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NEOPLASIA
From the Department of Biochemistry and Molecular
Biology, Medical College of Ohio, Toledo, OH, and the Department of
Immunopathology, St. Jude's Children's Research Hospital, Memphis,
TN.
Folate receptor (FR) type The human folate receptor (FR) is a single
polypeptide glycoprotein encoded by a family of 3 genes.1-4 FR- At present, the most effective treatment for myeloid leukemia is
retinoid differentiation therapy, for which acute promyelocytic leukemia (APL) is the paradigm, since in various studies 72% to 95%
of APL patients have shown complete remission owing to treatment with
all-trans retinoic acid (ATRA).18 Other types
of acute myeloid leukemia (AML), which constitute about 90% of
AML cases, do not respond well to ATRA therapy. Furthermore, in APL,
the initial favorable response to ATRA treatment is followed by a significant incidence of relapse, at which time the patients are refractory to ATRA differentiation therapy.18 In those
cases, ATRA resistance has frequently been related to mutations that cause blocks in the pathway of retinoid-induced
differentiation.19
We report here that in KG-1 myeloid leukemia cells and also in
leukemic blasts from patients with non-APL AMLs, ATRA induces FR- Cells and reagents
Treatment of cells with retinoid compounds or other
differentiation reagents
Treatment of leukemic blasts with ATRA Leukemic blasts from patient bone marrow aspirates, separated by Ficoll density gradient centrifugation, were received frozen from the Pediatric Oncology Group tissue bank (St. Jude's Children's Hospital, Memphis, TN). The cells were rapidly thawed under hot running water and immediately resuspended at 37°C in RPMI-1640 containing 30% FBS. The cells were sedimented at 400g for 10 minutes and seeded at a density of 2.5 × 105 cell/mL in RPMI-1640 medium containing 20% FBS with the inclusion of interleukin 3 (IL-3) (20 ng/mL), human stem cell factor (cKit ligand) (20 ng/mL), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/mL) (R&D Systems, Inc). The cells were treated with ATRA as described above.Folate binding assay Cells were washed with ice-cold acid buffer (10 mmol/L sodium acetate, pH 3.5, containing 150 mmol/L NaCl) and cold PBS sequentially. The cells were then incubated with 1 mL PBS containing a mixture of 5 pmol of [3H] folic acid (20.2 Ci/mmol) (Moravek Biochemicals, Brea, CA) and 25 pmol of unlabeled folic acid at 4°C for 30 minutes. After the incubation, the cells were washed twice with cold PBS. The acid buffer (1 mL) was used to extract bound [3H] folic acid, and the radioactivity was measured by liquid scintillation counting.Preparation of cell lysate and Western blot analysis Cells (1 × 106) were lysed at 37°C for 45 minutes in 30 µL of PBS containing 1% Triton X-100 and 1 mmol/L phenylmethanesulfonyl fluoride.20 After sedimenting the insoluble material by centrifugation at 10 000g for 10 minutes, we mixed 25 µL of the supernatant (cell lysate) with an equal volume of 2 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (62.5 mmol/L Tris-HCl, pH 6.8, containing 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.00125% bromophenol blue). The samples were electrophoresed on 12% SDS-PAGE gels and transferred to nitrocellulose filters. The blots were probed with affinity-purified rabbit antihuman FR- antibody followed by alkaline phosphatase-conjugated goat antirabbit immunoglobulin G (IgG) (Promega, Madison, WI) as previously described.2,20
Immunostaining for flow cytometry analysis Cells were washed and resuspended in PBS at a density of 1 × 107 cells per milliliter. Aliquots (100 µL) of the cell suspension were incubated with either rabbit anti-FR- antibody
or normal rabbit serum (1:20 dilution in PBS) on ice for 60 minutes
with intermittent gentle agitation. The cells were washed twice with PBS and incubated with fluorescein isothiocyonate (FITC)-conjugated goat antirabbit IgG (1:500 dilution) in 500 µL PBS on ice for 30 minutes with intermittent gentle agitation (Cappel, Durham, NC). After
2 more washes with PBS, the cells were fixed in 300 µL of 1%
paraformaldehyde and stored at 4°C. The samples were examined in a
flow cytometer within 24 hours.
MTT assay MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was purchased from Sigma (St. Louis, MO), and MTT cell proliferation assay was carried out following the supplier's protocol. Briefly, 90 µL aliquots of KG-1 or HL60 cells were added to each well of a 96-well plate followed by the addition of 10 µL of MTT stock solution (5 mg/mL) and incubation at 37°C for 3 to 4 hours. The reaction was stopped by adding 100 µL of 0.1 N HCl in isopropanol. The optical density was measured at 570 nm with background subtraction at 630 to 690 nm.Nitroblue tetrazolium reduction assay and -naphthyl acetate esterase (NAE) kit (Sigma) following the
manufacturer's protocol.
Treatment with phosphatidylinositol-specific phospholipase C Cells (1 × 106) were incubated in 500 µL of phosphatidylinositol-specific phospholipase C (PI-PLC) buffer (25 mmol/L Tris-HCl, pH 7.5, containing 250 mmol/L sucrose, 10 mmol/L glucose, and 1% bovine serum albumin) at 37°C for 1 hour in either the presence or the absence of 0.15 U of PI-PLC (Boehringer Mannheim Corp, Indianapolis, IN) with mild agitation. Cells were then washed twice with PBS, and cell lysates were prepared for Western blot analysis as described above. Alternatively, crude membranes were prepared from the PI-PLC-treated cells as described previously.20Isolation of total RNA and Northern blot analysis Total RNA was isolated with the use of Trizol reagent (Gibco-BRL) following the protocol provided by the vendor. Total RNA (30 µg) from each sample was analyzed by Northern blot as described previously.9 The blot was hybridized separately with [ -32P] deoxyadenosine triphosphate (3000Ci/mmol,
DuPont-New England Nuclear, Boston, MA) labeled complementary DNA
(cDNA) fragments of FR- (nucleotides 591 to 933), FR-
(nucleotides 558 to 900), or -actin.
Nuclear run-on assay This procedure is a modification of the one described by Garber et al.22 Briefly, cells (5 × 107) were lysed in 5 mL of lysis buffer A (15 mmol/L Hepes, 60 mmol/L KCl, 15 mmol/L NaCl, 0.15 mmol/L spermin, 0.5 mmol/L spermidine, 0.5 mmol/L egtazic acid, 2 mmol/L EDTA, 0.3 mol/L sucrose, and 14 mmol/L -mercaptoethanol) containing 0.1% nonidet P-40. The nuclei were
separated by centrifugation and finally resuspended gently in nuclear
storage buffer (50 mmol/L Tris, pH 8.3, containing 40% glycerol, 5 mmol/L MgCl2, and 0.1 mmol/L EDTA), snap-frozen on dry ice
and stored at 80°C up to 4 weeks. For each reaction, 107 nuclei were thawed on ice and resuspended with 300 µL
reaction buffer (50 mmol/L Tris, pH 8.0, containing 150 mmol/L KCl, 5 mmol/L MgCl2, 0.5 mmol/L MnCl2, 2 mmol/L
dithiothreitol, 0.1 mmol/L EDTA, 10% glycerol), and 60 U
RNase inhibitor from Roche Diagnostic Corp (Indianapolis, IN). The
reaction was initiated by the addition of ribonucleotides to a final
concentration of 0.5 mmol/L adenosine triphosphate, 0.5 mmol/L
guanosine 5'-triphosphate, 0.5 mmol/L cytidine
5'-triphosphate, and 100 µCi -32P] uridine
triphosphate (800 Ci/mmol, DuPont-New England Nuclear). The reaction
was first incubated in a water bath at 30°C followed by digestion
with 100 U DNase I (Roche Diagnostics Corp) and 33 µg of proteinase K
in 33 µL of 10 × second incubation buffer (100 mmol/L
Tris-HCl, pH 7.5, containing 5% SDS, 50 mmol/L EDTA) at 37°C or
42°C, respectively. Each incubation step lasted for 30 minutes. The
newly transcribed RNA was extracted with phenol-chloroform, precipitated with sodium acetate and ethanol, and purified on micro
bio-spin columns P-30 Tris (Bio-Rad Laboratories, Hercules, CA). The
purified RNA probes (1 × 106 to 3 × 106
cpm) were hybridized to FR- or -actin cDNA, which was immobilized on a nitrocellulose membrane, in 500 µL hybridization buffer at 45°C for 48 hours. Then the membrane was washed with 2 × SSPE at
45°C and sequentially washed with the same buffer containing either
RNase A (Roche Diagnostics Corp) or proteinase K at 37°C. The
membrane was then exposed to x-ray film.
DNA constructs For FR- promoter-luciferase constructs, the FR- genomic
DNA fragment 478nt to +64nt containing the transcription start-site at nucleotide +1 was inserted into the pGL3 basic vector (Promega) in
the polylinker immediately upstream of the luciferase reporter. The
retinoid acid receptor (RAR) , RAR , and RAR cDNA constructs were kindly provided by Dr Pierre Chambon (Institute de Genetique et de
Biologie Moleculaire et Cellulaire, Cude Strasbourg, France).
Transient transfection and luciferase assay We transfected 293 cells, at 50% to 70% confluence in 6-well tissue-culture plates, with 1 µg each of the FR-
promoter-luciferase construct, pSV- -gal (Promega), and RAR ,
RAR , or RAR cDNA constructs using lipofectamine (Life
Technologies, Inc, Gaithersburg, MD) according to the vendor's
protocol. At 8 hours after transfection was initiated, the medium
containing DNA complexes was removed and 2 mL of regular MEM medium
containing ATRA (1 µmol/L) or the vehicle alone was added to
each well. At 48 hours after transfection, the cells were harvested in
the reporter lysis buffer provided in the luciferase assay system
(Promega) and centrifuged at 14 000g for 2 minutes at room
temperature. The supernatant was assayed for luciferase,
-galactosidase activity, and total protein concentration, as
previously described.23
Stable transfection and isolation of transfectant clones We cotransfected 293 cells, at 60% confluence in a 100-mm plate, with 9 µg of the FR- promoter-luciferase construct and 3 µg of pcDNA1/Neo plasmid (Invitrogen, Carlsbad, CA) using
lipofectamine. At 40 hours after transfection, the cells were
transferred into MEM medium containing G418 (0.5 mg/mL) (Gibco-BRL). At
72 hours after transfection, the cells were split at different ratios
(1:3 to 1:24) and cultured in 100-mm plates. After about 2 more weeks, individual colonies were picked and cultured.
Specific and reversible up-regulation of FR- , which may be detected on the
cell membrane by FR- -specific antibodies (Figure
1). When the cells were grown in the
presence of ATRA, there was a dramatic dose-dependent increase in
FR- expression up to a concentration of 10 6 mol/L
ATRA. The elevation in FR- could be demonstrated by an increase in
the intensity of a protein of apparent molecular weight of
approximately 36 000 on a Western blot (Figure 1A) probed with a
rabbit antibody that is specific for FR- but that is not
cross-reactive with FR- .12 Flow cytometric quantitation
of the cell-surface-associated FR- showed an elevation of the
receptor by approximately 20-fold in the presence of 10 6
mol/L ATRA (Figure 1B). The ATRA-induced increase in FR- occurred progressively over a 5-day period (Figure 1C). Withdrawal of ATRA at
the end of a 5-day treatment resulted in an eventual decline in FR-
(Figure 1D), indicating that the effect of ATRA on FR- expression is
reversible. Treatment with 1 µmol/L ATRA for 5 days did not induce
FR- expression or appreciably alter FR- expression in a variety
of established cell lines, including myeloid leukemia cells, which
either did not express FR or expressed only FR- . The cell lines
tested and their FR expression pattern are indicated in Table
1.
PI-PLC sensitivity of ATRA-induced FR- expression and then treated with PI-PLC, there was a
quantitative decrease in the FR- detected by Western blot in the
cell lysate (results not shown). The ability of PI-PLC to release a
protein from the cell surface is a diagnostic test for the presence of a GPI membrane anchor in proteins including FR- . This result further confirms the cell surface localization and identity of the
ATRA-induced band on Western blots of cell lysates from KG-1 cells.
Effect of ATRA and other differentiating agents on growth,
differentiation, and FR- ) had no
apparent effect on FR- expression in KG-1 cells (Table 2).
Induction of FR- expression in leukemic cells in
the bone marrow of AML patients was tested under cell-culture
conditions in the presence of GM-CSF, IL-3, and cKit ligand to ensure
cell viability. Leukemic blasts and mononuclear cells were obtained from the patient bone marrow aspirates by Ficoll density gradient centrifugation and cultured in suspension in either the presence or
absence of 10 6 mol/L ATRA. The leukemic cells were
derived from 2 patients with French-American-British (FAB)-M2
type (AML with maturation) leukemia and 2 with FAB-M4 type
(acute myelomonocytic) leukemia. Subsequent Western blot analysis of
the cell lysates showed that ATRA induction of FR- expression
occurred in all of those samples (Figure
2). Furthermore, the increase in FR-
expression could be observed as early as 6 hours after the introduction
of ATRA (Figure 2A) and could persist up to 5 days of treatment (Figure
2D). These results extend the observed ATRA induction of FR- in the
KG-1 cell line to primary cultures of leukemic cells derived from
AML patients.
Effect of ATRA on FR- expression by ATRA in KG-1 cells was
accompanied by a specific increase in the FR- , but not FR- ,
transcript observed in Northern blots (Figure
3A). From nuclear run-on experiments, ATRA increased the transcription rate of the FR- gene in KG-1 cells
(Figure 3B). Under similar conditions, neither the mRNA level nor the
transcription rate was altered for a control housekeeping gene, the
-actin gene (Figure 3). It appears, therefore, that the induction of
FR- by ATRA in KG-1 cells may be accounted for, at least in part, by
direct or indirect modulation of the FR- gene by ATRA.
Modulation of FR- . The retinoid agonists 9-cis RA (pan-RAR, pan-retinoid X
receptors [pan-RXR]), TTNPB (pan-RAR), CD336
(RAR ), LG101093 (RAR and RAR ), and CD2781 (RAR ) induced
FR- expression, albeit to a lesser extent than ATRA (pan-RAR),
whereas CD417 (RAR ), CD2314 (RAR ), and LG100364 (pan-RXR) had no
detectable effect on the level of FR- (Figure
4; Table
3). Among the retinoid antagonists
tested, LG100629 (RAR ), CD2503 (RAR ), and CD2665 (RAR /RAR )
counteracted retinoid-agonist-induced FR- expression (Figure
4; Table 3). These results indicate that in KG-1 cells, up-regulation
of FR- by retinoids may be mediated by the nuclear receptors,
RAR , and/or RAR but not by RAR or by RXRs.
Interestingly, the induction of FR- Ability of RAR isoforms to mediate ATRA activation of the
FR- agonists to induce FR- expression in
KG-1 cells noted above could be attributed to the known lack of RAR
in KG-1 cells.24 To test whether RAR can mediate the
effect of ATRA on FR- expression, as well as to complement the
studies with retinoid compounds described in the previous section, it was of interest to test the effect of ATRA on the activity of the
FR- gene promoter and the effect of transiently expressing individual RAR isoforms on the action of ATRA. Owing to the technical difficulty of transfecting KG-1 cells, 293 cells were chosen for these
studies. For testing the FR- promoter activity, a luciferase reporter was attached downstream of a 542-base pair genomic fragment of FR- ( 478nt to +64nt), encompassing the transcription start site
at +1nt and the known upstream cis elements required for promoter
activity.25 ATRA treatment resulted in an increase in the
reporter activity in 293 cells transiently transfected with the
promoter construct (Figure 5A).
Co-expression of the nuclear receptors, RAR , RAR , and RAR , all
resulted in an enhancement of the ATRA effect (Figure 5A).
Overexpression of the nuclear receptors also resulted in relatively
small but significant increases in the FR- promoter activity, even
in the absence of ATRA treatment (Figure 5A); this effect is presumably
due to enhancement of the activity of endogenous retinoids in the cells
caused by overexpression of RARs. The results suggest that ATRA
induction of FR- may be mediated at least in part by activation of
the proximal promoter for FR- and that the RAR types , , and
are all capable of mediating this effect. Here, it may be noted
that in keeping with the narrow tissue specificity of FR- discussed
earlier, the endogenous FR- gene in 293 cells was not expressed
under any of the conditions in Figure 5A.
Response of the FR- promoter-luciferase
reporter fragment into the 293-cell genome, the majority of randomly selected recombinant 293 sublines (21 of 25) showed an induction of the
reporter luciferase activity upon ATRA treatment. In representative clones (Figure 5B), ATRA treatment produced up to a 10-fold increase in
luciferase activity progressively over a 5-day period, mimicking ATRA
induction of FR- in KG-1 cells. This result provides evidence that
modulation of the FR- promoter in the chromosome is a major mechanism of FR- induction by ATRA.
Even though FR- KG-1 cells cannot be induced to produce terminal differentiation by
ATRA,27,28 but do differentiate into macrophages in response to phorbol ester.29 Treatment of KG-1 cells with
ATRA for a 5-day period resulted in a progressive and dramatic increase in FR- The increase in the expression of FR- The effect of ATRA in promoting differentiation is mediated primarily
by the nuclear receptor isoform RAR The refractoriness of ATRA-resistant APL cells, as well as other
malignant cell types, to retinoid differentiation therapy has been
attributed to various resistance mechanisms. Reduced intracellular
availability of retinoids due to either accelerated in vivo
clearance37 or increased levels of cellular retinoic acid
binding protein38-40 may occur in the resistant cells. More recent studies have implicated alterations in the level of RAR Recent studies with variant APL-derived cell lines, as well as primary
cultures of non-APL myeloid leukemia cells, have characterized the cell
growth inhibition, terminal differentiation, and apoptosis resulting
from ATRA treatment of APL as independent events.49-51 The
therapeutic action of ATRA in APL is, therefore, the net result of its
pleiotropic actions in the cell. It follows that a block in any one
critical pathway of ATRA action leading to ATRA resistance should not
affect its ability to modulate a variety of genes. The studies
described in this article would predict that ATRA could
up-regulate FR-
We thank Dr Elizabeth Allegreto (Ligand Pharmaceuticals Inc, San Diego, CA) and Dr Uwe Reichert (Galderma Research and Development, Valbonne, France) for sharing various retinoid compounds; Dr Pierre Chambon (Institute de Genetique et de Biologie Moleculaire et Cellulaire, Cude Strasbourg, France) for providing the RAR cDNAs; Jenny Zak and Anna Chlebowski for typing the manuscript; and Thomas Sawyer for his invaluable help in the use of the flow cytometer.
Submitted March 27, 2000; accepted July 6, 2000.
Supported by National Institutes of Health R01 grants CA80183 and CA70873 to M.R.
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: Manohar Ratnam, Medical College of Ohio, Department of Biochemistry and Molecular Biology, 3035 Arlington Ave, Toledo, OH 43614-5804; e-mail: mratnam{at}mco.edu.
1. Lacey SW, Sanders JM, Rothberg KG, Anderson RG, Kamen BA. Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidylinositol. J Clin Invest. 1989;84:715-720. |