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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2484-2494
Type 4 Cyclic Adenosine Monophosphate Phosphodiesterase as a
Therapeutic Target in Chronic Lymphocytic Leukemia
By
Doo Ho Kim and
Adam Lerner
From the Department of Medicine, Section of Hematology and Oncology,
Boston Medical Center, Boston, MA.
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ABSTRACT |
Theophylline, a drug known to inhibit several classes of adenosine
3 5 cyclic monophosphate (cAMP) phosphodiesterases (PDEs), induces apoptosis in chronic lymphocytic leukemia (CLL) cells. Because
the PDE target for theophylline in CLL remains unknown, we examined the
ability of isoform-specific PDE inhibitors to increase cAMP levels and
induce apoptosis in primary CLL cells. Reverse transcriptase-polymerase
chain reaction of purified CLL cDNA amplified transcripts for PDE1B, 4A
and 4B. The type 4 PDE inhibitor rolipram but not the type 1 inhibitor
vinpocetine increased CLL cAMP levels. Rolipram-inhibitable (type 4)
but not calcium-calmodulin augmented (type 1) PDE enzyme activity was
detected in CLL samples. In samples from 13 of 14 CLL patients,
rolipram induced apoptosis in a dose-dependent fashion over a 48-hour
period. Interleukin-2 (IL-2)-cultured whole mononuclear cells (WMC)
and anti-Ig stimulated CD19+ B cells were resistant to
the induction of apoptosis by rolipram while unstimulated
CD19+ B cells, which had a high basal apoptotic rate,
were more sensitive. Rolipram stimulated elevations in cAMP levels in
all four of these cell populations, suggesting that they differed in
sensitivity to cAMP-induced apoptosis. Consistent with this hypothesis,
incubation with the cell permeable cAMP analog dibutyryl-cAMP induced
apoptosis in CLL cells and unstimulated B cells but not in
IL-2-cultured WMC or anti-Ig stimulated B cells. These data identify
PDE4 as a family of enzymes whose inhibition induces apoptosis in CLL cells.
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INTRODUCTION |
B-CELL CHRONIC lymphocytic leukemia (CLL)
is a CD5+, CD19+, CD23+ malignancy
that has not been associated with a single unifying molecular defect
but in which abnormalities in the induction of apoptosis appear to play
a role. Although CLL patients may have initial clinical responses to
alkylating agents such as chlorambucil or adenosine analogs such as
fludarabine, many ultimately become refractory to therapy and there is
a pressing need for the identification of novel approaches to this
disease. Mentz et al1 have reported that CLL cells undergo
apoptosis when exposed to theophylline, a methylxanthine known to
nonspecifically inhibit 3:5 cyclic adenosine
monophosphate (cAMP) phosphodiesterases
(IC50 = 200 µmol/L). Theophylline synergizes with
chlorambucil in inducing CLL apoptosis in vitro and a phase 2 trial
suggests that this observation may be clinically
relevant.2,3
The first observation that some lymphoid cells die after exposure to
agents that increase intracellular cAMP levels was made by Daniel et
al,4 who found that the murine lymphoma cell line S49.1
underwent cytolysis after 48 hours of exposure to the combination of
theophylline and a cell permeable 3:5 cAMP analog,
dibutyryl cAMP (dbcAMP). When mutant S49.1 clones resistant to the
cytolytic effects of dbcAMP were isolated in soft agar, they were shown to be defective in the regulatory subunit of protein kinase A, confirming that cytolysis occurred as a direct result of PKA-mediated phosphorylation of unknown lymphoid target proteins.5
Subsequent work has shown that cAMP-induced cytolysis occurs by
apoptosis.6 Certain normal T- and B-lymphoid subsets
express the same marked sensitivity to cAMP-induced toxicity as tumor
cell lines. Within the T lineage, CD4+CD8+
thymocytes appear to be more sensitive to the induction of apoptosis by
cAMP than mature T cells.6 Apoptosis in resting human B lymphocytes, which occurs spontaneously at a high rate in culture, can
be augmented by the addition of stimuli which elevate intracellular cAMP levels, such as the diterpene adenylate cyclase activator forskolin.7
Cyclic AMP is catabolized within cells to 5-AMP by
3:5 cAMP phosphodiesterases (PDE), a diverse group of
enzymes encompassing 15 gene products and 7 classes of enzymes which
have proven to be the target of successful pharmaceutical agents for
neurologic, cardiovascular, and inflammatory disorders.8
Despite this large array of cyclic nucleotide PDEs, only a subset of
these enzymes have been reported in human lymphoid cells. Among them,
the most commonly reported enzymes in human T cells are types 1, 3, and 4. Calcium-calmodulin-dependent type 1 PDE activity has been detected in phytohemagglutinin-stimulated, but not resting, peripheral blood
lymphocytes.9 One isoform from this family, PDE1B1, was recently detected in acute lymphocytic leukemia cells; inhibition of
this enzyme was reported to induce apoptosis.10 PDE1
enzymes, which can catalyze the degradation of both cAMP and cyclic
guanosine monophosphate (cGMP), are specifically
inhibited by vinpocetine (IC50 = 21 µmol/L).11 Two groups have reported both type 3 and type
4 PDE in human T lymphocytes; lectin-mediated proliferation was
completely suppressed only by treating cells with specific inhibitors
of both classes of enzymes.12,13 Although four human PDE4
genes have been cloned, only two of the isoforms (PDE4A and B) have
been identified in lymphocytes. Type 4 enzymes are specifically inhibited by rolipram
[4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone] (IC50 = 1 µmol/L) and the structurally related compound
RO20-1724 (IC50 = 2 µmol/L).14,15
Theophylline induces apoptosis in CLL cells, as noted above, but both
the clinical and research applications of theophylline are complicated
by its activity as an adenosine receptor antagonist.16 As
an alternate approach, we used transcript-specific probes, enzyme
assays, and isoform-specific nonmethylxanthine cAMP PDE inhibitors to
identify PDE targets in CLL cells and confirm that inhibition of
specific PDE enzymatic activities induces CLL apoptosis.
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MATERIALS AND METHODS |
Reagents.
The following reagents were obtained from commercial sources: alkaline
phosphatase, cAMP, dibutyryl cAMP, calmodulin, forskolin (Sigma
Chemical Co, St Louis, MO); vinpocetine (Alexis Biochemicals, San
Diego, CA); recombinant human interleukin-2 (rhIL-2; Genzyme, Boston,
MA); F(ab)2 fragment goat anti-human IgG and IgM
(Jackson Immunoresearch Laboratories, West Grove, PA); Hoechst 33342 (Molecular Probes, Eugene, OR). Rolipram (racemate of
4-[3-cyclopentyloxy-4-methoxyphenyl]-2-pyrrolidone) was
a gift from Dr Ronald Wohl (Berlex Laboratories, Wayne, NJ).
Patient selection.
After Institutional Review Board (IRB)-approved
informed consent, blood was drawn in heparinized tubes from patients
with flow cytometry-verified CLL who were either untreated or at least 1 month postchemotherapy. Patients with active infections or other serious medical conditions were not included in this study. Charts were
reviewed to establish patients' sensitivity to chemotherapy and the
stage of CLL. Resistance to a chemotherapeutic agent was defined as an
increase in peripheral leukemic cell count or progression of adenopathy
or splenomegaly before the initiation of the next scheduled cycle of
chemotherapy.
Cell purification and culture.
Mononuclear cells were obtained by density gradient centrifugation over
Histopaque 1077 (Sigma). As flow cytometry showed that CLL cells made
up more than 95% of the mononuclear cells so purified, both apoptosis
and cAMP assays were performed with these patient cell preparations.
For polymerase chain reaction (PCR) experiments on CLL cells or for all
experiments on normal circulating B cells, the whole mononuclear cells
were further purified by incubation with Dynal anti-CD19 magnetic beads
at a 1:1 bead:cell ratio, extensive washing with a magnetic particle concentrater, and elution with CD19 Detachabead reagent (Dynal, Lake
Success, NY). Leukemic cells from two CLL patients showed sensitivity
to rolipram-mediated apoptosis whether or not they were further
purified by anti-CD19 magnetic beads. Cells were cultured in RPMI 1640 media (Biowhittaker, Walkersville, MD) supplemented with 10% fetal
calf serum, 50 µmol/L 2-mercaptoethanol, 2 mmol/L L-glutamine, 10 mmol/L HEPES pH 7.4, 100 U/mL penicillin, and 100 U/mL
streptomycin (Sigma) at 37°C and 5% CO2 in air.
Reverse transcriptase (RT)-PCR and Northern analysis.
RNA was isolated from CLL or whole mononuclear cells using Ultraspec
reagent (Biotecx, Houston, TX). cDNA was synthesized from 10 µg of
total RNA using oligo d(T) primers and Maloney murine leukemia virus
reverse transcriptase in a final volume of 40 µL (Stratagene, La Jolla, CA). One microliter of the first-strand cDNA
product was then used as template for PCR amplification with AmpliTaq
DNA polymerase (Roche Molecular Systems, Branchburg, NJ) by 40 thermocycles of 94°C for 1 minute, 60°C for 1 minute, and
72°C for 1 minute. The PDE PCR assay products were as follows with
oligonucleotide sequences given 5  3: Human
PDE1B1 (Genbank accession no. U56976) was 430 bp (first base 1660;
sense = GTC TTC ATT GAG TCC AAA GTG , antisense = GAC CTG CCA GCT AAG ATC TGG).10 Human PDE3A (cGIP1, HSPDE3B) (X95520) was 340 bp (first base 2999, sense = GTA ACT CCT ATG ATG CTG CTG G, antisense = CTA TTC CTC TTC ATC TGC CTC).17,18 Of note, these PDE3 PCR oligonucleotides are selective for the human cGIP1 PDE, homologous to
rat PDE3A, as the amplified sequence has only 50% nucleotide homology
to the cardiac/platelet form of human PDE3 (cGIP2).19 Human
PDE4A (M37744) was 461 bp (first bp 1819, sense = GGA GGA AGA AAT ATC
AAT GGC CC, antisense = GAT GTG TCC TCC CCA AAT GTC).20
Human PDE4B (L20966) was 479 bp (first bp 2213, sense = ATT CTG AAG GAC
CTG AGA AGG, antisense = CAG TGA GTT CAG TCA CTG TCG).21
For hybridization to Northern blots, these PCR products were subcloned
into a plasmid vector (pCRII; Invitrogen, Carlsbad, CA) and
subsequently used for PCR-based amplification of  32P
dATP-labeled probes.
cAMP assay.
One million cells in 1 mL were treated for 2 hours with or without
drugs. 0.8 mL of cells were pelleted by spinning at 4,000 rpm (relative
centrifugal force [RCF] = 1,310) in a microcentrifuge tube. After discarding 0.7 mL, 400 µL of ethanol was added, vortexed, and left on ice for five minutes. Particulate cell debris was removed
by centrifugation at 14,000 rpm (RCF = 16,000). The supernatant was
stored at  20°C until the day of assay, at which time it was dried in a Speedivac (Savant, Farmingdale, NY) to a volume of 50 µL.
After 10-fold dilution in 10 mmol/L Tris pH 8.0, 1 mmol/L EDTA, the
cAMP sample was analyzed for cAMP concentration using a cAMP
radioimmunoassay (RIA) kit (New England Nuclear [NEN], Boston,
MA) according to the manufacturer's instructions using the nonacetylated protocol.
cAMP PDE assay.
The technique of Robicsek et al, itself adapted from an assay described
by Thompson and Appleman, was used in modified form.12,22 One hundred fifty million purified CLL cells were pelleted and sonicated (Branson 350 Sonifier with microtip probe;
Branson Ultrasonics, Danburg, CT; output = 2, 50% duty cycle) on ice
in 1.0 mL of a buffer which contained 20 mmol/L Tris (pH 6.8), 1 mmol/L
EDTA, aprotinin (50 U/mL), pepstatin (1 mg/mL),
phenylmethyl sulfonyl fluoride (PMSF) (1 mmol/L), and 3.75 mmol/L  -mercaptoethanol (-ME). Assay buffer
contained 100 mmol/L Tris (pH 8.0), 20 mmol/l MgCl2, 0.2%
bovine serum albumin (BSA), and 7.5 mmol/L -ME.
[3H]-cAMP (NEN) was incubated with PDE for 10 minutes at
30°C in 20-µL volumes (10 µL of sonication buffer and 10 µL
of assay buffer) which contained 0.22 U of alkaline phosphatase.
Rolipram, 10 µmol/L, or 0.2 mmol/L calcium/20 nmol/L calmodulin were
added to the assay buffer as appropriate. Reactions were halted by the
addition of 0.5 mL of a 1:3 slurry wt/vol slurry of AG1-X8 anion
exchange resin and a mixture of equal volumes of water and isopropanol. The resin bound the unreacted nucleotide but not the dephosphorylated nucleoside. Microcentrifuge tubes were spun at 3,000 rpm (RCF = 735)
for 15 minutes. The radiolabeled nucleosides in the supernatant were
counted using Ecoscint scintillation fluid (National Diagnostics, Atlanta, GA). Three to five enzyme dilutions were assayed to determine each velocity. Linearity of velocity with respect to enzyme
concentration and time were verified.
DNA ladder gel assay.
Ten million purified CLL cells were obtained by centrifugation after
exposure to drugs during a 72-hour tissue culture incubation. Cells
were lysed in 0.5 mL of 20 mmol/L Tris (pH 7.4), 0.4 mmol/L EDTA,
0.25% Triton X 100 (American Bioanalytical, Natick,
MA). After 15 minutes of incubation at room
temperature, nuclei were removed by centrifugation at 14,000 rpm (RCF = 16,000). The supernatant was transferred to a new tube and soluble DNA
precipitated overnight at 20°C following the addition of 55 µL of 5 mol/L NaCl and 550 µL of isopropanol. After centrifugation
at 14,000 rpm for 10 minutes, followed by a 70% ethanol wash, the
pellet was resuspended in 20 µL of 10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA, 0.1 mg/mL RNase, and incubated at 37°C for 30 minutes before electrophoresis on 1.6% Tris borate
EDTA agarose gels. DNA fragments were visualized by UV
light after staining the gels with ethidium bromide.
Hoechst 33342 apoptosis assay.
Hoechst 33342 was dissolved in water and frozen at 33 mg/mL at
20°C. One million cells per well were incubated in duplicate or triplicate in 48-well tissue culture plates (Costar, Cambridge, MA)
with or without drug treatment for 48 hours in 1 mL of culture media.
Cells were transferred to 12 × 75-mm polystyrene Falcon 2054 FACS
tubes (Becton Dickinson Labware, Lincoln Park, NJ) and incubated for 10 minutes at 37°C with Hoechst 33342 at a final concentration of 0.25 µg/mL.23 Cells were stored on ice until analysis on a
FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA). Hoechst
33342 dye fluorescence was excited with a UV laser and detected using a
450 bandpass filter. Data were analyzed using Cellquest software
(Becton Dickinson).
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RESULTS |
Identification of PDE targets in CLL cells.
As an initial approach to identify potential PDE targets in CLL, we
performed PCR on cDNA derived from unpurified mononuclear cells of a
CLL patient with oligonucleotides specific for human PDE1-1B, PDE3A,
PDE-4A, and PDE-4B, all of which have been previously identified in
various normal and malignant primary lymphoid cells. We detected
appropriate-sized PCR products from all four transcripts using this
template. To reduce the likelihood of amplifying nonleukemic cell
transcripts, we purified CD19+ cells from this and two
other CLL patients before synthesizing cDNA. PDE1-1B, PDE-4A, and
PDE-4B were still detected in these three templates, as they were in
cDNA derived from normal whole mononuclear cells
(Fig 1). Using the same four PCR products
as probes on Northern blots, only PDE-4B transcript was detectable in
10 µg of loaded RNA (Fig 2 and data not
shown). PDE-4B transcript levels were significantly higher in freshly
isolated CLL cells than in CLL cells which had been cultured for 6 hours. Addition of 10 µmol/L rolipram, a type 4 PDE-specific
phosphodiesterase inhibitor, significantly reduced this decrease in
PDE-4B transcript levels during the 6-hour culture period.
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| Fig 1.
CLL cells contain transcripts for PDE 1B1, PDE4A, and
PDE4B. PCR was performed on cDNA derived from WMC (purified from a
normal donor) or CLL cells for PDE1, PDE3A, PDE4A, and PDE4B. The cDNA
used in the PCR in the bottom three panels was derived from leukemic
cells from three different patients purified by positive selection for
CD19 expression. The lowest band in the MWM lane on the left in the
upper panel is 603 bp. Expected PCR product sizes for PDE1, 3A, 4A, and
4B are 430, 340, 461, and 470 bp, respectively.
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| Fig 2.
PDE4B levels decrease in CLL cells after culture but are
partially maintained by treatment with 10 µmol/L rolipram. RNA was
isolated from 20 million CLL cells (derived from patient no. 12)
immediately after cell purification (CT), or after 6 hours culture in
media alone (6Hr) or with addition of 10 µmol/L rolipram (Roli).
Equal loading and transfer of RNA was confirmed by hybridization with
an actin probe as shown. These results are representative of Northern
analysis performed on leukemic cells from two patients.
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To determine whether these PDE transcripts were translated into protein
with constitutive activity, we performed PDE enzyme assays on CLL cell
lysates. We found no evidence of type 1 PDE activity in CLL cells
because the basal PDE activity was not augmented by the addition of
calcium and calmodulin (Fig 3, left).
However, we were able to identify substantial constitutive type 1 PDE
activity in the murine B-lymphoma cell line Bal-17, as shown by an
increase in PDE activity with the addition of calcium and calmodulin
(Fig 3, right). In contrast, CLL PDE activity was markedly inhibited by
the type 4 specific inhibitor, rolipram in each of the three patients
tested (Fig 3, left and data not shown). Rolipram altered both
Km and the Vmax, consistent with its known
activity as a noncompetitive antagonist.14
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| Fig 3.
Type 1 and 4 PDE activity differs between a murine B
lymphoma cell line (Bal-17) and CLL cells. Lineweaver-Burk analysis of
PDE enzymatic activity in lysates of CLL cells and Bal-17 cells. (Left)
PDE activity in CLL cells was inhibited by 10 µmol/L rolipram but was
not augmented by the addition of 0.2 mmol/L calcium and 20 nmol/L
calmodulin. (Right) Bal-17 PDE activity is augmented by the addition of
calcium and calmodulin. The CLL data are representative of enzymatic
assays on three patients.
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To determine what role type 1 and type 4 PDE activity might play in the
catabolism of cyclic nucleotides in CLL cells, we measured cAMP levels
after culturing leukemic cells with varying concentrations of
PDE-isoform specific inhibitors, either alone or in conjunction with an
activator of adenylate cyclase, the diterpene forskolin. When incubated
with forskolin and the PDE-1 inhibitor vinpocetine, CLL cell cAMP was
not augmented above levels induced by forskolin alone
(Fig 4D). Incubation of Bal-17 cells with
vinpocetine reduced both basal and forskolin-stimulated cAMP levels, a
result in keeping with the reported primary effect of this drug on cGMP
rather than cAMP levels (data not shown).11 In contrast,
addition of the type 4 PDE inhibitor rolipram augmented CLL cAMP
levels, both when used alone and more dramatically when combined with
forskolin (Fig 4A through D). CLL cells were not unique with respect to
their response to these isoform-specific inhibitors. cAMP levels in
both a predominantly T-cell population (IL-2 supplemented normal whole
mononuclear cells; >90% CD3+ T cells by flow cytometry)
and magnetic-bead purified CD19+ B cells increased after
inhibition of type 4 but not type 1 PDE activity (Fig 4E and F, and
data not shown).
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| Fig 4.
Rolipram raises cAMP levels in CLL cells, WMC, and
resting B cells. One million freshly isolated cells were incubated with
the indicated drugs for 2 hours before analysis of cAMP in cell lysates
using a radioimmunoassay. Data are the mean of three individually
assayed culture wells. In (D), vinpocetine was added at 30 µmol/L.
The experimental conditions indicated with an asterisk had a greater
[cAMP] than, as appropriate, the untreated or forskolin-treated
control cells (t-test: one-tailed significance
level <.05).
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Type 4 PDE inhibition induces CLL apoptosis.
Having identified the type 4 cAMP phosphodiesterase family as an
important regulator of cAMP levels in CLL cells, we next turned to a
study of the activity of type 4-specific PDE inhibitors as inducers of
cAMP-mediated apoptosis in leukemic cells from CLL patients. CLL cells
were incubated for 72 hours either alone or with 10 µmol/L rolipram,
40 µmol/L forskolin, or both agents. We tested whether rolipram
induces internucleosomal DNA fragmentation characteristic of apoptosis
by isolating soluble DNA from the leukemic cells with detergent
treatment, then removing DNA from intact nonapoptotic nuclei by
centrifugation. As shown in Fig 5, while
culture of CLL cells in media alone resulted in only a faint DNA
"ladder," treatment with rolipram and/or forskolin resulted in pronounced internucleosomal DNA fragmentation.
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| Fig 5.
Rolipram and forskolin induce DNA fragmentation in CLL
cells. Soluble DNA was isolated from 10 million CLL cells cultured for
72 hours in media (CT), 10 µmol/L rolipram (Roli), 40 µmol/L
forskolin (Fsk) or a combination of the latter two agents (Ro/Fs). DNA
fragments were resolved by electrophoresis on a 1.5% agarose gel and
visualized with ethidium bromide. These data are representative of the
four leukemic cell samples tested.
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As a more quantitative analysis of CLL apoptosis, we used a flow
cytometry method in which apoptotic cells are distinguished both by
their reduced size (FSC) and their increased uptake of the lipophilic
UV fluorescent dye Hoechst 33342 (FL-4) when the intact, heterogeneous
cell population is incubated with a low concentration of the dye (0.25 µg/mL) for 10 minutes at 37°C (Fig 6).23 Previous reports of cAMP-induced lymphoid apoptosis
have noted that this form of programmed cell death may take 48 to 72 hours to develop maximally.6 Using the Hoeschst 33342 assay in a time-course experiment, we found that the combination of 10 µmol/L rolipram and 40 µmol/L forskolin induced significant CLL
apoptosis which plateaued 48 to 72 hours after the addition of these
drugs (Fig 7, left panel). Using the
72-hour culture period, we found a dose-dependent increase in CLL cell
apoptosis when leukemic cells were incubated with rolipram (Fig 7,
right panel). Treatment of CLL cells with forskolin alone induced
moderate apoptosis, but the combination of forskolin with even low
doses of rolipram resulted in a supra-additive effect on induction of CLL apoptosis (Fig 7, right panel). We obtained similar results with a
structurally distinct PDE4 inhibitor, RO20-1724, or when isoproterenol
or prostaglandin E2 were used to activate CLL adenylate cyclase rather than forskolin (data not shown).
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| Fig 6.
Rolipram-induced apoptosis in CLL cells is detectable by
Hoechst 33342 flow cytometry. Cells were cultured for 72 hours in media
(1), 1 µmol/L rolipram (2), 40 µmol/L forskolin (3), or a
combination of the two drugs. The abcissa reflects forward light
scatter and the ordinate Hoechst 33342 fluorescence. Apoptotic cells
are characterized by reduced forward light scatter and increased
Hoechst 33342 fluorescence.
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| Fig 7.
The type 4-specific cAMP phosphodiesterase inhibitor
rolipram induces apoptosis in CLL cells. (A) Time course: Rolipram (10 µmol/L) and forskolin (40 µmol/L) were added to cultures of 1 million CLL cells for the indicated time period before determination of
apoptosis by Hoechst 33342 flow cytometry. (B) Dose titration: CLL
cells were cultured for 72 hours in 1 mL of media with the indicated
concentration of rolipram with ( ) or without ( ) the addition of
40 µmol/L forskolin. The SEMs of triplicate cultures are indicated.
All of the rolipram or rolipram/forskolin-treated cultures had a
significantly greater percentage of apoptotic cells than the media or
forskolin-only-treated cultures, respectively (t-test:
one-tailed significance level <.05).
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When CLL cells were incubated with the type 1 PDE inhibitor
vinpocetine, they underwent apoptosis at dosages of 10 or 30 µmol/L but not at 2 µmol/L. Given that vinpocetine failed to augment cAMP
accumulation and that we were unable to detect type 1 PDE activity in
CLL cells, we suspect that this drug may induce apoptosis by a
mechanism unrelated to cAMP. Consistent with this hypothesis, the
kinetics of CLL apoptosis were different when vinpocetine was used with
peak apoptosis by 24 rather than 48 hours (data not shown).
Nonetheless, we cannot rule out either a temporally restricted or a
topologically compartmentalized cAMP-mediated apoptotic effect from
this drug.
Given that CLL is a clinically heterogeneous disease, we tested a total
of 14 CLL patients of varying clinical stage, treatment history, and
known resistance to chemotherapeutic agents for the sensitivity of
their cells to phosphodiesterase inhibitor-mediated apoptosis. Patients
were assessed for the apoptotic sensitivity of their leukemic cells to
rolipram, forskolin, or both drugs. In samples from 10 "rolipram-sensitive patients," treatment with 10 µmol/L
rolipram induced apoptosis in more than a third of the leukemic cells,
with overall apoptosis ranging from 44% to 80% (see
Table 1 for tabulated results). Among the
seven rolipram-sensitive patients whose cells were treated with both
conditions, 40 µmol/L forskolin as a single agent induced less
apoptosis than rolipram alone, suggesting that blockade of cAMP
catabolism induced a more potent apoptotic signal than further
augmentation of adenylate cyclase activity (P < .08, Wilcoxon
signed-ranks test for matched pairs). Among four relatively
rolipram-resistant patient samples (patient nos. 11 through 14; Table
1), the absolute increase in apoptotic cells was less than 33%, with
overall apoptosis ranging from 14% to 40%. Nonetheless, addition of
forskolin to rolipram augmented apoptosis (68% and 69%) in two of the
three patient samples examined within this group.
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Table 1.
Apoptosis of CLL Patients' Leukemic Cells After
Treatment With 10 µmol/L Rolipram, 40 µmol/L forskolin, or 100 µmol/L dbcAMP
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Effect of PDE4 inhibition on normal B and T cells.
Given that cAMP has been reported to be cytocidal to specific normal
lymphocyte subsets, we wished to determine if rolipram also induced
apoptosis in normal circulating human B- and T-cell populations. We
found that IL-2-cultured whole mononuclear cells (WMC) (>90% CD3+ T cells by flow
cytometry) were resistant to even high doses of rolipram and forskolin
(Fig 8, upper panel). In
contrast, magnetic bead purified CD19+ B cells were
sensitive to rolipram, although the increment in apoptosis observed was
superimposed on a high basal apoptosis rate that has previously been
reported in cultured human B cells.7 Given that
crosslinking of cell-surface Ig on resting B cells has been reported to
reduce basal and forskolin-induced apoptosis in culture, we also
stimulated CD19+ cells with a polyclonal
Fab2 anti-IgM/IgG reagent 30 minutes before the
addition of the phosphodiesterase inhibitor.7 Prior stimulation through surface Ig markedly reduced both basal apoptosis and the sensitivity of the B cells to rolipram (Fig 8, middle panel).
In contrast, anti-Ig stimulation of CLL cells derived from two patients
failed to protect these cells from rolipram-induced apoptosis, a result
that is consistent with reported defects in CLL cells in either surface µ heavy-chain expression or mutations in the B29 (CD79b) B-cell
receptor accessory protein (Fig 8, bottom panel).24
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| Fig 8.
CLL cells, WMC cultured with IL-2, and resting
and activated CD19+ B cells vary in their sensitivity to
rolipram-induced apoptosis. (Top) One million WMC were cultured with
the indicated concentration of rolipram with () or without () the
addition of 40 µmol/L forskolin for 72 hours in the presence of 2 U/mL IL-2. Apoptosis was determined by Hoechst 33342 flow cytometry.
(Middle) CD19+ peripheral B cells were purified by
adherence to anti-CD19 magnetic beads. These cells were then cultured
with the indicated concentration of rolipram with () or without
( ) 40 µmol/L forskolin. In addition, an identical set of cells
were stimulated through their surface Ig by the addition of 10 µg/mL
of Fab2 goat anti-human IgG/M 30 minutes before
addition of rolipram with () or without () forskolin. (Bottom)
CLL cells from two patients were cultured using an experimental design
similar to that described for the middle panel. Rolipram was used at 10 µmol/L and forskolin at 40 µmol/L. (), Media patient no. 1;
(), anti-Ig patient no. 1; (), media patient no. 2; (),
anti-Ig patient no. 2. The SEMs of triplicate cultures are
indicated.
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The alteration in rolipram sensitivity in anti-Ig-stimulated B cells
was not caused by a change in the ability of this drug to augment cAMP
levels at 2 hours in these cells, as rolipram increased cAMP levels
equivalently in unstimulated or stimulated CD19+ B cells
(Fig 9). To determine whether
the rolipram-sensitive cell populations had a more prolonged elevation
of cAMP than the insensitive cell populations following drug treatment,
we measured cAMP levels 6 or 24 hours after addition of rolipram, times
at which levels of apoptosis were still low even in sensitive
populations. For each of the four cell populations, at 2 or 6 hours
after drug treatment, cAMP levels were higher for
rolipram/forskolin-treated cells than for forskolin only treated cells
(t-test: one-tailed significance level <.05) (Fig 9). By 24 hours, there was no longer significant rolipram-induced augmentation of
forskolin-stimulated cAMP accumulation in any of the four cell
populations (Fig 9). Thus, the degree of cAMP augmentation by rolipram
did not predict the sensitivity of cell populations to induction of
apoptosis by this drug at any time point tested.
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| Fig 9.
Rolipram blocks cAMP catabolism in both
sensitive and resistant lymphoid populations. One million cells from
the leukemic cells of two CLL patients (top; [], patient no. 1;
[], patient no. 2), magnetic bead-purified CD19+ B
cells (middle; [], resting; [], stimulated), or IL-2
supplemented WMC (bottom panel) were cultured with media, 10 µmol/L
rolipram, 40 µmol/L forskolin, or both agents for 2, 6, or 24 hours,
as indicated. cAMP content was determined by radioimmunoassay. In the
case of the CD19+ cells, the cells were first cultured in
media or Fab2 anti-IgG/M for 30 minutes before
treatment with rolipram or forskolin. Data are the mean of duplicate
wells for each condition and are representative of two experiments with
similar results.
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A potential weakness of inhibitor studies is that, although experiments
may correlate drug effects (in this case, apoptosis) with inhibition of
a known target enzyme (in this case, reflected by increased cAMP
levels), the observed effect may still be caused by other, unrelated
activities of the inhibitor. In the experiments described above, we
have found that rolipram augments cAMP in both sensitive (CLL, resting
B cells) and insensitive (WMC, activated B cells) cell populations,
raising the concern that the observed cell death is the result of
non-cAMP-related rolipram activity. Therefore, to address the
variability of rolipram-induced apoptosis directly, without using the
inhibitor itself, we examined the sensitivity of these four populations
to the cell permeable cAMP analog, dibutyryl cAMP. As shown in
Fig 10, we found a strong correlation between rolipram- and dbcAMP-induced apoptosis. For CLL cells and
resting B cells, the percentage of apoptotic cells increased significantly relative to control cells after treatment with rolipram and forskolin or concentrations of dbcAMP  30 µmol/L
(t-test: one-tailed significance level <.05). For
anti-Ig-stimulated B cells or IL-2-cultured WMC, comparable
treatments did not significantly increase the percentage of apoptotic
cells. Consistent with this results, in the seven CLL patients studied
thus far, there has also been a correlation between sensitivity to
rolipram and sensitivity to dbcAMP (see Table 1). These data indicate
that type 4 PDE is the relevant target for rolipram in its induction of
apoptosis in CLL cells.
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| Fig 10.
Among four different lymphoid populations, sensitivity
to rolipram mirrors sensitivity to dbcAMP. One million CLL cells (),
resting () or anti-Ig activated () CD19+ cells, or
IL-2-supplemented WMC () were cultured for 72 hours with 10 µmol/L rolipram, 40 µmol/L forskolin, or the indicated dbcAMP
concentration (in µmol/L) before measurement of apoptosis by Hoechst
33342 flow cytometry. The SEMs of triplicate cultures are indicated.
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| |
DISCUSSION |
We have identified inhibition of type 4 cAMP phosphodiesterase activity
as a novel means by which to trigger apoptosis in CLL cells in vitro.
After finding type 4A and 4B transcripts as well as
rolipram-inhibitable PDE activity in CLL cells, we determined that
treatment of CLL cells with the PDE4 family-specific inhibitor rolipram
increased cAMP levels and induced apoptosis in a dose- and
time-dependent manner, an effect which correlated with apoptosis induced by dbcAMP.
Examination of treatment history and therapy resistance showed no clear
relationship between these parameters and the sensitivity of the
leukemic cells to rolipram-induced apoptosis. Five of the patients
whose cells were sensitive to rolipram had shown clinical resistance to
chlorambucil, cyclophosphamide, and/or fludarabine (see Table
1). In contrast, several chemotherapy naive patients were largely
resistant to rolipram as a single agent. Most patients' leukemic cells
had not undergone cytogenetic analysis to allow correlation with such
prognostically useful markers as 11q deletions, trisomy 12, or
abnormalities at 13q12 or 13q14.25 Of note, however, the
most rolipram-resistant patient had an elevated (20%) proportion of
cells with prolymphocytic morphology on peripheral smear as well as
multiple chromosomal abnormalities involving chromosomes 1, 2, 8, 9, 14-16, 18, and 22.
If PDEs are to be a useful pharmacologic target in the therapy of CLL,
drug dosages that trigger apoptosis in leukemic cells in vivo must have
clinically tolerable effects on other tissues. Type 4 PDEs are widely
distributed enzymes; transcripts for both PDE4A and PDE4B have been
detected by RT-PCR in all cell types tested, with the exception of the
PDE4B within the T lineage.26 PDE4C and PDE4D are also
widely distributed, although within the hematopoietic system PDE4C is
absent and PDE4D appears to be restricted to the myelomonocytic
lineage.26 Despite their widespread tissue distribution,
type 4 inhibitors have been used effectively as anti-inflammatory drugs
in animal models of asthma, inhibiting pulmonary eosinophil
accumulation after allergen challenge of sensitized
animals.27,28 Rolipram has been studied extensively as an
antidepressant in humans and is well tolerated, although higher dosages
are emetogenic. The clinical utility of type 4-specific PDE inhibitors
related to rolipram is likely to improve further with the introduction
of compounds, now identified by several groups, which maintain potency
as PDE inhibitors yet have reduced ability to displace high-affinity
[3H]rolipram binding to rat cortex. The high-affinity
binding of rolipram to brain is a characteristic of this drug which
appears to be separable from its activity as a PDE inhibitor, although central nervous system binding may be to an allosteric site on PDE4
enzymes.29
Although the feasability of type 4 PDE inhibition as a therapeutic
approach for lymphoid malignancies will require further preclinical
studies, we have made an effort in this work to determine whether
rolipram's ability to induce apoptosis in the leukemic cells of some
CLL patients is unique to this malignant population or shared by normal
circulating lymphocytes. We observed that IL-2 cultured WMC and
sIg-triggered B cells were largely insensitive to both rolipram- and
dbcAMP-induced apoptosis, whereas treatment of nonstimulated B cells
with rolipram or dbcAMP induced a moderate increase in apoptosis,
albeit superimposed on considerable basal apoptosis. All our studies on
B-cell apoptosis and cAMP metabolism were performed on cells positively
selected by adherence to anti-CD19 antibody-coated magnetic beads, a
technique that could alter the selected cells' subsequent responses.
This acknowledged, our findings of elevated basal levels of apoptosis
in nonstimulated human B cells and modest sensitivity to cAMP-mediated
apoptosis are in keeping with studies reported by Lomo et
al7 examining forskolin-induced apoptosis. In contrast,
Mentz et al2 reported low levels of both basal and
theophylline-induced apoptosis in their normal human B-cell controls.
Although formal reports on the sensitivity of human circulating T cells
to cAMP are scarce, murine peripheral T cells have been shown to be
insensitive to cAMP-mediated apoptosis, in contrast to their thymocyte
precursors.6 Whether the variable in vitro sensitivity of
these lymphoid populations to cAMP-induced apoptosis accurately
predicts the sensitivity of these cells in vivo will have to be
addressed in preclinical, animal studies.
Given the complexity of cell-cell interactions and the hormonal and
cytokine milieu of recirculating lymphocytes in vivo, cAMP metabolism
in leukemic cells within a patient may prove to be different from that
of cells maintained in tissue culture. Among those patients whose CLL
cells were sensitive to rolipram, at low dosages of the inhibitor, the
addition of the adenylate cyclase activator forskolin augmented
apoptosis, suggesting that the basal leukemic cell adenylate cyclase
activity in vitro was insufficient to adequately elevate cAMP levels.
The finding that transcript levels for PDE4B are significantly higher
in freshly isolated CLL cells than following culture is consistent with
the hypothesis that cAMP production is higher in vivo than in vitro, as
PDE4B transcript levels are known to be upregulated by elevated cAMP
levels. However, given the length of time necessary for purification of
lymphoid cells, it is difficult to prove or disprove this possibility experimentally. Although it is possible that the "flux-mediated" sensitivity of leukemic cells to treatment with rolipram as a single
agent may be higher in vivo than in vitro, the efficacy of PDE
inhibitors in CLL treatment might also be augmented by hormonal agents
that enhance adenylate cyclase activity in circulating lymphocytes.
The resistance to cAMP-mediated apoptosis observed in Ig-stimulated B
cells and IL-2-cultured WMC, as well as that observed in several CLL
patients, could result either from alterations in cAMP metabolism or
changes in the ability of PKA signaling to activate apoptotic signal
transduction. Our studies indicate that, at least with regard to normal
cell populations, insensitivity to cAMP-mediated apoptosis is not
likely to result from altered cAMP metabolism. We found that at early
time points, cAMP levels increased just as dramatically in the normal
cell populations that were insensitive to cAMP as in those that were
sensitive. Whether resistant CLL patients resemble sensitive ones with
regard to cAMP metabolism will require further study; thus far,
rolipram treatment has elevated cAMP levels in all CLL samples
examined.
If lymphoid populations sensitive or insensitive to cAMP-mediated
apoptosis differ with respect to apoptotic signaling by downstream
targets for PKA, the identity of the relevant apoptotic target remains
unknown. Transfection studies in a glucocorticoid receptor defective
line derived from the human T-ALL cell line, CEM.C7, have suggested
that functional glucocorticoid receptor signaling is required for
cAMP-mediated apoptosis.30 Although steroid resistance is
often noted clinically in CLL patients, deletions or alterations in the
gene for glucocorticoid receptor are uncommon in CLL. Nonetheless, the
glucocorticoid receptor remains a potential target for cAMP's
cytolytic activity in CLL and nondeletional alterations in this
receptor's signaling properties might underlie cAMP resistance.
BCL-2 and related proteins are likely to regulate sensitivity to
apoptosis in CLL and are also potential targets for cAMP-mediated signal transduction. Although less than 10% of CLL patients have chromosomal translocations involving BCL-2, hypomethylation and high-level BCL-2 transcription is common.31 In their
examination of theophylline's ability to synergize with chlorambucil
in the induction of apoptosis by CLL cells, Mentz et
al2 report that theophylline treatment
reduced BCL-2. These observations are now complicated by the fact that
McConkey et al32 have reported that incubation of CLL cells
in vitro leads per se to BCL-2 downregulation. In the same report,
downregulation of BAX with prolonged culture of CLL cells was found to
be predictive of resistance to glucocorticoid or
fludarabine/mitoxantrone-induced apoptosis.32 Whether such reduced BAX levels are also predictive of insensitivity to
cAMP-mediated apoptosis has not been addressed.
| |
FOOTNOTES |
Submitted February 12, 1998;
accepted June 4, 1998.
Supported by a Leukemia Society of America Translational Research Grant
Award and a grant from the American Cancer Society (IN97U).
Address correspondence to Adam Lerner, MD, Department of Medicine,
Section of Hematology and Oncology, Evans 539, Boston Medical Center,
88 E Newton St, Boston, MA 02118.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs Rita Blanchard, Mark Brauer, Evan Vosburgh, and Lewis
Weintraub for their help in obtaining blood samples from CLL patients;
Dr Keith Tornheim for advice regarding the PDE enzyme assay; Maris
Handley for technical assistance during the Hoechst dye apoptosis
assay; Kim Lui for excellent technical assistance; Dr Ronald Wohl for
providing rolipram (Berlex Laboratories, Wayne, NJ); and Drs Thomas
Rothstein, David Seldin, and Daniel Wright for helpful discussions
throughout this project.
| |
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