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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1497-1504
RAPID COMMUNICATION
Arsenic Trioxide and Melarsoprol Induce Programmed Cell Death in
Myeloid Leukemia Cell Lines and Function in a PML and PML-RAR
Independent Manner
By
Zhu-Gang Wang,
Roberta Rivi,
Laurent Delva,
Andrea König,
David A. Scheinberg,
Carlo Gambacorti-Passerini,
Janice L. Gabrilove,
Raymond P. Warrell Jr, and
Pier Paolo Pandolfi
From the Department of Human Genetics, Molecular Biology Program, and
the Molecular Therapeutics Program, the Sloan-Kettering Institute,
Graduate School of Medical Sciences, Cornell University, New York,
NY; the Leukemia and Developmental Chemotherapy Services,
the Department of Medicine, Memorial Sloan-Kettering Cancer Center, New
York, NY; and the Istituto Nazionale Tumori, Milan, Italy.
 |
ABSTRACT |
Inorganic arsenic trioxide (As2O3) and the
organic arsenical, melarsoprol, were recently shown to inhibit growth
and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and
chronic B-cell leukemia cell lines, respectively.
As2O3 has been proposed to principally target
PML and PML-RAR proteins in APL cells. We investigated
the activity of As2O3 and melarsoprol in a
broader context encompassing various myeloid leukemia cell lines,
including the APL cell line NB4-306 (a retinoic acid-resistant cell
line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To
examine the role of PML in mediating arsenical activity, we also tested
these agents using murine embryonic fibroblasts (MEFs) and bone marrow
(BM) progenitors in which the PML gene had been inactivated by
homologous recombination. Unexpectedly, we found that both compounds
inhibited cell growth, induced apoptosis, and downregulated bcl-2
protein in all cell lines tested. Melarsoprol was more potent than
As2O3 at equimolar concentrations ranging from
10 7 to 105 mol/L.
As2O3 relocalized PML and PML-RAR onto
nuclear bodies, which was followed by PML degradation in NB4 as well as
in HL60 and U937 cell lines. Although melarsoprol was more potent in
inhibiting growth and inducing apoptosis, it did not affect PML
and/or PML-RAR nuclear localization. Moreover, both
As2O3 and melarsoprol comparably inhibited
growth and induced apoptosis of PML+/+ and PML / MEFs, and
inhibited colony-forming unit erythroid (CFU-E) and CFU
granulocyte-monocyte formation in BM cultures of PML+/+ and
PML/ progenitors. Together, these results show that
As2O3 and melarsoprol inhibit growth and induce
apoptosis independent of both PML and PML-RAR expression in a
variety of myeloid leukemia cell lines, and suggest that these agents
may be more broadly used for treatment of leukemias other than APL.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THE USE OF ORGANIC arsenicals was
pioneered by Paul Ehrlich, who showed that Salvarsan (arsphenamine) was
an extremely effective antispirochetal agent.1 Most organic
arsenicals have been superseded by other drugs, but melarsoprol remains
an important therapy for certain tropical diseases, such as African
sleeping sickness.2 Moreover, potassium arsenite
(administered orally as Fowler's solution) was widely used in the West
for controlling leukocytosis in patients with chronic myelocytic
leukemia in the early 1900s.3 However, this therapy
gradually waned with the advent of radiotherapy in the 1930s and the
identification of potent antileukemic agents after the Second World
War.
Recently, inorganic arsenic trioxide (As2O3)
was reported to induce complete remission in a high proportion of
patients with refractory acute promyelocytic leukemia
(APL).4-6 Chen et al showed that
As2O3 induced apoptosis in the APL cell line,
NB4, which may be mediated through downregulation of bcl-2, and
modulation of the PML-RAR fusion protein, the specific product of
the t(15;17) of APL,7-10 and/or the PML
protein.11 More recently, the antileukemic effects of
As2O3 were proposed to be directly mediated by
its ability to induce the relocalization and degradation of PML, as well as the degradation of PML-RAR in APL cells.12-14 We
found that the organic arsenical, melarsoprol, exhibited effects
similar to As2O3 on chronic B-cell leukemic
cell lines,15 a finding which suggested that the cytotoxic
action of this drug may not be dependent on PML or PML-RAR
expression.
In the present study we have explored the effects of
As2O3 and melarsoprol in a variety of myeloid
leukemia cell lines in which the PML-RAR fusion protein is or is not
expressed. Furthermore, to examine whether PML mediated the growth
inhibitory and apoptotic activities of these compounds, we also
evaluated both As2O3 and melarsoprol in cells
from mice in which PML had been disrupted by homologous recombination,
using both murine embryonic fibroblasts (MEFs) and bone marrow (BM)
progenitors from PML/ mice.16-18
We unexpectedly found that As2O3 and
melarsoprol can inhibit growth and induce apoptosis in a PML and
PML-RAR independent manner, thus suggesting that both compounds may
be useful for treatment of leukemias other than APL.
| |
MATERIALS AND METHODS |
Reagents.
Arsenic trioxide (As2O3) was purchased from
Sigma Chemicals, Inc (Milwaukee, WI). A stock solution was made with
phosphate-buffered saline (PBS) at a concentration of
102 mol/L. Melarsoprol (Arsobal; purchased from
Rhône Poulenc Rorer, Paris, France) was dissolved in propylene
glycol at a stock concentration of 0.09 mol/L (36 mg/mL) and stored at
room temperature in the dark. Both solutions were diluted to working
concentrations before use.
Cell culture and cell viability assays.
Cell lines, including NB4 cells, the RA-resistant NB4 subclone 306, HL60, U937, and KG-1, were cultured in RPMI 1640 medium containing 10%
fetal bovine serum (FBS), 2 mmol/L L-glutamine, penicillin G (100 U/mL), and streptomycin (100 µg/mL). MEFs were isolated from
wild-type and PML/ mouse embryos at 13.5 days of
gestation and cultured in Dulbecco's Modification of Eagle's Medium
(DMEM) supplemented with 20% FBS. Early passage MEFs were used. For cell growth and viability assays, NB4, NB4.306, HL60, U937,
and KG-1 cells were seeded at an initial concentration of 1 × 105 cells/mL, and incubated in RPMI 1640 medium plus 10%
FBS with or without As2O3 or melarsoprol. Cell
growth and viability were assessed in triplicate by an automatic
counter (Zb1; Coulter Electronic, Miami, FL) and trypan blue exclusion
at indicated days. The percentage of viable cells in treated samples
was divided by the average viability of the untreated samples
(controls). Each growth curve was repeated at least three times.
In situ terminal deoxynucleotidyl transferase mediated dUTP-biotin
nick-end labeling (TUNEL).
The procedure for identification of programmed cell death in situ was
performed as described by Gavrieli et al,19 with minor modifications. In brief, NB4, HL60, and U937 cells were treated with
reagents described above for 24 hours, and then pelleted onto slides by
centrifugation. The slides were quickly air-dried and fixed in 4%
paraformaldehyde for 30 minutes at room temperature and washed with
PBS. To quench endogenous peroxidase, cells were treated with 0.1%
H2O2 in PBS for 15 minutes at room temperature and washed with dH2O and TdT buffer. The labeling reaction
was performed with TdT-biotin dUTP labeling mix for 1 hour in a humid chamber and stopped in 2× SSC (0.3 mol/L NaCl, 0.03 mol/L sodium citrate, pH 7.0) for 15 minutes. The slides were incubated
with Vectastain ABC reagent (Vector Laboratories, Burlingame, CA) for 30 minutes after blocking with 2% bovine serum albumin (BSA) in PBS.
TUNEL-positive cells were visualized by 3, 3 -diaminobenzidene (DAB) staining and counterstaining with hematoxylin. The
same assay was also performed with MEFs. In this case, 1 × 104 cells were plated in each chamber of Permanox slides
(Nalge Nunc International, Naperville, IL) in 1 mL of DMEM
medium with or without the above-mentioned reagents. After a
24-hour incubation, cells were fixed and the same procedures
described above were performed. At least 300 cells were counted for
each condition.
Western blot analysis.
Cells were lysed in a lysis buffer containing 20 mmol/L
Tris-HCl, 1 mmol/L EGTA, 50 mmol/L NaVO4, 50 mmol/L NaF, 0.01 U/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL
pepstatin, 1 mmol/L elastinal, and 1 mmol/L phenylmethylsulfonyl
fluoride (PMSF) (all from Sigma). The lysates were then sonicated using
a ultrasonic homogenizer (4710 series; Cole Parmer Instruments,
Chicago, IL), centrifuged at 7,500g (Sorvall Instruments,
Newtown, CT), and the protein content of the lysates was determined
(BioRad Protein Assay Kit I, Melville, NY) at 595 nm with a BSA
standard. A sample buffer containing 10% glycerol, 0.4% sodium
dodecyl sulfate (SDS), 0.3% bromphenol blue, 0.2% pyronin Y, in
1× stacking buffer (Tris base 0.5 mol/L, 0.8% SDS), 20%
2-mercaptoethanol, was added to the cell lysates which were
subsequently heat-denatured at 95°C for 3 minutes. Ten micrograms
per lane of protein was loaded on an SDS-polyacrylamide gel containing
12.5% polyacrylamide, and size-fractionated by electrophoresis.
Proteins were electroblotted onto Immobilon-P PVDF transfer membrane
(Millipore, Bedford, MA) and immunostained with a mouse anti-human
monoclonal bcl-2 antibody (clone 124, M0887; dilution 1:5,000; Dako,
Carpinteria, CA). Bound antibody was detected using the ECL
chemiluminescence detection system (Amersham, Arlington Heights, IL).
Protein bands were quantified by computer densitometry.
Immunofluorescence analysis.
Cells were cultured in medium with As2O3 or
melarsoprol at concentrations of 106 and
105 mol/L for 4 hours or 24 hours, and then pelleted
onto slides by cytospin and fixed in 4% paraformaldehyde. A 1:100
dilution of a mouse monoclonal antibody raised against the N-terminal
region of PML (clone PGM3, SC-966; Santa Cruz Biotechnology, Santa
Cruz, CA) was applied to the slides for 1 hour. Subsequently, a 1:200 dilution of a Texas Red-conjugated rabbit anti-mouse antibody (115-075-003; Jackson ImmunoResearch Laboratories Inc, West Grove, PA)
was used as secondary antibody to stain the PML protein.
In vitro BM culture.
BM cells from wild-type and PML/ mice were obtained by
flushing the femur and tibia BM in Iscove's MEM containing 15% FBS and plated at a concentration of 5 × 104 cells per
dish in "Complete" methycellulose medium (StemCell Technologies,
Vancouver, BC, Canada) with or without 106 mol/L of
As2O3 or melarsoprol. Colony-forming unit
erythroid (CFU-E) and CFU granulocyte-monocyte (GM) were counted at
days 2 and 6 after plating, respectively. The inhibition of colony formation was calculated by comparison with control.
| |
RESULTS |
Growth inhibition by arsenicals does not depend on
PML-RAR.
We tested the effects of As2O3 and melarsoprol
on the growth of NB4, HL60, U937, and KG-1 cells. At
concentrations of As2O3 or melarsoprol ranging
from 107 to 105 mol/L, both
compounds inhibited cell growth in all cell lines tested. The effective
concentration of As2O3 for growth inhibition was 106 mol/L, and at equimolar concentrations,
melarsoprol was more potent than As2O3 in all
cell lines but KG-1 (Fig 1). Inhibition of
growth and survival of cell lines was not dependent on the presence or
absence of PML-RAR whose expression is restricted to NB4 cells.
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| Fig 1.
Growth inhibition of NB4, HL60, U937, and KG-1 cell lines
after As2O3 or melarsoprol treatment. Various
concentrations of As2O3 and melarsoprol were
added to the media (Materials and Methods): Controls ( ),
105 mol/L ( ), 106 mol/L ( ), and
107 mol/L ( ). As2O3 at a
concentration of 104 mol/L ( ) was also used.
Representative results from one of three independent experiments are
shown as the mean ± SD of triplicates. Inhibition of cell growth and
loss of viability are comparable in four different cell lines in which
PML-RAR fusion protein is or is not present.
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Growth inhibition by arsenicals of the RA-resistant NB4 subclone 306.
Both drugs were then tested in the RA-resistant subclone 306 derived
from NB4 cells in which the intact PML-RAR protein is no longer
expressed.20 This line was sensitive to
As2O3 (106
mol/L) and melarsoprol (107 mol/L)
(Fig 2A and B). Growth inhibition was also
in this case both time and dose dependent. Surprisingly, NB4.306 seemed
to respond better than the parental NB4 cell line to both compounds. These results are in keeping with the notion that inhibition of growth
and survival of NB4 cell lines does not depend on the presence of
PML-RAR or its degradation, and suggest that both arsenicals can be
used in the treatment of both RA-sensitive and RA-resistant APL.
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| Fig 2.
As2O3 and melarsoprol induce
growth inhibition of the RA-resistant cell line NB4.306 cells.
Concentrations of As2O3 (A, growth curve; C,
viability) and melarsoprol (B, growth curve; D, viability) are the same
as depicted in Fig 1. As2O3 and melarsoprol
inhibit cell growth in a dose-dependent manner.
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Induction of apoptosis by arsenicals in NB4, HL60, and U937 cell
lines.
To show whether the growth inhibition by As2O3
or melarsoprol is caused by induction of apoptosis, in situ TdT
labeling or DAPI staining were performed 24 hours after cells were
treated with different concentrations of As2O3
or melarsoprol (see Materials and Methods). TUNEL-positive cells or
cells with nuclear fragmentation which represent apoptotic cells were
counted. Both As2O3 and melarsoprol induced
apoptosis in all cell lines tested. Maximal apoptotic effects were
observed at 105 mol/L with both drugs; however,
melarsoprol was again somewhat more potent at this concentration
(Fig 3A). At 106 mol/L
concentration, apoptotic cells accounted for only 6% to 10% of the
population examined. However, percent of mortality, in certain cases,
exceeded these values, suggesting that arsenicals may also induce cell
death through mechanisms other than apoptosis. The in situ TdT assay,
in these experiments, seemed more sensitive than the morphological
analysis (Fig 3). Apoptosis induced by these two compounds was
comparable among the three different cell lines tested, irrespective of
the expression of the PML-RAR fusion protein.
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| Fig 3.
As2O3 and melarsoprol induce
apoptosis in NB4, HL60, and U937 cell lines. Apoptotic cells were
identified by the in situ TdT assay, as well as morphological changes
of nuclei after DAPI staining. The percentages of TUNEL-positive cells
(A) and cells with nuclear fragmentation (B) were expressed as the mean ± SD. Morphology of TUNEL-positive cells are indicated with black
arrows and fragmented nuclei are indicated with white arrows, as shown
in panels (C) and (D).
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PML and PML-RAR are relocalized and degraded by
As2O3 but not melarsoprol.
PML is typically concentrated within discrete speckled nuclear
structures that have been variably named PML nuclear bodies (NBs), or
Kremer bodies, ND10, or PODs (for PML oncogenic
domains).21-25 The presence of PML-RAR leads to the
delocalization of PML from the NBs in APL cells, where it acquires a
microspeckled/granular nuclear localization. PML, RAR, RXR, and
the PML-RAR fusion protein colocalize in these aberrant structures.
Upon RA treatment, these proteins reacquire their natural
nuclear localization.23-25
A previous report suggested that As2O3-induced
relocalization of PML or PML-RAR onto nuclear bodies and subsequent
degradation of PML or PML-RAR was key in the induction of apoptosis
in NB4 cells by this compound.12 To test this hypothesis,
we cultured three different leukemic cell lines in the presence of
As2O3 or melarsoprol at a concentration of
106 (Fig 4) or
105 mol/L (not shown) for 24 hours, time by which
both compounds already induced cell death. Indirect immunofluorescence
staining of PML with a specific monoclonal antibody was performed in
control and treated NB4, HL60, and U937 cells after 4 and 24 hours. In NB4 cells, the microspeckled pattern of PML staining rapidly changed to
a speckled pattern upon As2O3 treatment,
followed by a reduction in the number of PML NBs (Fig 4). The normal
nuclear distribution of PML was seen in untreated HL60 and U937 cells,
as shown in Fig 4. However, after incubation with
As2O3, the number of PML NBs was dramatically
reduced also in these cells. Although melarsoprol was more potent in
inducing apoptosis, treatment with this drug did not alter PML or
PML-RAR nuclear localization patterns in NB4, HL60, and U937 cells
either after 4 hours (not shown) or after 24 hours of incubation at
both 106 (Fig 4) or 105 mol/L
(not shown) concentrations, suggesting that these agents do not
modulate PML or PML-RAR proteins in an identical manner.
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| Fig 4.
PML relocalization and degradation in APL or non-APL cell
lines after As2O3 and melarsoprol treatment.
As2O3 induces PML relocalization onto nuclear
bodies, followed by PML degradation not only in NB4 cells but also in
HL60 and U937 cell lines. Melarsoprol does not affect PML nuclear
localization.
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As2O3 and melarsoprol downregulate bcl-2.
To identify what would be a possible mechanism underlying the ability
of As2O3 and melarsoprol to induce apoptosis,
we performed a Western blot analysis of bcl-2 expression on NB4,
NB4-306, HL60, U937 cells treated with various concentrations of
As2O3 and melarsoprol (Materials and Methods).
Both compounds reduced the levels of bcl-2 protein, not only in NB4
cells but also in HL60, U937, and the RA-resistant NB4 subclone 306 upon 24-hour incubation with these drugs
(Fig 5). These changes were dose dependent.
Again, melarsoprol was more potent in this effect compared to
As2O3. Thus, both As2O3
and melarsoprol induce bcl-2 downregulation, and this process does not
depend on the presence of PML-RAR.
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| Fig 5.
Downregulation of bcl-2 by As2O3
and melarsoprol in myeloid cell lines irrespective of PML-RAR
expression. As2O3 and melarsoprol downregulate
bcl-2 expression at concentrations of 106 and
105 mol/L in all cell lines tested.
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Growth inhibition and induction of apoptosis by arsenicals in cells
devoid of PML.
To study the role of PML in growth inhibition or apoptosis by
arsenicals, we took advantage of mice in which the PML gene had been
ablated by homologous recombination.16-18 We treated
primary MEFs isolated from wild-type and PML/ mice with
different concentrations of As2O3 or
melarsoprol for 24 hours. Both compounds triggered apoptosis of MEFs at
concentrations of 106 and 105
mol/L. No differences in apoptosis between wild-type and
PML/ MEFs were observed (Fig
6A and B). Next, we performed in vitro cultures with BM cells from
wild-type and PML/ mice in the presence or absence of
As2O3 or melarsoprol (Materials and Methods).
Formation of CFU-E and CFU-GM from wild-type and PML/ BM
progenitors were comparably inhibited by 106 mol/L
concentration of both compounds (Fig 6C and D). Thus, both As2O3 and melarsoprol can inhibit cell growth
in a PML independent manner.
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| Fig 6.
As2O3 and melarsoprol induce
growth inhibition and apoptosis in a PML independent manner. Wild-type
and PML/ MEFs were incubated with the indicated concentrations of
As2O3 (A) and melarsoprol (B) for 24 hours, and
apoptotic cells were identified by in situ TdT. For each condition, at
least 300 cells were counted. No differences in apoptosis and growth
inhibition induced by As2O3 and melarsoprol
between wild-type and PML/ cells were found. For in vitro BM
cultures (Materials and Methods), wild-type and PML/ cells were
incubated with As2O3 and melarsoprol at a
concentration of 106 mol/L. At day 2 and day 6 CFU-E (C)
and CFU-GM (D) were counted in triplicate. The data are expressed as a
percentage of colony formation of arsenicals-treated versus untreated
cells: untreated = 100%. The bars indicate the mean values ± SD
from one representative experiment out of three.
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| |
DISCUSSION |
Clinical reports from two Chinese groups, confirmed by a preliminary
study in the United States, indicate that As2O3
is extremely effective for inducing complete remission in patients with
APL who are resistant to both cytotoxic chemotherapy and
all-trans retinoic acid.4-6 These studies have
suggested that almost all patients who express PML-RAR fusion mRNA
are clinically responsive. Recently, the cytotoxic effects of this
agent were linked to its ability to relocalize the aberrant PML-RAR
fusion protein onto a specific nuclear organelle, the PML nuclear body,
which was closely followed by degradation of PML-RAR and the PML
proteins.12,14 The direct implication of these results is
that the antileukemic effects of As2O3 would
thus be limited to APL patients who expressed the fusion protein.
However, we have recently tested both As2O3 and
melarsoprol (an organic arsenical) in various B-cell lymphoid cell
lines and observed striking apoptotic effects,15 suggesting that in these cell lines, PML-RAR expression was not essential to
the cytotoxic mechanism. Therefore, to further examine the dependency
of arsenicals on PML or PML-RAR expression, we evaluated the effects
of these agents in a number of myeloid cell lines that expressed
wild-type PML, with or without the fusion PML-RAR, and also in cells
in which wild-type PML had been knocked out by homologous
recombination.
Results from our experiments show that the cytotoxic effect of both
arsenicals in these cell lines is not mediated by mechanisms that are
dependent on PML or PML-RAR expression. In most lines, melarsoprol
was somewhat more potent compared with As2O3 in
inhibiting growth and inducing apoptosis, and the effects of both drugs
were dose dependent. As previously reported,12 we confirmed
that As2O3 relocalized PML protein onto nuclear
bodies and induced PML and PML-RAR degradation in NB4 cells while
triggering apoptosis. However, similar effects were also observed in
HL60 and U937 cells which do not harbor the PML-RAR fusion gene.
Moreover, melarsoprol induced apoptosis in all the cell lines tested
without altering PML and/or PML-RAR.
Similarly, the differentiating action of As2O3
and melarsoprol, which in our hands was negligible, did not appear to
depend on the expression and/or modulation of PML
and/or PML-RAR either. In fact, the small effect we observed
in long-term cultures (up to 2 weeks), was comparable in all the cell
lines tested with both compounds (data not shown).
We also found that bcl-2 downregulation, which has been previously
linked to the antileukemic effects of As2O3 in
APL, was also not dependent on expression of PML-RAR protein,
because it occurred in the NB4 subclone 306 in which the intact protein is not detectable. Finally, to test whether PML expression was essential to the antileukemic effects of arsenicals, we tested both
agents in mouse embryonic fibroblasts and BM cells from animals wherein
wild-type PML had been eliminated by homologous recombination. In these
cells wholly lacking PML expression,18 both
As2O3 and melarsoprol were equally effective in
inhibiting growth and inducing apoptosis, and both had similar effects
on normal CFU-E and CFU-GM colony formation. Moreover, no differences
between wild-type and PML/ cells were observed. Together,
these data strongly support the notion that the antileukemic effects of
these arsenicals occurs independently of both PML and PML-RAR
expression. These results are in keeping with the medicinal history of
arsenicals for diseases that are not characterized by alterations in
PML protein such as, for instance, chronic myelocytic
leukemia.3
Our results indicate that both As2O3 and
melarsoprol are broadly active as antileukemic agents in both myeloid
and lymphoid diseases. However, recent studies in solid tumor cell
lines show that this activity is not indiscriminate, and does not occur
merely as a nonselective cellular poison.26-28 Recent
attempts to extend the clinical utility of melarsoprol have been
constrained by previously unappreciated neurotoxicity.29
Although severe neurotoxicity has also been reported with doses of
As2O3 approximately fivefold in excess of that
recently reported to be effective in APL,30 this effect has
not been observed with the lower doses used clinically in recent
series.4-6
In conclusion, our data indicate that cytotoxic activity is not
mediated by the PML protein and therefore is not limited to diseases
that are associated with alterations in PML expression. Thus, assuming
a reasonable therapeutic index can be established clinically,
arsenicals may have a potentially broader therapeutic role that is not
confined to APL.
| |
FOOTNOTES |
Submitted February 24, 1998;
accepted June 5, 1998.
Supported in part by the National Cancer Institute, National Institutes
of Health (CA-77136 to R.P.W. and CA-71692 to P.P.P.); the US Food and
Drug Administration (FD-R-001364 to R.P.W.); and by a grant from the
Lymphoma Foundation. P.P.P. is a Scholar of the Leukemia Society of
America.
Address reprint requests to Pier Paolo Pandolfi, MD, PhD, Memorial
Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021;
e-mail: p-pandolfi{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" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract