Leukemia (2006) 20, 498–504 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu ORIGINAL ARTICLE

High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia

A Sainz-Perez1,3, H Gary-Gouy1,3, A Portier1, F Davi2, H Merle-Beral2, P Galanaud1 and A Dalloul1

1INSERM Unite´ 131, IFR 13, Universite´ Paris XI 32 Rue des Carnets, Clamart, France and 2Laboratoire d’He´matologie, Hoˆpital de la Pitie´-Salpeˆtrie`re, Paris, France

Chronic lymphocytic leukemia (CLL)-B-cells are quiescent stimulation with auto-antigen, a condition shown to induce differentiated cells that produce (IL)-10 and accu- anergy,13 and CD5 expression on B-cells in experimental models.14 mulate due to resistance to . The mechanisms Whether CD5 plays a role in CLL B-cell survival is not yet underlying such resistance are poorly understood. Herein we clear. We have however observed that, contrary to the majority show that all CLL B-cells tested (30/30) display high mRNA and À þ expression of the tumor suppressor Mda-7/IL-24, an of CD5 B cells, normal CD5 blood B cells displayed a low IL-10 family member, in comparison to normal B cells. A Ca2 þ response following anti-IgM stimulation,15 confirming the downstream Mda-7 signaling target, p38 mitogen-activated role of CD5 as a negative regulator of BCR signaling.16,17 Even protein kinase (MAPK) was highly phosphorylated in all CLL though BCR-mediated activation is often deficient in CLL, CD5 cells but not in normal B-cells. Mda-7 expression and p38 MAPK may act independently of the BCR by recruiting signaling phosphorylation diminished in culture and the latter could be molecules,18,19 and eliciting the production of B-cell survival reinduced by recombinant (r)-IL-24 or LPS and Mda-7 transfec- 15 tion. Mda-7/IL-24 siRNA specifically inhibited p38 MAPK factors such as interleukin (IL)-10. phosphorylation in CLL without affecting p38 MAPK, bcl2, or IL-10 is indeed produced by most CLL B-cells,20 improves the Lyn expression, further demonstrating the direct role of Mda-7/ survival of malignant CLL B-cells,21 and IL-10 serum levels in IL-24 in p38 MAPK activation. Both pharmacological inhibition CLL correlated with the severity of the disease.22 We compared of p38 MAPK and Mda-7 silencing augmented spontaneous mRNAs from vector- and CD5-transduced Daudi cells,15 by apoptosis by three-fold in CLL cells cultured in autologous means of Affymetrix DNA chips. We found that all IL-10 family serum, which was reversed by LPS and r-IL-24. We established 23 the role of p38 MAPK in CLL cell survival and demonstrated a , and receptors were upregulated in CD5-transfected vs paradoxical effect, whereby Mda-7 and IL-24, inducers of vector-transfected Daudi cells (article in preparation). Among apoptosis in diverse cells, promote the survival of CLL these , IL-24,24 is an alternative transcript of Mda-7, a B-cells through p38 MAPK activation. encoding a melanoma differentiation antigen that func- Leukemia (2006) 20, 498–504. doi:10.1038/sj.leu.2404073; tions as a tumor suppressor in the context of cancer cells.25 We published online 12 January 2006 Keywords: CLL; apoptosis; p38MAP kinase; Mda-7; Interleukin-24; confirmed by real-time PCR the augmented expression of Mda- CD5 7/IL-24 transcripts in CD5 þ Daudi cells, and in the CD5 þ B-cell population from normal adult blood in comparison to CD5-negative B-cells. Our DNA chips data were also validated by RT-PCR for IL-10, IL-19 and IL-20.23 Mda-7/IL-24 mRNA Introduction expression in CLL was more than 10-fold that in normal B-cells and a positive correlation between CD5 and Mda-7/RNA B-cell chronic lymphocytic leukemia (CLL) is characterized by expression was found on randomly analyzed CLL samples. This the progressive accumulation of circulating CD5 þ monoclonal prompted us to study the function of Mda-7/IL-24 in CLL. We B-cells.1,2 These cells contrary to normal B- are show herein that this gene acts as a survival factor in part refractory to activation by mitogens,3,4 and have quantitative through phosphorylation of the p38 mitogen-activated protein and qualitative deficiencies in the B-cell receptor (BCR) kinase (MAPK) protein and the importance of this latter pathway expression and signaling.1,2,5,6 As a consequence, these cells in CLL is highlighted in this work. display a resting phenotype, often respond poorly to activation and are mostly in G0/early G1 phase of the cell cycle,7–9 a characteristic that may protect them from drugs- and activation- Materials and methods induced apoptosis.10 CLL cells have been considered as anergic although this may be CLL samples especially true for those with mutated IgV genes. CLL B cells have Blood samples from patients (22 treated, eight untreated) and 18 membrane markers of mature antigen-experienced cells.11 More- samples from healthy donors were processed after they had over, in up to 70% of cases, their mutational status within Ig V given informed consent to the Pitie´-Salpeˆtrie`re university regions suggest that they have passed through germinal centers.12 In hospital. Patients were 18 males and 12 females, with ages ranging from 49 to 87 years old and counts ranging addition, their mature phenotype associated with a low BCR 3 expression suggest that they have been submitted to a chronic from 3940 to 113 000/mm , with % lymphocytes/PBMC ranging from 32.6 to 97% PBMC were isolated by density centrifugation 15 Correspondence: Dr A Dalloul, INSERM Unite´ 131, 32 Rue des and B-cells were negatively selected as described. Carnets, 92140 Clamart, France. E-mail: [email protected] 3These two authors contributed equally to this work. Cell culture and reagents Received 17 August 2005; revised 31 October 2005; accepted 7 Daudi cells and PBMC from healthy donors and patients were November 2005; published online 12 January 2006 cultured in RPMI medium supplemented with 10% FCS or 10% Mda-7/IL-24 and p38 MAP kinase activation in CLL A Sainz-Perez et al 499 autologous serum, Penicilline (100 U/ml), Streptomycine The schematic structure of the IL-24 gene (Figure 1) was (100 mg/ml), 2 mML-glutamate and 1 mM sodium pyruvate copied from SV Kotenko. The family of IL-10-related cytokines (Invitrogen, Cergy, France). Cells were incubated with r-IL-2, and their receptors: related but to what extent? and r-IL-24, (all from R&D systems, Lille, France) at respective final Reviews 2002; 13: 223–240. 0 concentrations of 50 ng/ml and 100 ng/ml. Affinipure F(ab )2 polyclonal Rabbit anti-human IgM (Jackson Immunotech, West Grove, USA) was used at 2 mg/ml and LPS from Escherichia coli RNA interference and gene transfer into CLL cells 6 (VWR International SAS, Fontenay sous bois, France) was used 10 Â 10 CLL-B cells/condition were transfected with 100 nM of at 10 mg/ml. SB 203580 in solution (lot#B64038) (Calbiochem) Mda-7/IL-24 siRNA (Dharmacon cat # M-007977-01) or control was used at doses ranging from 1 to 20 mM. For apoptosis siRNA; or with 10 mg of either empty vector or Mda7 inserted studies, cells were stained with the Apo 2.7 mAb (Beckman- into PCI-neo vector, using the Human Nucleofectort Kit Coulter, Villepinte, France). (AMAXA, Ko¨ln, Germany) following to the manufacturer’s instructions (selected program U-15). Cells were processed as indicated postnucleofection. Control siRNA or Human Mda-7/ Cell lines and RT-PCR IL-24 siRNA were synthesized annealed, desalted, 20 depro- Full-length cDNA for IL-24 and Mda-7 were amplified by PCR, tected and purified by Dharmacon Inc. (La Fayette Co.). To using the following primers: estimate transfection efficacy, cells were transfected with IL-24: Sense :ggctcgagctcaaatgcagatggttgtgctccc, fluorescent control siRNA: siGLO RISC-free siRNA Dharmacon Opp: aagaggccgctcagagcttgtagaatttctgcat. cat # D-001600-01, a fluorescently labeled siRNA with Mda-7: Sense :gctcgagcgatgaattttcaacagaggctgcaa, impaired ability for RISC interaction and with X4 mismatches Opp: aagcggccgctcagagcttgtagatttctgcat. with known human genes. PCR products were inserted into PCI-neo vector (Promega) at XhoI and NotI sites and cloned in Top-10 bacteria. Preps from empty vector or insert-containing vectors were linearized with Western blots BglII and Daudi cells (5 Â 106 cells) mixed with 20 mg of each CLL cells were processed from fresh blood and washed at room 6 plasmid and electroporated as described.15 1a-containing temperature in a serum-free RPMI–10 mM HEPES. Cells (2 Â 10 7 region was amplified by RT-PCR in CLL samples using the Daudi, or 10 PBMC or CLL) were lysed at 41C for 30 min in lysis following primers: Sense: gcctgattggtgaatggt, Opp : ctctccgca buffer (20 mM Tris-Hcl, pH 7.5, 140 mM NaCl, 1 mM EDTA, tccgagacgtt. Expression of the different IL-24 receptor subunits 50 U/ml aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate) were analyzed by classic polymerase chain reaction using PCR containing 1% nonidet-P-40 detergent. 60 mg /sample Master Mix (Promega, Charbonnie`res, France) and the following were separated on SDS-PAGE gels under reducing conditions, primers synthesized by Proligo (Paris, France): electrotransferred onto PVDF membranes (Amersham), and blotted with antiphospho-p38 MAPK (T180/Y182) rabbit poly- clonal antibody (R&D) then with anti p38 MAPK mAb (A-12, IL20R1, forward: ccctgtgtctctggtggttt, Santa-Cruz, Heidelberg, Germany) after stripping. Mda-7/IL-24 reverse: agtcctggcacactgcttct. proteins were blotted with anti-human IL-24 mAb (R&D). Mouse IL20R2, forward: caaactgagtctaccaaatgcaga, mAb IgG anti Bcl2 and Rabbit polyclonal anti-Lyn were from reverse: tccgcaaacctatgagatcc. Santa Cruz. Blots were revealed using Goat-anti-Mouse or Goat- IL22R, forward: ttggagggaagcagagagaa, anti-Rabbit HRP-conjugated (Bio-Rad Lanoratories) and ECL reverse: gcataggacagtggggagag. detection (Amersham).

Real-time PCR and Mda7 6 Deglycosylation of IL-24 Cells (2 Â 10 /ml) were incubated in complete medium for 24 h In total, 25 ng of human rIL24 (R&D) were diluted in 35 ml de- under various conditions, and RNA purified using the RNeasy ionized water, 10 mlof5Â reaction buffer pH 7.5 (250 mM Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was sodium phosphate) and 2.5 ml denaturation solution (2% SDS, carried out with random hexamers using Superscript first-strand 1 M b-mercaptoethanol) were added to the mixture, heated synthesis system for RT-PCR (Invitrogen). Transcript expression 10 min at 1001C, and 2.5 ml Triton X-100 (15% solution) þ 2.5 U was analyzed by PCR using the TaqMan technology. Results of peptide-N-glycosidase F (Sigma, 500 U/ml) added for 3 h at were analyzed using the ABI Prism 7700 sequence detection 371C and inactivated 5 min at 1001C. system software (PE Applied Bio System). Each reaction was normalized by the cycle threshold (Ct) of GAPDH cDNA expression. Relative expression was calculated by measuring the ELISA difference of normalized Ct between condition A vs condition B For IL-24 detection, 96 well flat-bottom plates were coated and applying the following formula: difference of expression overnight with an anti-IL-24 mAb (R&D) (100 ml at 2 mg/ml) in between A and B ¼ 2eÀ(CtAÀCtB). The following TaqMan Na carbonate coating buffer, washed with PBS, 0.05% Tween- specific primers and probes were selected using Primer Express 20, incubated for 1 h at RT with a blocking buffer (PBS, 1% BSA, Software (PE Applied Biosystems) and specificity verified using 0.2% Tween-20), and washed. Serial dilutions of rh IL-24 (R&D), NCBI Blast software: or serum (100 ml/well) from patients or controls were added for Mda-7/IL-24: Forward: gtaagttacatgaaggcagcagaatatt, Re- 2 h at RT. HRP-streptavidin (Biogenex, San Ramon) was added verse: ccacactgtggcaaggattg, Probe: tgccccatgcttctttacccctca. for 30 min, at RT. Reaction was developed by adding ABTS Mda-7: Forward: cagccctcaagcatcacttaca, Reverse: aaaattcatctctc (Roche, Meylan, France) in the dark for 10 min, and stopped agtctcgtgtt, Probe: tggccttcctcccaagcagcct. CD5: Forward: ggatggc with 1 N H2SO4. The OD was red at 450 nm (Beckman Coulter, acatggtttgca, Reverse: gcttaagggcaccccacagt, Probe: cgtcaaaagtctg Villepinte, France). Detection of IgG in sera was performed with cagcggctg. GAPDH: Forward: gaaggtgaaggtcggagtc, Reverse: a human IgG ELISA detection kit (Interchim, Montluc¸on, gaagatggtgatgggatttc, Probe: caagcttcccgttctcagcc. France).

Leukemia Mda-7/IL-24 and p38 MAP kinase activation in CLL A Sainz-Perez et al 500 a Mda-7 Mda-7/IL-24

ATG ATG STOP AAAAA Ex1b Ex1a Ex1 Ex2 Ex3 Ex4 Ex5

70 587 b c CLL B cell Daudi 200 180 P<0.001 650 Actin 160 140 Mda-7 120 518 100 365 IL-24 80 P<0.01 P<0.05 60 40 P<0.05 d

mRNA relative expression 20 CLL cc = 0.85 0 DauDau-CD5+ BB-CD5+ CLL

CD5 mRNA expression mda7/L24 mRNA expression

Figure 1 Conventional and real-time PCR shows that Mda-7 and IL-24 mRNA expression is augmented in CLL as compared to normal B-cells: correlation with CD5 expression. (a) Organization of the Mda-7/IL-24 cDNA: The IL-24 transcript is generated by of exon-1a. Primer sets and probes (arrows and line) specific for mda-7 only (50 region) and for both mda-7 and IL-24 (30 UTR region) transcripts were used for cDNA amplification by real-time PCR. (b) Real-time PCR was performed in vector-transfected (Dau), CD5-transfected (Dau-CD5 þ ) Daudi cells, blood B-cells (B), and FACS-sorted CD5 þ -B-cells (three samples), and 30 CLL. Y axis: relative expression of Mda-7 only (white histograms) and Mda-7/IL-24 (black histograms) mRNA, where 1 U represents the arbitrary value for empty vector-transfected Daudi cells. (c) The presence of Mda- 7 and IL-24 mRNA was determined by RT-PCR using primers (located at þ 70 and þ 587 base position Figure 1a, arrows) that amplify a 365 or a 518-bp products depending on the absence (IL-24) or presence (Mda-7) of exon 1a, respectively. Agarose gel (2%) with the 650 bp actin control. (d) Positive correlation between CD5 and Mda-7/IL-24 in CLL. CD5 (x-axis) and Mda-7/IL-24 (y-axis) expression in 18 CLL samples were amplified by real-time PCR. Correlation coefficient was calculated using the Excel program.

Statistical analysis ILI-24 mRNA amounts by real-time PCR analysis of 18 CLL All statistics were carried using the Mann–Whitney U-test. samples (Figure 1d). The 10-fold higher expression of both Mda-7 and IL-24 mRNAs in CLL cells than in normal adult blood B-cells prompted us to investigate the expression of the protein and Results the IL-24 receptor in CLL.

High expression of Mda-7 and IL-24 mRNA in CLL B-cells Characterization of Mda-7/IL-24 protein We analyzed IL-24 and Mda-7 transcripts in several B-cell types and IL-24-receptor expression in CLL by conventional and real-time PCR. IL-24 arises from Mda-7 We investigated the presence of Mda-7/IL-24 protein in lysates through alternative splicing of Exon 1a (Figure 1a). Two sets of from various B-cells by SDS-PAGE followed by Western blot. primers and internal probes were used, the first one located The protein was undetectable in Daudi cells, and weak or within exon 1a and another one at the 30UTR region of the undetectable in PBMC and purified B cells from normal blood transcript, specific for Mda-7 only or for both Mda-7 and IL-24, (Figure 2a). By contrast, it was always positive albeit with respectively (Figure 1a). The expression of mda-7/IL-24 mRNA variable intensity on all (30/30) CLL samples tested as shown was augmented upon introduction of CD5 into Daudi B cell line from a representative panel of three patients (Figure 2a). In CLL, (Po0.05) in keeping with our previously observed CD5-induced the Mda-7/IL-24 mAb recognized predominantly a 20 kDa IL-10 expression.15 Mda-7/IL-24 mRNA were higher in normal protein and higher molecular forms were present, whereas adult blood B cells than in Daudi cells (Po0.01), and were control r-IL-24, migrated as a single 35 kDa band reflecting the augmented 2.5-fold in the CD5 þ B-cell population. Most glycosylation of the recombinant cytokine. Indeed, in vitro importantly, Mda-7/IL-24 expression in 30/30 fresh CLL B cells deglycosylation with the endoglycanase, PNGase F 26 restored was augmented 10-fold as compared to normal blood B cells the 20 kDa protein size (Figure 2a left). Thus, CLL express Mda- (Po0.001) (Figure 1b). To show that both Mda-7 and IL-24 7/IL-24 protein in a predominantly nonglycosylated form. IL-24 transcripts were expressed in CLL, a set of primers located and IgG as control were measured by ELISA in the sera of 30 outside exon 1a was used to amplify this region generating patients and 18 healthy blood donors. IL-24 was undetectable in 365 bp in exon1a-negative (IL-24) and 518 bp PCR products in patient’s sera at variance with 2/3 of control sera in which exon 1a-positive (Mda-7) PCR products, respectively (Figure 1c), 4100 ng/ml were detected by average (Figure 2b). and both products amplified. Our results from normal and IL-24 receptors are heterodimers composed of a common Daudi B-cells suggested that Mda-7/IL-24 expression in CLL IL20-R2 chain associated to either IL20-R1 or IL22-R chain.26 could have been enhanced by CD5. In support of this we The presence of receptors in CLL was investigated by RT-PCR observed a 0.85 positive correlation between CD5 and Mda-7/ specific for the three chains, respectively, and was evidenced in

Leukemia Mda-7/IL-24 and p38 MAP kinase activation in CLL A Sainz-Perez et al 501 a a b rIL24 Cell Iysates Daudi CLL Daudi B CLL

35 IL20R1

20 IL20R2 0 10203040506070 0 10203040506070 0 1 2 3 4 0 1 2 3 4 PNGase - + Dau PBMC B1 5 17 10 10 10 10 10 10 10 10 10 10 CLL number IL22R IL20R1 - FITC b 600 20 000 Cell count 18 000 GAPDH 500 16 000

400 14 000 12 000

300 10 000 0 10203040506070 0 10203040506070 100 101 102 103 104 100 101 102 103 104 8 000 200 6 000 IL20R2 - FITC Serum IgG (mg/ml) Serum IL24 (pg/ml) 100 4 000 2 000 Figure 3 CLL cells express receptors for IL24. (a) RT-PCR amplifica- 0 - tion of IL22R, IL20R1, IL20R2 and GAPDH from Daudi, blood B cells ControlsCLL Controls CLL and CLL B cells. (b) FACS analysis of CLL and Daudi cells stained with anti-IL20R2-FITC or anti-IL20R1-FITC Abs (filled histogram: isotype- Figure 2 High intracellular Mda-7/IL-24 protein expression in CLL. specific control Ab). Representative of six CLL and controls. (a) High intracellular expression of Mda-7/IL-24 protein in CLL B cells. Right: cell lysates from Daudi B cells (Dau), fresh Peripheral blood mononuclear cells (PBMC) or purified B cells from healthy donors and CLL B cells samples were blotted with anti-Mda-7/IL-24 mAb. by r-IL-24 and by the canonical p38 MAPK activator LPS.28 Representative of experiments from 28 CLL and 12 healthy donors. Forced expression of Mda-7 also augmented p38 MAPK Left: as a positive control, 10 ng of native human recombinant IL-24 phosphorylation in CLL in comparison to empty vector- and 25 ng of deglycosylated (PNGase F þ ) r-IL-24 were analyzed by transfected cells (Figure 4d). Western blot. (b) IgG and IL-24 levels were measured by ELISA in sera from 30 CLL patients and 15 age-matched adult controls. Mean values A direct evidence of Mda-7/IL-24-driven p38 MAPK activa- (horizontal lines) are shown. tion in CLL was therefore needed and could be obtained by silencing Mda-7/IL-24 expression using Mda-7-specific siRNA. Cell scatter was comparable in both control and Mda-7- all CLL samples tested and also to a lesser extent in normal transfected samples. As shown in Figure 4e, we were able to 4 blood B cells, at variance with Daudi cell line which was transfect fresh CLL cells with a high 80% efficiency. At day 3, negative (Figure 3a). Surface expression of IL-20R2 and IL-20R1 Mda-7/IL-24 protein expression became almost undetectable in on CLL cells was formally demonstrated by direct staining with Mda-7/IL-24- but not in control siRNA-treated cells. In parallel, anti-IL20R1 and anti-IL20R2 Abs (Figure 3b) raising the the amount of p38 MAPK protein was comparable in Mda-7/ possibility that malignant cells may respond to exogenous IL-24. IL-24 and in control siRNA-treated cells, whereas phosphory- lated p38 MAPK diminished in Mda7-silenced cells but not in control cells. Moreover, silencing did not affect the amount of other important proteins in CLL such as Bcl2 or Lyn pointing to p38 MAPK is phosphorylated in fresh CLL cells and the specific effect of endogenous Mda-7/IL-24 on p38 MAPK activated by rIL-24 and by Mda7 pathway. Some of the Mda-7/IL-24-induced signaling events were previously characterized in various non-B cell types using Mda-7-adenoviral infection and GST-Mda-7 fusion proteins, and The role of p38 MAPK activation in CLL survival include induction of p38-MAP kinase activation.27 Transient The finding of a significant phosphorylated MAPK fraction in introduction of IL-24 or Mda-7 cDNAs into Daudi B-cells fresh CLL cells suggested that p38 MAPK activation was not (Figure 4a) did not modify total p38 MAPK expression but harmful. Whether its inhibition is detrimental was therefore enhanced p38 MAPK phosphorylation in comparison to empty investigated. We cultured all CLL samples in medium with 10% vector-transfected cells (Figure 4a). This effect is due to autologous serum to minimize spontaneous apoptosis, and intracellular IL-24 and Mda-7 proteins since Daudi cells lacked assessed cell death by staining with the mitochondrial marker IL-24R (Figure 3) ruling out an autocrine effect of secreted IL-24. Apo 2.7.29 FACS analysis was performed at days 4 and 7. We therefore investigated the p38 MAPK phosphorylation status Addition of SB 203580 at 10 mM to the culture induced cell in malignant cells. Western blots were performed and mem- death in all samples tested as early as day 4 as reflected by the branes incubated with anti-phospho-p38MAPK, stripped and cell-size decrease on cell scatter plot and a nearly three-fold reprobed with anti-p38 MAPK antibody. Phosphorylated p38- increase in apoptotic cell numbers on histogram (Figure 5a). In MAPK protein predominated in all fresh CLL cells by contrast to total, 12 samples from patients were cultured with or without normal B cells, (Figure 4b). Kinetic analysis of p38 MAPK r-IL-24, LPS, SB 203580 and theophylline. SB 203580-mediated protein expression showed that the phosphorylation of p38- apoptosis was reversed by LPS and by r-IL-24, whereas LPS or MAPK gradually diminished in culture (Figure 4c and d) r-IL-24 alone did not protect cells from spontaneous death although the amount of total p38 MAPK was constant overtime. (Figure 5b). The specificity of MAPK inhibition in this process is Mda-7/IL-24 expression also diminished in culture (Figure 4c). suggested by the action of SB 203580 and by the ability of the The phosphorylation of p38 MAPK protein could be reinduced MAPK activators LPS and IL-24 to reverse SB 203580-mediated

Leukemia Mda-7/IL-24 and p38 MAP kinase activation in CLL A Sainz-Perez et al 502 a bc

Mda7 Mda7

phospho phospho p38 MAPK p38 MAPK p38 MAPK p38 MAPK

emptyIL-24mda7 13 7101218 d0 d2 d6

Daudi B cell CLL number CLL 30 4

de10 3

control 10 2

siRNA 10 Mda7 1 SSC Height 10

LPS IL24 0 10 0 200 400 600 800 1000 phospho FSC Height phospho 4 p38 MAPK 10

p38 MAPK 3 p38 MAPK Mda7 10 2

p38 siRNA 10 1 SSC Height bc12 MAPK 10 0

10 0 200 400 600 800 1000 d0 d3 d3 d3 empty mda7 FSC Height Lyn 89% CLL 29 % si RNA Random Mda7-siRNA

transfer Counts CLL 25 0 5 10 15 20 100 101 102 103 104 PE

Figure 4 p38 MAP kinase is phosphorylated in fresh CLL cells and activated by rIL-24 and endogenous Mda-7/IL-24 protein. For all Western blot studies, the same membranes were hybridized sequentially with anti-phospho-p38 MAPK, stripped and rehybridized with anti-p38 MAPK (total protein) Ab, or other Abs as indicated. (a) Daudi cell line electroporated with empty vector or with vector-containing IL-24 or Mda-7 full-length cDNA was cultured for 48 h before protein extraction and Western blot. (b) p38 MAPK is phosphorylated in CLL but not in normal B-cells. Ex vivo fresh B cells from healthy blood donors (B cell) or CLL patients were purified and lysed. Extracted proteins (60 mg/well) were analyzed by Western blot. Representative CLL panel from six out of 24 different patients and one of six controls. (c) Progressive loss of p38 MAPK phosphorylation in culture (day 0 to day 6), representative from 10 patients. (d) p38 MAPK phosphorylation is inducible by rIL-24, LPS and Mda-7. Left: CLL cells were cultured for 72 h to allow progressive dephosphorylation of p38 MAPK, and were stimulated with 100 ng/ml r-IL-24 or 10 mg/ml LPS for 2 h before Western blot analysis. Right: cells were transfected with empty vector or with Mda7 cDNA and Western blot performed at day 2. Representative of three different experiments. (e) Mda-7/IL-24 mRNA-silencing in CLL specifically inhibits p38 MAPK phosphorylation. 107 CLL cells were transfected with 100 nM of either control or Mda-7/IL-24 small interfering RNA, and cultured for 3 days, before protein extraction. Left: cell scatters of control and Mda-7/IL-24 siRNA-transfected cells at day 1. Histogram shows the 89% efficacy of siRNA transfection. Right: Western blot analysis of cells transfected with random (control) siRNA (left) or Mda-7/IL-24-specific siRNA (right). The same membrane was revealed sequentially with anti-Mda-7, phospho p38 MAPK, total p38 MAPK, bcl2 and Lyn Abs. Representative of one experiment (CLL 25) out of three (samples 6, 18 and 25).

cell death. Another evidence is that the apoptosis induced by was observed in terminally differentiated melanocytes.25,27 Our theophylline in vitro29 was not antagonized by r-IL-24 further failure to detect the protein in serum may be due to a dilution pointing to the specificity of p38 MAPK in CLL survival. To effect by nonsecreting malignant cells that express the receptors definitively demonstrate the effect of intracellular Mda-7/IL-24 and should be able to internalize the cytokine. Alternatively, protein, apoptosis was studied in the three patients samples in malignant cells may inhibit the production of IL-24 by normal which the protein could be efficiently silenced. As shown mononuclear cells. If so, a deficit in IL-24, a pro-TH1 (Figure 5c), apoptosis was augmented in all three experiments in cytokine,26 may contribute to the functional T-helper and Mda-7/IL-24 siRNA-silenced cells as compared to control cells dendritic cell deficits in CLL.30 cultured for 7 days in medium þ autologous serum. Further- p38 MAPK is an important intermediate for the function of more, r-IL-24 and LPS protected cells against Mda-7/IL-24 Mda-7, and is activated by endogenous Mda-7/IL-24 protein or siRNA induced cell death. Thus, endogenous Mda-7/IL-24 by exogenous IL-24 in CLL. p38 MAPK can be proapoptotic in protein mediated its effect – at least partly – through p38 MAPK tumor cell lines,25,27 meanwhile it is constitutively active in phosphorylation. normal liver cells.31 It is obvious that phospho-p38 MAPK has no proapoptotic function in CLL since it is present in fresh cells, and is instead needed for the survival of malignant cells as Discussion inferred from the induction of apoptosis by the MAPK inhibitor SB 203580 and by Mda-7/IL-24 silencing, the latter which We have shown here that CLL B-cells constantly express high inhibited p38 MAPK phosphorylation. Addition of SB 203580 amounts of the tumor suppressor Mda-7/IL-24 protein and of inhibited IL2-mediated cell proliferation in a dose–response Mda-7 and IL-24 mRNA. The strong Mda-7/IL-24 expression in manner, with a 50% inhibition of thymidine incorporation at highly differentiated/activated CLL B-cells is reminiscent of what 2.5 mM (data not shown), whereas apoptosis was detectable at

Leukemia Mda-7/IL-24 and p38 MAP kinase activation in CLL A Sainz-Perez et al 503 4

a 4 10 10 3 3

200 250 11 39 200 250 10 10

2 M1 2 M1 10 10 SSC SSC Counts Counts 1 1 10 10 0 0 0 50 100 150 0 50 100 150

10 10 0 1 2 3 0 200 400 600 800 1000 100 101 102 103 104 0 200 400 600 800 1000 10 10 10 10 104 FSCApo2.7 FSC Apo2.7 Medium +Autologus serum +SB203580

bc100

90 80 80 p<0.05 70 70 60 60 p<0.01 50 50 40 40 30 30 % apoptotic cells 20 20 10 10 0 0 rIL-24 -- ++ -- -- ++ -- -- ++ 61825 LPS -- -- ++ -- -- ++ -- -- SB ------++ ++ ++ -- -- Theo ------++ ++ CLL number

Figure 5 p38 MAPK inhibition induces apoptosis in CLL. FACS analysis of % apoptotic cells as evaluated by staining with Apo 2.7 Ab. (a) Cells were cultured in medium þ 10% autologous serum, with or without 10 mM SB 203580, and stained at day 4 with Apo 2.7 Ab. FACS analysis shows the difference in cell scatter and the % of apoptotic cells in control- vs SB 203580-treated cells. (b) SB 203580-mediated apoptosis in CLL is reversed by r-IL-24 and LPS. In total, 12 CLL samples from distinct patients were cultured in medium þ autologous serum with or without combinations of 100 ng/ml r-IL-24, 10 mg/ml LPS, 10 mM SB 203580 and 100 mM theophylline and stained at day 4 (white histograms) and day 7 (black histograms). (c) Samples from patients 6, 18 and 25 were transfected with control or Mda-7/IL-24-specific siRNA and cultured with or without 100 ng/ml r-IL-24 or 10 mg/ml LPS and stained at day 7 with Apo 2.7 Ab. Histograms: White: control siRNA. Black: Mda-7/IL-24 siRNA. Hatched: Mda-7/IL-24 siRNA þ LPS. Dotted: Mda-7/IL-24 siRNA þ r-IL-24.

higher (45 mM) concentrations. The importance of p38 MAPK in In CLL, the BCR repertoire of malignant cells is suggestive of CLL survival is highlighted by the protective effect of IL-24 and selection by antigen.11 A chronic stimulation likely induces CD5 LPS on SB 203580-mediated apoptosis, inasmuch as LPS expression and cell anergy.15,16 We therefore propose that CLL induces IL-24 production by PBMC.24 Our results confirmed permanent triggering in vivo results in the upregulation of genes the previously published data demonstrating that p38 MAPK is such as CD5 and Mda-7/IL-24, the latter activating p38 MAPK constitutively active and exerts a protective role in CLL.32 We and inhibiting cell death. This suggests that CD5 silencing cannot totally exclude that IL-24 works also through a should be detrimental for CLL survival. In support of this, CD5 p38MAPK-independent mechanism; however, our data sug- ligation induced apoptosis in a CLL subgroup, possibly by gested a competition between IL-24 and SB 203580 for p38 uncoupling CD5 from the BCR or from BCR-associated signaling MAPK activation as evidenced by sequential incubations. molecules.36 We have been able to silence in Indeed, IL-24 augmented p38 MAPK phosphorylation when CLL with a fairly good efficiency. This technology will allow us added several hours after SB 203580, suggesting that the to study the role of selected molecules, upstream of p38 MAPK inhibitory effect of this drug is reversible (data not shown). on the survival of malignant cells. p38 MAPK is an important regulator of cell cycle and survival in CLL, possibly through direct interaction with, and phosphor- 33 ylation of p53 at specific serine sites. p38 MAPK activation by Acknowledgements IL-24 and other factors, such as TGF-b,34 may also contribute to 35 anergy in CLL similarly to that demonstrated in T cells. Since Franc¸oise Gaudin and Herve´ Strub are acknowledged for Mda-7 promoted cell survival, it could be expected that its technical help. This work was supported by two grants from the expression level correlates with the clinical/biological status in ‘Association de Recherche contre le Cancer’, ARC, and SIDAC- CLL, and a quantitative study of Mda-7 and phospho-p38 MAPK TION. should be carried out, although in preliminary studies we failed to observe a difference between Ig-mutated vs unmutated patients. The same question should be addressed for CD5. References Our results suggested that CD5 induced Mda-7 in CLL; however, a direct demonstration was not obtained due to our failure to efficiently silence CD5 either by RNA interference or in culture 1 Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med 1995; 333: 1052–1057; Erratum in: N Engl J Med 1995; with Cyclosporine-A or IL-4, which reversed the PMA-induced 333:1515. CD5 increment, but did not affect its basal level in malignant 2 Hamblin TJ, Oscier DG. Chronic lymphocytic leukaemia: the cells. nature of the leukaemic cell. Blood Rev 1997; 11: 119–128.

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