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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3368-3375
Human Monocytoid Leukemia Cells Are Highly Sensitive to Apoptosis
Induced by 2 -Deoxycoformycin and 2-Deoxyadenosine:
Association With dATP-Dependent Activation of Caspase-3
By
Nozomi Niitsu,
Yuri Yamaguchi,
Masanori Umeda, and
Yoshio Honma
From the Department of Chemotherapy, Saitama Cancer Center Research
Institute, Saitama; and the First Department of Internal Medicine, Toho
University School of Medicine, Tokyo, Japan.
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ABSTRACT |
The adenosine deaminase (ADA) inhibitor 2 -deoxycoformycin
(dCF) significantly inhibits the proliferation of leukemia and lymphoma
cell lines. When cells were incubated in the presence of both dCF and
2-deoxyadenosine (dAd), the concentration of dCF required to
induce apoptosis of monocytoid leukemia cells was much lower than that
required for myeloid, erythroid, or lymphoma cell lines. Among the cell
lines tested, U937 cells were the most sensitive to this treatment. The
concentration of dCF that effectively inhibited the proliferation of
U937 cells was 1/1,000 of that required for lymphoma cell lines, on a
molar basis. However, the uptake of dCF or dAd in U937 cells was
comparable with that in other leukemia and lymphoma cell lines. The
intracellular accumulation of dATP in U937 cells was only
slightly higher than that in other leukemia cells in dCF-treated
culture. Treatment with dCF plus dAd induced apoptosis in U937 cells at
low concentrations, and this apoptosis was reduced by treatment with
caspase inhibitors. Induction of caspase-3 (CPP32) activity accompanied
the apoptosis induced by dCF plus dAd. No activation of CPP32 was
observed in cytosol prepared from exponentially growing leukemia and
lymphoma cells. However, dATP effectively induced CPP32 activation in
cytosol from monocytoid cells, but not in that from nonmonocytoid
cells, suggesting that dATP-dependent CPP32 activation is at least
partly involved in the preferential induction of apoptosis in
monocytoid leukemia cells. The combination of dCF and dAd may be useful
for the clinical treatment of acute monocytic leukemia.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
WITH PROGRESS in chemotherapy, the
frequency of complete remission of acute myelogenous leukemia (AML) has
increased. However, acute monocytic leukemia is more resistant to
intensive chemotherapy than other types of AML, and its prognosis is
poor. Even when remission is achieved by treatment with conventional
cytotoxic antileukemic drugs, the median duration of remission is only
about 6 months.1 These results in acute monocytic leukemia
clearly call for improved therapies.
2-Deoxycoformycin (dCF), a nucleoside analog produced by
Aspergillus nidulans Y176-2,2 is a specific and
potent inhibitor of adenosine deaminase (ADA). dCF has been effectively
used to treat hairy cell leukemia,3 adult T-cell
leukemia/lymphoma,4 cutaneous T-cell lymphoma,5
acute lymphoblastic leukemia,6 chronic lymphocytic
leukemia,7 and other lymphoid malignancies. The mechanism
of action of dCF, either as an antitumor agent in vivo or as
an antiproliferative agent when combined with the ADA substrate
2-deoxyadenosine (dAd) in vitro, is not completely understood.
However, it is generally believed to be mediated through the
accumulation of dAd after ADA inhibition. These changes have been
reported to result in elevated levels of dATP,8 inhibition of S-adenosyl homocysteine (SAH) hydrolase,9 and
single-strand breaks in DNA.10
Recently, we showed that dCF induced functional and morphological
differentiation of myeloid leukemia HL-60 and NB4 cells in combination
with dAd, but not alone.11 Differentiation of these cells
was effectively induced by clinically applicable concentrations of dCF
in the presence of dAd. Although dCF has been reported to be less
effective in clinical trials against myeloid malignancies than against
lymphoid ones, dCF might be useful for treating some myeloid
malignancies. Preliminary results showed that human monocytic leukemia
U937 cells were much more sensitive to dCF than human lymphoma cell
lines with regard to the inhibition of cell proliferation. Therefore,
in the present study, we examined the growth-inhibitory effect of dCF
on several human myeloid, monocytoid, and lymphoid cell lines and the
possible mechanism(s) of selective apoptosis of monocytoid leukemia
cells.
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MATERIALS AND METHODS |
Materials.
dCF was obtained from The Chemo-Sero-Therapeutic Research Institute
(Kumamoto, Japan). dAd, thymidine (dT), deoxyguanosine (dG),
deoxycytidine (dC), 5-amino-5-dAd,
1-[ -D-arabinofuranosyl] cytosine (Ara C) and
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) were
purchased from Sigma Chemical Co (St Louis, MO). Polyethyleneimine (PEI)-cellulose thin-layer plastic sheets were purchased from Merck
(Darmstadt, Germany). [G-3H]dCF (179 GBq/mmol) and
[8-14C]dAd (2072 MBq/mmol) were obtained from Moravek
Biochemicals, Inc (Brea, CA). Acetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD),
acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD), and
benzyloxycarbonyl-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-Asp-CH2-DCB) were purchased from Peptide Laboratories
(Osaka, Japan). Monoclonal antibodies against human CPP32 (caspase-3) and cytochrome c were purchased from Transduction Laboratories (Lexington, KY) and PharMingen (San Diego, CA), respectively.
Cell lines and cell culture.
Human myeloid (HL-60 and NB4), monocytoid (U937, THP-1, and HEL/S),
erythroid (K562, KU812, and HEL) leukemia, and B-cell lymphoma (BALM3,
SKW-4, and U-698-M) cell lines were cultured in suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a
humidified atmosphere of 5% CO2 in air.12 The
B-cell lymphoma cell lines were donated by Dr Yoshinobu Matsuo (Hayashibara Biochemical Laboratories, Okayama, Japan).
Assay of cell growth and apoptosis.
Cells (105/mL) were suspended in 2 mL of culture medium and
cultured with or without test compounds in multidishes (Costar, Cambridge, MA). Cell numbers were counted with a Model ZM Coulter Counter (Coulter Electronics, Luton, UK) after culture for the indicated times. Cell viability in the above experiments was examined by MTT assay, as described previously.13 Morphological
changes were examined in cell smears stained with
May-Grünwald-Giemsa stain. The cellular DNA content was analyzed
using propidium iodide-stained nuclei.14
Measurement of ADA activity.
Cells were washed three times with phosphate-buffered saline (PBS) and
cell suspension (107/0.1 mL) was lysed by sonication for 30 seconds. After centrifugation at 2,000g for 10 minutes, the
supernatant fluid was used as a crude cell extract for assay of ADA
activity. Activity was measured in terms of the conversion of dAd to
deoxyinosine in the presence of cell extract.14
Uptake of dAd and dCF.
Cells were incubated with [14C]dAd or
[3H]dCF at final external concentrations of 10 to 50 µmol/L or 0.1 to 10 µmol/L for various periods of time ranging from
0.25 to 24 hours, respectively. At each time point, an aliquot of cells
was obtained by centrifugation and washed twice with PBS. The
radioactivity of the cell pellets was counted in a liquid scintillation
counter.
Measurement of dATP accumulation.
Cells (3 × 106 cells/5 mL) were preincubated with or
without 0.1 µmol/L dCF at 37°C for 2 hours. Triplicate cultures
were incubated with 50 µmol/L [14C]dAd for 1 hour. The
incubation was stopped by adding 5 vol of cold PBS and washed three
times with cold PBS. Nucleosides and nucleotides were then extracted
with tetrahydrofuran at 4°C for 2 hours.15 Ascending
chromatography on a PEI-cellulose plate was used to separate dAd and
its metabolites. Authentic compounds were added to the supernatant and
an aliquot was spotted on a chromatography sheet, which was developed
in a solvent system of 0.1 mol/L LiCl. The zones corresponding to
authentic compounds were evaluated by autoradiography with a Fuji
Bio-Image Analyzer BSA2000 (Fuji Film Co, Ltd, Tokyo, Japan).
Assay for ICE and CPP32 activity.
ICE (caspase-1) and CPP32 (caspase-3) activities were assayed with the
fluorogenic substrates acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (YVAD-MCA) and acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-MCA) (Peptide Institute, Inc, Osaka, Japan).16
Briefly, 107 cells were extracted with 1 mL of 10 mmol/L
Tris-HCl (pH 8.1) containing 9 mmol/L
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 5 mmol/L
dithiothreitol, 1 mmol/L phenylmethanesulfonyl fluoride, 100 µmol/L
leupeptin, 1.5 µmol/L pepstatin, 18.4 µmol/mL phosphoramidon, and 5 mmol/L EDTA at 0°C for 30 minutes, and then centrifuged at 12,000 rpm for 2 minutes. Extracts were stored at  80°C until use.
For assay, the extracts were mixed with the substrate (final, 100 µmol/L) and Tris-HCl (final, 10 mmol/L; pH 7.4), and incubated at
37°C for 90 minutes. An equal volume of 1 mol/L acetic acid was
added and the supernatants were analyzed by a fluorescence spectrophotometer (excitation at 370 nm and emission at 460 nm). Enzyme
activity was expressed as pmol aminomethylcoumarin/min/mg protein.17
Preparation of cytosol and immunoblot analysis.
Exponentially growing cells were obtained and washed three times with
cold PBS. The cells were suspended in 5 vol of cold extraction buffer
(20 mmol/L HEPES-KOH [pH 7.5], 10 mmol/L KCl, 1.5 mmol/L
MgCl2, 1 mmol/L sodium EDTA, 1 mmol/L sodium EGTA, 1 mmol/L
dithiothreitol, and 1 mmol/L phenylmethylsulphonyl fluoride). After sitting on ice for 15 minutes, the cells were broken by passing
10 times through a G28 needle. After centrifugation in a
microcentrifuge for 15 minutes at 4°C, the supernatants were further centrifuged at 105g for 60 minutes in a
table-top ultracentrifuge (Beckman, Palo Alto, CA). The
resulting supernatants were used to assay activation of CPP32. An
aliquot (10 µL) of cytosol (50 µg protein) was incubated in the
presence of dATP at 37°C for 1 hour in a final volume of 20 µL of
extraction buffer. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). Western blot analysis was performed as described
previously.18,19
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RESULTS |
Effects of dCF and dAd on the growth of several human leukemia and
lymphoma cell lines.
We examined the growth-inhibitory effects of dCF and dAd on several
leukemia and lymphoma cell lines (Fig 1).
dCF alone did not inhibit the growth of any of the cell lines tested,
even at a high concentration (data not shown), and dAd had only a
modest growth-inhibitory effect. When the cells were treated with 10 µmol/mL of dAd for 4 days, growth was inhibited by 0% to 5%. Cell growth was dose-dependently inhibited by dCF in the presence of 10 µg/mL dAd (Fig 1). Monocytic leukemia U937, THP-1, and HEL/S cells
were much more sensitive to the growth-inhibitory activity of dCF and
dAd than other cells: 9.2 to 9.7 nmol/L dCF in the presence of 10 µmol/mL dAd inhibited growth by 50% (IC50). The cells
were cultured with various concentrations of dCF in the presence of 10, 30, and 50 µmol/mL of dAd for 4 days, and IC50 concentrations of dCF were calculated. The IC50s of
monocytoid leukemia cells were three orders of magnitude lower than
those of the other leukemia and lymphoma cells. The synergistic effect of dCF and dAd was also clearly observed in the treatment of U937 cells
for 2 days (data not shown).
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| Fig 1.
Combined effects of dCF and dAd on the growth of several
human leukemia and lymphoma cells. Cells were cultured with various
concentrations of dCF in the presence of 10 µmol/L dAd for 4 days.
Monocytic: U937 ( ), THP-1 ( ), HEL/S ( ); myeloid: HL-60 ( ),
NB4 ( ); erythroid: K562 ( ), KU812 ( ), HEL ( ); B-lymphoma:
BALM3 ( ), SKW-4 ( ), U-698-M ( ).
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Reduction of the growth-inhibitory effect of dCF plus dAd on U937
cells by other nucleosides.
dCF effectively inhibits ADA, which catalyzes the conversion of
adenosine and dAd into inosine and deoxyinosine, respectively, by
deamination. If dCF is administered, the dAd level in the blood increases as a result of the inhibition of ADA activity, and dAd is
incorporated into cells where it undergoes triphosphorylation and is
converted into dATP. This strongly inhibits ribonucleotide reductase
(RNR) activity, leading to the inhibition of DNA
synthesis.20 In addition, dAd can inhibit the
methylation of DNA and RNA by inhibiting SAH hydrolase, an enzyme that
is essential for methylation.21,22 When U937 cells were
treated with 10 µmol/L each of dC, dT, and dG in the presence of dCF,
no significant inhibition of growth was observed. Adenosine was also
ineffective in inhibiting cell growth in combination with dCF,
suggesting that the combination of dAd and dCF is specific for the
growth-inhibitory effect. The growth-inhibitory effect of dAd plus dCF
was decreased by the addition of dC, dT, and dG
(Fig 2). The addition of 10 µmol/L dC
alone did not reduce the growth-inhibitory effect of dCF plus dAd (data
not shown). When neplanocin A, a potent inhibitor of SAH hydrolase
inhibitor,22 was applied along with various concentrations of dAd and/or dCF, dAd and/or dCF did not significantly
enhance the growth inhibition induced by neplanocin A, suggesting that the growth-inhibitory effect of dCF plus dAd on U937 cells is not
primarily mediated by the inhibition of SAH hydrolase activity.
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| Fig 2.
Effects of other nucleosides on the growth of
monocytoid (U937, THP-1), K562 erythroid, or HL-60 myeloid cell
lines treated with dCF and dAd. Cells were cultured with various
concentrations of dCF in the presence of 0 (), 10 µmol/L dAd
(), 10 µmol/L each of dC + dG + dT (), or 10 µmol/L each
of dAd + dC + dG + dT ( ) for 4 days.
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Effects of dAd analogs on the growth of several human leukemia cells.
Treatment with 5-amino-5-dAd, an inhibitor of adenosine
kinase,14 dose-dependently reduced the growth inhibition
induced by the combination of dCF and dAd in monocytoid U937 and THP-1 cells (Fig 3). A low concentration of this
drug (1 µmol/L) reversed the growth inhibition in the monocytoid
cells treated with 1 µmol/L dCF and 50 µmol/L dAd, suggesting that
adenosine phosphorylating activity may be involved in the
growth-inhibitory effect of dCF plus dAd.
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| Fig 3.
Reduction of the growth-inhibitory effect of dCF plus dAd
by 5-amino-5-dAd. Cells were cultured with 50 µmol/L
dAd and various concentrations of dCF in the presence of 0 (), 1 (), or 10 () µmol/L 5-amino-5-dAd for 4 days.
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Ara C is phosphorylated by deoxycytidine kinase, and it similarly
suppressed the growth of U937, THP-1, HL-60, K562, and BALM3 cells
(data not shown). Fludarabine and cladribine are ADA-resistant analogs
of dAd and need intracellular phosphorylation by deoxycytidine kinase
to form intracellular active metabolites.23,24 These drugs
similarly suppressed the growth of monocytoid and nonmonocytoid cells.
Hydroxyurea, a potent inhibitor of RNR activity, also similarly inhibited the growth of these cells (data not shown), suggesting that
RNR in U937 and THP-1 cells is as sensitive to hydroxyurea as that in
other leukemia cells.
Cellular uptake of dCF, ADA activity, and the accumulation of dATP in
cells treated with dCF.
Conversion of dAd to deoxyinosine is prevented when ADA is
substantially inhibited by dCF. dAd is converted via phosphorylation catalyzed by adenosine kinase into dATP, which effectively inhibits RNR
activity. Thus, this inhibition reduces the production of dGTP, dCTP,
and dTTP and causes derangement of DNA synthesis. dCF has been
considered to suppress cell growth through these processes. We measured
the dATP level in several leukemia cells and the effect of dCF on dATP
synthesis in the cells. U937 and K562 cells exhibited similar kinetics
in their uptake of [14C]dAd with respect to time (data
not shown). dCF was taken up much more slowly than natural nucleosides.
With [3H]dCF at an extracellular concentration of 10 nmol/L, a steady intracellular level was reached after 3 hours; ie,
4.98 and 2.47 pmol/107 cells for U937 and K562 cells,
respectively. However, in the presence of 50 µmol/L dAd this level
was 7.32 and 2.71 pmol/107 cells, respectively
(Fig 4). The intracellular uptake of dCF by
other monocytic leukemia cells was also slightly higher than that by
other types of leukemia cells (Fig 4).
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| Fig 4.
Uptake of dCF into monocytoid and nonmonocytoid cells.
Cells were incubated with 10 nmol/L [3H]dCF in the
presence or absence of 50 µmol/L dAd. U937 cells with () or
without () dAd, THP-1 cells with () or without () dAd, HL-60
cells with () or without ( ) dAd, and K562 cells with () or
without () dAd. Values are the mean for three separate
experiments.
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ADA activity of untreated U937 cells was similar to that of K562 cells,
and there was no significant difference in the sensitivity of ADA
activity in the cell lysates to dCF between U937 and K562 cells,
suggesting that ADA of U937 cells was quantitatively and qualitatively
similar to that of K562 cells (data not shown). Treatment with dCF for
24 hours concentration-dependently inhibited ADA activity of leukemia
cells, and ADA activity in dCF-treated U937 and THP-1 cells was
slightly lower than that in nonmonocytoid cells
(Fig 5).
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| Fig 5.
ADA activity of dCF-treated cells. U937 (), THP-1
(), HL-60 (), NB4 (), and K562 () cells were treated with
various concentrations of dCF for 24 hours. The cell extracts were
incubated with [14C]dAd at 37°C for 60 minutes.
Values are the mean for four separate experiments.
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The amount of dATP in several leukemia cells treated with dCF was
investigated. The formation of dATP in untreated U937 and K562 cells
was 22.3 and 26.2 pmol/106 cells/h, respectively, when
incubated with 50 µmol/L [14C]dAd. Treatment with dCF
resulted in the concentration-dependent accumulation of large amounts
of dATP in these cells. After incubation with 0.1 µmol/L dCF for 1 hour, the amount of dATP was 78.6 and 54.5 pmol/106 cells
in U937 and K562 cells, respectively. Lower levels of dATP accumulated
in BALM3, NB4, HL-60, and HEL cells (53.4, 49.8, 53.1, and 61.2 pmol/106 cells), and a higher accumulation of dATP was
observed in THP-1 and HEL/S cells (71.5 and 79.2 pmol/106
cells), although these differences were modest. Similar results were
obtained when intracellular dATP levels were measured by the DNA
polymerase assay method (data not shown).
Induction of apoptosis of U937 cells treated with dCF and dAd.
When exposed to dCF in the presence of10 µmol/L dAd for 2 days, the
number of viable U937 cells decreased in a dose-dependent manner,
whereas the viability of K562 cells was not affected. dCF effectively
inhibited the viability of U937 cells at a concentration of less than 5 nmol/L. After exposure to dCF for 2 days, a morphological analysis
showed shriveled cells, chromatin condensation, nuclear fragmentation
and cytoplasmic blebbing (data not shown). Induction of apoptosis in
treated U937 cells was confirmed by an analysis of DNA histograms
(Fig 6). When U937 cells were cultured with various concentrations of dCF in combination with 10 µmol/L dAd for 2 days, cells in sub G1 phase (apoptotic cells) appeared dose dependently. A significant increase in apoptotic cells was observed in
U937 cells treated with 1 nmol/L dCF in the presence of 10 µmol/L dAd
(Fig 6). Similar findings were observed in other monocytic leukemia
cells at this concentration, but not in K562 or HL-60 cells (Fig 6).
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| Fig 6.
DNA histogram of U937, HL-60, and K562 cells after
treatment with dCF and dAd. Cells were cultured with 1 (2) and 10 (3)
nmol/L dCF in the presence of 10 µmol/L dAd for 2 days. 1, Untreated
control cells. Cells were fixed and stained with propidium iodide, and
the DNA content was analyzed by flow cytometry. The apoptotic cell
population is shown by the first peak (Apo).
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Untreated U937 cells expressed Fas-antigen (22.5% positive cells), and
this expression increased to 60.3% after treatment with the
combination of 10 nmol/L dCF and 10 µmol/L dAd (data not shown).
However, THP-1 cells did not express the antigen, and expression was
not affected by the combined treatment.
An apoptotic signal requires activation of ICE and CPP32, members of a
family of cysteine proteases that are evolutionarily conserved
determinants of cell death.25,26 Using tetrapeptide inhibitors of ICE and CPP32, we examined the extent to which these proteases participate in the apoptosis of U937 cells caused by dCF plus
dAd. U937 cells were treated with YVAD-CHO, DEVD-CHO, or
Z-Asp-CH2-DCB for 2 hours, and then with dCF+dAd. Cell
viability was measured by the MTT assay after 48 hours.
Figure 7 shows that these peptides
suppressed apoptosis caused by dCF plus dAd, suggesting that ICE and
CPP32 are involved in the apoptosis induced by dAd plus dCF. YVAD had
the weakest suppressive effect. Next, we examined the activities of ICE
and CPP32 using YVAD-MCA and DEVD-MCA as substrates in the extract of
U937 or THP-1 cells treated with dAd plus dCF. Treatment with dCF in
the presence of 10 µmol/L dAd dose-dependently induced both
proteolytic activities, and the activity of DEVD-MCA cleavage was
slightly greater (Fig 8). Assessment of the
expression of apoptosis-associated mRNAs showed that bcl-2,
bcl-XL, and c-myc mRNA expression began
to decrease 24 hours after treatment with 10 nmol/L dCF plus 10 µmol/L dAd, but bax mRNA expression did not significantly change
(data not shown).
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| Fig 7.
Effect of caspase inhibitors on dCF-induced cell death.
U937 cells were incubated with culture medium alone (), 20 µmol/L
YVAD (), 20 µmol/L DEVD (), or 100 µg/mL
Z-Asp-CH2-DCB () for 2 hours and then further incubated
with various concentrations of dCF in the presence of 10 µmol/L dAd
for 2 days. Values are the mean SD for three separate experiments.
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| Fig 8.
Induction of caspase activity by dCF and dAd. Cell
lysates from U937 and THP-1 cells treated with various concentrations
of dCF and 10 µmol/L dAd for 2 days were assayed for protease
activity toward Ac-YVAD-MCA () or Ac-DEVD-MCA (). Values are the
mean SD for three separate experiments.
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dATP-dependent activation of CPP32 in cytosol of leukemia cells.
CPP32 in cytosol can be activated by dATP, but not by other
nucleotides.18 We prepared 100,000g cytosolic
supernatants from exponentially growing leukemia and lymphoma cells.
Because the activation of CPP32 is the result of cleavage of its
32-kD precursor into a 20-kD NH2-terminal fragment and an
11-kD COOH-terminal fragment,27 the activation of CPP32 in
the cytosol was monitored by Western blot analysis using a monoclonal
antibody against the 20-kD fragment of CPP32.
Figure 9 shows that dATP markedly
accelerates the activation of CPP32 in monocytic leukemia cell cytosol,
whereas it does not effectively activate CPP32 in nonmonocytic leukemia cell cytosol.
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| Fig 9.
dATP-dependent activation of CPP32 in vitro. An aliquot
(10 µL) of cytosol was incubated with 0, 0.1, or 1 mmol/L dATP at
37°C for 60 minutes. HL-60 cells (lanes 1 through 3), K562 cells
(lanes 4 through 6), BALM3 cells (lanes 7 through 9), U937 cells (lanes
10 through 12), THP-1 cells (lanes 13 through 15), HEL/S cells (lanes
16 through 18), ML-1 cells (lanes 19 through 21), and NB4 cells (lanes
22 through 24). P32; CPP32, and P20; 20-kD fragment of CPP32 (active
form).
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Recent reports link the release of mitochondrial cytochrome c to the
induction of apoptosis.19 Cytochrome c was detected in the
cytosol of monocytic leukemia cells, and the amounts were similar to
those in nonmonocytoid cells (data not shown). We also examined the
total amount of cytochrome c per cell in U937 and K562 cells. The
results indicated that these cells contain comparable amounts of
cytochrome c. Cytochrome c is a protein located in the intermembrane
space of mitochondria.28 Therefore, the presence of
cytochrome c in the cytosolic fraction may be the result of a ruptured
outer mitochondrial membrane caused by hypotonic shock during
preparation. To test this hypothesis, we also prepared cytosol from
U937 and K562 cells in the presence of 0.25 mol/L sucrose to preserve
mitochondrial integrity. The cells were gently broken by douncing in a
teflon homogenizer. Cytosol prepared this way contained little
cytochrome c compared with that used in the previous experiments, and
there was no significant difference between U937 and K562 cells (data
not shown). These results suggest that the release of cytochrome c does
not play a dominant role in the selective activation of CPP32 in the
cytosol of monocytoid cells, although cytochrome c is required for
activation of CPP32.
| |
DISCUSSION |
dCF, a heterocyclic purine analog of antimicrobial origin, is an
irreversible inhibitor of ADA with an inhibition constant (Ki) value of 0.1 nmol/L. As a result of the
high ratio of the phosphorylating enzyme (deoxy)adenosine kinase to the
dephosphorylating enzyme 5-nucleotidase in lymphocytes, adenosine and
dAd are converted to triphosphate metabolites. The accumulation of dATP
inhibits RNR, which in turn inhibits DNA synthesis. This is believed to be the mechanism of action of dCF in dividing cells. However, treatment
with dCF plus dAd is toxic to both resting lymphocytes and
phytohemagglutinin (PHA)-stimulated lymphocytes, which do not
synthesize DNA when their ADA is inhibited by dCF.20
Sylwestrowicz et al29 suggested that there may be two
mechanisms for the toxicity of dCF toward blastic cells. First, the
accumulation of dATP may inhibit RNR by reducing the supply of the
three other deoxynucleoside triphosphates needed for DNA synthesis.
Second, the accumulation of SAH may inhibit
S-adenosylmethionine-mediated reactions. The present study showed that
the growth-inhibitory effect by dCF plus dAd is decreased in the
presence of dC, dG, and dT, suggesting that the inhibition of RNR may
be involved in this growth-inhibitory effect. However, there was no
significant difference in the growth-inhibitory effects of hydroxyurea
on monocytoid and nonmonocytoid leukemia cells, suggesting that the
inhibition of RNR is not involved in the preferential effect of dCF
plus dAd on monocytoid cells. Neplanocin A (an SAH hydrolase inhibitor)
did not affect the growth inhibition produced by dCF plus dAd, and the
growth suppression produced by neplanocin A alone was similar among the
various cell lines, suggesting that the inhibition of SAH hydrolase
does not play a role in the preferential suppression of cell growth.
Therefore, the antiproliferative effect of dCF plus dAd on monocytoid
cells may be based on some mechanism(s) other than the suppression of RNR or SAH hydrolase.
ADA, a key enzyme in the purine salvage pathway, regulates
intracellular adenosine and dAd levels through the irreversible deamination of adenosine and dAd. ADA is widely distributed in mammalian tissues, with the highest activity found in lymphoid tissues,
including circulating lymphocytes, spleen, and thymus, although
significant activity is also found in the intestine and pancreas.30 ADA levels are higher in normal circulating T
cells than in B cells, and are much higher in the blast cells of
patients with thymic phenotype of acute lymphocytic leukemia than in
those of common non-B-cell and non-T-cell acute lymphocytic
leukemia.31 ADA activity is low in normal bone marrow
cells, whereas myeloid leukemic blast cells express high
levels.32 T lymphoblasts show high ADA activity and contain
a high concentration of dATP, and are thus very sensitive to the
cytotoxic effects of dAd.33,34 According to Camici et
al,14 the sensitivity of colon carcinoma cells increases
with adenosine kinase activity. In the present study, we investigated
the mechanism by which dCF + dAd produces marked suppression of the
growth of monocytic leukemia cell lines. First, we measured ADA
activity in the cells. The ADA activity of U937 cells was not
significantly different from that of nonmonocytoid K562 cells, nor was
there any remarkable difference in the suppression of ADA activities
after treatment with dCF + dAd. We also measured intracellular levels
of dAd and dATP in the cells. The uptake of dAd and accumulation of
dATP in dCF-treated K562 were comparable to those in U937 cells. dAd
kinase has been considered to have no role in the suppression of cell
growth by fludarabine or cladribine.23,24 These drugs are
also adenosine analogs, but are metabolized by deoxycytidine kinase to
triphosphates. Their cytotoxicity has been considered to be based on
the inhibition of RNR by these triphosphates. Fludarabine and
cladribine exert a cytotoxic effect of comparable strength against a
variety of cells. These results are consistent with the hypothesis that
the inhibition of RNR is not involved in the preferential suppression
of cell growth in monocytoid cells, although dATP formation is required
for the action of dAd and dCF on monocytoid cells.
Monocytic leukemia U937 cells have been reported to undergo apoptotic
cell death when treated with various agents, including anticancer
drugs. Although these agents act on different cellular targets, they
induce a similar pattern of apoptosis, suggesting a common signaling
pathway for apoptosis.35-37 CPP32 normally exists as an
inactive precursor that becomes activated proteolytically in cells
undergoing apoptosis.38,39 More direct evidence to support
the concept that the activity of CPP32 is required for apoptosis came
from an experiment in which a tetrapeptide aldehyde inhibitor that
specifically inhibits CPP32 activity was shown to block
apoptosis.35,40 The present experiments showed that YVAD-CHO, DEVD-CHO, and Z-Asp-CH2-DCB effectively
suppressed the apoptosis caused by dCF plus dAd, and the activities of
both ICE and CPP32 were increased in U937 cells 48 hours after
treatment with dCF plus dAd. These results suggest that caspase
activation is at least partly involved in the apoptosis of monocytoid
cells induced by dCF plus dAd.
Binding of Fas ligand to Fas/APO-1 receptor transduces an apoptotic
signal that requires activation of ICE and CPP32.25 U937
cells significantly expressed Fas-antigen, but the other leukemia cells
examined did not. Therefore, Fas-mediated signal transduction may be
not associated with the apoptosis induced by dCF plus dAd.
Recent reports indicate that caspase-9 is the most upstream member of
the apoptotic protease cascade that is triggered by dATP and cytochrome
c.18,41 In etoposide-induced apoptosis, the spectrum and
subcellular distribution of active caspase species differ between K562
and HL60 leukemia cells.42 In the present study, a
caspase-1 inhibitor blocked the dCF + dAd-induced apoptosis of U937
cells. Therefore, selective activation of several caspases, including
caspase-3 (CPP32), may be involved in the preferential induction of
apoptosis in monocytoid cells. The bcl-2 family of proteins consists of members with both survival-enhancing and proapoptotic activities.43,44 Expression of members of the bcl-2 family was also affected by treatment with dCF plus dAd. The
differential expression of bcl-2 family genes might contribute to the
difference in the sensitivity to dCF + dAd between monocytoid and
nonmonocytoid cells.
Acute monocytic leukemia is more refractory to conventional
chemotherapy than other types of acute myeloid leukemia.1
When dCF is administered along with dAd, the concentration required to
induce apoptosis of human monocytic leukemia cell lines is much lower
than that required to induce apoptosis of human lymphoma or other
leukemia cell lines. We are beginning to examine the apoptosis-inducing
effect of dAd and dCF in monocytoid and nonmonocytoid leukemia cells in
primary culture (4 cases of AML-M4 and M5, 6 cases of other AML, and 5 cases of lymphoid leukemia and lymphoma). In the presence of 10 µmol/L dAd, the IC50s of dCF were 0.03, 4,600, and 92 µmol/L, respectively. These results may provide useful information
regarding the value of this combination for the clinical treatment of
acute monocytic leukemia.
| |
FOOTNOTES |
Submitted February 17, 1998;
accepted June 21, 1998.
Supported in part by Grants for Cancer Research from the Ministry of
Education, Science, Sports and Culture and the Ministry of Health and
Welfare, Japan.
Address reprint requests to Yoshio Honma, PhD, Department of
Chemotherapy, Saitama Cancer Center Research Institute, Ina, Kita-adachi, Saitama 362, Japan.
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