Post-Transcriptional Regulation of Inducible Nitric Oxide Synthase In

Home , Inducer

Leukemia (2004) 18, 48–56 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions AC Tiscornia1, A Cayota2, AI Landoni3, C Brito1, P Oppezzo4, F Vuillier4, C Robello1, G Dighiero4, R Gabu´s3 and O Pritsch1

1Departamento de Bioquı´mica, de la Facultad de Medicina, Universidad de la Repu´blica, Montevideo, Uruguay; 2Departamento de Ba´sico de Medicina, de la Facultad de Medicina, Universidad de la Repu´blica, Montevideo, Uruguay; 3Servicio de Hematologı´a y Transplante de Me´dula O´ sea, Hospital Maciel, Montevideo, Uruguay; and 4Unite´ d’ Immunohe´matologie, Institut Pasteur, Paris, France

Functional inducible NOS (iNOS) may be involved in the implicated in several physiologic and pathophysiologic me- prolonged lifespan of chronic lymphocytic leukemia cells (B- chanisms.20 Recently, Zhao et al21 and Kolb et al22 reported that CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression inducible NOS (iNOS) protein can be detected in the cytoplasm in normal B lymphocytes and B-CLL cells in pro- and of B-CLL cells and proposed that this should provide further antiapoptotic conditions. Our results demonstrate: (1) The insights into the mechanism controlling proliferation and existence of a new splice variant characterized by a complete apoptosis in these tumor cells. deletion of exon 14 (iNOS 13–1614del), which was preferentially The regulation of iNOS expression is complex and occurs at detected in normal B lymphocytes and may represent an multiple levels, and includes transcriptional and post-transcrip- isoform that could play a role in the regulation of enzyme 23,24 activity. (2) The existence of another alternatively spliced iNOS tional mechanisms. The human iNOS gene corresponds to a mRNA transcript involving a partial deletion of the flavodoxin single 37 kb genomic DNA fragment and consists of 26 exons region (iNOS 13–16neg) was correlated to a decreased B-CLL and 25 introns located on chromosome 17 at position 17 cen– 25 26 cell viability. The 9-b-D-arabinofuranosyl-2-fluoradenine or q11.2 according to Chartrain et al. Recently, Eissa et al fludarabine (F-ara) treatment induced iNOS 13–16neg transcript have reported four sites of alternative splicing of iNOS mRNA, variants, whereas IL-4 enhanced both the transcription of and suggested that this mechanism may be involved in the variants, including these exons (iNOS 13–16pos), and the expression of a 122 kDa iNOS protein. These results suggest regulation of iNOS expression and/or activity. that in B-CLL, a regulation process involving nitric oxide (KNO) The purine analogue 9-b-D-arabinofuranosyl-2-fluoradenine levels could occur by a post-transcriptional mechanism (fludarabine or F-ara) is being increasingly used in the treatment mediated by soluble factors. Our results also provide an insight of lymphoproliferative disorders.27,28 It is proposed to be involved into a new complementary proapoptotic action of F-ara in B- in triggering apoptotic cell death through a complex mechanism, CLL by the induction of particular iNOS splice variants, leading including DNA damage (by incorporating a significant amount of to the activation of a caspase-3-dependent apoptotic pathway. F-ara AMP into DNA), Apaf-1 activation,29 inhibition of ribonu- Leukemia (2004) 18, 48–56. doi:10.1038/sj.leu.2403169 30,31 Published online 23 October 2003 cleotide reductase, DNA primase and DNA ligase. However, Keywords: B-CLL; iNOS; F-ara; IL-4; apoptosis; splicing whereas all these mechanisms have been related to proliferating leukemic cells, B-CLL cells are characterized by the relentless accumulation of tumoral cells with a low ratio of cycling cells blocked at the G1 phase of the cycle. Thus, the precise Introduction mechanism by which F-ara kills the indolent B-CLL cells remains unclear. This work aimed to characterize the iNOS expression in In chronic lymphocytic leukemia (B-CLL), the most common B-CLL cells and evaluate in vitro the influence of proapoptotic K form of leukemia in the Western world, the clonal accumulation (F-ara) and survival (IL-4) signal conditions on the NO pathway of B tumoral cells appears as a consequence of prolonged as a putative mechanism controlling cell survival in B-CLL. survival due to the inhibition of apoptotic cell death rather than increased proliferation.1 This defective apoptosis in B-CLL cannot be accounted by known genetic aberrations.2,3 Since Materials and methods in vitro cultures of purified B-CLL cells undergo spontaneous and rapid apoptosis,4 multiple humoral and cellular factors Patients controlling apoptosis have been postulated to play a role in the prolonged lifespan of B-CLL cells.5–7 Among these signals, After informed consent, according to international regulations, several cytokines, such as interferon alpha,8,9 interleukin (IL)- patient samples were provided by the Hematology and Bone 4,10,11 IL-13,12 IL-613 and IL-8,14 and different drugs have been Marrow Transplantation Service, Hospital Maciel, Montevideo, proposed. Therefore, the size of the B-CLL tumoral population is Uruguay. A total of 20 B-CLL patients (12 stage A, five stage B 32 likely to be regulated directly by extracellular signals that and three stage C) were classified according to Binet staging. determine whether the cell-death program is engaged. All patients were free from clinically relevant infectious Recent work postulated that nitric oxide (KNO), a free radical complications, and were untreated or had not received messenger produced from L-arginine by the action of nitric cytoreductive chemotherapy for at least 3 months prior to oxide synthase (NOS), plays an important role in the control of sample collection. Samples from healthy volunteers donors were apoptosis in various cell types,15–18 including lymphocytes.19 It provided by the National Blood Bank, Montevideo, Uruguay. has been reported that KNO is a potent biologic mediator

Correspondence: Dr A Tiscornia, Avda. Gral Flores 2125, Montevideo Reagents CP 11800, Uruguay; Fax: þ 598 2 924 03 95 Received 29 April 2003; accepted 4 September 2003; Published Dynabeads coated with goat anti-mouse IgG were purchased online 23 October 2003 from Dynal (Oslo, Norway). The monoclonal antibodies iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 49 (MoAbs) anti-T-cell CD3 (clone UCHT 1), antimonocyte CD14 specific upstream and downstream oligonucleotide primers (clone TUK 4), antinatural killer Cell CD-56 (clone MOC-1), (OPERON Technologies Inc.): (1) To amplify the middle region anti-B-cell CD 20 (clone B-Ly1), anti-B-cell CD 19 RPE (clone of iNOS mRNA, we have developed a nested PCR that in a first HD 37) and phycoerythrin-conjugated anti-mouse IgG were round amplified a fragment corresponding to exons 12–19 and purchased from DAKO SA (Paris, France). Tri-Reagent, diami- in a second round amplified a 317 bp fragment corresponding to nofluorescein diacetate (DAF 2DA) and 4,5 diaminofluorescein exons 13–16 by using the following primers: 12F,50-GGC AGG (DAF-2) were obtained from Sigma (St Louis, USA). Rabbit anti- ACG AGA AGC GGA GAC CC-30; 19R,50GCT CCC CCG GCA iNOS polyclonal antibodies (BD Transduction Laboratories, GGT AGT TCA GG-30; afterwards, a second round of PCR was Lexington, KY, USA) were raised against the carboxy-terminal performed by using 13F,50-GGC GTC CCG AGT CAG AGT region of mouse iNOS protein (amino acids 961–1144) and CAC C-30; and 16R,50-GAG CAA AGG CGC AGA ACC GAG crossreact against human iNOS. HRP conjugated anti-rabbit IgG GG-30. Conditions for the first round were as follows: 951C for immunoglobulins, (DAKO) and enhanced chemiluminescence 4 min, then 30 cycles of denaturation at 951C for 30 s, annealing detection Supersignal system (Pierce, Rockford, IL, USA) were at 581C for 1 min and extension at 721C for 1 min, followed by a used for Western blot detection of iNOS protein. Aminoguani- final extension at 721C for 10 min. In the second round, only the dine (AG), bicarbonate salt , No-nitro-D-arginine methyl ester annealing temperature at 601C was modified. (2) To amplify a hydrochloride (D-NAME), and No-nitro-L-arginine methyl ester fragment of 259 bp corresponding to exon 26 of iNOS: 26F,50- hydrochloride (L-NAME) were obtained from Sigma (St Louis, CGG TGC TGT ATT TCC TTA CGA GGC GAA GAA GG-30; and USA). Different concentrations of AG from a 200 mM stock 26R,50-GGT GCT GCT TGT TAG GAG GTC AAG TAA AGG solution of pH 7.4 were used as the iNOS inhibitor. IL-4 were GC-30. Conditions previously reported by Zhao et al21 were obtained from Pharmingen (San Diego, USA). The purine performed. analogue, F-ara was kindly provided by Schering Uruguaya. PCR products were resolved by 2% agarose-gel electrophor- esis and stained with ethidium bromide. In an attempt to control the cDNA synthesis, we performed the amplification of the PBMC isolation, B-lymphocyte purification and cell constitutive glyceraldehyde phosphate dehydrogenase (GAPD) culture mRNA under the same conditions as iNOS amplification in the first round using the following primers: GAPD-F,50-CTG AGA Peripheral blood mononuclear cells (PBMCs) were isolated by ACG GGA AGC TTG TC-30; and GAPD-R,50-CCT GCT TCA centrifugation over a Ficoll–Hypaque layer (Lymphoprep, CCA CCT TCT TG-30. GIBCO-Life Technologies). B-CLL lymphocytes were purified Amplified fragments were isolated and purified with Gene- by negative selection using magnetic beads (Dynabeads) coated Clean Kit (Bio 101) and cloned in pGEMs-T (Promega). with MoAbs anti-CD3, CD14 and CD56 according to the Electrocompetent XL1-Blue bacteries were transformed by manufacturer’s instructions. The nonattached cells (95–98% B electroporation (BTX Electroporation System 600; Genetronics lymphocytes as assessed by cytometric analysis) were either Biomedical Ltd). Positive colonies were expanded and small- processed for RNA or protein extraction or resuspended in RPMI scale plasmid purification was performed using the Wizards 1640 culture medium supplemented with 10% heat-inactivated Plus SV Minipreps DNA Purification System (Promega). Purified fetal bovine serum (LPS free; Life Technologies, Grand Island, plasmids were sequenced in an ABI 377 Sequencer with specific s NY, USA), 4 mM glutamine, 1 mM HEPES, 1 mM sodium primers for pGEM-T . pyruvate, 200 IU/ml penicillin and 50 mg/ml streptomycin (GIBCO-Life Technologies, Rockville, MD, USA). Cultures were followed up for 48 h in a 5% CO2 incubator in different iNOS protein detection by Western blot conditions: untreated (medium alone), in the presence of either 10 mg/ml of F-ara, 1000 U/ml of IL-4, different doses of AG and For Western blot analysis, pellets from cell suspensions were 5mM of L-NAME or D-NAME. B lymphocytes from healthy lysed in sample buffer (10 mM Tris-HCl, pH 6.8, 1% SDS and 5% donors were purified by positive selection by using magnetic glycerol), followed by boiling for 5 min, sonication and beads coated with MoAbs anti-CD19 or CD20, and immediately determination of protein concentration by the bicichoninic acid processed for RNA extraction. method (Pierce, Rockford, IL, USA). Samples were stored at The murine macrophage cell line J774, kindly provided by À701C until use. Total proteins (70 mg) were electrophoresed on Noel Alvarez, was used as a known cellular system that an 8% resolution SDS-polyacrylamide gel. Then, the proteins produces high fluxes of KNO when activated with 500 IU of were electrotransferred onto 0.45 mm pore size nitrocellulose IFN-g and 30 mg/ml of LPS. membranes, and nonspecific binding sites were blocked over- night in blocking buffer (5% BSA and 0.1% Tween in PBS). For iNOS immunodetection, nitrocellulose filters were probed with iNOS mRNA analysis the rabbit anti-iNOS polyclonal antibody diluted 1/1000 in blocking buffer for 2 h at room temperature. After three washes Total RNA was extracted from 5–10 Â 106 purified B lympho- with PBS–Tween 0.1%, blots were further incubated for 2 h with cytes by the guanidinium thiocyanate–phenol–chloroform HRP conjugated anti-rabbit IgG Ab diluted in PBS–BSA 1 mg/ml. method.33 cDNA synthesis was performed in a 20 ml reaction Finally, the filters were developed by using the enhanced volume at 421C for 50 min by using an oligo(dT)15À18 primer, chemiluminescence detection system (Supersignal, Pierce). 200 U of Superscriptt II RNAse HÀ Reverse Transcriptase (GIBCO BRL Life Technologies, Grand Island, NY, USA) and 0.5 mM of each deoxyribonucleotide. PCRs were performed on a Cell viability and caspase-3 activity by cytofluorometric Hybaid Thermocycler (Life Technologie) in a 25 ml tube reaction analysis containing PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl) with 1.5 mM MgCl2, 200 mM dNTP, 1 ml of cDNA, 0.625 U Taq DNA After 48 h of culture in different conditions, B-CLL cell viability polymerase (GIBCO) and 200 nM of each of the following and caspase-3 activity were analyzed on a FACScan flow

Leukemia iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 50 cytometer (Beckton Dikinson). To evaluate cell viability and DAF-DA was added in order to measure DAF-2T generation in apoptotic death, 1 Â 106 cells were washed in PBS, and stained the same conditions described above. Only 1 h of time assay with propidium iodide and Annexin V–FITC (Annexin V–FITC was required to obtain quantifiable values. Apoptosis Detection Kit (Sigma, St Louis, USA)) according to the The mean values for the first and last five measures at each manufacturer’s instructions. A viability index was expressed as well were calculated, and their differences were obtained. These the percentage of viable cells over the total cell count (live and values were interpolated into a calibration curve obtained dead cells). A PhiPhiLux G1D2 kit (OncoImmunin, Inc., previously with 1 mM DAF-2 and different doses of authentic K Gaithersburg, MD, USA) was used to evaluate caspase-3 activity NO diluted in PBS 100 mM to maintain the pH at 7.4. Then following the manufacturer’s instructions. Briefly, the purified B fluxes of KNO were calculated taking into account the time of cells were incubated at 371C for 60 min with 10 mM DEVD G1D2 the assay. substrate solution. Cells were then washed once with ice-cold cytometry dilution buffer and resuspended in 500 ml of fresh dilution buffer. Samples were analyzed on the FACSscan flow Statistical analysis cytometer using the FL1 channel. An activity index was expressed as percentages of cells with respect to caspase-3 For statistical analysis, the nonparametric Mann–Whitney rank activity of total cells (live and dead cells). test was used and P-value calculation was obtained.

K NO production in living cells Results

K In order to evaluate NO production by B lymphocytes, the B lymphocytes from healthy and leukemic patients membrane-permeable diaminofluorescein diacetate (DAF-DA) express different iNOS mRNA transcripts was used. DAF-DA is quickly transformed in DAF-2 by esterases in the cytosol, and this probe can react with N2O3 (produced by In order to detect iNOS mRNA, we have developed two PCR K NO and oxygen reaction) to form the highly fluorescent assays. One of them was reported previously21 and amplified a triazolofluorescein (DAF-2T), which could be detected by region included in exon 26 at the 30 end of the iNOS mRNA fluorescence microscopy and by fluorimeter equipment. (iNOS 26). As shown in Table 1 for purified B cells in basal Fluorescence microscopy: One to five million cells were conditions, 18 out of 20 (90%) patients displayed the expected incubated with 1 mM DAF-DA for 90 min at 371C in culture 259 bp fragment, whereas only three out of seven (43%) healthy medium without serum. After centrifugation, the pellet was donors were positive (data not shown). suspended in 100 ml of medium with mouse MoAb anti-human In our laboratory, we previously developed a nested PCR CD14 monocyte marker (DAKO), and incubated at 41C for strategy to amplify low levels of iNOS mRNA expression in 30 min. After washing with PBS, phycoerythrin-conjugated anti- lymphocyte cells. The first round of amplification spanning the mouse IgG was added to cells and incubated for 30 min at 41C. region from exons 12 to 19 and the second round amplified an The cells were finally washed and suspended in 50 ml of cold expected fragment of 317 bp from exons 13 to 16, as depicted in RPMI. In order to control the specificity of DAF-DA NO Figure 1(a). As shown in Table 1 and for representative cases in detection, PBMC obtained from B-CLL patients were cultured for Figure 1(b), 16 out of 20 (80%) patients in basal conditions 48 h in the presence of 5 mM of both NOS inhibitors: AG and displayed a highly represented 317 bp fragment (iNOS 13– L-NAME and 5 mM of D-NAME (inactive isomer of L-NAME). 16pos), whereas 12 out of 20 (60%) patients displayed an The cells were collected and incubated with 1 mM DAF-DA and additional 212 bp fragment. In only two cases (patients 9 and 13, anti-CD19-RPE MoAb for 60 min at 41C in culture medium Table 1), the fragment corresponding to exon 26 was not without serum. Images were obtained with a fluorescence amplified and this result can be explained by the difference in microscopy (NIKON Fluorescence Microscopy Optiphot) and the sensitivity of the two techniques (nested vs classical RT- Digital Camera (KodaK DC290 Zoom). PCR). In four out of 20 (20%) patients, no expression of 317 or Fluorimetric analysis: Purified B-CLL were suspended in Krebs 212 bp fragments was observed (iNOS 13–16neg), whereas the Ringer phosphate buffer supplemented with 5 mM arginine fragment corresponding to exon 26 was present in all these (KRP-Arg): 120 mM NaCl, 4.8 mM KCl, 0.54 mM CaCl2, 1.2 mM cases, indicating the presence of iNOS mRNA, and suggesting a MgSO4,11mM glucose and 15.9 mM sodium phosphate, pH deletion in this middle region. 7.2. A total of 50 000 cells were plated in a 96-well culture In contrast, in B lymphocytes from healthy donors, high levels plate, then 1 mM DAF-DA was added in a 200 ml final volume of the 212 bp fragment were detected in five out of seven (71%) and finally, the DAF-2T fluorescence was measured in a kinetic samples, whereas two out of seven (29%) expressed low levels mode on a plate fluorimeter (Fluostart Galaxy BMG Lab of the iNOS 13–16pos. Nucleotide sequence demonstrated that Technologies, Germany) setting the following conditions: all fragments were 100% homologous to reference iNOS excitation and emission at the top of the plate with wavelength sequence,25 but as shown in Figure 2, the 212 bp fragment at 485 and 520 nm, respectively, gain at 20, temperature displays a 105 bp deletion corresponding to complete exon 14 controlled at 371C and shaking before each measure. The pH (iNOS 13–1614del). These results show that a yet unreported of final DAF-DA solution was controlled and it was 7.4. When B alternative splice form involving a deletion of exon 14 is highly lymphocytes were tested, a long-time assay was required (5 h) represented in normal B lymphocytes. and cell viability was controlled at the final time assay by exclusion of trypan blue colorant (100% with respect to initial cell viability). As a positive control, we assayed the murine Proapoptotic culture conditions modulate the macrophage J774 cell line, which was cultured to confluence in expression of iNOS mRNA variants in B-CLL a 96-well culture plate, and then it was activated in culture medium for 6 h at 371C with LPS 30 mg/ml and IFN-g 500 U/ml. B-CLL cells undergo spontaneous apoptosis following exposure 4 The supernatant was removed and KRP-Arg containing 1 mM to culture conditions and this process is enhanced by F-ara in

Leukemia iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 51 Table 1 Clinical characteristics of B-CLL patients and mRNA iNOS expression

Patient Age (years) / Binet Lymphocytes/ml T ¼ 0 48 h untreated 48 h F-ara treatment no. sex stage Exons 13–16 (1) Exon 26 (2) Exons 13–16 (1) Exon 26 (2) Exons 13–16 (1) Exon 26 (2)

1 80/M C 35 000 + + À + À + 2 56/M B 45 000 ++++++ 3 70/M B 26 000 *+ +++++ 4 59/F A 53 000 *+ + + + À + 5 52/F A 34 000 ++++À + 6 76/M C 81 000 + + *+ + *+ + 7 77/F C 172 000 À + À + À + 8 78/F B 20 000 *+ + *+ + *+ + 9 68/F A 16 000 *+ À *+ À *+ + 10 73/M B 15 000 *+ + + + *+ + 11 83/F A 7000 *+ + + + *+ + 12 69/F A 26 000 *+ + À + À + 13 83/F B 46 000 *+ ÀÀ+ À + 14 53/M A 69 000 À +++++ 15 70/M A 21 000 *+ + À + À + 16 56/F A 45 000 *+ + À + À + 17 79/M A 110 000 *+ +++++ 18 70/M A 18 000 À +++À + 19 56/M A 15 000 À + À + À + 20 72/M A 20 000 *+ + *+ + *+ + M, male; F, female; T ¼ 0 (basal conditions). (1) (+) correspond to expected 317 bp fragment. (1) (*) correspond to 212 bp additional fragment. (2) (+) correspond to expected 259 bp.

Figure 1 Analysis of iNOS mRNA in purified B cells from B-CLL patients and healthy donors. (a) Schematic representation of iNOS domains (up) at the protein level and exon localization (down) at the mRNA level. Primers for nested PCR are represented (designed by a number indicating the exon followed by capital letter F for forward and R for reverse): 12F and 19R for the first round, and 13F and 16R for the second round amplifying the expected 317 bp fragment from exons 13 to 16 (iNOS 13–16); 26F and 26R were used to amplify 259 bp fragments from exon 26 (iNOS 26). (b) iNOS 13–16 PCR results from purified B cells of different representative B-CLL patients (1–8) and six healthy donors (9–14) in basal conditions. Samples 1, 2, 5, 6, 7, 8, 11 and 14 amplified the expected 317 bp fragment: (iNOS 13–16pos) whereas 1, 4, 5, 6, 7, 9, 10,11, 13 and 14 amplified a 212 bp fragment (iNOS 13–1614del).

29 vitro treatment. We have evaluated these two proapoptotic 20) were iNOS 13–16neg. Similar results were obtained in the culture conditions by analyzing cell viability and iNOS mRNA untreated group: 19 out of 20 (95%) patients displayed the expression. fragment corresponding to exon 26, and 13 out of 20 (65%) cell As shown in Table 1, the iNOS fragment corresponding to samples expressed the fragment iNOS 13–16pos, whereas seven exon 26 was constantly present in the 20 studied patients after out of 20 (35%) were iNOS 13–16neg. These results suggest that 48 h of F-ara treatment. However, in the same culture whereas all the cases displayed iNOS transcripts corresponding 0 conditions, the iNOS 13–16pos could only be detected in 10 to the 3 end of mRNA in proapoptotic conditions, the central out of 20 (50%) of these cell samples. The other 50% (10 out of region of iNOS mRNA could be ampliﬁed in only half of them.

Leukemia iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 52

Figure 2 Sequence comparison of iNOS PCR fragments ampliﬁed from exons 13 to 16: sequence alignment of the 317 and 212 bp ampliﬁed fragments was compared to a reference iNOS mRNA from Gene Bank (GI 687680). A deletion of 105 bp in the 212 bp fragment involves the complete exon 14.

Figure 3 B-CLL cell viability and iNOS 13–16 PCR results in proapototic culture conditions: purified B cells from B-CLL patients untreated (À) or treated with F-ara 10 mg/ml ( þ ) for 48 h. (a, b) Two representative kinetic analyses of iNOS 13–16 PCR results at 0, 24 and 48 h of culture. GAPD was amplified as control for cDNA. (c) Cell viability and iNOS 13–16 PCR results at 48 h of culture in vitro apoptotic conditions were compared. Two groups following iNOS 13–16 PCR results – iNOS 13–16neg or iNOS 13–16pos (the absence or presence of the amplified fragment, respectively) – were defined. Cell viability was evaluated by flow cytometry and a viability index was expressed as the percentage of viable cells over the total cell counts. Cell viability vs iNOS 13–16 PCR results for untreated conditions (left) and for treated cells with 10 mg/ml F-ara (right). Bars represent the % of cell viability index means7standard deviation (s.d.) and n indicates the number of patients included in each group.

7 Figure 3 displays the kinetics of two representative patients for the iNOS 13–16pos group and 15.9 8.6% for iNOS 13– studied for iNOS mRNA expression of B cells untreated and 16neg (P-value ¼ 0.0011). These results suggest that the expres- treated with F-ara at 0, 24 and 48 h of culture. In Figure 3(a), the sion of the iNOS variant displaying complete 13–16 exons is suppression of iNOS 13–16pos after 48 h of treatment with F-ara associated to higher cell survival. was representative for seven patients, whereas in Figure 3(b), the F-ara treatment did not affect the iNOS 13–16pos expression representing 10 patients. IL-4 treatment protects against in vitro apoptotic death According to the absence or the presence of iNOS 13–16 PCR and modulates the expression of iNOS variants products after 48 h of culture in proapoptotic conditions, samples were segregated into two groups. As depicted in To analyze the iNOS variant expression in different culture Figure 3(c), the mean index of viability associated to the conditions, five patients were evaluated. Purified B cells were 7 untreated condition was 55 16.2% for the iNOS 13–16pos cultured for 48 h: (a) untreated (medium alone), (b) with 10 mg/ 7 group, and 27.6 15.5% for the iNOS 13–16neg group ml F-ara, (c) with 1000 U/ml IL-4 and (d) with 1000 U/ml IL-4 (P ¼ 0.0063). In F-ara-treated B-CLL cells, it was 40.3713.1% and 10 mg/ml of F-ara. All five cases were evaluated for cell

Leukemia iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 53 viability, caspase-3 activity, detection of exons 13–16 and 26 from iNOS mRNA and iNOS protein expression. As shown in Figure 4(a), the mean index of viability after 48 h culture in the presence of IL-4 was 8772.4%, whereas for untreated cells it was 60719.1%. The mean indexes of viability of F-ara- and IL-4 þ F-ara-treated cells were 41719.0 and 64.872.4%, respectively. In all five cases, exon 26 expression from iNOS mRNA could always be detected whatever the culture condition. As for exons 13–16 expression in either the IL-4 or IL-4 þ F-ara groups, only the 317 bp fragment was detected in four out of five cases (data not shown). Protein expression was detected with a specific polyclonal antibody directed against the carboxy-terminal region of iNOS protein (amino acids 961–1144) corresponding to exons 21–25. Four different immunoreactive bands were detected. Although the theoretical mass of human iNOS protein is 131 kDa, the high molecular weight protein detected in purified B-CLL lysates was 122 kDa and displayed a slightly lower molecular weight than that detected in the 130 kDa positive control obtained from a lysate of murine macrophage J774 cell line stimulated with IFN- g and LPS. The other three bands corresponded to molecular weights of 69, 60 and 21 kDa. As depicted in Figure 4(b) for a representative case, both the IL-4 treatment and IL-4 þ F-ara treatment resulted in high iNOS protein expression. These results indicate that the survival signal resulting from IL- 4 treatment is associated with the detection of exons 13–16 by PCR and/or high levels of iNOS protein. To confirm that cell death in the five purified B-CLL cells was the consequence of apoptotic machinery activation, we assayed caspase-3 activity by cytometric analysis in all culture conditions. As shown in Figure 4(c), the mean values of active caspase-3-positive cells for five patients were 25 and 37.8% for untreated and F-ara- treated cells, respectively. IL-4 treatment resulted in a significantly lower percentage of cells with caspase-3 activity (5.6%), whereas the association of IL-4 þ F-ara treatment increased this percentage to 23.2%. These results confirmed that F-ara- induced cell death was through the induction of apoptosis and that IL-4 treatment was able to downregulate proapoptotic signals in these culture conditions, but was unable to avoid F-ara apoptotic cell death activation completely (IL-4 þ F-ara culture condition). Given that this latter condition results in the overexpression of the splice variant including exons 13–16 (Figure 4(b)), these results suggest that IL-4 induces the expression of this variant, succeeds to rescue a number of cells Figure 4 Cell viability, caspase-3 activity, iNOS mRNA and iNOS from F-ara-induced cell death, but is not sufficient to prevent protein detection after in vitro treatment of B-CLL with IL-4. (a) B-CLL apoptosis by F-ara completely. cell viability at 48 h in different culture conditions. Purified B-cells from five B-CLL patients were cultured for 48 h in different conditions – untreated (untreat.), with 10 mg/ml F-ara (F-ara), 1000 U/ml IL-4 (IL- K 4), 10 mg/ml F-ara and 1000 U/ml IL-4 (IL-4 þ F-ara) – and cell viability Low levels of NO produced by B-CLL could modulate was evaluated by cytometric analysis. Bars represent a viability index apoptotic pathway means7s.d. (percentage of viable cells over total cells count). (b) A representative case from the five above analyzed B-CLL patients: To evaluate KNO production in living PBMC cells, membrane- results for cell viability (up), PCR iNOS 13–16, iNOS 26 and GAPD K (middle), and Western blot (down) were aligned. Letters are used to permeable DAF-DA was used as a probe for NO detection name samples: A – activated murine macrophage sample (positive simultaneously to monocytes cells identification as known control for Western blot), B – molecular marker of Western blot, C – K NO-producing cells. Whereas in B-CLL cells we detected samples from purified B cells from a B-CLL patient in basal conditions highly green fluorescence from DAF-2T in lymphocyte cells, in (T ¼ 0). The following letters correspond to samples from 48 h culture PBMC from healthy donors positive CD-14 cells were mainly conditions: D – untreated, E – treated with 10 mg/ml F-ara, F – treated stained as depicted in Figure 5(a). These results suggested that B with 1000 U/ml IL-4 and G – treated with 10 mg/ml F-ara and 1000 U/ ml IL-4. (c) B-CLL caspase-3 activity at 48 h of different culture lymphocytes from B-CLL patients could produce increased K conditions. Purified B cells from five B-CLL patients were cultured for levels of NO compared to normal B cells. As shown in 48 h without drug treatment (untreated.), with 10 mg/ml F-ara (F-ara), Figure 5(b), the green fluorescence obtained on B lymphocytes 1000 U/ml IL-4 (IL-4), 10 mg/ml F-ara and 1000 U/ml IL-4 (IL-4 þ F- from CLL patients was inhibited in an important fraction of B ara), and caspase-3 activity was evaluated quantifying the fluorogenic cells with the NOS inhibitors AG and L-NAME, whereas no cleaved product by flow cytometry. Bars represent activity index means7s.d. (expressed as percentage of cells with caspase-3 activity inhibition was detected by using the D-NAME isomer. with respect to total cells).

Leukemia iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 54 Experiments evaluating KNO ﬂuxes produced by living B-CLL We selected an indirect method to evaluate iNOS activity in cells and activated murine macrophage J774 cells were the modulation of B-cell survival by using AG (a speciﬁc iNOS performed. KNO production in living B-CLL cells has been inhibitor). Cell viability and caspase-3 activity were measured reported previously (1.5 pmol KNO/h/106 cells).21 In agree- after 48 h of cell culture with different AG concentrations. ment, as shown in Figure 5(c), B-CLL cells showed a mean value Figure 5(d) shows that the percentage of cells with caspase-3 of 4 pmol KNO/h/106 cells, which contrasts with the 392 pmol/ activity directly correlates to increasing AG concentrations, h/106 cells KNO production of IFN-g-LPS-activated murine whereas the converse holds true for cell viability, suggesting that macrophage. These very low levels from B-CLL cells are at the iNOS activity could modulate apoptotic cell death in B-CLL. limit of the assay sensitivity and constitute a methodological Overall, our results in B-CLL cells showed that an increased limiting step in order to correlate the KNO produced by the F-ara apoptotic effect was associated with the expression of presence of the above different iNOS splice variants with cell iNOS variant without exons 13–16 (iNOS 13–16neg) and low viability. levels of iNOS protein. In contrast, IL-4 treatment resulted in the overexpression of iNOS variant containing exons 13–16 (iNOS 13–16pos), and an important increase of iNOS protein expression.

Discussion

KNO produced by a constitutively expressed iNOS has been proposed to be implicated in the prolonged lifespan of B lymphocytes in B-CLL patients.21 Upregulation of iNOS expression either by CD-2321 ligation or by IL-4 treatment34 is associated with the protection of cultured B-CLL cells from apoptotic death. The mechanisms proposed for KNO antiapoptotic action in lymphocytes (reviewed in Kolb35) are related to: (a) a dependent or independent cGMP pathway, (b) caspase- 3 inhibition by S-nitrosylation of the Cys-163 in the catalytic site, (c) prevention by KNO of caspase-8-mediated cleavage of the antiapoptotic protein bcl-2, as a way to avoid cytochrome c release and caspase-3 activation and (d) the mitochondrial hyperpolarization by inhibition of cytochrome c oxidase.36 The regulation of iNOS expression is complex and occurs at multiple sites, including transcriptional and post-transcriptional mechanisms.23,24 In this work, we have analyzed two regions of iNOS mRNA: the 30 end (exon 26) and a conserved central region (exons 13–16). In agreement with Zhao et al21 and Levesque et al,34 B lymphocytes from 90% of B-CLL patients constitutively expressed iNOS transcripts as assessed by RT-PCR of exon 26 in contrast to a 43% positivity observed for healthy donors. Different from normal B cells, these results suggest that there is an in vivo basal transcription for the iNOS gene, in the case of B-CLL cells. The role of this phenomenon in CLL pathogenesis and the regulation pathways involved in it remain to be elucidated. With respect to exons 13–16 expression, the expected 317 bp

K and an additional 212 bp fragments were amplified in B-CLL. Figure 5 NO production in living cells: (a) PBMC from healthy Interestingly, in normal B cells, this latter fragment, resulting donors (left) and B-CLL patients (right) were exposed to DAF-DA and from a complete exon 14 deletion, was highly expressed. In a then monocytes cells were marked with anti-CD14 MoAb and 26 phycoerythrin-conjugated antimouse IgG. The green fluorescence of previous work, Eissa et al reported four different splice variants DAF-2T and the red fluorescence by phycoerythrin-conjugated anti- for iNOS: (a) deletion of exon 5 that results in a truncated mouse IgG were obtained with appropriate filter in a fluorescence protein, (b) deletion of exons 8–9, (c) deletion of exons 9–11 and microscopy. Arrows in the picture identify a monocyte cell. (b) (d) deletion of exons 15–16. Thus, here we report a new splice Inhibition of NOS activity was performed by culturing PBMC from B- variant characterized by complete exon 14 deletion. This CLL patients for 48 h with 5 mM of AG, L-NAME or D-NAME and then K deletion involves the flavodoxin region, and could modify the exposing to 1 mM DAF-DA and anti-CD19-RPE. (c) – NO fluxes produced by living B-CLL cells in basal conditions and by activated FMN domain, which plays a role in electron transfer from the (LPS 30 mg/ml and IFN-g 500 U/ml) murine macrophage J774 cells for NADPH to the Hem domain. We can speculate that this iNOS 6 h were calculated from a quantitative fluorimeter assay using the variant could be a post-transcriptional mechanism implicated in membrane-permeable DAF-DA as described in Materials and meth- the downregulation of KNO levels. Ongoing work in our lab is 7 K ods. Bars correspond to means s.d. for triplicates of the activated directed to compare enzymatic activity and NO production by J774 cell line and for B-CLL cells from five different patients. (d) B-CLL recombinant full-length iNOS protein and iNOS protein with a cell viability and caspase-3 activity following incremental doses of an iNOS inhibitor. Purified B-CLL cells from a patient were cultured for deleted exon 14. 48 h with increasing doses of AG. Cell viability and caspase-3 activity Interestingly, in some B-CLL patients, we could detect the were measured by flow cytometry, as mentioned above. presence of exon 26 (iNOS 26), but neither the 317 bp nor the

Leukemia iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 55 212 bp fragment (NOS 13–16neg), which could suggest the than murine macrophage iNOS (131 kDa). The theoretical existence of iNOS splice variant, including deletion of exons molecular mass for human iNOS protein is 131 kDa, and the 15–1626 that causes an in-frame deletion also involving the 9 kDa mass difference could be explained either by alternative flavodoxin region.26 Zhao et al21 reported the spontaneous splicing or differences in glycosylation. Recent work45,46 expression of iNOS mRNA by two pairs of primers, one of them suggested that a novel mechanism involving iNOS ubiquitina- amplifying exon 26 and the other exons 8–11, whereas tion and proteasomic proteolysis could also play a role in the Levesque et al34 found similar results for primers amplifying regulation of iNOS activity. As previously proposed by Zhao exons 9–10. These results indicate that in B-CLL cells, splicing et al21 and Coers et al,47 the small molecular weight bands variants involving either exons 8–9 or 9–11 have never been revealed by Western blot could be considered as proteolytic detected. Interestingly, exon 8 plays a major role in the degradation products. However, further work in this direction dimerization process implicated in full enzymatic activity.37,38 should be performed in order to confirm this hypothesis. When purified B-CLL cells were cultured in proapoptotic In conclusion, we report a new iNOS splice variant conditions (untreated and F-ara treatment), exon 26 could be characterized by complete deletion of exon 14, which is highly detected in almost all cases, whereas exons 13–16 could be represented in normal B lymphocytes and might be related to amplified in only half of them. In both conditions, cell viability the downregulation of KNO production. Our results also favor was significantly higher in the group expressing exons 13–16 the existence of post-transcriptional mechanisms involved in when compared to nonexpressing cases. As for PBMC and iNOS regulation, which may be important in the modulation of purified B cells treated with F-ara for 48 h, cell viability cell life or cell viability in B-CLL. They also suggest a new appeared better sustained in PBMC, which was associated to complementary proapoptotic action of F-ara in B-CLL by the the presence of low levels of iNOS 13–16pos (data not shown). induction of a particular iNOS splice variant, leading to the These results suggest the presence of an iNOS expression post- activation of caspase-3-dependent apoptotic pathway. transcriptional control, mediated by other mononuclear cell delivered signals, which could prevent cell death by inducing iNOS mRNA variants containing exons 13–16. Acknowledgements IL-4 can prevent spontaneous apoptosis of cultured B-CLL cells and IL-4 and IFN-g can induce iNOS expression in these This work was supported in part by grants from Comisioń cells.5,34 iNOS expression is typically regulated in a synergistic Honoraria de Lucha contra el Cańcer, CSIC (Universidad de la manner by a combination of inducers of the NF-kB pathway and Repu´blica, Uruguay), Schering Uruguaya and Fundacioń Manuel by interferon–Jak–STAT pathways, except in the instance where Pe´rez. We thank Rafael Radi, Sergio Bianchi, Nicola´s Arrambide, single agents are effective for iNOS induction.24 The iNOS Gerard Dumas and Yuri Vasconcelos for critical discussion. promoter contains several interferon-g activation sites (GAS 39 elements) regulated by STAT-1 and STAT-6. Recent reports References have demonstrated that in B-CLL, STAT-1 is activated40 and 41 STAT-6 nuclear translocation is induced by IL-4 and controls 1 Dameshek W. Chronic lymphocytic leukemia an accumulative iNOS expression at the transcription level. Our results show that disease of immunolgically incompetent lymphocytes. Blood 1967; IL-4 treatment is correlated to the inhibition of apoptotic cell 29 (Suppl.): 566–584. death, the amplification of iNOS variants containing exons 13– 2 Reed JC. Molecular biology of chronic lymphocytic leukemia. 16 and increased iNOS protein expression. Semin Oncol 1998; 25: 11–18. 3 Keating MJ. Chronic lymphocytic leukemia. Semin Oncol 1999; These results suggest the existence of a possible fine control of K 26: 107–114. NO levels through post-transcriptional regulation mechanisms 4 Collins RJ, Verschuer LA, Harmon BV, Prentice RL, Pope JH, Kerr mediated by soluble factors like IL-4. This cytokine induces JF. Spontaneous programmed death (apoptosis) of B-chronic iNOS splice variants associated with cell survival. This is in lymphocytic leukaemia cells following their culture in vitro. Br J agreement with a recent report using oligonucleotide micro- Haematol 1989; 71: 343–350. arrays, demonstrating a five-fold increased expression of the IL-4 5 Mainou-Fowler T, Prentice AG. Modulation of apoptosis with cytokines in B-cell chronic lymphocytic leukaemia. Leukemia receptor in CLL B-cells when compared to normal tonsilar Lymphoma 1996; 21: 369–377. 42 CD5 þ B cells. On the other hand, a potent increase of IL-4 6 Panayiotidis P, Jones D, Ganeshaguru K, Foroni L, Hoffbrand AV. levels in T cells from CLL patients was also reported.43,44 Thus, Human bone marrow stromal cells prevent apoptosis and support iNOS mRNA variants induced by IL-4 signaling pathway could the survival of chronic lymphocytic leukaemia cells in vitro. Br J play a role in the survival advantage displayed by leukemic Haematol 1996; 92: 97–103. B-CLL cells. 7 Castejon R, Vargas JA, Romero Y, Briz M, Munoz RM, Durantez A. Modulation of apoptosis by cytokines in B-cell chronic lympho- As predicted, exon deletions implicated in the flavodoxin cytic leukemia. Cytometry 1999; 38: 224–230. region might produce either a suppression or a decrease of 8 Jewell AP, Worman CP, Lydyard PM, Yong KL, Giles FJ, Goldstone K NO production by these alternative splicing variants. The AH. Interferon-alpha up-regulates bcl-2 expression and protects B- methodological limiting step to quantify low levels of KNO was CLL cells from apoptosis in vitro and in vivo. Br J Haematol 1994; the reason why we could not confirm this idea. Methodological 88: 268–274. progress in quantification procedures may allow one in the 9 Panayiotidis P, Ganeshaguru K, Jabbar SA, Hoffbrand AV. Alpha- interferon (alpha-IFN) protects B-chronic lymphocytic leukaemia future to better analyze these subtle differences and to better cells from apoptotic cell death in vitro. Br J Haematol 1994; 86: define the role of the different iNOS splice variants. Also, the 169–173. results obtained with different AG concentrations showed that 10 Panayiotidis P, Ganeshaguru K, Jabbar SA, Hoffbrand AV. the inhibition of KNO production was also correlated to Interleukin-4 inhibits apoptotic cell death and loss of the bcl-2 apoptotic death. protein in B-chronic lymphocytic leukaemia cells in vitro. Br J Four different molecular weight-specific iNOS bands were Haematol 1993; 85: 439–445. 11 Dancescu M, Rubio-Trujillo M, Biron G, Bron D, Delespesse G, detected by Western blot with an antibody directed against the Sarfati M. Interleukin 4 protects chronic lymphocytic leukemic B carboxy terminus of the iNOS protein. In all cases, the higher cells from death by apoptosis and upregulates Bcl-2 expression. band, with a molecular weight of 122 kDa, was slightly smaller J Exp Med 1992; 176: 1319–1326.

Leukemia iNOS post-transcriptional regulation in B-CLL AC Tiscornia et al 56 12 Chaouchi N, Wallon C, Goujard C, Tertian G, Rudent A, Caput D nine 50-triphosphate. A mechanism of DNA synthesis inhibition. et al. Interleukin-13 inhibits interleukin-2-induced proliferation J Biol Chem 1993; 268: 7179–7185. and protects chronic lymphocytic leukemia B cells from in vitro 31 Yang SW, Huang P, Plunkett W, Becker FF, Chan JY. Dual mode of apoptosis. Blood 1996; 87: 1022–1029. inhibition of purified DNA ligase I from human cells by 9-beta-D- 13 Reittie JE, Yong KL, Panayiotidis P, Hoffbrand AV. Interleukin-6 arabinofuranosyl-2-fluoroadenine triphosphate. J Biol Chem 1992; inhibits apoptosis and tumour necrosis factor induced proliferation 267: 2345–2349. of B-chronic lymphocytic leukaemia. Leukemia Lymphoma 1996; 32 Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J 22: 83–90. et al. A new prognostic classification of chronic lymphocytic 14 Francia di Celle P, Mariani S, Riera L, Stacchini A, Reato G, Foa R. leukemia derived from a multivariate survival analysis. Cancer Interleukin-8 induces the accumulation of B-cell chronic lympho- 1981; 48: 198–206. cytic leukemia cells by prolonging survival in an autocrine fashion. 33 Chomczynski P, Sacchi N. Single-step method of RNA isolation by Blood 1996; 87: 4382–4389. acid guanidinium thiocyanate–phenol–chloroform extraction. 15 Brune B, von Knethen A, Sandau KB. Nitric oxide and its role in Anal Biochem 1987; 162: 156–159. apoptosis. Eur J Pharmacol 1998; 351: 261–272. 34 Levesque MC, Misukonis MA, O’Loughlin CW, Chen Y, Beasley 16 Dimmeler S, Zeiher AM. Nitric oxide and apoptosis: another BE, Wilson DL et al. IL-4 and interferon gamma regulate paradigm for the double-edged role of nitric oxide. Nitric Oxide expression of inducible nitric oxide synthase in chronic lympho- 1997; 1: 275–281. cytic leukemia cells. Leukemia 2003; 17: 442–450. 17 Nicotera P, Brune B, Bagetta G. Nitric oxide: inducer or suppressor 35 Kolb JP. Mechanisms involved in the pro- and antiapoptotic role of of apoptosis? Trends Pharmacol Sci 1997; 18: 189–190. NO in human leukemia. Leukemia 2000; 14: 1685–1694. 18 Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide 36 Beltran B, Mathur A, Duchen MR, Erusalimsky JD, Moncada S. The produced by human B lymphocytes inhibits apoptosis and Epstein– effect of nitric oxide on cell respiration: a key to understanding its Barr virus reactivation. Cell 1994; 79: 1137–1146. role in cell survival or death. Proc Natl Acad Sci USA 2000; 97: 19 Brito C, Naviliat M, Tiscornia AC, Vuillier F, Gualco G, Dighiero G 14602–14607. et al. Peroxynitrite inhibits T lymphocyte activation and prolifera- 37 Eissa NT, Haggerty CM, Palmer CD, Patton W, Moss J. Identifica- tion by promoting impairment of tyrosine phosphorylation tion of residues critical for enzymatic activity in the domain and peroxynitrite-driven apoptotic death. J Immunol 1999; 162: encoded by exons 8 and 9 of the human inducible nitric oxide 3356–3366. synthase. Am J Respir Cell Mol Biol 2001; 24: 616–620. 20 Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, 38 Eissa NT, Yuan JW, Haggerty CM, Choo EK, Palmer CD, Moss J. pathophysiology, and pharmacology. Pharmacol Rev 1991; 43: Cloning and characterization of human inducible nitric oxide 109–142. synthase splice variants: a domain, encoded by exons 8 and 9, is 21 Zhao H, Dugas N, Mathiot C, Delmer A, Dugas B, Sigaux F et al. critical for dimerization. Proc Natl Acad Sci USA 1998; 95: B-cell chronic lymphocytic leukemia cells express a functional 7625–7630. inducible nitric oxide synthase displaying anti-apoptotic activity. 39 Rao KM. Molecular mechanisms regulating iNOS expression in Blood 1998; 92: 1031–1043. various cell types. J Toxicol Environ Health B Crit Rev 2000; 3: 22 Kolb JP, Roman V, Mentz F, Zhao H, Rouillard D, Dugas N et al. 27–58. Contribution of nitric oxide to the apoptotic process in human B 40 Frank DA, Mahajan S, Ritz J. B lymphocytes from patients with cell chronic lymphocytic leukaemia. Leukemia Lymphoma 2001; chronic lymphocytic leukemia contain signal transducer and 40: 243–257. activator of transcription (STAT) 1 and STAT3 constitutively 23 Taylor BS, Geller DA. Molecular regulation of the human phosphorylated on serine residues. J Clin Invest 1997; 100: inducible nitric oxide synthase (iNOS) gene. Shock 2000; 13: 3140–3148. 413–424. 41 Kneitz C, Goller M, Seggewiss R, Yaman A, Serfling E, Tony HP. 24 Ganster RW, Geller DA. Regulation of the expression of STAT6 and the regulation of CD23 expression in B-chronic NOS isoform. In: Ignarro LJ (ed). Nitric Oxide: Biology lymphocytic leukemia. Leukemia Res 2000; 24: 331–337. and Pathobiology, 1st edn. San Diego: Academic Press, 2000, 42 Zheng Z, Venkatapathy S, Rao G, Harrington CA. Expression pp. 129–156. profiling of B cell chronic lymphocytic leukemia suggests deficient 25 Chartrain NA, Geller DA, Koty PP, Sitrin NF, Nussler AK, Hoffman CD1-mediated immunity, polarized cytokine response, altered EP et al. Molecular cloning, structure, and chromosomal localiza- adhesion and increased intracellular protein transport and proces- tion of the human inducible nitric oxide synthase gene. J Biol sing of leukemic cells. Leukemia 2002; 16: 2429–2437. Chem 1994; 269: 6765–6772. 43 Mu X, Kay NE, Gosland MP, Jennings CD. Analysis of blood T-cell 26 Eissa NT, Strauss AJ, Haggerty CM, Choo EK, Chu SC, Moss J. cytokine expression in B-chronic lymphocytic leukaemia: evi- Alternative splicing of human inducible nitric-oxide synthase dence for increased levels of cytoplasmic IL-4 in resting and mRNA. Tissue-specific regulation and induction by cytokines. J activated CD8T cells. Br J Haematol 1997; 96: 733–735. Biol Chem 1996; 271: 27184–27187. 44 Mainou-Fowler T, Miller S, Proctor SJ, Dickinson AM. The levels of 27 Keating MJ, O’Brien S, Lerner S, Koller C, Beran M, Robertson LE TNF alpha, IL4 and IL10 production by T-cells in B-cell chronic et al. Long-term follow-up of patients with chronic lymphocytic lymphocytic leukaemia (B-CLL). Leukemia Res 2001; 25: leukemia (CLL) receiving fludarabine regimens as initial therapy. 157–163. Blood 1998; 92: 1165–1171. 45 Kolodziejski PJ, Musial A, Koo JS, Eissa NT. Ubiquitination of 28 Robak T, Kasznicki M. Alkylating agents and nucleoside analogues inducible nitric oxide synthase is required for its degradation. Proc in the treatment of B cell chronic lymphocytic leukemia. Leukemia Natl Acad Sci USA 2002; 99: 12315–12320. 2002; 16: 1015–1027. 46 Musial A, Eissa NT. Inducible nitric-oxide synthase is regulated by 29 Genini D, Adachi S, Chao Q, Rose DW, Carrera CJ, Cottam HB the proteasome degradation pathway. J Biol Chem 2001; 276: et al. Deoxyadenosine analogs induce programmed cell death in 24268–24273. chronic lymphocytic leukemia cells by damaging the DNA and by 47 Coers W, Timens W, Kempinga C, Klok PA, Moshage H. directly affecting the mitochondria. Blood 2000; 96: 3537–3543. Specificity of antibodies to nitric oxide synthase isoforms in 30 Catapano CV, Perrino FW, Fernandes DJ. Primer RNA chain human, guinea pig, rat, and mouse tissues. J Histochem Cytochem termination induced by 9-beta-D-arabinofuranosyl-2-fluoroade- 1998; 46: 1385–1392.

Leukemia