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NEOPLASIA
From the Department of Medicine, Karmanos Cancer
Institute, Wayne State University, and the John D Dingell VA Medical
Center, Detroit, MI; the Burnham Institute, La Jolla, CA; College of
Pharmacy, Oregon State University, Corvallis; and Galderma R and D,
Sophia Antipolis, France.
We have recently described a novel retinoid
6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid
(CD437/AHPN) that induces apoptosis in a number of malignant cell
types. We now describe our studies examining the effects of CD437 and a nonretinoidal analog (MM002) on the in vitro proliferation of the
ALL-REH cell line, the in vitro and in vivo growth of a novel Epstein-Barr virus-negative (EBV B-cell chronic lymphocytic leukemia (B-CLL) is the
most common leukemia in the adult population.1 This
disease is characterized by the progressive accumulation of small
immature lymphocytes, which do not proliferate and remain predominately
(95%) in the G0 phase of the cell cycle.2
Expansion of the malignant clone of B-CLL cells appears to be due to an
underlying defect in its ability to undergo programmed cell
death, that is, apoptosis.2,3 High expression of the
antiapoptotic protein Bcl-2, a profound inhibitor of programmed cell
death, has been reported in the vast majority of B-cell
CLLs4-6 (see also references cited in McConkey et
al,4 Schena et al,5 and Thomas et
al6). Enhanced Bcl-2 expression in B-CLL cells
appears to be due to the hypomethylation of the Bcl-2 promoter region
rather than to a chromosomal translocation involving the Bcl-2
gene, which is seen in the follicular lymphomas.7 The
roles that high Bcl-2 expression play in the pathogenesis, and
specifically in the apoptotic defect of B-CLL cells, are still unclear.
A number of chemotherapy agents and regimens have been found to induce
apoptosis in these cells.3 However, no correlation between
the induction of B-CLL cell apoptosis by these agents and inhibition of
Bcl-2 expression has been found.3
Acute lymphoblastic leukemia (ALL), while much less prevalent than
B-CLL, has a grim prognosis in adults.8 Increased
expression of Bcl-2, as well as other members of the Bcl-2 family, that
is, Bcl-XL and Mcl-1, has been documented in ALL
cells.9-11 The role of these antiapoptotic proteins in the
etiology or chemoresistance of this disease is undefined.
We have recently found that the novel retinoid
6-[3-(1-adamantyl)- 4-hydroxyphenyl]-2-naphthalenecarboxylic
acid (CD437/AHPN) is a potent inducer of apoptosis in a number of cell
types.12-16 Retinoids exert their biological action
through their binding to and activation of specific retinoic acid
nuclear receptors (RARs) and retinoic acid X nuclear receptors (RXRs);
these receptors when complexed with ligand and bound to specific
regions in the promoters of genes designated as retinoid response
elements (RAREs and RXREs) modulate gene
expression.17 CD437 does not bind to the RXRs and
is an extremely poor binder and transactivator of the RAR subtype We examined the ability of CD437 to induce apoptosis in leukemia cells
of lymphoid origin, that is, B-CLL and ALL cells. A B-CLL cell line
(WSU-CLL) that, although Epstein-Barr virus-negative (EBV Materials
Acetylation
Olefination
Deacetylation To the ethyl cinnamate (6.89 g, 25.6 mmol) in methanol (50 mL) was added K2CO3 (7.00 g, 50.6 mmol). This mixture was stirred for 4 hours, then worked up (10% hydrochloric acid wash) to afford ethyl (E)-3-chloro-4-hydroxycinnamate as a white solid (5.05 g, 87% yield): mp, 104° to 106°C; Rf, 0.22 (20% ethyl acetate/hexane); 1H NMR spectrum (300 MHz, CDCl3) 1.44 ppm (t,
J = 7.1 Hz, 3, CH3), 4.44 ppm (q,
J = 7.1 Hz, 2, CH2), 5.75 ppm (s, 1, OH), 6.31 ppm (d, J = 16.0 Hz, 1, HC=CCO), 7.03 ppm (d,
J = 8.8 Hz, 1, ArH), 7.37 ppm (dd, J = 8.8,
2.0 Hz, 1, ArH), 7.51 ppm (d, J = 2.0 Hz, 1, ArH), 7.57 ppm (d, J = 16.0 Hz, 1, C=CHCO).
Triflate formation To the ethyl hydroxycinnamate (5.02 g, 22.1 mmol) and pyridine (4.0 mL, 50.0 mmol) in dichloromethane (50 mL) at 0°C under argon was added trifluoromethanesulfonic anhydride (4.0 mL, 23.7 mmol) (Aldrich) over a 30-minute period. The mixture was stirred for 4 hours, warmed to 20°C, then worked up (10% hydrochloric acid and 5% NaHCO3 washes) to afford ethyl (E)-3-chloro-4-(trifluoromethanesulfonyloxy)cinnamate as a white solid (7.90 g, 98% yield): mp, 59° to 61°C; Rf, 0.49 (20% ethyl acetate/hexane); 1H NMR spectrum (300 MHz, CDCl3) 1.35 ppm (t, J = 7.1 Hz, 3, CH3), 4.28 ppm (q, J = 7.1 Hz, 2, CH2), 6.45 ppm (d, J = 16.0 Hz, 1, HC=CCO),
7.38 ppm (d, J = 8.5 Hz, 1, ArH), 7.48 ppm (dd, J = 8.5, 1.8 Hz, 1, ArH), 7.59 ppm (d,
J = 16.0 Hz, 1, C=CHCO), 7.67 ppm (d, J = 1.9
Hz, 1, ArH).
Biaryl coupling Aqueous Na2CO3 (1.4 mL, 2.0 M) was added to the ethyl (trifluoromethane-sulfonyloxy)cinnamate (0.55 g, 1.53 mmol), 3-(1-adamantyl)-4-benzyloxyphenylboronic acid (0.50 g, 1.38 mmol) [1H NMR spectrum (300 MHz, CDCl3) 1.77 ppm, 2.26 ppm (2 s, 12, AdCH2), 2.07 ppm (s, 3, AdCH),
5.21 ppm (s, 2, CH2), 7.06 ppm (d, J = 8.2 Hz,
1, ArH), 7.3 to 7.5 ppm (multiplet [m], 5, ArH), 8.03 ppm
(d, J = 7.8 Hz, 1, ArH), 8.19 ppm (s, 1, ArH)],
tetrakis(triphenylphosphine)palladium (0.16 g, 0.14 mmol) (Aldrich),
and lithium chloride (0.13 g, 3.1 mmol) in dimethoxyethane (12 mL)
under argon. The mixture was heated at reflux (80° to 85°C)
overnight to achieve the biaryl coupling, then worked up, and
chromatographed (10% ethyl acetate/hexane) to give ethyl
(E)-4-[3-(1-adamantyl)-4-benzyloxyphenyl]-3-chlorocinnamate as as a white solid (0.58g, 79%): mp, 148° to 150°C;
Rf, 0.61 (20% ethyl acetate/hexane); 1H NMR
spectrum (300 MHz, CDCl3) 1.73 ppm, 2.17 ppm (2 s, 12, AdCH2), 2.04 ppm (s, 3, AdCH), 1.33 ppm (t,
J = 7.1 Hz, 3 CH3), 4.26 ppm (q,
J = 7.1 Hz, 2, CH2), 5.17 ppm (s, 2, CH2), 6.46 ppm (d, J = 15.9 Hz, 1, HC=CCO),
7.00 ppm (d, J = 8.2 Hz, 1, ArH), 7.3-7.5 ppm (m, 8, ArH),
7.52 ppm (d, J = 7.1 Hz, 1, ArH), 7.62 (s, 1, ArH), 7.65 ppm (d, J = 15.4 Hz, 1, C=CHCO).
Debenzylation To the ethyl benzyloxyphenylcinnamate (0.50 g, 0.95 mmol) in dichloromethane (10 mL) at 78°C under argon was added boron tribromide in dichloromethane (3.0 mL, 1.0 M) (Aldrich) over a 30-minute period. The mixture was stirred for 2 hours, worked up, and
chromatographed (20% ethyl acetate/hexane) to yield ethyl (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamate
as a pale-yellow solid (0.38 g, 92%): mp, 216° to 218°C;
Rf, 0.37 (20% ethyl acetate/hexane); 1H NMR
spectrum (300 MHz, CDCl3) 1.79 ppm, 2.15 ppm (2 s, 12, AdCH2), 2.09 ppm (s, 3, AdCH), 1.35 ppm (t,
J = 7.1 Hz, 3, CH3), 4.28 ppm (q,
J = 7.1 Hz, 2, CH2), 4.93 ppm (s, 1, OH), 6.46 ppm (d, J = 15.4 Hz, 1, HC=CCO), 6.72 ppm (d,
J = 7.8 Hz, 1, ArH), 7.19 ppm (d, J = 7.8 Hz,
1, ArH), 7.32 ppm (s, 1, ArH), 7.36 ppm (d, J = 8.1 Hz, 1, ArH), 7.44 ppm (d, J = 8.2 Hz, 1, ArH), 7.62 ppm (s, 1, ArH) 7.64 ppm (d, J = 15.5 Hz, 1, C=CHCO).
Ester hydrolysis To the ethyl ester (0.35 g, 0.80 mmol) in aqueous ethanol (40 mL, 75%) was added NaOH (1 pellet). This mixture was stirred at 85°C for 2 hours, acidified (10% hydrochloric acid), then worked up to afford (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid as a pale-tan solid (0.28 g, 85%): mp, 257° to 259°C; Rf, 0.42 (75% ethyl acetate/hexane); 1H NMR spectrum (300 MHz, dimethyl sulfoxide-d6) 1.73 ppm, 2.09 ppm (2 s, 12, AdCH2), 2.03 ppm (s, 3, AdCH),
6.62 ppm (d, J = 16.4 Hz, 1, HC=CCO), 6.85 ppm (d,
J = 8.3 Hz, 1, ArH), 7.14 ppm (d, J = 8.1 Hz,
1, ArH), 7.17 ppm (s, 1, ArH), 7.41 ppm (d, J = 8.0 Hz, 1, ArH), 7.59 ppm (d, J = 15.9 Hz, 1, C=CHCO), 7.69 ppm (d,
J = 7.9 Hz, 1, ArH), 7.88 ppm (s, 1, ArH); mass spectrum (electron-impact high-resolution) for
C25H25ClO3: calculated, 408.1492;
found, 408.1492.
Cell lines and growth conditions. The WSU-CLL and ALL-REH cell lines22,23 were cultured in RPM1 1640 supplemented with 5% FBS and gentamycin (25 µg/mL). B-CLL and ALL cells were isolated from peripheral blood as we have previously described.19 Cells were then incubated in the RPMI medium as described above.Western blots Western blots were performed according to our previously published protocol.25 Logarithmically growing cells were treated with CD437 for various times, and cells were harvested and lysed in Laemmli lysis buffer (0.5 M Tris-HCl, [tris(hydroxymethyl)aminomethane-HCl] [pH 6.8], 0.002 M EDTA [ethylenediaminetetraacetic acid], 10% glycerol, 10% sodium dodecyl sulfate, and 5% -mercaptoethanol). Protein lysates
(50 µg per lane) were electrophoresed on 12% sodium dodecyl
sulfate-polyacrylamide gels and transferred to nitrocellulose membranes. Filters were blocked with 5% nonfat dried milk in 1 ×
phosphate-buffered saline (PBS)/0.5% Tween 20 and then
incubated with the appropriate antibodies. Horseradish
peroxidase-conjugated rabbit antimouse immunoglobulin G (IgG)
(Bio-Rad Laboratories, Hercules, CA) was used as the secondary
antibody, and the bands were developed by means of the Amersham
(Arlington Heights, IL) electrogenerated chemiluminescence
(ECL) nonradioactive method following the manufacturer's instructions.
Apoptosis quantification Staining of apoptotic cells was performed as previously described.26,27 Briefly, after exposure to the agent, cells were harvested, washed with PBS, and resuspended at 1 × 106 cells per milliliter. Fifty microliters of cell suspension was stained with 5 mL acridine orange solution (100 mg/mL in PBS) in the dark. Cells displaying fragmented DNA were detected by means of a fluorescent microscope. Annexin V staining was performed by means of the Apo Direct Kit (Transduction Laboratory, Lexington, KY) according to the manufacturer's directions. Activation of caspase-1, caspase-2, caspase-3, caspase-6, caspase-8, and caspase-9 was assessed by means of a caspase activation kit (Bio Visions, Palo Alto, CA).CD437 inhibition of in vivo growth Small fragments (approximately 30 mg) of WSU-CLL xenografts were transplanted into Fox Chase CB 17 SCID mice obtained from Taconic Laboratory (Germantown, NY) by means of a 12-gauge trocar. Once palpable tumors developed, groups of 5 animals were removed randomly for a treatment and a control group. Each animal in the treatment group received an intravenous injection of CD437 (20 mg/kg) via tail vein daily for a total of 5 days. If the animals' total tumor burden reached 1500 mg, they were killed to eliminate discomfort. Tumor-doubling time in the SCID mice was approximately 7.3 days. The endpoints for assessing antitumor activity were the following: (1) tumor weight (mg) = (A × B2)/2, where A and B are the tumor length and width in millimeters, respectively; (2) tumor growth inhibition ( ) where
= [1 (T/C)], where T is the median tumor weight in the
treated group when the median tumor weight in the control group, C,
reached approximately 900 mg; (3) tumor growth delay, (T C),
where T is the median time (in days) required for the treatment group
tumors to reach 900 mg and C is the median time (in days) for the
control group tumors to reach the same weight; (4) tumor log cell kill
(log10) = (T C) (duration of treatment in
days)/(3.32) × (Td), where Td is the time (in days) required for
the tumor to double its weight during the exponential growth phase.
Growth inhibition and apoptosis Exposure of WSU-CLL and ALL-REH cells to CD437 results in a marked inhibition of growth (Figure 2). The addition of progressively increasing concentrations of CD437 to the 2 cell lines results in increasing inhibition of cell proliferation, with 500 nM CD437 completely blocking all growth with an actual loss in cell numbers. The decrease in the WSU-CLL and ALL-REH cell numbers following exposure to 500 nM of CD437 suggested the induction of apoptosis in these cells. Several parameters were examined to substantiate CD437-mediated apoptosis. Nuclear fragmentation is a hallmark of apoptosis.27 Cells exposed to 500 nM CD437 were assessed for nuclear fragmentation as a function of time. Following CD437 exposure, 30% and 80% to 100% of the cells displayed fragmented nuclei at 1 and 2 days, respectively (Figure 3A-B).
Disruption of membrane phospholipid symmetry has been found to occur
early in the apoptotic process, resulting in exposure of
phosphatidylserine on the outer leaflet of the cytoplasmic membrane and
the subsequent binding of annexin V.28 Therefore, we
examined CD437-mediated annexin staining in the WSU-CLL and REH-ALL
cell lines. An approximately 2- to 3-fold increase in annexin
V-positive but propidium iodide-negative cells (apoptotic but not
necrotic cells) was observed on exposure to 500 nM CD437 (Table
1). To assess whether CD437 induces
apoptosis of these cells through an RAR pathway, we examined the
antiproliferative effects of t-RA and the RAR
Caspase activation Apoptosis is associated with the activation of specific cysteine proteases (caspases)29,30 We therefore examined caspase activation during CD437-mediated WSU-CLL and ALL-REH cell apoptosis by means of a spectrophotometric assay. Marked activation of caspase-2 and caspase-3 was noted at 24 and 48 hours following the addition of CD437 in both cell lines (Figure 4). Levels of activated caspase-2 and caspase-3 increased 13-fold and 15-fold, respectively, in WSU-CLL cells and approximately 6-fold and 14-fold, respectively, in ALL-REH cells at 24 and 48 hours following exposure to CD437, whereas activation of caspase-5, caspase-6, caspase-8 and caspase-9 was minimal (Figure 4).
Caspases have been demonstrated to cleave numerous substrates following
the induction of apoptosis and their activation.31 PARP is
an important enzyme involved in DNA repair and has been demonstrated to
be cleaved by caspase-3 early in the apoptotic process.32
Since CD437 and MM002 (data not shown) markedly activated caspase-3 in
both the WSU-CLL and the ALL-REH cells, we investigated whether PARP
cleavage occurs following activation of caspase-3 in these cell lines.
Generation of the 85-kD fragment indicative of PARP cleavage was noted
as early as 8 hours following exposure to MM002 in both cell lines
(Figure 5); 24 hours of exposure to CD437
was required for PARP cleavage in the WSU-CLL cell line, and while
there was a decrease in the 115-kD PARP band, there was only a minimal
increase in the 85-kD cleaved product (Figure 5A).
Mediators of apoptosis Numerous mediators of apoptosis have now been identified.33 This is especially true of the Bcl-2 family of proteins in which 19 members of mammalian origin have been characterized.33 While sharing similar motifs, Bcl-2 family members have dramatically different effects on apoptosis, with some members being identified as proapoptotic and others as antiapoptotic.33 Bcl-2 and the Bcl-2 family member Mcl-1 both inhibit apoptosis and play important roles in the survival of malignant hematopoietic cells.6,7,34 While Bcl-2 and Mcl-1 are strongly expressed in WSU-CLL and ALL-REH cells, their expression was not modulated by CD437 during CD437-mediated apoptosis (data not shown).A genetic homolog of Bcl-2, Bcl-X encodes 2 proteins,
Bcl-XL and Bcl-XS, owing to alternate splicing
of bcl-x mRNA.35 While Bcl-XL is a potent
inhibitor of apoptosis, Bcl-XS is a potent inducer of
apoptosis.35 Following exposure to 0.5 µM CD437, there
was a rapid cleavage of Bcl-XL to an 18-kD product, with concomitant reduction in the level of the 26-kD form in the WSU-CLL and
ALL-REH cells (Figure 6). This 18-kD
cleavage product of Bcl-XL has been found to be the result
of caspase-3 cleavage of Bcl-XL in cytotoxic T-lymphocyte
line (CTLL) cells and results in the generation of a molecule
that now enhances apoptosis.36 We therefore examined
whether caspase activation is necessary for Bcl-XL cleavage in the WSU-CLL and ALL-REH cells. The addition of the pan-caspase inhibitor Z-VAD-fmk resulted in complete inhibition of
CD437-mediated increased cleavage of Bcl-XL in both cell
lines (Figure 7). We used the caspase-3
inhibitor, Z-DVED-fmk, to assess whether caspase-3 is also
responsible for Bcl-XL cleavage in the WSU-CLL and ALL-REH cells. The addition of Z-DEVD-fmk not only inhibited CD437- and MM002-mediated BCL-XL cleavage, but also markedly inhibited
CD437- and MM002-mediated apoptosis in both cells lines (Figure
8). The 18-kD cleavage product generated
following exposure to CD437 and MM002 can be identified by Western blot
with the use of an antibody directed to an epitope located at the
C-terminal region (Santa Cruz Biotechnology antibody) but not by the
Trevigen antibody directed to an epitope located in the N-terminal
region (between amino acids 3 and 14). The Bcl-2 homology 4 (BH4) domain of Bcl-XL is located between residues
4 and 24.37 This result would strongly suggest that the
amino-terminal BH4 domain is lost in this 18-kD product generated
following CD437 exposure.
MAPK kinase activation We previously found that CD437 activated the p38 and JNK MAPK pathways in HL-60R cells within 2 hours following the addition of CD437.19 The activation of p38 and JNK were dependent and independent, respectively, of caspase activation, suggesting that JNK activation may play a role in the initial events in CD437-mediated apoptosis.38 We therefore examined p38 and JNK activation in WSU-CLL and ALL-REH cells following treatment with CD437. Rapid activation of both the kinases occurred within 2 hours of CD437 addition (Figure 9). Activation of p38 and JNK was not dependent upon caspase activation since Z-VAD-fmk did not block their activation (data not shown). Inhibition of CD437-mediated activation of p38 by means of the p38 inhibitor SB203580 also failed to block CD437-mediated apoptosis in these cells (data not shown).
In vivo CD437 activity The ability of CD437 to inhibit the growth of palpable WSU-CLL tumors in SCID mice was examined. Treatment with CD437 resulted in an 85.3% reduction of tumor weight (T/C = 14.3%), marked tumor log kill (log10 kill = 2.1), and prolongation in animal survival (T C) of 21 days, with one animal being cured (Table
2). The mice did not display toxicity
during or following treatment with CD437. There was no evidence of
weight loss, decreased appetite, or decreased activity.
CD437-mediated apoptosis in primary B-CLL and all cultures B-CLL cells obtained from 4 patients were exposed to CD437 for varying periods of time, and apoptosis was assessed (Table 3). Exposure to 1 or 2 µM CD437 did not enhance the apoptotic frequency, but exposure to 4 µM CD437 resulted in an approximately 1.8- to 2.5-fold enhancement of the apoptotic frequency over that noted in the B-CLL cells treated only with vehicle (Table 3). The marked discrepancy between the concentration of CD437 required to induce apoptosis in the WSU-CLL cells and in the primary B-CLL cells may be due to the fact that the B-CLL cells do not proliferate but remain in G0 during the incubation period. The ability of CD437 to induce apoptosis in primary ALL cell cultures was also examined (Figure 10); 1 µM CD437 induced apoptosis in more than 80% of the cells after a 4-day exposure.
In this study, we demonstrate that a novel retinoid CD437 and
MM002 induce apoptosis in WSU-CLL and ALL-REH cells independently of
the RAR pathway. The ability of retinoids to mediate apoptosis has been
well documented. The receptor pathways involved in retinoid-mediated apoptosis appear to vary according to the cell type involved. In the
HL-60 human myeloblastic leukemia cell line and the SPOC-1 rat tracheal
cell line, the RAR The addition of CD437 or MM002 to the B-CLL and ALL cells resulted in the induction of apoptosis. Following exposure to these agents, cells developed fragmented nuclei and condensation and segregation of chromatin at the margin of the nuclear matrix, with the plasma membrane remaining intact, conditions that are hallmarks of apoptosis. Caspase activation has also been intimately associated with the induction of apoptosis.30,31 Exposure to CD437 and MM002 resulted in marked activation of caspase-2 and caspase-3 in these cells. Activation of caspase-3 was essential to the apoptotic process since inhibition of caspase-3 activity markedly blocked the induction of apoptosis by these 2 compounds. Several studies suggest that caspase-3 plays an important role in B-CLL apoptosis.48,49 Immunochemical analysis of B-CLL cells from patients revealed the presence of caspase-3.48 In addition, similar to our observations following exposure to CD437 or MM002, dexamethasone induction of apoptosis in primary cultures of B-CLL cells obtained from patients resulted in PARP cleavage; PARP is an important substrate of activated caspase-3.32,49 We examined the expression of a number of antiapoptotic and proapoptotic proteins following the addition of CD437 to the WSU-CLL and ALL-REH cells. While Bcl-2 was found in both the cell lines, we found that its levels were not modulated during CD437-mediated apoptosis; this was also true for the antiapoptotic protein Mcl-1 and the proapoptotic protein Bax (not shown). Bcl-XL expression was also found in both cell lines. ALL cell lines as well as patient samples have been found to express Bcl-XL; however, expression of this protein was not found in B-CLL specimens obtained from previously untreated patients.50 Acquisition of Bcl-XL expression in the WSU-CLL cells may be due to the fact that this cell line was obtained from a previously treated patient, who displayed resistance to a number of chemotherapy agents. Expression of drug resistance has been shown to be related to Bcl-XL expression.51,52 Expression of Bcl-XL was found in all of the specimens we examined. The Bcl-2 family of proteins can be subclassified according to their shared motifs and functional attributes: (1) antiapoptotic proteins with a transmembrane domain and 3 or 4 BH domains (BH1 to BH3/BH4), (2) proapoptotic proteins with a transmembrane domain and domains BH1 to BH3, and (3) proapoptotic proteins with only the BH3 domain.53 Expression of the BH4 domain appears to be essential for the antiapoptotic function of some Bcl-2 family members. Deletion of BH4 from Bcl-2 results in loss of its antiapoptotic activity.54,55 The BH4 domain in Bcl-XL has been shown to be the motif responsible for its ability to bind and sequester CED-4, an important caspase activator in Caenorhabditis Elegans and thus, antagonize apoptosis.56 Loss of the BH4 domain in Bcl-XL has been shown to result in a molecule that is now proapoptotic.36 Growth-factor depletion of murine hematopoietic cells has been shown to result in caspase-1 and caspase-3 cleavage of Bcl-XL at aspartate 61, resulting in an approximately 16- to 18-kD product with loss of the BH4 domain36,56; this 18-kD form displays proapoptotic activity in these cells. Exposure of the WSU-CLL and ALL-REH cell lines to CD437 or MM002 also results in the cleavage of Bcl-XL to an 18-kD product with loss of the BH4 domain, as indicated by the loss of the specific-antibody epitope located between residues 4 and 24. Formation of this product during CD437 and MM002 induction of apoptosis is also mediated by caspase-3 since its production is inhibited by the caspase-3 inhibitor Z-VED-fmk. The initial steps in CD437-mediated apoptosis have not yet been
discerned. One of the earliest events noted thus far is the activation of the MAP kinases p38 and JNK. The addition of CD437 results in JNK activation within 1 hour in both leukemia cell types.
Many inducers of apoptosis, including ceramide, ionizing radiation,
hydrogen peroxide, ultraviolet light, tumor necrosis factor- CD437 was a potent inducer of WSU-CLL cell death in vitro. To assess whether CD437 could kill these cells in vivo, we tested its efficacy in SCID mice with palpable WSU-CLL tumors. CD437 dramatically inhibited tumor growth, resulting in a significant tumor kill, prolongation of survival of the animals, and even the cure of one mouse. An 86% inhibition of tumor growth and log10 kill of 2.1 were noted following treatment with CD437, indicating that CD437 is highly active against this tumor according to National Cancer Institute (NCI) criteria.62 Mice were treated only for 5 days and displayed no toxicity. We were not able to evaluate the efficacy of CD437 against the in vivo growth of ALL-REH cells since these cells do not grow in mice. We also tested the ability of CD437 to induce apoptosis in primary cultures of B-CLL and ALL cells obtained from patients; exposure to CD437 significantly enhanced apoptosis in these cells. A much higher concentration of CD437 was required to induce apoptosis in the primary cultures of B-CLL cells. Whether this is due to the fact that these cells do not proliferate and remain in the G0 phase of the cell cycle, as we have found, is not clear. Chronic lymphocytic leukemia displays a wide spectrum of aggressiveness and morbidity. While the course of early-stage disease may be extremely indolent, those patients younger than 50 years old will experience a shortened survival by 19 years on the average.63,64 Patients with advanced-stage disease have only a median survival of 18 months to 3 years.65,66 Several new agents have been developed for the treatment of B-CLL and ALL. We found that CD437 was a potent inducer of cell death in the WSU-CLL cells both in vitro and in vivo as well as in primary B-CLL and ALL leukemia cells obtained from patients. CD437 and its analogs may play a potential role in the treatment of these diseases.
Submitted December 7, 2000; accepted September 27, 2001.
Supported by grants for Medical Research Services of the Department of Veteran Affairs (J.A.F.), Leukemia Society of America (J.A.F.), and National Institutes of Health (P01 CA51993) (M.I.D., J.A.F., M.L., X.-K.Z.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Marcia I. Dawson, Burnham Institute, 10901 Torrey Pines Rd, La Jolla, CA 92037; or Joseph A. Fontana, John D Dingell VA Medical Center, Oncology 11M-HO, 4646 John R St, Detroit, MI 48201; e-mail: marcia_i_dawson{at}hotmail.com or joseph.fontana{at}med.va.gov.
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Top
Abstract
Introduction
) B-cell
chronic lymphocytic leukemia (B-CLL) cell line (WSU-CLL), and
primary cultures of human B-CLL and acute lymphoblastic leukemia (ALL) cells. CD437 and MM002 induce apoptosis in both cell
lines, as indicated by the activation of caspase-2 and
caspase-3, cleavage of poly(adenosine diphosphate-ribose)
(poly(ADP-ribose)) polymerase, increase in annexin V binding,
and subsequent nuclear fragmentation. CD437-mediated apoptosis was not
associated with the modulation of Bcl-2, Bax, or Mcl-1 levels,
but was associated with the cleavage of the antiapoptotic
protein Bcl-XL to a proapoptotic 18-kD form. This cleavage
of Bcl-XL was dependent on caspase-3 activation since Bcl-XL cleavage and apoptosis were inhibited by the
caspase-3 inhibitor Z-DVED-fmk. CD437 markedly inhibited the
growth of WSU-CLL cells in severe combined immunodeficiency
(SCID) mice. Tumor growth inhibition, growth delay, and log
cell kill were 85.7%, 21 days, and 2.1, respectively, in the
treated mice. Moreover, 1 of the 5 treated mice was tumor-free longer
than 150 days and thus was considered cured. Exposure of primary
cultures of both B-CLL and ALL cells obtained from patients to CD437
and MM002 resulted in their apoptosis. These results suggest that CD437
and MM002 analogs may have a potential role in the treatment
of B-CLL and ALL.
(Blood. 2002;100:2917-2925)
.
