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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Br J Haematol. Author manuscript; available in PMC 2010 November 1.
Published in final edited form as:
PMCID: PMC2778754
NIHMSID: NIHMS146585

Preclinical activity of 8-chloroadenosine with mantle cell lymphoma: Roles of energy depletion and inhibition of DNA and RNA synthesis

Abstract

8-Chloroadenosine (8-Cl-Ado), an RNA-directed nucleoside analog, is currently being evaluated in phase I clinical trials for treatment of chronic lymphocytic leukemia. In the current study, the efficacy of 8-Cl-Ado was evaluated using mantle cell lymphoma (MCL) cell lines: Granta 519, JeKo, Mino, and SP-53. After continuous exposure to 10 μM 8-Cl-Ado for 24 h, loss of mitochondrial transmembrane potential and PARP cleavage were detected in 3 of 4 cell lines. Reduced ATP levels (30 to 60% reduction) and concurrent 8-Cl-ATP accumulation were highly associated with cell death (P < 0.01). The intracellular 8-Cl-ATP concentrations were also highly correlated with inhibition of global transcription (50 to 90%, r2 = 0.90, P < 0.01). However, the inhibition of transcription only accounted for 30 to 40% of cell death as determined by equivalent inhibition with actinomycin D. Likewise, short-lived mRNAs, those encoding cyclin D1 and Mcl-1, were not consistently reduced after treatment. Unique to MCL as compared to other hematological malignancies, 8-Cl-Ado inhibited the rates of DNA synthesis and selectively depleted dATP pools (50 to 80%). We conclude that the DNA and RNA directed actions of 8-Cl-Ado in combination with depleted energetics may promote cell death and inhibit growth of MCL cell lines.

Keywords: 8-chloroadenosine, mantle cell lymphoma, ATP, dATP

Introduction

Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by enhanced cell proliferation and impaired apoptosis (Smith 2008). Both conditions are thought to be promoted by overexpression of key cell cycle and anti-apoptotic proteins. Specifically, aggressive cell growth is believed to be influenced by overexpression of cyclin D1 caused by a t(11;14) (q13;q32) chromosomal translocation and subsequent disregulation of the G1-S phase checkpoint (Yatabe et al, 2000). Likewise, resistance to apoptosis is in part caused by overexpression of anti-apoptotic Bcl-2 protein family members including Bcl-2 and Mcl-1 (Khoury et al, 2003; Tucker et al, 2008).

As previously demonstrated for cyclin-dependent kinase inhibitors (Lacrima et al, 2005; Venkataraman et al, 2006), inhibition of transcription may reduce the expression of short-lived proteins, such as cyclin D1 and Mcl-1, leading to growth inhibition and apoptosis in MCL. Global transcription inhibitors including fludarabine (a DNA and RNA targeting nucleoside analog) and flavopiridol (a cyclin-dependent kinase inhibitor) have shown moderate clinical activity in MCL as single agents (Kouroukis et al, 2003; Lenz et al, 2004). We chose to evaluate the efficacy of a different class of transcription inhibitors, the 8-substituted adenosine analogs, using 8-chloroadenosine (8-Cl-Ado) as a model compound.

8-Cl-Ado is an RNA-directed adenosine analog and is currently in a phase I clinical trial for the treatment of chronic lymphocytic leukemia (CLL). The mechanisms of action and the metabolism of 8-Cl-Ado were previously described for CLL and multiple myeloma (MM) (Balakrishnan et al, 2005; Gandhi et al, 2001; Stellrecht et al, 2007; Stellrecht et al, 2003). Two major mechanisms of cell death for 8-Cl-Ado have been proposed: 1) transcription inhibition of short-lived antiapoptotic proteins and 2) reduction of intracellular ATP. For both causes of death, 8-Cl-Ado is sequentially phosphorylated by adenosine kinase and other enzymes to its ATP-analog, 8-Cl-ATP (Gandhi et al, 2001). Intracellular 8-Cl-ATP can then reduce mRNA transcripts by inhibiting RNA polymerase II by directly incorporating into RNA as a chain terminator or by inhibiting polyadenylation of full-length mRNA transcripts (Chen and Sheppard 2004; Stellrecht et al, 2003). Subsequent apoptosis is promoted by reduction of short-lived survival proteins, such as Mcl-1 in CLL and Met in MM (Balakrishnan et al, 2005; Stellrecht et al, 2007). In addition, intracellular 8-Cl-ADP may compete with ADP and consequently reduce intracellular ATP concentrations. As demonstrated for energy metabolism inhibitors (Hernlund et al, 2008; Mukherjee et al, 2008) and for inhibitors of de novo purine synthesis (Lu et al, 2000) the reduction of intracellular ATP can itself promote apoptosis.

8-Cl-Ado mechanisms of action are unique compared to the agents currently utilized in the treatment plan for MCL (R-HyperCVAD) (Romaguera et al, 2005). By inhibiting global transcription and reducing intracellular ATP concentrations, 8-Cl-Ado may overcome resistance to agents currently used to treat MCL by promoting cell death in all stages of the cell cycle. Positive preclinical evaluations of 8-Cl-Ado with other B-cell malignancies (CLL and MM) led us to hypothesize that 8-Cl-Ado may also be active in MCL (Balakrishnan et al, 2005; Gandhi et al, 2001; Stellrecht et al, 2003).

In the current study, the actions of 8-Cl-Ado were evaluated with 4 previously characterized MCL cell lines (Amin et al, 2003). 8-Cl-Ado inhibited growth of all cell lines and promoted apoptosis in 3 cell lines. Although global transcription was inhibited as previously reported for other hematological malignancies (Balakrishnan et al, 2005; Gandhi et al, 2001; Stellrecht et al, 2007; Stellrecht et al, 2003), 8-Cl-Ado actions with MCL were not exclusively RNA directed; the rates of DNA synthesis at early time points were also reduced. While the exact mechanisms require further study, we present evidence that reductions in ATP and dATP levels may contribute to the cytostatic and cytotoxic actions of 8-Cl-Ado in MCL.

Materials and Methods

Cell culture

Granta 519, JeKo, Mino, and SP-53 MCL cell lines were a gift from Hesham Amin (U.T. M. D. Anderson). These cell lines overall are representative of MCL primary cells (Amin et al, 2003); they overexpress the characteristic proteins of MCL including cyclin D1, cyclin E, retinoblastoma (Rb), c-Myc, p21Waf-1, p27Kip-1, Mcl-1, Bcl-2, Bax, and Bcl-xL. Only certain cell lines express cyclin D3 (JeKo and SP-53), p53 (Granta 519, Mino, and SP-53), and p16INK4a (JeKo and Mino) (Amin et al, 2003). Granta 519 was maintained in Dulbecco Modified Eagle Medium (DMEM) supplemented with 20% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). JeKo, Mino, and SP-53 were maintained in Roswell Park Memorial Institute (RPMI) Medium 1640 supplemented with FBS (10% for JeKo, 20% for Mino and SP-53). All cells were routinely tested for Mycoplasma infection using a MycoTect Kit (Invitrogen).

Chemicals and antibodies

8-Cl-adenosine and 8-Cl-ATP were purchased from BioLog (Bremen, Germany). [5,6-3H]uridine, [methyl-3H]thymidine, [methyl- 3H]dTTP, and [3H]dATP were procuredfrom Moravek Biochemicals, Inc. (Brea, CA). Protease Inhibitor Cocktail tablets were purchased from Roche Diagnostics Corporation (Indianapolis, IN). 3,3-dihexyloxacarbocyanineiodine (DiOC6) was from Molecular Probes (Eugene, OR). The antibodies to Mcl-1 (S-19), cyclin D1 (M-20), and Bcl-2 (100) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) while those for poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP), and annexin V-FITC were from BD Biosciences Pharmingen (San Diego, CA). IR DYE 680CW conjugated goat anti-mouse IgG and IR DYE 800CW conjugated goat anti-rabbit IgG secondary antibodies were procured from LI-COR Biosciences (Lincoln, NE). The β-actin antibody and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Growth inhibition

Exponentially growing MCL cells (2 to 4 × 105/mL) were incubated with 10 μM 8-Cl-Ado. Cell concentrations at various time points were quantified using a particle count and size analyzer (Beckman Coulter, Inc., Fullerton, CA).

Cell death/apoptosis assays

Cells were incubated with DiOC6 and propidium iodide in RPMI at 37°C for 20 min. Alternatively, cells were incubated with annexin V-FITC per the manufacturer’s instructions. Flow cytometry data were acquired within 30 min (FACScalibur, Becton Dickinson) and analyzed using CellQuest software (BD Biosciences).

Intracellular ATP and 8-Cl-ATP quantification

The intracellular nucleotides were extracted using 0.4N perchloric acid. The nucleoside triphosphates were separated on an analytical ion-exchange column (Partisil-10 SAX, 4.6 × 250 mm, Whatman, Maidstone, England) and quantified using external authentic standards at 256 nm. The compositions of the mobile phases were 5 mM NH4H2PO4, pH 2.8 (mobile phase A) and 0.75 M NH4H2PO4, pH 3.7 (mobile phase B). The analytes were eluted at 1.5 ml/min using the following gradient conditions: 0 min 50% B, 0–15 min linear increase to 55% B, 15–25 min linear increase to 100% B, and 25–35 min 100% B. The intracellular concentrations of nucleotides were estimatedfrom the total cell count and the median cell volume.

Intracellular dNTP quantification

The intracellular nucleotides were extracted using 60% methanol at 37°C for 15 min. The dATP concentrations of thecell extracts were quantified by a DNA polymerase assay as described previously with slight modifications (Sherman and Fyfe 1989). The dried nucleotide extract was reconstituted in buffer (20 mM HEPES, 2 mM MgCl2, pH 7.3) and co-incubated with the Klenow fragment of DNA polymerase I lackingexonuclease activity (United States Biochemical Corporation, Cleveland, OH), [methyl-3H]dTTP (25 μM, 10 μCi/mL), and a template primer (250 nM) at a final volume of 0.1 mL for 60 min at room temperature. The incubation solutions were dried on filter paper and washed with 5% sodium monophosphate, water, and ethanol. The remaining radioactive counts onthe filter papers were quantified by liquid scintillation counting (Tri-Carb 1900CA, Packard, Shelton, CT). The same procedure was used to quantify dCTP, dTTP, and dGTP using the appropriate oligonucleotide template primers and [3H]dATP. Standard curves were simultaneously generated for all dNTPs with a low limit of quantification of 0.5 pmol/well.

RNA and DNA synthesis

After a designated time of continuous incubation with 8-Cl-Ado or actinomycin D (ActD), MCL cells were incubated with [3H]uridine or [3H]thymidine (1 μCi/mL) at 37μC for 45 min to quantify the rates of RNA and DNA synthesis, respectively. The cells were washed with phosphate buffered saline (PBS) and lysed with 0.4 N perchloric acid. The pellet was dissolved in 0.5N KOH, and the total radioactivity values were quantified by liquid scintillation counting.

Cell cycle analysis

After a designated time, cells were fixed in 70% ethanol, permeabilized, and incubated with propidium iodide at room temperature for 30 min. Cells were analyzed by flow cytometry using CellQuest software (BD Biosciences).

Quantification of Proteins

MCL cells were lysed in a PBS buffer containing 100 mM NaF, 0.3% sodium pyrophosphate, 0.5% Triton X-100, 5 mM EDTA (ethylenediaminetetraacetic acid, pH 8), 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and one protease inhibitor tablet per 50 mL. The protein contents ofthe lysates were quantified using the Bio-Rad DC Protein Assaykit according to the manufacturer instructions (Bio-Rad, Hercules, CA). Protein lysates (20 μg) were separated by SDS-polyacrylamidegel electrophoresis and then electrotransferred onto nitrocellulosemembranes (0.22 μm, Osmonics, Inc., Minnetonka, MN). The membraneswere blocked for 1 h in PBS containing 5% nonfat dried milk, incubated with primary antibodies for at least 3 h, and then incubated with secondary antibodies for 1 h. The blots were visualized by scanning using a LI-COR Odyssey Imager (LI-COR Biosciences, Lincoln, NE).

Quantification of mRNA levels

Total cellular RNA was extracted from whole cells using the RNeasy kit and the QIAcube (Qiagen, Valencia, CA) and quantified using a spectrophotometer (NanoDrop 1000, Thermo Scientific, Wilmington, DE). Total RNA RT-PCR was performed with 50 ng of RNA/well in a 96-well plate using ABIPrism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) as described previously (Chen et al, 2005). Predesigned Taqman Gene Expression Primer and Probe assays were utilized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Mcl-1, and Bcl-2 (Applied Biosystems). Cyclin D1 primers and probes were synthesized by Sigma Genosys (Woodlands, TX) as follows: forward primer, 5′-ACCTGAGGAGCCCCAACAA; reverse primer, 5′-TCTGCTCCTGGCAGGCC; and probe, 5′-FAM-TCCTACTACCGCCTCACACGCTTCCTC-DBH1. The relative mRNA concentrations were calculated using the comparative Ct method with GAPDH as the endogenous control.

Statistical analysis

Correlations and t-tests were calculated using the GraphPad Prism5 software (GraphPad Software, Inc. San Diego, CA). P-values of < 0.05 were considered statistically significant.

Results

8-Cl-Ado induced cell death

To evaluate the efficacy of 8-Cl-Ado, four MCL cell lines were continuously incubated with 10 μM 8-Cl-Ado. This concentration was chosen because 10 μM 8-Cl-Ado is likely a therapeutically achievable plasma concentration based on data from previous clinical trials with 8-Cl-cAMP (Tortora et al, 1995). Cell death was specifically evaluated by loss of mitochondrial transmembrane potential (DiOC6 staining), membrane permeability to propidium iodide, annexin V binding, and PARP cleavage. 8-Cl-Ado promoted the loss of mitochondrial transmembrane potential after 12 h for JeKo and Mino and after 24 h for SP-53 and Granta 519 (P < 0.05, Fig 1A). However, for Granta 519 cells, the amplitude of response for apoptosis was much lower compared to the other 3 cell lines (13% cell death at 48 h); higher concentrations of drug (20 and 40 μM 8-Cl-Ado) were unable to increase the cell death (data not shown). DiOC6 staining was chosen as a measure of apoptosis because the annexin V negative and DiOC6 positive staining values were almost identical for paired samples from all cell lines with 8-Cl-Ado treatment (Fig 1B). Concurrently with the loss of DiOC6 staining, PARP cleavage was observed for all responsive cells: JeKo (6 h), Mino (12 h), and SP-53 (24 h) (Fig 1C). The cell death responses for JeKo, Mino, and SP-53 were also concentration dependent; EC50 values were approximately 8, 4, and 2 μM 8-Cl- Ado, respectively (data not shown).

Fig 1
8-Cl-Ado promoted cell death of MCL cells

8-Cl-ATP accumulation and ATP reduction were associated with cell death

Consistent with previous reports (Balakrishnan et al, 2005; Gandhi et al, 2001; Stellrecht et al, 2007; Stellrecht et al, 2003), 8-Cl-ATP accumulated while the ATP concentrations were reduced; CTP, UTP, and GTP concentrations were unchanged (Fig 2A). 8-Cl-ATP accumulation was also time dependent (Fig 2B) and reached a plateau after 6 to 12 h. Although all MCL cell lines accumulated 8-Cl-ATP, the steady-state intracellular concentrations of 8-Cl-ATP for Mino and JeKo cells were the highest reported for hematological malignancies (> 1 mM 8-Cl-ATP) (Balakrishnan et al, 2005; Gandhi et al, 2001; Stellrecht et al, 2003).

Fig 2
8-Cl-ATP accumulation was associated with ATP depletion and cell death

To evaluate the association between intracellular 8-Cl-ATP and ATP concentrations, the paired values from the cell lines were plotted for all time points (Fig 2C). As reported in previous studies (Balakrishnan et al, 2005; Gandhi et al, 2001), ATP reduction was directly related to accumulation of 8-Cl-ATP (Fig 2C). Two differences were observed between the cell lines: the maximum concentrations of 8-Cl-ATP and the endogenous ATP levels (y-intercept values). While SP-53 and Granta 519 accumulated similar concentrations of intracellular 8-Cl-ATP (approximately 0.5 mM), only SP-53 underwent apoptosis. SP-53 may have been more sensitive to cell death because the endogenous ATP pool of SP-53 was lower than that of Granta 519 (2 mM versus 3 mM).

To understand the role of ATP loss in cell death, for two representative cell lines (JeKo and SP-53) treated with multiple concentrations of 8-Cl-Ado, cell death at 24 h was correlated with loss of ATP and formation of 8-Cl-ATP (Fig 2D, r2 0.90, P < 0.01). Because less ATP depletion was required for cell death in SP-53 compared to JeKo cells, the lower endogenous ATP concentration of SP-53 cells may have increased its sensitivity to 8-Cl-Ado.

Inhibition of transcription by 8-Cl-Ado did not exclusively explain cell death

The rates of RNA synthesis, as quantified by uridine incorporation, were reduced after 8-Cl-Ado exposure for MCL cell lines (Fig 3A). To evaluate the relationship between the 8-Cl-ATP accumulation and the rates of RNA synthesis, the 8-Cl-ATP concentrations as shown in Fig 2A were plotted against the corresponding uridine incorporation data (Fig 3B). The relative rates of RNA synthesis were correlated to the 8-Cl-ATP levels up to 0.8 mM (r2 = 0.90, P < 0.01, Fig 3B). The rates of DNA synthesis were also inhibited by 8-Cl-Ado treatment albeit the responses were somewhat delayed compared to RNA synthesis inhibition. For all cell lines, reduced rates of DNA synthesis were observed within 3 h of 8-Cl-Ado treatment (Fig 3A), preempting any cell death, and were strongly associated with 8-Cl-ATP formation 8-Cl-ATP (r2 = 0.80, P < 0.01, Fig 3B).

Fig 3
8-Cl-Ado inhibited DNA synthesis, inhibited transcription, and promoted more cell death as compared to ActD with equivalent transcription inhibition

To evaluate the role of transcription inhibition on cell death in MCL, an RNA polymerase inhibitor, ActD, was evaluated in a paired experiment with 8-Cl-Ado (Fig 3C). At drug concentrations that inhibited uridine incorporation equally, 8-Cl-Ado promoted more cell death compared to ActD (>2-fold higher). Likewise, at equivalent cell death values, the uridine incorporation remained higher for 8-Cl-Ado treated cells compared to ActD treated cells (>2-fold higher). These results imply that global transcription inhibition by 8-Cl-Ado was not the primary mechanism of death for MCL cell lines.

To understand whether the reduction of individual antiapoptotic proteins by transcription inhibition contributed to cell death with 8-Cl-Ado, the protein levels of Mcl-1 and Bcl-2 were quantified by Western blot (Fig 4A). While the levels of Bcl-2 were consistently higher after treatment, the levels of Mcl-1 were reduced for JeKo, Mino, and SP-53 (40 to 50% of the 0 h control at 24 h). This reduction may have been caused in part by caspase activation and cleavage of Mcl-1; a secondary lower molecular weight band (24 kDa) was detected with the Mcl-1 antibody for JeKo and Mino by 12 h (Supplementary Fig S1). To determine if the effect on protein expression was due to transcription suppression, the mRNA levels of Bcl-2 and Mcl-1 were quantified by RT-PCR in two cell lines, JeKo and Granta 519 (Fig 4B). Consistent with an increase in Bcl-2 protein, the Bcl-2 mRNA transcripts for JeKo and Granta 519 were induced by 8-Cl-Ado. In contrast to the protein levels (Fig 4A), the mRNA levels of Mcl-1 were not reduced in JeKo; the mRNA levels were more than 2-fold higher than those of the untreated cells. Thus, the Mcl-1 protein was not reduced by transcription inhibition; either the protein was degraded by caspase activation (consistent with PARP cleavage) or another mechanism of post-transcriptional regulation.

Fig 4
Protein and mRNA levels of Mcl-1, cyclin D1, and Bcl-2 after 8-Cl-Ado treatment

Antiproliferative response by 8-Cl-Ado was the result of DNA synthesis inhibition, not a cell cycle effect

The reduced rates of DNA synthesis, as quantified by thymidine incorporation (Fig 3A), were consistent with the observed growth inhibition; with 10 μM 8-Cl-Ado, all cell lines were growth inhibited compared to their untreated controls (P < 0.05, Fig 5A). Even the growth of Granta 519, a cell line without significant cell death, was inhibited more than 50% by 8-Cl-Ado. To further understand the mechanism of growth inhibition by 8-Cl-Ado, the effects of cell cycle were quantified by propidium iodide staining (Table I). The percentages of cells in S-phase were not substantially lower after 24 h of treatment; the decreases in S-phase cells were less than or equal to 20%. Because the percentages of S-phase cells were minimally affected by drug treatment, the growth inhibition of the cells could not be explained by a shift in cell cycle.

Fig 5
Growth inhibition and apparent reduction of dATP concentrations after 8-Cl-Ado treatment
Table I
Cell cycle effects of 8-Cl-Ado

Consistent with minimal effects on cell cycle, 8-Cl-Ado did not consistently promote the loss of cyclin D1 (a protein overexpressed in MCL); the protein levels after 24 h were reduced only with JeKo cells (60% reduction, Fig 4A). The cyclin D1 mRNA levels were also reduced approximately 50% during this time as shown by RT-PCR (Fig 4B). Thus, in JeKo cells, transcription inhibition of cyclin D1 mRNA may have contributed to the loss of cyclin D1 protein. Consistent with a reduction in cyclin D1, a transient G0/G1 arrest was observed as the cyclin D1 levels were reduced for JeKo at 12 h (+18% of cells in G0/G1, Table I). Even so, the role of the G0/G1 arrest in growth inhibition was likely minor because the majority of JeKo cells accumulated in S-phase at 24 h (65%, Table I).

8-Cl-Ado promoted loss of dATP concentrations

To further evaluate the cause of DNA synthesis inhibition, the dNTP concentrations after 24 h of 8-Cl-Ado treatment were quantified by an enzymatic DNA polymerase assay (Fig 5B). Although the TTP concentrations were unchanged, the dATP concentrations were significantly reduced in all cell lines (50 to 80% reduction, P < 0.02). These results were unexpected because 8-Cl-Ado is neither a deoxynucleoside analog nor previously reported to reduce dATP levels (Gandhi et al, 2001). Consistent with a higher accumulation of 8-Cl-ATP, the reduction in dATP concentration for JeKo was significantly higher than those of the other cell lines (P < 0.01). While dATP was the only dNTP consistently depleted, the concentrations of other dNTPs were reduced in certain cell lines; dCTP levels dropped 80% for JeKo (P < 0.01), and dGTP levels dropped 30 to 50% for SP-53 and Mino, respectively (P < 0.02).

Discussion

The primary objectives of this study were to evaluate the efficacy of 8-Cl-Ado (cell death and growth inhibition) and heterogeneity of response with MCL cell lines. Unlike studies with other malignancies that utilized only one or two cell lines (Gandhi et al, 2001; Langeveld et al, 1997; Stellrecht et al, 2003), we completed a thorough investigation of 8-Cl-Ado using 4 simultaneously characterized MCL cell lines (Amin et al, 2003): Granta 519, JeKo, Mino, and SP-53. Although the findings of the current study will need to be validated with primary cells, these cell lines are reported to be representative of MCL cells in vivo (Amin et al, 2003).

Cell death was the first biological endpoint evaluated with 8-Cl-Ado treatment. At therapeutic concentrations of 8-Cl-Ado, apoptosis was observed by 24 h for 3 of the 4 cell lines (Fig 1A). To assess mechanism of action, the role of transcription inhibition was evaluated because reduced rates of transcription by 8-Cl-Ado were previously reported to affect cell survival for other hematological malignancies (Balakrishnan et al, 2005; Gandhi et al, 2001; Stellrecht et al, 2007). However, by comparing the actions of 8-Cl-Ado to ActD (a prototypical transcription inhibitor) with two responsive MCL cell lines, we demonstrate for the first time quantitatively that transcription inhibition by 8-Cl-Ado may not be the primary mechanism of death (Fig 3C). While 8-Cl-Ado did inhibit transcription, for the same degree of transcription inhibition, the MCL cells were more sensitive to 8-Cl-Ado than ActD (16 versus 5% death for JeKo, 29 versus 13% death for SP-53). In support of this finding, “short-lived” mRNAs and proteins of specific interest in MCL (Mcl-1 and cyclin D1) were not consistently inhibited by 8-Cl-Ado (Fig 4). Taken together, we conclude the mechanism of MCL cell death by 8-Cl-Ado was not related to reduced Mcl-1 or cyclin D1 mRNA transcripts and not exclusively global transcription inhibition.

As an alternative mechanism of death, the effects of 8-Cl-Ado on cellular energetics were evaluated by quantifying 8-Cl-ATP formation and ATP depletion. While we have previously demonstrated formation of 8-Cl-ATP and depletion of ATP with 8-Cl-Ado treatment (Balakrishnan et al, 2005; Gandhi et al, 2001; Stellrecht et al, 2007), this study is the first to quantitatively associate the maximum steady-state concentrations of 8-Cl-ATP and the corresponding ATP pool concentrations with cell death (Fig 2D). Although the relative roles of 8-Cl-ATP accumulation and ATP depletion could not be differentiated with the presented data, the heterogeneous responses of the cell lines were more closely associated with absolute ATP concentration than 8-Cl-ATP accumulation. First, JeKo cells required more ATP depletion (% of initial) for death than SP-53, a cell line with a lower endogenous ATP concentration (Fig 2D). Second, even though the 8-Cl-ATP concentrations of Granta 519 were equivalent to those of SP-53, cell death was not observed for Granta 519 (Fig 2C). While multiple explanations are possible for resistance to 8-Cl-Ado including dependence on Bcl-2 expression (Chen et al, 2008; Paoluzzi et al, 2008), Granta 519 like JeKo had a higher endogenous concentration of ATP as compared to SP-53. We conclude that ATP endogenous levels and depletion by 8-Cl-Ado may contribute to cell death in MCL.

In addition to cell death, we also evaluated the efficacy of 8-Cl-Ado on growth inhibition. By 3H-thymidine incorporation, significant growth inhibition was observed for all cell lines within 3 hours (Fig 3A). Although we do not know the mechanism, the variable responses of the cell lines (JeKo > Mino > SP-53 Granta 519) were associated with 8-Cl-ATP accumulation (Fig 3B). Also, as confirmed by cell cycle analysis (Table I), growth inhibition of all cell lines was related to inhibition of DNA synthesis. This result is in contrast to previous studies with hematological malignancies that did not report growth inhibition without apoptosis or a reduction in the S-phase fraction of cells (Gandhi et al, 2001; Zhu et al, 2006). Thus, the response of MCL to 8-Cl-Ado may be more similar to that of solid tumors; in glioma cells, 8-Cl-Ado has been reported to inhibit 3H-thymidine incorporation, and similar to JeKo cells, the glioma cells accumulated in S-phase (Langeveld et al, 1997).

Unique to MCL and not previously reported with 8-Cl-Ado, inhibition of DNA synthesis with 8-Cl-Ado was accompanied by a selective reduction in dATP pools (50 to 80% inhibition) resulting in a dNTP pool imbalance. While this imbalance may have been caused by an overall reduction of the adenylate pool, the actions of 8-Cl-Ado on dATP concentrations were surprisingly similar to the actions of other DNA-directed nucleoside analogs including clofarabine, cladribine, and gemcitabine (Gandhi et al, 2002; Hirota et al, 1989; Seymour et al, 1996; Xie and Plunkett 1996). Like the tri-phosphate metabolites of these compounds, 8-Cl-Ado metabolites may promote loss of dATP by inhibiting ribonucleotide reductase. Alternatively, as suggested for glioma cells (Langeveld et al, 1997), the 8-Cl-Ado metabolites, including perhaps 8-Cl-dATP, may directly misincorporate into DNA or inhibit DNA polymerase. Overall, the reduction in dATP levels by 8-Cl-Ado may contribute to the observed growth inhibition and requires further study to understand how 8-Cl-Ado can be used in combination with other DNA-directed agents.

In the current report, 8-Cl-Ado was able to induce cell death and inhibit growth of MCL cell lines. While 8-Cl-Ado inhibited global transcription as previously reported for other malignancies, the actions of 8-Cl-Ado were not those of a typical transcription inhibitor; mRNA transcripts encoding Mcl-1 and Bcl-2 were induced, and cell death occurred with less inhibition of global transcription than that required for death with ActD. In contrast, cell death was highly associated with a reduction in ATP, and growth inhibition was concurrent with a drop in dATP pool concentrations. Given that 8-Cl-Ado is efficacious with multiple MCL cell lines and has unique mechanisms of action directed at cellular energetics, we conclude that 8-Cl-Ado warrants further investigation in the treatment of MCL.

Supplementary Material

Fig s1

Fig S1. Mcl-1 immunoblots of whole-cell protein extracts (20 μg) after continuous exposure to 10 μM 8-Cl-Ado. The cleaved Mcl-1 band was observed at approximately 24 kDa. For Granta 519, the blot was also exposed to cyclin D1 primary antibody.

Acknowledgments

Supported in part by grant CA 85915 from the NCI and Lymphoma SPORE CA136411.

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