|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1758-1767
Targeting of PML/RAR Is Lethal to Retinoic Acid-Resistant
Promyelocytic Leukemia Cells
By
Kathryn Nason-Burchenal,
Janet Allopenna,
Agnes Bègue,
Dominique Stéhelin,
Ethan Dmitrovsky, and
Patrick Martin
From the Laboratory of Molecular Medicine, Department of Medicine and
Molecular Pharmacology and Therapeutics Program, Memorial
Sloan-Kettering Cancer Center, New York, NY; and CNRS UMR319 and CNRS
EP560, Institut Pasteur de Lille, Lille, France.
 |
ABSTRACT |
Acute promyelocytic leukemia (APL) cells, containing the t(15;17)
rearrangement, express the fusion protein, PML/RAR . Clinically, patients respond to all-trans retinoic acid (ATRA) through
complete remissions associated with myeloid maturation of leukemic
cells. This clinical ATRA response of APL is linked to PML/RAR
expression. Unfortunately, these remissions are transient and relapsed
APL is often ATRA-resistant. The role PML/RAR plays in the growth and maturation of these APL cells with acquired ATRA resistance has not
been fully explored. This study uses an ATRA-resistant NB4 cell line
(NB4-R1) to investigate the contribution of PML/RAR expression to
ATRA resistance. Targeting of PML/RAR in NB4-R1 cells was undertaken
using two approaches: homologous recombination and hammerhead
ribozyme-mediated cleavage. Reducing PML/RAR protein in NB4-R1 cells
rendered these cells more sensitive to ATRA. These cells were
growth-inhibited in ATRA, apoptosis was induced, and there was no
apparent signaling of differentiation. Sequence analysis identified a
mutation in the ligand binding domain (LBD) of the RAR portion of
PML/RAR. Results show that these retinoid-resistant NB4 cells
require persistent PML/RAR expression for leukemic cell growth.
Taken together, these findings can account for why these cells do not
respond to ATRA and how reduction of PML/RAR abrogates the
antiapoptotic effect it confers to these leukemic cells.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ACUTE PROMYELOCYTIC leukemia (APL) is
characterized by the t(15:17) that is specific for the
French-American-British (FAB) M3 subset of acute
leukemias.1 The t(15;17) yields a fusion product,
PML/RAR, which is linked to the induced clinical remissions of APL
patients after all-trans retinoic acid (ATRA) differentiation therapy.2-4 These complete remissions are not durable, and
relapse is often associated with clinical ATRA
resistance.3,4 Whereas variant translocations exist and
also involve RAR on chromosome 17, these are much less frequent than
t(15;17) APL cases.5,6 The precise role PML/RAR plays in
the ATRA response of APL remains to be defined. Studies indicate a
dominant-negative interference of this fusion protein on RAR -, PML-, or RXR -dependent pathways.7-11 It is known that
PML/RAR can homodimerize12-14 or heterodimerize with
PML10 or RXR.13 It is established that
PML/RAR can bind ATRA14 and activate transcription
through recognition of retinoic acid response elements
(RAREs).15 Transfection experiments indicate that
PML/RAR acts as an antiapoptotic factor in non-APL myeloid leukemic
cells. When U937 cells were transfected with PML/RAR, growth
factor-limited apoptosis was antagonized.11 This
finding suggests PML/RAR functions as an
antiapoptotic factor in retinoid-sensitive myeloid leukemic cells or
perhaps in retinoid-sensitive APL cells. Less is known of the role of
PML/RAR in regulating the growth and maturation states of
retinoid-resistant APL cells.
The causes of clinical retinoid resistance are under active study.
Studies have highlighted several potential retinoid resistance mechanisms. Pharmacokinetic studies in patients undergoing ATRA therapy
show that drug plasma concentrations decrease during continuous ATRA
therapy.16 Cellular retinoic acid-binding protein (CRABP) is upregulated during ATRA administration,17 which could
account for the decreased levels of ATRA measured in plasma. Other
studies report that defects in retinoic acid metabolism may account for resistance.18 Some laboratories have highlighted the role
of the multidrug resistance gene product in retinoid resistance. P-glycoprotein (Pgp) expression is reported as low in leukemic cells of
newly diagnosed APL patients,19 although treatment of APL
cells isolated from relapsed ATRA-resistant patients with the Pgp
antagonist verapamil allows these cells to mature.18
Because PML/RAR expression is tightly linked to the initial ATRA
clinical response in APL,20 this protein has been analyzed in ATRA-resistant NB4 cells.21-23 In one of these lines, a
dominant negative mutation in the ligand binding domain of the RAR
portion of PML/RAR is reported.24 This mutation leads to
altered binding to the ligand. In another ATRA-resistant NB4 line,
PML/RAR protein expression is undetected.22 In a
different reported line, PML/RAR expression is quite low until cells
are treated with ATRA.23 Other reported ATRA-resistant NB4
lines constitutively express PML/RAR protein.23
Using a de novo-derived ATRA-resistant NB4 cell line that we isolated
and designated NB4-R1,23 we sought to eliminate or reduce
PML/RAR expression and to identify potential PML/RAR mutations
through sequence analysis to assess its contribution to ATRA
resistance. To accomplish this, PML/RAR DNA was targeted using an
homologous recombination vector designed to eliminate this gene
product. Clones surviving drug selection displayed a reversal in
resistance to the growth-inhibitory effects of ATRA. Unexpectedly,
these clones could not be maintained in long-term culture, perhaps due
to the growth requirement for persistent PML/RAR expression. An
alternative approach taken to evaluate this dependency was to target
PML/RAR mRNA using a site-directed hammerhead ribozyme that
preferentially cleaves this mRNA four nucleotides downstream from the
fusion junction. The expression levels of the catalytic and
noncatalytic ribozymes were regulated by the dose of hygromycin used
for selection of the episomal vector-based ribozyme-transfected NB4-R1
cells. The engineering of the catalytic ribozyme, APL 1.1, and the
noncatalytic, antisense control ribozyme, APL 5.0, was previously
reported based on in vitro catalytic properties.25 The
current study extends this prior work by demonstrating a dependency on
persistent PML/RAR expression for growth of retinoid-resistant APL
cells. Whereas myeloid maturation is not triggered by targeting PML/RAR expression in NB4 ATRA-resistant cells, apoptosis is signaled and enhanced by ATRA treatment. Sequence analysis identified a
3-bp deletion in the ligand binding domain (LBD) of the RAR portion
of PML/RAR. This deletion eliminated a phenylalanine, but left the
reading frame intact. The implications of these findings for
retinoid-sensitive and -resistant APL cell growth are discussed.
| |
MATERIALS AND METHODS |
Cell lines and culture conditions.
NB4, a human APL cell line bearing the t(15;17), was a gift of Dr M. Lanotte (Paris, France). Two independent clones were isolated from
parental NB4 cells: an ATRA-sensitive clone, designated NB4-S1, and a
de novo isolated ATRA-resistant clone, designated NB4-R1.23
These lines were used in these studies. Cells were cultured in RPMI
containing 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L
glutamine, and 10% fetal calf serum. Cells transfected with the
knockout plasmid were selected and maintained in RPMI containing 500 µg/mL Geneticin (G-418 Sulfate; GIBCO BRL, Grand Island, NY).
Ribozyme-transfected cells were selected and maintained in RPMI
containing 65 µg/mL hygromycin. A selection/expression strategy for
episomal vector-transfected NB4 cells has been previously reported.26
Genomic DNA library.
Genomic DNA was extracted from NB4-S1 cells using standard techniques
and then partially digested with Sau3A I. Genomic DNA ranging
from 15 to 20 kb was selected on a sucrose gradient, ligated into  DASH II (Stratagene, La Jolla, CA) arms purified after a BamHI
digest, packaged into empty capsids (Stratagene), and amplified in
SRB(P2) bacteria (Stratagene).
Isolation of PML and RAR genomic clones.
RAR and PML genomic clones were selected using two cDNA-derived
radiolabeled probes. The RAR probe was a 500-bp Kpn
I-Sst I fragment from the 5 end of the coding sequence
corresponding to exons 3, 4, and 5. The PML probe was a 700-bp
Bgl II-Sst I fragment from the 5 end of the
coding sequence corresponding to exons 1, 2, and 3. For further
analysis and subcloning, one representative phage was chosen from each
set of positive clones. These two fragments were independently
subcloned into Bluescript SK+ (Stratagene) or pUC19 plasmids (American
Type Culture Collection, Rockville, MD) for detailed sequencing and
mapping analyses. A partial sequence analysis of a coding region from
both the PML and RAR selected fragments confirmed that these loci
corresponded to the previously published sequences for each
gene.7,27 The detailed restriction maps that were derived
(Fig 1) were in accord with previously
published maps for PML, RAR, and the breakpoint regions in
APL.28,29
View larger version (11K):
[in this window]
[in a new window]
| Fig 1.
Physical map of the PML/RAR homologous recombination
vector. Genomic DNA was isolated from NB4 cells as described in
Materials and Methods and detailed restriction maps were constructed in
the breakpoint region 3 (bcr3) for PML and RAR, as shown in this
schematic. The knockout vector was constructed using a 1.2-kb 5
sequence homologous to PML, followed by an in-frame, promotorless,
ATGless, neo gene and then by a 4.2-kb 3 sequence
homologous to RAR. The plasmid backbone is Bluescript SK+, and the
vector was linearized with EcoRI before transfection into
NB4-R1 cells.
|
|
Construction of PML/RAR homologous recombination
plasmid.
The plasmid used to knockout the PML/RAR-translocated locus was
constructed in Bluescript SK+ (Stratagene). A 5 region
homologous to PML consisted of a 1.2-kb Xba I-Sph I
fragment from intron 1 and a portion of the second coding exon (exon
2). This sequence was followed by the neomycin resistance gene,
neo, lacking both a promoter and an initiation codon, which was
inserted in frame with the AUG of PML. This in frame substitution of
the neo coding sequence, confirmed by sequence analysis, was
made in the second coding exon to eliminate the maximum amount of the
targeted gene product. The 3 homologous sequence was a 4-kb
EcoRI-Xba I fragment containing exons 5 and 6 of
RAR. These vectors were linearized with EcoRI before
transfection.
Control vectors.
Three control vectors were constructed and used. The first one tested
for a functional neo gene in the event of a successful homologous recombination in the PML/RAR locus. The first 86 amino acids of PML (EcoRI to klenow-treated Sph I) were fused
to the neo gene30 lacking the 10 amino-terminal
amino acids: this PML fragment was ligated to a Bluescript SK+ vector
(Stratagene) containing the neo gene and digested with
klenow-treated Eag I and EcoRI. The second control
vector assessed the incidence of random insertions or recombinations
that might occur with the homologous recombination vector that would
allow for a functional neo gene. To construct this, the splice
acceptor and 36 amino acids from exon 3 of RAR were fused to the
above described ATGless neo vector. The third control vector,
pKJ-1, was used to control for insertional mutagenesis and for the
phenotype of cells maintained in high doses of G418. The pKJ-1 vector
(a gift of Dr Jean-Christophe Bories, Paris, France) is a pUC 18 backbone containing the phosphoglycerate kinase (PGK) promoter driving
neo. This was linearized with HindIII before transfection.
Ribozymes and expression vector.
The hammerhead ribozymes used in this study were ribozyme APL 1.1, which is known to catalyze cleavage of PML/RAR mRNA, and APL 5.0, a
noncatalytic, antisense control, as described previously.25 These ribozymes, contained in self-cleaving cassettes, were cloned into
the HindIII/Not I sites of the Eboplpp vector. The
episomal Eboplpp vector contains the hygromycin resistance gene for
drug selection and is useful to regulate expression of the inserted ribozymes or other genes, as described previously.26
Transfections.
NB4-R1 cells (5 × 106) were washed once
and resuspended in phosphate-buffered saline (PBS)
(Ca2+/Mg2+ free). Twenty micrograms of plasmid
DNA was then added and the cells were electroporated at 0.3 kV and 25 µF.
Protein isolation and Western analysis.
NB4-R1 cells (107) were pelleted, resuspended in 1 mL of
lysis buffer (150 mmol/L NaCl; 50 mmol/L Tris, pH 8.0; 1% NP-40; 0.5% deoxycholate; 0.1% sodium dodecyl sulfate [SDS]; 0.5 µg aprotinin, leupeptin, pepstatin, antitrypsin, and chymostatin; and 1 mmol/L phenylmethyl sulfonyl fluoride [PMSF]), and incubated for 30 minutes at 0°C. The samples were centrifuged at 12,000 rpm, and the
supernatant was stored on ice. Protein concentration was assessed with
the Bradford assay, and 50 µg protein per sample was electrophoresed on a 10% SDS-polyacrylamide gel. The protein was electrotransferred from the gel to supported nitrocellulose (Amersham International, Little Chalfont, UK). The ECL Western blotting protocol and kit (Amersham International) were used for antibody labeling and detection and membranes were exposed to high-speed film (FR Images, Mamaroneck, NY). The RAR (F region) RP(F) antibody (provided by Dr Pierre Chambon, Strasbourg, France) recognizes the PML/RAR protein. Membranes were stained with Ponceau-S (Sigma, St Louis, MO) to control
for equal protein loading.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
Thiazolyl blue (MTT) proliferation assay.
On each day of the MTT assay,31 100 µL of cells was taken
from each of the culture conditions and placed, in triplicate, in a
96-well plate. Fifty micrograms of MTT was added to each well and this
mix was incubated for 4 hours at 37°C. At the end of incubation,
100 µL of 0.04 N HCl in 2-propanol was mixed thoroughly into each
well. Plates were read on a Molecular Devices microplate reader
(Sunnyvale, CA) at a wavelength of 570 nm, with a background reading at
650 nm subtracted. Triplicate readings for each sample were averaged.
Trypan blue dye (Sigma) exclusion was used to assess viability.
Nitroblue tetrazolium (NBT) reduction assay.
For each cell sample, 200 µg NBT solution (Sigma), 105
cells, and 100 ng phorbol 12-myristate 13-acetate (PMA; Sigma) were mixed gently. Samples were incubated at 37°C for 15 minutes and then at room temperature for 15 minutes. Subsequently, 0.1 mL of cells
from each sample was cytospun, fixed in methanol, and stained with
Safranin-O (Fisher Scientific, Pittsburgh, PA). A minimum of 100 cells
was scored microscopically as NBT negative or positive (blue).
Terminal deoxynucleotidyl transferase (Tdt) assay.
For fixation, 106 cells were centrifuged, resupended in 1%
formaldehyde, incubated on ice for 15 minutes, washed in PBS, and resuspended in 70% ethanol. To perform this assay, cells were centrifuged, rehydrated in PBS, and pelleted. The cell pellet was
resuspended in Tdt reaction buffer (40 mmol/L cacodylate buffer; 2.5 mmol/L CoCl2; 7.5 U Tdt enzyme [Boehringer Mannheim,
Indianapolis, IN]; and 0.5 nmol/L biotin dUTP [Boehringer
Mannheim]) and incubated for 30 minutes at 37°C.
After incubation, PBS was added and cells were centrifuged. The pellet
was resuspended in 100 µL staining buffer (4× SSC, 0.1% Triton
X-100, 5% dry milk, and 25 µg/mL fluoresceinated avidin) and
incubated for 30 minutes at room temperature in the dark. A wash
solution of PBS with 0.1% Triton X-100 was added and the sample was
centrifuged. The cells were washed again and finally resuspended in 0.5 mL propidium iodide stain (5 µg/mL propidium iodide + 0.1% RNAseA),
incubated overnight at room temperature, and stored at 4° C until
analysis. Samples were analyzed on a FACScan (Becton Dickinson,
Franklin Lakes, NJ).
Sequence analysis.
A reverse transcription-polymerase chain reaction (RT-PCR) was
performed on DNase (Promega, Madison, WI) -treated total cellular RNA
with a 21mer homologous to a sequence in the 3UTR of RAR. PCR
was performed on the resultant cDNA using a set of nested primers. For
both sets of inner and outer primers, the 5 primers were located
in PML, 5 of the fusion junction. The outer 3 primer was
the same as was used for RT-PCR and the inner 3 primer was just
internal to that, also located in the 3 UTR of RAR. Three independent PCRs were performed and the PCR reactions were cloned into
the TOPO TA Cloning kit as per the manufacturer's instructions (Invitrogen, Carlsbad, CA). One clone from each reaction was sequenced using the ABI Sequencing kit and the ABI 373A Automated Sequencer (Perkin Elmer, Applied Biosystems Division, Foster City, CA). Both
strands of each PML/RAR clone were sequenced. The ABI Autoassembler and Prism Sequencing softwares (Perkin Elmer) were used in the sequence
analysis.
| |
RESULTS |
NB4-R1 cells transfected with the PML/RAR recombination
vector reverse ATRA resistance.
To confirm that the neo gene would be functional if there were
an effective recombination, NB4-S1 cells (an ATRA-sensitive NB4 clone)
were transfected with a control vector that was engineered to contain
the expected fusion sequence of PML/neo should the anticipated
homologous recombination occur. The PML/neo gene proved functional by allowing transfected NB4-S1 cells to proliferate in doses
of G418 that selected against the nontransfected cells.
A second control vector was designed to assess the background incidence
of random integration or recombination that would result in an in-frame
alignment of the knockout vector with a gene other than PML/RAR.
This vector consisted of a splice acceptor fused to a promotorless,
ATGless neo. No clones were derived using this vector despite
multiple transfections, indicating that precise random integration was
rare.
A third vector (pKJ-1) was used to control for phenotypic changes in
cells due to insertional mutagenesis or to culture in high doses of
G418. This vector was efficiently transfected and integrated into
NB4-R1 cells and pools of these transfectants served as controls for
these recombination experiments.
Multiple transfections were performed with the NB4-R1 cells using the
PML/RAR homologous recombination vector depicted in Fig 1. Rare
clones were derived from these transfections. Of the 15 clones isolated
after G418 selection, 5 displayed a marked sensitivity to ATRA and the
other 10 were similar to the parental NB4-R1 cells, with little to no
growth-suppressive response to ATRA treatment.
This reversal in ATRA response of the NB4-R1 cells was initially
determined using the MTT proliferation assays
(Fig 2). The graph on the left of Fig 2
depicts the response of NB4-S1 and NB4-R1 cells to
10 5 mol/L ATRA. This is a 10-fold higher dose than
is necessary to growth-inhibit and differentiate ATRA-sensitive NB4-S1
cells. This dose was selected to emphasize the ATRA-resistant
properties of NB4-R1 cells and to contrast this ATRA response to that
observed in transfectants. As shown, NB4-S1 cells are growth-inhibited in the presence of ATRA, whereas NB4-R1 cells are not. The graph on the
right of Fig 2 shows that NB4-R1 cells transfected with the control
vector (pKJ-1) continue to proliferate in ATRA (but a representative
putative knockout clone reversed this resistant phenotype) and became
sensitive to the growth-inhibitory effects of ATRA.
View larger version (21K):
[in this window]
[in a new window]
| Fig 2.
MTT proliferation assay performed on control and knockout
NB4-R1 cells. The MTT assay was performed on the indicated days (X
axis) in the absence ( ATRA) or presence (+ ATRA) of 105 mol/L ATRA. The Y axis depicts the
optical density (O.D.). The growth curves in the left graph depict
untransfected NB4-S1, an ATRA-sensitive NB4 clone, and NB4-R1, the de
novo ATRA-resistant clone, from which the knockout subclones were
derived. The right graph depicts the growth curves for NB4-R1 cells
transfected with the pKJ-1 vector (Control) as a control for
insertional mutagenesis and growth in G418 and for one representative
knockout subclone (K.O.) that shows a reversal in resistance to growth
inhibition by ATRA treatment.
|
|
When this phenotypic change was observed, molecular analysis commenced.
Genomic DNA was isolated from nontransfected NB4-S1 and NB4-R1 cells to
serve as a negative control. Genomic DNA was also isolated from NB4-R1
cells transfected with the homologous recombination vector, from clones
that showed no apparent reversal in phenotype, and from others that did
reverse phenotype. Southern analysis was performed on genomic DNA
digested with Sst I. When probed with an RAR Sal
I/Sau I genomic DNA fragment, a unique approximately 4.2-kb
band would be present if RAR genomic sequence had been perturbed.
This band was identified only in cells that had undergone a reversal in
phenotype (data not shown). A neo probe also hybridized to this
same band, indicating the homologous recombination vector was present
in this approximately 4.2-kb fragment (data not shown). To fully
diagnose a recombination and the precise site of recombination,
multiple restriction endonuclease digestions were to be coupled with
appropriate probes used in Southern analyses. Unexpectedly,
transfectants anticipated to have undergone DNA recombination and
reversal of retinoid resistance survived only transiently in culture,
even in the absence of ATRA. The limited genomic DNA available did not
allow for additional analyses. This suggested that targeting PML/RAR
was lethal to NB4-R1 cells. It also indicated an alternative strategy
was needed to target PML/RAR, as discussed below.
Targeting of PML/RAR mRNA with a site-specific
ribozyme.
To circumvent the observed instability of the putative
PML/RAR-targeted NB4-R1 cells, another targeting strategy of this fusion gene product was undertaken. The use of a catalytic ribozyme that cleaves PML/RAR mRNA was reported previously.25 It
was hypothesized that effective recombination of PML/RAR accounted for the reversal of ATRA resistance in NB4-R1 cells. To learn whether
regulated reduction of PML/RAR could be accomplished in NB4-R1
cells, the PML/RAR catalytic ribozyme, designated APL 1.1, and a
noncatalytic control, designated APL 5.0, were used. Ribozyme APL 5.0 served as an antisense control, because it would bind to, but not
cleave PML/RAR mRNA. The use of episomal vector-based expression of
these ribozymes permitted regulation of their expression. Figure 3 depicts schematically the
ribozymes as they would hybridize to PML/RAR mRNA, spanning the
junction between PML and RAR. As noted, the cleavage site of APL 1.1 is four nucleotides downstream of the junction.
View larger version (14K):
[in this window]
[in a new window]
| Fig 3.
Schematic of the catalytic hammerhead ribozyme, APL 1.1, and the noncatalytic control, APL 5.0, hybridizing to PML/RAR mRNA.
The two hybridizing arms (15 and 14 nucleotides [nt], respectively)
of APL 1.1 are shown in dark black flanking the catalytic core (22 nt).
The noncatalytic control ribozyme, APL 5.0, is identical to APL 1.1 except for one nucleotide substitution and one nucleotide deletion, as
indicated. The PML ( )/RAR ( ) mRNA is depicted, with the
cleavage site indicated.
|
|
Western analysis of ribozyme transfected NB4-R1 cells.
The ribozyme-transfected NB4-R1 cells were selected and maintained in
65 µg/mL G418 before analyses. To assess cleavage of the PML/RAR
mRNA, cells were cultured in 65 or 195 µg/mL hygromycin for 5 days in
the absence () or presence (+) of 106 mol/L
ATRA for 5 days (Fig 4). Total cellular
protein was extracted and Western analysis was performed. An RAR
antibody was used to detect the PML/RAR protein, as described in
Materials and Methods. Even at the maintenance dose of 65 µg/mL
hygromycin, it is evident from Fig 4 that there is less PML/RAR
protein in cells containing the catalytic ribozyme than in those with
the control ribozyme. This decrease is augmented when the dose of hygromycin is increased to 195 µg/mL. It is notable that APL 1.1 transfectants have a further decrease in PML/RAR protein expression after exposure to ATRA, at both doses of hygromycin. This is consistent with findings obtained using ATRA-sensitive NB4 cells, which have a
decrease in the level of PML/RAR protein after exposure to ATRA.23
View larger version (14K):
[in this window]
[in a new window]
| Fig 4.
Western analysis for PML/RAR expression in
ribozyme-transfected NB4-R1 cells. NB4-R1 cells transfected with either
the control ribozyme, APL 5.0, or the catalytic ribozyme, APL 1.1, were
cultured in 65 or 195 µg/mL hygromycin (hygro) in the absence ()
or presence (+) of 106 mol/L ATRA for 5 days. Total
cellular protein was extracted and analyzed by Western analysis, using
an anti-RAR antibody to detect PML/RAR protein. Relative to APL
5.0 transfectants, this immunoblot indicates that a marked reduction of
PML/RAR expression occurs in APL 1.1-transfected NB4-R1 cells,
especially after ATRA treatment and selection at the 195 µg/mL
hygromycin dosage.
|
|
NB4-R1 cells transfected with a PML/RAR site-directed
catalytic ribozyme acquire sensitivity to ATRA.
NB4-R1 cells, selected and maintained in 65 µg/mL hygromycin, were
cultured in the same or 195 µg/mL hygromycin in the absence ()
or presence (+) of 106 mol/L ATRA for 6 days. MTT
proliferation assays were performed on days 3 through 6, as shown in
Fig 5A. The left graph shows that, in 65 µg/mL hygromycin, cells transfected with APL 5.0, the noncatalytic
control, continue to grow, even in ATRA, whereas APL 1.1 transfectants
proliferated poorly in the absence of ATRA and did not proliferate in
the presence of ATRA. In the graph on the right of Fig 5A, results are
displayed for the transfected cells cultured in 195 µg/mL in the
absence () or presence (+) of ATRA. APL 1.1 transfectants
proliferated poorly in the absence or presence of ATRA, whereas cells
containing APL 5.0 grew well in the absence of ATRA, with some growth
suppression in the presence of ATRA, perhaps due to antisense effects
that cooperate with retinoid signals at the higher doses of hygromycin.
It is noteworthy that cell cycle analysis of the growth inhibited cells
did not indicate that there was arrest at a particular stage in the
cell cycle (data not shown).
View larger version (20K):
[in this window]
[in a new window]
View larger version (37K):
[in this window]
[in a new window]
| Fig 5.
MTT proliferation assay and analysis of apoptosis in
ribozyme-transfected NB4-R1 cells. (A) Cells transfected with either
the control ribozyme, APL 5.0, or the catalytic ribozyme, APL 1.1, were
cultured in 65 or 195 µg/mL hygromycin in the absence () or
presence (+) of 106 mol/L ATRA for 5 days. The MTT
assay was performed on days 3 through 6 (X axis). The Y axis depicts
the optical density (O.D.). These data are from one experiment
representative of four independent analyses. (B) Untransfected NB4-R1
cells and cells transfected with either the control ribozyme, APL 5.0, or the catalytic ribozyme, APL 1.1, were cultured without hygromycin (0 µg/mL) or with 65 or 195 µg/mL hygromycin (hygro) in the absence
() or presence (+) of 106 mol/L ATRA for 4 days.
The Tdt assay was used to assses induction of apoptosis () on day 4. Viability was concurrently measured using trypan blue dye exclusion
( ). These data are from one experiment representative of four
independent analyses.
|
|
The observed growth inhibition was not associated with induced
differentiation, as shown in Table 1. The
NBT assay, which measures functional maturation of myeloid cells,
indicated in none of the treatment groups was there an appreciable
maturation response, as compared with 85% NBT positivity in the
ATRA-treated NB4-S1 cells.
A response of NB4-R1 transfectants to the catalytic ribozyme was
induction of apoptosis that was enhanced by ATRA treatment, as seen in
Fig 5B. For the Tdt assay, cells were cultured in 65 or 195 µg/mL
hygromycin in the absence () or presence (+) of ATRA
(106 mol/L) for 4 days. The gray bars in Fig 5B
depict the percentage of apoptosis (left Y axis) on day 4. The basal
level of apoptosis in nontransfected NB4-R1 cells was 2%, and ATRA
treatment did not induce apoptosis (1%). There was a low level of
apoptosis measured in cells transfected with APL 5.0 in the absence of
ATRA in 65 µg/mL (3%) and 195 µg/mL hygromycin (3%). In ATRA,
there was a slight increase in apoptosis at 65 µg/mL hygromycin (5%) and at 195 µg/mL hygromycin (6%). In contrast, NB4-R1 cells
transfected with APL 1.1 in the absence of ATRA at 65 µg/mL had an
increased level of apoptosis (10%), and when treated with ATRA,
apoptosis increased to 12%. When the dose of hygromycin was increased
to 195 µg/mL, in the absence of ATRA, there was 12% apoptosis; in the presence of ATRA, this increased to 26%. The viability remained high (>88%) in all of the APL 5.0-transfected cells, but decreased in APL 1.1-expressing cells at 65 µg/mL in the absence (63%) and presence (57%) of ATRA and at 195 µg/mL in the absence (54%) and presence (27%) of ATRA.
DNA sequence analysis identifies a mutation in the ligand binding
domain of PML/RAR.
The entire RAR portion of PML/RAR from NB4-R1 cells was sequenced
and compared with the published DNA sequence from parental NB4
cells.7 Sequence analysis identified a 3-bp deletion in the
LBD of RAR present in each of the clones sequenced. As depicted in
Fig 6, the last base pair in codon 778, a
threonine (ACC), and the first 2 bp in codon 779, a
phenyalanine (TTC), were deleted. The resulting ACC from the
3-bp deletion retained a threonine, but eliminated a phenylalanine. The
reading frame was preserved.
View larger version (12K):
[in this window]
[in a new window]
| Fig 6.
Sequence analysis of the RAR portion of PML/RAR.
(A) RT-PCR was performed on total cellular RNA from NB4-R1 cells using
a primer in the 3 UTR of RAR as indicated in this figure.
Three independent PCR reactions were performed on the cDNA generated
from the RT-PCR reaction. Nested primers were used as indicated in the
figure: both inner and outer 5 primers were in PML and both
3 primers were in the RAR UTR. The 3 outer primer was
the same as was used for the RT-PCR reaction. (B) Sequence analysis
showed a 3-nucleotide deletion: C was deleted from codon 778 and TT
from codon 779. This eliminated a phenylalanine but retained a
threonine. The reading frame was not disrupted.
|
|
| |
DISCUSSION |
Although ATRA treatment of t(15; 17) APL cases induces complete
clinical remissions, these remissions are transient. After single-agent
ATRA treatment of APL, clinical ATRA resistance often follows.3,4 In the in vivo setting, this ATRA resistance is
associated with reduced ATRA levels after continuous ATRA
therapy.16 This is associated with defects in retinoic acid
metabolism18 and with upregulation of CRABP.17
In the in vitro setting, ATRA APL resistance has also been
investigated. Retinoid-resistant NB4 cells were derived by several
groups, including our laboratory.21-23 In one NB4
retinoid-resistant line, a dominant-negative mutation was found within
the ligand binding domain of the RAR region of
PML/RAR.24 This results in altered ATRA binding to this receptor. In another ATRA-resistant NB4 line, PML/RAR protein, but
not mRNA expression, is undetected.22 In contrast, in the NB4-R1 line examined in this study,23 PML/RAR protein
expression is detected both before and after ATRA treatment.
This study was undertaken to explore directly the role of PML/RAR
expression in the growth and maturation states of the
retinoid-resistant NB4 line, NB4-R1. To explore the biologic effects of
PML/RAR in retinoid-resistant NB4-R1 cells, two genetic strategies
were taken to target PML/RAR expression. The first strategy was to introduce an homologous recombination vector that would target PML/RAR. In the results displayed within Fig 2, the findings obtained using this targeting vector indicated that a subset of isolated clones displayed an acquired sensitivity to ATRA-mediated growth suppression. Unexpectedly, these clones could not be
continuously maintained in culture and could not be genetically
characterized in detail. This suggested reduction of PML/RAR
expression conferred a major growth disadvantage to these
transfectants. Consistent with this view is the finding that two
control vectors designed to assess background incidence of random
integration failed to elicit this reversal of ATRA resistance. From
these experiments, it was speculated that a growth disadvantage existed
for these putative PML/RAR-targeted NB4-R1 transfectants and that
acquired ATRA sensitivity was linked to reduced PML/RAR expression.
Further experiments were undertaken to explore this possibility.
The second strategy taken to target PML/RAR expression involved
hammerhead ribozymes engineered to cleave PML/RAR mRNA. Our prior
work reported that PML/RAR transcripts are efficiently cleaved by
the hammerhead ribozyme, APL 1.1.25 In contrast, a control
hammerhead ribozyme, designated APL 5.0, will hybridize to PML/RAR
mRNA but will not cleave this transcript. These two hammerhead
ribozymes were used in transfection experiments involving the
retinoid-resistant APL line, NB4-R1. The results of experiments displayed in Fig 4 indicate that PML/RAR expression is markedly repressed in the APL 1.1 versus APL 5.0 transfectants. When the PML/RAR catalytic but not control ribozymes are introduced into NB4-R1 cells, findings show that NB4-R1 cell growth depends on the
selective pressure used to express APL 1.1 hammerhead ribozymes in
transfectants. Whereas a reduction of PML/RAR expression was observed at the lower hygromycin dosage for APL 1.1 (see Fig 4), at the
higher hygromycin dosage used for this transfectant (195 µg/mL), an
even greater reduction of PML/RAR expression was detected. Notably,
after ATRA treatment, a further reduction of PML/RAR expression was
observed. A tight link was found to exist between this reduced
PML/RAR expression and the growth properties of this transfectant,
as shown in Fig 5. As expected, the control hammerhead ribozyme, APL
5.0, which can hybridize to PML/RAR but not cleave this transcript,
had growth properties similar to parental NB4-R1 cells23
(data not shown). For APL 1.1 transfectants maintained at even higher
hygromycin dosages, the observed growth suppression was even more
prominent, suggesting that a greater reduction of PML/RAR expression
was incompatible with the maintenance of leukemic cell growth. This
view is supported by the experiments conducted using the homologous
recombination vector described in Fig 1.
It is notable that the observed growth suppression of the NB4-R1 cells
transfected with the APL 1.1 hammerhead ribozyme was due to reduced
viability at the higher dose of hygromycin selection. This reduced
viability was at least partly due to the signaling of apoptosis, as
shown in Fig 5B. Cell cycle analysis did not indicate arrest in a
specific phase of the cell cycle coincident with cell death (data not
shown). This observation is consistent with other studies of apoptosis.
For example, N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in
HL-60 cells did not block cells in a particular phase of the cell
cycle.32 As anticipated by the Western analysis for
PML/RAR expression in APL 1.1 transfectants treated with ATRA, an
even greater signaling of apoptosis followed ATRA treatment of these
transfectants. This suggests that reduced PML/RAR expression in
ATRA-resistant APL cells restores at least some of the retinoid
sensitivity to growth suppression triggered by ATRA treatment. It is
interesting to note that maturation was not triggered in these
transfectants having reduced PML/RAR expression, as shown in Table
1. This finding argues for an important role for persistent PML/RAR
expression to maintain basal leukemic cell growth. These findings are
consistent with the view that events in addition to PML/RAR are
required to trigger the full retinoid differentiation program in these
ATRA-resistant cells.
To explore PML/RAR mutations that may contribute to the phenotype of
these ATRA-resistant NB4 cells, the entire RAR portion of PML/RAR
isolated from these cells was sequenced. A 3-bp mutation in the ligand
binding domain of RAR that eliminated a phenylalanine was
identified, as shown in Fig 6. This provides a possible explanation for
the ATRA resistance of these NB4-R1 cells and is consistent with other
published work of ATRA-resistant myeloid leukemic
cells.24,33,34 Although these cells are resistant to ATRA,
the mutated PML/RAR appears to continue to act as an antiapoptotic
factor, because the elimination or reduction of this product through
the targeting strategies reported here abrogates this antiapoptotic
effect.
The findings presented in this study indicate that PML/RAR functions
as an antiapoptotic translocation product in these ATRA-resistant NB4
cells. That PML/RAR antagonizes an apoptotic program has precedence
in prior published work. For instance, induced overexpression of
PML/RAR in U937 myeloid leukemic cells leads to an antagonism of
growth factor-limited apoptosis.11 This work, highlighting an antiapoptotic role for PML/RAR, was accomplished using a non-APL myeloid leukemic cell line. The current study extends this prior work
by performing an analysis of the PML/RAR role in an APL cell
context. The current study complements prior work in this field by
conducting loss-of-function experiments using two approaches: (1)
homologous recombination and (2) hammerhead ribozymes, which target
PML/RAR mRNA expression. Both approaches led to similar biologic
outcomes, a lethal phenotype, due at least partly to the triggering of
apoptosis. That two genetic approaches, each directed towards reducing
PML/RAR expression, lead to a similar phenotype provides independent
confirmation of these results. The triggering of apoptosis in
retinoid-resistant myeloid leukemic cell lines also has precedence. One
study reports that apoptosis can be induced in ATRA-resistant NB4 cells
when primed with retinoic acid and then triggered with
cAMP.35 Prior work demonstrates that treatment with 4HPR
triggers apoptosis even in a myeloid leukemic cell line bearing an
RAR mutation.32,36 Whether acquired mutations within the
LBD of RAR are a general feature of retinoid-resistant promyelocytic
cell lines is the subject of work currently in
progress.
In summary, the findings reported in this study indicate two strategies
to target PML/RAR expression yield similar findings in
retinoid-resistant APL cells. This is the requirement for persistent PML/RAR expression to sustain leukemic cell growth. This requirement is linked, at least partly, to the antiapoptotic function of PML/RAR in NB4-R1 APL cells. That reduction of PML/RAR expression is incompatible with leukemic cell growth, even within an established APL
cell line, raises the prospect that targeting PML/RAR will have
therapeutic potential in retinoid-resistant or -sensitive APL cells.
Future work will explore this hypothesis.
| |
FOOTNOTES |
Submitted January 12, 1998;
accepted April 29, 1998.
Supported by the National Institutes of Health Grants No. RO1 CA
62275-04 (E.D.), NRSA F32 CA61646-01A1 (K.N.-B.), and P01 CA 29502-14A2
(K.N.-B.) and by grants from the Centre National pour la Recherche
Scientifique and the Institut Pasteur de Lille.
Address reprint requests to Kathryn Nason-Burchenal, PhD, Memorial
Sloan-Kettering Cancer Center, Box 305, 1275 York Ave, New York, NY
10021; e-mail: k-nason-burchenal{at}ski.mskcc.org.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr M. Lanotte (INSERM, Paris, France) for the gift of
the NB4 cell line from which we derived the NB4-R1 cells. We thank Dr
A. Selvakumar (Memorial Sloan-Kettering Cancer Center, New York,
NY) for help and advice with the DNA sequencing. We
thank Dr A. Goldberg and colleagues at Innovir
Laboratories (New York, NY) for helpful consultation
and for use of the hammerhead ribozymes 1.1 and 5.0. We thank Dr P. Chambon (INSERM, Strasbourg, France) for release of the anti-RAR
antibody used for Western analysis.
| |
REFERENCES |
1.
Larson RA,
Kondo K,
Vardiman JW,
Butler AE,
Golomb HM,
Rowley JD:
Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia.
Am J Med
76:827,
1984[Medline]
[Order article via Infotrieve]
2.
Huang M-E,
Ye Y-C,
Chen S-R,
Chai J-R,
Lu J-X,
Zhoa L,
Gu L-J,
Wang Z-Y:
Use of all-rans retinoic acid in the treatment of acute promyelocytic leukemia.
Blood
72:567,
1988[Abstract/Free Full Text]
3.
Castaigne S,
Chomienne C,
Daniel MT,
Ballerini P,
Berger R,
Fenaux P,
Degos L:
All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results.
Blood
76:1704,
1990[Abstract/Free Full Text]
4.
Warrell RP Jr,
Frankel SR,
Miller WH Jr,
Scheinberg DA,
Itri LM,
Hittelman WN,
Vyas R,
Andreeff M,
Tafuri A,
Jakubowski A,
Gabrilove J,
Gordon MS,
Dmitrovsky E:
Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans retinoic acid).
N Engl J Med
324:1385,
1991[Abstract]
5.
Chen Z,
Brand NJ,
Chen A,
Chen S-J,
Tong J-H,
Wang Z-Y,
Waxman S,
Zelent A:
Fusion between a novel Krüppel-like zinc finger gene and the retinoic acid receptor- locus due to a variant t(11;17) translocation associated with acute promyelocytic leukaemia.
EMBO J
12:1161,
1993[Medline]
[Order article via Infotrieve]
6.
Redner RL,
Rush EA,
Faas S,
Rudert WA,
Corey SJ:
The t(5;17) variant of acute promyelocytic leukemia expresses a nucleophosmin-retinoic acid receptor fusion.
Blood
87:882,
1996[Abstract/Free Full Text]
7.
de Thé H,
Lavau C,
Marchio A,
Chomienne C,
Degos L,
Dejean A:
The PML-RAR fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR.
Cell
66:675,
1991[Medline]
[Order article via Infotrieve]
8.
Kakizuka A,
Miller WH Jr,
Umesono K,
Warrell RP Jr,
Frankel SR,
Murty VVVS,
Dmitrovsky E,
Evans RM:
Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR with a novel putative transcription factor, PML.
Cell
66:663,
1991[Medline]
[Order article via Infotrieve]
9.
Rousselot P,
Hardas B,
Patel A,
Guidez F,
Gäken J,
Castaigne S,
Dejean A,
de Thé H,
Degos L,
Farzaneh F,
Chomienne C:
The PML-RAR gene product of the t(15,17) translocation inhibits retinoic acid-induced granulocytic differentiation and mediated transactivation in human myeloid cells.
Oncogene
9:545,
1994[Medline]
[Order article via Infotrieve]
10.
Kastner P,
Perez A,
Lutz Y,
Rochette-Egly C,
Gaub M-P,
Durand B,
Lanotte M,
Berger R,
Chambon P:
Structure, localization and transcriptional properties of two classes of retinoic acid receptor fusion proteins in acute promyelocytic leukemia (APL): Structural similarities with a new family of oncoproteins.
EMBO J
11:629,
1992[Medline]
[Order article via Infotrieve]
11.
Grignani F,
Ferrucci PF,
Testa U,
Talamo G,
Fagioli M,
Alcalay M,
Mencarelli A,
Grignani F,
Peschle C,
Nicoletti I,
Pelicci PG:
The acute promyelocytic leukemia-specific PMLRAR fusion protein inhibits differentiation and promotes survival of myeloid precursor cells.
Cell
74:423,
1993[Medline]
[Order article via Infotrieve]
12.
Jansen JH,
Mahfoudi A,
Rambaud S,
Lavau C,
Wahli W,
Dejean A:
Multimeric complexes of the PML-retinoic acid receptor fusion protein in acute promyelocytic leukemia cells and interference with retinoid and peroxisome-proliferator signaling pathways.
Proc Natl Acad Sci USA
92:7401,
1995[Abstract/Free Full Text]
13.
Perez A,
Kastner P,
Sethi S,
Lutz Y,
Reibel C,
Chambon P:
PMLRAR homodimers: Distinct DNA binding properties and heteromeric interactions with RXR.
EMBO J
12:3171,
1993[Medline]
[Order article via Infotrieve]
14.
Nervi C,
Poindexter EC,
Grignani F,
Pandolfi PP,
Lo Coco F,
Avvisati G,
Pelicci PG,
Jetten AM:
Characterization of the PML-RAR chimeric product of the acute promyelocytic leukemia-specific t(15;17) translocation.
Cancer Res
52:3687,
1992[Abstract/Free Full Text]
15.
Evans RM:
The retinoid receptors
, in Sporn MB,
Roberts AB,
Goodman DS
(eds):
The Retinoids: Biology, Chemistry, and Medicine.
New York, NY, Raven
, 1994
, p 319
16.
Muindi J,
Frankel SR,
Miller WH Jr,
Jakubowski A,
Scheinberg DA,
Young CW,
Dmitrovsky E,
Warrell RP Jr:
Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: Implications for relapse and retinoid "resistance" in patients with acute promyelocytic leukemia.
Blood
79:299,
1992[Abstract/Free Full Text]
17.
Cornic M,
Delva L,
Guidez F,
Balitrand N,
Degos L,
Chomienne C:
Induction of retinoic acid-binding protein in normal and malignant human myeloid cells by retinoic acid in acute promyelocytic leukemia patients.
Cancer Res
52:3329,
1992[Abstract/Free Full Text]
18.
Kizaki M,
Ueno H,
Yamazoe Y,
Shimada M,
Takayama N,
Muto A,
Matsushita H,
Nakajima H,
Morikawa M,
Koeffler HP,
Ikeda Y:
Mechanisms of retinoid resistance in leukemic cells: Possible role of cytochrome P450 and P-glycoprotein.
Blood
87:725,
1996[Abstract/Free Full Text]
19.
Paietta E,
Andersen J,
Racevskis J,
Gallagher R,
Bennett J,
Yunis J,
Cassileth P,
Wiernik PH:
Significantly lower P-glycoprotein expression in acute promyelocytic leukemia than in other types of acute myeloid leukemia: Immunological, molecular and functional analyses.
Leukemia
8:968,
1994[Medline]
[Order article via Infotrieve]
20.
Miller WH Jr,
Kakizuka A,
Frankel SR,
Warrell RP Jr,
DeBlasio A,
Levine K,
Evans RM,
Dmitrovsky E:
Reverse transcription polymerase chain reaction for the rearranged retinoic acid receptor clarifies diagnosis and detects minimal residual disease in acute promyelocytic leukemia.
Proc Natl Acad Sci USA
89:2694,
1992[Abstract/Free Full Text]
21.
Duprez E,
Ruchaud S,
Houge G,
Martin-Thouvenin V,
Valensi F,
Kastner P,
Berger R,
Lanotte M:
A retinoid acid `resistant' t(15;17) acute promyelocytic leukemia cell line: Isolation, morphological, immunological, and molecular features.
Leukemia
6:1281,
1992[Medline]
[Order article via Infotrieve]
22.
Dermime S,
Grignani F,
Clerici M,
Nervi C,
Sozzi G,
Talamo GP,
Marchesi E,
Formelli F,
Parmiani G,
Pelicci PG,
Gambacorti-Passerini C:
Occurrence of resistance to retinoic acid in the acute promyelocytic leukemia cell line NB4 is associated with altered expression of the pml/RAR protein.
Blood
82:1573,
1993[Abstract/Free Full Text]
23.
Nason-Burchenal K,
Maerz W,
Albanell J,
Allopenna J,
Martin P,
Moore MAS,
Dmitrovsky E:
Common defects of different retinoic acid resistant promyelocytic leukemia cells are persistent telomerase activity and nuclear body disorganization.
Differentiation
61:321,
1997[Medline]
[Order article via Infotrieve]
24.
Shao W,
Benedetti L,
Lamph WW,
Nervi C,
Miller WH Jr:
A retinoid-resistant acute promyelocytic leukemia subclone expresses a dominant negative PML-RAR mutation.
Blood
89:4282,
1997[Abstract/Free Full Text]
25.
Pace U,
Bockman JM,
MacKay BJ,
Miller WH Jr,
Dmitrovsky E,
Goldberg AR:
A ribozyme which discriminates in vitro between PML/RAR, the t(15;17)-associated fusion RNA of acute promyelocytic leukemia, and PML and RAR, the transcripts from the nonrearranged alleles.
Cancer Res
54:6365,
1994[Abstract/Free Full Text]
26.
Ahn M-J,
Nason-Burchenal K,
Moasser MM,
Dmitrovsky E:
Growth suppression of acute promyelocytic leukemia cells having increased expression of the non-rearranged alleles: RAR or PML.
Oncogene
10:2307,
1995[Medline]
[Order article via Infotrieve]
27.
Giguere V,
Ong ES,
Segui P,
Evans RM:
Identification of a receptor for the morphogen retinoic acid.
Nature
330:624,
1987[Medline]
[Order article via Infotrieve]
28.
Pandolfi PP,
Alcalay M,
Fagioli M,
Zangrilli D,
Mencarelli A,
Diverio D,
Biondi A,
Lo Coco F,
Rambaldi A,
Grignani F,
Rochette-Egly C,
Gaube M-P,
Chambon P,
Pelicci PG:
Genomic variability and alternative splicing generate multiple PML/RAR transcripts that encode aberrant PML proteins and PML/RAR isoforms in acute promyelocytic leukaemia.
EMBO J
11:1397,
1992[Medline]
[Order article via Infotrieve]
29.
Alcalay M,
Zangrilli D,
Pandolfi PP,
Longo L,
Mencarelli A,
Giacomucci A,
Rocchi M,
Biondi A,
Rambaldi A,
Lo Coco F,
Diverio D,
Donti E,
Grignani F,
Pelicci PG:
Translocation breakpoint of acute promyelocytic leukemia lies within the retinoic acid receptor locus.
Proc Natl Acad Sci USA
88:1977,
1991[Abstract/Free Full Text]
30.
Le Mouellic H,
Lallemand Y,
Brulet P:
Targeted replacement of the homeobox gene Hox-3.1 by the Escherichia coli lacZ in mouse chimeric embryos.
Proc Natl Acad Sci USA
87:4712,
1990[Abstract/Free Full Text]
31.
Mosmann T:
Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.
J Immunol Methods
65:55,
1983[Medline]
[Order article via Infotrieve]
32.
Delia D,
Aiello A,
Lombardi L,
Pelicci PG,
Grignani F,
Grignani F,
Formelli F,
Menard S,
Costa A,
Veronesi U,
Pierotti MA:
N-(4-hydroxyphenyl) retinamide induces apoptosis of malignant hemopoietic cell lines including those unresponsive to retinoic acid.
Cancer Res
53:6036,
1993[Abstract/Free Full Text]
33.
Li Y-P,
Said F,
Gallagher RE:
Retinoic acid-resistant HL-60 cells exclusively contain mutant retinoic receptor-.
Blood
83:3298,
1994[Abstract/Free Full Text]
34.
Robertson KA,
Emami B,
Collins SJ:
Retinoic acid-resistant HL-60R cells harbor a point mutation in the retinoic acid receptor ligand-binding domain that confers dominant negative activity.
Blood
80:1885,
1992[Abstract/Free Full Text]
35.
Bruel A,
Benoit G,
De Nay D,
Brown S,
Lanotte M:
Distinct apoptotic responses in maturation sensitive and resistant t(15,17) acute promyelocytic leukemia NB4 cells. 9-cis retinoic acid induces apoptosis independent of maturation and Bc1-2 expression.
Leukemia
9:1173,
1995[Medline]
[Order article via Infotrieve]
36.
Collins SJ,
Robertson KA,
Mueller L:
Retinoic acid-induced granulocytic differentiation of HL-60 myeloid leukemia cells is mediated directly through the retinoic acid receptor (RAR-).
Mol Cell Biol
10:2154,
1990[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
N. A. PAPANIKOLAOU
Rational Targeting in Acute Promyelocytic Leukemia
In Vivo,
January 1, 2010;
24(1):
21 - 27.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Quere, A. Baudet, B. Cassinat, G. Bertrand, J. Marti, L. Manchon, D. Piquemal, C. Chomienne, and T. Commes
Pharmacogenomic analysis of acute promyelocytic leukemia cells highlights CYP26 cytochrome metabolism in differential all-trans retinoic acid sensitivity
Blood,
May 15, 2007;
109(10):
4450 - 4460.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Ma, S. Fiering, C. Black, X. Liu, Z. Yuan, V. A. Memoli, D. J. Robbins, H. A. Bentley, G. J. Tsongalis, E. Demidenko, et al.
Transgenic cyclin E triggers dysplasia and multiple pulmonary adenocarcinomas
PNAS,
March 6, 2007;
104(10):
4089 - 4094.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Kitareewan, B. D. Roebuck, E. Demidenko, R. D. Sloboda, and E. Dmitrovsky
Lysosomes and Trivalent Arsenic Treatment in Acute Promyelocytic Leukemia
J Natl Cancer Inst,
January 3, 2007;
99(1):
41 - 52.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. B. Cheung, J. Bell, A. Raif, A. Bohlken, J. Yan, B. Roediger, A. Poljak, S. Smith, M. Lee, W. D. Thomas, et al.
The Estrogen-responsive B Box Protein Is a Novel Regulator of the Retinoid Signal
J. Biol. Chem.,
June 30, 2006;
281(26):
18246 - 18256.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Cote, S. McNamara, D. Brambilla, A. Bianchini, G. Rizzo, S. V. del Rincon, F. Grignani, C. Nervi, and W. H. Miller Jr
Expression of SMRT{beta} promotes ligand-induced activation of mutated and wild-type retinoid receptors
Blood,
December 15, 2004;
104(13):
4226 - 4235.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Oshima, H. Kawasaki, Y. Soda, K. Tani, S. Asano, and K. Taira
Maxizymes and Small Hairpin-Type RNAs That Are Driven by a tRNA Promoter Specifically Cleave a Chimeric Gene Associated with Leukemia in Vitro and in Vivo
Cancer Res.,
October 15, 2003;
63(20):
6809 - 6814.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Cote, A. Rosenauer, A. Bianchini, K. Seiter, J. Vandewiele, C. Nervi, and W. H. Miller Jr
Response to histone deacetylase inhibition of novel PML/RARalpha mutants detected in retinoic acid-resistant APL cells
Blood,
September 18, 2002;
100(7):
2586 - 2596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Kitareewan, I. Pitha-Rowe, D. Sekula, C. H. Lowrey, M. J. Nemeth, T. R. Golub, S. J. Freemantle, and E. Dmitrovsky
UBE1L is a retinoid target that triggers PML/RARalpha degradation and apoptosis in acute promyelocytic leukemia
PNAS,
March 19, 2002;
99(6):
3806 - 3811.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Mologni, E. Marchesi, P. E. Nielsen, and C. Gambacorti-Passerini
Inhibition of Promyelocytic Leukemia (PML)/Retinoic Acid Receptor-{alpha} and PML Expression in Acute Promyelocytic Leukemia Cells by Anti-PML Peptide Nucleic Acid
Cancer Res.,
July 1, 2001;
61(14):
5468 - 5473.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Wang, X. Zheng, F. G. Behm, and M. Ratnam
Differentiation-independent retinoid induction of folate receptor type beta , a potential tumor target in myeloid leukemia
Blood,
November 15, 2000;
96(10):
3529 - 3536.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Cote, D. Zhou, A. Bianchini, C. Nervi, R. E. Gallagher, and W. H. Miller Jr
Altered ligand binding and transcriptional regulation by mutations in the PML/RARalpha ligand-binding domain arising in retinoic acid-resistant patients with acute promyelocytic leukemia
Blood,
November 1, 2000;
96(9):
3200 - 3208.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Naoe, K. Kitamura;, M. Fanelli, and P. G. Pelicci
Relationship Between Degradation of PML-RARalpha and Differentiation
Blood,
August 15, 1999;
94(4):
1478 - 1479.
[Full Text]
[PDF]
|
|
|
|
|
|
|
G.-X. Cheng, X.-H. Zhu, X.-Q. Men, L. Wang, Q.-H. Huang, X. L. Jin, S.-M. Xiong, J. Zhu, W.-M. Guo, J.-Q. Chen, et al.
Distinct leukemia phenotypes in transgenic mice and different corepressor interactions generated by promyelocytic leukemia variant fusion genes PLZF-RARalpha and NPM-RARalpha
PNAS,
May 25, 1999;
96(11):
6318 - 6323.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Melnick and J. D. Licht
Deconstructing a Disease: RAR{alpha}, Its Fusion Partners, and Their Roles in the Pathogenesis of Acute Promyelocytic Leukemia
Blood,
May 15, 1999;
93(10):
3167 - 3215.
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. E. Gallagher
Arsenic -- New Life for an Old Potion
N. Engl. J. Med.,
November 5, 1998;
339(19):
1389 - 1391.
[Full Text]
|
|
|
|
|
Abstract
Introduction
Abstract