m, cultured for 104, Japan Received 29 October 1996; accepted 24 January 1997 1 h, then collected for RNA isolation.