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NEOPLASIA
From the Department of Pharmacology, Yale
University School of Medicine, New Haven, CT; and Memorial
Sloan-Kettering Cancer Center, New York, NY.
Previous studies have demonstrated that combinations of
all-trans retinoic acid (ATRA) with either
granulocyte-colony stimulating factor (G-CSF) or lithium chloride
(LiCl) produced synergistic terminal differentiation of WEHI-3B
myelomonocytic leukemia (D+) cells. It was found that
steady-state retinoic acid receptor alpha (RAR Successful chemotherapy of the acute leukemias
requires the use of cytotoxic drugs to kill the neoplastic cells.
Because these agents lack selectivity for leukemia cells, their use is
often accompanied by serious adverse side effects for the patient.
Clearly, alternatives to the use of cytotoxic regimens are desirable.
One such approach involves the concept that a leukemia cell is one that
fails to complete its normal maturation program, thereby retaining
infinite proliferative capacity. If the block or defect in the
maturation process were overcome, the cell could possibly mature to a
functional end-stage cell with a finite life span.1 Perhaps the most compelling evidence supporting the induction of
differentiation as a viable mode of cancer therapy arises from clinical
trials with all-trans retinoic acid (ATRA), in which complete remissions were attained in patients with acute promyelocytic leukemia (APL)2 in a process that clearly involved
terminal differentiation.3
Tempering the success of ATRA-based differentiation therapy are several
problems that attend its use.4 Serious side effects occur
in some patients treated with ATRA, but these are usually successfully
managed with steroid therapy.5 Of greater concern is the
fact that the remissions produced in patients treated with ATRA alone
are of short duration because of the rapid development of
resistance6 and the inability of ATRA to convert the
entire leukemic cell population to mature end-stage cells. Another
limitation of the differentiation therapy of the leukemias using ATRA
is that its effectiveness is limited to APL cells carrying the t(15;17) rearrangement,7 which fuses the PML (promyelocytic
leukemia-associated) gene to the retinoic acid receptor alpha (RAR Clinically, the development of resistance to ATRA is associated with
decreases in the plasma concentration of drug while patients are
actively treated.9 This may result from increased
catabolism or increased sequestration of ATRA by retinoid binding
proteins (see Warrell6 for references). In studies in cell
culture, relatively low concentrations of ATRA are capable of inducing the terminal differentiation of leukemia cells when combined with granulocyte-colony stimulating factor (G-CSF),10-15 a
cytokine that, through the binding of its receptor (G-CSFR), regulates the production of neutrophils and enhances their maturation, or LiCl,16 which also improves neutrophil production (see
Boggs and Joyce17 for review). Thus, it is conceivable
that the inclusion of G-CSF or LiCl in treatment regimens may allow the
effective use of relatively low levels of ATRA, possibly reducing
toxicity, delaying the development of retinoid resistance, and
extending the duration of remissions produced by ATRA through the
terminal differentiation of a larger proportion of the neoplastic cell population. Given that, as a single agent, ATRA has produced clinical usefulness only in patients with t(15;17) APL, it is of potential importance that major differentiation responses to the retinoid have
been obtained in vitro with several subtypes of myeloid
leukemia An understanding of the molecular basis for the synergistic induction
of terminal differentiation of malignant cells exhibited by mixtures of
agents such as G-CSF or LiCl and ATRA is essential for the optimum use
of differentiation therapy regimens. Retinoids such as ATRA are known
to exert most of their effects through the binding of 2 classes of
nuclear receptors, RARs and RXRs.18,19 The natural ligands
for the RARs and RXRs are ATRA and 9-cis retinoic acid
(9-cis-RA), respectively.18,20 Association of
these receptors, usually as RAR/RXR heterodimers, with specific DNA
sequences (retinoic acid response elements, RAREs) in the promoter
regions of target genes provides for the ligand-dependent modulation of
gene expression. Cells that do not express RARs and RXRs, or that
express mutant forms of the receptors, are unable to respond
appropriately to retinoids. The current mechanistic concept of retinoid
receptor regulation of gene expression involves the recruitment to the RARE of chromatin remodeling multiprotein complexes whose constituents and activities differ depending on ligand binding by the retinoid receptors (for reviews, see Johnson and Turner,21 Minucci
and Pelicci22). Because G-CSF also exerts its effects
through the binding of its receptor, it is conceivable that the
complementary modulation of receptor concentration or activation by
these agents could contribute to the synergistic effects observed with
these combinations. We now report that the modulation of RAR Cell culture and differentiation
The capacity of cells to undergo functional maturation was assessed by
nitro blue tetrazolium (NBT) dye reduction. Approximately 1 × 106 cells were collected by centrifugation at
300g for 5 minutes and resuspended in 1.0 mL complete medium
containing 0.1% NBT and 1.0 µmol/L of
12-O-tetradecanoylphorbol 13-acetate (TPA). The cell
suspension was incubated at 37°C for 30 minutes, and the percentage
of cells containing blue-black formazan deposits, indicative of a
TPA-stimulated respiratory burst, was determined by microscopic
visualization of at least 200 cells using a hemacytometer. To determine
whether the interaction between G-CSF or LiCl and ATRA is truly
synergistic, experiments were conducted in which the concentrations of
G-CSF or LiCl and ATRA were each varied differently in combination, and
the extent of differentiation of D+ cells was measured by
determining the percentage of NBT-positive cells after 72 hours of
incubation. The data were analyzed by isobologram and combination index
(CI) methodologies using computer-based programs.23,24
Northern blotting
Western blotting For Western blot analysis, 3 × 106 cells were collected by centrifugation and resuspended in Tris-buffered saline (10 mmol/L Tris-HCl, pH 7.6, 0.1 mol/L NaCl, 1 mmol/L EDTA) containing a mixture of protease inhibitors (2 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, and 1 µg/mL aprotinin), lysed by the addition of an equal volume of 2 × sodium dodecyl sulfate (SDS) gel-loading buffer (100 mmol/L Tris-HCl, pH 6.8, 200 mmol/L dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol), placed in a boiling water bath for 5 minutes, and vortexed vigorously. Extracts were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% dry milk in TBST (20 mmol/L Tris-HCl, pH 7.6, 137 mmol/L NaCl, 0.01% Tween 20) for 30 minutes, incubated overnight with rabbit polyclonal anti-RAR
or anti-RXR antibody, diluted to 1 µg/mL in TBST containing 5%
milk, washed with 3 changes of TBST for a total of 30 minutes, and then
incubated with horseradish peroxidase-conjugated donkey antirabbit IgG
for 1 hour and washed for 30 minutes with TBST (3 changes).
Immunoreactive proteins were visualized by enhanced chemiluminescence
(ECL; Amersham, Arlington Heights, IL).
Chemicals and antibodies ATRA and 9-cis-RA were purchased from Biomol (Plymouth Meeting, PA). NBT and Ultrapure LiCl were purchased from Sigma (St. Louis, MO). The RAR-specific ligand (TTNPB; 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) and the RXR-specific ligand (LG100346; 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) carbonyl]benzoic acid methoxy-oxime) were synthesized by Stacie Canan-Koch and kindly provided by Dr Elizabeth Allegretto (Ligand Pharmaceuticals, San Diego, CA). Retinoid receptor subfamily-specific antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Biomol.
Differentiation responses of D+ cells to G-CSF or LiCl and ATRA In previous studies we observed an enhanced induction of the differentiation of D+ cells by the combination of ATRA and G-CSF that permits the use of exceedingly low doses of ATRA to achieve a terminally differentiated state.13 A similar phenomenon is exhibited in HL-60 and AML-193 cells and in cells from patients with APL and AML.10,12,26 To determine whether the interaction between G-CSF and ATRA is truly synergistic, we have conducted experiments in which the concentrations of G-CSF and ATRA were each varied differently in combination and the extent of differentiation of D+ cells was measured by determining the percentage of NBT-positive cells. Isobologram and CI analyses of data from one of these experiments, demonstrating the particularly strong synergism exhibited by this combination, is shown in Figure 1. Note that the synergistic interaction of G-CSF and ATRA is so strong that all the points shown in the isobologram obtained with the combination are clustered in the lower left corner of the plot (Figure 1A). Analysis of data generated from an alternative effect-oriented study by CI methodology confirmed the strong synergism exhibited between these agents to produce terminal differentiation (Figure 1B). These studies also show that the success of retinoid-based differentiation therapy may not be limited to t(15;17) rearranged APL. Thus, the differentiation responses in this case were demonstrated in WEHI-3B cells, which is a non-APL myeloid leukemia cell line, when ATRA was combined with the differentiation-inducing cytokine, G-CSF.
Millimolar concentrations of LiCl have previously been shown to induce
terminal differentiation of both HL-60 and D+
cells.16 Furthermore, in these studies, the
differentiation-inducing effects of LiCl were markedly enhanced by the
addition of low levels of ATRA. We evaluated the interaction between
LiCl and ATRA in producing differentiation of D+ cells
using the NBT reduction assay. Isobologram and CI evaluation of the
results, shown in Figure 2, indicated
that synergistic differentiation induction was clearly produced.
Notably, when used in an admixture, significant differentiation
responses were achieved at concentrations of these agents that are
attainable in vivo.27
Effects of G-CSF and ATRA on G-CSFR expression in D+ cells Because both G-CSF and ATRA exert their effects through the binding of their cognate receptors, receptor presence and abundance in/on target cells are important determinants of cellular response and may correlate with the success of treatment with these agents. We were, therefore, interested in whether complementary modulation of receptor concentration was produced by these agents. It has been reported that ATRA increased G-CSFR mRNA in NB4 leukemia cells and in HL-60 leukemia cells.14,15 We were unable to duplicate this result in the HL-60 cell line routinely used in our laboratory. Moreover, increased G-CSFR mRNA expression was not induced by ATRA in our studies with WEHI-3B leukemia cells. Thus, Northern blot analyses showed that ATRA, over a range of concentrations that effectively produced terminal differentiation, did not significantly alter the expression of G-CSFR mRNA in D+ cells after 48 hours of treatment (Figure 3A). Even after 24 to 72 hours of exposure to the optimum concentration of ATRA, producing differentiation of D+ cells when used as a single agent (7 µmol/L), G-CSFR mRNA was not increased but appeared to be slightly decreased (Figure 3B). G-CSFR expression was also not significantly affected in D+ cells treated with the combination of G-CSF and ATRA (data not shown).
Effects of G-CSF and ATRA on
steady-state RAR 
protein was not detected in these cells either before or after
treatment. RAR , expressed almost exclusively in epithelial tissues,25 was not examined in these studies. RAR was
detected, however, and its levels were modulated by treatment. Figure
4A shows a representative RAR Western
blot of G-CSF- and ATRA-treated D+ cells. As a single
agent, G-CSF did not appreciably alter levels of RAR protein in
D+ cells, and only a weak differentiation response was
induced (Table 1). The effects of ATRA alone were more notable. The
retinoid alone produced a modest differentiation response in the
D+ cells and caused a reduction in the steady-state levels
of RAR protein such that the protein was barely detectable after 24 hours of treatment. Of greater interest, when G-CSF was used in
combination with ATRA, the ATRA-induced loss of RAR protein was
decreased and a synergistic differentiation response was elicited. The
basis for the attenuation of the ATRA-induced loss of RAR by
coexposure to G-CSF was investigated by Northern blot analyses of
RAR in drug-treated D+ cells (Figure 4B). Exposure to
ATRA alone induced an increase in the steady-state levels of RAR
mRNA even though RAR protein levels were markedly reduced.
Surprisingly, exposure to the combination of ATRA and G-CSF produced an
increase in the steady-state levels of RAR mRNA to a degree similar
to that found in cells exposed to ATRA alone. Recent studies have
definitively shown that ATRA induces proteasome-dependent degradation
of RAR in a variety of cell types.28 The
transcriptional up-regulation of RAR may be the cells' response to
counter this loss of receptor protein. Because the addition of G-CSF
with ATRA did not produce a further increase in RAR mRNA yet
protected the RAR pool, G-CSF signaling may interfere with some
aspect of the proteasomal degradation cascade.
Effects of G-CSF and ATRA on
steady-state levels of RXR was
detected in D+ cells, but its steady-state levels were not
changed after treatment with G-CSF, ATRA, or the combination thereof
(data not shown). Modest changes in the levels and mobility of RXR
protein, however, were detected in ATRA-treated cells (see Figure
5 for a representative blot). Laser
densitometric scanning of films exposed to Western blots (ECL
detection) indicated that the steady-state levels of RXR protein in
D+ cells treated for 24 hours with ATRA or G-CSF + ATRA
were increased by approximately 50% over those in untreated control
cells or in cells treated with G-CSF alone. The migration of most
RXR protein from cells incubated with ATRA was also slightly slower in the SDS-polyacrylamide gel than that from either untreated control
cells or from cells treated with G-CSF alone. Adding G-CSF to ATRA did
not appear to enhance or diminish this effect.
Evaluation of the induction of terminal differentiation by retinoid receptor-specific ligands RARs bind either ATRA or 9-cis-RA, whereas RXRs bind only 9-cis-RA. However, because these naturally occurring retinoids may be interconverted in target tissues,20 it was not possible to ascertain whether RARs, RXRs, or both contributed to the synergistic induction of differentiation produced by G-CSF and ATRA. Retinoid receptor subfamily-specific agonists and antagonists (see Fitzgerald et al29 for references) permitted a dissection of the roles of RARs and RXRs in the synergism exhibited by G-CSF and ATRA. Thus, RAR (TTNPB; 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) and RXR (LG100346; 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic acid methoxyoxime) -specific agonists provided evidence that both RAR and RXR participation were essential for induction of the differentiation of D+ cells (Figure 6). Given that neither RAR nor RXR activation alone was sufficient to produce differentiation of these cells, a heterodimer of RAR/RXR is likely to be the species involved. Moreover, these results suggest that both monomer partners must be bound by their respective ligands. This assumption is supported by the superior effectiveness of 9-cis-RA, which can bind and activate RARs and RXRs.
Effects of LiCl on retinoid receptor expression in ATRA-treated D+ cells When combined with ATRA, LiCl, like G-CSF, produced synergistic differentiation of D+ cells. We ascertained whether the effects of LiCl on RAR expression were comparable to those produced
by G-CSF. Western blot analyses showed that LiCl was at least as
effective as G-CSF in preventing ATRA-induced loss of RAR protein in
these cells (Figure 7A). Like G-CSF, LiCl
did not noticeably alter the levels of RAR mRNA (Figure 7B), and the
expression of RXR protein was also not affected by LiCl (Figure 7C).
Interestingly, both G-CSF and LiCl were capable of protecting RAR
pools in ATRA-treated cells by what appeared to be a nontranscriptional
mechanism while producing synergistic terminal differentiation of the
leukemia cells.
Effects of the prevention of the loss of RAR ) could
markedly limit the capacity of ATRA to produce terminal differentiation
of these cells. Thus, we postulated that the attenuation of the
ATRA-induced loss of RAR protein by coexposure to G-CSF or LiCl
could extend the response to the retinoid and thereby lead to the
synergistic induction of differentiation. Support for this concept
would be provided if prevention of the loss of RAR protein by other
means resulted in a heightened response to ATRA. To accomplish this,
D+ cells were transfected with an expression plasmid
containing RAR cDNA. Northern and Western blot analyses of 3 separate transfections are shown in Figure
8. Populations of these cells had a 6- to 8-fold increase in steady-state levels of RAR mRNA compared to vector-transfected control cells (Figure 8A) and showed a small but
significant increase (2- to 3-fold) in RAR protein (Figure 8B).
Single-cell clones were derived from these transfected populations by
flow cytometry, and clones with high RAR protein expression were
chosen for further evaluation. ATRA treatment was found to cause a
reduction in, but not a complete loss of, RAR protein in these
clones (Figure 9), which were found to be
considerably more responsive than parental WEHI-3B cells to ATRA as a
single agent (Table 2). These findings
support the concept that the basis for the synergistic interaction
between G-CSF or LiCl and ATRA in producing terminal differentiation of
D+ cells is at least in part caused by the prevention by
G-CSF and LiCl of the complete loss of RAR protein in
ATRA-treated cells.
Numerous investigations have established correlations between the
loss of retinoic acid receptors and malignant
progression.29-38 Similar to previous studies using other
cell lines,28 we have shown that ATRA induces a marked
decrease in the levels of RAR The RXRs also play important roles in retinoid action, and their
activation appears to be required for the maturation of D+
cells exposed to the retinoid. Western blot analyses of RXR G-CSF, initially identified by Nicola et al39 as a factor that induced the terminal differentiation of D+ cells, is widely used to stimulate neutrophil production after chemotherapy and in other syndromes accompanying neutropenia.40,41 Cases have been reported in which G-CSF, when used in combination with ATRA and chemotherapy, contributed to remission induction in patients with refractory APL,42-44 presumably because of the induction of terminal differentiation.42,45 The combination of these agents has also been used recently with some success in the treatment of myelodysplastic syndromes.46,47 These clinical responses, however modest, justify the search for more effective regimens of differentiation therapy. Lithium salts have long been known to have the capacity to
improve neutrophil production (see Boggs and Joyce17 for a
review). Thus, the use of this monovalent cation has been proposed as
an approach to minimize the myelosuppressive effects of anticancer and
antiviral therapies in humans. Previous work from our laboratory examined the possibility that lithium increased the formation of
granulocytes from immature leukemic precursors.16 LiCl, at millimolar concentrations, was found to induce the maturation of both
HL-60 human promyelocytic leukemia and D+ murine
myelomonocytic leukemia cells in culture. In preliminary studies of the
mechanism(s) involved, we found that KCl, but not NaCl or
MgCl2, could antagonize the differentiation-inducing
effects of LiCl alone or when combined with ATRA (data not shown). The significance of this observation is unclear. LiCl has been demonstrated to act on second messengers, blocking phosphatidyl inositol signaling pathways (see Berridge48 for a review). More recent
studies with lithium in the developmental field have shown that some of the effects of lithium are attributable to its inhibition of glycogen synthase kinase-3 One criticism of in vitro differentiation studies is that the concentrations of inducers required to elicit meaningful responses are not attainable in vivo. The use of agents such as G-CSF and LiCl in combination with retinoids may favorably impact on this limitation of differentiation therapy by allowing relatively low concentrations to be used. Drug combinations that include G-CSF are limited to blood cells that express G-CSFR. Combinations having LiCl as a constituent presumably will not be subject to the same limitation. This possibility entices speculation that retinoid-based differentiation therapy regimens for other forms of leukemia or for tumors originating from other tissue types (eg, lung, squamous cells of the head and neck) may be markedly improved by including LiCl to protect receptor pools.
Submitted January 6, 2000; accepted May 18, 2000.
Supported in part by United States Public Health Service grant CA-02817 from the National Cancer Institute. R.A.F. is a Special Fellow of the Leukemia and Lymphoma Society.
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.
Reprints: Alan C. Sartorelli, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520; e-mail: alan.sartorelli{at}yale.edu.
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Science.
2000;287:1606-1609
© 2000 by The American Society of Hematology.
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  T.-C. Chou Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies Pharmacol. Rev., September 1, 2006; 58(3): 621 - 681. [Abstract] [Full Text] [PDF]
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 A. Yen, R. Fenning, R. Chandraratna, P. Walker, and S. Varvayanis A Retinoic Acid Receptor {beta}/{gamma}-Selective Prodrug (tazarotene) Plus a Retinoid X Receptor Ligand Induces Extracellular Signal-Regulated Kinase Activation, Retinoblastoma Hypophosphorylation, G0 Arrest, and Cell Differentiation Mol. Pharmacol., December 1, 2004; 66(6): 1727 - 1737. [Abstract] [Full Text] [PDF]
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 A. M. Rice and A. C. Sartorelli Inhibition of 20 S and 26 S Proteasome Activity by Lithium Chloride. IMPACT ON THE DIFFERENTIATION OF LEUKEMIA CELLS BY ALL-TRANS-RETINOIC ACID J. Biol. Chem., November 9, 2001; 276(46): 42722 - 42727. [Abstract] [Full Text] [PDF]
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 M. Gianni', Y. Kalac, I. Ponzanelli, A. Rambaldi, M. Terao, and E. Garattini Tyrosine kinase inhibitor STI571 potentiates the pharmacologic activity of retinoic acid in acute promyelocytic leukemia cells: effects on the degradation of RAR{alpha} and PML-RAR{alpha} Blood, May 15, 2001; 97(10): 3234 - 3243. [Abstract] [Full Text] [PDF]
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C. D. Mao, P. Hoang, and P. E. DiCorleto Lithium Inhibits Cell Cycle Progression and Induces Stabilization of p53 in Bovine Aortic Endothelial Cells J. Biol. Chem., July 6, 2001; 276(28): 26180 - 26188. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
including, but not limited to, t(15;17) APL
