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.
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, 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.
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.
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.
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).
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).
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).
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.
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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)
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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
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