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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2672-2682
NEOPLASIA
From the Laboratory of Molecular Biology and the Department of
Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milano; the
Laboratory of Biology and Treatment of Metastasis, Laboratori Negri
Bergamo; the Division of Hematology, Ospedali Riuniti di Bergamo,
Bergamo, Italy; and Galderma Research and Development, Sophia
Antipolis, France.
6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid
(CD437) is a novel compound that represents the prototype of a new
class of synthetic retinoids with apoptogenic properties in acute
promyelocytic leukemia (APL) and other types of leukemia. In this
article, using SCID mice xenografted with APL-derived NB4 cells, we
demonstrate that CD437 has significant antileukemic activity in vivo.
In addition, we report on the isolation and characterization of an APL
cell line (NB4.437r) resistant to CD437. The cell line retains
expression of PML-RAR
6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene
carboxylic acid (CD437) is the prototype of a new and unique class of
synthetic retinoids1-10 that may find clinical application
in the first- and second-line treatment of acute promyelocytic leukemia
(APL) and other types of acute myelogenous leukemia. The retinoid is likely to exert its apoptogenic effects through the activation of
intracellular pathways that are distinct from those stimulated by
all-trans retinoic acid (ATRA) and many of the available
chemotherapeutic agents.3,6 In vitro, we and others
recently demonstrated that the compound induces programmed cell death
(PCD)2 in ATRA-sensitive and ATRA-resistant APL and acute
myelogenous leukemia cell lines3,6 and in freshly isolated
leukemia cells.3 In vivo, though no data on preclinical
models of leukemia are available, the retinoid is well tolerated and
shows antitumor activity on other types of neoplasia.9,11
The mechanism underlying the apoptogenic action of CD437 is still
obscure, but it does not entail activation of the nuclear retinoic acid
receptor.3,6 In APL cells, the process of PCD does not
require active protein synthesis. It is accompanied by the release of
cytochrome c (cyt c) from the mitochondria and the activation of
caspase-3 and caspase-7, which results in the degradation of many
proteins, including the PML-RAR In this article, we report on the in vivo antileukemic activity of
CD437 and on the isolation and biochemical characterization of a novel
APL cell line, NB4.437r, made resistant in vitro to the apoptogenic
action of the retinoid.
Reagents and cell lines
In vivo experiments
Isolation of the NB4.437r cell line To isolate the CD437-resistant cell line, NB4 cells were cultured for approximately 6 months in medium containing increasing concentrations of CD437. The selection protocol was started at 10 9 mol/L CD437, and the concentration of the
compound was progressively raised to 105 mol/L. At
the end of the selection protocol, we isolated a cell line that
survived in CD437 at 105 mol/L and was routinely
passaged in medium containing the retinoid at 106
mol/L. The CD437-resistant derivative line (NB4.437r) was cloned by
limiting dilution and has been cultured for almost 2 years without
phenotypic changes. Resistance to CD437 is stable; the line has been
subcultured in the absence of the retinoid for up to 4 months without
loss of its characteristic insensitivity to the retinoid. The number of
chromosomes determined by karyotypic analysis of NB4.437r cells (66 < n < 92) is not significantly different from that of the
parental counterparts (68 < n < 90). Before each
experiment, the NB4.437r cell line was cultured for 24 hours in the
absence of CD437.
Uptake and intracellular distribution of CD437 Uniformly 3H-labeled CD437 (specific activity, 50 Ci/mmol) was synthesized by Galderma Research and Development and used for the uptake and intracellular distribution experiments. For the uptake experiments, cells were incubated with 107
mol/L 3H-CD437, washed once with phosphate-buffered saline
and centrifuged, and the pellet was solubilized in Soluene (Packard,
Meriden, CT) before liquid scintillation counting. For the
intracellular distribution studies, cells were incubated in
107 mol/L 3H-CD437 for 1 hour and washed once with phosphate-buffered saline. Cytosolic,
microsomal, mitochondrial, and nuclear fractions from NB4 and NB4.437r
cells were obtained by ultracentrifugation according to standard
procedures and subjected to liquid scintillation counting.
Cytodifferentiation of NB4 and NB4.437r cells The surface markers CD11a, CD11b, CD11c, CD13, and CD33 were measured by flow cytometry using specific rhodamine- or fluorescein-conjugated antibodies (Becton Dickinson, Sunnyvale, CA) as previously described.3 The NBT reduction assay was performed on PMA-stimulated cells.15Cell viability, apoptosis, and determination of DEVD-amc, VEID-amc, and IETD-amc hydrolytic activity Cell viability was determined by counting the percentages of red and white cells after staining with erythrosin.3 For the determination of the apoptotic index, cells were fixed with methanol and stained with DAPI as described.3 The annexin-V assay was performed by flow cytometry (FACSORT system; Becton Dickinson) with a commercially available kit (Annexin- V-FLUOS staining kit; Boheringer Mannheim, Mannheim, Germany). The level of expression of PCNA was evaluated by biparametric flow cytometry after permeabilization and staining with PI and anti-PCNA fluorescein-conjugated antibodies.16 Determination of DEVD-amc hydrolytic activity was performed on NB4 and NB4.437r cell extracts as previously reported3 after normalization for the protein content.17 An identical protocol was used for the determination of VEID-amc and IETD-amc hydrolytic activity.Western blot analysis, cytochrome c intracellular redistribution assay, and determination of the mitochondrial membrane potential In the case of polyADP ribose polymerase (PARP), caspase isozymes, and cyt c, Western blot analysis3 was performed on cytosolic extracts from NB4 or NB4.437r with the after antibodies: caspase-3 (CPP32 p20 [N-19]; Santa Cruz Biotechnology, Santa Cruz, CA); PARP (PARP [N-20]; Santa Cruz Biotechnology); caspase-8 (Biomedia GmbH, Baesweiler, Germany); and caspase-6, caspase-7, caspase-9, and cyt c (Pharmingen, San Diego, CA). In the case of Akt, Western blot analysis experiments were performed on total cellular extracts with antibodies specific for the phosphorylated form of the protein and control antibodies, using a commercially available kit (New England Biolabs, Beverly, MA) according to the instructions of the manufacturer. The antibodies and the protocol used for PML-RAR and
actin detection have already been described.3
Immunoreactive protein bands were visualized with the ECL detection kit
(Amersham, Little Chalfont, UK). The transmembrane potential was
assessed by flow cytometry after loading cells with the fluorescent dye 3,3'-dihexyloxadicarbocyanine iodide (DiOC6;
Sigma).18
Jun N-terminal kinase activity and electrophoresis mobility shift assays Jun N-terminal kinase (JNK) activity was determined on immunoprecipitates obtained after challenge of NB4 or NB4.437r cell extracts with agarose-immobilized anti-JNK antibodies (Santa Cruz Biotechnology). Immunoprecipitates were incubated with gst-Jun (Santa Cruz Biotechnology) as a substrate in the presence of 32P-ATP (Amersham). The whole procedure was carried out
as described by Lee et al.19 The reaction mixture was
subjected to polyacrylamide gel electrophoresis and autoradiography.
The amount of JNK protein present in the immunoprecipitates was
determined by Western blot analysis using an anti-JNK-1 antibody
(Santa Cruz Biotechnology).
CD437 has antileukemic activity in SCID mice xenografted with NB4 cells and induces apoptosis in the ATRA-resistant NB4.R1 cell line in vitro To establish whether CD437 has antileukemic activity in vivo, we inoculated SCID mice with NB4 cells and determined the survival of vehicle- and CD437-treated animals (Figure 1A). CD437 administration caused a dose-dependent increase in the lifespan of NB4-xenografted animals, which was significant at the 15-mg/kg and the 30-mg/kg doses. This effect was primarily the consequence of the rapid and strong apoptogenic action of CD437. In fact, harvesting the ascitic fluid 18 hours after a single administration of the retinoid (30 mg/kg) demonstrated an approximately 5-fold decrease in the number of leukemic blasts compared with what was observed in vehicle-treated animals (Figure 1B). This was associated with a remarkable increase in the number of blasts showing signs of apoptosis, such as nuclear fragmentation (apoptotic index) (Figure 1C, left panel) and plasma membrane phosphatidyl serine externalization (annexin-V positivity) (Figure 1C, right panel). Treatment with CD437 caused arrest in the proliferation of the residual viable cells (Figure 1D), as demonstrated by the dramatic decrease in the level of expression of PCNA, a proliferation-associated nuclear protein.16 The decrease in PCNA was evident for the cells in the G1 phase and less dramatic for the cells in the G2/M phase of the cycle. In untreated animals, the majority of blasts in the S phase were PCNA+. Most of the cells in this phase of the cycle disappeared in CD437-treated animals, suggesting death or transit and arrest in the G2/M phase of the cycle. CD437-induced PCD and growth arrest were not the result of a granulocytic maturation effect because treatment of the animals with CD437 did not alter the level of expression of a number of myeloid markers. In fact, regardless of CD437 treatment, the majority of xenografted NB4 cells were CD13-positive (vehicle = 73.2% ± 4.1%, CD437 = 61.3% ± 3.0%; mean ± SD of 4 animals), CD11b-negative (vehicle = 6.5% ± 1.7%, CD437 = 7.7% ± 0.9%; mean ± SD of 4 animals), and CD11c-negative (vehicle = 4.3% ± 2.3%, CD437 = 6.7% ± 1.9%; mean ± SD of 4 animals) and were unable to reduce NBT after PMA stimulation (data not shown). Interestingly, the administration of CD437 (30 mg/kg) for 3 consecutive days resulted in a further decrease in the amount of leukemic cells from the ascitic fluid (8.0% ± 3.5% of those present in vehicle-treated animals; mean ± SD of 4 animals).
NB4-derived NB4.437r cell line resistant to apoptosis induced by CD437 Challenge of NB4 promyelocytes with increasing concentrations of CD437 resulted in the isolation of the resistant cell line NB4.437r. As shown in Figure 2A, when cultured in standard conditions, the morphology of NB4.437r cells was promyelocytic and was not different from that of the parental cells. The NB4 parental cell line underwent rapid PCD in 106 mol/L CD437,
whereas the resistant counterpart did not show any morphologic sign of
toxicity. NB4.437r blasts have similar steady-state levels of the
PML-RAR oncogene and the normal RAR protein relative to the
original NB4 cell line (Figure 2B). In addition, the PML-RAR protein
synthesized in NB4.437r cells was structurally identical to that
expressed in the parental cell line, at least as far as its junctional
breakpoint and RAR portions.25 In fact, sequence analysis after polymerase chain reaction (PCR) amplification of PML-RAR messenger RNA (mRNA) (from nucleotide 1546 of PML to nucleotide 2956 of the RAR) from NB4.437r did not demonstrate the
presence of any nucleotide substitution relative to what has been
reported for NB4 cells.25 As expected,3 on
treatment with CD437, PML-RAR was degraded in NB4 blasts, but a
similar effect was not observed in NB4.437r cells. The growth curves of NB4 and NB4.437r (either in the presence or in the absence of CD437)
blasts were similar (Figure 2C). The saturation density of the NB4.437r
cell line was slightly higher (1.2 × 106 cells/mL)
than that of the parental counterpart (1.0 × 106 cells/mL).
NB4.437r cells maintain sensitivity to the growth-inhibitory and apoptogenic effects of ATRA, whereas retinoid-dependent cytodifferentiation is partially impaired Treatment of NB4 or NB4.437r promyelocytes with ATRA (106 mol/L) resulted in an antiproliferative effect
that was similar (Figure 3A). Furthermore,
the retinoid induced the same level of apoptosis in both NB4 and
NB4.437r cells (Figure 3B). The effect of ATRA on various markers of
granulocytic differentiation was more complex (Figures 3C and 3D). The
increase in NBT-reducing activity and the down-regulation of CD33 were
equivalent in NB4 and NB4.437r cells. By contrast, the induction of
CD11a, CD11b, and CD11c observed in the NB4 parental cell line was
dramatically reduced in the CD437-resistant line. Surface expression of
CD13 was high and left unaffected by ATRA, regardless of the cell line
taken into consideration. Similar effects were observed with the
synthetic retinoid AM580, which has been shown to activate specifically RAR, PML-RAR, or both in NB4 cells.27 As expected,
AM580 at 108 mol/L was slightly more potent than
ATRA at 106 mol/L in inducing NBT reducing activity
(Figure 3C) and the surface expression of CD11a and CD11b (Figure 3D)
in NB4 cells. By contrast, the compound was as active as the natural
retinoid in up-regulating CD11c and in down-regulating CD33,
respectively, and did not affect the level of expression of CD13
(Figure 3D).
NB4.437r cell line sensitive to various chemotherapeutic drugs and apoptogenic compounds We investigated whether induced resistance in NB4.437r cells to CD437 is specific or is associated with an alteration in the apoptogenic response to other stimuli (Figure 4). NB4.437r blasts show complete cross-resistance to CD2325, a CD437 structural analog.3 By contrast, the chemopreventive and antitumoral retinoid fenretinide 28-30 is equally effective in causing apoptosis in the parental and the CD437-resistant cell line, suggesting that this compound and CD437 have different mechanisms of action. The 2 cell types are equally susceptible to challenge with doxorubicin, a drug inducing apoptosis through DNA intercalation and damage.31 This is similar to what is observed for taxol, an active antitumor agent that disorganizes the cytoskeleton.32 Relative to the parental counterpart, the NB4.437r cell line shows limited cross-resistance to AraC, an antimetabolite contained in various polychemotherapeutic protocols,33 and to cisplatin, a widely used anti-neoplastic agent that damages DNA.34 Staurosporine, a protein kinase C inhibitor35 and a strong apoptogenic agent,36 caused the same level of DNA fragmentation in both the NB4 and the NB4.437r cell lines. Arsenic, an antileukemic compound targeting the PML-RAR oncogene,37,38 was slightly more effective in the NB4.437r than in the parental NB4 cell line. Finally, in conditions in which
tumor necrosis factor- induces PCD in U937 cells, the cytokine was
totally ineffective in killing both the parental and the
CD437-resistant cells (data not shown).
NB4.437r resistance to CD437 is not explained by decreased uptake or altered subcellular localization of the compound A widespread form of induced drug resistance (multidrug resistance [MDR]) is caused by activation of a membrane pump that effectively decreases the intracellular levels of compounds, such as doxorubicin and vincristine.39 To test possible differences in the intracellular levels of CD437 in NB4 and NB4.437r blasts, the rate of uptake and subcellular distribution of the 3H-labeled compound were determined. Both cell lines showed a rapid and similar uptake of 3H-CD437, which plateaued in less than 5 minutes and remained constant for at least 4 hours. At this last time point, the concentrations of 3H-CD437 in the NB4 and NB4.437r cell lines were 4.0 ± 0.4 and 3.6 ± 0.4 pmol/1 × 106 cells (mean ± SD of 3 culture dishes), respectively. The subcellular distribution of the radiolabeled compound was almost superimposable in NB4 and NB4.437r cells, and more than 90% of the intracellular radioactivity localized in the nuclei (1.1 ± 0.1 versus 1.2 ± 0.1 pmol/1 × 106 cells) and in the mitochondria (0.5 ± 0.1 versus 0.4 ± 0.1 pmol/1 × 106 cells). The residual amount of 3H-CD437 localized in the microsomes (0.023 ± 0.002 versus 0.022 ± 0.001 pmol/1 × 106 cells) and cytosol (0.10 ± 0.02 versus 0.12 ± 0.06 pmol/1 × 106 cells). Most of the radioactivity that accumulated in the cells was caused by intact CD437; high-performance liquid chromatography analysis of cellular extracts demonstrated that the retinoid was not metabolized at a significant level (data not shown).CD437 induces degradation and activation of various caspase isoenzymes in NB4 but not in NB4.437r blasts In Figure 5A, the levels of the precursors and relative degradation products of caspase-3, -6, -7, -8, and -9 were determined in NB4 cells before and after treatment with CD437. Whereas the amounts of all pro-caspases were constant throughout the course of the experiment in untreated cells, a reduction in the levels of the caspase zymogens was already evident 2 hours after challenge with CD437. Pro-caspase-6, -7, and -9 disappeared rapidly, whereas pro-caspase-3 and -8 persisted for longer times. Disappearance of the zymogens was blocked by the caspase inhibitor z-VAD, indicating that proteolytic degradation is the consequence of an autocatalytic process or the result of a cascade activation of caspases. Specific degradation products were visible in the case of caspase-3 (Figure 5C), -7, -8, and -9, and the apparent molecular weights of these proteolytic bands were consistent with those of the respective active enzymes. To support further CD437-dependent activation of the various caspase isoforms, we determined the ability of NB4 cell extracts to hydrolize peptide fluorogenic substrates (Figure 5B). Stimulation of DEVD-amc (specific for caspase-3 and -7),40 IETD-amc (specific for caspase-8),41 and VEID-amc (specific for caspase-6)42 hydrolytic activity was evident in cytosolic extracts of CD437-treated cells relative to vehicle-treated extracts. Although DEVD-amc hydrolytic activity increased linearly between 2 and 21 hours of CD437 treatment, degradation of VEID-amc and IETD-amc peaked at 9 hours. Neither degradation of pro-caspase-3, -6, -7, and -8 (Figure 5C) nor stimulation of DEVD-amc, IETD-amc, or VEID-amc hydrolytic activity (data not shown) was observed in NB4.437r cells after treatment with CD437. This indicated that the molecular lesion(s) responsible for the CD437-resistance phenotype of NB4.437r cells lays upstream of caspase activation.
CD437-dependent release of cytochrome c into the cytosol is not dependent on changes in mitochondrial membrane potential in NB4 cells and is not observed in NB4.437r blasts Cyt c relocalization from the mitochondria into the cytosol plays an important role in the activation of downstream caspases, such as caspase-3, -6, and -7, through the formation of a DISC complex with Apaf-1 and caspase-9.43 In basal conditions, no significant differences in the amounts of total or mitochondria-associated cyt c were observed between NB4 and NB4.437r cells (data not shown). As expected,3 treatment of NB4 cells with CD437 resulted in increased amounts of cyt c present in the cytosol compared with what was observed in control conditions; however, a similar effect was not observed in NB4.437r cells (Figure 6A). In NB4 cells, the surge of cytosolic cyt c was associated with a dramatic augmentation in the level of apoptosis.
CD437-dependent induction of NFkb and AP1 nuclear complexes are observed in NB4 but not in NB4.437r cells In NB4.437r cells, AP1 induction is dissociated from JNK activation. The transcriptional complex NFkb seems to play a role in the resistance to apoptosis observed in various types of tumors,47-49 whereas activation of AP1 has been involved as a possible mediator of the CD437 apoptogenic action.5,9 As shown in Figure 7A, in basal conditions NB4 cells contained detectable amounts of NFkb and AP1 nuclear complexes, which were increased by CD437 treatment. The effect was quantitatively similar to that observed in the presence of PMA used as a positive control. Induction of the 2 nuclear complexes was specific because CD437 did not affect the amounts of the SP1 complex observed in control conditions. Although 2 bands that can be competed away by cold oligonucleotides were visible in the EMSAs, corresponding to both NFkb and AP1 complexes, only the upper band was specific. As to NFkb, the upper band contained the p65 component of the p50/p65 heterodimer because it could be supershifted by antibodies recognizing this protein but not by anti-STAT1 antibodies used as a negative control. Similarly, the upper band of the EMSA corresponding to AP1 could be supershifted by anti-Jun antibodies (data not shown). In NB4.437r cells, the basal levels of active NFkb and AP1 transcription factors were higher than those in NB4 promyelocytes and were not modulated by the addition of CD437 to the growth medium. In NB4 cells, pretreatment with cycloheximide (CHX) had only marginal effects on the basal or CD437-induced levels of NFkb and AP1 transcriptional complexes. By contrast, in NB4.437r cells, CHX caused a substantial decrease of both complexes regardless of CD437 treatment. AP1 activity is often regulated by JNK-dependent phosphorylation of Jun.50 As demonstrated in Figure 7B, JNK-1 immunoprecipitates from NB4 cells treated for 1 and 2 hours with CD437 contained significant amounts of a kinase activity that phosphorylates Jun in vitro. After treatment with the retinoid, the levels of kinase activity were much higher than those observed in basal conditions (visible only on longer exposure of the autoradiograms) and were not explained by differences in the amounts of immunoprecipitated JNK protein. Interestingly, treatment of NB4.437r cells with the retinoid for 1 hour resulted in a similar activation of the JNK protein. This appeared to be the only marker of CD437-activity similarly regulated in the sensitive and resistant cell lines.
CD437 causes phosphorylation of Akt in NB4 but not in NB4.437r cells Wortmannin inhibits phosphorylation of Akt without affecting sensitivity or resistance to CD437-triggered apoptosis. Akt kinase is activated by survival factors through phosphatidyl-inositol-3-kinase (PI3K)-dependent phosphorylation.51 Although NB4 and NB4.437r cells synthesized significant amounts of Akt in basal conditions (Figures 8A and 8B), the levels of this kinase were slightly lower in the CD437-resistant than in the CD437-sensitive cell line. Consistent with this, a background level of phosphorylation was evident in the NB4 cell line, whereas longer exposures of films were required to highlight a similar level of phosphorylation in the CD437-resistant counterpart (data not shown). Challenge of NB4 blasts with CD437 for 1 hour resulted in increased Akt phosphorylation. Phosphorylation was not secondary to CD437-triggered caspase activation because the phenomenon was not affected by the caspase inhibitor z-VAD. CD437-dependent Akt phosphorylation was transitory; it returned to baseline levels by 4 hours. This last effect was at least partially explained by a caspase-dependent cleavage of the Akt protein, which was observed at 4 hours and was blocked by z-VAD (Figure 8A). Treatment of NB4.437r cells with CD437 for 1 hour did not result in Akt phosphorylation or alteration in the total levels of the protein (Figure 8B).
The major findings of this study are the demonstration of the in vivo activity of CD437 using a preclinical model of APL and the development and biochemical characterization of a novel APL cell line made resistant to the retinoid.
We thank Prof S. Garattini and Dr M. Salmona for critical reading of the article.
Submitted May 17, 1999; accepted December 20, 1999.
Supported by a grant from the Associazione Italiana and Fondazione Italiana per la Ricerca contro il Cancro (R.G. and E.G.) and by a grant from the Istituto Superiore di Sanità. I.P. is the recipient of a fellowship from La Via di Natale.
I.P. and M.G. contributed equally to the results presented in this article.
Reprints: Enrico Garattini, Laboratory of Molecular Biology, Istituto di Ricerche Farmacologiche Mario Negri, via Eritrea, 62, 20157 Milano, Italy; e-mail: egarattini{at}irfmn.mnegri.it.
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.
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Top
Abstract
Introduction
and is approximately 33-fold more resistant
than the parental counterpart to the apoptogenic effects of the
retinoid. Resistance is relatively specific to CD437 and structural
congeners because the NB4.437r cell line is still sensitive to various
types of apoptogenic compounds. The CD437-resistant cell line maintains
sensitivity to the antiproliferative and apoptotic action of
all-trans-retinoic acid, AM580, and fenretinide, though it shows
partial resistance to the cytodifferentiating effects of the first 2 compounds. Resistance to CD437 lays upstream of the CD437-induced
release of cytochrome c from the mitochondria and the activation of
caspase-3, -7, -8, and -9. Furthermore, NB4.437r cells are deficient in
the CD437-dependent activation of nuclear NFkb and AP1-binding
activities and in the phosphorylation of the protein kinase Akt. In the
case of AP1, deficient assembly of the complex is not caused by the
lack of activation of the Jun N-terminal kinase (JNK) family of
kinases. The novel cell line will be useful in the elucidation of the
molecular mechanisms underlying the apoptogenic action of CD437 and
structurally related retinoids.
(Blood. 2000;95:2672-2682)
) or the presence (+) of the JNK substrate
gst-Jun. Equivalent amounts of the reaction mixtures were subjected to
PAGE under denaturing conditions and subsequent autoradiography. To
ensure that the same amount of JNK was present, Western blot
analysis was performed on each immunoprecipitate using a second
antibody specific to JNK-1. Data are representative of at least 2 independent experiments with similar results.
