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Experimental Therapeutics |
Department of Experimental Therapeutics and Leukemia [V. G.] and the Newman Department of Experimental Therapeutics [M. A., R. A. N.], the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611 [R. G. H., N. L. K., S. T. R.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first series of studies suggests that the antiproliferative effect of 8-Cl-cAMP involves modulation of the intracellular level of two isoforms of the cAMP-dependent PKA holoenzyme (PKAI and PKAII; 7
). 8-Cl-cAMP is suggested to function by binding to the regulatory subunit of type I or II cAMP-dependent protein kinases, causing differential regulation of their activity (1
, 2) . Additional reports suggested that 8-Cl-cAMP can decrease transforming growth factor-
mRNA levels without changing the epidermal growth factor level (8)
. In essence, these observations identified cAMP-mediated pathways as the target for the action of 8-Cl-cAMP.
The second group of investigations has provided evidence that, in several cell lines, the effect of 8-Cl-cAMP cannot be explained by its action as a cAMP analogue; rather, tumor-cell cytotoxicity is mediated via the product (8-Cl-Ado). This product may work as a conventional antimetabolite agent, acting through the inhibition of DNA and/or RNA polymerases (9, 10, 11) . Data from our group (11) and others (4) suggested that 8-Cl-Ado production occurred in the presence of active fetal bovine serum. Serum phosphodiesterase and 5'-nucleotidase activity converted 8-Cl-cAMP into 8-Cl-Ado in medium (11 , 12) . In the absence of serum in the medium, 8-Cl-cAMP was not cytotoxic to cell lines (4) suggesting that its conversion was necessary for cytotoxicity (13) .
Pharmacokinetic investigations during a Phase I study of 8-Cl-cAMP in breast cancer patients provided additional evidence for the conversion of 8-Cl-cAMP to 8-Cl-Ado (5) . Although 8-Cl-Ado was not observed in the plasma, it was present at relatively high concentrations in tumor biopsy samples. Importantly, 8-Cl-cAMP was not detected in tumor, indicating its conversion in plasma before transport to the cells. Although these investigations provided evidence for a biochemical change of 8-Cl-cAMP, they did not provide the metabolic fate of recently generated 8-Cl-Ado.
Mechanistic investigations of 8-Cl-Ado in itself suggested that its cytotoxicity is not mediated through the PKA pathway (11) . Similarly, the growth-inhibitory activity of 8-Cl-Ado is not attributable to the interaction with the classical adenosine receptors (13) . A cAMP-independent pathway has been suggested for the action of 8-Cl-Ado that results in a modulatory effect on PKA-subunit mRNA and protein concentrations in mouse lung epithelial cells. Although the exact mechanism of this down-regulation is not known, an RNA-directed action has been suggested (10 , 14) . Taken together, this group of investigations delineate that the role of 8-Cl-cAMP is to generate 8-Cl-Ado, which in turn acts as a nucleoside analogue to exert cytotoxicity.
Neither the studies of 8-Cl-cAMP nor those of 8-Cl-Ado describe their metabolic fate in either cell or in culture medium. This is especially important if one considers that the action of 8-Cl-cAMP is attributable to 8-Cl-Ado. This compound resembles a classical nucleoside analogue, which must be converted to its phosphorylated form before incorporation into nucleic acids or actions on other cellular targets. With that in mind, the objective of the present investigation was to identify the metabolites of 8-Cl-cAMP and 8-Cl-Ado, determine the cytotoxic metabolite, and monitor the fate of these metabolites.
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MATERIALS AND METHODS |
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Drugs and Other Chemicals.
For in vitro investigations, 8-Cl-Ado and 8-Cl-cAMP were purchased initially from Bio Log (La Jolla, CA) and then obtained from Dr. Vishnuvajjala Rao at the Drug Development Branch of the National Cancer Institute (Frederick, MD). For identification of metabolites, [3H]8-Cl-Ado was purchased from Moravek Biochemicals (Brea, CA). For HPLC analyses, 8-Cl-ATP was custom-synthesized by Bio Log (La Jolla, CA). dCF was obtained from Dr. Ven L. Narayanan at the National Cancer Institute. IBMX was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were reagent grade.
Accumulation of 8-Cl-Ado Metabolites.
To identify the metabolites of 8-Cl-Ado, the exponentially growing MM cells were incubated with 1, 3, and 10 µM [3H]8-Cl-Ado for 3 h. The ADA inhibitor dCF (10 µM) was added to prevent deamination of 8-Cl-Ado. The medium was saved and then stored at -20°C until analysis. The cellular nucleotides were extracted using the perchloric acid extraction procedure (18)
and analyzed on a gradient that separates free nucleoside, mono-, di-, and triphosphate forms (18)
. The radioactive peaks were collected, and the amount of radioactivity in each peak was calculated to determine the total concentration of metabolite.
Accumulation and Elimination of 8-Cl-ATP.
Exponentially growing MM cells were incubated with the indicated concentrations of 8-Cl-Ado in the presence or absence of 10 µM dCF. Incubations were maintained for 3–12 h, and aliquots (5 x 106–2 x 107 cells) were removed at the indicated times. The nucleotides were extracted and quantitated as described below. For 8-Cl-ATP elimination studies, cells were incubated with 8-Cl-Ado for 3 h and then washed into drug-free medium. Aliquots were taken every 2 h for up to 8 h and then at 18, 22, 24, 48, and 72 h. The aliquots were extracted for quantitation of cellular nucleotides using the perchloric acid extraction procedure (18)
and analyzed as described in "Measurement of intracellular nucleoside triphosphate by HPLC."
Accumulation of 8-Cl-ATP in Ado Kinase Variant Cell Lines.
L1210 and its variant lacking Ado kinase were incubated with 10 µM [3H]8-Cl-Ado or [3H]8-Cl-cAMP in the presence of dCF for 3 h. The cells were extracted for quantitation of cellular nucleotides using the perchloric acid extraction procedure (18)
and analyzed as described in "Measurement of intracellular nucleoside triphosphate by HPLC."
Metabolism of 8-Cl-cAMP.
To determine the culture medium pharmacokinetics of 8-Cl-Ado, 8-Cl-inosine, and 8-Cl-cAMP, media were obtained before treatment and at 4, 8, and 12 h after incubation with either 8-Cl-cAMP or 8-Cl-cAMP plus IBMX. The cell culture medium was examined for the relative content of 8-Cl-cAMP and/or 8-Cl-Ado using validated HPLC assays. Aliquots of the cell culture medium (100 µl) were directly injected onto a reverse-phase HPLC analytical column [Phenomenex Sphericlone; 5 µ ODS 2: (250 x 4.6 mm)]. Compounds were eluted using a gradient mobile phase consisting of (a) 98% 80 mM ammonium acetate and 2% acetic acid (2% solution); and (b) 90% methanol, 8% ammonium acetate, and 2% acetic acid. The gradient was linear over 20 min and was run at a flow rate of 0.8 ml/min. Compounds were detected using a UV monitor set at 265 nm. The lower detection limit for both compounds was 7.8 ng (on column). For the cellular pharmacokinetics, on the other hand, the cells were diluted and processed for nucleotide extraction. Ribonucleotides and 8-Cl-ATP were separated using an anion-exchange Partisil-10 SAX column (Waters Corp., Milford, MA).
Measurement of Intracellular Nucleoside Triphosphates by HPLC.
Nucleotides were extracted using perchloric acid, and the extracts were neutralized with KOH as described previously (18)
and stored at -20°C until analyzed. The neutralized extracts were applied to an anion-exchange Partisil-10 SAX column and eluted at a flow rate of 1.5 ml/min with a 50-min concave gradient (curve 7; Waters 600E System Controller; Waters Corp.) from 60% 0.005 M NH4H2PO4 (pH 2.8) and 40% 0.75 M NH4H2PO4 (pH 3.6) to 100% 0.75 M NH4H2PO4 (pH 3.6). The column eluate was monitored by UV absorption at 256 nm, and the nucleoside triphosphates were quantitated by electronic integration with reference to external standards. The analogue triphosphate (8-Cl-ATP) was identified by comparing its retention profile and absorption spectrum with those of an authentic standard. The intracellular concentration of nucleotides contained in the extract was calculated from a given number of cells of a determined mean volume. The cell number was determined using a Coulter counter (Coulter Electronics, Hialeah, FL). This equipment is attached to a channelizer, which was used to estimate the mean volume of cells in a given cell population. This volume was used to quantitate the concentration of nucleotides. This calculation assumed that the nucleotides were uniformly distributed in a total cell volume. The lower limit of sensitivity of this assay was 10 pmol in an extract of 5 x 106 cells corresponding to a cellular concentration of 1 µM.
When radioactive 8-Cl-Ado was used, the HPLC was done using a Radiomatic Flow-through HPLC system (Packard, Downers Grove, IL). The eluate from the anion exchange column passed through an automatic radiometric detector along with liquid scintillation fluid (Ultima Flo; Packard) in a ratio of 1:3, and tritium counts were recorded for each radioactive peak.
Cell Proliferation Assay.
This assay was performed as described previously (11)
. Briefly, myeloma or L1210 and ED2 cells were cultured into 96-well dishes at a concentration of 25,000 cells/well and incubated with either 8-Cl-Ado or 8-Cl-cAMP. After 5 days of incubation, cell proliferation was determined using the MTS Cell Titer Aqueous assay, (Promega, Madison, WI), which measured the conversion of a tetrazolium compound into formazan by a mitochondrial dehydrogenase enzyme in live cells. The amount of formazan was measured spectrophotometrically and was linear with the cell number. Each data point was the average of four independent determinations, and the error bars represented the SD. The data were expressed as the percentage of formazan produced by cells treated with the control medium in the same assay. Each assay was representative of a minimum of three independent experiments.
Determination of the Intracellular dATP Pool.
MM cells were incubated with 10 µM 8-Cl-Ado and 10 µM dCF. Nucleotides were extracted using 60% methanol for determination of cellular dATP pool. The DNA polymerase assay as modified by Sherman and Fyfe (19)
was used to quantitate dATP in the cell extracts. The Klenow fragment of DNA polymerase I lacking exonuclease activity (United States Biochemical Corporation, Cleveland, OH) was used to start a reaction in a mixture that contained 100 mM HEPES buffer (pH 7.3), 10 mM MgCl2, 7.5 µg of BSA, and synthetic oligonucleotides of defined sequences as templates annealed to a primer, [3H]dTTP and either standard dATP or the extract from 1 or 2 x 106 MM cells before and after 8-Cl-Ado treatment. Reactants were incubated for 1 h and applied to filter discs; after they were washed, the radioactivity on the disks was determined by liquid scintillation counting and compared with that in the standard dATP samples.
Inhibition of Nucleic Acid Synthesis by 8-Cl-Ado.
Exponentially growing cells were incubated with 10 µM 8-Cl-Ado and 10 µM dCF for up to 12 h. One h before removal of the aliquot, 2 µCi/ml [3H]thymidine or [3H]uridine were added to these cultures, and incubation was continued for an additional 1 h in a multiscreen assay system (Millipore Corp., Bedford, MA). The cells were then collected on multiscreen GV filters under vacuum and washed four times each with ice-cold 8% trichloroacetic acid, water, and 100% ethanol. The radioactivity in the acid-insoluble material retained on the filters was measured by scintillation counting and expressed as the percentage of control (untreated) value of cells.
Flow Cytometry.
To determine the distribution of cells within the cell cycle, aliquots of cells (1 x 106 each) were pelleted (1500 rpm x 5 min at 4°C), and washed twice in ice-cold PBS (8.1 g of NaCl, 1.14 g of Na2HPO4, 0.22 g of KCl, and 0.25 g/liter KH2PO4), fixed in ice-cold 70% ethanol, and stored at 4°C until analyzed. Before analysis by flow cytometry, the fixed cells were pelleted, washed in PBS, and resuspended in ice-cold flow buffer (PBS containing 0.5% Tween 20, 15 µg/ml propidium iodide, and 5 µg/ml DNase-free RNase). The stained cells were analyzed using an Epics Profile II flow cytometer (Coulter Electronics, Inc.).
Calculations and Statistical Analysis.
The half-life of 8-Cl-ATP was determined by linear regression analysis of the concentration versus time data. Linear regression analyses for r and P for statistical correlation were done using the Prism software program (GraphPad Software, Inc., San Diego, CA). The same software program was used to evaluate differences using Student’s paired one-tailed t test.
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RESULTS |
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Concentration-dependent Metabolism of 8-Cl-Ado in MM Cells.
Exponentially growing MM cells were incubated with 1, 3, 10, and 30 µM 8-Cl-Ado for 3 h. HPLC analysis demonstrated a dose-dependent accumulation of 8-Cl-ATP, which reaches to 
150 µM with exogenous drug concentration. These analyses also revealed that there was a dose-dependent decrease in the endogenous level of ATP. At 3 h with 30 µM 8-Cl-Ado, the cellular concentration of ATP was decreased by 25% of the control value (data not shown). To determine the effect of ADA inhibitor on the accumulation of 8-Cl-ATP, parallel cultures were incubated with same concentrations of 8-Cl-Ado in the presence of 10 µM dCF. The levels of intracellular 8-Cl-ATP were similar in the presence or absence of dCF. Similarly, the decrease in ATP pool in each case was identical (data not shown). These data suggested that in MM cell lines, 8-Cl-Ado was not deaminated by ADA; however, to prevent any deamination attributable to longer incubations, dCF was added to all cultures with 8-Cl-Ado incubations.
Time-dependent Metabolism of 8-Cl-Ado in MM Cells.
Previous clinical investigations using 8-Cl-cAMP have suggested that at the maximum tolerated dose, the plasma concentration of 8-Cl-Ado is 1–5 µM (5
, 6)
. We expect that similar or higher concentrations of 8-Cl-Ado will be achieved when this nucleoside analogue is directly infused to patients. Hence, additional studies were done using 10 µM 8-Cl-Ado. As shown in Fig. 1A
, there was a time-dependent increase in the intracellular level of 8-Cl-ATP when cells were incubated with 10 µM 8-Cl-Ado. In inverse proportion to the 8-Cl-ATP pool, there was a decline in the level of endogenous ATP. At 12 h, the ATP level was <40% of the control level (Fig. 1A)
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250 µM. Cells were washed and incubated in drug-free medium, and samples were taken at various time points (Fig. 1B)
. The exponential decay characteristics showed that there was a biphasic elimination of 8-Cl-ATP. The first phase, which lasted up to 18 h after drug removal, eliminated 8-Cl-ATP with a half-life of 4.5 h. The remaining 8-Cl-ATP was degraded slowly in the cells, having a half-life of about 56 h. With this kinetic profile, 72 h after drug removal, there was a detectable (30 µM) concentration of analogue triphosphate.
Whereas the 8-Cl-ATP pool declined, the ATP pool was recovered (Fig. 1B)
. As observed previously (Fig. 1A)
, the 4-h incubation of 8-Cl-Ado resulted in a 40–45% decline in the cellular ATP pool. However, as cells were eliminating 8-Cl-ATP, the ATP pool started to increase. By 18 h after drug removal, the ATP pool size reached 75% of that of the control. After 3 days, >90% of ATP was recovered, and the ATP pool size was similar to that in the untreated control cells. These data further demonstrated that there was a relationship between the accumulation of 8-Cl-ATP and the decline in the ATP pool that was recovered when the analogue triphosphate was eliminated from the cells.
8-Cl-cAMP and 8-Cl-Ado-mediated Toxicity in MM Cells.
Cells were incubated with 1, 3, 10, and 30 µM 8-Cl-cAMP or 8-Cl-Ado for 5 days. At the end of the incubation time, cytotoxicity was measured using the MTS cell titer assay. We found that 8-Cl-Ado was more potent than 8-Cl-cAMP, especially at low concentrations (Fig. 2)
. Ninety percent inhibition of growth was observed with 10 µM 8-Cl-cAMP and 3 µM 8-Cl-Ado. Other assays for cytotoxicity, such as the oligonucleosomal DNA fragmentation assay (data not shown), were consistent with the growth inhibition assay, suggesting that the inhibition was not attributable to cytostatic growth; rather, it is attributable to apoptosis (11)
. Similar results were obtained using the steroid-insensitive MM.IRL cell line as well as in IM-9 and U266 cell lines (data not shown).
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. All cultures contained dCF to prevent deamination of 8-Cl-Ado. There was a time-dependent increase in the concentration of 8-Cl-ATP, although the intracellular levels were different in each case. With 8-Cl-Ado, the concentration reached 200 µM by 4 h and >400 µM by 12 h. In contrast, at 4 and 12 h, the concentrations were 40 and <200 µM, respectively, when the cells were incubated with 8-Cl-cAMP. In parallel, the medium used during the incubations with 8-Cl-cAMP was analyzed for 8-Cl-cAMP and 8-Cl-Ado. In the absence of IBMX, during the first 4 four h of incubation, there was a substantial (>30%) decline in the original concentration of 8-Cl-cAMP. There was no measurable concentration of the 8-Cl-Ado in the medium at the start of the experiment, however, after 4 h, the 3 µM level was achieved, and this concentration in the medium was fairly stable for 12 h (Table 1)
. Hence, treatment with 10 µM 8-Cl-cAMP, in reality, reflected incubation with 3 µM 8-Cl-Ado. Inhibition of phosphodiesterase by IBMX inhibited the production of 8-Cl-Ado in the medium. Additionally, at all time points, the level of 8-Cl-cAMP remained at 85% of the control level (Table 1)
. The presence of this inhibitor, however, abrogated accumulation of 8-Cl-ATP by >80%, resulting in an 8-Cl-ATP concentration of <30 µM at 12 h. Taken together, these data (Fig. 3
and Table 1
) suggest that, metabolically, 8-Cl-cAMP is converted to 8-Cl-Ado and then is phosphorylated to 8-Cl-ATP, and that this process is inhibited by IBMX.
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. At the end of the incubation time, cytotoxicity was measured using the MTS assay. We found that both 8-Cl-Ado and 8-Cl-cAMP were effective to inhibit cell growth in L1210 cells, but both of these drugs were ineffective in the ED2 variant. To determine that this was attributable to the lack of accumulation of 8-Cl-ATP in the ED2 cell line, both cell types were incubated for 3 h with either 10 µM 8-Cl-Ado or 10 µM 8-Cl-cAMP. As shown in Fig. 4B
, after incubation with 8-Cl-Ado, the level of 8-Cl-ATP was 40 µM in L1210 cells. In contrast, the ED2 cells accumulated 0.14 µM of 8-Cl-ATP. Accumulation of 8-Cl-ATP from 8-Cl-cAMP was much slower, and L1210 accumulated 8 µM of the triphosphate. Again the ED2 cells had <0.2 µM 8-Cl-ATP. These data strongly suggest that the presence of Ado kinase is necessary for the accumulation of 8-Cl-ATP from 8-Cl-Ado or 8-Cl-cAMP.
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may lead to a decline in the cellular dATP pool. Additionally, 8-Cl-ATP resembled ATP and dATP, which play a major role in global activation or inactivation of the ribonucleotide reductase, the enzyme responsible for de novo synthesis of deoxynucleotides. Hence, it was important to determine the effect of 8-Cl-Ado incubations on the cellular dATP concentration. For this, cells were incubated with 10 µM 8-Cl-Ado for up to 12 h, and the dATP pools in treated and untreated cells were quantitated (data not shown). Data from three experiments done in quadruplicate showed an endogenous dATP concentration of 1.62 ± 0.1 µM in the untreated cells. Treatment with 8-Cl-Ado did not affect this pool size, as evident by the intracellular level of 1.8, 1.9, 1.7, 1.5, 1.7, and 1.6 µM at 2, 4, 6, 8, 10, and 12 h, respectively. Because of this negative result, additional deoxynucleotide triphosphates were not evaluated.
Because 8-Cl-ATP is an analogue of ATP, we investigated whether it had any effect on nucleic acid synthesis. Cells were incubated with 10 µM 8-Cl-Ado and 10 µM dCF for the indicated times; however, during the last hour of incubation, the DNA and RNA precursors thymidine and uridine were added to the medium. There was no significant change in the inhibition of DNA synthesis, but there was a time-dependent decline in RNA synthesis resulting in a 50% decrease by 12 h (Fig. 5)
. The slope of RNA synthesis inhibition was significantly different from the 0 value (P < 0.0001).
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DISCUSSION |
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Once inside the cell, 8-Cl-Ado is phosphorylated to the cytotoxic moiety of 8-Cl-ATP (Fig. 1)
. The first step in this conversion is the phosphorylation to the respective monophosphate, 8-Cl-AMP. The fact that cells lacking Ado kinase were resistant to 8-Cl-Ado and did not accumulate 8-Cl-ATP (Fig. 4, A and B)
indicated that Ado kinase is necessary for this step. In addition, in vitro investigation using purified enzymes has demonstrated that this step is catalyzed by Ado kinase (9)
. This study indicated that 8-Cl-Ado is a favorable substrate for phosphorylation by Ado kinase [Km = 7 µM versus to 1.7 µM for the natural substrate (Ado) with a 50% Vmax compared with Ado]. Furthermore, the kinetic value of ADA for metabolic inactivation of 8-Cl-Ado suggested that phosphorylation may be a preferred route of 8-Cl-Ado metabolism. This is based on the fact that 8-Cl-Ado is not a good substrate for deamination by ADA (Km = 830 µM versus to 29 µM for Ado with a 1.8% Vmax). With these kinetic values, it could be expected that 8-Cl-Ado will be quickly converted to its monophosphate without the adverse effect of ADA. The observation that the presence or absence of dCF (a potent inhibitor of ADA) in the medium did not make any difference in the accumulation of 8-Cl-ATP from 8-Cl-Ado (Fig. 1
and data not shown) is consistent with this postulate. However, the stability of 8-Cl-Ado in human plasma must be examined before its use as a chemotherapeutic agent.
Our metabolite analyses using radioactive 8-Cl-Ado demonstrated that although free nucleoside, mono-, di-, and triphosphates are accumulated in the cells, the major metabolites are mono- and triphosphates. This is consistent with other adenosine and deoxyadenosine analogues, such as cladribine and clofarabine (18 , 20) ; however, it differs from arabinosylguanine, cytarabine, and gemcitabine, which accumulate primarily as triphosphates. Similar to other purine nucleoside analogues, the triphosphate is also the cytotoxic metabolite of 8-Cl-Ado.
Mechanistically, there is a major difference between clinically used purine nucleoside analogues and 8-Cl-Ado. From our studies, it appears that the action of 8-Cl-Ado is directed toward RNA synthesis, whereas DNA synthesis is not affected (Fig. 5)
. Because MM cells in patients are generally quiescent, they provide a great opportunity to investigate RNA-directed action of 8-Cl-Ado and drug-induced programmed cell death. Additionally, the utility of 8-Cl-Ado is not limited to MM; in fact, several studies using 8-Cl-cAMP and 8-Cl-Ado have suggested effectiveness in other tumors, such as human colon carcinomas, gliomas, and human mammary carcinomas (2, 3, 4, 5, 6)
.
The exact mechanism of 8-Cl-Ado-mediated cell death is not well understood at this time. Our data suggest that the mechanism of 8-Cl-Ado-mediated apoptosis is not cAMP-mediated pathways but is related to the accumulation of purine metabolites. It is possible that 8-Cl-Ado-mediated inhibition of RNA synthesis plays a role directly or indirectly in the induction of apoptosis. 8-Cl-Ado provides a dual function for inhibition of RNA synthesis. First, it decreases the cellular ATP pool, which is needed for RNA synthesis. Cellular ATP pool depletion has been associated with drug-induced cytotoxicity (21) . Second, mechanisms of cytotoxicity may interfere by inhibiting RNA polymerases or being incorporated into RNA polymers. This may lead to inhibition of specific RNA species or transcripts. Studies of these mechanisms are being done to determine the incorporation of 8-Cl-ATP in m-, t-, and r-RNA and inhibition of these RNA species. Additional studies are in progress identifying the actions of 8-Cl-ATP on gene-specific mRNA transcript inhibition.
In contrast to RNA-directed actions, upon metabolism, 8-Cl-Ado may enter the deoxynucleotide pool as 2'-deoxy-8-Cl-Ado inhibiting and become inserted into replicating DNA strands or block DNA replication by inhibition of DNA polymerases. However, at present, this pathway does not seem to be the case, because metabolites such as 8-Cl-dATP were not detected in the cell.
A major objective of the present study was to prove, based on biochemical evidence, that 8-Cl-cAMP is converted to 8-Cl-Ado, and that this nucleoside analogue then enters the cell and is phosphorylated to accumulate 8-Cl-ATP. In previous study, although incubation with 8-Cl-cAMP with subsequent HPLC analysis showed peaks that eluted in the region of 8-Cl-Ado-mono-, di-, and triphosphates, additional studies including quantitation were not done because of the lack of 8-Cl-Ado peaks (22)
. One reason for the absence of 8-Cl-Ado from the medium or the cell may be its efficient conversion to monophosphate by Ado kinase (9)
. In addition, cell line-specific differences may occur regarding metabolism of 8-Cl-cAMP and 8-Cl-Ado. The following observations in the present investigation provide strong support to our postulate that 8-Cl-cAMP is converted to 8-Cl-Ado and then phosphorylated to triphosphate. First, our extracellular data (Table 1)
as well as intracellular data (Fig. 3)
clearly demonstrated formation of 8-Cl-Ado in medium containing 8-Cl-cAMP and accumulation of 8-Cl-ATP in cells incubated with 8-Cl-cAMP. Second, and consistent with the first observation, blockage of phosphodiesterase by IBMX significantly eliminated the formation of 8-Cl-ATP (Fig. 3)
. Third, cells lacking Ado kinase were completely resistant to 8-Cl-cAMP (Fig. 4A)
, suggesting a role of this kinase for cytotoxicity. Fourth, the cells lacking Ado kinase did not accumulate 8-Cl-ATP from either 8-Cl-Ado or 8-Cl-cAMP (Fig. 4B)
. Taken together, these biochemical and pharmacological investigations demonstrate that 8-Cl-cAMP serves as a prodrug for 8-Cl-Ado.
As mentioned previously, clinical trials have used 8-Cl-cAMP as an anticancer agent. On the basis of our data, 8-Cl-cAMP may be considered a prodrug for 8-Cl-Ado. Although the prodrug could be used in clinical trials, the following points provide rationale for the use of 8-Cl-Ado directly. First, the conversion of 8-Cl-cAMP in fetal bovine serum (Table 1)
was a slow process, and at the end of 12 h, 57% of 8-Cl-cAMP still remained in the medium. This resulted in a slower rate of 8-Cl-ATP accumulation compared with that of 8-Cl-Ado (Fig. 3)
. Hence, to facilitate the rate of 8-Cl-ATP accumulation, direct infusion of 8-Cl-Ado may be recommended. Second, the activity of phosphodiesterases is variable among species, with efficiencies ranging from 28% to 72% when incubated for 3 days at 37°C. Also, relative to fetal bovine serum, human serum is low in phosphodiesterase activity (11
, 12)
; thus, the effectiveness of 8-Cl-cAMP as a chemotherapeutic agent may be variable and would be dependent on the activity of this enzyme in individual patients. Third, the favorable kinetic values of Ado kinase in phosphorylating 8-Cl-Ado suggest that high-levels of plasma 8-Cl-Ado will result in a rapid conversion of 8-Cl-Ado to its monophosphate without achieving saturation kinetics (9)
. Such high concentrations of 8-Cl-Ado may be achieved by direct infusion of 8-Cl-Ado. In summary, these data strongly suggest development of 8-Cl-Ado as a chemotherapeutic agent to be used in lieu of 8-Cl-cAMP. In collaboration with the National Cancer Institute, we are pursuing such efforts through a Rapid Access to Intervention Development (RAID) grant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work is supported in part by Grant CA85915 from the National Cancer Institute, Department of Health and Human Services; a Translational Research Award from the Leukemia Society of America; and contracts N01 CM-07109 and CM-57203 from the National Cancer Institute. This work was also supported by the Rapid Access to Intervention Development (RAID) program, Developmental Therapeutics, National Cancer Institute. 
2 To whom requests for reprints should be addressed, at Department of Experimental Therapeutics, Box 71, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 Phone: (713) 792-2989; Fax: (713) 794-4316; E-mail: vgandhi{at}notes.mdacc.tmc.edu
3 The abbreviations used are: cAMP, cyclic adenosine monophosphate; 8-Cl-cAMP, 8-chloro-cyclic AMP; 8-Cl-Ado, 8-chloro-adenosine; PKA, protein kinase A; MM, multiple myeloma; HPLC, high-performance liquid chromatography; 8-Cl-ATP, 8-chloro-adenosine triphosphate; dCF, deoxycoformycin; IBMX, 3-isobutyl-1-methylxanthine; dATP, deoxyadenosine triphosphate; ADA, adenosine deaminase.
Received 7/ 5/00. Accepted 5/16/01.
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subunit of cAMP-dependent protein kinase leading to type IIß holoenzyme-dependent growth inhibition and differentiation of HL-60 leukemia cells. J. Biol. Chem., 268: 5774-5782, 1993.This article has been cited by other articles:
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  A. J. Robinson-White, I. Bossis, H.-P. Hsiao, M. Nesterova, W. W. Leitner, and C. A. Stratakis 8-Cl-Adenosine Inhibits Proliferation and Causes Apoptosis in B-Lymphocytes via Protein Kinase A-Dependent and Independent Effects: Implications for Treatment of Carney Complex-Associated Tumors J. Clin. Endocrinol. Metab., October 1, 2009; 94(10): 4061 - 4069. [Abstract] [Full Text] [PDF]
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 M. Shanmugam, S. K. McBrayer, J. Qian, K. Raikoff, M. J. Avram, S. Singhal, V. Gandhi, P. T. Schumacker, N. L. Krett, and S. T. Rosen Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine J. Biol. Chem., September 25, 2009; 284(39): 26816 - 26830. [Abstract] [Full Text] [PDF]
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A. J. Robinson-White, H.-P. Hsiao, W. W. Leitner, E. Greene, A. Bauer, N. L. Krett, M. Nesterova, and C. A. Stratakis Protein Kinase A-Independent Inhibition of Proliferation and Induction of Apoptosis in Human Thyroid Cancer Cells by 8-Cl-Adenosine J. Clin. Endocrinol. Metab., March 1, 2008; 93(3): 1020 - 1029. [Abstract] [Full Text] [PDF]
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 C. M. Stellrecht, C. J. Phillip, F. Cervantes-Gomez, and V. Gandhi Multiple Myeloma Cell Killing by Depletion of the MET Receptor Tyrosine Kinase Cancer Res., October 15, 2007; 67(20): 9913 - 9920. [Abstract] [Full Text] [PDF]
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 K. Balakrishnan, W. G. Wierda, M. J. Keating, and V. Gandhi Mechanisms of Cell Death of Chronic Lymphocytic Leukemia Lymphocytes by RNA-Directed Agent, 8-NH2-Adenosine Clin. Cancer Res., September 15, 2005; 11(18): 6745 - 6752. [Abstract] [Full Text] [PDF]
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Y.-H. Ahn, J. M. Jung, and S. H. Hong 8-Chloro-Cyclic AMP-Induced Growth Inhibition and Apoptosis Is Mediated by p38 Mitogen-Activated Protein Kinase Activation in HL60 Cells Cancer Res., June 1, 2005; 65(11): 4896 - 4901. [Abstract] [Full Text] [PDF]
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 K. Balakrishnan, C. M. Stellrecht, D. Genini, M. Ayres, W. G. Wierda, M. J. Keating, L. M. Leoni, and V. Gandhi Cell death of bioenergetically compromised and transcriptionally challenged CLL lymphocytes by chlorinated ATP Blood, June 1, 2005; 105(11): 4455 - 4462. [Abstract] [Full Text] [PDF]
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 K. Ghias, C. Ma, V. Gandhi, L. C. Platanias, N. L. Krett, and S. T. Rosen 8-Amino-adenosine induces loss of phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Akt kinase: Role in induction of apoptosis in multiple myeloma Mol. Cancer Ther., April 1, 2005; 4(4): 569 - 577. [Abstract] [Full Text] [PDF]
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 C. M. Stellrecht, L. S. Chen, and V. Gandhi Inhibition of Oncogene Expression by RNA-Directed Agents Am. Assoc. Cancer Res. Educ. Book, April 1, 2005; 2005(1): 338 - 343. [Full Text] [PDF]
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N. L. Krett, K. M. Davies, M. Ayres, C. Ma, C. Nabhan, V. Gandhi, and S. T. Rosen 8-Amino-adenosine is a potential therapeutic agent for multiple myeloma Mol. Cancer Ther., November 1, 2004; 3(11): 1411 - 1420. [Abstract] [Full Text] [PDF]
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L. S. Chen and T. L. Sheppard Chain Termination and Inhibition of Saccharomyces cerevisiae Poly(A) Polymerase by C-8-modified ATP Analogs J. Biol. Chem., September 24, 2004; 279(39): 40405 - 40411. [Abstract] [Full Text] [PDF]
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C. M. Stellrecht, C. O. Rodriguez Jr., M. Ayres, and V. Gandhi RNA-Directed Actions of 8-Chloro-Adenosine in Multiple Myeloma Cells Cancer Res., November 15, 2003; 63(22): 7968 - 7974. [Abstract] [Full Text] [PDF]
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K. C. Anderson and W. S. Dalton Synopsis of a Research Roundtable Presented on Cell Signaling in Myeloma: Regulation of Growth and Apoptosis--Opportunities for New Drug Discovery Mol. Cancer Ther., December 1, 2002; 1(14): 1361 - 1365. [Abstract] [Full Text] [PDF]
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) and ATP (
). B, for elimination studies, cells were incubated with 10 µM 8-Cl-Ado for 3 h in the presence of dCF. The cells were washed in drug-free medium and incubated for an additional 12 h. Aliquots were taken at the indicated times, and cellular levels of 8-Cl-ATP (
and 
and •) for 3 days in the presence of dCF. At the end of incubation, cytotoxicity was determined using the MTS assay as described in "Materials and Methods." Data points, mean and SD from four determinations in one experiment. The experiment was repeated three times with similar results. B, L1210 (L) or ED2 (E) cells were incubated with 10 µM 8-Cl-Ado or 8-Cl-cAMP for 3 h. The ADA inhibitor, dCF, was added in all cultures during the incubation period. Cell extracts were analyzed for cellular accumulation of 8-Cl-ATP. Bars, SD from three identical experiments.