Vav promotes differentiation of human tumoral myeloid precursors

Experimental Cell Research 306 (2005) 56 – 63 www.elsevier.com/locate/yexcr

Vav promotes differentiation of human tumoral myeloid precursors Valeria Bertagnoloa, Federica Brugnolia, Carlo Mischiatib, Alessia Serenib, Alberto Bavellonic, Cinzia Carinia, Silvano Capitania,d,* a

Signal Transduction Unit-Laboratory of Cell Biology, Section of Human Anatomy, Department of Morphology and Embryology, University of Ferrara, Via Fossato di Mortara, 66, 44100 Ferrara, Italy b Department of Biochemistry and Molecular Biology, University of Ferrara, Italy c Laboratory of Cell Biology and Electron Microscopy, IOR, Bologna, Italy d MIUR ICSI (Interdisciplinary Center for the Study of Inflammation), University of Ferrara, Italy Received 17 June 2004, revised version received 30 November 2004 Available online 17 March 2005

Abstract Vav is one of the genetic markers that correlate with the differentiation of hematopoietic cells. In T and B cells, it appears crucial for both development and functions, while, in non-lymphoid hematopoietic cells, Vav seems not involved in cell maturation, but rather in the response of mature cells to agonist-dependent proliferation and phagocytosis. We have previously demonstrated that the amount and the tyrosine phosphorylation of Vav are up-regulated in both whole cells and nuclei of tumoral promyelocytes induced to granulocytic maturation by ATRA and that tyrosine-phosphorylated Vav does not display any ATRA-induced GEF activity but contributes to the regulation of PI 3-K activity. In this study, we report that Vav accumulates in nuclei of ATRA-treated APL-derived cells and that the down-modulation of Vav prevents differentiation of tumoral promyelocytes, indicating that it is a key molecule in ATRA-dependent myeloid maturation. On the other hand, the overexpression of Vav induces an increased expression of surface markers of granulocytic differentiation without affecting the maturation-related changes of the nuclear morphology. Consistent with an effect of Vav on the transcriptional machinery, array profiling shows that the inhibition of the Syk-dependent tyrosine phosphorylation of Vav reduces the number of ATRA-induced genes. Our data support the unprecedented notion that Vav plays crucial functions in the maturation process of myeloid cells, and suggest that Vav can be regarded as a potential target for the therapeutic treatment of myeloproliferative disorders. D 2004 Elsevier Inc. All rights reserved. Keywords: Vav; Acute promyelocytic leukemia (APL); Granulocytic differentiation; All-trans retinoic acid (ATRA); Tyrosine phosphorylation

Introduction Vav, which is predominantly expressed during adult life, is one of the genetic markers that correlate with the differentiation of the hematopoietic cells [1]. Its functional importance has been extensively studied in T and B cells, in which it appears crucial for both development and functions [2]. In non-lymphoid hematopoietic cells, on the contrary, Vav does not show any obvious role in cell maturation, * Corresponding author. Signal Transduction Unit/Laboratory of Cell Biology, Section of Human Anatomy-Department of Morphology and Embryology, Via Fossato di Mortara, 66, 44100 Ferrara, Italy. Fax: +39 0532 207351. E-mail address: [email protected] (S. Capitani). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.12.001

being mainly involved in the response of mature cells to a variety of stimuli that lead to spatial and temporal reorganization of the actin-based cytoskeleton during proliferation and phagocytosis [3]. Data obtained in our laboratory indicate that Vav is upregulated during granulocytic differentiation of HL-60 cells treated with All-trans Retinoic Acid (ATRA) [4] and that during ATRA treatment, Vav is tyrosine-phosphorylated by Syk, an event that is essential for the changes of the nuclear shape that occur during neutrophil-like maturation of these tumoral cells [5]. In contrast with the best-known function of Vav as a tyrosine-phosphorylation-modulated GDP/GTP exchange factor (GEF) for Rho/Rac proteins [6], it has recently been suggested that some functions of Vav in lymphocytes are

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independent on its GEF activity [7]. Similarly, in retinoidtreated HL-60, we have found that tyrosine-phosphorylated Vav does not display any ATRA-induced GEF activity but contributes to the regulation of PI 3-K activity [8]. Consistent with a direct effect on the transcriptional machinery, Vav is also present in the cell nucleus [4,9,10] where it is part of a transcriptionally active complex that binds the NF-AT binding site of the IL-2 promoter [9] and associates with the Ku-70 component of the DNA-dependent protein kinase complex as well as with the heterogeneous nuclear ribonucleoprotein (hnRNP) [10]. In this study, we report that Vav promotes neutrophil differentiation of neoplastic promyelocytes, and that its tyrosine phosphorylation may be essential to complete the maturation process. Our data indicate a crucial role for Vav in modulating the maturation of non-lymphoid cells and suggest that Vav is a potential target for gene therapy of myeloproliferative disorders.

Materials and methods Cell culture and differentiation HL-60 (American Type Culture Collection, ATCC CCL240, Rockville, MD), NB4 (German Collection of Microorganisms and Cell Cultures, Braunschweing, Germany), and the ATRA-resistant NB4 clone (NB4-R, kindly provided by Dr. Carlo Gambacorti-Passerini, Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy) were cultured and treated with ATRA (Sigma, St Louis, MO) as previously reported [4,11]. To inhibit Syk kinase activity, cells were treated with piceatannol, alone or in combination with ATRA, as previously described [5]. Primary blasts were obtained from patients with APL, at diagnosis, before any therapy, after obtaining informed consent according to the Helsinki declaration of 1975 and were cultured and treated with ATRA as previously reported [11]. For the isolation of primary CD34+ hematopoietic cells, informed consent to the study was obtained according to the Helsinki declaration of 1975 from normal blood adult donors. Cells were purified and induced to granulocytic differentiation, as previously reported [11]. The degree of granulocytic differentiation was monitored morphologically by means of a specific nuclear staining with 4V-6-diamidino-2-phenylindole (DAPI) and by evaluating the phenotypic expression of the CD11b myeloid surface marker after direct staining with PE-conjugated mAb, as previously reported [4,5]. Morphometric analysis of HL-60 cell nuclei was performed after staining with DAPI and evaluation of nuclear shape by computer-assisted image analysis (IBAS 20, Kontron Company, Kaufbeuzen, Germany). Form factor of nuclei of control and differentiating cells was calculated as described by He et al. [12].

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RNA interference assays Small interfering RNA (siRNA) sequences targeting the Vav gene (National Center for Biotechnology Information accession number NM 5428) were synthesized by QIAGEN (QIAGEN S.p.A., Milan, Italy). The specific sequence requirements were as follows: AA(N19)dTdT (N is any nucleotide); 21-nt sense and 21-nt antisense strand; ~50% G/C content; and a symmetric 2-desoxythymidine 3V overhang. The lyophilized siRNA was dissolved in sterile Suspension Buffer (100 mM potassium acetate, 30 mM Hepes-KOH, 2 mM magnesium acetate, pH 7.4) to obtain a 20-AM solution. Then, oligonucleotides were heated to 908C for 1 min followed by 1 h at 378C. As a control, nonsilencing fluorescein-labeled duplex siRNA, also purchased from QIAGEN, was used. Exponentially growing HL-60 and NB4 cells were transfected in contemporary with 8 Ag of all siRNA duplexes using electroporation procedure, with a ElectroSquarePorator apparatus (Genetronics Inc., San Diego, CA), according to the manufacturer’s protocols. Briefly, 32 Ag of siRNA in suspension buffer was mixed with 2  106 cells in 100 Al of RPMI plus 20% FBS. Following electroporation, cells were allowed to recover in 600 Al of RPMI culture medium with 20% FBS. After 5 h, cells transfected with fluoresceinlabeled siRNA were subjected to cytofluorimetric analisys to determine transfection efficiency, that resulted about 60%. Cells transfected with silencing siRNAs were treated with ATRA and subjected to Western blot analysis and evaluation of their degree of differentiation. Overexpression of Vav In the transfection experiments, we used the plasmid pEF Vav-myc, containing the Vav entire coding sequence (kindly provided by Dr. Germani, ICGM-Hopital Cochin-Inserm, Paris, France). Exponentially growing HL-60 and NB4 cells were transfected by electroporation with an ElectroSquarePorator apparatus, according to the manufacturer’s protocols. After 24 h from transfection, cells were either treated or not with 1 AM ATRA for 3 days. Daily, cells were collected and their degree of granulocytic differentiation was measured. To evaluate the efficiency of transfection and to identify the overexpressing cells, the same procedure was performed cotransfecting cells with both pEF Vav-myc and pEGFP plasmids (9:1 ratio). After 24 h from transfection, the cells expressing the EGFP protein, evaluated by inverted fluorescence microscopy, were from 15 to 25%. Cell viability, evaluated by Trypan Blu exclusion, was about 60%. Preparation of total homogenates, nuclei, and immunoprecipitates Cell homogenates and nuclei depleted of nuclear membranes were prepared following a previously described

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procedure [4]. Nuclear purity was assessed by ultrastructural analysis, marker enzyme assays, and absence of h-tubulin as previously reported [4]. For immunoprecipation experiments, cells (10  106) and nuclei (100  106) were lysed and supernatant was incubated with the indicated antibodies, as previously reported [4]. Immunochemical analysis Total lysates (50 Ag protein) from cells (1  106) and nuclei (10  106) and immunoprecipitates (from 1 mg protein) were separated on 7.5% polyacrylamide denaturing gels and blotted to nitrocellulose membranes (Amersham Life Science, Little Chalfont, U.K.). The blots were then incubated with the antibodies and the final detection was performed using the ECL system (DuPont, NEN Research Products, Boston, MA), according to the manufacturer’s instructions. Densitometric analysis was performed on the ImageQuant TL (Amersham Bioscience, Little Chalfont, U.K.). Gene profiling RNA was extracted with the TRIzol reagent (Invitrogen SRL, S.Giuliano Milanese, Italy) and the polyadenylated fraction selected with the Atlas Pure kit (Clontech, Palo Alto, CA). The Atlas Human Cytokine/Receptor nylon array filters (Clontech) were used for the cDNA array experiments. Single-strand 32P-labeled cDNA probes were synthesized from poly(A+) RNA as suggested by the manufacturer. Video imaging was performed with the Molecular Analyst GS670 phosphorimager (BIORAD, Richmond, CA). Microarray data were quantitated with the ATLAS IMAGE 2.0 analysis software (Clontech). Reverse transcription and Q-PCR RNAs (2 Ag) were reverse transcribed (RT) with the Superscript II reverse transcriptase (Life Technologies Ltd., Paisley, U.K.) using oligo-dT primers in a standard 20 Al reaction. Then, cDNAs (1 Al) were amplified by quantitative real-time PCR (Q-PCR) using gene-specific primers. Q-PCR reactions were performed in a 50-Al volume with 0.5 AM primers and MgCl2 concentration was optimized between 2 and 5 mM. A typical protocol included a 30-s denaturation step followed by 40 cycles including a 958C denaturation for 30 s, a 608C annealing for 30 s, and a 728C extension for 30 s. Annealing and extension temperatures and detailed amplification protocols were calculated with the MacVector DNA analysis software (Oxford Molecular Group, Inc., Campbell, CA). Q-PCR was performed in the presence of the SYBRR Green I dye (Molecular Probes Inc., Invitrogen), and the amounts of accumulating amplimers were quantitated in the ABI PRISMR 7700 Sequence Amplification and Detection

System (PE Biosystems, Foster City, CA). All determinations were run in quadruplicate wells. Well-to-well variability was minimized by normalization with the ROX internal passive reference. Relative quantitation of a target template in the samples was based on CT values (CT: threshold cycle, i.e., the number of cycles required to detect a specific amplimer) of control and treated samples. The DCT values (the difference between treated cells CT and control cells CT values) and the DDCT values (the difference between the DCT values for each target gene and the mean DCT value of tubulin, selected as calibrator to normalize different samples) were then calculated. The relative quantitation value was expressed as 2 DDCT. The effects of Vav knock-down siRNA molecules on the expression of selected genes in ATRA-treated HL-60 cells was determined by using as reference the 2 DDCT value obtained in cells treated only with ATRA (relative expression).

Results and discussion Vav increases during granulocytic differentiation of both normal and tumoral myeloid precursors As shown in Fig. 1A, variable basal levels of Vav can be found in promyelocytic cells blocked at different steps of their neutrophil differentiation. However, treatment with differentiating doses of ATRA induces a significant increase of the expression of Vav in the promyelocytic cell line HL-60 as well as in NB4 cells and in primary blasts obtained from bone marrow of APL patients. A similar increase of Vav was observed when normal CD34+ hematopoietic progenitors were treated with a cytokine cocktail promoting granulocytic differentiation. As reported in Fig. 1A, Vav expression remained low up to 8 days of culture, when most CD34-derived cells were approximately at the stage of promyelocytes, and largely increased at day 21, matching the acquisition of a fully differentiated granulocytic phenotype [11]. These data indicate that an adequate expression of Vav has to be achieved along with neutrophil maturation of both normal and poorly differentiated neoplastic cells, and suggest that Vav may be implicated in the path leading myeloid precursors to acquire the mature phenotype of differentiated neutrophils. In APL-derived NB4 cells, as we have already reported in HL-60 [4] and as also described for other cell types [9,10], we demonstrated that Vav shows also an intranuclear localization (Fig. 1B) and that the basal amount of this protein found in nuclei purified from unstimulated cells largely increases following ATRA treatment. Compared to the scanty basal phosphorylation level, a large increase in the phosphotyrosine content of Vav occurs in nuclei of differentiated HL-60 and NB4 cells. Concerning the amount of tyrosine-phosphorylated Vav in whole cells, only a slight increase due to

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Fig. 1. The amount of Vav increases in granulocytic differentiation of myeloid precursors. In A, lysates from control ( ) or differentiated (+, 4 days of ATRA treatment) HL-60, NB4, and APL blasts, as well as from CD34+-derived cells induced to differentiate along the granulocytic lineage for the indicated times, were subjected to Western blot and analyzed with the indicated antibodies. In B, the immunochemical analyses were performed, with the indicated antibodies, on anti-Vav immunoprecipitates from cells and highly purified nuclei, in control conditions ( ) and after treatment with ATRA for 4 days (+) of the indicated cell lines. Lysates from cells and nuclei under the same experimental conditions were analyzed for h-tubulin content. In C, the same cells, cultured in control or differentiating conditions, were incubated with a fluorescent antibody directed against CD11b and analyzed by flow cytometry. The data are representative of 3 separate experiments, performed in duplicate.

ATRA treatment was observed, if compared with the correspondent increase of the total Vav protein (Fig. 1B). The accumulation of tyrosine-phosphorylated Vav inside the nucleus seems to be a distinctive feature of APL-derived cells that accomplish myeloid differentiation, as further suggested by the observation that neither the amount nor the phosphorylation level of nuclear Vav are appreciably affected by retinoid treatment of the NB4-R clone, which

shows a minimal capability to differentiate under ATRA treatment (Figs. 1B,C). Down-modulation of Vav prevents granulocytic differentiation Developmental roles of Vav have been described for immune cells in Vav-deficient mice, in which it has been

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reported that Vav / immature B and T cells fail to complete their maturation and activation [2]. On the contrary, a role for Vav in modulating maturation of nonlymphoid cells is unprecedented. Our data address the question of whether the increase of the amount of Vav we have observed in myeloid differentiation is merely designed to the function of Vav in mature cells or, more intriguingly, it is functionally relevant to the maturation mechanism. In this latter event, Vav could play an active role during ATRA-driven differentiation of tumoral cells,

thus constituting a molecular target for therapeutic intervention. To elucidate this point, we have down-modulated the expression of Vav during ATRA treatment of both HL-60 and NB4 cells. By means of siRNA sequences, a reduced amount of Vav protein was obtained in control cells as well as in differentiating cells, in which Vav expression was forcedly silenced in the aim of counteracting the ATRAinduced increase in Vav protein (Fig. 2A). As reported in Fig. 2B, the reduced expression of Vav protein results in a

Fig. 2. Down-modulation of Vav expression impairs granulocytic differentiation. HL-60 and NB4 cells, subjected to RNA interference assay in control ( ) or differentiating conditions for 3 days (+), were subjected to immunochemical analysis to evaluate Vav expression (A) and to cytofluorimetric determination to estimate their differentiation level (B). In C, the amount of Vav in HL-60 under the different experimental conditions, determined by densitometric scanning of autoradiograms, is reported. The value of control cells was conventionally fixed to 100. The percentage of differentiated cells, evaluated after nuclear staining with DAPI in the same culture conditions, is reported on the right. The data are representative of 3 separate experiments performed in duplicate.

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large decrease of the granulocytic differentiation level in both cell lines. The analysis of nuclear morphology of differentiating HL-60, that shifts from round to lobated shape, indicates that a forced reduction of Vav expression during ATRA treatment results in a decrease in differentiated cells (Fig. 2C). This set of data demonstrates that a reduced expression of Vav considerably impairs the differentiative potential of ATRA and supports the contention that Vav is not

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dispensable for the progression of tumoral promyelocytes along the granulocytic lineage. Overexpression of Vav promotes differentiation of tumoral myeloid precursors A further set of experiments was aimed to assess whether a forced expression of Vav is capable to promote the maturation of tumoral myeloid precursors along the gran-

Fig. 3. Overexpression of Vav induces phenotypical differentiation. HL-60 and NB4 were transiently co-transfected with a EGFP construct and a plasmid containing the full-length cDNA of human Vav (EGFP/Vav), cultured for 3 days and subjected to immunochemical analysis with the indicated antibodies (A). After 24 h from transfection, cells were treated with ATRA for 48 h and their level of granulocytic differentiation was estimated by cytofluorimetric analysis of CD11b expression (B) and, only for HL-60 cells, by monitoring the modifications of nuclear morphology after nuclear staining with DAPI (C). Transfection with the EGFP construct allowed to identify the overexpressing cells (green cells). The data are representative of 2 separate experiments performed in duplicate.

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ulocytic lineage. To this end, HL-60 and NB4 cells were transfected with a plasmid containing the full-length cDNA of human Vav, cultured for 3 days and evaluated for their content of Vav and for their level of granulocytic differentiation (Figs. 3A,B). Co-transfection with a EGFP construct allowed us to identify the overexpressing cells, by both fluorescence microscopy and flow cytometry. As reported in Fig. 3B, flow cytometric analysis of the EGFPlabeled cells showed that the overexpression of Vav induced a significant increase of CD11b expression only in HL-60. On the contrary, morphometrical analysis of nuclear shape, performed on HL-60 after specific nuclear staining with DAPI, failed to identify any role of the overexpressed Vav in modulating nuclear architecture (Fig. 3C). When transfected cells were treated with ATRA, an additional increased expression of surface markers was found in EGFP/Vav-labeled HL-60 cells and a significant rise of the differentiation level reached by NB4 cells was observed (Fig. 3B), indicating that the overexpression of Vav potentiates the differentiative action of ATRA in both tumoral cell lines. However, none of the Vav-overexpressing HL-60, showing higher levels of CD11b, displayed a more differentiated nuclear morphology (Fig. 3C), suggesting that the sole overexpression of Vav is not sufficient to promote the complete maturation of early myeloid precursors and that additional events are necessary. Among these, the tyrosine phosphorylation of Vav seems to be a lacking step, in view of the fact that we have already demonstrated that full myeloid differentiation of HL-60 requires tyrosine phosphorylation of Vav [5] and that, here, we demonstrate that the overexpressed Vav does not contain appreciable levels of phosphotyrosines (Fig. 3A). Table 1 Genes up-regulated by ATRA whose expression is negatively affected by piceatannol GenBank accession no.

Description (gene/protein name)

Function (gene/protein classification)

M37435

macrophage-specific colony-stimulating factor (MCSF) glia maturation factor h (GMF-h) neurotrophin 3 (NT3) trk-T3 platelet-derived growth factor A subunit (PDGFA) notch homolog

Growth factors, cytokines, and chemokines

M86492 X53655 X85960 X06374

M99437 D16431 M92381

hepatoma-derived growth factor (HDGF) thymosin h 10 (TMSB10)

Growth factors, cytokines, and chemokines Proteins neuropeptides Protein kinase receptors Growth factors, cytokines, and chemokines Oncogenes and tumor suppressors Growth factors, cytokines, and chemokines Growth factors, cytokines, and chemokines

cDNAs from HL-60 cells treated with ATRA for 4 days and differentiated in the presence of the inhibitor of tyrosine phosphorylation of Vav were subjected to cDNA array experiments using Human Cytokine/Receptor nylon array filters. After quantitation of microarray data, ATRA-induced genes whose expression was reduced by piceatannol were identified.

Fig. 4. Down-modulation of Vav reduces the expression of ATRA-induced genes. RNAs from differentiated cells (ATRA) and ATRA-treated HL-60 cells in which the expression of Vav was silenced (siRNA + ATRA) were reverse transcribed and cDNAs were amplified by quantitative real-time PCR (Q-PCR) using specific primers for the indicated genes. All determinations were run in quadruplicate.

Tyrosine-phosphorylated Vav is crucial for the ATRA-induced gene expression Based on the evidence that a nuclear accumulation of tyrosine-phosphorylated Vav correlates with the maturation of tumoral promyelocytes (Fig. 1B), we tried to assess if tyrosine-phosphorylated Vav may have a role in modulating ATRA-induced gene expression. By means of array analysis, and starting from the reported evidence that ATRA induces the expression of a number of cytokines [13,14] involved in the differentiative program of HL-60, we evaluated the effects of the inhibition of the tyrosine phosphorylation of Vav on the ATRA-induced expression of genes encoding for cytokine and cytokine receptors. As reported in Table 1, in the presence of the Syk kinase inhibitor Piceatannol, that we have previously demonstrated to be able to down-modulate the tyrosine phosphorylation of Vav in HL-60 [5], the expression of 8 ATRA-induced genes was modulated down to the levels of untreated cells. Among them, thymosin beta-10 (TMSB10) gene encodes for a small G-actin binding protein that induces depolymerization of intracellular F-actin pools modulating actin architectures [15,16] and the gene for Notch homolog that encodes for a molecule playing a role in mediating cell fate decisions during hematopoiesis [17]. In order to ascertain whether tyrosine phosphorylation of Vav is crucial for the ATRA-induced gene expression, and to exclude that the use of Syk inhibitor might have a more general effect on protein phosphorylation independently of its specific effect on Vav, we evaluated the expression of ATRA-induced genes in differentiating cells in which Vav protein was forcedly silenced. As reported in Fig. 4, the expression of some genes, randomly selected from those reported in Table 1, was down-modulated in cells in which the amount of Vav was reduced during ATRA treatment, confirming that an adequate amount of Vav is necessary for the ATRA-induced gene expression. RT-PCR performed on S100A, a gene overexpressed by ATRA but not downmodulated in the presence of piceatannol during differ-

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entiation, confirmed that there is a clear relationship between the effect of piceatannol and the effect of downmodulation of Vav on gene expression happening in the course of ATRA-induced differentiation. The data we have obtained collectively indicate that the expression of Vav not only is necessary for, but promotes the maturation of, tumoral myeloid precursors along the granulocytic lineage, constituting the first evidence of the requirement of Vav for maturation of non-lymphoid cells. On the other hand, Vav bper seQ is not sufficient for activating the complete maturation mechanism, since both expression and tyrosine phosphorylation of this protein seems to be necessary for completion of the maturation program.

[6] [7]

[8]

[9]

[10]

Acknowledgments This research was supported by grants from MIUR Cofin (2003), Ministero della Salute (Programma Speciale Emopoiesi, 2002), FIRB (2001), and Interdisciplinary Center for the Study of Inflammation (ICSI), University of Ferrara (Italy) to S. Capitani.

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