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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 711-718
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
From the Division of Experimental Oncology 1, Centro di
Riferimento Oncologico, Aviano, Italy.
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Abstract |
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 
receptor (RAR) selective agonist. RAR 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)
© 2000 by The American Society of Hematology.
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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-cis-RA, and ATRA powerfully inhibit LCL proliferation at concentrations
corresponding to therapeutically achievable plasma levels
(10 6 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 up-regulation; decreased levels of cyclins D2, D3,
and A; and inhibition of CDK2, CDK4, and CDK6 activity, which
ultimately 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.
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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 RAR 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 101 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  20°C and protected from light and
oxygen. The following human recombinant cytokines were used:
interleukin-1 (IL-1), specific activity 107 U/mg, and
IL-6, specific activity 2 × 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% heat-inactivated 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 × 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 × 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 EBV
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 × 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  -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/Tryton- X-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)
(Transduction Laboratories, Lexington, KY); GR (E-20, 1:1000), RAR
(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 × 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 × 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 80°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).
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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 × 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 (105 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.
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-1, 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 growth-arrested by RA (not shown). Conversely,
administration of the glucocorticoid hormones Dex and HC at physiologic
concentrations (106 to 107 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.
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| Fig 1.
Glucocorticoids are able to release LCLs from DAA-3 cells
growth-arrested by RA.
DAA-3 cells were treated with 105 mol/L
13-cis-RA for 7 days and then recultured without RA in medium
alone or supplemented with 2 different concentrations of Dex
(106 and 107 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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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, RA-mediated CD19 and CD21 down-regulation and CD38 up-regulation 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.
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| Fig 2.
Immunophenotypic profile of DAA-3 cells growth-arrested
by RA and reversed by HC.
Cells were treated with 106 mol/L 13-cis-RA
for 6 days and then replated either in medium alone or with
106 mol/L HC (indicated as RA + 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.
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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
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
106 to 108 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 107 mol/L HC, administration of
supraphysiologic doses (105 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 106 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).
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| Fig 3.
Glucocorticoids enhance LCL proliferation.
Treatment with Dex (106 to 108
mol/L) or HC (105 to 108 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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Glucocorticoids antagonize the antiproliferative activity exerted on
LCLs by RA
To assess whether glucocorticoids could interfere with RA-mediated
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 105 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 106 to
107 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 RA-induced growth inhibition are of general
relevance in the LCL system.
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| Fig 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 106 mol/L 13-cis-RA
(RA), 106 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.
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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 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
106 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 growth-promoting 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 106 mol/L Dex on the expression of
the same cell cycle regulatory proteins (as given above) in HDE-14
cells exposed to 106 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, Dex-treated 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 down-regulation 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).
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| Fig 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 105 to
107 mol/L) in the presence or absence of
106 mol/L Dex (left panel) or 107
mol/L HC (right panel). (B) The panel depicts the effects of different
concentrations of RU486 (from 105 to
107 mol/L) on the antagonistic activity exerted by
106 mol/L Dex (left panels) or
107 mol/L HC (right panels) against DAA-3 and HDE-14
cell growth inhibition induced by 105 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.
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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 105 to 107 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
5A, RU486 was able to abrogate the growth-promoting stimulus exerted by
Dex and HC on DAA-3 LCLs. In particular, 105 to
106 mol/L RU486 was active against
106 mol/L Dex, and 105 mol/L
RU486 was active against 107 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 107 mol/L efficiently counteracted the activity
of 106 mol/L Dex and 107 mol/L HC
in HDE-14 cells (Figure 5B). In DAA-3 cells, RU486 concentrations higher than 106 mol/L completely inhibited the
effects of 106 mol/L Dex. However, the activity of
107 mol/L HC was suppressed by RU486 in a
dose-dependent fashion, with maximal inhibition observed at
105 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 GR and GR isoforms, showed that HDE-14 and
DAA-3 LCLs constitutively expressed detectable amounts of GR (not
shown). The GR isoform, a physiologic antagonist of
GR,10 was not expressed in these cells. While
13-cis-RA did not affect GR expression levels, exposure to
106 mol/L Dex induced a marked GR 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 GR
(not shown).
Glucocorticoids antagonize the growth inhibition mediated by an
RAR-selective agonist without affecting RAR expression
levels
We have previously demonstrated that RA-induced LCL growth arrest is
mediated by RAR.11 As shown in Figure
6, both Dex and HC were able to antagonize
the growth inhibition induced in DAA-3 cells by the RAR selective
agonist Ro 40-6055 at all concentrations. To assess whether the
antagonistic effects exerted by glucocorticoids were mediated by
changes in RAR expression, RAR protein levels were investigated
in HDE-14 and DAA-3 cells cultured in steroid-free medium with either
106 mol/L Dex, 105 mol/L
13-cis-RA, or both for 2, 4, and 7 days. No significant change
in RAR expression levels was observed in cells exposed to Dex at all
time-points considered, whereas a decrease in the amount of RAR
protein was seen in 13-cis-RA-treated cells since day 2 (not
shown). Cells treated with both Dex and 13-cis-RA showed RAR
expression levels similar to those observed in cells exposed to
13-cis-RA alone (not shown).
View larger version (37K):
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| Fig 6.
Glucocorticoids antagonize LCL growth inhibition induced
by the RAR selective agonist Ro 40-6055.
Concentrations of Dex and HC, ranging from 106 to
107 mol/L, efficiently antagonized the
antiproliferative effects exerted by 107 and
108 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.
|
|
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 = .0003)
(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 = .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 growth-promoting
cytokines such as IL-1, 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 (105
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
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
RAR,11 and here we report that glucocorticoids also
efficiently antagonized the antiproliferative activity of the RAR
selective agonist Ro 40-6055. While both RA and glucocorticoids down-regulated their own relevant receptors (RAR and GR,
respectively), probably by homologous regulation,2,17,23
glucocorticoids had no effect on RAR protein levels. These findings
indicate that the interference exerted on RA signaling by
glucocorticoids probably occurs at a level beyond the modulation of
RAR concentration. However, further studies are required to
elucidate the mechanisms underlying the cross-talk between RAR and
GR 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.
| |
Footnotes |
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.
Reprints: Mauro Boiocchi, Division of Experimental Oncology 1, Centro di Riferimento Oncologico, via Pedemontana Occidentale 12, 33081 Aviano (PN), Italy; e-mail: mboiocchi{at}ets.it.
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.
| |
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Abstract
Introduction