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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 143-151
Cancer Procoagulant and Tissue Factor Are Differently Modulated
by All-trans-Retinoic Acid in Acute Promyelocytic Leukemia
Cells
By
A. Falanga,
R. Consonni,
M. Marchetti,
G. Locatelli,
E. Garattini,
C. Gambacorti Passerini,
S.G. Gordon, and
T. Barbui
From the Hematology Division, Ospedali Riuniti, Bergamo; Mario Negri
Institute for Pharmacological Research, Milan; Istituto Nazionale
Tumori, Milan, Italy; and the Department of Biology, San Diego State
University, San Diego, CA.
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ABSTRACT |
All-trans-retinoic acid (ATRA) downregulates the expression
of two cellular procoagulants, tissue factor (TF) and cancer
procoagulant (CP), in human promyelocytic leukemia cells. To evaluate
whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have
characterized the effect of ATRA on the TF and CP expression by NB4
cells, an ATRA maturation-inducible cell line, and two NB4-derived cell
lines resistant to ATRA-induced maturation, the NB4.306 and NB4.007/6
cells. Next, we evaluated the effect on the PCAs of the NB4 parental
cells of three synthetic retinoid analogues, ie: AM580 (selective for
the retinoic acid receptor [RAR]  ), capable to induce
the granulocytic differentiation of NB4 cells; and CD2019 (selective
for RAR ) and CD437 (selective for RAR ), both lacking this
capability. Cells were treated with either ATRA or the analogues
(10 6 to 108 mol/L) for 96 hours. The
effect on cell differentiation was evaluated by morphologic changes,
cell proliferation, nitro blue tetrazolium reduction assay, and flow
cytometry analysis of the CD33 and CD11b surface-antigen expression.
PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the
one-stage clotting assay of normal and FVII-deficient plasmas. Further
TF and CP have been characterized and quantified in cell-sample
preparations by chromogenic and immunological assays. In the first
series of experiments, ATRA downregulates both TF and CP in NB4
parental cells, as expected. However, in the differentiation-resistant
cell lines, it induced a significant loss of TF but had little or no
effect on CP. In a second series of experiments, in the NB4 parental
cells, the RAR agonist (AM580) induced cell maturation and reduced
91% CP expression, whereas CD437 and CD2019 had no
cyto-differentiating effects and did not affect CP levels. On the other
hand, in the same cells the TF expression was reduced by ATRA and
AM580, but also by the RAR agonist CD2019, which did not induce cell
maturation. These data indicate that in NB4 cells, ATRA modulation of
CP occurs in parallel with signs of cell differentiation, while the
regulation of TF appears to be at least in part independent from these
processes, and involves both and nuclear retinoid receptors.
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INTRODUCTION |
ALL-TRANS-RETINOIC ACID (ATRA)
induces the differentiation of acute promyelocytic leukemia (APL) cells
into mature granulocytes. APL is a variety of acute myeloid leukemia
(AML-M3 in the French-American-British classification) characterized by
the association with a life-threatening coagulation/bleeding
syndrome1,2 and by the balanced reciprocal t(15;17)
chromosomal translocation with breakpoints in the retinoic acid receptor (RAR) gene on chromosome 17 and the promyelocyte (PML) gene
on chromosome 15. A PML/RAR chimeric gene encoding a fusion
PML/RAR protein is formed as a result of the translocation. The
presence of PML/RAR confers to leukemic cells a unique sensitivity to ATRA-induced cyto-differentiation. ATRA therapy for remission induction of APL represents one of the most important advances in the
field of leukemia therapy, because it induces the complete remission in
greater than 90% of APL patients and a simultaneous rapid resolution
of the bleeding symptoms.3-5
Among the mechanisms responsible for the intravascular clotting
activation in APL is the release of procoagulant activities (PCA) by
the promyelocytic blast cells. APL cells possess at least two
procoagulants: (1) tissue factor (TF), a transmembrane glycoprotein of
normal and malignant cells which forms a complex with factor VII (FVII)
to activate coagulation factor X (FX)6-8; and (2) cancer
procoagulant (CP), a cysteine proteinase procoagulant from fetal and
malignant tissues, which directly activates FX in the absence of
FVII.9-12 ATRA treatment significantly depresses both TF
and CP expression in human APL cells in vitro and in
vivo.13-16 Furthermore, in APL patients, the decrement of
the bone marrow (BM) cells' PCA parallels the improvement of clotting
parameters, including the increase of platelet count and plasma
fibrinogen, and the decrease of circulating markers of
hypercoagulation/hyperfibrinolysis.15 This suggests that
modulating blast cell PCA may have a role for the control of the
coagulopathy.
Mechanisms of ATRA-induced changes of APL cell PCAs are not clarified.
We have designed this study to define whether these changes correlate
to ATRA-induced cell differentiation. To this purpose we have followed
changes in the expression of TF and CP and in cell differentiation
features : (1) in response to ATRA treatment in APL cell lines
sensitive and resistant to ATRA-induced cyto-differentiation; and (2)
in response to a series of retinoid analogues with a different capacity
to induce differentiation in ATRA-sensitive cells.
First, we have compared the effect of ATRA on the expression of TF and
CP by NB4 cells, a human APL cell line sensitive to ATRA-induced
maturation (S-NB4),17 to the effect on TF and CP expressed
by two NB4-derived cell lines, resistant to the ATRA-induced maturation
(R-NB4), NB4.306, and NB4.007/6 cell
lines.18,19 S-NB4 and R-NB4 cells contain the
t(15;17) chromosome translocation and show the PML/RAR hybrid DNA;
however, they differ in that R-NB4 cells do not possess the complete
form of the PML/RAR protein.18,19
Second, we have exposed S-NB4 cells to three retinoid derivatives,
selective for each of the nuclear RAR subfamily members, RAR,
RAR, and RAR.20,21 ATRA does not have any selectivity for the RAR subtypes. The use of these compounds has shown that the
RAR agonist AM580 is a potent inducer of the granulocytic differentiation of leukemic promyelocytes, whereas CD2019 and CD437,
the RAR and agonists, respectively, lack this
capacity.22 The effect of AM580, CD2019, and CD437 on CP
and TF expression by S-NB4 cells was evaluated. The results of this
study show that while CP downregulation by retinoids correlates with
phenotype differentiation features, TF modulation occurs at least in
part independently from these processes and involves and retinoic acid receptors. The different regulation of these procoagulant proteins by ATRA suggests new implications for ATRA/PCA interactions in
human malignancy.
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MATERIALS AND METHODS |
Cell Lines and Reagents
The following human leukemic cell lines were used: (1) The NB4 parental
line, kindly provided by Dr M. Lanotte's laboratory (St Louis Hopital,
Paris, France). This line, established in vitro from an APL
patient,17 shows the typical t(15;17) chromosomal translocation, expresses the PML/RAR fusion protein, and is
sensitive to ATRA-induced cell differentiation (S-NB4). (2) Two cell
lines derived in vitro from S-NB4, which are resistant to ATRA-induced differentiation (R-NB4): (a) NB4.306 cell line, obtained by mutagenesis with low-dose radiation and then selected with increasing ATRA concentrations (108 to 106
mol/L),18 and (b) NB4.007/6 cell line, obtained by
long-term culture with increasing ATRA concentrations
(108 to 106 mol/L).19
NB4.306 and NB4.007/6 cells contain the t(15;17) chromosome
translocation, but express no detectable amount of the intact
PML/RAR protein, a property considered relevant for the resistence
to ATRA.18,19
Cells were maintained in RPMI 1640 containing 10% fetal calf serum,
penicillin (100 U/mL), and streptomycin (100 µg/mL). In the first
series of experiments, S- and R-NB4 cells were resuspended in fresh
medium (2 × 105 cells/mL) and cultured for 96 hours
in the absence or presence of 106 mol/L ATRA
(Hoffman-La Roche, Basel, Switzerland), the optimal condition to induce
cell differentiation.17 In the second series of
experiments, S-NB4 cells were cultured for 96 hours either with ATRA or
the following synthetic agonists of RARs: (1) the RAR agonist AM580
{[4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carboxamido] benzoic acid), (2) the RAR agonist CD2019
{6-[3-(1-methylcyclohexyl)-4-methoxyphenyl]-2-naphthoic acid},
and (3) the RAR agonist
CD437{6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid] (CIRD
Galderma, Sophia Antipolis, Valbonne, France).20-22 The
RAR agonist AM580 induces the granulocytic differentiation of NB4
cells,22 whereas both the RAR agonist CD2019 and RAR agonist CD437 lack this capacity. In most of the experiments the three
compounds were used at a final concentration of 108
mol/L, the highest concentration showing selective binding for the
receptors and not affecting cell viability.
In some experiments RAR was blocked by the RAR synthetic
inhibitor Ro 41-5253 (p-[(E)-2-[3 ,4-dihydro-4,4-dimethyl-7-(heptyloxy)-2H-1-benzothiopyran-6-yl]propenyl] benzoic acid 1, 1-dioxide) (Hoffman-La
Roche),23 while RAR was blocked by an RAR antagonist
CD2665 (CIRD Galderma). S-NB4 cells were cultured with
106 mol/L Ro 41-5253 or 106 mol/L
CD2665 or both plus 108 mol/L ATRA or
108 mol/L AM580. Stock solutions of the various
retinoids (102 mol/L) were prepared in
dimethylsulfoxide under dimmed light, stored at  80°C, and
protected from light until use. After treatment with ATRA or
RARs-agonists and antagonists, cell viability, evaluated by Tripan Blue
(Crle Erbe, Milan, Italy) exclusion dye test, was more
than 95%.
Analysis of Cell Differentiation
Cell diffentiation was evaluated after 96 hours treatment with ATRA or
retinoids or the vehicle by different criteria. (1) Morphological
changes (appearance of nuclear segmentation and granules formation)
were analyzed by microscopic examination of histologic slides after
May-Grünwald-Giemsa and cytochemical stainings. (2) Cell
proliferation was evaluated by direct cell counting (trypan blue
exclusion method) using a hematocytometer chamber. (3) The capability
to reduce nitro blue tetrazolium (NBT), a property associated with
myeloid cell maturation, was evaluated spectrophotometrically at 540 nm, as previously described.24 (4) Analysis of myeloid
cell-surface markers included the quantification of the number of
CD33+ and CD11b+ cells using a FACScan analyzer
(Becton Dickinson, Mountain View, CA). CD33 and CD11b are cellular
surface antigens of early and late myeloid differentiation,
respectively.25 Increase in CD11b and decrease in CD33
expressions correlate with cell differentiation in S-NB4 and R-NB4
cells.17-19,22,24,26 Determination of the surface markers
was performed by a direct immunofluorescence assay using the following
fluorescence-conjugated monoconal antibodies (MoAbs) (Becton
Dickinson): phycoerythrin (PE)-conjugated Leu15 (anti-CD11b) and Leu M9
(anti-CD33).
Samples
After 96 hours of treatment with ATRA or the retinoids, cells were
washed three times in sterile phosphate-buffered saline (PBS) and
different types of samples were ad hoc prepared according to the
optimal assay conditions required for each type of procoagulant (CP or
TF). The following samples were prepared:
Cell extracts in Veronal buffer (VB).
Sample preparation consisted of protein extraction in a
low ionic strength aqueous buffer (Veronal, BDH Chemicals ltd, Poole, UK), which is the optimal condition to extract CP, as
described.9-13 Cells (40 × 106
cells/mL) were extracted in two changes of 20 mmol/L VB, pH 7.8, at
4°C.
VB cell extracts were centrifuged at 10,000g for 10 minutes and
the supernatants were assayed for: (1) total and factor
VII-independent PCA by the coagulation assay; (2) CP activity by the
CP functional chromogenic assay; and (3) CP antigen by the CP
immunological assay.
Cell lysates.
Cell lysates were prepared to measure the activity of TF, a membrane
glycoprotein, by the TF functional chromogenic assay. Specifically,
cells (33 × 106 cells/mL) were resuspended in 10 mmol/L HEPES buffer, pH 7.45 (containing 137 mmol/L NaCl, 4 mmol/L KCl,
11 mmol/L -D glucose, 5 mg/mL ovalbumin, 2.5 mmol/L
CaCl2), and lysed by three cycles of
freezing/thawing.27
Cell extracts in Tris Buffer/Triton (TBT).
TBT cell extracts consisted of cell membrane
solubilization with Triton. This type of sample was to quantify TF
antigen, as is the most efficient condition for the recovery of this
antigen.27 Cells (20 × 106 cells/mL) were
resuspended in Tris Buffer, pH 7.5 (containing 50 mmol/L Tris, 100 mmol/L NaCl, 1% Triton X-100 [Merck-Darmstadt, Frankfurt, Germany]),
disrupted by three cycles of freezing/thawing and extracted on ice for
3 hours. Samples were centrifuged at 10,000g for 10 minutes and
supernatants assayed for TF antigen content by the TF immunological
assay.
Coagulation Assay
Total PCA of VB cell extracts was measured by the one-stage
recalcification assay of normal human plasma (NHP, containing the
coagulation factors, including factor VII [FVII]), as previously described.9-13 Briefly, 0.1 mL of cell extract was
incubated with 0.1 mL of NHP for 1 minute at 37°C. The reaction was
started by the addition of 0.1 mL of 25 mmol/L CaCl2 and
the clotting time was recorded. To identify an FVII-independent PCA, a
characteristic of CP, the recalcification assay of human plasma
congenitally deficient of FVII (FVII-DP; Behringwerke, AG, Marburg,
Germany) was performed. PCA was expressed as Russell's Viper Venom
(RVV) units per milligram of protein. Units were calculated on a
calibration curve obtained with different dilutions (from
101 to 106) of RVV (Sigma
Chemical Co, St Louis, MO); 1 unit = activity of 1 mEq/mL RVV in the
one-stage clotting assay.9-13 Protein content was
determined by the Bradford assay method.
To enzymatically characterize the VB cell extract PCA, an inhibition
study was conducted by assaying the samples' clotting activity in the
presence of three cysteine proteinase inhibitors, including
HgCl2 (Sigma), Iodoacetic acid (Sigma), and
Z-Ala-Ala-peptidyl diazomethyl-ketone (Z-Ala-Ala-CHN2;
Enzyme System Products, Dublin, CA), which inhibit CP, and of two TF
inhibitors, including Concanavalin A (Con A) and anti-TF MoAb
(anti-human TF TF9-9B4; American Diagnostica Inc, Greenwich, CT). Cell
extracts were incubated with 0.1 mmol/L HgCl2, 1 mmol/L
Iodoacetic acid and 0.2 mmol/L Z-Ala-Ala-CHN2 and with
anti-TF (0.1 mg/mL final concentration) for 30 minutes at 25°C, and
with 200 µg/mL Con A for 50 minutes at 37°C, before the
coagulation assay, as described.9-13,27
CP Functional Assay
CP functional activity was determined by a three-stage chromogenic
assay.28 VB cell extract (100 µL) was mixed with 10 µL of bovine FX (100 mg/mL; Sigma) and 30 µL of 25 mmol/L
CaCl2 in 50 mmol/L bis-Tris propane buffer (pH 6.7). After
30 minutes of incubation, 10 µL of bovine prothrombin (1 mg/mL;
Enzyme Research Laboratories Inc) and 30 µL of rabbit brain cephalin
(RBC)/Ca2+ mix were added to the samples
[RBC/Ca2+ mix = 1 part of a 1:10 dilution RBC + 1 part of 50 mmol/L CaCl2 + 2 parts of 100 mmol/L
bis-Tris propane buffer (pH 7.8)]. After a further 30-minute
incubation at 37°C, 200 µL 50 mmol/L Tris buffer, pH 7.8, and 50 µL thrombin substrate Sar-Pro-Arg-p-nitroanilide (2 mmol/L in 10%
dimethyl sulfoxide [DMSO; Sigma]) were added. Color development at
405 nm was recorded in the time (from 0 to 30 minutes). Samples' CP
content was expressed as mUnits per milliliter (1 Unit = the amount of enzyme responsible for releasing 1 µmol of
p-nitroaniline from the subtrate in 1 minute). RVV, a serine proteinase
FX activator, was the standard control to calibrate the assay.
Under these conditions, thrombin formation is totally inhibited by
incubation of the VB extracts (30 minutes at 25°C)
with 1 mmol/L HgCl2.
CP Antigen Assay
CP antigen was measured with a standard dot-blot analysis against
calibrated CP standards purified from human amnion-chorion tissue.29 Five CP standard concentrations (from 0 to 41 µg of CP/mL) and cell extract samples were diluted to 0.4 mL in VB
and 100-µL samples were added to the wells of the dot-blot apparatus containing nitrocellulose. After 30 minutes' incubation of the sample
solution with the nitrocellulose, remaining solution was removed by
applying a vacuum. Then the nitrocellulose sheet was removed and
blocked overnight by Tris-bufferd saline (pH 7.6) (TBS) containing 10%
nonfat dried milk. The membrane was first incubated with anti-CP IgG in
TBS-0.1% Tween 20 (TBST) containing 1% nonfat dried milk for 2 hours
at 25°C, and then, after washings with TBST, was incubated with an
antimurine IgG alkaline phosphatase conjugate goat antibody for 2 hours
at 25°C. Finally, after washing again, the nitrocellulose was
incubated in BCIT/NBT alkaline phosphatase substrate at room
temperature until the color development was of satisfactory intensity.
The nitrocellulose image was scanned into a computer and the dot
intensity was determined by SigmaScan. CP antigen content was
calculated from a calibration curve obtained with the different
concentrations of pure CP.
TF Functional Assay
TF activity of cell lysates was measured as the rate of FX hydrolysis
using a spectrophotometric assay for FXa, as described.27 Briefly, 30 µL of lysed cells were 1:6 diluted with 10 mmol/L HEPES
buffer (pH 7.45) and incubated with 1 nmol/L FVII for 10 minutes at
37°C to allow binding to TF. The reaction of FX hydrolysis was
started by the addition of 0.1 µmol/L FX. After 0, 2.5, 5, 10, and 20 minutes, 30-µL sub-samples were taken from the reaction mixture and
diluted in 20µL ice-cold 50 mmol/L Tris/EDTA (pH 7.5) stopping
buffer. To measure FXa content, each sub-sample was 1:1 diluted with 50 mmol/L Tris-NaCl buffer and the reaction started by adding 0.2 mmol/L
S-2337 chromogenic substrate (Ortho Diagnostic System, Milan, Italy).
After 30 minutes, the reaction was stopped by 50 mmol/L benzamidine and
the 405 nm absorbance was recorded. The assay was calibrated by active
site-titrated FXa and results expressed as rate of FX hydrolysis (pmol
FXa/min/106 cells).
Under these conditions, the FXa formation was completely dependent on
the presence of FVII and totally inhibited by antibody against human TF
(0.1 mg/mL final concentration MoAb anti-human TF TF9-9B4, American
Diagnostica Inc).
TF Antigen Assay
TF antigen was measured in a Tris/NaCl buffer-1% Triton X-100 (TBT)
solubilized cell samples by a double-antibody enzyme-linked immunosorbent assay (ELISA).27 Briefly, microtiter plates
were coated with the anti-TF MoAb TF8-10H10 (American Diagnostica Inc) and incubated overnight at 4°C (0.25 µg/well). After three
washings, 100 µL sample or standard TF dilutions (0 to 400 pmol/L;
Recomboplastin S, Baxter Diagnostica Inc, Deerfield, IL) were added to
each well and incubated overnight at 4°C. Plates were then washed
and 100 µL of biotinylated anti-TF MoAb TF9-9B4 (2 µg/mL/well;
American Diagnostica Inc) were added. After 4 hours of incubation at
25°C, a 1:1,000 dilution Avidine-horse radish peroxidase conjugate
(Sigma) was added. After 3 hours at 25°C, bound peroxidase activity
was detected by the substrate tetra-methyl benzidine (100 µg/mL). The
color reaction was stopped after 10 minutes by 100 µL 4 N H2SO4 and the absorbance at 450 nm was read.
Results were calculated on the reference curve of standard TF and
expressed as femtomoles of TF per 106 cells.
In this case the results were expressed per cell number, as appropriate
for this type of sample and because protein could not be technically
determined on account of interference by the detergent (Triton) in this
sample.
Statistical Analysis
Statistical analysis of the data was performed by the unpaired and
paired Student's t-tests.
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RESULTS |
Untreated and ATRA-treated S-NB4 and the two R-NB4 cells (NB4.306 and
NB4.007/6) were first assayed for PCA by the coagulation assay
(Fig 1). Total PCA is the activity measured
with the clotting assay using NHP, and FVII-independent (CP-like) PCA
is the activity measured with the clotting assay using FVII-DP. All the
cell lines were assayed for both total and FVII-independent PCA.
Treatment of S-NB4 cells with 106 mol/L ATRA for 96 hours significantly decreased (59%) the total PCA from the control
level of 12.2 ± 6.2 RVV units/mg protein to the ATRA-treated level
of 5 ± 4 RVV units/mg protein (P < .01); the total PCA of
the NB4.306 and NB4.007/6 cells decreased 44% and 42%, respectively
(P < .05) (Fig 1, left panel). In contrast, ATRA treatment
virtually abolished the FVII-independent PCA of S-NB4 cells from a
control level of 5.46 ± 2.83 RVV units/mg protein to an
ATRA-treated level of 0.006 ± 0.024 RVV units/mg protein, while
there was little or no effect on the activity of the FVII-independent PCA of ATRA-resistant cells (NB4-306 showed a 15% decrease and NB4-007/6 showed no decrease in PCA) (Fig 1, right panel).
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| Fig 1.
PCA of normal human plasma (A) and FVII-deficient plasma
(B) of cell extracts from NB4 cells, sensitive to ATRA-induced
differentiation, and two NB4-derived cell lines, resistant to
ATRA-induced differentiation (ie, NB4.306 and NB4.007/6). Cells were
cultured with 106 mol/L ATRA ( ) or the vehicle ( )
for 96 hours. Results, expressed as RVV units/mg total proteins, are
the mean of at least three experiments. Statistical analysis of PCA of
untreated versus treated samples was performed by the paired Student's
t-test; *P < .05, **P < .01.
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The PCA of untreated and ATRA-treated samples was further characterized
by testing the sensitivity to three cysteine proteinase inhibitors,
known to inhibit CP (ie, HgCl2, Iodoacetic acid and Z-Ala-Ala-CHN2), and to Con A and anti-TF MoAb, as TF
inhibitors (Fig 2). As previously
observed,13 in S-NB4 cells, the PCA of untreated samples
was highly sensitive to HgCl2 (P < .0001),
Iodoacetic acid (P < .05), and Z-Ala-Ala-CHN2
(P < .005), while the ATRA-treated counterparts were
unaffected by the cysteine proteinase inhibitors. In addition untreated
and ATRA treated S-NB4 cells were significantly affected by Con A (26%
and 15% inhibition, respectively) and anti-TF MoAb (65% and 93%,
respectively). In contrast, in R-NB4 cell extracts the PCA of either
untreated or ATRA-treated samples were significantly inhibited by the
three cysteine proteinase inhibitors and the anti-TF MoAb (Fig 2).
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| Fig 2.
Sensitivity to three cysteine proteinase inhibitors (0.1 mmol/L HgCl2, 0.2 mmol/L ZAA-diazomethyl-ketone, and 1 mmol/L IodoAcetic acid [IA]), and to the TF inhibitor anti-TF MoAb
(0.1 mg/mL final concentration) of the PCA of S-NB4 (left) and
R-NB4-306 cells (right) treated with 106 mol/L ATRA
() or the vehicle () for 96 hours. VB cell extracts were
incubated with cysteine proteinase inhibitors or the anti-TF MoAb for
30 minutes or the vehicle (control), before the clotting assay.
Statistical analysis compares the inhibitor-treated sample with the
untreated (vehicle treated) corresponding control. *P < .05, **P < .01.
|
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These results prompted us to further characterize the effect of ATRA on
the different procoagulants. Thus, we measured CP and TF activities and
antigens by specific chromogenic and immunological assays in optimized
experimental conditions. Untreated and ATRA-treated S- and R-NB4 cells
were subdivided in aliquots to prepare different samples for the
separate analysis of CP and TF activities and antigens.
Figure 3 shows TF chromogenic activity of
cell lysates (3A) and TF antigen content of TBT extracts (3B) from
S-NB4, R-NB4.306, and R-NB4.007/6 cells. TF chromogenic activity was
differently, but significantly, downregulated by ATRA treatment in all
three cell lines (61%, 39%, and 39% decrease in TF activity,
respectively). A similar profile was observed for TF antigen reduction
(55%, 38%, and 45% reduction in TF antigen, respectively).
Measurements of CP chromogenic activity and antigen in VB extracts from
the same cell lines are shown in Fig 3C and D. After ATRA treatment, there was a significant decrease of 85% in the CP activity of the
S-NB4 cells (P < .01), but there was little or no reduction in the two R-NB4 lines. Accordingly, CP antigen showed a 79% reduction in the S-NB4 cells (P > .01) but no significant changes in
the R-NB4 cell lines.
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| Fig 3.
Effect of ATRA on cellular procoagulants analyzed by
chromogenic and immunological assays for TF and CP expressed by S-NB4 cells and the two R-NB4 cell lines, NB4.306 and NB4.007/6, cultured for
96 hours with 106 mol/L ATRA or the vehicle. TF
functional activity (A) was measured as rate of FX hydrolysis by the
TF/FVII complexes in cell lysates. TF antigen (B) was measured in TBT
cell extracts by ELISA, using an anti-human TF MoAb. CP functional
activity (C) was measured in VB cell extracts by a three-stage
chromogenic assay. CP antigen (D) was immunologically identified by a
dot-blot assay using a pure anti-CP monoclonal IgG. The results are the
mean of at least three experiments. Statistical analysis as in Fig 1.
*P < .05, **P < .01.
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ATRA-induced cell maturation of NB4 cells was evaluated by microscopic
examination, cell proliferation, NBT reduction assay, and the
expression of CD33 and CD11b surface antigens, the two markers of early
and late myeloid differentiation,25 respectively. In NB4
parental cells, ATRA treatment caused growth inhibition (fold increase:
untreated v treated cells: 4.174 ± 2.28 v
2.02 ± 0.77; P < .01). The analysis of cell morphology,
evaluated on May-Grünwald-Giemsa-stained cells, showed that ATRA
treated S-NB4 cells became morphologically similar to metamyelocytes
and polynuclear neutrophils, as described.17 Morphologic
maturation corresponded to increased capacity to reduce NBT and to
changes in myeloid differentiation marker expression. The results of
the NBT reduction assay and the cell-surface markers analysis are
reported in Table 1. In all the
experiments, in the NB4 parental cells, ATRA increased the capacity of
reducing NBT (from 32 ± 4 to 110 ± 10  /h; P < .01) and the number of CD11b+ cells
(control, 5.8% ± 3%; +ATRA, 31.6% ± 5%, P < .01),
whereas it significantly decreased the CD33+ cells
(control, 96.8% ± 4.8%; +ATRA, 83.0% ± 8.85%, P < .01). The same treatment did not induce significant NBT reduction
capacity and surface antigen changes in the two R-NB4 cell lines.
In further experiments we focused our attention on the effect of three
synthetic RAR agonists (RAR, RAR, and RAR) on the PCA
expression by S-NB4 cells. The RAR agonist AM580 induces granulocytic differentiation of NB4 cells,22 while neither
the RAR agonist CD2019 nor the RAR agonists CD437 have the
capacity to induce differentiation. Table 1 also shows the effect of
AM580, CD2019, and CD437 on the NBT reduction capacity and the
expression of the surface differentiation antigens, CD11b and CD33, by
S-NB4 cells. Only AM580 was able to significantly affect both the NBT assay (from 29 ± 6 to 190 ± 12 OD/h; P < .01), and
the CD11b and CD33 expression. A low percentage (ranging from 5.3% to
6.1%) of the undifferentiated cells expressed CD11b; the RAR
agonist induced the CD11b expression up to 56%, a ninefold increase
over the control NB4 cells (P < .01). The expression of CD33
in the undifferentiated cells (ranging from 97% to 92% of the cells) decreased to 73% with the agonist AM580 (P < .01).
S-NB4 cell maturation induced by AM580 was accompanied by a significant
91% loss (P < .01) of CP chromogenic activity (from 11.48 ± 2.9 mU/mL to 1 ± 0.8 mU/mL), while treatment of the NB4 cells
with CD2019 or CD437 did not modulate CP expression
(Fig 4A). In contrast, cellular TF
chromogenic activity was significantly decreased not only by the RAR
agonist AM580 (58% decrease, P < .01), but also by the
RAR agonist CD2019 (36% decrease, P < .05); CD437 (RAR
agonist) had no effect (Fig 4B).
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| Fig 4.
Effect of AM580 (RAR agonist), CD2019 (RAR
agonist), and CD437 (RAR agonist) on CP chromogenic activity (A) and
TF activity (B) in S-NB4 cells. Cells were cultured ATRA
(106 mol/L) or synthetic retinoids (108
mol/L) for 96 hours. (), Untreated; (), treated. The results are
the mean of at least three experiments. Statistical analysis as in
Fig 1. *P < .05, **P < .01.
|
|
To evaluate the role of RAR in the downregulation of the two
procoagulants, in some experiments RAR was blocked by the RAR antagonist Ro 41-5253 (Fig 5). Treatment of
S-NB4 cells with Ro 41-5253 + ATRA completely prevented the
ATRA-induced CP downregulation, and only partially counteracted the TF
downregulation, confirming a role of other receptor(s) in the
regulation of TF expression. In contrast, the treatment of S-NB4 cells
with Ro 41-5253 in addition to AM580 completely prevented the
AM580-induced downregulation of both CP and TF. In these conditions, CP
chromogenic activity was 100% inhibited by treatment of the sample
with HgCl2 and TF chromogenic activity was 100% abolished
by the anti-TF MoAb.
View larger version (25K):
[in this window]
[in a new window]
| Fig 5.
Effect of the RAR antagonist Ro 41-5253 in blocking
the ATRA-induced and AM580-induced reduction of CP and TF expression in
S-NB4 cells. The cells were cultured for 96 hours with
108 mol/L ATRA ± 106 mol/L Ro 41-5253 or with 108 mol/L AM580 ± 106 mol/L Ro
41-5253. Results are the mean of at least three experiments. Statistical analysis to compare untreated and treated samples as in Fig
1. *P < .05; **P < .01 versus untreated control
samples.
|
|
To further investigate the role of RAR in the modulation of TF, we
have studied the effect of ATRA and AM580 in the presence of RAR and
RAR antagonists. As shown in Table 2,
the RAR antagonist slightly counteracted the action of ATRA, whereas
the simultaneous presence of both the RAR and RAR antagonists
completely abolished its effect. The RAR antagonist did not affect
the activity of AM580.
View this table:
[in this window]
[in a new window]
|
Table 2.
Downregulation of TF Expression by ATRA and RAR
Agonist AM580 in S-NB4: Inhibition of the Effect by RAR and RAR
Selective Antagonists
|
|
| |
DISCUSSION |
Several studies have shown ATRA's capability to downregulate the PCA
of APL cells.13-16 This property may be one of the
mechanisms by which ATRA facilitates a beneficial effect on the
coagulopathy associated with the early phase of APL. We have observed
that both procoagulants, CP (a cysteine proteinase) and TF (a membrane glycoprotein), are decreased by ATRA in the BM cells of patients under
treatment.15 However, whether this decrease is produced by
a direct mechanism or is a part of the differentiation process is not
known. To address this question, we have designed this study primarily
to follow the changes in the expression of the two procoagulants in
response to retinoids, in the presence or absence of retinoids-induced
cyto-differentiation. Specifically, two experimental conditions were
exploited, the availability of ATRA maturation-resistant NB4-derived
cell lines18,19 and the use of synthetic retinoid analogues
with different granulocytic differentiating capacity on NB4
cells.20-22 The results show that CP downregulation
parallels cell differentiation, whereas TF occurs, at least in part,
independently from this mechanism.
All of the experiments to evaluate the modifications of CP and TF
expression were uniformly conducted treating the cells with retinoids
for 96 hours to allow the time necessary for cell
differentiation.17,22 The effect of retinoid treatment on
cell maturation was confirmed by the study in the same cells of
morphological changes, growth arrest, NBT reduction capacity, and by
the analysis of surface expression of CD11b and CD33 markers of myeloid
differentiation.25 The surface CD11b increase and CD33
decrease induced by retinoids highly correlate, in previous studies,
with morphological changes, growth arrest, and NBT reduction capacity
in S- and R-NB4 cells.17-19,22,24,26 They also correlate
with the expression of alkaline phosphatase, another marker of
granulocytic differentiation, in NB4 cells treated with
ATRA.22
PCA was identified in the study by four different
criteria: (1) the clotting activity by the one-stage clotting assay of
NHP and FVII-DP; (2) selective inhibition by agents known to block either TF or CP PCA; (3) the chromogenic assays for CP and TF activity;
(4) the immunological identification of the two procoagulants by
specific anti-TF and anti-CP MoAbs. The clotting assay of NHP and
FVII-DP allows identification of total PCA (including all possible
procoagulants present) and the proportion of FVII-independent PCA. We
choose to perform these plasma assays in the VB extracts because these
conditions have been used in previous studies to characterize
PCA9-13,15 and, therefore, permit comparisons of the
results. In agreement with our previous findings in the same
cells13 and in cells from APL patients,10,12,15
we found that in NB4 cells, before treatment, a large proportion (45%)
of total PCA is FVII independent. The use of inhibitors of cysteine
proteinases or of TF provides a sensitive way to identify the presence
of CP or TF or both.
In the first part of the study, after ATRA, the total PCA was
significantly reduced in all cell lines, regardless of their sensitivity to ATRA-induced differentiation. However, the
FVII-independent CP-like PCA was virtually abolished only in the S-NB4
cells that underwent differentiation, but not in the resistant cells
that did not differentiate. The assay of PCA's sensitivity to
inhibitors confirmed the persistence, after ATRA, of a cysteine
proteinase procoagulant in the R-NB4, but not in the S-NB4, cells.
This first observation suggested that ATRA might differently affect the
two procoagulants. Therefore, we further characterized the two
procoagulants in the treated and untreated cell samples by specific
chromogenic and immunological assays. Because the VB aqueous extract is
the optimal condition for the CP recovery, ad hoc samples from S- and
R-NB4 cells were prepared to optimize the detection of TF as well.
Specifically, cell lysates were prepared for the chromogenic assay of
TF activity and cell extract in Tris/NaCl buffer containing 1% Triton
were prepared for the ELISA of TF; these are the most efficient
condition for TF antigen recovery.15,27 These further
analyses, by different assays, confirmed the occurrence of CP reduction
in association with cell differentiation features, while TF modulation
was, at least in part, independent from this process. TF was
downregulated by ATRA in both S- and R-NB4 cells. Because NB4.306 and
NB4.007/6 contain no or very low levels of the PML/RAR
protein,18,19 these data indicate that the reduction of CP
by retinoids requires the presence of detectable PML/RAR protein
levels.
These results are in agreement with known characteristics of the two
procoagulants. TF is a procoagulant of malignant cells, but it is also
the cellular procoagulant found in normally differentiated cells that
activates normal blood coagulation.6,30 CP has been
described in extracts of neoplastic cells or in amnion-chorion tissues,
but not in extracts of normally differentiated
cells.10-12,31-33 In patients with acute myeloid leukemias,
CP was detected in the BM mononuclear cells at the onset of the
disease, but not in samples from the same subjects during complete
remission.12 All of these findings support the thesis that
CP may be expressed by undifferentiated fetal and dedifferentiated
malignant cells and, once normal differentiation occurs, the expression
of this enzyme is repressed. On the other hand, TF has been shown to be
downregulated by ATRA in leukemic cells other than APL, not expressing
the PML/RAR and not sensitive to ATRA-induced
cyto-differentiation,34 and also in normal human endothelial cells and monocytes.35-37
To confirm the relation to cell maturation of the two procoagulants
modulating mechanisms, we used a second experimental approach, the
treatment of NB4 cells with retinoic acid analogues that selectively act on the RAR subtypes and possess different cyto-differentiating capacity. The RAR gene family comprises three subtypes , , and .20 The classification of these receptors is based on
differences in amino acid sequence, responsiveness to different
retinoids, and the ability to modulate the expression of different
target genes. ATRA binds to all members of this family with the same affinity. Therefore, the multiple effects of retinoic acid are better
analyzed by using ligands selective for the known
receptors.21 Using this strategy RAR has been shown to
play a major role in the granulocytic differentiation of leukemic
promyelocytes.22 In our study the compound AM580, which
selectively binds to RAR and induces cell differentiation, produced
a significant reduction of CP levels, whereas the CD2019 and CD437
retinoid-analogues, which selectively bind to RAR and RAR,
respectively, neither showed an effect on modulation granulocyte
differentiation nor affected the expression of CP in the NB4 cells. In
contrast, TF was modulated not only by AM580, but also by CD2019,
regardless of the cell maturation status. The RAR agonist CD437 had
no effect on either TF or CP regulation. These data suggest a major
role for RAR in CP downregulation by retinoids, whereas both RAR and RAR appear to be implicated in the regulation of TF expression in promyelocytic leukemia cells. A major role for RAR in the CP
downregulation by retinoids is also shown by the experiments with the
RAR antagonist Ro41-5253. Cotreatment of cells with this compound
completely blocked the downregulation of CP induced by both ATRA and
AM580. On the other hand, the same agent completely prevented the
reduction of TF induced by AM580, the RAR agonist, but counteracted
only partially the reduction of TF induced by ATRA, which binds to all
RARs. The cotreatment of cells with both the RAR and RAR
antagonists completely blocked the ATRA-induced TF downregulation
(Table 2). This provides evidence for a role of RAR in the
downregulation of TF by retinoids. The involvement of RAR in the
regulation of TF (in leukemic cells and normal endothelial cells) has
also been recently demonstrated by Shibakura et
al.38
It is known that AM580 is a more powerful cyto-differentiating agent
than ATRA in NB4 cells.22 In our study AM580 is more potent
than ATRA in downregulating TF in the S-NB4 cells (see Table 2). In
addition, treatment of NB4 cells with AM580 results in a significant
reduction of TF expression at concentrations 100-fold lower than those
necessary to obtain the same effect with ATRA (as shown in Fig 4). In
contrast, AM580 and ATRA have the same effect in the R-NB4.306 line
(data not shown). These data confirm that, while AM580 is an active
retinoid in cells expressing RAR, its potential is enhanced in cells
containing PML/RAR.
The different pathways of regulation of the cell procoagulants, CP and
TF, by ATRA, have important new implications for these proteins during
ATRA therapy in human malignancy. It supports the concept that CP is a
differentiation dependent protein that is expressed by malignant and
fetal cells. Further, we postulate that the CP loss, besides reducing
the procoagulant capacity of APL cells, might provide an important new
tool (marker) to monitor leukemic cell (and possibly other cancer
cells) maturation. Furthermore, the TF loss even in non-ATRA
maturation-sensitive tumor cells might help to improve clotting
complications in other malignant diseases different from APL treated
with ATRA.
| |
FOOTNOTES |
Submitted August 11, 1997;
accepted February 13, 1998.
M.M. is the recipient of a fellowship from the Associazione Italiana
Ricerca sul Cancro (AIRC).
Address reprint requests to A. Falanga, MD, Hematology
Division, Ospedali Riuniti, Largo Barozzi 1, 24100 Bergamo, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr M. Lanotte (INSERM U-301, Hopital Saint-Louis,
Paris, France) for providing the NB4 cells, and Drs B. Shroot and U
Reichert (CIRD Galderma, Sophia Antipolis, Valbonne, France) for
providing the synthetic retinoid analogues.
| |
REFERENCES |
1.
Tallman MS,
Kwaan HC:
Reassessing the hemostatic disorders associated with acute promyelocytic leukemia.
Blood
79:543,
1992[Free Full Text]
2.
Rodeghiero F,
Avvisati G,
Castaman G,
Barbui T,
Mandelli F:
Early deaths and anti-hemorrhagic treatments in acute promyelocytic leukemia. A GIMEMA retrospective study in 268 consecutive patients.
Blood
75:2112,
1990[Abstract/Free Full Text]
3.
Huang ME,
Ye UC,
Chen SR,
Chai JR,
Lu JX,
Lin Z,
Gu LJ,
Wang ZY:
Use of all-trans-retinoic acid in the treatment of acute promyelocytic leukemia.
Blood
72:567,
1988[Abstract/Free Full Text]
4.
Grignani F,
Fagioli M,
Alcalay M,
Longo L,
Pandolfi PP,
Donti E,
Biondi A,
Lo Coco F,
Grignani F,
Pelicci PG:
Acute promyelocytic leukemia: From genetics to treatment.
Blood
83:10,
1994[Free Full Text]
5.
Castaigne S,
Chomienne C,
Daniel MT,
Ballerini P,
Fennaux P,
Degos L:
All-trans-retinoic acid as a differentiation therapy for acute promyelocytic leukemia.
Blood
76:1704,
1990[Abstract/Free Full Text]
6.
Nemerson Y:
The tissue factor pathway of blood coagulation.
Semin Hematol
29:170,
1992[Medline]
[Order article via Infotrieve]
7.
Goualt-Heilmann M,
Chardon E,
Sultan C,
Josso F:
The procoagulant factor of leukemic promyelocytes. Demonstration of immunologic cross-reactivity with human brain tissue factor.
Br J Haematol
30:151,
1975[Medline]
[Order article via Infotrieve]
8.
Kubota T,
Andoh K,
Sadakata H,
Kobayashi N:
Tissue factor released from leukemic cells.
Thromb Haemost
65:59,
1991[Medline]
[Order article via Infotrieve]
9.
Falanga A,
Gordon SG:
Isolation and characterization of cancer procoagulant: A cysteine proteinase from malignant tissue.
Biochemistry
24:5558,
1985[Medline]
[Order article via Infotrieve]
10.
Falanga A,
Alessio MG,
Donati MB,
Barbui T:
A new procoagulant in acute leukemia.
Blood
71:870,
1988[Abstract/Free Full Text]
11.
Alessio MG,
Falanga A,
Consonni R,
Bassan R,
Minetti B,
Donati MB,
Barbui T:
Cancer procoagulant in acute lymphoblastic leukemia.
Eur J Haematol
445:78,
1990
12.
Donati MB,
Falanga A.,
Consonni R,
Alessio MG,
Bassan R,
Buelli M,
Borin L,
Catani L,
Pogliani E,
Gugliotta L,
Masera G,
Barbui T:
Cancer procoagulant in acute non lymphoid leukemia: Relationship of enzyme detection to disease activity:
Thromb Haemost
64:11,
1990[Medline]
[Order article via Infotrieve]
13.
Falanga A,
Consonni R,
Marchetti M,
Mielicki WP,
Rambaldi A,
Lanotte M,
Gordon SG,
Barbui T:
Cancer procoagulant in the human promyelocytic cell line NB4 and its modulation by retinoic acid.
Leukemia
8:156,
1994[Medline]
[Order article via Infotrieve]
14.
Koyama T,
Hirosawa S,
Kawamata N,
Tohda S,
Aoki N:
All-trans retinoic acid upregulates thrombomodulin and downregulates tissue factor expression in acute promyelocytic leukemia cells: Distinct expression of thrombomodulin and tissue factor in human leukemic cells.
Blood
84:3001,
1994[Abstract/Free Full Text]
15.
Falanga A,
Iacoviello L,
Evangelista V,
Belotti D,
Consonni R,
D'Orazio A,
Robba L,
Donati MB,
Barbui T:
Loss of blast cell procoagulant activity and improvement of hemostatic variables in patients with acute promyelocytic leukemia administered all-trans retinoic acid.
Blood
86:1072,
1995[Abstract/Free Full Text]
16.
De Stefano V,
Teofili L,
Sica S,
Mastrangelo S,
Di Mario A,
Rutella S,
Salutari P,
Rumi C,
d'Onofrio G,
Leone G:
Effect of all-trans retinoic acid on procoagulant and fibrinolytic activities of cultured blast cell from patients with acute promyelocytic leukemia.
Blood
86:3535,
1995[Abstract/Free Full Text]
17.
Lanotte M,
Martin-Thouvenin V,
Najman S,
Ballerini P,
Valensi F,
Berger R:
NB4, a maturation inducible line with t(15;17) marker isolated from human acute promyelocytic leukemia (M3).
Blood
77:1080,
1991[Abstract/Free Full Text]
18.
Dermine S,
Grignani F,
Clerici M,
Nervi S,
Sozzi G,
Talamo GP,
Marchesi E,
Formelli F,
Parmiani G,
Pelicci PG,
Gambacorti-Passerini C:
Occurrence of resistence to retinoic acid in acute promyelocytic leukemia cell line NB4 is associated with altered expression of the pml/RAR protein.
Blood
82:1573,
1993[Abstract/Free Full Text]
19.
Dermime S,
Grignani F,
Rogaia D,
Liberatore C,
Marchesi E,
Gambacorti-Passerini C:
Acute promyelocytic leukemia cells resistant to retinoic acid show further perturbation of the RAR signal transduction system.
Leuk Lymphoma
16:289,
1995[Medline]
[Order article via Infotrieve]
20.
Pemrick SM,
Lucas DA,
Grippo JF:
The retinoid receptors.
Leukemia
8:1797,
1994[Medline]
[Order article via Infotrieve]
21.
Martin B,
Bernardon JM,
Cavey MT,
Bernard B,
Carlavan I,
Charpentier B,
Pilgrim WR,
Shroot B,
Reichert U:
Selective synthetic ligands for human nuclear retinoid acid receptors.
Skin Pharmacol
5:57,
1992[Medline]
[Order article via Infotrieve]
22.
Giannì M,
Li Calzi M,
Terao M,
Guiso G,
Caccia S,
Barbui T,
Rambaldi A,
Garattini E:
AM580, a stable benzoic derivative of retinoic acid, has powerful and selective cyto-differentiating effect on acute promyelocytic leukemia cells.
Blood
87:1520,
1996[Abstract/Free Full Text]
23.
Apfel C,
Bauer F,
Crettaz M,
Forni L,
Kamber M,
Kaufmann F,
LeMotte P,
Pirson W,
Klaus M:
A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects.
Proc Natl Acad Sci USA
89:7129,
1992[Abstract/Free Full Text]
24.
Giannì M,
Terao M,
Gambacorti-Passerini C,
Rambaldi A,
Garattini E:
Effects of 1,25-dihydroxy vitamin D3 on all-trans retinoic acid sensitive and resistant acute promyelocytic leukemia cells.
Biochem Biophys Res Comm
224:50,
1996[Medline]
[Order article via Infotrieve]
25.
Terstappen LWMM,
Safford M,
Loken MR:
Flow cytometric analysis of human bone marrow III. Neutrophil maturation.
Leukemia
4:657,
1990[Medline]
[Order article via Infotrieve]
26.
Benedetti L,
Grignani F,
Scicchitano BM,
Jetten AM,
Diverio D,
Lo Coco F,
Avvisati G,
Gambacorti-Passerini C,
Adamo S,
Levin AA,
Pelicci PG,
Nervi C:
Retinoid-induced differentiation of acute promyelocytic leukemia involves PML/RAR-mediated increase of type II transglutaminase.
Blood
87:1939,
1996[Abstract/Free Full Text]
27.
Consonni R,
Bertina RM:
Antigenic and functional expression of tissue factor in endotoxin stimulated U937 cells: Regulation of activity by calcium ionophore A23187.
Thromb Haemost
74:904,
1995[Medline]
[Order article via Infotrieve]
28.
Mielicki WP,
Gordon SG:
Three-stage chromogenic assay for the analysis of activation properties of factor X by cancer procoagulant.
Blood Coagul Fibrinol
4:441,
1993[Medline]
[Order article via Infotrieve]
29.
Mielicki WP,
Mielicka E,
Gordon SG:
Cancer procoagulant activity studies using synthetic peptidyl substrates.
Thromb Res
87:251,
1997[Medline]
[Order article via Infotrieve]
30.
Contrino J,
Hair G,
Kreutez DL,
Rickles FR:
In situ detection of tissue factor in vascular endothelial cells: Correlation with the malignant phenotype of human breast disease.
Nature Med
2:209,
1996[Medline]
[Order article via Infotrieve]
31.
Donati MB,
Gambacorti-Passerini C,
Casali B,
Falanga A,
Vannotti P,
Fossati G,
Semeraro N,
Gordon SG:
Cancer procoagulant in human tumor cells: Evidence from melanoma patients.
Cancer Res
46:6471,
1986[Abstract/Free Full Text]
32.
Falanga A,
Gordon SG:
Comparison of properties of cancer procoagulant and human amnion-chorion procoagulant.
Biochem Biophys Acta
831:161,
1985[Medline]
[Order article via Infotrieve]
33.
Gordon SG,
Franks JJ,
Lewis LB:
Comparison of procoagulant activities in extracts of normal and malignant tissue.
J Natl Cancer Inst
62:773,
1979
34.
Saito T,
Koyama T,
Nagata K,
Kamiyama R,
Hirosawa S:
Anticoagulant effects of retinoic acid on leukemic cells.
Blood
87:657,
1996[Abstract/Free Full Text]
35.
Ishii H.,
Horie S.,
Kizaki K.,
Kazama M:
Retinoic acid counteracts both the downregulation of thrombomodulin and the induction of tissue factor in cultured human endothelial cells exposed to tumor necrosis factor.
Blood
80:2556,
1992[Abstract/Free Full Text]
36.
Falanga A,
Marchetti M,
Giovanelli S,
Barbui T:
All-trans retinoic acid counteracts endothelial cell procoagulant activity induced by human promyelocytic leukemia cells (NB4).
Blood
87:613,
1996[Abstract/Free Full Text]
37.
Conese M,
Montemurro P,
Fumarulo R,
Giordano D,
Riccardi S,
Colucci M,
Semeraro N:
Inhibitory effect of retinoids on the generation of procoagulant activity by blood mononuclear phagocytes.
Thromb Haemost
66:662,
1991[Medline]
[Order article via Infotrieve]
38.
Shibakura M,
Koyama T,
Saito T,
Shudo K,
Miyasaka N,
Kamiyama R,
Hirosawa S:
Anticoagulant effects of synthetic retinoids mediated via different receptors on human leukemia and umbilical vein endotelial cells.
Blood
90:1545,
1997[Abstract/Free Full Text]
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Abstract
Introduction
Abstract