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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2893-2898
8Cl-cAMP Cytotoxicity in Both Steroid Sensitive and Insensitive
Multiple Myeloma Cell Lines Is Mediated by 8Cl-Adenosine
By
Robert G. Halgren,
Ann E. Traynor,
Shafali Pillay,
Joann L. Zell,
Kimberly F. Heller,
Nancy L. Krett, and
Steven T. Rosen
From the Lurie Comprehensive Cancer Center and Department of
Medicine, Northwestern University, Chicago, IL.
 |
ABSTRACT |
We have examined the cytotoxic effects of cyclic
adenosine-3 ,5-monophosphate (cAMP) derivatives on
multiple myeloma cells lines and determined that the 8-Chloro
substituted derivative (8Cl-cAMP) is one of the most potent. We report
here that 8Cl-cAMP is cytotoxic to both steroid sensitive and
insensitive myeloma cells with a half maximal concentration of
approximately 3 µmol/L. 8Cl-cAMP toxicity in myeloma cells is
dependent on phosphodiesterase activity in the serum of cell culture
medium. A metabolite of 8Cl-cAMP, 8-Chloro-adenosine (8Cl-AD), kills
myeloma cells as effectively as 8Cl-cAMP. Adenosine deaminase (ADA)
converts 8Cl-AD into 8Cl-inosine and abrogates the cytotoxic effects of
8Cl-cAMP, 8Cl-AMP, and 8Cl-AD, as does 5-(p-Nitrobenzyl)-6-Thio-Inosine (NBTI), an inhibitor of nucleoside uptake. These data
suggest that 8Cl-cAMP must be converted to 8Cl-AD and that 8Cl-AD is
the compound that enters the cell. Contrary to glucocorticoid-mediated cell death in myeloma cells, the pathway of 8Cl-AD-mediated cell death
appears to be independent of interleukin-6 (IL-6) actions. Although the
exact mode of action for this agent is currently unknown, its ability
to kill steroid sensitive and insensitive multiple myeloma cells in an
IL-6 independent fashion may offer exciting new therapeutic options.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
MULTIPLE MYELOMA IS a neoplasia of
antibody-secreting plasma cells and represents approximately 10% of
all hematologic malignancies. The median survival time of all myeloma
patients is 2.5 to 3 years. This median survival time has not changed
substantially in over 3 decades.1 Marrow ablation followed
by bone marrow or stem cell infusion may produce a prolonged remission,
but this approach has been limited to young patients and the potential
for a cure is not proven.2 One of the best therapeutic
options for multiple myeloma is treatment with glucocorticoids,
however, some patients do not initially respond to glucocorticoids, and
those that do will inevitably become resistant to such therapy. To
investigate the mechanism of hormone resistance, we have previously
isolated and characterized a multiple myeloma cell line,
MM.1,3 which was further selected into glucocorticoid
sensitive and glucocorticoid resistant subtypes. The MM.1S cell line
contains a normal compliment of functional glucocorticoid receptors and
is killed by submicromolar concentrations of the synthetic
glucocorticoid dexamethasone, whereas the MM.1RL cell line
has no measurable expression of glucocorticoid receptors and is highly
resistant to dexamethasone.4,5
Membrane permeable cyclic adenosine-monophosphate derivatives have been
shown to have a wide variety of effects in multiple cell types and may
share some common distal events with the glucocorticoid receptor-mediated pathway. For example, selection of a WEHI thymoma cell line for dexamethasone resistance resulted in one clone that was
also resistant to cyclic adenosine-3,5-monophosphate
(cAMP)-induced cell death.6 To investigate the possibility
that cAMP analogs could be cytotoxic to both steroid sensitive and
resistant MM.1 cells, we examined the effect of dibutyryl-cAMP and
found that it causes apoptosis in both the MM.1S and MM.1R
sublines.7 Because the cytotoxic concentration of Bu2-cAMP
in vitro is too high to be used clinically, several other cAMP analogs
were tested.
The 8-Chloro derivative of cAMP (8Cl-cAMP) is a very potent,
site-selective cAMP analog that is under intensive investigation as a
potential chemotherapeutic agent. Phase I clinical studies have been
performed against solid tumors,8 demonstrating that it can
be given safely, with evidence for clinical response. The mechanism of
action of 8Cl-cAMP has been the subject of some debate. One potential
mode of action is through modulation of protein kinase A (PKA)
activity. Another theoretical pathway requires the conversion of
8Cl-cAMP by serum enzymes to 8Cl-adenosine. Here we report that
8Cl-cAMP kills both steroid sensitive and insensitive multiple myeloma
cells in vitro; that this killing is dependent on its conversion to
8Cl-AD; and that this phenomenon cannot be reversed by the addition of
exogenous interleukin-6 (IL-6). This represents the first report of
cytotoxicity in multiple myeloma cells mediated by 8Cl-cAMP, a
therapeutic agent with activity against both glucocorticoid sensitive
and resistant disease.
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MATERIALS AND METHODS |
Cell culture.
MM.1 cells were previously established from the peripheral blood of a
multiple myeloma patient.3 These cells were cultured in
RPMI-1640 (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal
bovine serum (GIBCO BRL), 2 mmol/L glutamine, 100 U/mL of penicillin,
100 µg/mL streptomycin, and 2.5 µg/mL fungizone. Cells were grown
in a 37°C incubator with 5% CO2.
Chemicals.
IL-6 was purchased from R & D Systems (Minneapolis, MN), 8-chloro-cAMP,
8-chloro-AMP, and 8-chloro-adenosine were purchased from BioLog (La
Jolla, CA), and dibutyryl-cAMP, 5-(p-Nitrobenzyl)-6-Thio-Inosine (NBTI), ADA, 3-isobutyl-1-methylxanthine (IBMX),
camptothecin, and dexamethasone were purchased from Sigma (St Louis,
MO).
Cell proliferation assay.
A total of 25,000 cells per well was plated into 96-well dishes in a
final volume of 0.1 mL RPMI-1640 growth media containing drugs as
described in the figure legends. The plates were removed after 1, 3, and 5 days of incubation for assay. Cell proliferation was determined
by the Cell Titer Aqueous assay of Promega (Madison, WI),
which measures the conversion of a novel tetrazolium compound into
formazan by mitochondrial dehydrogenase enzymes in live cells. The
amount of formazan was measured spectophotometrically at 490 nm and in
the range observed is linear with the number of live cells in the
assay. Each data point is the average of four independent determinations, and error bars represent standard deviation. The data
are expressed as a percentage of the formazan produced by cells treated
with control medium in the same assay. Each assay is representative of
a minimum of three independent experiments.
DNA isolation.
Cells were grown in 25 cm2 flasks with 5 µmol/L
8Cl-adenosine. The DNA was isolated as described by Gong et
al.9 Briefly, the cells were treated for the indicated
times, harvested, and counted by trypan blue exclusion. A total of 5 × 106 cells were collected and fixed with 70%
ethanol at  20°C overnight. The ethanol was removed and the
cells resuspended in 40 µL of phosphate-citrate buffer
(0.192 mol/L Na2HPO4, 4 mmol/L
citric acid, pH 7.8) and incubated at room temperature for 30 minutes. The cells were pelleted by centrifugation, the supernatant removed to a
fresh tube, and concentrated to approximately 30 µL by 15 minutes of
vacuum drying. In this fashion, only the low molecular weight DNA is
recovered because the cells were permeablized and not lysed. The
majority of the high molecular weight DNA remains associated with the
cell pellet. The isolated DNA was treated with RNase (0.1 mg/mL) for 30 minutes at 37°C in the presence of 0.025% sodium dodecyl sulfate
(SDS), followed by a 30-minute 37°C incubation with 0.1 mg/mL
proteinase K. The isolated DNA was then fractionated on a 1% agarose
gel containing ethidium bromide for 4 hours at 2 V/cm. One million
HL-60 cells, treated with 0.15 µmol/L camptothecin for 3 hours,
served as a positive control.9
Flow cytometric analysis.
A total of 5 × 106 cells were treated with the
indicated concentration of drug for either 24 or 48 hours, then washed
with cold phosphate-buffered saline (PBS) and fixed in 40% cold
ethanol at 4°C overnight. Cells were then washed with PBS,
resuspended in 50 µg/mL RNase A in PBS for 30 minutes at 37°C,
and then resuspended in 15 µg/mL propidium iodide in 38 mmol/L sodium
citrate buffer. Flow cytometry was performed on a FACsort instrument
and data analyzed using the CellQuest software package (Becton
Dickinson, Bedford, MA). Data are presented as an average
of three independent determinations.
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RESULTS |
8Cl-cAMP and 8Cl-AD concentration dependence curves.
To determine the ability of 8Cl-cAMP to act as a cytotoxic agent in
multiple myeloma cells, concentration dependence curves were generated
for both 8Cl-cAMP and a potential metabolite of 8Cl-cAMP, 8Cl-AD
(Fig 1). 8Cl-cAMP is effective against both
MM.1S and MM.1R cell lines, with a concentration of 10 µmol/L causing a greater than 90% reduction in cell number. Interestingly, the 8Cl-cAMP metabolite, 8Cl-AD, is more potent than the parent compound. Both steroid sensitive and insensitive cell lines are growth inhibited, but the concentration required for 90% inhibition is approximately 3 µmol/L. MM.1R cells are only slightly less sensitive to both agents
than their MM.1S counterparts. Additional myeloma cell lines were
examined, including IM-9 and U266, and these also demonstrated similar
dose-dependent decreases in proliferation upon treatment with either
8Cl-cAMP or 8Cl-AD (data not shown). The data presented are for 5 days
of continuous treatment with these drugs; however, the
antiproliferative effects are evident after shorter treatments and are
readily apparent at 3 days.
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| Fig 1.
Concentration dependence of 8Cl-cAMP- and
8Cl-AD-mediated growth inhibition in MM.1S and MM.1R cells. Cells are
treated with the indicated concentration of drug for 5 days and then
cell number is assayed and presented as described in Materials and
Methods. MM.1S treated with 8Cl-cAMP ( ); MM.1S treated with 8Cl-AD
( ); MM.1R treated with 8Cl-cAMP ( ); MM.1R treated with 8Cl-AD
( ).
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Metabolic processing of 8Cl-cAMP.
Because 8Cl-AD was more potent than 8Cl-cAMP and could be a metabolite
of that drug, we analyzed the possibility that 8Cl-cAMP exerts its
effects through an 8Cl-AD metabolite by examining key steps in the
metabolic pathway. The first step examined was the activity of
phosphodiesterase (PDE), which is present in serum and converts cAMP
into AMP. IBMX is a potent inhibitor of PDE activity. When 8Cl-cAMP is
given concurrently with 50 µmol/L IBMX to myeloma cells, the
antiproliferative effect is greatly reduced (Fig 2). IBMX had no effect on the activity
of 8Cl-AD. Therefore, extracellular PDE activity is required for
activation of 8Cl-cAMP. This is further evidenced by the fact that
8Cl-cAMP activity in serum-free medium is greatly reduced
(Fig 3).
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| Fig 2.
8Cl-cAMP and 8Cl-AD activity in the presence of IBMX or
ADA. MM.1S (A) or MM.1R (B) cells are treated with either control
medium, medium containing 3 µmol/L 8Cl-cAMP, or medium containing 3 µmol/L 8Cl-AD for 5 days and cell number is assayed and presented as
described in Materials and Methods. Where indicated, IBMX is added
concurrently with treatment medium at a concentration of 50 µmol/L.
Where indicated, ADA is added concurrently with treatment medium at a
concentration of 2 U/mL.
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| Fig 3.
ADA inhibits 8Cl-AMP activity. MM.1S cells are treated
with 8Cl-AMP for 5 days at the indicated concentrations in the presence
(light bars) or absence (dark bars) of 2 U/mL of ADA. The assay is done
in normal growth medium that does not contain serum. 8Cl-cAMP
(5 µmol/L) and 8Cl-AD (5 µmol/L) are included as controls. Cell
number is assayed and presented as described in Materials and
Methods.
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The activity of PDE on 8Cl-cAMP results in the formation of
8Cl-adenosine monophosphate, which is subsequently converted into 8Cl-AD by 5-nucleotidase. The reverse reaction (8Cl-AD to
8Cl-AMP) can also occur. To investigate the possibility that 8Cl-AMP is the cytotoxic metabolite, we examined the ability of 8Cl-AMP to kill
myeloma cells. Concentration dependence curves for this agent against
MM.1 cells were generated and demonstrate that 8Cl-AMP inhibits myeloma
cells in a dose-dependent fashion similar to 8Cl-cAMP (data not shown).
We next addressed the possibility that 8Cl-AMP is active without
further metabolic processing. To examine this, we treated cells
concurrently with 8Cl-AMP and 2 U/mL ADA in serum-free conditions.
Serum-free medium was used to eliminate the influence of serum
nucleotidases (Fig 3). 8Cl-AD-mediated growth inhibition is greatly
reduced by the addition of ADA, as is 8Cl-AMP-mediated growth (Figs 2
and 3). This indicates that 8Cl-AMP must be converted to 8Cl-AD to
exert its effect, as AMP is not a substrate for ADA. 8Cl-inosine, the
product of ADA activity on 8Cl-AD, is inactive in this system.
Furthermore, because the experiment was performed in serum-free
conditions, the MM.1 cells must be producing 5-nucleotidase.
This is not unexpected, as other cells of B-cell lineage have been
shown to express ecto-5-nucleotidase (CD73), which has been
implicated as having a role in mediating lymphocyte binding to
endothelial and follicular dendritic cells.10 Because
myeloma cells are antibody-producing plasma cells, it is reasonable to
expect that they would also express this marker.
Taken together, these data strongly suggest that 8Cl-cAMP must be
converted to 8Cl-AMP by serum phosphodiesterases, which is then
converted to 8Cl-AD by removal of the phosphate. 8Cl-AD is the ultimate
extracellular metabolite, as further metabolic products are not active.
8Cl-AD entry into the cell is required for activity.
NBTI, an inhibitor of nucleoside uptake,11,12 was used to
determine if the drug must enter into the cell to exert its cytotoxic effect. In the presence of this inhibitor, the activity of both 8Cl-cAMP and 8Cl-AD were greatly reduced
(Fig 4), indicating that nucleoside uptake
is required for cytotoxic activity. Unsubstituted adenosine is not
cytotoxic to MM.1 cell lines in concentrations up to 1 mmol/L (data not
shown). It is expected that adenosine would be able to activate surface
adenosine receptors, thereby activating adenylate cyclase and producing
intracellular cAMP. However, because adenosine has no effect on MM.1
cells, we believe that such signaling is not involved in
8Cl-AD-mediated toxicity. Together, these observations would indicate
that signaling through surface adenosine receptors is not the mechanism
by which 8Cl-AD acts, and that the drug must enter the cell to be
effective. Its fate after it enters the cell is as yet undetermined.
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| Fig 4.
NBTI inhibits 8Cl-cAMP and 8Cl-AD activity. MM.1S cells
are treated with 8Cl-cAMP for 5 days at the indicated concentrations in
the presence (light bars) or absence (dark bars) of 50 µmol/L
NBTI. MM.1S cells treated with 3 µmol/L 8Cl-AD are also
included. Cell number is assayed and presented as described in
Materials and Methods.
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IL-6 does not inhibit 8Cl-cAMP or 8Cl-AD.
IL-6 is a potent growth factor for multiple myeloma, and it has been
reported that glucocorticoids exert a large part of their antiproliferative effect through downregulation of both the cytokine and its receptor.13 Adding exogenous IL-6 to
dexamethasone-treated MM.1S cells can partially inhibit
glucocorticoid-induced cell death (Fig 5A).
Dexamethasone treatment reduces cell number by approximately 90%, but
in the presence of 500 pg/mL IL-6, there is only a 60% decrease . MM.1R cells are not affected by dexamethasone treatment (Fig 5B). In
contrast, cells treated with 3 µmol/L 8Cl-cAMP or 8Cl-AD show a
greater than 90% reduction in cell number that is not rescued by the
addition of exogenous IL-6, indicating that it is unlikely that
downregulation of this cytokine is the mechanism of action of
8Cl-AD-mediated cytotoxicity.
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| Fig 5.
IL-6 does not inhibit 8Cl-cAMP or 8Cl-AD activity. MM.1S
(A) and MM.1R (B) cells are treated for 5 days with control medium
only, dexamethasone (1 µmol/L), 3 µmol/L 8Cl-cAMP, or 3 µmol/L
8Cl-AD in the presence (light bars) or absence (dark bars) of 500 pg/mL
IL-6. Cell number is assayed and presented as described in Materials
and Methods.
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8Cl-AD causes apoptosis in MM.1S and MM.1R cell lines.
To determine if the antiproliferative effects of 8Cl-AD were due to its
ability to induce apoptosis, DNA was isolated from MM.1S and MM.1R
cells after treatment with 8Cl-AD and examined for the appearance of a
DNA chromatin "ladder pattern" associated with apoptotic cell
death.14 A very faint ladder is detected after 16 hours of
treatment, which increases in intensity with increasing duration of
drug incubation. A very intense ladder pattern is seen after 48 hours
of treatment (Fig 6). The viability of
these cells (assayed by trypan blue exclusion) decreases in parallel
with DNA ladder formation (data not shown). While chromatin ladder
formation is not the sole defining characteristic of
apoptosis,15 it is a good marker for this process and
certainly indicates that 8Cl-AD is acting as a cytotoxic, not a
cytostatic, agent.
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| Fig 6.
8Cl-AD induces chromatin ladder formation. Five million
MM.1S cells are treated for the indicated times with 3 µmol/L 8Cl-AD
and low molecular weight DNA is obtained as described in Materials and
Methods. Markers are the 123-bp DNA ladder (GIBCO BRL). DNA is
fractionated through 1% agarose. HL-60 cells treated with 0.15 µmol/L camptothecin serve as a positive control for the DNA isolation
protocol.
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To further confirm that 8Cl-AD is acting through an apoptotic
mechanism, flow cytometric analysis was performed with propidium iodide
(Table 1). Apoptotic cells were defined as
those with subdiploid DNA content and are presented as the percentage
of total counted cells in the assay. Dexamethasone, used as a positive control for apoptosis, shows a dramatic increase in apoptotic cells by
24 hours. The apparent decrease in apoptotic cells by 48 hours of
dexamethasone treatment may be due to the fact that most of the cells
have already completed the apoptotic program. After 24 hours of 8Cl-AD
treatment, approximately 30% of the cells are apoptotic, as compared
with approximately 5% in the control. As further evidence that
8Cl-cAMP needs metabolic activation, only 13% of cells are apoptotic
when treated with this drug for 24 hours, yet after 48 hours, the
activity 8Cl-cAMP approaches that of 8Cl-AD at 24 hours. Also, 1 U/mL
of ADA completely abrogates the affect of 8Cl-AD at both 24 and 48 hours, further demonstrating that this is indeed the active metabolite.
Interestingly, ADA does not completely inhibit the activity of 8Cl-cAMP
at 48 hours, which may indicate that there is a small proportion of
cell death mediated by the native 8Cl-cAMP molecule.
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DISCUSSION |
8Cl-cAMP-mediated cytotoxicity in multiple myeloma cell lines is
dependent on its extracellular conversion to 8Cl-AD. This conversion
requires the activity of serum PDE and 5-nucleotidase. If PDE is
inhibited, or if the conversion of 8Cl-AD to 8Cl-inosine is enhanced,
8Cl-cAMP is no longer effective, indicating that 8Cl-AD is the active
agent. This finding was surprising, as we have previously demonstrated
that agents, which are known to signal through PKA, including dibutyryl
cAMP and forskolin, can be effective at inducing cell death in a
dose-dependent fashion.7 The inability of ADA to completely
inhibit 8Cl-cAMP-induced apoptosis, as demonstrated in the flow
cytometric assay, may be due to its ability to mediate a small portion
of its activity through PKA. This is consistent with our previous
observations.7 However, the predominant cytolytic actions
of 8Cl-cAMP are through the 8Cl-AD metabolite. This is especially
apparent over longer time periods, as demonstrated by the proliferation
assay data. 8Cl-AD is not causing cell death by stimulating surface
adenosine receptors, thereby causing activation of adenylate cyclase
and subsequent production of cAMP, as adenosine is unable to achieve
the same effect. Furthermore, a nucleoside uptake inhibitor blocks
8Cl-AD action, indicating that the agent must be taken up into the cell
for activity.
8Cl-cAMP inhibits the growth of several cell types in vivo, possibly
due to its ability to influence the ratio of PKA isozymes in the
cell.16,17 Two basic isoforms of the regulatory subunit have been identified (RI and RII), leading to formation of two types of
the PKA holoenzyme, PKAI and PKAII. 8Cl-cAMP binds with high efficiency
to one of the two sites of the RII type subunit, which is insufficient
to cause release of the catalytic subunits. However, 8Cl-cAMP binds
with similar efficiency to both sites of the RI subunit, leading to the
release of the catalytic subunit and subsequent downregulation of the
PKAI holoenzyme. The ratio of PKAI to PKAII has been shown to correlate
with the differentiation state of several cell types. When PKAI is high
in relation to PKAII, the cells tend to be in a proliferative state,
while increased PKAII levels is associated with a differentiated or
growth arrested state (reviewed in Cho-Chung18).
Consequently, the selective degradation of PKAI by 8Cl-cAMP could be
responsible for the toxicity of this agent.
8Cl-cAMP has also been reported to act through conversion to its 8Cl-AD
metabolite in several systems, including mouse lung epithelial cell
lines,19 human glioma,17 human mammary
carcinoma,20 and human colon tumor cell
lines.21 The exact mechanism of 8Cl-AD-mediated cell death
is not well understood at this time. It is possible that 8Cl-AD
inhibits DNA or RNA polymerase directly. It has been shown in human
glioma cell lines that both 8Cl-cAMP and 8Cl-AD partially inhibit DNA
and RNA synthesis, possibly through incorporation into these
macromolecules.22 However, Lange-Carter et al19 demonstrate that while 8Cl-cAMP conversion to 8Cl-AD is required for
effectiveness, 8Cl-AD treatment results in a decrease of the levels of
the RI subunit of PKA. It is possible that while the drug must be
converted to the adenosine metabolite for entry into the cell, it may
be reconverted into 8Cl-cAMP after entry by the cellular machinery. We
are currently investigating all of these potential mechanisms of action
in multiple myeloma cells.
A potential benefit to using 8Cl-cAMP (or 8Cl-AD) as an agent against
multiple myeloma is its ability to act independently of IL-6. Hardin et
al13 have shown that glucocorticoids can downregulate both
this cytokine and its receptor in myeloma cells. Furthermore,
glucocorticoid cytotoxicity to myeloma cells in vitro can be inhibited
by addition of exogenous IL-6.13 A possible mechanism for
this effect is through modulation of NF- B activity. Glucocorticoids
have been shown to regulate the production of the inhibitor of NF-B,
IB , thus inhibiting the ability of NF-B to translocate to the
nucleus and stimulate the production of cytokines, such as
IL-6.23,24 IL-6 is abundant in the tumor millieu and is
overproduced in approximately one third of multiple myeloma patients.
This overproduction is correlated with a poor prognosis.25
We have demonstrated that addition of exogenous IL-6 cannot inhibit the
cytotoxic activity of 8Cl-cAMP or 8Cl-AD. The ability of 8Cl-cAMP to
kill myeloma cells in an IL-6 independent fashion may have significant
therapeutic implications.
The requirement for 8Cl-AD production may also have clinical
significance. Relative to fetal bovine serum, human serum is low in PDE
activity, thus the effectiveness of 8Cl-cAMP as a chemotherapeutic agent may be variable dependent on the PDE activity of the individual patient. 8Cl-cAMP added to tissue culture medium containing 20% human
serum was unable to promote killing of myeloma cells, whereas 8Cl-AD
remained effective (data not shown). Consequently 8Cl-cAMP, as a
prodrug, may not be converted to the active metabolite in sufficient
concentrations to be maximally effective in vivo. Studies of the
pharmacokinetics of 8Cl-cAMP in both human breast cancer patients and
nude mice bearing colon tumor xenografts indicate that the 8Cl-AD
metabolite, while in low concentration in the plasma, was in a
significantly higher concentration in tumor biopsies.26 Therefore, while the effectiveness of 8Cl-cAMP may be dependent on PDE
activity in the serum or tumor cell density, administration of 8Cl-AD
directly can circumvent this problem and may enhance the efficacy of
this therapeutic approach. We are currently pursuing the development of
8Cl-AD as a therapeutic alternative.
| |
FOOTNOTES |
Submitted January 7, 1998;
accepted June 12, 1998.
Supported by the Northwestern Memorial Foundation to N.L.K., by
Grant No. CA 60061 to S.T.R. and N.L.K., and Grant No. P30 CA60553 to the Robert H. Lurie Cancer Center from the National Cancer Institute.
Address reprint requests to Robert G. Halgren,
Northwestern University Medical Center, Lurie Comprehensive Cancer
Center, 8-340 Olson Pavilion, 303 E Chicago Ave, Chicago, IL 60611;
e-mail: r-halgren{at}nwu.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract