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Experimental Therapeutics |
Institut National de la Santé et de la Recherche Médicale (INSERM U496), Institut Universitaire d’Hématologie (Université Paris 7), Hôpital Saint-Louis (AP-HP), 75475 Paris (Cedex 10), France
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ABSTRACT |
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fusion
protein. We established that the four ATRA metabolites were indeed
formed by the NB4 cells in vitro. NB4 cell growth was
inhibited (69–78% at 120 h) and cell cycle progression in the
G1 phase (82–85% at 120 h) was blocked by ATRA and
all of the metabolites at 1 µM concentration. ATRA and
its metabolites could induce NB4 cells differentiation with similar
activity, as evaluated by cell morphology, by the nitroblue
tetrazolium reduction test (82–88% at 120 h) or by the
expression of the maturation specific cell surface marker CD11c. In
addition, nuclear body reorganization to macropunctated
structures, as well as the degradation of PML-RAR, was found to be
similar for ATRA and all of its metabolites. Comparison of the relative
potency of the retinoids using the nitroblue tetrazolium reduction test
showed effective concentrations required to differentiate 50% of cells
in 72 h as follows: ATRA, 15.8 ± 1.7
nM; 4-oxo-RA, 38.3 ± 1.3 nM;
18-OH-RA, 55.5 ± 1.8 nM; 4-OH-RA,
79.8 ± 1.8 nM; and 5,6-epoxy-RA,
99.5 ± 1.5 nM. The ATRA metabolites were
found to exert their differentiation effects via the RAR nuclear
receptors, because the RAR-specific antagonist BMS614 blocked
metabolite-induced CD11c expression in NB4 cells. These data
demonstrate that the principal ATRA Phase 1 metabolites can elicit
leukemia cell growth inhibition and differentiation in
vitro through the RAR signaling pathway, and they suggest
that these metabolites may play a role in ATRA antileukemic activity
in vivo. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ß, 
), with
various isoforms of each subtype (2)
. These receptors
belong to the superfamily of nuclear hormone receptors and act as
ligand-activated transcription factors of numerous genes
(reviewed in Refs. 2
and 3
). Retinoid
receptors interact as either homo- or heterodimers on specific hormone
response elements (RA responsive elements or RX responsive elements) of
target gene promoters (4, 5, 6)
. The transcriptional activity
of these receptors is further positively (holo-receptor) or negatively
(apo-receptor) modulated by ligand binding (reviewed in Refs.
7
and 8
). In addition, intracellular CRABPs
play a regulatory function in RA signaling (9)
. Biological
responses to ATRA may, therefore, be modulated by the type of nuclear
receptors present in cells, by nuclear cofactors, by the CRABPs,
and by the availability of specific retinoid ligands (reviewed in Ref.
10
).
The differentiating properties of ATRA have led to the development of
therapies for the prevention and treatment of various human cancers
(11)
including APL, in which retinoids are particularly
active (12)
. APL is a subtype of myeloid leukemia (M3),
characterized by the accumulation of cells blocked at the promyelocytic
stage. This leukemia exhibits a specific chromosomal translocation
t(15;17) involving the PML locus on chromosome 15 and the RAR
locus on chromosome 17, thus generating a chimeric gene
PML-RAR, translated into a chimeric nuclear receptor
PML-RAR (13, 14, 15, 16, 17)
. PML-RAR functions as an aberrant
receptor and is considered to be the cause of APL (18)
.
Although several clinical studies have established that ATRA can induce
leukemia cell differentiation and remission in almost all patients
(12
, 19)
, resistance to this therapy develops rapidly
(20)
. This acquired resistance appears to be
attributable in part to the decline in ATRA plasma levels below
therapeutic concentrations after repeated administration, presumably
caused by the induction of hepatic cytochrome P450s that may
increase its clearance (21, 22, 23)
. It has also been proposed
that increased CRABPs contribute to resistance by decreasing nuclear
availability of ATRA (24)
. More recently, mutations in the
E-ligand-binding region of the RAR chimeric protein PML-RAR have
been linked to ATRA resistance (25, 26, 27, 28, 29)
.
ATRA metabolism is complex and only partially understood at the present time (reviewed in Ref. 30 ). It can be oxidized by cytochrome P450s to various metabolites including 4-OH-RA, 4-oxo-RA, 18-OH-RA, and 5,6-epoxy-RA, which may be further metabolized to water-soluble glucuronides. Although ATRA metabolites are often considered to be catabolic products possessing low biological activity, some metabolites have been reported to be active in embryogenesis and to inhibit the growth of certain cancer cell lines (31, 32, 33, 34, 35, 36) . However, metabolism also seems to be involved in the response of certain cancer cells to ATRA, as shown by a study of breast cancer cell lines in which cell lines sensitive to ATRA growth inhibition (T47D and MCF7) could metabolize ATRA to polar metabolites (e.g., 4-oxo-RA and 4-OH-RA), whereas resistant cell lines (MDA-MB231 and MDA-MB418) metabolized ATRA poorly (35) . Similar results involving ATRA metabolism to polar compounds by other sensitive cancer cells have also been reported (37) .
As the metabolism of ATRA appears to be involved in its mechanism of
action in certain cancer cell lines, the purpose of this study was to
evaluate the in vitro effects of the principal oxidized ATRA
metabolites using the human NB4 promyelocytic leukemia cell line that
expresses the characteristic APL t(15;17) chromosomal translocation
(38)
. Our results show that NB4 cells can metabolize ATRA
to polar metabolites (4-OH-RA, 4-oxo-RA, 18-OH-RA, and 5,6-epoxy-RA),
and that these metabolites are active retinoids in vitro
with regard to cell growth inhibition, cell cycle arrest in
G1 phase, cell differentiation, NB
reorganization, and degradation of the chimeric protein PML-RAR. In
addition, when using a specific RAR antagonist, ATRA metabolites are
shown to act via the RAR nuclear receptor signaling pathway, as does
the parent compound.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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antagonist BMS614 was synthesized
by Bristol-Myers Squibb and kindly donated by Dr. H. Gronemeyer
(Institut de Génétique et de Biologie
Moléculaire et Cellulaire, Illkirch, France). Stock solutions of
retinoids were prepared in ethanol at 10-2
M and stored protected from light at -20°C.
Cell Culture.
The NB4 human APL cell line, developed in our laboratory
(38)
, was maintained in RPMI 1640 (Life Technologies,
Inc.) supplemented with 10% FCS (Bayer Diagnostics, Puteaux,
France), 2 mM L-glutamine, 50 units/ml
penicillin G, and 50 µg/ml streptomycin (Life Technologies, Inc.).
The cells were cultured at 37°C in a humidified atmosphere containing
5% CO2. Cell density was assessed with an
electronic particle counter and size analyzer (Coulter Electronics,
Hialeah, FL). Stock solutions of retinoids (at
10-2 M in ethanol) were further
diluted in culture medium to the final concentration indicated in each
experiment. Final concentration of ethanol in culture medium did not
exceed 0.01%. Stock solutions of retinoids were regularly checked for
purity using reversed-phase HPLC analysis and did not show any
contamination by ATRA. Cell cultures were diluted with fresh medium
every 48 h, and drug concentrations were adjusted accordingly. All
of the experimental procedures were light protected.
ATRA Metabolism in NB4 Cells and HPLC.
Cells (107) were incubated at 37°C in 2 ml of
the above medium with 1 µM ATRA for 24 h. After
acidification (HCl, 1%), cells and medium were extracted with ethyl
acetate (39)
, the organic phase recuperated by
centrifugation, and evaporated under a gentle nitrogen stream. The dry
residue was reconstituted in 100 µl of methanol, and 50 µl was
injected onto a HPLC system composed of a Varian 5000 chromatograph, a
C18 reversed-phase column (Beckman, Ultrasphere 5
µm, 4,6 mm x 250 mm), protected by a precolumn, a UV
detector set at 340 nm (Waters 484), and a peak integrator (Varian
4400). Elution was accomplished using a 25-min linear gradient starting
at 65% methanol/35% water with 1% ammonium acetate, and ending at
90% methanol/10% water pumped at 1 ml/min.
NBT Reaction.
Cells (5 x 105) were added to 450
µl of 1 mg/ml NBT solution containing 3 x 10-7 M PMA in PBS buffer, and
incubated at 37°C for 25 min. Cytospin preparations of 200 µl of
the cell suspensions (Cytospin; Shandon) were allowed to air-dry. The
NBT-positive and -negative cells were scored under phase contrast
microscopy examination (Leica) at x40. A minimum of 400 cells per
slide, scored with at least two scores, were performed per experimental
condition.
May-Grünwald Giemsa Staining.
Cytospin preparations of 2 x 105
cells were allowed to air-dry, incubated in pure May-Grünwald
solution for 5 min, then in 50% May-Grünwald/water for 10 min,
washed in water, and incubated in a 20% Giemsa/water solution for 20
min. The slides were then washed in water, air-dried, and examined
under immersion microscopy (Leica, x63).
Flow Cytometry Analysis of Cell Cycle Distribution.
Cells (1 x 106) were washed in
PBS and resuspended in 200 µl of PBS. The cells were fixed in 2 ml of
75% ethanol/water for 2 min, centrifuged, and resuspended in 1 ml of
PBS solution with 40 µg of propidium iodide and 100 µg of RNase A
for 30 min at 37°C. Propidium iodide (red) fluorescence was measured
using a FACScan flow cytometer (Becton Dickinson) with
104 cells acquired per sample.
Flow Cytometry Analysis of CD11c Cell Surface Integrin
Expression.
Cells (1 x 106) were incubated
with an anti-CD11c-PE, or the isotype control IgG2a-PE (Becton
Dickinson), for 20 min on ice in the dark. The cells were washed once
with PBS and resuspended in 500 µl of PBS. PE (red)
fluorescence was measured using a FACScan flow cytometer (Becton
Dickinson) with 104 cells acquired per sample.
Immunofluorescence.
Cytospin preparations of 106 cells were allowed
to air-dry for 24 h, fixed with acetone for 10 min at 4°C, and
air-dried for 20 min. The slides were rehydrated for 10 min in PBS and
then incubated with a polyclonal rabbit antiserum directed against a
PML recombinant protein (40)
at a dilution of 1:500 in PBS
for 1 h at room temperature. The slides were then incubated with
fluorescein-coupled antirabbit secondary antibody (Sigma) at a dilution
of 1:200 for 30 min at room temperature in the dark. All of the
incubations were followed by three washes in PBS for 5 min.
Preparations were examined under fluorescence microscopy (Leica, x63).
Western Blot.
Crude cytoplasmic and nuclear extracts were prepared from untreated and
treated NB4 cells. The cells were collected, washed once with cold PBS,
centrifuged at 1300 rpm at 4°C, and resuspended in buffer A [10
mM HEPES (pH 7.9), 1.5 mM MgCl2, 10
mM KCl, and 0.5 mM DTT] for 10 min at 4°C.
After centrifugation for 1 min at 5000 rpm at 4°C, cells were lysed
for 1 min on ice in buffer A containing 0.2% NP40, 1.5 µg/ml
protease inhibitor cocktail, and 0.2% phenylmethylsulfonyl
fluoride, and centrifuged 1 min at 5000 rpm. Supernatants corresponding
to cytoplasmic proteins were collected, and the nuclear proteins
contained in the pellets were extracted with buffer B [20
mM HEPES (pH 7.9), 0.2 mM EDTA, 0.5
mM DTT, 0.42 M NaCl, 25% v/v glycerol, 1.5
µg/ml protease inhibition cocktail, and 0.2 mM
phenylmethylsulfonyl fluoride) for 20 min on ice, and centrifuged 1 min
at 5000 rpm at 4°C. Cytoplasmic and nuclear extracts were frozen at
-80°C. Nuclear protein extracts (10 µg) were boiled in the
presence of 5% ß-mercaptoethanol and loaded onto 8%
SDS-polyacrylamide gels, electrophoresed, and blotted onto
polyvinylidene difluoride membranes (Immobilon P; Millipore). The
membranes were blocked with 4% nonfat milk in PBS for 3 h at room
temperature, then incubated with primary human antibody anti-RAR
[RP(F); 1:5000; Ref. 41
] in PBS-T (PBS and 0.1%
Tween 20) and 0.5% milk for 18 h at 4°C. Membranes were
washed five times for 5 min each with PBS-T, incubated with
horseradish peroxidase-coupled antirabbit antibody (1:10000; Jackson
Laboratories) for 30 min in PBS-T at room temperature, and washed five
times for 5 min with PBS-T. Detection was performed as described in the
ECL kit (Amersham; Ref. 42
).
Determination of Relative Potency of the Retinoids.
NB4 cells (2 x 105 cells/ml) were
exposed to ATRA or its metabolites at concentrations ranging from 7
nM to 1 µM, the NBT test was performed at
72 h, and the positive cells were scored as described above. The
data from three separate experiments were plotted on a semilogarithmic
scale to determine the EC50 that elicited half
maximal NBT-positive cells. Nonlinear regression analysis was carried
out using a sigmoidal dose-response curve and GraphPad software.
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RESULTS |
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shows the ATRA metabolites formed after 24 h in the presence of 1
µM ATRA. The metabolites formed were 4-oxo-RA, 4-OH-RA,
18-OH-RA, and 5,6-epoxy-RA, which presented concentrations of 0.06,
0.03, 0.08, and 0.39 µM, respectively. The stereoisomers
13-cis-RA and 9-cis-RA presented concentrations
of 0.07 and 0.16 µM, respectively, and ATRA of
0.19 µM.
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. Compared with untreated cells, the growth inhibition was
found to be similar for ATRA and the 4 metabolites tested, yielding
50% growth inhibition between 72 and 96 h. At 120 h, cell
growth was still similar for all compounds, and ranged from 22 to 31%
of untreated cells. Cell growth declined to 11–13% of controls for
ATRA and its metabolites at 168 h (data not shown). In parallel
experiments, the influence of ATRA or its metabolites on the cell cycle
distribution was assessed for 5 days (Fig. 2B)
. A
time-dependent cell cycle accumulation in the G1
phase was observed for all compounds tested, yielding 82–84%
G1 accumulation at day 5, at the expense of the S
and G2-M phases. These data are in agreement with
the observed growth arrest.
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. ATRA and its metabolites induced similar kinetics of
differentiation with comparable efficacy at 1
µM reaching 82–88% NBT positive cells in 5
days (Fig. 3B)
. Moreover, ATRA and its metabolites induced
the expression of the maturation-specific cell surface marker CD11c in
NB4 cells 24 h after retinoid exposure (Fig. 3C)
; at 48
or 72 h, there was little change in the percentage of CD11c
positive cells compared with that at 24 h, and no difference was
noted between ATRA and its metabolites with regard to the level of
CD11c expression (Table 1)
.
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Protein Degradation by ATRA
Metabolites. proteins in NB4
promyelocytic leukemia cells exposed to ATRA or its four metabolites at
1 µM for 5 days was analyzed by immunocytochemistry. The
nuclei from control cells presented fine and numerous labeled dots
containing PML (Fig. 4A)
, corresponding to the micropunctated NBs typically
found in APL cells. When the cells were exposed to ATRA, the
morphological reorganization was characterized by an increase in NB
size corresponding to macropunctated NBs, and a decrease in
their number. A similar pattern of NB reorganization to that seen with
ATRA was observed for all of the metabolites tested (Fig. 4A)
. As early as 24 h, NB reorganization could be seen,
which reached a maximum at 72 h. No major difference in NB size
was observed between 72 h and 120 h for ATRA or its
metabolites (data not shown). Concomitant to NB reorganization,
degradation of the PML-RAR chimeric protein by ATRA metabolites was
also assessed. ATRA metabolites treatment induced PML-RAR
degradation as did ATRA (Fig. 4B)
. The protein became barely
detectable at 48 h, with the emergence of a lower molecular weight
band corresponding to degradation of the chimeric protein (Fig. 4B)
.
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. ATRA was more efficient than its four metabolites in the induction of
differentiation, with effective concentrations to elicit 50%
(EC50) of NBT positive cells of 15.8 ± 1.7 nM. The metabolites yielded
EC50 values of 38.3 ± 1.3
nM for 4-oxo-RA, 55.5 ± 1.8 nM
for 18-OH-RA, 79.8 ± 1.8 nM for 4-OH-RA,
and 99.5 ± 1.5 nM for 5,6-epoxy-RA.
However, at higher concentrations (from 0.25 to 1 µM),
ATRA and its metabolites showed similar differentiation efficiency of
about 80% NBT positive cells.
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Antagonist on the Induction of Differentiation by
ATRA Metabolites. receptor, we therefore
blocked this pathway using the RAR-specific antagonist BMS614. NB4
cells were simultaneously treated with ATRA or its metabolites at 0.1
µM, in the absence or presence of the RAR-specific
antagonist BMS614 (1 µM) for 48 h (Fig. 6)
. As expected, BMS614 abolished ATRA-induced CD11c expression as shown
by the decrease in fluorescence intensity to near control values.
Likewise, induction of CD11c expression by the ATRA metabolites was
blocked by BMS614, which indicated that the metabolites induced
maturation through the RAR signaling pathway, as does the parent
compound.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It can be differentiated by ATRA treatment
and may be used to elucidate mechanisms of differentiation in APL, a
paradigm of a disease sensitive to differentiation therapy. Our results show that NB4 cells metabolize ATRA to the more polar metabolites 4-oxo-RA, 4-OH-RA, 18-OH-RA, and 5,6-epoxy-RA. ATRA metabolism is cytochrome P450-dependent and appears to involve several isoforms including CYP2C8 (45) and CYP26 (46 , 47 ; reviewed in Ref. 48 ). More recently, Marill et al. (49) have identified several other human cytochrome P450s significantly involved in the metabolism of ATRA.
ATRA and its metabolites were found to be equally active in terms of
growth inhibition of NB4 leukemia cells at 1-µM
concentration. These data are in agreement with those of van der Leede
et al. (35)
and van Heusden et
al. (33)
, who also observed growth inhibition of
breast cancer cell lines with ATRA metabolites (4-oxo-RA,
4-OH-RA, and 5,6-epoxy-RA). Rat rhabdomyosarcoma cells have been
reported to be sensitive to growth inhibition by 4-oxo-RA, 4-OH-RA, and
5,6-epoxy-RA (34)
. However, the NB4 leukemia cell line
used in this report appears to be more sensitive than solid tumor cell
lines, because 
90% growth inhibition was obtained in these cells
compared with 40–50% reported in MCF-7 cells or rhabdomyosarcoma
cells (33, 34, 35)
. To our knowledge, this is the first report
on the growth inhibitory activity of the principal Phase 1 metabolites
of ATRA in a promyelocytic leukemia cell line and, also, the first
account of the activity of the 18-OH-RA.
The ATRA metabolites were also shown to be active in regard to differentiation induction in the NB4 cells as evaluated by several criteria including cell morphology, NBT reduction test, and CD11c integrin expression. These metabolites were as active as the parent compound at concentrations between 0.2 and 1 µM. At lower concentrations, differences were seen between ATRA and its metabolites in their relative potency (EC50) to induce maturation of NB4 cells in the NBT reduction test. Although ATRA was found to be the most potent retinoid tested (EC50, 15.8 nM), 4-oxo-RA (EC50, 38.3 nM) is also very active, inasmuch as only a 2.4-fold difference was observed between this metabolite and the parent compound. It is of interest to note that the EC50 found for 4-oxo-RA is achievable in vivo in humans after ATRA administration, because concentrations in the range of 19–30 ng/ml (61–96 nM) can be reached (50) . In some studies, 4-oxo-RA has also been reported to be more active than ATRA in the modulation of positional specification in Xenopus laevis embryos (31) . With regard to the EC50 found for 4-oxo-RA, 4-OH-RA, and 5,6-epoxy-RA, similar relative potency data have been reported in the differentiation of rat rhabdomyosarcoma cells (34) . The relative potency of these natural retinoids may, therefore, be dependent on the cell type and on the physiological phenomenon considered. Comparison of the relative potency of ATRA metabolites may also be biased in the sense that the metabolites were applied exogenously to the cells, as opposed to the physiological situation in which the metabolites are formed intracellularly, via the cytochrome P450s.
Concomitant to the induction of differentiation in the NB4 cells by the
ATRA metabolites, we observed a reorganization of the nuclear
PML-containing bodies, and a degradation of the PML-RAR chimeric
protein, as has been previously observed with ATRA
(51, 52, 53)
. These data suggest that the metabolites share
the same signaling pathway as ATRA. To verify this, we used a
RAR-specific antagonist (BMS614) in combination with the
metabolites. CD11c expression analysis showed a complete inhibition of
CD11c expression induction when NB4 cells were cotreated with BMS614,
which indicated that ATRA metabolites act through the RAR pathway
for differentiation induction.
In summary, these data demonstrate new biological properties of the
ATRA metabolites and indicate that they are not ineffective catabolic
products. These metabolites may participate in the ATRA mechanism of
action on cell growth inhibition and differentiation via the RAR
receptor signaling pathway. In conclusion, the principal ATRA Phase I
metabolites are active retinoids which, provided that manageable
toxicity and in vivo pharmacological concentrations were
achievable, could be of interest for differentiation therapy of
sensitive human cancers.
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ACKNOWLEDGMENTS |
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antibody; Dr. Marcel Koken (Unité Propre de
Recherche 9051 CNRS, Paris) for the PML antibody; Dr. H.
Gronemeyer (IGBMC, Illkirch, France) for kindly providing the
RAR antagonist BMS614; and Christelle Doliger and Michel Schmid of
the Cellular and Molecular Imaging Service of our Institute for expert
assistance in image analysis. |
FOOTNOTES |
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1 Supported by the Institut National de la
Santé et de la Recherche Médicale (INSERM), the Centre
National de la Recherche Scientifique (CNRS), the Association pour la
Recherche sur le Cancer (ARC, Villejuif), and the Ligue Nationale
Contre le Cancer (Paris). N. I. was supported by a fellowship from the
Ministère de l’Education Nationale, de la Recherche et de la
Technologie. M. F. was supported by the Association Claude Bernard
(Paris). The results presented herein were presented in part at the
91st Annual Meeting of the American Association for Cancer
Research, San Francisco, CA, April 1–5, 2000 (Proc. Am. Assoc.
Cancer Res., 41: 14, abstract 92, 2000). 
2 To whom requests for reprints should be
addressed, at Institut Universitaire d’Hématologie, INSERM U496,
Centre Hayem, Hôpital Saint-Louis, 1 avenue Claude-Vellefaux,
75475 Paris (Cedex 10), France. Phone: 33-1-53-72-21-30; Fax:
33-1-42-40-95- 57; E-mail: gchabot{at}chu-stlouis.fr
3 The abbreviations used are: RAR,
retinoic acid receptor; RA, retinoic acid; APL, acute
promyelocytic leukemia; ATRA, all-trans RA; 4-OH-RA,
4-hydroxy-RA; 18-OH-RA, 18-hydroxy-RA; RXR, retinoids X receptor;
CRABP, cellular RA binding protein; HPLC, high-performance liquid
chromatography; NBT, nitroblue tetrazolium; NB, nuclear body; PMA,
phorbol 12-myristate 13-acetate; PE, phycoerythrin.
Received 5/16/00. Accepted 11/14/00.
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 M. G. Villani, V. Appierto, E. Cavadini, A. Bettiga, A. Prinetti, M. Clagett-Dame, R. W. Curley, and F. Formelli 4-Oxo-Fenretinide, a Recently Identified Fenretinide Metabolite, Induces Marked G2-M Cell Cycle Arrest and Apoptosis in Fenretinide-Sensitive and Fenretinide-Resistant Cell Lines. Cancer Res., March 15, 2006; 66(6): 3238 - 3247. [Abstract] [Full Text] [PDF]
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 L. Liu, J. B. Berletch, J. G. Green, M. S. Pate, L. G. Andrews, and T. O. Tollefsbol Telomerase inhibition by retinoids precedes cytodifferentiation of leukemia cells and may contribute to terminal differentiation Mol. Cancer Ther., August 1, 2004; 3(8): 1003 - 1009. [Abstract] [Full Text] [PDF]
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R. Qi, H. An, Y. Yu, M. Zhang, S. Liu, H. Xu, Z. Guo, T. Cheng, and X. Cao Notch1 Signaling Inhibits Growth of Human Hepatocellular Carcinoma through Induction of Cell Cycle Arrest and Apoptosis Cancer Res., December 1, 2003; 63(23): 8323 - 8329. [Abstract] [Full Text] [PDF]
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 L. Yang, H. Zhao, S.-W. Li, K. Ahrens, C. Collins, S. Eckenrode, Q.-g. Ruan, R. A. McIndoe, and J.-X. She Gene Expression Profiling during All-trans Retinoic Acid-Induced Cell Differentiation of Acute Promyelocytic Leukemia Cells J. Mol. Diagn., November 1, 2003; 5(4): 212 - 221. [Abstract] [Full Text] [PDF]
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S.-W. Li, D. Tang, K. P. Ahrens, J.-X. She, R. C. Braylan, and L. Yang All-trans-retinoic acid induces CD52 expression in acute promyelocytic leukemia Blood, March 1, 2003; 101(5): 1977 - 1980. [Abstract] [Full Text] [PDF]
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C. Pisano, P. Kollar, M. Gianni, Y. Kalac, V. Giordano, F. F. Ferrara, R. Tancredi, A. Devoto, A. Rinaldi, A. Rambaldi, et al. Bis-indols: a novel class of molecules enhancing the cytodifferentiating properties of retinoids in myeloid leukemia cells Blood, November 15, 2002; 100(10): 3719 - 3730. [Abstract] [Full Text] [PDF]
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N. Idres, J. Marill, M. A. Flexor, and G. G. Chabot Activation of Retinoic Acid Receptor-dependent Transcription by All-trans-retinoic Acid Metabolites and Isomers J. Biol. Chem., August 23, 2002; 277(35): 31491 - 31498. [Abstract] [Full Text] [PDF]
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Y. Jing, L. Xia, and S. Waxman Targeted removal of PML-RARalpha protein is required prior to inhibition of histone deacetylase for overcoming all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia Blood, July 18, 2002; 100(3): 1008 - 1013. [Abstract] [Full Text] [PDF]
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