Chronic Lymphocytic Leukemia, Normal B and T Cells

Thymidine-phosphorothioate oligonucleotides induce activation and apoptosis of CLL cells independently of CpG motifs or BCL-2 gene interference

Article metrics

Abstract

We compared antisense phosphorothioate oligonucleotides (PS-ODN) that target BCL-2 such as Genasense® (G3139-PS), with other PS-ODN or phosphodiester-ODN (PO-ODN) in their relative capacity to induce apoptosis of chronic lymphocytic leukemia (CLL) B cells in vitro. Surprisingly, we found that thymidine-containing PS-ODN, but not PO-ODN, induced activation and apoptosis of CLL cells independent of BCL-2 antisense sequence or CpG motifs. All tested thimidine-containing PS-ODN, irrespective of their primary sequences, reduced the expression of Bcl-2 protein and increased the levels of the proapoptotic molecules p53, Bid, Bax in CLL cells. Apoptosis induced by thymidine-containing PS-ODN was preceded by cellular activation, could be blocked by the tyrosine-kinase inhibitor imatinib mesylate (Gleevec®), and was dependent on ABL kinase. We conclude that thymidine-containing PS-ODN can activate CLL cells and induce apoptosis via a mechanism that is independent of BCL-2 gene interference or CpG motifs.

Introduction

Oligodeoxynucleotides (ODN) can interfere with specific gene expression when they are complementary to the target gene's mRNA.1 Such antisense ODN can activate RNase H-mediated selective mRNA degradation and/or interference, thereby reducing the stability and expression of the target RNA.2, 3 As phosphorothioate ODN (PS-ODN) resist nuclease degradation and can achieve higher concentrations in vivo than native phosphodiester ODN (PO-ODN), antisense PS-ODN appear better suited than PO-ODN for clinical applications.4

A very attractive target gene for such antisense PS-ODN is BCL-2, an oncogene that is overexpressed in most human cancers, including B-cell chronic lymphocytic leukemia (CLL).5, 6 Tumor cells that overexpress BCL-2 are resistant to apoptosis induced by chemotherapy, radiation therapy or immune effector mechanisms.6 BCL-2 antisense PS-ODN potentially could lower Bcl-2 expression and thereby render tumor cells susceptible to apoptosis.7, 8, 9 One such BCL-2 antisense PS-ODN is Oblimersen sodium (G3139-Genasense®), which is an 18-mer complementary to the first six codons of the human BCL-2 mRNA.8 Clinical trials combining G3139-Genasense® with chemotherapy in different malignancies such as prostate cancer, small cell lung cancer and acute leukemia have reported encouraging results.10, 11, 12

However, the concentrations of antisense PS-ODN that can be achieved in vivo appear lower than that required to interfere with BCL-2 gene expression in vitro.13 Also, mechanical measures, such as cell electroporation or cationic lipid adjuvants, commonly are required to achieve effective cellular uptake of antisense ODN in vitro.1 In addition, it is possible that antisense PS-ODN could have direct effects on cells other than that of gene interference. G3139-Genasense®, for example, has CpG motifs that could stimulate immune function via a mechanism that is independent of interference with BCL-2 gene expression.14 As a result of these considerations, we compared the effects of G3139-Genasense® on primary CLL cells in vitro with other PS-ODN or PO-ODN, with or without CpG motifs or sequence homology for BCL-2.

Materials and methods

Chronic lymphocytic leukemia-samples and cell culture conditions

Peripheral blood mononuclear cells (PBMC) from patients with CLL were obtained from the CLL Research Consortium (CRC) tissue bank. The blood samples were collected after obtaining a written informed consent from the patients and PBMC were isolated by density gradient centrifugation over Histopaque 1077 as described.15 All patients met criteria for CLL.16 The samples that were used had more than 95% positive cells for CD19 and CD5, as assessed by flow cytometry. Chronic lymphocytic leukemia samples were incubated in RPMI media 10% fetal calf serum (FCS) at 37°C with 5% CO2. Normal B cells were purified from healthy volunteers using positive isolation with Dynabeads® CD19 (Dynal Biotech, Oslo, Norway) the purity was 95% assessed by flow cytometry analysis.

In some experiments, Staurosporine (Sigma-Aldrich Corp., St Louis, MO, USA), LY-294,002 (Sigma, St Louis, MO, USA), Gö6976 and Z-VAD-FMK (Calbiochem, San Diego, CA, USA), imatinib mesylate Gleevec® (Novartis, Basel, Switzerland), were added to the culture media. Titration of the optimal concentration from each reagent was performed.

Oligodeoxynucleotides

Oligodeoxynucleotides, were synthesized by Integrated DNA Technologies (Skokie, IL, USA), using phosphodiester (PO) and phosphorothioate (PS) backbones. PS-ODN were used at a concentration of 1 μg/ml and PO-ODN at 10 μg/ml (those concentration were selected after performing titrations of the optimal concentration of each ODN and also because they reflected serum concentrations obtained in vivo after intravenous administration of PS-ODN).17 The following ODN were used in our experiments:

  • R20 (an ODN without CpG motifs or homology with BCL-2)18 5′-IndexTermCCTGTCTGTTCAGACATGTC-3′

  • R1060 (an ODN with CpG motifs but without homology with BCL-2)19 5′-IndexTermCCAGTCGTACAGGAAACATGCGTTCTAGATGTTCGGGGC-3′

  • G3139-Genasense® (a BCL-2 anti-sense ODN with CpG motifs)8 5′-IndexTermTCTCCCAGCGTGCGCCAT-3′

  • Homopolymers with 20 nucleotide sequences (Poly A, T, C, without CpG motifs or homology with BCL-2).18

  • ODN with substitution of CpG for GpC dinucleotides. G3139 Δ-CpG and R1060 Δ-CpG.

  • G3139-PS (T/Cx). G3139-PS-ODN with substitution of thimidine nucleotides for cytidine. X represents the nucleotide number where the substitution was made.

Flow cytometry and apoptosis cytometry bead assay

Flurochrome-conjugated monoclonal antibodies specific for CD40 and Bcl-2 were purchased from Pharmingen, San Diego, California for flow cytometry using a FACScalibur (Becton-Dickinson, Franklin Lakes, NJ, USA), as described.15 Data were analyzed using Flow-Jo 3.6 software (Stanford University-Tree Star Inc., San Francisco, CA, USA).

To perform the apoptosis cytometry bead assay (CBA), we followed the manufacturer's instructions. Briefly, the samples were lysed and mixed with the capture beads (coated with antibodies against activated caspase 3, PARP-1 and Bcl-2). After incubation for 1 h at room temperature the PE detection reagent was added. Then the samples were washed and analyzed in a FACScalibur machine.

Immunoblot and immunoprecipitation

Chronic lymphocytic leukemia B cells were harvested at different time points as indicated in each experiment and protein lysates were prepared using RIPA buffer supplemented with protease inhibitors (10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin and 1 mM phenylmethylsulfonyl fluoride) and in some cases phosphatase inhibitors (1 mM Na-Vanadate and 10 mM β-Glycerophosphate). After SDS-PAGE immunoblots membranes were probed with different antibodies including the following: rabbit polyclonal antisera raised against peptides (Bcl-2)20 or recombinant protein produced in bacteria (caspase 3, 8),21 anticaspase 9 (clone 5B4, Medical & Biological Laboratories, Nagoya, Japan), anti-p53 antibody (clone DO-1, Oncogene, Cambridge, MA, USA), anti-β actin (clone AC-15, Sigma, St Louis, MI, USA), rabbit polyclonal anti-BID antibody (Cell Signaling, Beverly, MA, USA), rabbit polyclonal anti-PUMA (Oncogene, Cambridge, MA, USA), rabbit polyclonal anti-c-ABL (Santa Cruz, CA, USA), rabbit polyclonal anti-Bim (Imgenex, San Diego, CA, USA), mouse anti-PARP (Poly(ADP-ribose) Polymerase) (clone C2-10, PharMingen, La Jolla, CA, USA), anti-Noxa (clone 114C307, Alexis Biochemicals, Lausen, Switzerland), mouse antiphosphotyrosine (Clone 4G10-Upstate, Waltham, MA, USA). Immnuno-detection was accomplished by using horseradish peroxidase (HRP) conjugated secondary antibodies and the reaction was developed with Chemiluminescent Substrate kit-ECL Plus from Amersham-Biosciences (Piscataway, NJ, USA).

Lysates for immunoprecipitation were prepared as described above. Protein-G Sepharose beads (Zymed, San Francisco, CA, USA) were preblocked with 5% BSA. The cell lysates were precleared by incubating with 50 μl of protein-G Sepharose beads (50% slurry). c-Abl antibody (Santa Cruz, Santa Cruz, CA, USA) was added to 100 μg of the precleared cell lysate, and incubated by rotating overnight at 4°C. Precleared beads (20 μl, 50% slurry) were then added and incubated by rotating for 2 h at 4°C. Bound beads were centrifuged at 2000 g and then washed twice in lysis buffer. We added SDS-sample buffer and then incubated the samples for 5 min at 95°C. Bound samples were analyzed by SDS-PAGE and immunoblot using the indicated antibodies. Immuno-detection was developed as described above.

Detection of apoptosis

Apoptotic and viable cells were discriminated via flow cytometry of cells stained with 3,3′ dihexyloxacarbocyanine iodide (DiOC6) (Molecular Probes, Eugene, OR, USA) and propidium iodine (Sigma, St Louis, MO, USA), as described.22 Using this method viable cells exclude PI and stain brightly positive for DiOC6.

Generation and transduction of chronic lymphocytic leukemia-B cells using adenovirus – mutant c-ABL

We generated an adenovirus vector encoding c-ABL mutant resistant for STI571. We cloned the cDNA encoding mouse c-ABL gene with a point mutation (T315I) into the CMV promoter and polyadenylation signal of pcDNA3. This construct was then subcloned into the shuttle vector MCS(SK)pXCX2 as described before.23 This construct was designated c-ABL (T315I) pXCX2 (FLAG peptide sequence tag). c-ABL(T315I) pXCX2 was co-transfected with pJM17 into 293 cells using the calcium phosphate method. Isolated adenovirus plaques were expanded by reinfecting 293 cells. High titer adenovirus preparations were obtained, as described.23 The virus titer was determined by counting the number of formed plaques after infecting 293 cells with serial dilutions of the purified adenovirus. We tansduced CLL-B cells during 24 h using the adenovirus vectors with an MOI of 500. A control transduction was performed using an adenovirus-LacZ vector.

Results

PS-ODN induce apoptosis in chronic lymphocytic leukemia B cells but not normal B cells

We examined whether PS-ODN with (G3139-Genasense®) or without (R1060) BCL-2 antisense sequence homology, could induce apoptosis of B cells from healthy donors or CLL patients. We observed that either PS-ODN could induce significant killing of CLL B cells within 48–60 h with similar efficacy (Figure 1a). These effects were time and dose dependent. On the other hand, PS-ODN did not induce apoptosis of B cells from healthy donors.

Figure 1
figure1

Phosphorothioate oligonucleotides (PS-ODN) induce apoptosis in chronic lymphocytic leukemia (CLL) B cells but not normal B cells. (a) Primary leukemia B cells from CLL patients (n=8) and normal B cells (n=8) were incubated with G3139-Genasense®, R1060-PS ODN and media only as a control. The samples were harvested at different time points (24, 60 and 120 h). Cell viability was measured by flow cytometry using propidium iodine (PI) exclusion and mitochondrial membrane potential changes using DiOC6. The data shows the results of samples analyzed in duplicates, error bars represent the s.d. These results were reproduced several times. (b) Samples from CLL patients were incubated for 60 h with G3139-Genasense®, R1060-PS and permutated ODN with substitution of CpG nucleotides for GpC (ΔCpG – ODN). Apoptosis in CLL cells was assessed by flow cytometry using PI and DiOC6 staining (contour plots). Viable cells are represented in the right lower quadrant and stain brightly with DiOC6 and are negative for PI. In addition, cells were labeled with antiBcl-2 and CD40 antibodies (shaded histograms). Control isotype antibodies were used to assess not specific antibody binding (open histograms). Leukemia cells cultured in media only were used as control for spontaneous apoptosis and basal level of CD40 and Bcl-2 expression. These data correspond to a representative experiment of multiple ones with results that were reproducible.

PS-ODN induce phenotypic changes and apoptosis of chronic lymphocytic leukemia cells independent of sequence homology with BCL-2 or CpG motifs

We treated CLL B cells with different ODN: G3139-Genasense® (CpG antisense BCL-2 PS-ODN), R1060 (CpG PS-ODN without BCL-2 sequence homology), or control versions of G3139-Genasense® and R1060 lacking CpG nucleotides, G3139 Δ-CpG or R1060 Δ-CpG, respectively. Incubation with G3139-Genasense® or R1060-PS at concentrations of 1 μg/ml resulted in loss of leukemia-cell viability relative to that control cultures without ODN (Figure 1b). Apoptosis at 60 h was preceded by morphologic and phenotypic changes similar to those induced by agents that trigger lymphocyte activation.24 Activation changes were observed within 12 h of incubation and were characterized by increased forward- and side-angle light scatter by flow cytometry and enhanced expression of CD40, CD54, CD80, CD86 and major histocompatibility complex (MHC) class I and II antigens (Figure1b and data not shown). However, control PS-ODN lacking CpG motifs, namely G3139 Δ-CpG or R1060 Δ-CpG, also had these effects on CLL cells at the same concentrations, indicating that the effects on CLL cells were not secondary to BCL-2 sequence homology or CpG motifs. Additionally, we observed that Bcl-2 expression was lower in all treated samples undergoing apoptosis (Figure 1b).

Determination of oligodeoxynucleotides composition required for activation and apoptosis of chronic lymphocytic leukemia cells reveals a significant role of thymidine nucleotides

We compared G3139-Genasense® with other ODN lacking homology to BCL-2 for their activity on CLL B cells. PO-ODN were used at 10-fold higher concentrations to compensate for their short half-life due to increased sensitivity to nuclease degradation. These oligomers included: (a) R20, random ODN without CpG motifs or homology to BCL-218 (5′-IndexTermCCTGTCTGTTCAGACATGTC-3′); (b) R1060, ODN with CpG motifs but without homology to BCL-219 (5′-IndexTermCCAGTCGTACAGGAAACATGCGTTCTAGATGTTCGGGGC-3′); (c) G3139-Genasense®, BCL-2 antisense ODN with CpG motifs8 (5′-IndexTermTCTCCCAGCGTGCGCCAT-3′) and (d) Homopolymers with 20 nucleotide sequences (Poly-A, T, C) without CpG motifs or BCL-2 homology.18 Poly-G ODN was not used due to difficulties in its synthesis resulting from the high rate of aggregate formation.

We cultured CLL samples (n=15) with each ODN for 60 h and examined for apoptosis and expression of CD40 in the viable cell population. This viable population was gated based on parameters of forward- and side-angle light scatter and PI exclusion by flow cytometry22 (Figure 2a). Treatment with G3139-Genasense® and other PS-ODN (R20, R1060, Poly-T) induced CLL cells from each patient to express significantly higher levels of CD40. On the other hand, poly-A or poly-C PS-ODN did not induce CLL cells to undergo such changes.

Figure 2
figure2

ODN induced cellular activation and apoptosis in chronic lymphocytic leukemia (CLL)-B cells is determined primarily by the presence of thymidine nucleotides and phosphorothioate backbone in the primary sequence. Samples from CLL patients (n=15) were incubated during 60 h with PO and PS ODN. (a) Column scatter histogram shows the level of regulation of CD40 measured by flow cytometry on samples treated with different ODN. (b) Percentage of viable cells determined by staining of PI and DiOC6. Mean values are shown for each group of treated samples. This figure shows a representative experiment that has been reproduced several times. (c) CLL cells were treated for 48 h with G3139-PS and modified versions of this phosphorothioate oligonucleotides (PS-ODN) in which each of the thymidine nucleotides were substituted by cytidines residues (T/C1, T/C2, T/C3, T/C4); we also tested one PS-ODN in which all four thymidines were substituted (T/C1–4). Flow cytometry was used to assess apoptosis using DiOC6 and PI. Error bars represent the standard deviation of duplicate experiments. (*P<0.05). P-values were calculated using one-way analysis of variance (ANOVA) with Bonferroni post-test analysis.

The majority of CLL samples (60%; n=15) treated with G3139-Genasense® underwent apoptosis after 60 h of treatment (Figure 2b). Similarly, all thymidine PS-ODN tested induced apoptosis with a similar efficiency as G3139-Genasense®. Additional experiments showed that apoptosis induced by thymidine-PS-ODN, did not depend on the concentration of T cells in the cell preparation, and also that Fas (CD95) was not induced in CLL B cells following exposure to thymidine-PS-ODN (data not shown). To further characterize the role of thymidine nucleotides in PS-ODN induced apoptosis, we treated CLL cells with variants of G3139-PS in which the thymidine residues were substituted for cytidines (Figure 2c). Using this modified PS-ODN, we observed that substitutions of all four thymidine residues but not single substitutions in G3139-PS significantly decrease the induction of apoptosis on CLL cells compared with the unmodified G3139-PS ODN.

R1060-PO was the only PO-ODN that induced activation of CLL cells. However, the relative increase in CD40 observed on R1060-PO treated cells was significantly less (P<0.001 Bonferroni's multiple comparison test) than that observed on CLL cells treated with thymidine PS-ODN, even when R1060-PO was used at higher concentrations. G3139-PO, a PO-ODN containing CpG motifs with a BCL-2 antisense sequence identical to G3139-Genasense®, did not have such activity.

Apoptosis of chronic lymphocytic leukemia cells induced by thymidine-containing PS-ODN was associated with activation of caspases and reduction in Bcl-2 expression and was inhibited by imatinib mesylate (Gleevec®)

We examined whether treatment of CLL cells with thymidine PS-ODN induced caspase activation. Chronic lymphocytic leukemia cells treated with thymidine PS-ODN underwent activation of caspase 3 and 9, generating their cleaved forms p17 and p30-35, respectively. We did not observe processing of caspase 8 (Figure 3a). The pan-caspase inhibitor Z-VAD-FMK inhibited thymidine PS-ODN to induce apoptosis of CLL cells, confirming that caspase activation is induced by such PS-ODN (Figure 4a).

Figure 3
figure3

Thymidine-phosphorothioate oligonucleotides (PS-ODN) induced apoptosis in chronic lymphocytic leukemia (CLL)-B cells requires activation of caspases from the mitochondrial- ‘intrinsic’ apoptosis pathway and is associated with Bcl-2 downregulation. (a) CLL cells were incubated at different time points (24, 60 and 120 h) with R1060-PS and G3139-PS ODN. Protein lysates from those cultures were analyzed by immunoblot using specific antibodies that recognize caspase 9, 8, and 3 (proform and cleaved forms). Anti-β-actin antibody was used to assess equal loading of the gels. This experiment was reproduced several times with different CLL samples. (b) Quantitative evaluation of Bcl-2, active caspase 3 and PARP-1 cleavage was performed on protein lysates using the apoptosis cytometric bead assay (CBA) from Beckton Dickinson-La Jolla, CA. The samples were incubated for 24 h (open bars) and 60 h (black bars) with each of the indicated PS-ODN and media only as a control. The samples were analyzed by flow cytometry. Error bars represent the s.d. of duplicate experiments. (*P<0.05).

Figure 4
figure4

Thymidine-phosphorothioate oligonucleotides (PS-ODN) induced apoptosis in chronic lymphocytic leukemia (CLL)-B cells can be inhibited by the pan-caspase inhibitor Z-VAD-FMK and imatinib mesylate (Gleevec®) but no other kinase inhibitors. (a) Leukemia samples from CLL patients were incubated during 60 h with a thymidine PS-ODN (R1060) or kept in media as control. Staurosporine, LY-294,002, Gö6976 and imatinib mesylate were added to the PS-ODN treated samples simultaneously with each PS-ODN. Cell viability was assessed by DiOC6/PI staining and then normalized with respect to the highest value obtained at each experiment. Kinase inhibitors were titrated and used at concentrations previously reported or those that induced the lowest degree of apoptosis in CLL B cells. (b) CLL cells were incubated with R1060-PS for 60 h. To these samples, Imatinib mesylate (Gleevec®) was added to at different time points as indicated. Viability was measured at 60 h by DiOC6/PI staining. Base line viability of CLL cells incubated either in media only or treated with R1060-PS is shown in the left hand side of the figure (open and black bars). These are representative experiments that have been reproduced more than three times.

Using a sensitive and quantitative cytometric bead assay (CBA),25 we found that Bcl-2 levels were significantly reduced in the samples treated with thymidine PS-ODN, but not in control samples treated with Poly-C PS-ODN or media alone. We also found that thymidine PS-ODN induce active caspase 3, cleaved poly ADP-ribose polymerase (PARP-1) and their activity did not depend on BCL-2 antisense sequence homology (Figure 3b).

To examine whether kinase activity was required for initiation of apoptosis in CLL cells treated with thymidine PS-ODN, we treated CLL cells with PS-ODN along with each of several different kinase inhibitors, including Staurosporine (inhibitor of phospholipid/calcium-dependent protein kinase),26 LY-294,002 (phosphatidylinositol 3-kinase inhibitor),27 Gö6976 (protein kinase C inhibitor),28 or Imatinib mesylate (Gleevec®) (inhibitor of c-ABL, Bcr-ABL, PDGF, c-Kit).29 Imatinib mesylate, but not the other kinase inhibitors blocked the capacity of thymidine PS-ODN to induce apoptosis of CLL cells (Figure 4a). Inhibition of apoptosis by imatinib mesylate was optimal at concentrations achieved in patients treated with this drug (1-3 μ M).30 Imatinib mesylate was effective at inhibiting apoptosis of CLL cells only when added to the cultures within the first 5 h of culture with PS-ODN (Figure 4b).

Apoptosis of chronic lymphocytic leukemia cells induced by thymidine PS-ODN is associated with upregulation of p53, Bid and phosphorylation of c-ABL

We examined CLL cells for expression of p53 protein before and after treatment with thymidine PS-ODN. Chronic lymphocytic leukemia cells cultured with either G3139-Genasense®, R1060-PS or Poly T-PS, expressed increased levels of P53 mRNA and p53 protein relative to controls; this process was inhibited by imatinib mesylate (Figure 5a and data not shown).

Figure 5
figure5

Apoptosis in chronic lymphocytic leukemia (CLL)-B cells induced by thymidine PS-ODN is associated with upregulation of p53, Bid and phosphorylation of c-ABL. (a) Protein lysates from primary leukemia cells from a patient sensitive (CLL-1) and resistant (CLL-2) to thymidine containing PS-ODN induced apoptosis, were analyzed by immunoblot using different antibodies as shown. The samples were harvested after 60 h in culture. Imatinib mesylate was added to the cultures when indicated. Cells incubated in media only were used as a control. (b) CLL-B cells were infected with adenovirus encoding a mutated c-ABL (T315I) protein that is resistant to the inhibitory activity of imatinib mesylate (Gleevec®). Transgene expression was evaluated by using anti-FLAG and anti c-ABL antibodies. CLL samples were incubated with adenovirus encoding LacZ As a transduction control. Both adenovirus were used at MOI=500. Expression of Bid and c-ABL was evaluated with specific antibodies; phosphorilated c-ABL was detected using the 4G10 antibody. These immunoblots are representative of several experiments performed using different sensitive and resistant CLL B cells to PS-ODN induced apoptosis.

As a result of these findings, we evaluated whether treatment of CLL cells with such PS-ODN altered the expression of p53 regulated proapoptotic molecules such as the BH-3 only proapoptotic proteins (Bid, Bim, Puma, Noxa), and the multidomain Bcl-2 member Bax. Chronic lymphocytic leukemia cells that underwent apoptosis expressed high levels of Bid and Bax in response to the thymidine PS-ODN. The upregulation of these proapoptotic molecules was observed early (12 h) and lasted for more than 60 h (Figure 5a and data not shown). We did not observe significant changes in the expression of other Bcl-2 family member proteins in response to ODN. Addition of imatinib mesylate to these cultures inhibited expression of Bid and Bax (Figure 5a). We did not observe upregulation of p53, Bid or Bax in CLL cases that were resistant to the proapoptotic activity of thymidine PS-ODN (Figure 5a).

Using immunoprecipitation, we found that c-ABL protein was phosphorylated in CLL samples incubated with thymidine PS-ODN but not in controls. Phosphorylation of c-ABL was observed after 12 h of incubation and was present after 60 h of treatment in vitro. In addition, phosphorylation of this kinase was inhibited by imatinib mesylate (Figure 5a). We did not detect phosphorylated c-ABL in the lysates of CLL cells that did not undergo apoptosis in response to treatment with the thymidine PS-ODN. To confirm that c-ABL and no other kinases inhibited by imatinib mesylate was involved in this process, we transduced CLL-B cells with an adenovirus expressing a mutated c-ABL that was resistant to imatinib mesylate (Adeno c-ABL(T315I)). We found that CLL transduced to express c-ABL(T315I), but not CLL cells infected with a control adenovirus (Adeno-LacZ), were induced to express Bid and to undergo phosphorylation of c-ABL by thymidine PS-ODN even in the presence of inhibitory concentrations of imatinib mesylate (Figure 5b).

Discussion

Our results show that thymidine PS-ODN can induce apoptosis of CLL B cells as effectively as the BCL-2 antisense PS-ODN, G3139-Genasense®. This proapoptotic effect was not observed in B cells of healthy donors (Figure 1a). R1060, a PS-ODN without homology to BCL-2, induced cellular activation and apoptosis of CLL cells as efficiently as G3139-Genasense® even when the CpG motifs were permuted to GpC (Figure 1a and b). The common features of ODN capable of inducing activation and apoptosis of CLL B cells were the presence of a PS chemical backbone and a nucleotide composition in which 23% or more were thymidine residues (Figure 2). Furthermore, we have shown that substitution of thymidine nucleotides for cytidines abrogates the process of apoptosis induced by G3139-Genasense®. We conclude that the presence of a PS backbone and thymidine nucleotides are both necessary and sufficient for the noted activity of PS-ODN on CLL B cells.

The results of our study contrast with those of previous reports in which the activity of antisense BCL-2 ODN such as G3139-Genasense® was related to specific binding and degradation of BCL-2 mRNA in various types of malignant cells.9 One potential explanation is that our study was performed with primary leukemia cells from patients and not with cells lines that overexpressed Bcl-2.8, 9 However, other studies have also suggested the lack of specificity of antisense ODN including G3139-Genasense®. For example, Pepper et al.31 reported proapoptotic activity of 2009, a BCL-2 antisense PS-ODN, on CLL B cells. They noted that even the scrambled control PS-ODN induced apoptosis of CLL cells in 14 out of 18 patients examined.31 Although the authors acknowledged this ‘cytotoxic effect’ of the control PS-ODN, the killing achieved with the BCL-2 antisense PS-ODN was assumed due to BCL-2 gene interference. Another recent study in prostate cancer cells, also raises questions about the specificity of G3139-Genasense® and suggest that the activity of this compound is BCL-2 independent, as they found that this ODN was still active inducing apoptosis in cells that had complete downregulation of BCL-2 mediated by siRNA prior to treatment with G3139-Genasense®.32

We demonstrate here that thymidine PS-ODN, independently of CpG motifs, induced morphological and phenotypic changes in CLL cells suggestive of cellular activation (Figures 1b and 2a). Other studies have shown that CpG motifs are responsible for the immune stimulatory activity of PS-ODN on normal or leukemia B cells.14, 33 However, we and others have previously reported the presence of CpG-independent activity of PS-ODN.18, 34 Contrary to the report of Vollmer et al.,34 we did not find differences in the activity of CpG compared with non-CpG PS-ODN (Figures 1 and 2a).

The effects of the thymidine PS-ODN do not appear secondary to contamination of the PS-ODN preparations with endotoxin or other factors related to their synthesis and/or handling. All PS-ODN and PO-ODN were handled in similar manner and these compounds lacked significant levels of endotoxin (data not shown).

Immunoblot analysis of CLL cells treated with thymidine-PS-ODN, including G3139-Genasense®, revealed activation of caspase 9 and 3, but not caspase 8. The processing of these caspases was similar in CLL cells treated with G3139-Genasense® or other thymidine PS-ODN, such as R1060-PS (Figure 3a). Since caspase 9 is the apical protease in the mitochondrial pathway for apoptosis,35 we conclude that thymidine PS-ODN induce apoptosis of CLL cells via this ‘intrinsic’ pathway, as opposed to the TNF/Fas death receptor ‘extrinsic’ pathway where activation of caspase 8 plays a critical role.

Quantitative analyses using the CBA assay revealed that the samples treated with thymidine PS-ODN, but not those treated with control poly-C PS-ODN, were induced to activate caspase 3, cleave PARP-1 and to express low levels of Bcl-2. These effects were time dependent and were observed with other techniques, such as immunoblot or flow cytometry (Figures 1b, 3a and 5a and data not shown). Collectively, these data demonstrate that thymidine PS-ODN with no antisense sequence homology to BCL-2 or CpG motiffs can downregulate Bcl-2 protein levels as effectively as G3139-Genasense®, suggesting that mechanisms other than antisense modulation of BCL-2 mRNA are responsible for the noted proapoptotic effect of these molecules on CLL cells. In this regard, treatment of CLL cells with thymidine PS-ODN may reduce the level of Bcl-2 expression nonspecifically in a similar way to other agents such as chemotherapy, nonsteroidal antiinflammatory drugs, gamma or ultraviolet irradiation.36, 37

Using immunoblot analysis and real-time PCR, we found that thymidine PS-ODN treated CLL cells express high levels of P53 within 24 h after treatment (Figure5a and data not shown). Induced-expression of p53 typically was associated with the response to agents that induce DNA damage and/or genotoxic stress.38 Recent studies have found that other nongenotoxic stimuli, such hypoxemia, cytokines (TNF-α), hyperthermia or treatment with proteasome inhibitors, can regulate the expression of p53.39, 40 The data presented here show that thymidine-PS-ODN can induce CLL cells to express p53 independent of whether the PS-ODN has BCL-2 antisense sequence homology or not. PS-ODN mediated p53 upregulation, could account for the noted synergistic activity of G3139-Genasense® with other anticancer treatments, including radiation or chemotherapy.9

We tested various protein kinase inhibitors and a caspase inhibitor to elucidate the pathways involved in the induction of apoptosis by thymidine-PS-ODN. We found that the pan-specific caspase inhibitor z-VAD-FMK and the tyrosine kinase inhibitor imatinib mesylate could block apoptosis induced by thymidine PS-ODN (Figure 4a). These observations confirm that apoptosis of CLL induced by thymidine PS-ODN is caspase-dependent and suggest that c-ABL tyrosine kinase is involved in this process.

Imatinib mesylate inhibited PS-ODN induced apoptosis of CLL cells only when added during the first 5 h of culture. This suggests that early signal transduction events regulated by c-ABL kinase are required for the activity of PS-ODN in CLL cells (Figure 4b). Furthermore, we observed that thymidine PS-ODN induced auto phosphorylation of c-ABL and that this could be inhibited by imatinib mesylate. CLL cells transduced with a vector encoding a mutant c-ABL protein resistant to imatinib mesylate were able to induce upregulation of Bid and undergo apoptosis after treatment with thymidine PS-ODN despite of coincubation with imatinib mesylate (Figure 5b). This confirms that c-ABL, and not other kinases inhibited by imatinib mesylate, is involved in the proapoptotic activity of thymidine PS-ODN in CLL.

The c-ABL tyrosine kinase is activated by genotoxins or TNF-α and contributes to apoptosis responses.41, 42 ABL can activate the proapoptotic function of p53 and p73,42, 43 and activation of nuclear ABL kinase can induce apoptosis in p53-null cells, but not in p73-null cells.44 Both ABL and p73 contribute to p53-independent apoptosis in response to cisplatin and TNF-induced apoptosis.41, 42, 43, 44, 45 Whether p73 is involved in PS-ODN-induced activation and apoptosis of CLL cells waits further investigation. Inhibition of ABL by imatinib mesylate interferes with the proapoptotic activity of thymidine PS-ODN as we have demonstrated here. Additionally, imatinib mesylate also interferes with apoptosis induced by chemotherapy in several human tumor cell lines.46 Therefore, careful consideration of this possible interaction should be taken for clinical protocols that include the combination of imatinib mesylate with other agents.

We observed that thymidine PS-ODN could induce, in sensitive CLL cells, expression of the proapoptotic proteins Bid and Bax. We did not observe changes in the levels of expression of other BH3-only proteins such as Bim, Puma or Noxa (Figure 5a). Bid interacts with Bcl-2, inhibiting its formation, while also binding to and activating the proapoptotic proteins Bax and Bak.47, 48 Interestingly, p53 could interact synergistically with Bid as it has been shown that p53 possess both transcriptional and nontranscriptional proapoptotic activity, including the ability to translocate into the cytosol and activate Bax and Bak directly.49, 50 Thus, several parallel mechanisms for overcoming Bcl-2 may be induced by thymidine PS-ODN.

In conclusion, we have found new active requirements and activities of PS-ODN on primary leukemia cells from patients with CLL. ODN-containing thymidine nucleotides with a PS backbone-induced cellular activation in CLL-B cells followed by apoptosis. The activation-induced changes preceded cell death and were not dependent upon the presence of CpG motifs. Downregulation of Bcl-2 protein levels occurred after incubation with thymidine PS-ODN regardless of whether the oligonucleotides had sequence homology with BCL-2. Moreover, apoptosis of CLL cells induced by thymidine PS-ODN was associated with upregulation of several proapoptotic molecules such as p53, Bid and Bax, involved activation of cellular caspases, and was dependent upon activation and phosphorylation of c-ABL. These findings have important implications for understanding the activities of PS-ODN and may assist in design of active PS-ODN for the treatment of leukemia and possibly other types of cancer.

References

  1. 1

    Wagner RW . Gene inhibition using antisense oligodeoxynucleotides. Nature 1994; 372: 333–335.

  2. 2

    Donis-Keller H . Site specific enzymatic cleavage of RNA. Nucleic Acids Res 1979; 7: 179–192.

  3. 3

    Crooke ST . Progress in antisense technology. Annu Rev Med 2004; 55: 61–95.

  4. 4

    Stephens AC, Rivers RP . Antisense oligonucleotide therapy in cancer. Curr Opin Mol Ther 2003; 5: 118–122.

  5. 5

    Hanada M, Delia D, Aiello A, Stadtmauer E, Reed JC . bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia. Blood 1993; 82: 1820–1828.

  6. 6

    Reed JC . Dysregulation of apoptosis in cancer. J Clin Oncol 1999; 17: 2941–2953.

  7. 7

    Reed JC, Stein C, Subasinghe C, Haldar S, Croce CM, Yum S et al. Antisense-mediated inhibition of BCL2 protooncogene expression and leukemic cell growth and survival: comparisons of phosphodiester and phosphorothioate oligodeoxynucleotides. Cancer Res 1990; 50: 6565–6570.

  8. 8

    Cotter FE, Johnson P, Hall P, Pocock C, al Mahdi N, Cowell JK et al. Antisense oligonucleotides suppress B-cell lymphoma growth in a SCID-hu mouse model. Oncogene 1994; 9: 3049–3055.

  9. 9

    Klasa RJ, Gillum AM, Klem RE, Frankel SR . Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Dev 2002; 12: 193–213.

  10. 10

    Rudin CM, Kozloff M, Hoffman PC, Edelman MJ, Karnauskas R, Tomek R et al. Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell lung cancer. J Clin Oncol 2004; 22: 1110–1117.

  11. 11

    Marcucci G, Stock W, Dai G, Klisovic RB, Liu S, Klisovic MI et al. Phase I study of oblimersen sodium, an antisense to Bcl-2, in untreated older patients with acute myeloid leukemia: pharmacokinetics, pharmacodynamics, and clinical activity. J Clin Oncol 2005; 23: 3404–3411.

  12. 12

    Tolcher AW, Chi K, Kuhn J, Gleave M, Patnaik A, Takimoto C et al. A phase II, pharmacokinetic, and biological correlative study of oblimersen sodium and docetaxel in patients with hormone-refractory prostate cancer. Clin Cancer Res 2005; 11: 3854–3861.

  13. 13

    Reed JC . Promise and problems of Bcl-2 antisense therapy. J Natl Cancer Inst 1997; 89: 988–990.

  14. 14

    Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995; 374: 546–549.

  15. 15

    Kato K, Cantwell MJ, Sharma S, Kipps TJ . Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells. J Clin Invest 1998; 101: 1133–1141.

  16. 16

    Matutes E, Owusu-Ankomah K, Morilla R, Garcia Marco J, Houlihan A, Que TH et al. The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia 1994; 8: 1640–1645.

  17. 17

    Raynaud FI, Orr RM, Goddard PM, Lacey HA, Lancashire H, Judson IR et al. Pharmacokinetics of G3139, a phosphorothioate oligodeoxynucleotide antisense to bcl-2, after intravenous administration or continuous subcutaneous infusion to mice. J Pharmacol Exp Ther 1997; 281: 420–427.

  18. 18

    Castro JE, Motta M, Wu C, Carson DA, Kipps TJ . Phosphorothioate oligodeoxynucleotides activate chronic lymphocytic leukemia B cells through a CpG independent mechanism. Blood 2002; 100: 1463a.

  19. 19

    Wu CC, Castro JE, Motta M, Cottam HB, Kyburz D, Kipps TJ et al. Selection of oligonucleotide aptamers with enhanced uptake and activation of human leukemia B cells. Hum Gene Ther 2003; 14: 849–860.

  20. 20

    Krajewski S, Bodrug S, Gascoyne R, Berean K, Krajewska M, Reed JC . Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes. Am J Pathol 1994; 145: 515–525.

  21. 21

    Svingen PA, Karp JE, Krajewski S, Mesner Jr PW, Gore SD, Burke PJ et al. Evaluation of Apaf-1, and procaspases-2, -3, -7, -8, and -9 as potential prognostic markers in acute leukemia. Blood 2000; 96: 3922–3931.

  22. 22

    Chu P, Deforce D, Pedersen IM, Kim Y, Kitada S, Reed JC et al. Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002; 99: 3854–3859.

  23. 23

    Cantwell MJ, Sharma S, Friedmann T, Kipps TJ . Adenovirus vector infection of chronic lymphocytic leukemia B cells. Blood 1996; 88: 4676–4683.

  24. 24

    Baumgarth N . A two-phase model of B-cell activation. Immunol Rev 2000; 176: 171–180.

  25. 25

    Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J et al. Cytometric bead array: a multiplexed assay platform with applications in various areas of biology. Clin Immunol 2004; 110: 252–266.

  26. 26

    Radloff M, Gercken G . Protein kinase C activity and phosphoprotein pattern in stimulated alveolar macrophages. Toxicol Lett 1996; 88: 139–145.

  27. 27

    Vlahos CJ, Matter WF, Hui KY, Brown RF . A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994; 269: 5241–5248.

  28. 28

    Qatsha KA, Rudolph C, Marme D, Schachtele C, May WS . Go 6976, a selective inhibitor of protein kinase C, is a potent antagonist of human immunodeficiency virus 1 induction from latent/low-level-producing reservoir cells in vitro. Proc Natl Acad Sci USA 1993; 90: 4674–4678.

  29. 29

    Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Druker BJ et al. Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res 1996; 56: 100–104.

  30. 30

    Bakhtiar R, Lohne J, Ramos L, Khemani L, Hayes M, Tse F . High-throughput quantification of the anti-leukemia drug STI571 (Gleevec) and its main metabolite (CGP 74588) in human plasma using liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 768: 325–340.

  31. 31

    Pepper C, Thomas A, Hoy T, Cotter F, Bentley P . Antisense-mediated suppression of Bcl-2 highlights its pivotal role in failed apoptosis in B-cell chronic lymphocytic leukaemia. Br J Haematol 1999; 107: 611–615.

  32. 32

    Raffo A, Lai JC, Stein CA, Miller P, Scaringe S, Khvorova A et al. Antisense RNA down-regulation of bcl-2 expression in DU145 prostate cancer cells does not diminish the cytostatic effects of G3139 (Oblimersen). Clin Cancer Res 2004; 10: 3195–3206.

  33. 33

    Krieg AM . From A to Z on CpG. Trends Immunol 2002; 23: 64–65.

  34. 34

    Vollmer J, Janosch A, Laucht M, Ballas ZK, Schetter C, Krieg AM . Highly immunostimulatory CpG-free oligodeoxynucleotides for activation of human leukocytes. Antisense Nucleic Acid Drug Dev 2002; 12: 165–175.

  35. 35

    Reed JC . Apoptosis-targeted therapies for cancer. Cancer Cell 2003; 3: 17–22.

  36. 36

    Gibson LF, Fortney J, Magro G, Ericson SG, Lynch JP, Landreth KS . Regulation of BAX and BCL-2 expression in breast cancer cells by chemotherapy. Breast Cancer Res Treat 1999; 55: 107–117.

  37. 37

    Husain SS, Szabo IL, Tamawski AS . NSAID inhibition of GI cancer growth: clinical implications and molecular mechanisms of action. Am J Gastroenterol 2002; 97: 542–553.

  38. 38

    Wang JY . DNA damage and apoptosis. Cell Death Differ 2001; 8: 1047–1048.

  39. 39

    Lain S, Lane D . Improving cancer therapy by non-genotoxic activation of p53. Eur J Cancer 2003; 39: 1053–1060.

  40. 40

    Pluquet O, Hainaut P . Genotoxic and non-genotoxic pathways of p53 induction. Cancer Lett 2001; 174: 1–15.

  41. 41

    Chau BN, Chen TT, Wan YY, DeGregori J, Wang JY . Tumor necrosis factor alpha-induced apoptosis requires p73 and c-ABL activation downstream of RB degradation. Mol Cell Biol 2004; 24: 4438–4447.

  42. 42

    Wang JY . Regulation of cell death by the Abl tyrosine kinase. Oncogene 2000; 19: 5643–5650.

  43. 43

    Goldberg Z, Vogt Sionov R, Berger M, Zwang Y, Perets R, Van Etten RA et al. Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation. EMBO J 2002; 21: 3715–3727.

  44. 44

    Vella V, Zhu J, Frasca F, Li CY, Vigneri P, Vigneri R et al. Exclusion of c-Abl from the nucleus restrains the p73 tumor suppression function. J Biol Chem 2003; 278: 25151–25157.

  45. 45

    Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin Jr WG, Levrero M et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 1999; 399: 806–809.

  46. 46

    Truong T, Sun G, Doorly M, Wang JY, Schwartz MA . Modulation of DNA damage-induced apoptosis by cell adhesion is independently mediated by p53 and c-Abl. Proc Natl Acad Sci USA 2003; 100: 10281–10286.

  47. 47

    Yi X, Yin XM, Dong Z . Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed. J Biol Chem 2003; 278: 16992–16999.

  48. 48

    Wang K, Yin XM, Chao DT, Milliman CL, Korsmeyer SJ . BID: a novel BH3 domain-only death agonist. Genes Dev 1996; 10: 2859–2869.

  49. 49

    Leu JI, Dumont P, Hafey M, Murphy ME, George DL . Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol 2004; 6: 443–450.

  50. 50

    Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M et al. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 2004; 303: 1010–1014.

Download references

Acknowledgements

This work was supported by a National Institutes of Health K08 Grant CA106 605-01 (JEC) and PO1 Grant CA 81534 for the CLL Research Consortium (TJK). The authors acknowledge the helpful technical support provided by Dr Anissa Agadir from Pharmingen, La Jolla, CA and Dr Laura Rassenti from the Chronic lymphocytic leukemia Research Consortium (C.R.C)

Author information

Correspondence to T J Kipps.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Castro, J., Prada, C., Aguillon, R. et al. Thymidine-phosphorothioate oligonucleotides induce activation and apoptosis of CLL cells independently of CpG motifs or BCL-2 gene interference. Leukemia 20, 680–688 (2006) doi:10.1038/sj.leu.2404144

Download citation

Keywords

  • apoptosis
  • leukemia
  • oligonucleotides
  • CpG motifs
  • Bcl-2

Further reading