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Prepublished online as a Blood First Edition Paper on December 19, 2002; DOI 10.1182/blood-2002-09-2730.
NEOPLASIA
From U348 Institut National de la Santé
et de la Recherche Médicale (INSERM), IFR Circulation
Lariboisière, Hôpital Lariboisière, Paris,
France; School of Biosciences, University of
Birmingham, United Kingdom; and Molecular Biochemistry and
Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milano,
Italy.
Sarco-endoplasmic reticulum calcium ATPase
(SERCA) enzymes control calcium-induced cellular activation by
accumulating calcium from the cytosol into the endoplasmic reticulum
(ER). To better understand the role of SERCA proteins and cellular
calcium homeostasis in all-trans retinoic acid
(ATRA)-induced differentiation, we investigated the effect of
pharmacologic inhibition of SERCA-dependent calcium uptake into the ER
on ATRA-induced differentiation of the HL-60 myelogenous and the NB4
promyelocytic cell lines. SERCA inhibitors
di-tert-butyl-benzohydroquinone (tBHQ), thapsigargin, and
cyclopiazonic acid significantly enhanced the induction of nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase activity and
CD11b marker expression induced by suboptimal concentrations of ATRA
(50 nM) in both cell lines. Analysis of cellular calcium homeostasis revealed that a 60% mobilization of the total
SERCA-dependent intracellular calcium pool was necessary to
obtain enhancement of ATRA-dependent differentiation by tBHQ. Moreover,
after 3 days of ATRA treatment in combination with tBHQ, NB4 cells
showed a significantly decreased calcium mobilization compared with
treatments with tBHQ or ATRA alone, suggesting that enhanced
differentiation and calcium mobilization are causally related.
Interestingly, several ATRA-resistant NB4-derived cell lines were
partially responsive to the differentiation-inducing effect of the
combination of the 2 drugs. In addition, we found that retinoic acid
receptor In vitro, the HL-60 myelogenous and the NB4
promyelocytic cell lines can be induced toward neutrophil granulocytic
differentiation exhibiting increased nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase activity, growth arrest, and induction of
CD11b expression by exposure to all-trans retinoic acid
(ATRA), dimethyl sulfoxide (DMSO), or adenosine 3':5'-cyclic
monophosphate (cAMP) analogs.1-5 The highly
pleiotropic effects of retinoic acid are mediated by 2 families of
ligand-dependent transcriptional regulators: the retinoic acid
receptors (RAR Calcium accumulation into intracellular calcium storage organelles or
calcium pools is accomplished by sarco-endoplasmic reticulum calcium
ATPase (SERCA)-type calcium pumps that transport calcium from
the cytosol into the endoplasmic reticulum (ER) using ATP as a
source of energy.16,17 In response to external signals, second messenger-induced calcium mobilization from the ER into the
cytosol is a key component of intracellular signaling networks controlling cell activation,18 leading subsequently to
calcium influx across the plasma membrane.19 The depletion
of intracellular calcium storage in the ER can be pharmacologically
achieved by the direct inhibition of SERCAs by permeable agents such as
di-tert-butyl-benzohydroquinone (tBHQ),20
thapsigargin (TG),21 or cyclopiazonic acid
(CPA).22 These agents release calcium into the cytosol,
mimicking second messenger-induced calcium mobilization, and activate
capacitative calcium influx from the extracellular
medium.23 Depending on the cell type, the direct and
specific inhibition of SERCAs can induce cell activation leading to
differentiation,24,25 growth arrest,26
apoptosis,27,28 or enhanced HIV production,29 indicating that SERCAs play a key role in the control of cell activation. By modulating the spatio-temporal characteristics of
calcium transients, SERCAs participate in the modulation of calcium-dependent cell activation and permit specific and selective activation of target enzymes or transcription
factors.30,31
We previously observed that HL-60 and NB4 cells, as well as fresh
leukemia cells isolated from APL patients, coexpress simultaneously several isotypes of SERCA (SERCA2b and SERCA3) and that upon
ATRA-induced differentiation of cells, the expression of SERCA3 is
selectively induced approximately 4-fold.32 These results
raise the question concerning the involvement of the modulation of
SERCA function in leukemia cell differentiation and suggest that a
relation may exist between calcium homeostasis and retinoic
acid-induced differentiation.
In the present work, we studied the effect of pharmacologic inhibition
of SERCA activity on ATRA-induced differentiation of HL-60 and NB4
cells and of their differentiation-resistant sublines. We quantitated
intracellular calcium mobilization and ensuing calcium influx required
for cell differentiation induced by SERCA inhibitors. We also measured
the levels of intracellular calcium concentration in response to SERCA
inhibitors in NB4 and ATRA-resistant NB4-derived cell lines. Then, we
examined the stability of RAR Cell culture and induction of differentiation
Prior to experiments, exponentially growing cells were harvested and
resuspended in complete medium at a density of 2 × 105
cells/mL. The cyclic AMP analog 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (8-CPT-cAMP) and ritodrine were purchased from Sigma (St Louis, MO). ATRA, tBHQ, TG, or CPA (Sigma) were added to
the cells from concentrated stock solutions in DMSO (Sigma). The amount
of DMSO vehicle added to the cells did not exceed 0.1%, was included
in control experiments, and did not interfere with the assays. To
prevent oxidation, tBHQ stock solutions were stored in the vapor phase
over liquid nitrogen. NB4-R1 cells were pretreated for 6 days with
10 Cell differentiation was assessed by (1) measuring NADPH oxidase
activity using nitroblue-tetrazolium (NBT; Sigma) as substrate as
described previously,32,39 (2) detecting growth arrest by trypan blue exclusion and cell counting, and (3) detecting surface CD11b antigen expression determined by flow cytometry analysis on a
fluorescence-activated cell-sorter (FACS) EPICSXL-MCL
(Beckman/Coulter, Paris, France) using fluorescein
isothiocyanate-conjugated monoclonal antibody Bear-1 directed against
CD11b (integrin Cell lysates and Western blot analysis
Measurement of intracellular calcium concentration Intracellular calcium concentration [Ca2+]i was measured as the change in Fura-2 fluorescence. After treatment with differentiation-inducing agents, cells (2 × 106/mL) were loaded with 1 µM Fura-2 acetoxymethyl ester in phenol red-free RPMI-1640 medium (Gibco/Invitrogen) at 37°C in the dark for 30 minutes, washed twice, and resuspended in a solution consisting of 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH = 7.4), 120 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 40 µM CaCl2, 10 mM NaHCO3, 10 mM glucose, and 5 mM Na2HPO4, at 106 cells/mL, and then used for the experiments. Dual excitation, alternating at 340 and 380 nm, was provided by spectrofluorophotometers (models LS-50B [Perkin-Elmer, Milano, Italy] or RF-1501 [Shimadzu, Darmstadt, Germany]) equipped with 2 excitation monochromators. To eliminate extracellular calcium, 0.5 mM EGTA (ethylene glycol tetraacetic acid) was added to each sample before the addition of SERCA inhibitors. The fluorescence signals were calibrated as described previously40 using the following equation: [Ca2+]i = Kd[(F Fmin)/(FmaxF)]b,
in which F is the measured fluorescence; Fmin and
Fmax are the values of the F at
[Ca2+]i less than 0.1 nM and more
than 1 mM, respectively; and b is the ratio of the emission intensities
(at 480 nm with the LS-50B model and 510 nm with the RF-1501) with
excitation at 380 nm when the [Ca2+]i is less
than 0.1 nM and more than 1 mM. Kd for
Fura-2 was taken as 224 nM. The temperature was fixed at 37 ± 1°C.
Triton X-100 and 50 mM EGTA were used to obtain the maximal and minimal Ca2+ levels, respectively. Maximal calcium release and
subsequent capacitative influx values were determined following
addition of tBHQ at supramaximal concentrations depending on the cell
type (12 µM for HL-60 cells and 40 µM for NB4 cells). As determined in control experiments, ATRA did not induce any appreciable
[Ca2+]i increase in acute experimental
settings in HL-60 or NB4 cells (not shown).
Experiments shown in this work were performed 3 or more times (specified in figure legends). Data are presented as mean ± standard error of the mean (SEM).
Enhancement of differentiation in the presence of ATRA and the SERCA inhibitor tBHQ in HL-60 cells To investigate the effect of Ca2+ on ATRA-induced granulocytic differentiation, we treated HL-60 cells with various concentrations of ATRA in the absence or presence of tBHQ, a drug known to specifically inhibit SERCA enzymes.20,41 In accordance with data in the literature,3 1 µM ATRA induced terminal granulocytic differentiation of HL-60 cells, as reflected by the acquisition of NADPH oxidase activity measured by NBT reduction (4.3-fold increase; Figure 1A), growth arrest (Figure 1B), and an approximately 3-fold induction of CD11b expression (Figure 1C). A concentration of 50 nM ATRA alone did not have a strong differentiation-inducing effect. When 3 µM tBHQ was added to cells in the presence of 50 nM ATRA, a marked enhancement of the differentiation markers, without affecting viability (not shown), was observed (Figure 1A-C). This treatment resulted in the induction of NADPH oxidase activity (4.4-fold), growth arrest, and a 4-fold induction of CD11b expression similar to that obtained with 1 µM ATRA alone. These data indicate that cotreatment of HL-60 cells by ATRA and 3 µM tBHQ resulted in an approximately 20-fold decrease of the maximally effective ATRA concentration (50 nM versus 1 µM). At concentrations in which ATRA is already maximally effective alone (1 µM), the cotreatment with tBHQ did not enhance the NADPH oxidase activity (Figure 1A). A concentration of 2 µM tBHQ had no effect on ATRA-induced NADPH oxidase activity (not shown), whereas at higher concentrations (more than 4 µM), tBHQ alone induced an increase of NADPH oxidase activity (Figure 1D). The enhancement of the differentiation-inducing effect of 50 nM ATRA by 3 µM tBHQ was also studied in time-course experiments. Whereas 50 nM ATRA or 3 µM tBHQ was ineffective when applied alone, their combination resulted in the induction of differentiation of the cells with a rate comparable to 1 µM ATRA alone (Figure 1E). These results strongly suggest a role of SERCA inhibition in the ATRA-induced differentiation of HL-60 cells.
Enhancement of ATRA-induced differentiation of HL-60 cells in the presence of other SERCA inhibitors To study the specificity of the SERCA-inhibitory activity on the enhancement of the ATRA-induced differentiation, 2 other structurally unrelated SERCA inhibitors, CPA and TG, were used. HL-60 cells were treated with ATRA, CPA alone, TG alone, and 50 nM ATRA together with CPA or TG. As a positive control, cells treated with 1 µM ATRA were used. NBT assays were performed after 3 days of treatment (Figure 2).
SERCA inhibitors when applied alone at low concentrations did not
change the state of differentiation of HL-60 cells. However, when the
SERCA inhibitors TG or CPA were applied in combination with 50 nM ATRA,
induction of differentiation of HL-60 cells was as effective as the
differentiation with 1 µM ATRA as a single drug, confirming the
above results and indicating that the
differentiation-enhancing effect is due to the specific
SERCA-inhibitory activity of the drugs. We also tested ritodrine, a
Analysis of the calcium homeostasis of HL-60 and NB4 cell lines treated with SERCA enzyme inhibitors SERCA inhibition leads to Ca2+ mobilization from the ER Ca2+ pools and a subsequent capacitative Ca2+ influx across the plasma membrane. To gain insight into the functional consequences of SERCA inhibition on calcium homeostasis during ATRA-induced differentiation, we investigated cytosolic Ca2+ concentration after SERCA inhibition. tBHQ induced a dose-dependent calcium release and ensuing calcium influx in HL-60 cells (the effect of 12 µM tBHQ is shown in Figure 3A). A concentration of 3 µM tBHQ, which exhibits an optimal potentializing effect on ATRA-dependent differentiation (Figure 1), induced approximately 60% of Ca2+ release of the total SERCA-dependent intracellular calcium pool (Figure 3B), and this was followed by a capacitative calcium influx corresponding to 70% of the value obtained by complete SERCA inhibition (Figure 3C).
The effect of tBHQ on the differentiation induced by 50 nM ATRA in NB4
cells was then investigated. In contrast with HL-60 cells, no
significant differentiation-enhancing effect was detectable in NB4
cells at 3 µM tBHQ (not shown). However, when measured by calcium
fluorimetry (Figure 4A), this
concentration of tBHQ mobilized approximately only 30% of the calcium
stored in SERCA-dependent intracellular pools in NB4 cells, and 60%
mobilization could be achieved by only a higher concentration (8 µM)
of tBHQ. This result may be explained by the observation that the level
of total SERCA protein expression was higher in NB4 than in HL-60 cells
(not shown), suggesting that the SERCA-dependent calcium pool is larger in NB4 cells. This is in accordance with the observation that a higher
concentration of tBHQ is needed to have a comparable level of
Ca2+ release in NB4 than in HL-60 cells. When NB4 cells
were treated with 50 nM ATRA in combination with 8 µM tBHQ, a
significant enhancement of ATRA-induced differentiation was observed
with a level comparable with that of the maximally active ATRA
concentration (1 µM; Figure 4B). Taken together, these results
suggest that the release of approximately 60% of Ca2+ from
ER Ca2+ pools is necessary and sufficient to enhance
ATRA-induced differentiation in HL-60, as well as in NB4 cells. This
corresponds to different absolute levels of SERCA inhibition,
presumably due to differences in SERCA expression or activity levels
and ER calcium pool sizes in the 2 cell lines.
Interaction between tBHQ and ATRA partially reverts ATRA resistance of the NB4-007/6 and NB4-306 cell lines On the basis of the data obtained on HL-60 and NB4 cells, indicating that the ATRA-induced differentiation can be enhanced by the presence of tBHQ, we examined, in a clinical interest, whether SERCA inhibition could contribute to rescue the resistance to ATRA of differentiation-resistant cells. We used 2 ATRA-resistant derivatives of NB4, the NB4-007/6 and the NB4-306 cell lines. In accordance with previous data,38 both cell lines were refractory to the cyto-differentiating action of 50 nM to 1 µM ATRA, and, as shown in Figure 5A, 8 µM tBHQ alone was ineffective as well. However, when cells were treated simultaneously with 50 nM ATRA and 8 µM tBHQ, a significant increase in NBT-reducing activity was observed. This result indicates that the addition of a SERCA inhibitor to ATRA partially reverted the ATRA-resistant character of the NB4-007/6 and the NB4-306 cell lines. This is an encouraging observation considering that NB4 and primary APL cells behave very similarly. This effect, however, is restricted to NB4-derived cells, as ATRA-resistant derivatives of HL-60 cells, Ast4 and HL-60RES, were refractory to treatments by ATRA in combination with tBHQ (Figure 5B). The resistance of HL-60 derivatives to the cotreatment by ATRA plus tBHQ in contrast to NB4 variants may be explained by the conditions of the Ca2+ pools, as suggested above, and/or by the different mechanisms of the resistance. Indeed, whereas in these NB4-derived cells ATRA resistance may be due to enhanced proteolytic degradation of PML-RAR ,44 ATRA
resistance of HL-60RES cells is due to an invalidating
mutation in the RAR gene.34
To gain insight into the functional consequences of SERCA inhibition on calcium homeostasis upon ATRA with or without tBHQ treatment in ATRA-resistant NB4 cell sublines, we examined the increase of Ca2+ concentration in the cytosol [Ca2+]i acutely induced by 20 µM tBHQ in NB4, NB4-007/6, and NB4-306 cell lines in the absence and in the presence of a 3-day- long pretreatment by ATRA with or without 8 µM tBHQ (Figure 5C). [Ca2+]i increase induced by 20 µM tBHQ, corresponding to Ca2+ mobilization from SERCA-dependent Ca2+ pools, was lower (212% increase compared with the basal Ca2+ concentration) in NB4 cells treated for 3 days with tBHQ compared with untreated (620% increase) or ATRA-treated NB4 cells (500%), confirming that Ca2+ pools were not filled completely as a partial quantity of SERCA was inhibited for 3 days. When NB4 cells were treated for 3 days by ATRA in combination with tBHQ, [Ca2+]i increase induced by 20 µM tBHQ was severely reduced (9%). These results suggest that a cross-talk between Ca2+ homeostasis and ATRA signaling may control the availability of Ca2+ from the SERCA-dependent Ca2+ pools. In untreated NB4-007/6 and NB4-306 cell lines, the SERCA-dependent [Ca2+]i increase by an acute treatment with 20 µM tBHQ was much lower (200% and 248%, respectively) than in untreated wild-type NB4 cells (620%), suggesting that Ca2+ accumulation by SERCA is less effective in ATRA-resistant cells (Figure 5C). In contrast with NB4 cells treated with 8 µM tBHQ, [Ca2+]i increase after 20 µM tBHQ was not significantly modified in ATRA-resistant NB4 sublines treated with 8 µM tBHQ, suggesting again a difference in the filling of SERCA-dependent Ca2+ pools between the wild-type and the ATRA-resistant NB4 cells. However, a little, but significant modification of [Ca2+]i increase could be observed in NB4-007/6 (102%) and in NB4-306 (118%) cell lines following cotreatment by ATRA plus tBHQ, confirming that the partial enhancement of the differentiation observed on NBT reduction (Figure 5A) paralleled the modifications of Ca2+ homeostasis. These results also indicate that the resistance of ATRA-resistant NB4 cell sublines may be partially reverted by SERCA inhibition. Enhancement of differentiation induction by tBHQ is ATRA-specific In addition to ATRA, the granulocytic differentiation of HL-60 cells can also be induced by other agents such as cAMP analogs5 or by high concentrations of DMSO.4 To determine whether the enhancement by SERCA inhibitors is specific to ATRA-induced differentiation, HL-60 cells were treated with various concentrations of 8-CPT-cAMP (Figure 6A) or DMSO (Figure 6B) in the absence or presence of tBHQ. No significant enhancement of differentiation of HL-60 cells was observed when the differentiation of the cells was induced by 8-CPT-cAMP or DMSO in the presence of tBHQ. These results suggest that the potentializing effect of SERCA inhibition is specific to ATRA-induced differentiation.
Protection of RAR in HL-60
cells and on RAR and PML-RAR in ATRA-sensitive and ATRA-resistant NB4 variants. HL-60 cells were treated with ATRA in the presence or
absence of tBHQ, and RAR protein was detected by immunoblotting. In
accordance with previous data,45 RAR has been detected
as a doublet corresponding to phosphorylated and nonphosphorylated receptor proteins. Although ATRA alone induced an almost complete degradation of RAR at day one (Figure 7A, left
panel), in accordance with previous
reports on ligand-activated degradation of receptors by the
ubiquitin-proteasome system,10,11 when HL-60 cells were treated with ATRA in the presence of tBHQ, RAR persisted at a level
comparable with that observed in untreated cells, whereas tBHQ alone
had no significant effect (Figure 7A, right panel). Protection from
ATRA-induced degradation of RAR by tBHQ was also observed in NB4
cells. ATRA-sensitive wild-type NB4 cells were treated for one day with
ATRA in the presence or absence of tBHQ, and RAR as well as
PML-RAR protein were detected by immunoblotting (Figure 7B, top left
panel).10 When NB4 cells were treated with ATRA alone,
PML-RAR as well as RAR protein levels were
decreased.10,11 Treatment with tBHQ alone had no
significant effect on the stability of either RAR or PML-RAR (not
shown). However, when NB4 cells were treated with ATRA in the presence
of tBHQ, the PML-RAR protein, as well as RAR, persisted in the
cells. These results show that treatment by SERCA inhibitors in
combination with ATRA leads to the protection of RAR and PML-RAR
proteins from degradation in ATRA-sensitive cells, suggesting that
SERCA inhibition increases the stability of RAR and PML-RAR
proteins in the presence of ATRA, leading to enhanced differentiation.
To further investigate the effect of SERCA inhibitors on the protection
of PML-RAR As summarized in Table 1, the partial
differentiation by SERCA inhibition combined with ATRA treatment
observed in NB4-306 and NB4-007/6 cells may be due to the stabilization
mainly of RAR
Treatment of the HL-60 and NB4 leukemic cell lines and of fresh
APL cells with ATRA results in a 4-fold overexpression of the SERCA3
gene products, indicating that SERCA enzymes are involved in the
acquisition of the mature granulocytic phenotype.32 On the
other hand, the direct and specific inhibition of SERCA enzymes has
been shown to lead to differentiation in other experimental systems.24,25 The data presented in this report show that
a cross-talk may operate between cellular calcium homeostasis modulated by pharmacologic SERCA inhibitors and the ATRA-dependent signaling pathway during ATRA-induced differentiation, and that Ca2+
homeostasis may be involved in the regulation of the ATRA-induced degradation of the RAR SERCA enzymes are directly involved in the control of cytosolic and intra-endoplasmic reticulum calcium levels and can modulate transplasma membrane calcium influx indirectly, because of capacitative calcium influx, a mechanism controlled by the filling state of the ER. Capacitative calcium influx can also be activated by the depletion of ER calcium content by SERCA inhibitors.23 Since an increase of the cytosolic calcium concentration and calcium influx47,48 can enhance myeloid differentiation as suggested in earlier works using calcium ionophores,49,50 it was of interest to determine whether calcium pumping into the ER exerts a functional effect on the differentiation process. Our results indicate that a partial inhibition of calcium pumping into the ER significantly enhances ATRA-induced differentiation of cells that are sensitive to the effect of ATRA, suggesting that SERCA activity may be able to fine-tune the sensitivity of the cell to the differentiation-inducing activity of ATRA. An approximately 60% Ca2+ release of the total SERCA-dependent calcium pools, which leads to an approximately 70% capacitative calcium influx, was required for the potentialization of the differentiation-inducing effect of ATRA. Although potentialization of differentiation could be obtained in a relatively narrow SERCA inhibitor concentration range, cell type-dependent differences of inhibitor concentration were observed (3 and 8 µM tBHQ required to enhance differentiation of HL-60 and NB4, respectively). This is in agreement with the observation that NB4 cells are larger than HL-60 and express approximately 2 times more SERCA proteins (not shown). As shown previously,32 ATRA-induced granulocytic differentiation is accompanied by the induction of SERCA3 expression, whereas, as shown in the present study, inhibition of SERCA activity enhances differentiation. This paradox can be reconciled by hypothesizing that SERCA activity exerts a negative feedback control on the differentiation process, presumably by decreasing the stability of retinoic acid receptors, as shown in the present work. However, the calcium affinity of SERCA3 is weaker than that of SERCA2b,51 which is also expressed in myeloid cells. Therefore, induction of SERCA3 expression, and a parallel decrease of SERCA2b levels, reported previously during granulocytic differentiation32 may lead to facilitated second messenger-induced calcium release, which, in turn would increase retinoic acid receptor half-lives in intact cells, similar to that seen upon SERCA inhibition presented in the present paper. In addition, data in the literature show that the size of the intracellular calcium pool that can be mobilized by second messengers such as inositol-1,4,5-trisphosphate (IP3), as well as the magnitude of calcium transients that can be elicited in the cells by extracellular stimuli, increase during myeloid differentiation.47,52,53 Moreover, SERCA3 is specifically associated with the IP3-sensitive intracellular calcium pool.54 Induction of SERCA3 expression during granulocytic differentiation may therefore reflect the formation of an excitable intracellular calcium compartment in the cell, involved in the control of the differentiation process, as well as in the responsiveness of the mature cell to extracellular stimuli. In this work, we found that SERCA inhibition enhanced ATRA differentiation specifically through the ATRA-dependent signaling system, since the effect of other differentiation inducers such as cAMP analog or DMSO was not potentialized by calcium pump inhibition. The lack of potentialization of the differentiation-inducing effect of DMSO by SERCA inhibitors is compatible with the notion that they may share the same target (ie, calcium homeostasis). Indeed, previous observations show that DMSO induces a transient increase of the cytosolic calcium concentration of HL-60 cells55 and that DMSO enhances ATRA-dependent differentiation.3,56 These data suggest that calcium mobilization by DMSO may be involved in the differentiation-inducing activity of the molecule. In acute promyelocytic leukemia, the t(15;17) translocation
fuses the RAR In ATRA-sensitive wild-type NB4 cells, granulocytic differentiation
induced by ATRA in combination with the tyrosine kinase inhibitor
ST1571 results in a decrease in ATRA-induced degradation of RAR | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
and calcium-dependent signaling
Top
Abstract
Introduction
, and 
) and the retinoid X receptors (RXR
6 M ATRA prior to experiments (primed cells) as
described previously.
) or in the absence (
) of 3 µM
tBHQ, and cellular NADPH oxidase activity, as measured by NBT
reduction, was determined. Data presented are the mean ± SEM
(n = 3). (B-C) HL-60 cells were treated for 3 days with vehicle (lane
1), 3 µM tBHQ (lane 2), 50 nM ATRA (lane 3), 3 µM tBHQ plus 50 nM
ATRA in combination (lane 4), and 1 µM ATRA (lane 5). (B) The
inhibition of cell proliferation was determined by counting the number
of cells after 3 days of the various treatments (mean ± SEM;
n = 10). (C) Expression of CD11b was determined by FACS analysis and
reported as fluorescence intensity in arbitrary units; control Ig
), 50 nM ATRA (
); and cellular NADPH
oxidase activity was determined by NBT reduction. Mean ± SEM
(n = 3).
.01 and *P 
PML-RAR