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(Journal of Leukocyte Biology. 2002;72:83-92.)
© 2002 by Society for Leukocyte Biology

Modulation of malignant B cell activation and apoptosis by bcl-2 antisense ODN and immunostimulatory CpG ODN

B. Jahrsdörfer*, R. Jox*, L. Mühlenhoff*, K. Tschoep*, A. Krug*, S. Rothenfusser*, G. Meinhardt{dagger}, B. Emmerich{dagger}, S. Endres* and G. Hartmann*

* Division of Clinical Pharmacology and
{dagger} Division of Hematology and Oncology, Department of Internal Medicine, University of Munich, Germany

Correspondence: Gunther Hartmann, M.D., Department of Internal Medicine, University of Munich, Ziemssenstr. 1, 80336 Munich, Germany. E-mail: ghartmann{at}lrz.uni-muenchen.de


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ABSTRACT
 
Inhibition of bcl-2 expression by antisense oligodeoxynucleotides (ODN) might render bcl-2 overexpressing malignant B cells more susceptible to chemotherapy. ODN containing unmethylated CG dinucleotides (CpG) are known to activate B cells. We studied the effects of two bcl-2 antisense ODN, with (G3139) or without CG dinucleotides (NOV 2009) within the sequence, and the effects of a nonantisense, CpG-containing ODN (ODN 2006) on activation and apoptosis of malignant B cell lines and primary B-CLL cells. Without cationic lipids, no antisense-mediated inhibition of bcl-2 synthesis was achieved with G3139 and NOV 2009. Instead, G3139, but not NOV 2009, induced similar changes as ODN 2006 in proliferation, expression of costimulatory and antigen-presenting molecules, as well as in bcl-2 and bcl-xL levels of primary B-CLL cells. G3139 and ODN 2006 inhibited in vitro, spontaneous apoptosis in B-CLL cells of patients with high serum thymidine kinase activity (s-TK, marker for proliferative activity of malignant B cells), whereas in patients with low s-TK activity, apoptosis was induced. In conclusion, our results suggest that modulation of malignant B cell apoptosis by G3139 depends on its immunostimulatory properties rather than on antisense-mediated reduction of bcl-2 expression. Immunostimulatory CpG ODN may have a therapeutic potential in patients with B-CLL, especially those with low s-TK activity.

Key Words: oligodeoxynucleotide • lymphoma • cationic lipids • high-serum thymidine kinase


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INTRODUCTION
 
bcl-2 homologues represent a major apoptosis-regulatory gene family [1 ]. The apoptosis suppressor bcl-2 is overexpressed in more than half of all human cancers [2 ]. bcl-2 was discovered as a proto-oncogene at the breakpoints of chromosomal translocations t(14;18) in low-grade B cell non-Hodgkin’s lymphomas. High levels of bcl-2 have been found in most follicular lymphomas, in chronic lymphocytic leukemia (B-CLL), and in some cases of diffuse large-cell lymphomas. The oncogenic impetus of bcl-2 has been verified in bcl-2 transgenic mice, which show polyclonal expansion of B cells with prolonged survival [3 , 4 ]. The bcl-2 family has been implicated not only in the pathogenesis of cancer, but also in resistance to therapy [2 , 5 6 7 ]. Anticancer drugs and radiation ultimately kill cancer cells by inducing the apoptotic cell-suicide pathway. There is evidence that bcl-2 represents a multidrug resistance protein, which prevents apoptosis induced by radiation and chemotherapy.

It has been shown that the level of thymidine kinase activity in the serum (s-TK) of patients with B-CLL correlates with the proliferative activity of B-CLL cells [8 ]. s-TK is a cellular enzyme involved in a "salvage pathway" for DNA synthesis. Elevated systemic levels of s-TK predict a high risk of disease progression in patients with low-grade non-Hodgkin’s lymphoma and B-CLL [9 10 11 12 ].

Antisense oligodeoxynucleotides (ODN) are short, synthetic, single-stranded DNA molecules that can interfere theoretically with gene expression via heteroduplex formation with complementary sequences within the target messenger RNA [13 14 15 16 ]. Several antisense sequences against bcl-2 have been designed [17 18 19 20 ]. One of them (G3139) is being investigated in clinical trials as single therapy or in combination with chemotherapy for the treatment of chronic lymphocytic leukemia (B-CLL; phases II and III) [21 , 22 ] and for the treatment of solid tumors such as malignant melanoma (phase III), and prostate, colon, and small-cell lung carcinoma (phase II). However, antisense ODN may display other than antisense effects depending on the chemically modified backbone used to protect the ODN from rapid degradation and on particular sequence motifs [15 , 23 , 24 ]. This problem could be considerably reduced by the development of 2'-O-methoxyethyl-modified ODN [19 , 25 , 26 ] and other modifications [13 ]. Although new backbone modifications show higher and more specific antisense effects in vitro, most antisense ODN in clinical trials, including the bcl-2 antisense ODN, still use the "first generation" phosphorothioate backbone.

The phosphorothioate backbone of ODN costimulates human monocytes independently of the ODN sequence [27 ]. Furthermore, particular immunostimulatory sequence motifs have been identified, which contain a central, unmethylated CG dinucleotide within certain base contexts (CpG motifs). ODN, which contain such CpG motifs (CpG ODN), mimic bacterial DNA [28 29 30 31 32 33 ]. In vertebrate DNA, such CpG motifs are underrepresented and methylated [34 ]. This enabled the vertebrate-immune system to detect CpG motifs as a molecular pattern associated with bacterial DNA [35 ]. CpG ODN stimulate B cells and dendritic cells and provide excellent immune adjuvants for priming antigen-specific B and T cell responses [24 , 32 , 36 37 38 ].

CpG ODN not only stimulate normal B cells but also malignant B cells [39 40 41 ]. Although up-regulation of surface markers, such as costimulatory and antigen-presenting molecules, supports the immune system to recognize and eliminate malignant cells, concurrent induction of proliferation of malignant B cells may have the opposite effect. It has been shown that CpG ODN inhibit apoptosis of murine lymphoma cell lines [42 43 44 ], but the influence on apoptosis of primary human malignant B cells is not known.

The bcl-2 antisense ODN being tested in clinical trials contain two CG dinucleotides and a TC dinucleotide at the 5' end, both of which have been shown to promote immunostimulatory activity of a CpG ODN [33 ]. We speculated that this particular ODN interacts with malignant B cells, such as an immunostimulatory CpG ODN, rather than an antisense ODN. In the present study, we examined the effects of this ODN (G3139; antisense to bcl-2, two CG dinucleotides) on apoptosis and activation of human malignant B cells. The contribution of an antisense- versus a CpG-mediated mechanism was determined by including a well-established bcl-2 antisense ODN without CG dinucleotides (NOV 2009) and an established immunostimulatory CpG ODN (ODN 2006). We found that antisense-mediated inhibition of bcl-2 expression in malignant B cells by G3139 and by NOV 2009 was poor. In contrast, ODN-mediated modulation of malignant B cell activation and apoptosis depended on the presence of CG dinucleotides within the sequence and was correlated with s-TK activity of individual patients.


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MATERIALS AND METHODS
 
Patient blood specimens
Peripheral blood from 13 patients with the diagnosis of B-CLL, according to clinical and immunphenotypic criteria, was obtained with their informed consent (Table 1 ). The local ethics committee approved blood sampling. All patients were untreated or had not received cytoreductive chemotherapy for at least 2 years before the investigation. s-TK was determined with a commercially available radioimmunoassay (Prolifigen, Sangtec Medical, Bromma, Sweden). Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation on Ficoll-Hypaque (Biochrom KG, Berlin, Germany) as described previously [27 ].


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  		Table 1. Spontaneous and ODN-Induced Changes of Apoptosis and Associated Markers of B-CLL Samples

Cell lines and cell culture conditions
For some experiments, we used the pre-B cell, acute lymphocytic leukemia cell line GM697 (DSMZ, Braunschweig, Germany) and the Burkitt’s lymphoma cell line Daudi JP (kindly provided by Dr. Fisch, Freiburg, Germany). All cell lines as well as freshly isolated PBMC were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C, 30 min) fetal calf serum, 2 mM L-glutamine (both from Gibco-BRL, Paisley, Scotland), and 100 U/ml penicillin, and 100 µg/ml streptomycin (both from Sigma Chemical Co., Munich, Germany). All compounds were purchased endotoxin-tested. Cells were incubated for various time periods at 37°C in a fully humidified, 5% CO2-containing atmosphere in the presence of oligonucleotides as indicated.

ODN
Phosphorothioate-modified ODN were provided by the Coley Pharmaceutical Group (Wellesley, MA) or purchased from Operon Technologies (Alameda, CA). Endotoxin levels in all ODN were <0.075 EU/ml, as determined by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). The sequences were as follows (TC dinucleotide at 5' end and CG dinucleotides appear in bold letters): G3139 (antisense to bcl-2) [45 ]: 5'-TCTCCCAGCGTGCGCCAT-3'; NOV 2009 (antisense to bcl-2) [18 ]: 5'-AATCCTCCCCCAGTTCACCC-3'; ODN 2006 [33 ]: 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'; ODN d(C20) (CpG-negative control): 5'-CCCCCCCCCCCCCCCCCCCC-3'. All ODN were stored in TE buffer (10 mM Tris, 1 mM ethylenediaminetetraacetate, pH 8.0) at -20°C. ODN were added in a final concentration of 0.08 µg/ml–30 µg/ml, as indicated in the figure legends. In antisense experiments, GM697 cells were preincubated for 30 min with ODN (final concentration, 1 µM) and the cationic lipid lipofectin (final concentration, 27.2 µg/ml; Gibco-BRL), in a polystyrol tube (room temperature; ODN and lipofectin added at 20x concentration).

Immunofluorescence assays
At the time points indicated, cells were washed in ice-cold phosphate-buffered saline (PBS) and were stained for surface antigens as described previously [46 ]. Monoclonal antibodies (mAb) against CD5 (UCHT2), CD19 (SJ25C1), CD20 (2H7), CD40 (5C3), CD54 (HA 58), CD69 (FN50), CD80 (L307.4), CD86 (IT2.2), major histocompatibility complex (MHC) I (G46-2.6), MHC II (TÜ39), and appropriate isotype controls were purchased from PharMingen (San Diego, CA). More than 80% of PBMC from patients with B-CLL were malignant B cells (positive for CD19 and CD5). Viable cells were gated according to their typical forward/side-scatter characteristics and propidium iodide (PI) staining, and B cells were identified by CD19 expression. For intracellular staining of apoptosis-related proteins, 1 x 106 cells were fixed and permeabilized ("Fix & Perm," Caltag, Burlingame, CA). Briefly, cells were washed with PBS (400 g, 4°C, 5 min), incubated for 15 min with fixation buffer A, washed again, and then incubated with permeabilization buffer B, together with 10 µl of fluorescein isothiocyanate (FITC)-labeled anti-bcl-xL mAb (7B2.5; Southern Biotechnology Associates, Birmingham, AL) and phycoerythrin-labeled anti-bcl-2 mAb (6CR) or the corresponding isotype antibodies (all from PharMingen). Finally, the cells were washed twice (400 g, 4°C, 5 min), resuspended in PBS, and analyzed immediately by flow cytometry using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). Spectral overlap was corrected by appropriate compensation. Data were analyzed with the fluorescein-activated cell-sorter evaluation program FlowJo (version 2.7.8, Tree Star, Stanford, CA).

Apoptosis was assessed by annexin V and PI staining on all cells counted with an appropriate forward scatter (FSC) threshold. Approximately 1 x 106 cells were resuspended in 195 µl annexin V-binding buffer (10 mM Hepes/NaOH, 140 mM NaCl, 2.5 mM CaCl, pH 7.4) together with 5 µl FITC-labeled annexin V. After 15 min of incubation at room temperature in the dark, cells were washed with PBS and stained with PI (2 µg/ml) immediately before flow cytometry. The increase in the cell fraction positive for annexin V and PI compared with control samples was considered the increase in apoptotic cells.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
B-CLL cells (3–5x105/sample) were lysed, and RNA was extracted (High-Pure lysis solution, total RNA isolation kit, Roche, Mannheim, Germany) and reverse transcribed (First Strand cDNA synthesis kit, Roche). For quantitation of bcl-2 and bcl-xL mRNA, target sequences were amplified by quantitative real-time PCR using the TaqMan® system (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol.

Statistics
Data are expressed as means ± SEM. Statistical significance of differences was determined by the paired two-tailed Student’s t-test or Wilcoxon test on absolute values. A P value of <0.05 was considered significant. Regression analysis was performed using the Spearman rank test. Statistical analysis was performed using the program StatView D-4.5 (Abacus Concepts, Berkeley, CA).


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RESULTS
 
The bcl-2 antisense oligonucleotides G3139 and NOV 2009 fail to down-regulate bcl-2 expression in malignant B cells in the absence of cationic lipids
Antisense-mediated inhibition of bcl-2 expression is expected to be beneficial in patients with B cell malignancies. In general, cationic lipids are required to provide sufficient uptake of antisense ODN into cells in vitro. In initial experiments, we found that the use of the cationic lipid lipofectin increased uptake of fluorescein-labeled ODN in cells of the B-cell leukemia cell line GM697 as determined by flow cytometry. As described earlier, efficacy of ODN uptake was highly dependent on the ratio of ODN and cationic lipids [46 ]. Optimal ODN uptake was achieved when 1 µM (=6 µg/ml) fluorescein-labeled ODN was combined with 27.2 µg/ml lipofectin.

We tested the ability of two well-established bcl-2 antisense ODN, G3139 [45 ] and NOV 2009 [18 ], to down-regulate bcl-2 expression in GM697 cells. Cells were incubated in the presence of ODN with or without lipofectin. Expression of bcl-2 was analyzed by flow cytometry. Inhibition of bcl-2 expression required the presence of lipofectin (Fig. 1 ). In the absence of lipofectin, bcl-2 levels in this particular cell line showed no major changes for both ODN.



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  		Figure 1. Inhibition of bcl-2 synthesis by bcl-2 antisense ODN requires the use of cationic lipids. GM697 cells were incubated with two different bcl-2 antisense ODN (non-CG-containing: NOV 2009; CG-containing ODN: G3139; all at 1 µM) with or without lipofectin (27.2 µg/ml). In samples with lipofectin, cells were washed after 4 h, and fresh medium was added for the remaining 12 h. After a total of 16 h, bcl-2 expression was examined by flow cytometry (MFI, mean fluorescence intensity, with baseline expression in the absence of ODN set as 100%). Bars indicate the mean ± range of three (medium control and G3139) or two (NOV 2009) independent experiments.

The bcl-2 antisense oligonucleotide G3139, but not NOV 2009, induces bcl-xL in primary B-CLL cells
In contrast to cells within solid tissues, ODN uptake of primary malignant B cells is expected to be similar in vitro and within the circulation in vivo. Recent studies indicate that NOV 2009 at high concentrations inhibits bcl-2 expression in primary B-CLL cells in vitro, even in the absence of cationic lipids [47 ]. Applying the same experimental conditions (5 µM, 48 h), we compared the effects of G3139 and NOV 2009 on bcl-2 expression in primary B-CLL cells in vitro.

PBMC were isolated from 13 patients with B-CLL (Table 1) . PBMC of all CLL patients contained >80% CD5- and CD19-positive, malignant B cells. PBMC were cultured in the presence of G3139 or NOV 2009 (30 µg/ml=5 µM). In preliminary experiments, we found a significant spontaneous uptake of fluorescein-labeled ODN in B-CLL cells. Unexpectedly, only 5 out of 13 patients showed inhibition of bcl-2 protein in response to G3139. On average, G3139 and NOV 2009 did not significantly change bcl-2 expression in B-CLL cells (G3139: P=0.65; Fig. 2A ). To determine if G3139-induced changes in individual patients were bcl-2-specific, we examined bcl-xL as a control protein. G3139, but not NOV 2009, increased bcl-xL expression consistently (Fig. 2A) . Changes on the protein level were confirmed by quantitative analysis of mRNA expression levels of bcl-2 and bcl-xL (Fig. 2B) .



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  		Figure 2. Modulation of bcl-2 and bcl-xL protein and mRNA in primary B-CLL cells by G3139 and ODN 2006 in the absence of cationic lipids. PBMC from patients with B-CLL were incubated for 48 h in the presence of the non-CG-containing bcl-2 antisense ODN (NOV 2009), the CG-containing bcl-2 antisense ODN (G3139), and the CpG ODN 2006, all at 30 µg/ml. The levels of bcl-2 and bcl-xL expression were determined on CD19+ B cells (>80% CD5+ malignant B cells). (A) bcl-2 protein analyzed by flow cytometry. Data are presented as means (±SEM) of relative values of 13 patients (G3139 and ODN 2006) or 11 patients (NOV 2009). (B) Quantitative real-time PCR. Data are presented as means (±SEM) of increased transcript numbers as compared with the control without ODN (five patients). Statistical evaluation was performed on absolute values using the Wilcoxon test.

Up-regulation of bcl-xL by G3139 depends on immunostimulatory CG dinucleotides
To confirm that up-regulation of bcl-xL by G3139 was independent of a potential antisense-mediated modulation of bcl-2, we examined the effects of G3139 on bcl-xL expression in the Burkitt lymphoma cell lines Daudi JP, which expresses bcl-xL but no detectable levels of bcl-2. G3139 up-regulated bcl-xL expression in Daudi cells, further supporting the concept of an underlying, non-antisense mechanism (Fig. 3 ).



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  		Figure 3. CpG-mediated up-regulation of bcl-xL and activation markers by G3139 in bcl-2-negative Burkitt lymphoma cells. The Burkitt lymphoma cell line Daudi JP, which does not express detectable levels of bcl-2, was incubated in the presence of G3139 (bcl-2 antisense) or ODN 2006, both at 6 µg/ml. After 24 h, expression of bcl-xL, CD54, CD80, and CD86 was measured by flow cytometry. Results are shown as means (±SEM) of six independent experiments.

G3139 contains two CG dinucleotides within the sequence and a TC dinucleotide at the 5' end, both of which have been demonstrated to increase the immunostimulatory potential of ODN [33 ]. We speculated that modulation of bcl-2 and bcl-xL by G3139 depends on immunostimulatory activity of this ODN rather than an antisense mechanism. The sequence ODN 2006 represents an immunostimulatory ODN with high activity on human B cells and dendritic cells [30 , 32 ]. Like G3139, ODN 2006 increased the level of bcl-xL consistently in B-CLL cells (Fig. 2A) and Daudi JP cells (Fig. 3) .

G3139 stimulates B-CLL cells in a similar way as ODN 2006
CpG ODN have been demonstrated to stimulate human malignant B cells to express higher levels of costimulatory and antigen-presenting molecules [39 40 41 ]. If G3139 shared immunostimulatory properties with ODN 2006, G3139 should not only induce bcl-xL but should also activate human malignant B cells. Indeed, at the relatively high concentration tested, G3139 as well ODN 2006 stimulated the expression of high levels of CD54, CD80, and CD86 in Daudi JP cells (Fig. 3) . Furthermore, cells increased in size and granularity in response to G3139 and ODN 2006 (not shown).

Both bcl-2 antisense sequences, G3139 and NOV 2009, are completely phosphorothioate-modified ODN. To exclude the possibility that the phosphorothioate backbone itself rather than the specific CG dinucleotide-containing sequence of G3139 is responsible for the immunostimulatory properties, we compared G3139 and ODN 2006 with the non-CpG ODN NOV 2009 for their ability to increase the expression of a panel of surface markers (CD20, CD40, CD69, CD80, CD86, CD54, MHC I, and MHC II) in B-CLL cells from nine different patients. G3139 as well as ODN 2006 up-regulated all markers tested, and NOV 2009 was essentially inactive (Fig. 4A ). The entire population of CD19- and CD5-positive malignant B cells responded to G3139 (Fig. 4B) and ODN 2006 (not shown). These results confirmed the immunologic properties of G3139 in primary malignant B cells.



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  		Figure 4. Activation of primary B-CLL cells by G3139 and ODN 2006, but not NOV 2009. PBMC from patients with B-CLL were incubated with the bcl-2 antisense ODN G3139 or NOV 2009, both at 6 µg/ml. After 48 h, viable CD19-positive cells were analyzed by flow cytometry. (A) Results represent means of nine independent experiments (±SEM). (B) The analysis of G3139-induced CD86 expression in PBMC of one representative patient with B-CLL is shown. The upper panel identifies subpopulations of cells within PBMC based on the expression of CD5 and CD19. Numbers indicate the percentage of gated cells of all viable cells. In the lower panel, CD86 expression of malignant B cells (CD5- and CD19-positive; black curves) and CD5-positive/CD19-negative cells (gray curves) is depicted.

G3139 is a CG-containing ODN with low activity compared with the established immunostimulatory sequence ODN 2006
In general, the immune-activating effect of CG-containing immunostimulatory ODN is dose-dependent. A potent CG-containing ODN is active at much lower concentrations than a CG-containing ODN with weaker activity. We examined the dose response of ODN 2006, G3139, NOV 2009, and the control ODN d(C20) to stimulate CD86 expression on primary B-CLL cells (Fig. 5 ). ODN 2006 is already active at concentrations as low as 0.3 µg/ml, where G3139 is still inactive. NOV 2009 and the control ODN d(C20) were inactive even at concentrations up to 5 µg/ml. Thus, the use of concentrations higher than 5 µg/ml, as used in the other experiments, allowed a qualitative comparison of ODN regarding immunostimulatory effects but not a quantitative estimate of the specific immunologic activity of the ODN.



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  		Figure 5. Dose-dependent activation of primary B-CLL cells by G3139 and ODN 2006, but not by ODN, without CpG motif. Primary B-CLL cells were incubated with increasing concentrations of G3139, NOV 2009, ODN 2006, and the control ODN d(C20). After 48 h, CD86 expression on CD5/CD19 double-positive B-CLL cells was determined by flow cytometry. Data are shown as MFI. Results represent the means of two independent experiments.

Modulation of apoptosis of B-CLL cells by G3139 is dependent on the presence of CG dinucleotides and is linked to s-TK activity
For individual patients, we observed a strong correlation between the effects of G3139 and ODN 2006 for bcl-2 expression; i.e., patients in whose cells G3139 induced high bcl-2 also showed high induction by ODN 2006 (and vice versa for low induction; Table 1 and Fig. 6 ). This parallel response to G3139 and ODN 2006 was also observed for bcl-xL expression, apoptosis, proliferation, CD86, and MHC II, suggesting that both ODN affected B-CLL cells by an identical mechanism of action (Fig. 6) .



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  		Figure 6. Correlation of G3139- and ODN 2006-induced changes of apoptosis-associated markers, costimulatory- and antigen-presenting molecules. PBMC from 13 patients with B-CLL were incubated with G3139 and ODN 2006 (30 µg/ml). After 48 h, the expression of bcl-2 and bcl-xL (MFI), the rate of apoptosis and proliferation, as well as CD86 and MHC II expression were measured. ODN-induced changes (for bcl-2, bcl-xL, apoptosis, and proliferation-baseline expression in the absence of ODN was set as 100%) for G3139 (x-axis) and ODN 2006 (y-axis) are compared. Statistical analysis was performed using the Spearman rank test.

There were no significant changes of the mean of apoptosis with G3139 and ODN 2006 (Fig. 7A ). We were interested in whether the proliferative activity of B-CLL cells as represented by s-TK might be linked to the effect of G3139 and ODN 2006 on apoptosis and proliferation of B-CLL cells. G3139 and ODN 2006 strongly stimulated proliferation of normal B cells from healthy donors (Table 2 ). Although most B-CLL samples examined (proliferation available in six patients) did not show spontaneous proliferative activity in vitro, the two samples with the highest s-TK level showed considerable spontaneous proliferation, which was further increased in the presence of CG-containing ODN (Table 3 ). In agreement with proliferation, we found a statistically significant negative correlation between the systemic level of s-TK and the rate of apoptosis induced by G3139 (Fig. 7B) . As expected, the rate of apoptosis in response to G3139 negatively correlated with G3139-induced proliferation of B-CLL cells (Fig. 7C) .



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  		Figure 7. s-TK activity and CpG ODN-induced modulation of apoptosis in B-CLL. B-CLL cells were incubated in the presence of G3139 and ODN 2006 at 30 µg/ml for 48 h. (A) Induction of apoptosis by G3139 and ODN 2006 (x-fold increase compared with control without ODN; not statistically significant). (B) Correlation of G3139-induced changes in apoptosis and the level of s-TK. (C) Correlation of G3139-induced changes of apoptosis and proliferation (proliferation values available for six patients; see Table 2 ).


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  		Table 2. ODN-Induced B-Cell Proliferation in Healthy Subjects (%)*


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  		Table 3. Percentage of Proliferating Cells in Patients with B-CLL (%)*

As demonstrated in Figure 2A , bcl-xL was up-regulated consistently in B-CLL cells by G3139 (P=0.007) and ODN 2006 (P=0.006) and thus, unlikely accounts for the differences in CpG-induced modulation of apoptosis in individual donors. However, there was some indication that changes of bcl-2 are associated with modulation of B-CLL apoptosis (Table 1) . In five out of six patients with marked CpG-mediated induction of apoptosis, expression of bcl-2 was reduced. In contrast, in the samples in which CpG inhibited apoptosis, bcl-2 was strongly up-regulated. Thus, CpG seems to modulate cell death of B-CLL via bcl-2 rather than bcl-xL.


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DISCUSSION
 
Overexpression of the antiapoptotic protein bcl-2 in many tumors [2 ] forms the rationale for the development of antisense ODN against bcl-2, which ideally would provide a tool for specific inhibition of bcl-2 synthesis [14 , 48 , 49 ]. Although a recent study investigating the biologic and clinical effects of G3139 in patients with non-Hodgkin’s lymphoma showed clinical responses, no consistent reduction of bcl-2 expression could be found [21 ]. Unlike other tumor cells such as malignant melanoma cells, malignant B cells are highly sensitive to immunostimulatory properties of ODN [39 40 41 ]. Furthermore, CpG ODN have been shown to inhibit apoptosis of murine B-cell lymphoma cell lines [42 43 44 ]. The important question as to whether CpG ODN also affect apoptosis of human primary malignant B cells has not been answered. We speculated that ODN 2006 ODN and bcl-2 antisense ODN might share immunostimulatory properties by which both may influence apoptosis of human primary malignant B cells in a similar way.

We compared two well-established bcl-2 antisense ODN: the sequence G3139, which is tested currently in clinical trials, and the sequence NOV 2009, which inhibits bcl-2 in vitro but for which no antitumor activity in animal models has been demonstrated. Antisense-mediated inhibition of bcl-2 expression was not observed unless ODN delivery was increased by cationic lipids. Without cationic lipids, NOV 2009 showed no effect on bcl-2 expression of B cell leukemia cell lines or primary B-CLL cells.

There was no significant change of bcl-2 expression in primary B-CLL cells. Unexpectedly, bcl-xL, which was used as a control protein to test antisense specificity, was up-regulated consistently by G3139 but not by NOV 2009. The concept that the biological effects of G3139 are a result of immunostimulatory properties rather than antisense effects was further supported by the observation that G3139 but not NOV 2009 strongly increased the expression of a panel of surface markers, including costimulatory molecules and antigen-presenting molecules as well as CD20. All of these markers have been shown to be up-regulated specifically by CpG ODN [39 40 41 ]. Although qualitatively, G3139 and ODN 2006 showed the same biological properties, dose-response experiments demonstrated that ODN 2006 was active at lower concentrations than G3139.

ODN 2006 and G3139 had identical biological activities on B-CLL cells regarding bcl-2, bcl-xL, apoptosis, proliferation, and activation markers, suggesting an identical mechanism of action of both ODN. The level of systemic s-TK activity as a measure for the proliferative state of the disease in individual patients correlated negatively with the influence of CG-containing ODN on apoptosis. Similarly, G3139 and ODN 2006 increased spontaneous apoptosis of B-CLL cells from patients with low s-TK (low proliferative state) and decreased apoptosis in patients with high s-TK activity. However, because no linear regression between both parameters was found, the extent of apoptosis modulation is not predicted by the level of s-TK.

The mechanism by which immunostimulatory CG-containing ODN influences apoptosis of B-CLL cells is most likely a result of modulation of apoptosis-associated proteins. Of the two anti-apoptotic proteins examined in this study, bcl-xL was up-regulated consistently by CpG ODN. In contrast, increased bcl-2 was found in all B-CLL samples in which CpG ODN inhibited apoptosis, and decreased bcl-2 levels were observed in samples in which CpG ODN induced apoptosis. Therefore, modulation of bcl-2 rather than bcl-xL seemed to play a major role in the modulation of apoptosis by CpG ODN.

Several groups have demonstrated antisense-mediated inhibition of bcl-2 expression in vitro [17 18 19 , 45 , 50 51 52 53 54 ]. In most of these studies, ODN were delivered to tumor cell lines by cationic lipids. One group has demonstrated bcl-2 inhibition in primary B-CLL cells in vitro using high concentrations of the bcl-2 antisense NOV 2009 [47 ]. In our study, we were unable to show antisense-mediated inhibition of bcl-2 synthesis in B-cell lymphoma cell lines and primary B-CLL cells in the absence of cationic lipids. Antisense-mediated inhibition of bcl-2 by G3139 in primary B-CLL seems to be complicated by non-antisense-mediated up-regulation of bcl-2 in some patients, which abrogates the desired antisense effect. This is not an issue in the case of other malignancies, such as malignant melanoma, because these tumor cells, in contrast to B cells, are not susceptible to immunostimulation.

Although it has been demonstrated that antisense ODN can specifically inhibit gene expression, recent investigations have raised puzzling questions regarding what factors govern their biological activity. For antisense ODN to be active, efficient delivery of ODN into the target cell must be achieved. In vivo, however, antisense ODN are apparently active when given in the free form [20 , 45 , 51 , 55 , 56 ]. It is therefore unclear to what extent therapeutic activity can be attributed to specific antisense-mRNA interactions rather than to systemic immune effects. It is important to note that for phosphorothioate-modified ODN, in contrast to unmodified phosphodiester ODN, the presence of CG dinucleotides within the ODN sequence does not precisely predict the immunostimulatory activity of an ODN, and thus the activity has to be determined empirically [32 ]. Consequently, to examine the possible contribution of immune stimulation in a tumor model, the correct ODN to control for a CpG effect are established CpG ODN (for which immunostimulatory properties have been demonstrated) rather than random CG-containing sequences.

All of the in vivo studies but one [20 ] have been performed with the bcl-2 antisense ODN G3139 [45 , 51 , 55 , 56 ]. This ODN has been tested in tumor models in severe combined immunodeficient (SCID) mice, suggesting that T cells are not required for the observed anti-tumor activity. SCID/Rag-2 mice, which lack B cells as well as T cells, still rejected the tumor in response to systemic anti-bcl-2 antisense ODN administration [45 ]. As CpG ODN are known to activate natural killer (NK) cells [29 ], which are still present in SCID mice, Klasa et al. [45 ] tested G3139 in pfp/Rag-2 mice, which lack perforin-mediated killing of NK cells in addition to the lack of B and T cells. In these mice, anti-tumor activity of G3139 was reduced greatly but was still detectable. Because these mice lack perforin-mediated killing, the remaining activity in this model may be a result of Fas- or TRAIL-mediated killing mechanisms. Alternatively, CpG-activated macrophages or macrophage-derived cytokines such as type I interferon (IFN) [29 , 57 ] may contribute to anti-tumor activity in these mice by a direct effect on tumor cells. Type I IFN is approved for the therapy of non-Hodgkin’s lymphoma [58 , 59 ] and has shown cytoreduction, but no clinical benefit, in patients with early B-CLL [60 , 61 ].

In a phase I clinical trial, G3139 has been tested in patients with bcl-2-positive, relapsed non-Hodgkin’s lymphoma. Because the decline in bcl-2 levels was small and no control proteins were measured, the contribution of bcl-2 inhibition to the clinical effects is still unclear [15 ]. It is interesting that the major side effects of G3139 included fever, skin inflammation at the site of subcutaneous injection, and painful enlargement of local lymph nodes [21 , 22 ]. These observations suggest that G3139 has immunologic activity in humans in vivo. Plasma levels of G3139 associated with dose-limiting toxicity were higher than 4 µg/ml, and thus were within the concentration range, which, in our hands, activated malignant B cells in vitro. Waters et al. [21 ] were unable to detect immune activation by G3139 when performing intracellular cytokine staining for interleukin-2, IFN-{gamma}, IL-4, and perforin in peripheral blood of four patients as a measure of a systemic T helper cell type 1 response or NK cell activation. However, CpG ODN-mediated activation of T cells requires the presence of a specific antigen triggering the T cell receptor [62 ]. Furthermore, activated immune cells usually leave the circulation and thus are not readily accessible by peripheral blood sampling.

Given that ODN 2006 as well as G3139 have a direct, stimulatory effect on malignant B cells, the question remains whether this effect is clinically beneficial or not. Our results demonstrate that, at least for B-CLL, depending on the systemic level of s-TK, ODN 2006 and G3139 may enhance or inhibit apoptosis of malignant B cells. In addition to s-TK-associated regulation of apoptosis by ODN 2006 and G3139, increased expression of costimulatory and antigen-presenting molecules may improve immune recognition and elimination of malignant B cells by immune effector cells. Although it is difficult to predict if this general activation of anti-tumor immunity will outweigh undesired inhibition of apoptosis in patients with high s-TK activity, patients with low s-TK activity are more likely to benefit from CpG ODN therapy.

CpG ODN have not been shown to have any direct effect on tumor cells that do not derive from immune cells. Nevertheless, immunostimulatory properties of ODN may support the immune system to gain control over progressive tumor growth. Anti-tumor activity of CpG ODN has been demonstrated in animal models in which CpG ODN were administered alone or as an adjuvant in conjunction with a tumor vaccine or in combination with tumor-specific antibodies [63 64 65 66 ]. It is difficult to assess to what extent immunostimulatory properties of G3139 contribute to the therapeutic effects seen in animal models and in clinical studies. If immunostimulation indeed contributes to the major part of the anti-tumor activity of G3139, potent CpG ODN are likely to be more effective. In this context, it is interesting to note that many antisense ODN, which are tested in clinical trials, contain CG dinucleotides [67 ] and that some diseases targeted in these trials, such as viral infections and cancer, would most likely benefit from immune stimulation.


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CONCLUSIONS
 
Our studies offer important information for the further development of ODN-based therapy of malignancies. G3139, which was developed originally as a bcl-2 antisense ODN, shares immunologic properties with CpG ODN. The immunologic activity of G3139, rather than an antisense-mediated inhibition of bcl-2 protein, is most likely to be responsible for the anti-tumor effects in vivo. CG-containing ODN stimulate B-CLL cells and modulate apoptosis of primary B-CLL cells directly, depending on the proliferative state of the disease, as reflected by s-TK levels. s-TK levels may help to identify B-CLL patients, who may respond to CpG ODN therapy. Tumors other than B cell malignancies may respond to treatment with CG-containing ODN, not by a direct effect on the tumor cells, but by increased activity of the immune system.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the University of Munich, FöFoLe Nr. 44; Dr. Mildred Scheel-Stiftung, 10-1309-En2; and the German-Israeli Foundation, Nr. I-021-203.05/96. This work is part of the thesis of R. J. and L. M. at LMU Munich. B. J. and R. J. contributed equally to this work.

Received August 25, 2001; revised December 8, 2001; accepted February 4, 2002.


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