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 Bandoh1, T Tsukada1, K Maruyama1, N Ohkura2 and K Yamaguchi1
Growth Factor Division, National Cancer Center Research Institute, Tokyo; and 2Terumo R&D Center, Kanagawa, Japan
1
NOR-1, NGFI-B and Nurr1 are closely related transcription factors that constitute a distinct subfamily within the nuclear
receptor superfamily. Genes for these proteins are immediate–
early genes, and are inducible in diverse cell types by various
stimuli. In the present study, we investigated the effect of
mechanical agitation on the gene expression of these transcription factors in cultured suspension cells by the quantitative reverse transcription-polymerase chain reaction. We found
that mechanical agitation transiently induced NOR-1, NGFI-B
and Nurr1 mRNAs in several leukemic cell lines in a dosedependent manner. This induction was most pronounced in the
HL-60 promyelocytic leukemia cell line, but also occurred to a
lesser extent in other cell lines including KG-1, THP-1 and U937
cells. The induction was attenuated by serum or albumin, which
are shear stress protectants for suspension culture cells.
These reagents did not suppress forskolin-induced NOR-1
gene expression. These findings suggest the involvement of
fluid shear stress in agitation-induced immediate–early gene
expression. Since even moderate agitation could cause the
induction, investigators should be cautious when evaluating
the expression of immediate–early genes in some leukemic
cell lines.
Keywords: agitation; immediate–early gene; HL-60; NOR-1; NGFIB; Nurr1
inducible in vivo under various conditions including organ
regeneration,6,11 seizure22 and inflammation.23
Several leukemic cell lines serve as good models of hematopoietic cell differentiation. These include HL-60, KG-1, U937,
K562 and THP-1 cells. During experiments in which these
cells were treated with various reagents, we found that mechanical agitation induced NOR-1 gene expression in some of
them. In the present study, we investigate the effect of agitation on the gene expression of NOR-1, NGFI-B and Nurr1
in various cell lines by quantitative reverse transcriptionpolymerase chain reaction (RT-PCR).20,21 The agitationinduced gene expression was dose dependent and most pronounced in HL-60 cells, one of the most widely used cell lines
for studying differentiation and immediate–early gene
expression. The effect was attenuated by adding serum or
albumin to the culture medium, suggesting that the induction
may be caused by fluid shear stress24–26 evoked by the mechanical agitation. Although it remains to be clarified whether
mechanical agitation results in significant biological consequences, investigators should be cautious when evaluating the
expression of immediate–early genes in certain cell lines.
Introduction
Materials and methods
NOR-1 is a putative transcription factor originally identified
in the fetal rat brain,1 that has close structural homology to
NGFI-B2 (also called Nur773 and TR34) and Nurr15 (also
called RNR-16 and NOT7), which constitute a distinct subfamily within the nuclear receptor superfamily.8 Since specific
ligands for these molecules have not yet been identified, they
are often called orphan nuclear receptors. NOR-1, NGFI-B
and Nurr1 have been implicated in neuroendocrine regulation,5,9 neuronal differentiation,2,10 liver regeneration6,11 and
T cell apoptosis.12,13 However, little is known about the molecular mechanisms by which these transcription factors are
involved in such diverse biological processes. NGFI-B and
Nurr1 form heterodimers with RXR, a receptor for 9-cis retinoic acid.14,15 NOR-1 fuses to the truncated EWS gene product to form an anomalous chimeric protein in human chondrosarcomas.16,17 These findings suggest that these orphan
receptors are involved in the control of cell growth and differentiation by modulating the retinoic acid signaling pathway.
We have shown that NOR-1, NGFI-B and Nurr1 genes are
immediate–early genes that are transiently induced in diverse
cell types by a variety of stimuli such as forskolin, 12-O-tetradecanoylphorbol-13 acetate and growth factors.18,19 These
immediate–early genes are ubiquitously expressed in vivo, but
predominantly in the nervous, endocrine and immune systems
under basal conditions.20,21 Expression of these genes is also
Cell cultures
Correspondence: Shuji Bandoh, Growth Factor Division, National
Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo
104, Japan
Received 29 October 1996; accepted 24 January 1997
HL-60 (promyelocytic leukemia), THP-1 (monocytic leukemia), U937 (histiocytic lymphoma), K562 (chronic myelogenous leukemia), Raji (Burkitt lymphoma), KG-1 (acute
myelogenous leukemia) and KATO III cells (gastric carcinoma)
were obtained from Human Science Research Resources
Bank, Tokyo, Japan. All cell lines were cultured in RPMI-1640
medium containing 10% fetal bovine serum (FBS) (Mitsubishi
Kasei, Tokyo, Japan) and maintained at 37°C in 5% CO2. For
agitation studies, 5 × 105 (HL-60, THP-1, U937, K562, Raji
and KG-1) or 3 × 105 (KATO III) cells were plated in 10 ml of
growth medium in 75 cm2 tissue culture flasks (Iwaki, Tokyo,
Japan) 1 day before study.
Agitation of cells and total RNA isolation
Cells were agitated by manually inclining the culture flask.
One cycle of agitation consisted of continuously moving the
flask from an upright, to a horizontal position and back again.
After defined repetitions of this procedure (1 cycle/s), cells
were cultured, then total RNA was isolated using a kit
(RNeasy; Qiagen, Hilden, Germany). To study the inhibiting
effect of albumin, cells were cultured in growth medium containing 10% FBS plus 0, 0.5 or 1% (w/v) of bovine albumin
(Fraction V: Daiichi Pure Chemicals, Tokyo, Japan). Cells
were agitated or stimulated with forskolin (Calbiochem, La
Jolla, CA, USA) at a final concentration of 1 mm, cultured for
1 h, then collected for RNA isolation.
Agitation-induced NOR-1 gene expression
S Bandoh et al
1454
Quantitative analysis of NOR-1, NGFI-B and Nurr1
mRNAs by RT-PCR
The amounts of NOR-1, NGFI-B and Nurr1 mRNA were measured by means of quantitative RT-PCR using the internal standard RNA as described20,21 with the following modifications.
The cDNAs of NOR-1, NGFI-B and Nurr1 mRNA were generated in a single tube by reverse transcription of total cellular
RNA (2 mg) in the presence of 0.05, 0.5 and 0.05 attomoles
of NOR-1, NGFI-B and Nurr1 internal standard RNA, respectively, with the mixture of the specific antisense primers for
NOR-1, NGFI-B and Nurr1 as described.20,21 The cDNA of
each mRNA was amplified separately by PCR with specific
primers as described,20,21 of which the sense primer was labeled with fluorescein isothiocyanate. The conditions were
1 min at 95°C, 2 min at 66°C and 3 min at 72°C (23 cycles
for NGFI-B and 28 cycles for NOR-1 and Nurr1, respectively),
with a final extension at 72°C for 10 min. The amount of the
fluorescent PCR product was measured using a capillary DNA
sequencer (ABI PRISM310; Perkin-Elmer, Foster City, CA,
USA). The amounts of NOR-1, NGFI-B and Nurr1 mRNA were
determined by multiplying the concentration of the internal
standard RNA by the ratio of the fluorescence of the authentic
NOR-1, NGFI-B and Nurr1 PCR products and comparing it to
that derived from the corresponding internal standard RNA.
The data were compared by the two-way analysis of variance
followed by a Student’s t-test.
Results
Evaluation of the quantitative RT-PCR for NOR-1,
NGFI-B and Nurr1 mRNAs
We measured NOR-1, NGFI-B and Nurr1 mRNAs by a modification of the described quantitative RT-PCR,20,21 which used
in vitro-synthesized RNA as internal standards. The modification included the synthesis of cDNA in a single tube with
mixed specific antisense primers and the use of fluoresceinlabeled PCR primers.
We examined the linearity and sensitivity of the modified
quantitative RT-PCR assay. In the presence of 0.5 attomoles
of the NGFI-B and 0.05 attomoles of the NOR-1 and Nurr1
internal standard RNA, the molar ratio of the PCR product
derived from the authentic mRNA to that from the internal
standard was proportional to the amount of the total cellular
RNA over the ratio range of 0.13–17.9 for NOR-1 (Figure 1a),
0.06–1.3 for NGFI-B (Figure 1b) and 0.68–47.3 for Nurr1
(Figure 1c), which corresponded to the range of 0.0065–0.895
(0.13 × 0.05–17.9 × 0.05) attomoles of NOR-1 mRNA, 0.03–
0.65 (0.06 × 0.5–1.3 × 0.5) attomoles of NGFI-B mRNA and
0.034–2.365 (0.68 × 0.05–47.3 × 0.05) attomoles of Nurr1
mRNA, respectively. These findings validated this RT-PCR as
a means of quantifying NOR-1, NGFI-B and Nurr1 mRNA.
Two micrograms of total RNA from all cell lines generated
specific NOR-1, NGFI-B and Nurr1 PCR products. The
amount of the specific PCR products fell within the linear
range of the RT-PCR assay (NOR-1: 3.3–448 attomoles/mg
total RNA, NGFI-B: 15–325 attomoles/mg total RNA and
Nurr1: 17–1183 attomoles/mg total RNA). Neither the blank
samples prepared by the RT-PCR in the absence of cellular
RNA nor those prepared in the absence of reverse transcriptase generated specific PCR products (data not shown).
Time course and dose–response of NOR-1, NGFI-B
and Nurr1 mRNA induction by mechanical agitation
Cells were agitated manually in polystyrene culture flasks.
Mechanical agitation rapidly increased the amount of mRNAs
of NOR-1, NGFI-B and Nurr1 within 30 min in HL-60 cells
(Figure 2). The mRNA levels reached a maximum at 1 h after
agitation, then decreased rapidly. NOR-1 mRNA was similarly
induced in cells agitated in polypropyrene culture tubes (data
not shown).
We next examined the dose effect of mechanical agitation
on NOR-1, NGFI-B and Nurr1 gene expression in HL-60 cells
1 h after agitation (Figure 3). The increase in the dose of mechanical agitation up to 10 cycles (see Materials and methods)
caused an increase in the mRNA levels of all these genes in a
dose-dependent manner. The induction was maximal in cells
agitated for 10 cycles. The mean maximum increase over the
non-agitated control was 4.5-fold (NOR-1), 2.7-fold (NGFI-B)
and 3.6-fold (Nurr1), respectively. A further increase (20 and
40 cycles) in the agitation dose resulted in the submaximal
induction of NOR-1 and Nurr1 genes (P , 0.05 vs cells agitated for 10 cycles, n = 4).
NOR-1 mRNA induction in various cell lines by
mechanical agitation
We investigated NOR-1 mRNA induction in various leukemic
and non-leukemic cell lines 1 h after 10 cycles of agitation
(Figure 4). The induction was most pronounced in HL-60
cells. KG-1, U937 and THP-1 cells showed less pronounced,
but significant increase in NOR-1 mRNA levels (1.9-, 1.6- and
1.4-fold, respectively, P , 0.05, n = 4). On the other hand,
K562, Raji and KATO III cells did not show significant NOR1 induction. Induction of NGFI-B and Nurr1 mRNA also
showed similar cell-specific profiles with the highest induction
being in HL-60 cells (data not shown).
Inhibition of agitation-induced NOR-1 expression by
bovine albumin
To understand the mechanism of the agitation-induced
expression of immediate–early genes, we performed several
experiments. Adding conditioned medium from the agitated,
to non-agitated HL-60 cells did not induce NOR-1 mRNA
(data not shown), excluding the involvement of chemical
mediators released from the agitated cells. We found that HL60 cells cultured in medium containing 20% FBS showed an
attenuated induction of NOR-1 mRNA after mechanical agitation (data not shown). We examined whether bovine albumin exhibited similar effects. Bovine albumin in the culture
medium significantly inhibited the NOR-1 mRNA induction
(P , 0.05, n = 3) in a dose-dependent manner (Figure 5a). In
contrast, forskolin-induced NOR-1 gene expression was not
suppressed by albumin (Figure 5b). These findings indicate
that the inhibitory effect of albumin on the agitation-induced
NOR-1 gene expression was not caused by the general suppression of cell signalling and transcription.
Discussion
NOR-1, NGFI-B and Nurr1 are closely related putative transcription factors that constitute a distinct subfamily within the
Agitation-induced NOR-1 gene expression
S Bandoh et al
1455
Figure 1
Relationship between the amount of cellular RNA and the ratio of the fluorescence of the authentic PCR products to that derived
from the internal standard RNA. Serially diluted total cellular RNA isolated from culture cells was reverse-transcribed in a single tube in the
presence of 0.05, 0.5 and 0.05 attomoles of NOR-1, NGFI-B and Nurr1 internal standard RNA, respectively, and a mixture of the specific
antisense primers for NOR-1, NGFI-B and Nurr1. One-twentieth of the cDNA mixture was amplified by PCR with specific fluorescein-labeled
primers. The ratio of the fluorescence of the PCR products derived from the authentic mRNA to that from the internal standard RNA was plotted
against the amount of the cellular RNA. (a) NOR-1; (b) NGFI-B; (c) Nurr1. Each point represents the mean of two samples.
Figure 2
Time course of NOR-1 (a), NGFI-B (b) and Nurr1 (c) mRNA levels after mechanical agitation. HL-60 cells were agitated manually
by 10 cycles (see Materials and methods) at time 0. The open and closed circles indicate non-agitated control and agitated cells, respectively.
Each point represents the mean of two samples.
Figure 3
Dose–response relationships of mechanical agitation-induced gene expression. HL-60 cells were not agitated (Control), or agitated
for 3, 5, 10, 20 or 40 cycles, cultured for 1 h, then collected for RNA isolation. (a) NOR-1; (b) NGFI-B; (c) Nurr1. Values are means ± s.e.
(n = 4). *P , 0.05 vs non-agitated control. †P , 0.05 vs cells agitated for 10 cycles.
Agitation-induced NOR-1 gene expression
S Bandoh et al
1456
Figure 4
NOR-1 gene expression induced by mechanical agitation in various cell lines. Cells were not agitated (Control), or agitated for 10
cycles, cultured for 1 h, and then collected for RNA isolation. (a) HL-60; (b) KG-1; (c) U937; (d) THP-1; (e) K562; (f) Raji and (g) KATOIII cells.
Values are means ± s.e. (n = 4). *P , 0.05 vs control.
Figure 5
Effect of albumin on agitation-induced and forskolin-induced NOR-1 induction. HL-60 cells were cultured in medium containing
10% FBS in the absence (0) or presence of 0.5 or 1.0% (w/v) bovine albumin. (a) Agitation. Cells were not agitated (Control), or agitated for
10 cycles (Agitation). (b) Forskolin stimulation. Forskolin at a final concentration of 1 mm (Forskolin) or vehicle (Control) was gently added to
the cell culture. Cells were collected for RNA isolation 1 h after treatment. Values are means ± s.e. (n = 3). *P , 0.05 vs 0% albumin.
steroid hormone receptor superfamily, and also belong to the
category of the immediate–early genes.27 A variety of stimulants, including cAMP, phorbol ester, growth factors and a
membrane depolarizer, induce the expression of these genes
through several signalling pathways. In the present study, we
demonstrated that NOR-1, NGFI-B and Nurr1 were induced
by mechanical agitation in a dose-dependent manner. The
rapid and transient induction of these genes was typical of
immediate–early genes. Dose–response analysis revealed
attenuated responses to excessive stimuli, as is often seen in
other biological systems.
Although the exact mechanism of the agitation-induced
expression of immediate–early genes remains elusive, the
findings that serum and albumin suppressed this induction
Agitation-induced NOR-1 gene expression
S Bandoh et al
suggest the involvement of fluid shear stress,24–26 because
these reagents act as shear stress protectants in suspension culture cells.28,29 Fluid shear stress, the tractive force acting on
the cell surface as a result of fluid flow, induces a number of
morphological and functional changes in the endothelium.24–26
These include transcriptional activation of immediate–early
genes, c-fos and c-myc.30 Shear stress-induced biological
responses have also been identified in suspension culture
cells.31 It is notable that the promoter regions of NOR-1
(Ohkura et al, manuscript in preparation) contains the 6-bp
sequence, 5′-GAGACC-3′, which is defined as a shear stress
response element in the platelet-derived growth factor-B
gene.32
A variety of leukemic cell lines have been established and
used to study leukemic cell growth and differentiation. Among
them, HL-60 cells have been used extensively for the investigation of differentiation and immediate–early gene expression.
The present study demonstrated that this cell line is very sensitive to mechanical agitation and that even moderate agitation
caused a significant induction of immediate–early genes.
Although the mechanism and biological consequences of the
agitation-induced gene expression remain to be elucidated,
these findings emphasize the need for caution when evaluating immediate–early gene expression in some leukemic cell
lines because inappropriate handling of cells may inadvertently induce these genes.
Acknowledgements
This study was supported in part by a research grant from the
Princess Takamatsu Cancer Research Fund; by a grant-in-aid
from the Ministry of Health and Welfare for the 2nd Term
Comprehensive 10-Year Strategy for Cancer Control; by a
grant-in-aid for Cancer Research (6-29) from the Ministry of
Health and Welfare; by the Special Coordination Funds from
the Science and Technology Agency for Promoting Science
and Technology and by a grant-in-aid from Tokyo Biochemical Research Foundation, Japan. S Bandoh and K Maruyama
are awardees of a Research Resident Fellowship from the
Foundation for the Promotion of Cancer Research, Japan.
References
1 Ohkura N, Hijikuro M, Yamamoto A, Miki K. Molecular cloning
of a novel thyroid/steroid receptor superfamily gene from cultured
rat neuronal cells. Biochem Biophys Res Commun 1994; 205:
1959–1965.
2 Milbrandt J. Nerve growth factor induces a gene homologous to
the glucocorticoid receptor gene. Neuron 1988; 1: 183–188.
3 Hazel TG, Nathans D, Lau LF. A gene inducible by serum growth
factors encodes a member of the steroid and thyroid hormone
receptor superfamily. Proc Natl Acad Sci USA 1988; 85: 8444–
8448.
4 Chang C, Kokontis J, Liao S, Chang Y. Isolation and characterization of human TR3 receptor: a member of steroid receptor superfamily. J Steroid Biochem 1989; 34; 391–395.
5 Law SW, Conneely OM, DeMayo FJ, O’Malley BW. Identification
of a new brain-specific transcription factor, NURR1. Mol Endocrinol 1992; 6: 2129–2135.
6 Scearce LM, Laz TM, Hazel TG, Lau LF, Taub R. RNR-1, a nuclear
receptor in the NGFI-B/Nur77 family that is rapidly induced in
regenerating liver. J Biol Chem 1993; 268: 8855–8861.
7 Mages HW, Rilke O, Bravo R, Senger G, Kroczek RA. NOT, a
human immediate–early response gene closely related to the
steroid/thyroid hormone receptor NAK1/TR3. Mol Endocrinol
1994; 8: 1583–1591.
8 Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G,
Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans
RM. The nuclear receptor superfamily: the second decade. Cell
1995; 83: 835–839.
9 Davis IJ, Lau LF. Endocrine and neurogenic regulation of the
orphan nuclear receptors Nur77 and Nurr1 in the adrenal glands.
Mol Cell Biol 1994; 14: 3469–3483.
10 Ohkura N, Hijikuro M, Miki K. Antisense oligonucleotide to NOR1, a novel orphan nuclear receptor, induces migration and neurite
extention of cultured forebrain cells. Mol Brain Res 1996; 35:
309–313.
11 Petropoulos I, Part D, Ochoa A, Zakin MM, Lamas E, NOR-2
(neuron-derived orphan receptor), a brain zinc finger protein, is
highly induced during liver regeneration. FEBS Lett 1995; 372:
273–278.
12 Woronicz JD, Calnan B, Ngo V, Winoto A. Requirement for the
orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas.
Nature 1994; 367: 277–281.
13 Liu ZG, Smith SW, McLaughlin KA, Schwartz LM, Osborne BA.
Apoptotic signals delivered through the T-cell receptor of a T-cell
hybrid require the immediate–early gene nur77. Nature 1994;
367: 281–284.
14 Perlmann T, Jansson L. A novel pathway for vitamin A signaling
mediated by RXR heterodimerization with NGFI-B and NURR1.
Genes Dev 1995; 9: 769–782.
15 Forman BM, Umesono K, Chen J, Evans RM. Unique response
pathways are established by allosteric interactions among nuclear
hormone receptors. Cell 1995; 81: 541–550.
16 Labelle Y, Zucman J, Stenman G, Kindblom LG, Knight J, TurcCarel C, Dockhorn-Dworniczak B, Mandahl N, Desmaze C, Peter
M, Aurias A, Delattre O, Thomas G. Oncogenic conversion of a
novel orphan nuclear receptor by chromosome translocation.
Hum Mol Genet 1995; 4: 2219–2226.
17 Clark J, Benjamin H, Gill S, Sidhar S, Goodwin G, Crew J, Gusterson BA, Shipley J, Cooper CS. Fusion of the EWS gene to CHN,
a member of the steroid/thyroid receptor gene superfamily, in a
human myxoid chondrosarcoma. Oncogene 1996; 12: 229–235.
18 Maruyama K, Tsukada T, Bandoh S, Sasaki K, Ohkura N, Yamaguchi K. Expression of NOR-1 and its closely related members of
the steroid/thyroid hormone receptor superfamily in human neuroblastoma cell lines. Cancer Lett 1995; 96: 117–122.
19 Bandoh S, Tsukada T, Maruyama K, Ohkura N, Yamaguchi K.
Gene expression of NOR-1, a neuron-derived orphan receptor, is
inducible in neuronal and other cell lineages in culture. Mol Cell
Endocrinol 1995; 115: 227–230.
20 Bandoh S, Tsukada T, Maruyama K, Ohkura N, Yamaguchi K. Differential expression of NGFI-B and RNR-1 genes in various tissues
and developing brain of the rat: comparative study by quantitative
reverse transcription-polymerase chain reaction. J Neuroendocrinol 1997; 9: 3–8.
21 Maruyama K, Tsukada T, Bandoh S, Sasaki K, Ohkura N, Yamaguchi K. Expression of the putative transcription factor NOR-1 in the
nervous, the endocrine, the immune systems and the developing
brain of the rat. Neuroendocrinology 1997; 65: 2–8.
22 Watson M, Milbrandt J. The NGFI-B gene, a transcriptionally
inducible member of the steroid receptor gene superfamily: genomic structure and expression in rat brain after seizure induction.
Mol Cell Biol 1989; 9: 4213–4219.
23 Hayashi K, Ohkura N, Miki K, Osada S, Tomino Y. Early induction
of the NGFI-B/Nur77 family genes in nephritis induced by antiglomerular basement membrane antibody. Mol Cell Endocrinol
1996; 123: 205–209.
24 Franke R-P, Gräfe M, Schnittler H, Seiffge D, Mittermayer C.
Induction of human vascular endothelial stress fibres by fluid shear
stress. Nature 1984; 307: 648–649.
25 Diamond SL, Eskin SG, McIntire LV. Fluid flow stimulates tissue
plasminogen activator secretion by cultured human endothelial
cells. Science 1989; 243: 1483–1485.
26 Malek AM, Greene AL, Izumo S. Regulation of endothelin 1 gene
by fluid shear stress is transcriptionally mediated and independent
of protein kinase C and cAMP. Proc Natl Acad Sci USA 1993; 90:
5999–6003.
27 Herschman HR. Primary response genes induced by growth factors and tumor promoters. Annu Rev Biochem 1991; 60: 281–319.
28 Lee GM, Savinell JM, Palsson BO. Serum can act as a shear pro-
1457
Agitation-induced NOR-1 gene expression
S Bandoh et al
1458
tecting agent in agitated hybridoma cell cultures. Hybridoma
1989; 8: 639–645.
29 Zhang Z, Chisti Y, Moo-Young M. Effects of the hydrodynamic
environment and shear protectants on survival of erythrocytes in
suspension. J Biotechnol 1995; 43: 33–40.
30 Hsieh HJ, Li NQ, Frangos JA, Pulsatile and steady flow induces
c-fos expression in human endothelial cells. J Cell Physiol 1993;
154: 143–151.
31 Naess A, Halstensen A, Solberg CO. Enhancement of leukocyte
membrane receptor expression after mechanical agitation. Int
Arch Allergy Appl Immunol 1986; 81: 235–237.
32 Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey CF Jr,
Gimbrone MA Jr. Platelet-derived growth factor B chain promoter
contains a cis-acting fluid shear-stress-responsive element. Proc
Natl Acad Sci USA 1993; 90: 4591–4595.