NEOPLASIA
Glucocorticoids promote the proliferation and antagonize the retinoic
acid–mediated growth suppression of Epstein-Barr
virus–immortalized B lymphocytes
Michele Quaia, Paola Zancai, Roberta Cariati, Silvana Rizzo, Mauro Boiocchi, and Riccardo Dolcetti
Glucocorticoids are able to release Epstein-Barr virus–immortalized (EBV-immortalized) lymphoblastoid B cell lines
(LCLs) from the persistent growth arrest
induced in these cells by retinoic acid
(RA). Moreover, physiologic concentrations of glucocorticoids efficiently antagonized LCL growth inhibition induced by
13-cis-RA; 9-cis-RA; all-trans-RA; and Ro
40-6055, an RA a receptor (RARa) selective agonist. RARa expression levels,
however, were not affected by glucocorticoids. Glucocorticoids, but not other
steroid hormones, directly promote LCL
proliferation, a phenomenon that was
mainly mediated by down-regulation of
the cyclin-dependent kinase (CDK) inhibitor p27Kip-1. Moreover, glucocorticoids
contrasted the up-regulation of p27Kip-1,
which was underlying the RA-induced
LCL growth arrest, thereby indicating that
glucocorticoids and RA signalings probably converge on p27Kip-1. Both antagonism of RA-mediated growth inhibition
and promotion of LCL proliferation were
efficiently reversed by the glucocorticoid
receptor (GR) antagonist RU486, indicating that all of these effects were mediated
by GR. Of note, RU486 also proved to be
effective in vivo and, in mice, was able to
significantly inhibit the growth of untreated LCLs as well as LCLs growth-
arrested by RA in vitro. These findings
provide a rational background to further
evaluate the possible role of glucocorticoids in the pathogenesis of EBV-related
lymphoproliferations of immunosuppressed patients. Moreover, GR antagonists deserve further consideration for
their possible efficacy in the management
of these disorders, and the use of schedules, including both RA and a GR antagonist, may allow a more thorough evaluation of the therapeutic potential of RA in
this setting. (Blood. 2000;96:711-718)
r 2000 by The American Society of Hematology
Introduction
Retinoids, including retinol (vitamin A) and its natural and
synthetic derivatives, are a class of compounds of crucial importance in the regulation of numerous physiologic processes such as
embryonal morphogenesis, visual response, reproduction, growth,
cell differentiation, and immune function. The pleiotropic effects
induced by retinoids are mediated by the binding to and activation
of 2 different families of nuclear receptors, the retinoic acid (RA)
receptors (RARs) and the retinoid X receptors (RXRs), which
belong to the steroid–thyroid hormone receptor superfamily.1,2 Extensive data have provided evidence of a retinoid role in the prevention or
reversal of premalignant lesions of the upper aerodigestive tract, skin,
and cervix.3 Retinoids are also effective in inhibiting the proliferation of neoplastic cell lines of various origins in vitro3,4 and, in
clinical settings, all-Trans RA (ATRA) was shown to induce
complete remission in most patients with acute promyelocytic
leukemia (APL).5 Moreover, retinoids, alone or in combination
with other drugs, have shown some activity in other hematologic
malignancies including juvenile chronic myeloid leukemia, myelodysplastic syndrome, and cutaneous T-cell lymphoma.4,6
Despite these promising findings, however, the clinical usefulness of retinoids is limited. This is mainly due to the wide
heterogeneity of cellular responses to these drugs. In fact, either
among different types of cancers or within a single tumor histotype,
not all transformed cells are sensitive to the antiproliferative effects
of these compounds, and the growth of some malignancies may
even be enhanced by retinoid treatment.4,6 The mechanisms
underlying these phenomena are still unclear. In particular, it is
presently unknown whether these contrasting effects are, at least in
part, due to host factors that are able to modulate and/or interfere
with retinoid-mediated signaling. Elucidation of this issue is,
however, of relevance not only to gain insight into the physiopathology of retinoids but also to improve the efficacy of these drugs in
the clinical setting.
Epstein-Barr virus–immortalized (EBV-immortalized) lymphoblastoid B cell lines (LCLs) are a suitable in vitro model for the
study of EBV-related lymphoproliferative disorders of immunosuppressed patients. We have previously shown that 9-cis-RA, 13-cisRA, and ATRA powerfully inhibit LCL proliferation at concentrations corresponding to therapeutically achievable plasma levels
(1026 mol/L).7 The antiproliferative effects of RA were not
dependent on the induction of a terminal differentiation, and they
were not mediated by a direct modulation of EBV-encoded latent
antigen expression.7 We have also shown that RA treatment of
EBV-immortalized B lymphocytes is associated with multiple
effects on the G1 regulatory proteins including p27Kip-1 upregulation; decreased levels of cyclins D2, D3, and A; and
inhibition of CDK2, CDK4, and CDK6 activity, which ultimately
From the Division of Experimental Oncology 1, Centro di Riferimento Oncologico, Aviano, Italy.
Reprints: Mauro Boiocchi, Division of Experimental Oncology 1, Centro di
Riferimento Oncologico, via Pedemontana Occidentale 12, 33081 Aviano (PN),
Italy; e-mail: [email protected].
Submitted December 3, 1999; accepted March 13, 2000.
Supported in part by a grant (R.D.) from the Italian Association for Cancer
Research (AIRC), Milan, Italy. P.Z. is the recipient of a fellowship from the
Italian Foundation for Cancer Research (FIRC), Milan, Italy.
BLOOD, 15 JULY 2000 • VOLUME 96, NUMBER 2
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
r 2000 by The American Society of Hematology
711
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QUAIA et al
results in reduced pRb phosphorylation and G0/G1 protein
growth arrest.8
Interestingly, the strong growth inhibitory effect exerted by
13-cis-RA, 9-cis-RA, and ATRA on LCLs persisted in vitro for
more than 10 days following drug withdrawal.7 However, LCLs
persistently growth-arrested by RA treatment in vitro were able to
recover their proliferative activity following inoculation into severe
combined immunodeficiency (SCID) mice. On these grounds, our
experimental model appears particularly useful to identify factors
able to antagonize RA-mediated antiproliferative effects. In this
study, we show that glucocorticoids are able to release LCLs from
the RA-mediated proliferative block both in vitro and in vivo and to
efficiently antagonize RA-induced growth inhibition. These effects
were also clearly evident at physiologic concentrations of glucocorticoids and were inhibited by the glucocorticoid receptor (GR)
antagonist RU486. Moreover, we also demonstrated that glucocorticoids directly convey growth-promoting signals to LCLs that may
contribute to sustain the proliferation of these cells both in vitro and
in vivo. These findings provide a rational background for the design
of new strategies that are potentially useful to improve the
management of EBV-related lymphoproliferations of immunosuppressed patients.
Materials and methods
Reagents
Reagents used in the study included ATRA and 9-cis-RA (Roche, Basel,
Switzerland); 13-cis-RA, mifepristone (RU486), dexamethasone (Dex),
hydrocortisone (HC), estradiol, and testosterone (Sigma Chemical Co,
Milan, Italy); and the RARa agonist Ro 40-6055 (also known as Am580)9
(gift from Dr W. Bollag, Hoffman-LaRoche, Basel, Switzerland). Retinoids
and RU486 were dissolved in dimethylsulfoxide (DMSO) at 1021 mol/L
and diluted in culture medium to a final concentration of less than 0.01%
(vol/vol). RA was handled under subdued light, and the stock solutions
were stored at 220°C and protected from light and oxygen. The following
human recombinant cytokines were used: interleukin-1a (IL-1a), specific
activity 107 U/mg, and IL-6, specific activity 2 3 108 U/mg (Boehringer
Mannheim GmbH, Mannheim, Germany), and IL-4, specific activity 107
U/mg (Genzyme, Cambridge, MA).
Cell lines and culture conditions
Establishment and characterization of DAA-3 and HDE-14 LCLs have
been described elsewhere.7 The cell lines were cultured in Roswell Park
Memorial Institute medium (RPMI 1640) supplemented with 10% heatinactivated fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL
streptomycin, and 20 mmol/L L-glutamine. They were maintained in a
humidified 5% carbon dioxide (CO2) incubator at 37°C. All experiments
with steroid hormones were performed with cells cultured in a medium
containing 15% charcoal-stripped FCS (HyClone, Logan, UT).
Cell surface immunofluorescence analysis
Cell surface immunofluorescence was performed as previously described.7
Briefly, after preincubation at 4°C for 30 minutes in binding buffer (10%
rabbit serum in phosphate-buffered saline [PBS]), 5 3 105 cells were
incubated with saturating concentrations of the primary monoclonal
antibody (mAb) at 4°C for 30 minutes. After 3 washes in PBS, the samples
were incubated at 4°C for an additional 30 minutes, with optimal dilutions
of fluorescence isothiocyanate–conjugated (FITC-conjugated) second-step
antibody. The samples were then washed 3 times with PBS and fixed in 1%
buffered paraformaldehyde. Isotype-matched controls were used to determine nonspecific binding. All flow cytometric analyses were performed on
a fluorescence activated cell sorter (FACS) (FACScan using Lysis II
software; Becton Dickinson, Milan, Italy). The expression of the EBV-
encoded latent membrane protein-1 (LMP-1) and EBV nuclear antigen-2
(EBNA-2) antigens was investigated by immunofluorescence on cells
permeabilized and fixed using ORTHO PermeaFix (Ortho Biotech, Milan,
Italy). Briefly, 5 3 105 cells were incubated with 2 mL ORTHO PermeaFix
(1:2 dilution) at room temperature for 40 minutes.
After centrifugation at 400g for 10 minutes, the supernate was aspirated,
and the pellets were resuspended and kept at room temperature for 10
minutes in 2 mL 10% PBS/BSA. Cells were then centrifuged, the supernate
was discarded, and staining was performed as described above. Optimal
dilutions of anti–LMP-1 and anti–EBNA-2 antibodies were determined by
using the EBV2 Burkitt’s lymphoma-derived cell line DG75 as a negative
control. We used the following mAbs for immunophenotypic studies:
CD21, CD23, and CD71 (Becton Dickinson); phycoerythrin-conjugated
(PE-conjugated) CD19 (Biosource, Camarillo, CA); PE-conjugated CD38
(PharMingen, San Diego, CA); CD39 (Serotec, Oxford, England); anti–
surface immunoglobulin (sIg) (Ortho Biotech); and FITC-conjugated
CD30, anti–LMP-1 (CS1.4), and anti–EBNA-2 (PE2) (brand names in
parentheses; Dako, Milan, Italy). We also used isotypic controls (mouse
IgG1, IgG1-PE, and IgG2a) and FITC-conjugated goat antimouse Ig
(Becton Dickinson).
Cell proliferation assay
Proliferation assays were performed in 96-well plates in quadruplicate
cultures. Cells were seeded at an initial density of 104 cells per well in 200
µL of medium. Appropriate dimethyl sulfoxide (DMSO) dilutions were
used as controls. DMSO did not affect proliferation of any cell line.
Proliferative responses to B-cell growth-promoting cytokines were evaluated in serum-free medium. At the time-points indicated, cultures were
pulsed with 0.037 MBq (1 µCi) 3H-methyl thymidine (specific activity,
92.5 3 1010 Bq/mmol/L [25 Ci/mmol/L]) (Amersham International, Bucks,
England) for 6 hours and subsequently harvested (Unifilter-96, GF/C filter
plates; Packard, Meriden, CT). Radioactivity was measured in a liquid
scintillation counter (Top Count NXT, Packard), and the results were
expressed as mean counts per minute (cpm) plus or minus SD of
quadruplicate wells. In some experiments, proliferation was also evaluated
by counting the number of viable cells (9 aliquots per time-point) in a
Bürker chamber in the presence of trypan blue dye exclusion.
Western blot analysis
Whole cell extracts were prepared by lysing 107 cells in a buffer containing
50 mmol/L Tris-HCl (tris[hydroxymethyl] aminomethane hydrogen chloride) (pH 7.5), 150 mmol/L sodium chloride (NaCl), 2 mmol/L ethylenediamine tetraacetic acid (EDTA), 2 mmol/L ethyleneglycotetraacetic acid
(EGTA), 25 mmol/L sodium fluorine (NaF), 25 mmol/L b-glycerolphosphate, 0.1 mmol/L sodium orthovanadate, 0.1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, 1 µg/mL aprotinin, 0.2% Tryton-X-100,
and 0.3% Nonidet P-40 (lysis buffer). After 20 minutes of incubation at 0°C,
the extracts were centrifuged at 12 000 rpm for 30 minutes at 4°C. The
protein concentration in the lysate was determined by the Bio-Rad protein
assay kit (Bio-Rad Laboratories, Richmond, CA). Aliquots of the supernatant were mixed with 2 times sodium dodecyl sulfate (SDS) sample buffer
(150 mmol/L Tris, 30% glycerol, 3% SDS, 1.5 mg/100 mL bromophenol
blue dye, and 100 mmol/L dithiothreitol) and denatured at 100°C for 5
minutes. Equivalent amounts (40 µg) of protein were separated on 12.5%
SDS-PAGE (polyacrylamide gel electrophoresis) and transferred onto a
nitrocellulose membrane (Schleicher and Schuell, Keene, NH). Ponceau S
staining was performed to confirm that equal amounts of total protein were
present in all the lanes.
The membrane was blocked with 0.5% casein in PBS for 1 hour at room
temperature and incubated with the appropriate antibody overnight at 4°C.
After 3 washes with 0.5% casein for 5 minutes, the membranes were
incubated at room temperature for 1 hour with an appropriate horseradish
peroxidase–linked secondary antibody to a final concentration of 1:1000.
Final washes were performed in 0.5% casein for 15 minutes, PBS/TrytonX-100 for 5 minutes (3 times), and distilled water for 5 minutes.
Immunolabeled bands were detected with the ECL Western blot detection
system (Amersham). The following antibodies were used (brand names and
concentrations noted in parentheses): p27Kip-1 (1:2500) and CDK2 (1:2500)
BLOOD, 15 JULY 2000 • VOLUME 96, NUMBER 2
(Transduction Laboratories, Lexington, KY); GR (E-20, 1:1000), RARa
(C-20, 1:1000), cyclin E (C-19, 1:1000), cyclin A (H-432, 1:2000), cyclin H
(C-18, 1:1000), CDK4 (H-22, 1:1000), and CDK6 (C-21, 1:1000) (Santa
Cruz Technologies, Santa Cruz, CA); and CDK7 (Ab-1, 1:100) (Calbiochem, Oncogene Research, Cambridge, MA).
In vivo experiments
Female CB.17 SCID/SCID mice, 4 weeks old (Harlan-Nossan, Milan,
Italy), were kept under conventional conditions during the experiments.
Groups of 12 mice were used, and a suspension of 10 3 106 cells in 200 µL
saline buffer was given as subcutaneous (s.c) inoculations in the right flank.
RU486 (0.1 mol/L in DMSO) was dissolved in 2.5% Cremophor EL (Fluka
Chemie AG, Buchs, Switzerland) in water. Twenty-four hours after s.c. LCL
transplantation, RU486 was administered at a dose of 0.5 mg per day per
animal (in a volume of 200 µL) for 35 days. An equal volume of the vehicle
alone was administered to control mice with the same schedule. Mice were
inspected weekly for the appearance and progressive growth of tumor
masses. The size of s.c. tumors was measured with calipers, and tumor
volumes were calculated by using the following formula: (length 3
width2) / 2. Statistical significance was calculated by using the 2-tailed
Fisher exact test. Aliquots of tumor tissue were either formalin-fixed and
paraffin-embedded or snap-frozen and stored at 280°C. For further in vitro
analyses, single-cell suspensions from s.c. masses of mice with advanced
tumors were purified using Ficoll-Hypaque density gradient (Pharmacia,
Uppsala, Sweden).
Results
LCLs, persistently growth-arrested by RA in vitro, recover their
proliferative activity following transplantation into SCID mice
As a first step, we assessed whether the proliferative block induced
by RA on LCLs in vitro also persisted in vivo. To this end, groups
of 12 SCID mice received s.c. injections with DAA-3 LCLs
(10 3 106 cells per mouse). The LCLs were previously treated for 7
days with the solvent alone (0.001% DMSO) or with 13-cis-RA at a
concentration capable of inducing a persistent (1025 mol/L) growth
arrest in vitro.7 In all groups of mice, transplanted cells gave rise to
tumor masses that grew noninvasively at the site of inoculation.
Although s.c. tumors induced by RA-treated DAA-3 cells appeared
slightly later compared with controls, 35 days after inoculation,
both groups of mice showed masses larger than 4 cm3 (not shown).
DAA-3 cells purified from s.c. tumors were recultured in vitro in
the presence of various concentrations of 13-cis-RA. These cells
showed a responsiveness to RA-mediated growth inhibition similar
to that of the parental ones (not shown), ruling out the fact that the
in vivo growth of DAA-3 cells could be due to the appearance of
RA-resistant variants. These findings demonstrate that the growth
arrest induced by RA on LCLs, although persistent in vitro upon
drug withdrawal, is reversible in vivo. These findings also indicate
that host factors may allow the recovery of LCL proliferation.
EFFECT OF GLUCOCORTICOIDS ON EBV B LYMPHOCYTES
713
glucocorticoid hormones Dex and HC at physiologic concentrations (1026 to 1027 mol/L) induced a prompt recovery of DAA-3
cells previously growth-inhibited by RA (Figure 1 and data not
shown). These findings indicate that glucocorticoids are able to
relieve the proliferative block induced by RA on LCLs.
To verify whether glucocorticoids preferentially induced the
outgrowth of phenotypically distinct cell clones, we investigated
the expression of several differentiation and activation markers on
the DAA-3 LCLs released by Dex or HC from RA-induced growth
arrest. The analysis revealed that consistent with the recovery of
LCL proliferation, Dex and HC reversed RA-induced CD71
down-regulation (Figure 2 and data not shown). Moreover, while
the expression of CD23, CD30, CD39, and sIg tended to return to
basal levels after the decrease induced by RA treatment, RAmediated CD19 and CD21 down-regulation and CD38 upregulation persisted in cells recovered by Dex and HC (Figure 2
and data not shown). Nevertheless, separate experiments showed
that glucocorticoids administered to untreated LCLs induced a
marked decreased of CD19 and CD21 expression, which occurred
concomitantly with up-regulation of CD38 (not shown). There was
an apparent lack of reversibility of RA-induced changes relative to
CD19, CD21, and CD38 antibodies, and this irreversibility was
probably due to the direct effect exerted on the expression of these
markers by glucocorticoids. These findings, together with the
observation that all these effects were reproducibly induced by
glucocorticoids in a large panel of LCLs, including those monoclonal for Ig gene rearrangements (not shown), argue against the
possibility that Dex and HC may stimulate the preferential growth
of distinct cell clones.
Glucocorticoids, but not other steroid hormones, enhance LCL
proliferation in vitro
To gain further insight into the effects exerted on LCLs by glucocorticoids, we investigated whether various steroid hormones,
particularly Dex and HC, were able to affect LCL proliferation in
Glucocorticoids, but not B-cell growth-promoting cytokines,
induce a proliferative recovery of LCLs persistently
growth-arrested by RA
As a next step, we determined whether cytokines known to
promote B-cell proliferation were able to interfere with the
antiproliferative activity of RA and, particularly, to release LCLs
from RA-induced proliferative block. Preliminary experiments
indicated that 100 U/mL IL-1a, 0.5 µg/mL IL-4, or 100 U/mL IL-6
enhanced the proliferation of DAA-3 cells in vitro. Nevertheless,
these cytokines, either singularly or in combination, failed to
recover the proliferation of DAA-3 cells persistently growtharrested by RA (not shown). Conversely, administration of the
Figure 1. Glucocorticoids are able to release LCLs from DAA-3 cells growtharrested by RA. DAA-3 cells were treated with 1025 mol/L 13-cis-RA for 7 days and
then recultured without RA in medium alone or supplemented with 2 different
concentrations of Dex (1026 and 1027 mol/L). Proliferation was evaluated at different
time-points by 3H-thymidine uptake. The results of 1 representative experiment out of
3 are shown. Each point represents the mean plus or minus SD of values obtained
from triplicate wells. Similar findings were obtained with HC.
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BLOOD, 15 JULY 2000 • VOLUME 96, NUMBER 2
Glucocorticoids antagonize the antiproliferative activity
exerted on LCLs by RA
To assess whether glucocorticoids could interfere with RAmediated LCL growth inhibition, we investigated the effects of
different concentrations of various steroid hormones (Dex, HC,
progesterone, estradiol, and testosterone) on the antiproliferative
activity exerted on DAA-3 LCLs by 1025 mol/L 13-cis-RA.
Evaluation of 3H-thymidine uptake over a 7-day period of time
showed that only Dex and HC were able to significantly counteract
RA-induced LCL growth inhibition. Antagonistic activity of both
Dex and HC was clearly evident at all concentrations investigated,
with more pronounced effects at 1026 to 1027 mol/L (not shown).
Similar findings were obtained with ATRA and 9-cis-RA, the 2
other RA isomers active on LCLs (not shown). The antagonistic
effect exerted by glucocorticoids on the antiproliferative activity of
RA was also confirmed by further cotreatment experiments in
which proliferation was evaluated by counting viable cells by
trypan blue dye exclusion (not shown). Moreover, the analysis of
an additional group of 9 LCLs yielded similar results (not shown),
indicating that the effects exerted by glucocorticoids on RAinduced growth inhibition are of general relevance in the LCL
system.
Effects of glucocorticoids on cell cycle regulatory proteins
To better understand the mechanisms of action of glucocorticoids
involved in the promotion of LCL growth, we investigated the
Figure 2. Immunophenotypic profile of DAA-3 cells growth-arrested by RA and
reversed by HC. Cells were treated with 1026 mol/L 13-cis-RA for 6 days and then
replated either in medium alone or with 1026 mol/L HC (indicated as RA 1 HC).
Immunophenotype was evaluated after 7 days of culture, when cells exposed to HC
fully recovered their proliferation. Data relative to the percentage of positive cells (A)
and mean fluorescence intensity (B) are shown. The results of 1 representative
experiment out of 3 are reported. Similar findings were observed with Dex.
vitro. Because normal FCS contains variable concentrations of
steroid hormones, including glucocorticoids, we first investigated
whether deprivation of steroid hormones in the culture medium had
any effect on LCL proliferation. These experiments showed that
DAA-3 and HDE-14 cells grown in steroid-free medium proliferated less efficiently than those cultured with normal FCS, with a
35%-40% decrease in 3H-thymidine incorporation on day 3 of
culture (not shown). This indicates that LCL proliferation is
enhanced by FCS-derived steroid hormones. Glucocorticoids probably accounted for most of the growth-promoting activity exerted
by FCS-derived steroids because only Dex and HC (Figure 3), but
not progesterone, estradiol, or testosterone, enhanced LCL proliferation in steroid-free medium (not shown). As shown in Figure 3, the
enhancement of LCL proliferation induced by various concentrations (from 1026 to 1028 mol/L) of both Dex and HC was largely
dose-dependent, with slightly more pronounced effects induced by
HC. Compared with the 3H-thymidine uptake induced by 1027
mol/L HC, administration of supraphysiologic doses (1025 mol/L)
of this steroid did not result in any further increase in LCL
proliferation rates (Figure 3). These findings were confirmed on a
larger panel of LCLs (not shown). We also verified that the
growth-promoting effect exerted on LCLs by glucocorticoids was
associated with changes in the expression of EBV-encoded latent
antigens. While the levels of LMP-1 were substantially unaffected
by 1026 mol/L Dex and HC, HDE-14 and DAA-3 cells exposed to
these steroids showed a slight EBNA-2 up-regulation and an
increase in mean fluorescence intensity of usually less than
20%-30% compared with controls (not shown).
Figure 3. Glucocorticoids enhance LCL proliferation. Treatment with Dex (1026 to
1028 mol/L) or HC (1025 to 1028 mol/L) induced a dose-dependent increase of
3H-thymidine uptake in DAA-3 and HDE-14 cells cultured in steroid-free medium
(SFM). The results of 1 representative experiment out of 3 are shown. Each point
represents the mean plus or minus SD of values obtained from triplicate wells.
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715
expression of several cell cycle regulatory proteins in DAA-3 and
HDE-14 cells cultured for 2, 4, and 7 days in steroid-free medium
with or without 1026 mol/L Dex. At all time-points, cells treated
with Dex showed significantly increased amounts of the phosphorylated form of CDK2, whereas there was no observed change in the
levels of CDK4, CDK6, and CDK7 (Figure 4 and data not shown).
Also, the expression of cyclin E, A, and H was not modulated by
Dex (Figure 4 and data not shown). Of note, Dex induced a marked
down-regulation of p27Kip-1 that was evident since day 2 of
treatment (Figure 4). These findings indicate that the decreased
availability of this CDK inhibitor in Dex-treated LCLs probably
accounted for the enhanced phosphorylation of CDK2 in these cells
and resulted in enhanced CDK2 kinase activity and accelerated
G1-to-S transition. Thus, Dex-induced p27Kip-1 down-regulation
probably constitutes the key factor responsible for the growthpromoting activity exerted on LCLs by glucorticoids.
To gain insights into the mechanisms underlying the antagonism of glucocorticoids on RA-mediated growth inhibition, we
also investigated the effects of 1026 mol/L Dex on the expression of
the same cell cycle regulatory proteins (as given above) in HDE-14
cells exposed to 1026 mol/L 13-cis-RA. The analysis showed that
treatment with Dex contrasted with, although not completely,
RA-induced p27Kip-1 up-regulation (Figure 4). Consistently, Dextreated cells also showed higher levels of the phosphorylated active
form of CDK2 (Figure 4). These effects were evident since day 2 of
treatment (Figure 5). Moreover, RA-induced cyclin A downregulation observed on day 4 was almost entirely abrogated by
Dex, which is consistent with the full recovery of the proliferative
activity of these cells (Figure 4). Similar findings were also
observed in the DAA-3 LCLs (not shown).
Glucocorticoid-mediated effects are abrogated by the GR
antagonist RU486
To determine whether the effects exerted by Dex and HC on LCLs
are mediated by GRs, we investigated the ability of the GR
antagonist RU486 to suppress the activity of these steroids.
Preliminary experiments showed that 1025 to 1027 mol/L RU486
alone had no significant effect on the proliferation of LCLs grown
in steroid-free medium (not shown). Also, the expression of LMP-1
and EBNA-2 was not affected by RU486 (not shown). As shown in
Figure 4. Immunoblot analysis of cyclin A, CDK2, and p27Kip-1 proteins in DAA-3
cells. The cells were cultured for 2 and 4 days in SFM (center) alone or supplemented
with either 1026 mol/L 13-cis-RA (RA), 1026 mol/L Dex, or a combination of these 2
drugs. For CDK2, faster migrating bands represent the phosphorylated active forms
of this kinase. Dex induced a decrease in the amount of p27Kip-1 protein and a
markedly contrasted p27Kip-1 up-regulation induced by RA. Dex also increased the
levels of the phosphorylated active forms of CDK2 and antagonized an RA-induced
decrease of CDK2 phosphorylation. Similar findings were obtained with the HDE-14
LCLs (not shown). We subjected 50 µg extract proteins from each lysate to
immunoblot analysis. The cellular proteins visualized in each panel are indicated to
the left.
Figure 5. The GR antagonist RU486 fully reversed the LCL growth-promoting
effects and RA antagonism mediated by glucocorticoids. (A) DAA-3 cells were
incubated in SFM (CTR, control) for the indicated times with or without RU486 (from
1025 to 1027 mol/L) in the presence or absence of 1026 mol/L Dex (left panel) or 1027
mol/L HC (right panel). (B) The panel depicts the effects of different concentrations of
RU486 (from 1025 to 1027 mol/L) on the antagonistic activity exerted by 1026 mol/L
Dex (left panels) or 1027 mol/L HC (right panels) against DAA-3 and HDE-14 cell
growth inhibition induced by 1025 mol/L 13-cis-RA. Proliferation was evaluated at
different time-points by 3H-thymidine uptake. The results of 1 representative experiment out of 3 are shown. Each point represents the mean plus or minus SD of values
obtained from triplicate wells.
Figure 5A, RU486 was able to abrogate the growth-promoting
stimulus exerted by Dex and HC on DAA-3 LCLs. In particular,
1025 to 1026 mol/L RU486 was active against 1026 mol/L Dex, and
1025 mol/L RU486 was active against 1027 mol/L HC, confirming
the stronger effects exerted by this latter steroid hormone. Similar
results were obtained with the HDE-14 LCL (not shown). Consistently, RU486 also suppressed the antagonism exerted by glucocorticoids on RA-mediated LCL growth inhibition. In fact, RU486
concentrations as low as 1027 mol/L efficiently counteracted the
activity of 1026 mol/L Dex and 1027 mol/L HC in HDE-14 cells
(Figure 5B). In DAA-3 cells, RU486 concentrations higher than
1026 mol/L completely inhibited the effects of 1026 mol/L Dex.
However, the activity of 1027 mol/L HC was suppressed by RU486
in a dose-dependent fashion, with maximal inhibition observed at
1025 mol/L (Figure 5B). These findings indicate that the effects of
glucocorticoids reported here were mediated by GRs. Western blot
analysis, which was carried out with a polyclonal antibody specific
for both GRa and GRb isoforms, showed that HDE-14 and DAA-3
LCLs constitutively expressed detectable amounts of GRa (not
shown). The GRb isoform, a physiologic antagonist of GRa,10 was
not expressed in these cells. While 13-cis-RA did not affect GRa
expression levels, exposure to 1026 mol/L Dex induced a marked
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QUAIA et al
GRa protein down-regulation that was evident since day 2 of
treatment (not shown); 13-cis-RA had no effect on Dex-induced
down-regulation of GRa (not shown).
Glucocorticoids antagonize the growth inhibition mediated
by an RARa–selective agonist without affecting RARa
expression levels
We have previously demonstrated that RA-induced LCL growth
arrest is mediated by RARa.11 As shown in Figure 6, both Dex and
HC were able to antagonize the growth inhibition induced in
DAA-3 cells by the RARa selective agonist Ro 40-6055 at all
concentrations. To assess whether the antagonistic effects exerted
by glucocorticoids were mediated by changes in RARa expression,
RARa protein levels were investigated in HDE-14 and DAA-3
cells cultured in steroid-free medium with either 1026 mol/L Dex,
1025 mol/L 13-cis-RA, or both for 2, 4, and 7 days. No significant
change in RARa expression levels was observed in cells exposed
to Dex at all time-points considered, whereas a decrease in the
amount of RARa protein was seen in 13-cis-RA–treated cells since
day 2 (not shown). Cells treated with both Dex and 13-cis-RA
showed RARa expression levels similar to those observed in cells
exposed to 13-cis-RA alone (not shown).
RU486 decreases both the in vivo recovery of LCLs
persistently growth-arrested by RA and the growth of untreated
LCLs transplanted into SCID mice
The effects exerted on LCLs by glucocorticoids in vitro and
particularly their ability to counteract RA-mediated growth inhibition at physiologic concentrations suggested a likely relevant role
of these steroids in the prompt in vivo recovery of LCLs
persistently growth-arrested by RA in vitro. To directly address this
issue, we investigated the in vivo effects of RU486 on the growth of
RA-treated DAA-3 cells following s.c. inoculation into SCID mice.
Administration of 0.5 mg per day RU486 significantly reduced
both the number and the volume of s.c. tumor masses (Table 1)
without evidence of toxic effects. In particular, after 35 days of
treatment, only 3 of 12 animals (25%) treated with RU486 carried
s.c. masses as large as those grown in control mice (P 5 .0003)
Figure 6. Glucocorticoids antagonize LCL growth inhibition induced by the
RARa selective agonist Ro 40-6055. Concentrations of Dex and HC, ranging from
1026 to 1027 mol/L, efficiently antagonized the antiproliferative effects exerted by
1027 and 1028 mol/L Ro 40-6055 on DAA-3 LCLs. Cell proliferation was evaluated
after 7 days in SFM alone (center). The results of 1 representative experiment out of 3
are shown. Each histogram represents the mean plus or minus SD of values obtained
from triplicate wells.
Table 1. Effects of the GR antagonist RU486 on tumor formation in SCID mice
2 RU486
Mice,
n/total
.4
1 RU486
%
Size of s.c.
Masses, cm3
Mice,
n/total
12/12
100
.4
3/12
#4
—
—
#4
4/12*
33.3
None
—
—
None
5/12
41.7
Size of s.c.
Masses, cm3
%
25
The size of tumor masses was evaluated 35 days after s.c. inoculation of 10 3
106 DAA-3 cells persistently growth-arrested in vitro by a 7-day treatment with 1025
mol/L 13-cis-RA.
*Indicates that these animals carried masses markedly smaller than 4 cm3
(between 0.04 cm3 and 0.5 cm3). Mice treated with RU486 developed significantly
fewer s.c. masses greater than 4 cm3 (P 5 .0003, 2-tailed Fisher exact test).
(Table 1). Of note, 5 of 12 treated mice (42%) showed no evidence
of tumor formation whereas 4 of 12 animals (33%) showed the
growth of very small s.c. masses (from 0.04-0.5 cm3). These
findings indicate that endogenous glucocorticoids probably constitute the main host factors responsible for the in vivo recovery of
LCLs that are persistently growth-arrested by RA.
We also investigated whether the in vivo growth of untreated
LCLs was influenced by endogenous glucocorticoids. To this end,
we evaluated the effects of 0.5 mg per day RU486 on the growth of
untreated DAA-3 cells following transplantation into SCID mice.
These experiments showed that RU486 also markedly reduced the
growth of untreated DAA-3 cells. In fact, after 35 days of
treatment, approximately 42% of the animals exposed to RU486
carried s.c. masses significantly smaller than those of controls
(P 5 .019), with 2 of 12 mice (16.7%) showing no evidence of
tumor formation.
Discussion
In the present study, we demonstrate that glucocorticoids exert
antagonistic effects on RA-mediated LCL growth inhibition both in
vitro and in vivo. In fact, Dex and HC, but not B-cell growthpromoting cytokines such as IL-1a, IL-4, and IL-6, were able to
recover the in vitro proliferation of LCLs persistently arrested in
G0/G1 protein by RA. Moreover, the recovery of these cells
occurring in vivo was significantly reduced by treating the SCID
mice with the steroid antagonist RU486, which indicates that
endogenous glucocorticoids are probably the most relevant host
factors responsible for the release of LCLs from RA-induced
proliferative block. Consistently, physiologic concentrations of
glucocorticoids directly antagonized the antiproliferative activity
exerted by 13-cis-RA, 9-cis-RA, and ATRA, even when these
retinoids were administered at high doses (1025 mol/L). It is worth
noting that glucocorticoids efficiently antagonized RA-mediated
growth inhibition in a large panel of LCLs, indicating that this is a
generalized effect in the LCL system.
Glucocorticoids are known to influence B-cell survival, activation, and proliferation by inducing variable effects depending on
the functional and differentiation status of these cells.12-14 Nevertheless, the biologic effects exerted by these steroids on preactivated B
cells, such as EBV-immortalized B lymphoblasts, are still poorly
defined. Here we show that glucocorticoids directly promote LCL
proliferation by conveying stimulatory signals that dominate over
the growth-inhibitory stimulus induced by RA. In particular, we
found that the proliferation of a large panel of LCLs cultured in
steroid-free medium was enhanced by Dex or HC but not by other
steroid hormones, which indicates that glucocorticoids probably
BLOOD, 15 JULY 2000 • VOLUME 96, NUMBER 2
EFFECT OF GLUCOCORTICOIDS ON EBV B LYMPHOCYTES
account for most of the growth-promoting activity exerted on LCLs
by FCS-derived steroids. Moreover, we provide evidence indicating that endogenous glucocorticoids also have a contributory role
in sustaining LCL growth in vivo. In fact, administration of RU486
markedly reduced both the number and size of s.c. tumor masses
induced by transplantation of normal LCLs into SCID mice.
The LCL growth-promoting activity of glucocorticoids may be,
at least in part, due to the slight increase in the levels of EBNA-2
expression induced by these steroids, although this issue awaits
further elucidation. It is unlikely, however, that glucocorticoids
antagonize the effects of RA by up-regulating EBNA-2. RA does not
require a direct modulation of EBV-latent antigens to inhibit LCL
growth and probably acts downstream to the signaling(s) activated
by these viral proteins. To gain further insight into the mechanisms
underlying the growth-promoting activity exerted on LCLs by
glucocorticoids, we investigated the effects of these steroids on cell
cycle regulatory proteins. Our findings strongly suggest that glucocorticoids enhance LCL proliferation mainly by down-regulating
p27Kip-1. In fact, in LCLs treated with glucocorticoids, the reduced
levels of this inhibitor were associated with and were probably
responsible for the increased amount of the active phosphorylated
form of CDK2, a phenomenon that is relevant for the enhancement
of CDK2 kinase activity and, ultimately, for cell cycle progression.15
These results are consistent with recent findings indicating that
p27Kip-1 is one of the targets modulated by glucocorticoid signaling
to regulate lymphocyte proliferation.16 The observation that p27Kip-1
down-regulation underlies the growth-promoting effects exerted on
LCLs by glucocorticoids is intriguing. This is particularly true in
light of our previous findings indicating that up-regulation of
p27Kip-1 has a central role in mediating the antiproliferative effects
induced by RA on the same cells.8 Besides strengthening the
relevance of p27Kip-1 in controlling LCL growth, the results
presented herein also suggest that RA and glucocorticoid signaling
probably converge on p27Kip-1 to differentially modulate the
proliferation of these cells.
Both the promotion of LCL proliferation and antagonism of
RA-mediated growth inhibition exerted by glucocorticoids were
reversed by the GR antagonist RU486. These findings strongly
suggest that all these effects were mediated by GR. Glucocorticoids
and RA exert their biologic activities through nuclear receptors that
share a similar structural organization and belong to the same
family of ligand-activated transcription factors.17 Considering that
both RA and glucocorticoids can cross-regulate the expression of
several members of the same nuclear receptor superfamily,18-22 it
appeared of interest to assess whether glucocorticoids antagonized
the activity of RA by affecting the expression of relevant RARs or
RXRs. We have recently shown that RA-induced LCL growth
inhibition was mainly mediated by RARa,11 and here we report that
glucocorticoids also efficiently antagonized the antiproliferative
717
activity of the RARa selective agonist Ro 40-6055. While both RA
and glucocorticoids down-regulated their own relevant receptors
(RARa and GRa, respectively), probably by homologous regulation,2,17,23 glucocorticoids had no effect on RARa protein levels.
These findings indicate that the interference exerted on RA
signaling by glucocorticoids probably occurs at a level beyond the
modulation of RARa concentration. However, further studies are
required to elucidate the mechanisms underlying the cross-talk
between RARa and GRa in the LCL system.
EBV-immortalized LCLs are the in vitro counterpart of the cells
that give rise to EBV-related lymphoproliferations of immunosuppressed patients.24,25 In particular, LCLs have biologic features that
are highly reminiscent of posttransplantation lymphoproliferative
disorders (PTLDs).24-26 PTLD, which occurs in 1%-10% of all
cases, constitutes a life-threatening complication that may arise
after transplantation of solid organs.24,26 Several lines of evidence
indicate that the functional impairment of EBV-specific cytotoxic T
lymphocytes (CTLs) due to immunosuppressive therapy is the
major factor responsible for the uncontrolled proliferation of
EBV-immortalized B lymphocytes occurring in the early phases of
PTLD development.27 Nevertheless, the demonstration that physiologic concentrations of glucocorticoids promote LCL growth both
in vitro and in vivo suggests that endogenous glucocorticoids may
be involved in the pathogenesis of PTLD. Our findings are in fact
consistent with the possibility that after EBV immortalization, the
in vivo growth of EBV-infected B lymphoblasts may be at least in
part promoted and sustained by endogenous glucocorticoids.
Similar growth-promoting effects could also be induced by synthetic glucocorticoids administered to these patients in combination
with other immunosuppressive drugs to prevent or control graft
rejection. These relevant issues deserve to be directly addressed by
further studies. Moreover, although the regression of most PTLDs
occurring after reduction or withdrawal of immunosuppressive
therapy is largely due to restoration of EBV-specific CTL responses,27,28 it would be of interest to verify whether a decreased
glucocorticoid-mediated growth-promoting stimulus may also contribute to this phenomenon. Finally, our results also may have other
implications for the management of EBV-associated lymphoproliferations of immunocompromised patients, and the use of schedules
including both RA and a GR antagonist, such as RU486,29 may
allow a more thorough evaluation of the therapeutic potential of RA
in this setting.
Acknowledgments
The authors thank Prof Werner Bollag (Hoffmann-La Roche) for
supplying Ro 40-6055 and Dr P. Tonel and Mrs P. Pistello for help
with the manuscript.
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