Original Article
Notch Affects the Prodifferentiating Effect of Retinoic Acid and PMA on Leukemic Cells Maja Matulic,1 Josipa Skelin,2 Delfa Radic-Kristo,3 Ika Kardum-Skelin,3 Danka Grcevic,4 Mariastefania Antica2*
1
Department of Molecular Biology, Faculty of Science, Zagreb, Croatia
2
Division of Molecular Biology, Rudjer Boskovic Institute, Zagreb, Croatia
3
Department of Medicine, Merkur University Hospital and School of Medicine University of Zagreb, Zagreb, Croatia
4
Department of Physiology and Immunology, University of Zagreb School of Medicine, Zagreb, Croatia.
Received 17 January 2014; Revised 15 May 2014; Accepted 7 October 2014 Grant sponsor: Croatian Science Foundation, Grant number: 7140; Grant sponsor: Croatian Academy of Sciences and Arts; Grant sponsor: European Union’s Seventh Program for Research, Technological Development and Demonstration, within the THYMISTEM; Grant number: 602587. Additional Supporting Information may be found in the online version of this article. *Correspondence to: Prof. Mariastefania Antica, PhD, Division of Molecular Biology, Rudjer Boskovic Institute, Bijenicka 54, HR-10000 Zagreb, Croatia. E-mail: [email protected] Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22582 C 2014 International Society for V
Advancement of Cytometry
Cytometry Part A 00A: 0000, 2014
Abstract Notch proteins determine cell fate decisions in the development of diverse tissues. Notch has been initially found in T-ALL but its role has been also studied in myelopoiesis and myeloid leukemias. Studies in different model systems have led to a widespread controversy as to whether Notch promotes or blocks myeloid differentiation. In this work, we evaluated the influence of Notch activation on leukemic cell differentiation along the monocytic and myelocytic pathway induced by phorbol 12myristate 13-acetate (PMA) or all-trans retinoic acid (ATRA). We observed that differentiation of the human myeloblastic cell line HL-60 can be retarded or blocked by Delta/Notch interaction. ATRA induces complete remission in patients with acute promyelocytic leukemia, but it cannot completely eliminate the leukemic clone and to be effective it should be combined with chemotherapy. Our findings suggest that Notch signaling may contribute to the incomplete elimination of the leukemic cells after PMA or ATRA treatment and the blockage of Notch pathway may be beneficial in the treatment of myeloid leukemia. VC 2014 International Society for Advancement of Cytometry
Key terms Notch; leukemic cell; ATRA; PMA; differentiation
HEMATOPOIETIC cell differentiation is a complex process regulated very tightly. It has been shown that one of the major regulators of lymphocyte development is Notch signaling. The first Notch mutation causing wing notching has been described in Drosophila but over the years several functions and regulatory mechanisms of Notch signaling in other organisms has been uncovered (1). Notch is a transmembrane protein with a critical role in T versus B cell commitment (2,3). However, the role of Notch in hematopoiesis is not limited to lymophopoiesis but its involvement in myelopoieis and myeloid leukemias has also been described (4–6). The human Notch signaling is triggered by binding to its ligands, specifically Notch receptor (Notch1–4) interaction with either Delta-like ligands (DLL1, 3 and 4) or Jagged family ligands (Jagged 1, 2) is followed by cleavage of the Notch intracytoplasmic domain (NIC) and its translocation to the nucleus where NIC acts as a transcription factor. The human myeloblastic cell line HL-60 can differentiate upon phorbol myristate acetate (PMA) (7) or all-trans retinoic acid (ATRA) treatment into macrophages or granulocytes, respectively (8), and therefore, it has been used extensively to examine the factors involved in differentiation. By the use of this model system, here we tested for a novel effect of DLL1 on PMA or ATRAinduced differentiation. We studied the changes of the stimulated HL-60 cells when cocultured with the Notch activating OP9 stromal cells expressing Delta ligands (OP9-DL1). Here we show a protective role of Notch activation in myeloid differentiation process.
Original Article
MATERIALS AND METHODS Antibodies and Reagents Immunofluorescence analysis was performed as previously described (9,10) using phycoerythrin (PE)-conjugated anti-CD4 and fluorescein isothiocyanate (FITC) conjugated anti-CD11b monoclonal antibodies, isotypic control antibodies, IgG1-PE, and IgG2a-FITC, all products from DAKO (Glostrup, Denmark). The stimulators used were phorbol 12-myristate 13-acetate (PMA) and ATRA from Sigma (USA). For the detection of respiratory burst activity, the cell-permeable fluorogenic probe 20 ,70 dichlorohydrofluorescein diacetate (DCFH-DA) was employed. A gamma secretase inhibitor N-[(3,5-difluorophenyl)acetyl]-Lalanyl-2-phenyl]glycine-1,1-dimethylethylester (DAPT) for blocking Notch signaling, a cell proliferation reagent WST-1 (Boehringer, Mannheim, Germany), RPMI-1640 medium, alpha Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich Chemie GMBH, Taufkirchen, Germany), and fetal calf serum (FCS; Gibco Invitrogen, Grand Island, New York) were used for the experiments. All other reagents employed were laboratorygrade chemicals from Kemika (Zagreb, Croatia). Cell Cultures Human cell lines HL-60, U937, and Jurkat obtained from the German Cell Culture Collection (DSMZ), NALM-1 chronic myeloid leukemia ((CML) in blast crisis), and MOLT4 (T-lineage acute lymphoblastic leukemia [T-ALL]) were a kind gift from Professor A. Boyd (Queensland Institute of Medical Research Brisbane, Australia). Cells were cultured in suspension in RPMI-1640 medium supplemented with 10% FCS. For a typical experiment, cells were seeded at a density of 105 cells/ml, in 35-mm diameter Petri dishes. OP9 and OP9-DL1 cells were a kind gift from Professor J. C. Zuniga-Pfl€ ucker (Department of Immunology, University of Toronto, Canada). The stromal cell line OP9, established from a newborn B6C3F1-op/op mouse calvaria, was stably transfected with the human Notch-1 ligand Delta-like-1 (3). The cells were cultured according to the protocol in OP9 medium (alphaMEM supplemented with 10% heat inactivated FCS supplemented with L-glutamine) as indicated in the original article (3). The cells were harvested after incubation in trypsin and seeded on fresh culture petri dishes till confluent. The cells of interest were plated on nonconfluent OP9-DL1 cultures and incubated in the presence or absence of PMA (80 ng/ml) or ATRA (1 mM). Cocultivation HL-60 cells were cultured alone or in the presence of OP9-DL1 or control OP9 stromal cell line. OP9 (105/ml) cells were seeded at Day 1 in 1 ml of OP9 medium. At Day 2, after OP9 cells became adherent, the medium was removed and 1 ml of fresh OP9 medium with resuspended HL-60 cells (105/ml) was added. After 24 and 48 h of coculture, HL-60 cells were easily harvested by gentle pipetting, centrifuged, and tested for cell viability by trypan blue dye exclusion test, WST-1 cell proliferation test, and flow cytometry. Determination of Antigen Expression Cells were seeded and induced to differentiate with PMA or ATRA as described and evaluated for differentiation 2
Figure 1. Flow cytometric analysis of DCF fluorescence as indicator of ROS generation in PMA stimulated HL-60 cells. Timedependent response compared to the untreated control (dotted line). Cellular fluorescence is shown: Basal conditions are designated in the first time point (gray histogram) and 20 min stimulation with PMA is the second time point (black full line). The figure expresses representative results of two independent experiments.
markers at indicated time points. Differentiation morphology (increase in cell volume, granularity, and appearance of irregularly shaped cellular processes) was examined microscopically. Viable cells were scored by trypan blue exclusion. Immunofluorescence analysis was performed as previously described (9,10) using monoclonal antibodies and analyzed by flow cytometer (FACSCalibur, Becton Dickinson, New Jersey). Dead cells were excluded from flow cytometric analysis by propidium iodide staining. Superoxide Production Reactive oxygen species (ROS) production was monitored by DCFH-DA fluorescence probe as originally described by Bass et al. (11). The DCFH-DA probe has been used to evaluate the endogenously generated intracellular H2O2. DCFH-DA diffuses into cells, where it is cleaved by cellular esterases, leaving impermeant nonfluorescent DCFH trapped inside the cells and susceptible to H2O2 oxidation. Intracellular oxidation of nonfluorescent DCFH leads to formation of dichlorofluorescein (DCF). In this study, aliquots of cell suspensions were first preincubated with DCFH-DA (5 picomolar (pM)) in the dark with gentle agitation at 37 C for 15 min, washed, resuspended in PBS buffer and then stimulated with PMA. Stimulated and unstimulated unstained cells served as controls in each experiment. All samples were finally resuspended in a propidium iodide (PI) (1 mg/ml) containing buffer. PI labeled dead cells were excluded from the analysis. Cell Proliferation Assay The assay for quantification of cell proliferation and viability was used according to the manufacturer’s instructions (F. Hoffman-La Roche Ltd, Basel, Switzerland). The cell proliferation assay is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Briefly, 10 ml/well of WST-1 reagent is mixed with cells (ratio 1:10) grown for 3 days in a-MEM medium, supplemented with 10% of FCS (Gibco) or as described with PMA or ATRA. The cells are incubated for 2 h with WST-1 reagent and the formazan dye, produced by viable cells, can be quantified by a multiwell spectrophotometer (microplate reader) by measuring the absorbance of the dye solution at 440 nm (Biorad, Hercules, California, USA). All samples were measured in quadruplicates. Notch Affects Leukemic Cell Differentiation
Original Article
Figure 2. Morphology of the cultivated HL-60 cells treated with PMA. Cells were cultured for 24 h with PMA (B, D, and F) or without treatment (A, C, and E) and analyzed by microscopy. The right panel shows cocultures of HL-60 with OP9 (C and D) or OP9-DL1 (E and F). The arrows are showing the differentiated cells in the PMA stimulated HL-60 OP9 control cultures. (Scale bars 510 mm). A: control HL-60; B: PMA treated HL-60; C: control HL-60 cocultured on OP9; D: control PMA treated HL-60 cocultured on OP9; E: control HL-60 cocultured on OP9-DL1; F: PMA treated HL-60 cocultured on OP9-DL1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Immunoblotting Cells were centrifuged and lysed using Carin lysis buffer (20 mM Tris-HCl pH 5 7.4, 1% NP40, 138 mM NaCl, 2 mM EDTA, 10% glycerol) and proteinase inhibitor cocktail (1:20,
1 ml of protease inhibitors/106 cells; Roche) and frozen in liquid nitrogen. Protein concentrations in the cleared lysates were assessed by Bradford protein assay. The lysates were mixed with 4 3 Laemmli buffer and resolved by 12% SDS-
Figure 3. Flow cytometric analysis of CD11b and CD4 antigen expression at 30 min, 24, and 48 h PMA stimulation of HL-60 cells. The cells were double-stained with FITC-conjugated anti-CD11b and PE conjugated anti-CD4 antibodies. A: FL1, CD11b; (B) FL2, CD4. Control studies were performed with IgG1 isotype antibodies (gray shadowed histograms). Dead cells as detected by PI staining were excluded from the analysis.
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Original Article Immunoblots were developed by means of Signalfire (Cell Signaling Technology) and imaged on Medical X-ray film (Fujifilm, Tokyo, Japan). Reverse Transcription Polymerase Chain Reaction (RT-PCR) R RNA isolation was performed using TRIZOLV reagent according to the manufacturer’s instructions. About 1 mg of R RNA was reversely transcribed using SuperScriptV III Reverse Transcriptase and the cDNA amplified by PCR using AmpliR Taq GoldV DNA Polymerase (all reagents from Life Technologies, Thermo Fisher Scientific, Waltham, MA USA). Primers were designed manually as previously described (12). Briefly, all primers were 20 bp long with the same frequency of GC as AT content. All primers were checked for unspecific binding, as well as for dimer structures with the freely available Amplify software version 3.0 (13). In addition to that primers were checked for intron/exon boundaries to avoid genomic amplification. Statistical Analysis To assess statistical significance, a non-parametric Mann– Whitney test was used and threshold (P) was set to less than 0.05. All experiments were performed at least in triplicate unless otherwise noted.
RESULTS
Figure 4. Proliferation test and trypan blue exclusion analysis. Cells were incubated for 3 days with or without PMA and cocultured with OP9-DL1 or OP9 GFP cells. After 3 days, cells were counted using (A) trypan blue exclusion method and (B) WST proliferation test. This graph shows summarized data from at least three independent experiments. WST1 readings of all samples were performed in quadruplicates and arithmetic mean of each sample was used for further analysis.
PAGE. The proteins were transferred for 15 h at 4 C to nitrocellulose membranes (GE Healthcare, Piscataway, New Jersey; Uppsala, Sweden; Life Sciences Advanced Technologies, St Petersburg, FL, USA) and blocked in tris buffered saline with tween 20 (TBST) containing 5% bovine serum albumin (BSA; Sigma). Membranes were incubated overnight with primary antibodies for human b-Actin (13E5, 1:10,000) or Notch1 (D1E11, 1:2,000) XPV Rabbit mAb (both Cell Signaling Technology, Danvers, MA, USA) and washed three times for 5 min. The membranes were than incubated for 1 h with antirabbit IgG HRP-linked secondary antibody (1:5,000) (Cell Signaling Technology) and washed four times for 10 min. R
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HL-60 Stimulation and Differentiation Intracellular ROS formation as a first response to PMA activation was measured with the DCFH-DA fluorescent probe. DCFH-DA diffuses into cells where it is hydrolyzed into nonfluorescent DCFH. The H2O2 produced during the cell oxidative response oxidizes nonfluorescent intracellular DCFH into a highly fluorescent DCF. Since H2O2 is a secondary product of superoxide anion, this method is an adequate probe to assess intracellular O22 production (14). We evaluated the fluorescence level for 20 min and analyzed the cells after PMA stimulation. Figure 1 shows that intracellular hydrogen peroxide was detected by an increased fluorescence level of DCFH-loaded HL-60 cells. In order to confirm that the cells were stimulated we also checked their morphology as a typical sign of differentiation and analyzed the cells by light microscopy. As shown in Figures 2 and 3, PMA stimulated HL-60 cells changed their morphology and antigen expression already after 24 h in culture as previously established. Inhibition of Differentiation by OP9-DL1 Cocultivation To determine whether there is a relationship between Notch Delta interaction and HL-60 differentiation, we set up cocultures of HL-60 and OP9-DL1 and stimulated the cells with ATRA and PMA. After 24 h, PMA treated HL-60 had a typical morphology as the differentiated cells and the absence of the increase of the cell number confirmed the proliferation arrest. While OP9-DL1 cells alone do not influence the growth of HL-60 cells, upon PMA treatment the HL-60 cells demonstrated impaired differentiation. As a control we used PMA Notch Affects Leukemic Cell Differentiation
Original Article
Figure 5. Flow cytometric analysis of apoptosis of HL-60 cells with (lower row) or without (upper row) the presence of OP9-DL1. Cells were treated with PMA and ATRA and the level of Annexin V and PI binding was measured by flow cytometry. OP9-DL1 cells were excluded from the analysis according to GFP expression. Dot plots are representative of three independent experiments.
Figure 6. Notch expression in human leukemia cell lines. Immunoblot of T-ALL and AML cell lines probed for Notch1 and b-actin (upper panel). mRNA expression of Notch1, Hes1, and HPRT by reverse transcription PCR analysis of HL-60 cells. Representative of at least two blots.
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treated cells grown on the control OP9 cells. The control cell cultures proliferate and increase in cell number compared to the number of cells stimulated with PMA and ATRA. The coculture of HL-60 cells with OP9 cells impact cell proliferation. However, when stimulated with PMA, there was no statistically significant difference (Mann–Whitney test, P > 0.05) between cocultured and control HL-60 cells (Fig. 4). After the cells were treated with PMA or ATRA in cocultures the level of Annexin V and PI binding was measured by flow cytometry in order to establish the degree of apoptosis (Fig. 5). There was no significant difference between HL-60 cells cocultured with OP9-DL1 and the control cultures. We found Notch1 and the downstream target Hairy Enhancer of Split (Hes1) mRNA expression by the promyelocytic cell line HL-60 (Fig. 6). Further, by Western blot analysis we compared the Notch1 protein level expression among TALL and AML cell lines and HL-60 cells. All examined cell lines express Notch1 at the protein level (Fig. 6). We further analyzed the expression of CD11b and CD4 upon stimulation. Unstimulated HL-60 cells express CD4 and already 30 min after PMA stimulation. CD4 expression decreases markedly and stays low further 24 and 48 h. On the contrary, CD11b which is not expressed on unstimulated cells readily shifts 24 h upon stimulation and its expression further increases after 48 h (Fig. 7). Cells in cocultures with OP9-DL1 5
Original Article
Figure 7. Flow cytometric analysis of CD11b and CD4 antigen expression of (A) HL-60 cells cocultured with either OP9 (open black histogram) or OP9-DL1 cells (open red histogram) and treated with PMA or ATRA. Expression of CD11b and CD4 on HL-60 cells cocultured but untreated are represented by shadowed histograms. B: PMA treated HL-60 cells cultured either alone or in cocultures with OP9, OP9-DL1, and OP9-DL1 with DAPT. The histograms show the level of CD11 expression. DAPT 5 Gamma secretase inhibitor. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
showed only a weak response to PMA and ATRA, considering expression of CD11b and CD4. The cells do not differentiate when cocultured with OP9-DL1 cells and the CD4 and CD11b level is very close to the level of control unstimulated cells (Fig. 7A). Figure 7B shows that this phenomenon is specific since it can be inverted by the gamma secretase inhibitor DAPT which inhibits cleavage and consequently activation of Notch. 6
DISCUSSION Notch proteins are involved in the cell fate determination in various systems. They consist of a family of transmembrane receptors that are responsive to binding of ligand of the Delta or Jagged families. Ligand binding leads to the release of activated intracellular Notch domain, its binding to CSL and coactivation and expression of downstream target genes, such
Notch Affects Leukemic Cell Differentiation
Original Article as Hes1 and Hes5 (15,16). Notch has been initially found in T-ALL but it has been shown to be an important regulator of other hematopoietic cells as well. Notch signaling is essential, on one side, to induce T-cell development and, on the other side, to stop the development of B-cells. It has been shown that multipotent lymphoid progenitor cells derived from the bone marrow develop into B-cells in the thymus of Notch1 knockout mice (2,17–19). Ectopic expression and lack of Notch, respectively, disbalance the induction of B versus T cells (2,20). Taghon et al. analyzed the responses of multipotent precursors to Notch signaling. Hes1 was upregulated within 1 day, while other T lineage differentiation gene expression was delayed until about Day 3 of DLL1 signaling (GATA-3 and TCF-1, CD25) when CD44CD25 DN2 thymocytes appeared. Stable induction of a T cell development was dependent on the duration of the contact, density of ligands, and modulating proteins (such as Fringe) (21). A role for Notch in T cell leukemias was demonstrated in animal models, as well as in 50% of human T-ALL with activating mutations in Notch1 (22). Schmitt et al. (3) established a cell line that supports T cell development in vitro by transfecting the OP9 stromal cells with Delta1. The OP9-DL1 culture system stimulates the differentiation of T lymphocytes from hematopoietic stem cells from bone marrow or umbilical cord (23). However, deregulation of Notch signaling was found to have a role in the progression of chronic myeloid leukemia (24). The role of Notch in myelopoiesis and in myeloid leukemia has been studied in the recent years (4–6,25). These studies investigated Notch effects in different model systems and perhaps as a consequence of the four closely related receptors the conclusions drawn were often conflicting as to whether Notch promotes or blocks myeloid differentiation. Our results show that Notch/Delta interaction can influence myelocytic differentiation. We investigated the regulation of the CD11b (integrin) gene in the promyeloblastic leukemic cell line, HL60, following differentiation along the monocytic pathway with PMA and ATRA. The steady-state level of CD11b increased markedly over 48 hr from the undetectable level present before differentiation. Our results indicate that OP9DL1 inhibits PMA induced CD11b expression on HL-60 cells as a marker of HL-60 differentiation. We show here a protective role of Notch activation in the differentiation process. As an additional control we also followed the expressionof CD4 on differentiated cells. Our results indicate that rapid downregulation of CD4 upon PMA stimulation may be independent of Notch/DLL1 interaction. On the other hand, ATRA alone does not influence the expression of CD4, but interestingly this was achieved in the presence of DLL1. CD4 expression in regard to Notch1/DLL1 interaction is a matter of further investigation. A reversion of a blockage stage in certain leukemia types is a base for treatment with different stimulators of differentiation. ATRA is a potent inducer of differentiation and inducer of proliferation and has been widely used in the treatment of acute promyelocytic leukemia (APL). ATRA induces complete remission in patients with APL, but cannot completely eliminate the leukemic clone and to be effective is Cytometry Part A 00A: 0000, 2014
combined with chemotherapy. Since, ATRA alone is not sufficient to eliminate the leukemic clone it is possible that some cells remain protected from differentiation induction if interacted with a Delta expressing tissue. Our findings suggest that the cells which do not differentiate after ATRA stimulation, and therefore cannot be deleted are protected through the Notch pathway. While this paper was in revision, additional evidence for Notch involvement in acute myelogenous leukemia has been published (26,27). The results on primary leukemia cell samples confirm our findings and validate the conclusions on cell lines. The authors propose the development of Notch agonists as a potential therapeutic approach in AML. Our study focused on the effects of Notch on ATRA or PMA treated cells and we suggest that HL-60 cells can be a useful tool for analysis of the mechanisms involved in Notch/ Delta1 interactions in cell differentiation.
ACKNOWLEDGMENTS Authors would like to thank Professor J. C. ZunigaPfl€ ucker (Department of Immunology, University of Toronto, Canada) for his generous gift of the transfected OP9 and OP9-DL1 cells used in our experiments and Professor A. Boyd (Queensland Institute of Medical Research Brisbane, Australia) for his generous gift of the NALM-1 and MOLT-4 cells. Authors also thank M. Paradzik for providing technical assistance in the preparation of the manuscript, Prof. H. Sill and Prof. L. Cicin-Sain for critically reading the manuscript.
AUTHOR CONTRIBUTIONS M.A. designed and performed research and wrote the manuscript; M. M., and J. S. performed research, D. R.-K., I. K-S., D. G. and M. A. contributed to experimental design, analyzing data and writing of the manuscript.
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Original Article 13. Engels W. Contributing software to the internet: The amplify program. Trends Biochem Sci 1993;18:448–450. 14. Yoshida H, Inoue S, Yoshida K, Nakajima O, Mizuno S. Stimulation of hydrogen peroxide production by drinking water contaminants in HL-60 cells sensitized by retinoic acid. Environ Health Prev Med 1998;3:106–112. 15. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, Israel A. Signaling downstream of activated mammalian Notch. Nature 1995;377:355–358. 16. Davis RL, Turner DL. Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning. Oncogene 2001;20:8342–8357. 17. Pui JC. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 1999;11:299–308. 18. Radtke F. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 1999;10:547–558. 19. Radtke F, Wilson A, Mancini SJ, MacDonald HR. Notch regulation of lymphocyte development and function. Immunity 2004;5:247–253. 20. Taghon TN, David E-S, Zuniga-Pflucker JC, Rothenberg EV. Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling. Genes Dev 2005;19:965–978. 21. Weerkamp F, Luis TC, Naber BAE, Koster EEL, Jeannotte L, van Dongen JJM, Staal FJT. Identification of Notch target genes in uncommitted T-cell progeni-
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Notch Affects Leukemic Cell Differentiation
Notch Affects the Prodifferentiating Effect of Retinoic Acid and PMA on Leukemic Cells Maja Matulic,1 Josipa Skelin,2 Delfa Radic-Kristo,3 Ika Kardum-Skelin,3 Danka Grcevic,4 Mariastefania Antica2*
1
Department of Molecular Biology, Faculty of Science, Zagreb, Croatia
2
Division of Molecular Biology, Rudjer Boskovic Institute, Zagreb, Croatia
3
Department of Medicine, Merkur University Hospital and School of Medicine University of Zagreb, Zagreb, Croatia
4
Department of Physiology and Immunology, University of Zagreb School of Medicine, Zagreb, Croatia.
Received 17 January 2014; Revised 15 May 2014; Accepted 7 October 2014 Grant sponsor: Croatian Science Foundation, Grant number: 7140; Grant sponsor: Croatian Academy of Sciences and Arts; Grant sponsor: European Union’s Seventh Program for Research, Technological Development and Demonstration, within the THYMISTEM; Grant number: 602587. Additional Supporting Information may be found in the online version of this article. *Correspondence to: Prof. Mariastefania Antica, PhD, Division of Molecular Biology, Rudjer Boskovic Institute, Bijenicka 54, HR-10000 Zagreb, Croatia. E-mail: [email protected] Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22582 C 2014 International Society for V
Advancement of Cytometry
Cytometry Part A 00A: 0000, 2014
Abstract Notch proteins determine cell fate decisions in the development of diverse tissues. Notch has been initially found in T-ALL but its role has been also studied in myelopoiesis and myeloid leukemias. Studies in different model systems have led to a widespread controversy as to whether Notch promotes or blocks myeloid differentiation. In this work, we evaluated the influence of Notch activation on leukemic cell differentiation along the monocytic and myelocytic pathway induced by phorbol 12myristate 13-acetate (PMA) or all-trans retinoic acid (ATRA). We observed that differentiation of the human myeloblastic cell line HL-60 can be retarded or blocked by Delta/Notch interaction. ATRA induces complete remission in patients with acute promyelocytic leukemia, but it cannot completely eliminate the leukemic clone and to be effective it should be combined with chemotherapy. Our findings suggest that Notch signaling may contribute to the incomplete elimination of the leukemic cells after PMA or ATRA treatment and the blockage of Notch pathway may be beneficial in the treatment of myeloid leukemia. VC 2014 International Society for Advancement of Cytometry
Key terms Notch; leukemic cell; ATRA; PMA; differentiation
HEMATOPOIETIC cell differentiation is a complex process regulated very tightly. It has been shown that one of the major regulators of lymphocyte development is Notch signaling. The first Notch mutation causing wing notching has been described in Drosophila but over the years several functions and regulatory mechanisms of Notch signaling in other organisms has been uncovered (1). Notch is a transmembrane protein with a critical role in T versus B cell commitment (2,3). However, the role of Notch in hematopoiesis is not limited to lymophopoiesis but its involvement in myelopoieis and myeloid leukemias has also been described (4–6). The human Notch signaling is triggered by binding to its ligands, specifically Notch receptor (Notch1–4) interaction with either Delta-like ligands (DLL1, 3 and 4) or Jagged family ligands (Jagged 1, 2) is followed by cleavage of the Notch intracytoplasmic domain (NIC) and its translocation to the nucleus where NIC acts as a transcription factor. The human myeloblastic cell line HL-60 can differentiate upon phorbol myristate acetate (PMA) (7) or all-trans retinoic acid (ATRA) treatment into macrophages or granulocytes, respectively (8), and therefore, it has been used extensively to examine the factors involved in differentiation. By the use of this model system, here we tested for a novel effect of DLL1 on PMA or ATRAinduced differentiation. We studied the changes of the stimulated HL-60 cells when cocultured with the Notch activating OP9 stromal cells expressing Delta ligands (OP9-DL1). Here we show a protective role of Notch activation in myeloid differentiation process.
Original Article
MATERIALS AND METHODS Antibodies and Reagents Immunofluorescence analysis was performed as previously described (9,10) using phycoerythrin (PE)-conjugated anti-CD4 and fluorescein isothiocyanate (FITC) conjugated anti-CD11b monoclonal antibodies, isotypic control antibodies, IgG1-PE, and IgG2a-FITC, all products from DAKO (Glostrup, Denmark). The stimulators used were phorbol 12-myristate 13-acetate (PMA) and ATRA from Sigma (USA). For the detection of respiratory burst activity, the cell-permeable fluorogenic probe 20 ,70 dichlorohydrofluorescein diacetate (DCFH-DA) was employed. A gamma secretase inhibitor N-[(3,5-difluorophenyl)acetyl]-Lalanyl-2-phenyl]glycine-1,1-dimethylethylester (DAPT) for blocking Notch signaling, a cell proliferation reagent WST-1 (Boehringer, Mannheim, Germany), RPMI-1640 medium, alpha Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich Chemie GMBH, Taufkirchen, Germany), and fetal calf serum (FCS; Gibco Invitrogen, Grand Island, New York) were used for the experiments. All other reagents employed were laboratorygrade chemicals from Kemika (Zagreb, Croatia). Cell Cultures Human cell lines HL-60, U937, and Jurkat obtained from the German Cell Culture Collection (DSMZ), NALM-1 chronic myeloid leukemia ((CML) in blast crisis), and MOLT4 (T-lineage acute lymphoblastic leukemia [T-ALL]) were a kind gift from Professor A. Boyd (Queensland Institute of Medical Research Brisbane, Australia). Cells were cultured in suspension in RPMI-1640 medium supplemented with 10% FCS. For a typical experiment, cells were seeded at a density of 105 cells/ml, in 35-mm diameter Petri dishes. OP9 and OP9-DL1 cells were a kind gift from Professor J. C. Zuniga-Pfl€ ucker (Department of Immunology, University of Toronto, Canada). The stromal cell line OP9, established from a newborn B6C3F1-op/op mouse calvaria, was stably transfected with the human Notch-1 ligand Delta-like-1 (3). The cells were cultured according to the protocol in OP9 medium (alphaMEM supplemented with 10% heat inactivated FCS supplemented with L-glutamine) as indicated in the original article (3). The cells were harvested after incubation in trypsin and seeded on fresh culture petri dishes till confluent. The cells of interest were plated on nonconfluent OP9-DL1 cultures and incubated in the presence or absence of PMA (80 ng/ml) or ATRA (1 mM). Cocultivation HL-60 cells were cultured alone or in the presence of OP9-DL1 or control OP9 stromal cell line. OP9 (105/ml) cells were seeded at Day 1 in 1 ml of OP9 medium. At Day 2, after OP9 cells became adherent, the medium was removed and 1 ml of fresh OP9 medium with resuspended HL-60 cells (105/ml) was added. After 24 and 48 h of coculture, HL-60 cells were easily harvested by gentle pipetting, centrifuged, and tested for cell viability by trypan blue dye exclusion test, WST-1 cell proliferation test, and flow cytometry. Determination of Antigen Expression Cells were seeded and induced to differentiate with PMA or ATRA as described and evaluated for differentiation 2
Figure 1. Flow cytometric analysis of DCF fluorescence as indicator of ROS generation in PMA stimulated HL-60 cells. Timedependent response compared to the untreated control (dotted line). Cellular fluorescence is shown: Basal conditions are designated in the first time point (gray histogram) and 20 min stimulation with PMA is the second time point (black full line). The figure expresses representative results of two independent experiments.
markers at indicated time points. Differentiation morphology (increase in cell volume, granularity, and appearance of irregularly shaped cellular processes) was examined microscopically. Viable cells were scored by trypan blue exclusion. Immunofluorescence analysis was performed as previously described (9,10) using monoclonal antibodies and analyzed by flow cytometer (FACSCalibur, Becton Dickinson, New Jersey). Dead cells were excluded from flow cytometric analysis by propidium iodide staining. Superoxide Production Reactive oxygen species (ROS) production was monitored by DCFH-DA fluorescence probe as originally described by Bass et al. (11). The DCFH-DA probe has been used to evaluate the endogenously generated intracellular H2O2. DCFH-DA diffuses into cells, where it is cleaved by cellular esterases, leaving impermeant nonfluorescent DCFH trapped inside the cells and susceptible to H2O2 oxidation. Intracellular oxidation of nonfluorescent DCFH leads to formation of dichlorofluorescein (DCF). In this study, aliquots of cell suspensions were first preincubated with DCFH-DA (5 picomolar (pM)) in the dark with gentle agitation at 37 C for 15 min, washed, resuspended in PBS buffer and then stimulated with PMA. Stimulated and unstimulated unstained cells served as controls in each experiment. All samples were finally resuspended in a propidium iodide (PI) (1 mg/ml) containing buffer. PI labeled dead cells were excluded from the analysis. Cell Proliferation Assay The assay for quantification of cell proliferation and viability was used according to the manufacturer’s instructions (F. Hoffman-La Roche Ltd, Basel, Switzerland). The cell proliferation assay is based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Briefly, 10 ml/well of WST-1 reagent is mixed with cells (ratio 1:10) grown for 3 days in a-MEM medium, supplemented with 10% of FCS (Gibco) or as described with PMA or ATRA. The cells are incubated for 2 h with WST-1 reagent and the formazan dye, produced by viable cells, can be quantified by a multiwell spectrophotometer (microplate reader) by measuring the absorbance of the dye solution at 440 nm (Biorad, Hercules, California, USA). All samples were measured in quadruplicates. Notch Affects Leukemic Cell Differentiation
Original Article
Figure 2. Morphology of the cultivated HL-60 cells treated with PMA. Cells were cultured for 24 h with PMA (B, D, and F) or without treatment (A, C, and E) and analyzed by microscopy. The right panel shows cocultures of HL-60 with OP9 (C and D) or OP9-DL1 (E and F). The arrows are showing the differentiated cells in the PMA stimulated HL-60 OP9 control cultures. (Scale bars 510 mm). A: control HL-60; B: PMA treated HL-60; C: control HL-60 cocultured on OP9; D: control PMA treated HL-60 cocultured on OP9; E: control HL-60 cocultured on OP9-DL1; F: PMA treated HL-60 cocultured on OP9-DL1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Immunoblotting Cells were centrifuged and lysed using Carin lysis buffer (20 mM Tris-HCl pH 5 7.4, 1% NP40, 138 mM NaCl, 2 mM EDTA, 10% glycerol) and proteinase inhibitor cocktail (1:20,
1 ml of protease inhibitors/106 cells; Roche) and frozen in liquid nitrogen. Protein concentrations in the cleared lysates were assessed by Bradford protein assay. The lysates were mixed with 4 3 Laemmli buffer and resolved by 12% SDS-
Figure 3. Flow cytometric analysis of CD11b and CD4 antigen expression at 30 min, 24, and 48 h PMA stimulation of HL-60 cells. The cells were double-stained with FITC-conjugated anti-CD11b and PE conjugated anti-CD4 antibodies. A: FL1, CD11b; (B) FL2, CD4. Control studies were performed with IgG1 isotype antibodies (gray shadowed histograms). Dead cells as detected by PI staining were excluded from the analysis.
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Original Article Immunoblots were developed by means of Signalfire (Cell Signaling Technology) and imaged on Medical X-ray film (Fujifilm, Tokyo, Japan). Reverse Transcription Polymerase Chain Reaction (RT-PCR) R RNA isolation was performed using TRIZOLV reagent according to the manufacturer’s instructions. About 1 mg of R RNA was reversely transcribed using SuperScriptV III Reverse Transcriptase and the cDNA amplified by PCR using AmpliR Taq GoldV DNA Polymerase (all reagents from Life Technologies, Thermo Fisher Scientific, Waltham, MA USA). Primers were designed manually as previously described (12). Briefly, all primers were 20 bp long with the same frequency of GC as AT content. All primers were checked for unspecific binding, as well as for dimer structures with the freely available Amplify software version 3.0 (13). In addition to that primers were checked for intron/exon boundaries to avoid genomic amplification. Statistical Analysis To assess statistical significance, a non-parametric Mann– Whitney test was used and threshold (P) was set to less than 0.05. All experiments were performed at least in triplicate unless otherwise noted.
RESULTS
Figure 4. Proliferation test and trypan blue exclusion analysis. Cells were incubated for 3 days with or without PMA and cocultured with OP9-DL1 or OP9 GFP cells. After 3 days, cells were counted using (A) trypan blue exclusion method and (B) WST proliferation test. This graph shows summarized data from at least three independent experiments. WST1 readings of all samples were performed in quadruplicates and arithmetic mean of each sample was used for further analysis.
PAGE. The proteins were transferred for 15 h at 4 C to nitrocellulose membranes (GE Healthcare, Piscataway, New Jersey; Uppsala, Sweden; Life Sciences Advanced Technologies, St Petersburg, FL, USA) and blocked in tris buffered saline with tween 20 (TBST) containing 5% bovine serum albumin (BSA; Sigma). Membranes were incubated overnight with primary antibodies for human b-Actin (13E5, 1:10,000) or Notch1 (D1E11, 1:2,000) XPV Rabbit mAb (both Cell Signaling Technology, Danvers, MA, USA) and washed three times for 5 min. The membranes were than incubated for 1 h with antirabbit IgG HRP-linked secondary antibody (1:5,000) (Cell Signaling Technology) and washed four times for 10 min. R
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HL-60 Stimulation and Differentiation Intracellular ROS formation as a first response to PMA activation was measured with the DCFH-DA fluorescent probe. DCFH-DA diffuses into cells where it is hydrolyzed into nonfluorescent DCFH. The H2O2 produced during the cell oxidative response oxidizes nonfluorescent intracellular DCFH into a highly fluorescent DCF. Since H2O2 is a secondary product of superoxide anion, this method is an adequate probe to assess intracellular O22 production (14). We evaluated the fluorescence level for 20 min and analyzed the cells after PMA stimulation. Figure 1 shows that intracellular hydrogen peroxide was detected by an increased fluorescence level of DCFH-loaded HL-60 cells. In order to confirm that the cells were stimulated we also checked their morphology as a typical sign of differentiation and analyzed the cells by light microscopy. As shown in Figures 2 and 3, PMA stimulated HL-60 cells changed their morphology and antigen expression already after 24 h in culture as previously established. Inhibition of Differentiation by OP9-DL1 Cocultivation To determine whether there is a relationship between Notch Delta interaction and HL-60 differentiation, we set up cocultures of HL-60 and OP9-DL1 and stimulated the cells with ATRA and PMA. After 24 h, PMA treated HL-60 had a typical morphology as the differentiated cells and the absence of the increase of the cell number confirmed the proliferation arrest. While OP9-DL1 cells alone do not influence the growth of HL-60 cells, upon PMA treatment the HL-60 cells demonstrated impaired differentiation. As a control we used PMA Notch Affects Leukemic Cell Differentiation
Original Article
Figure 5. Flow cytometric analysis of apoptosis of HL-60 cells with (lower row) or without (upper row) the presence of OP9-DL1. Cells were treated with PMA and ATRA and the level of Annexin V and PI binding was measured by flow cytometry. OP9-DL1 cells were excluded from the analysis according to GFP expression. Dot plots are representative of three independent experiments.
Figure 6. Notch expression in human leukemia cell lines. Immunoblot of T-ALL and AML cell lines probed for Notch1 and b-actin (upper panel). mRNA expression of Notch1, Hes1, and HPRT by reverse transcription PCR analysis of HL-60 cells. Representative of at least two blots.
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treated cells grown on the control OP9 cells. The control cell cultures proliferate and increase in cell number compared to the number of cells stimulated with PMA and ATRA. The coculture of HL-60 cells with OP9 cells impact cell proliferation. However, when stimulated with PMA, there was no statistically significant difference (Mann–Whitney test, P > 0.05) between cocultured and control HL-60 cells (Fig. 4). After the cells were treated with PMA or ATRA in cocultures the level of Annexin V and PI binding was measured by flow cytometry in order to establish the degree of apoptosis (Fig. 5). There was no significant difference between HL-60 cells cocultured with OP9-DL1 and the control cultures. We found Notch1 and the downstream target Hairy Enhancer of Split (Hes1) mRNA expression by the promyelocytic cell line HL-60 (Fig. 6). Further, by Western blot analysis we compared the Notch1 protein level expression among TALL and AML cell lines and HL-60 cells. All examined cell lines express Notch1 at the protein level (Fig. 6). We further analyzed the expression of CD11b and CD4 upon stimulation. Unstimulated HL-60 cells express CD4 and already 30 min after PMA stimulation. CD4 expression decreases markedly and stays low further 24 and 48 h. On the contrary, CD11b which is not expressed on unstimulated cells readily shifts 24 h upon stimulation and its expression further increases after 48 h (Fig. 7). Cells in cocultures with OP9-DL1 5
Original Article
Figure 7. Flow cytometric analysis of CD11b and CD4 antigen expression of (A) HL-60 cells cocultured with either OP9 (open black histogram) or OP9-DL1 cells (open red histogram) and treated with PMA or ATRA. Expression of CD11b and CD4 on HL-60 cells cocultured but untreated are represented by shadowed histograms. B: PMA treated HL-60 cells cultured either alone or in cocultures with OP9, OP9-DL1, and OP9-DL1 with DAPT. The histograms show the level of CD11 expression. DAPT 5 Gamma secretase inhibitor. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
showed only a weak response to PMA and ATRA, considering expression of CD11b and CD4. The cells do not differentiate when cocultured with OP9-DL1 cells and the CD4 and CD11b level is very close to the level of control unstimulated cells (Fig. 7A). Figure 7B shows that this phenomenon is specific since it can be inverted by the gamma secretase inhibitor DAPT which inhibits cleavage and consequently activation of Notch. 6
DISCUSSION Notch proteins are involved in the cell fate determination in various systems. They consist of a family of transmembrane receptors that are responsive to binding of ligand of the Delta or Jagged families. Ligand binding leads to the release of activated intracellular Notch domain, its binding to CSL and coactivation and expression of downstream target genes, such
Notch Affects Leukemic Cell Differentiation
Original Article as Hes1 and Hes5 (15,16). Notch has been initially found in T-ALL but it has been shown to be an important regulator of other hematopoietic cells as well. Notch signaling is essential, on one side, to induce T-cell development and, on the other side, to stop the development of B-cells. It has been shown that multipotent lymphoid progenitor cells derived from the bone marrow develop into B-cells in the thymus of Notch1 knockout mice (2,17–19). Ectopic expression and lack of Notch, respectively, disbalance the induction of B versus T cells (2,20). Taghon et al. analyzed the responses of multipotent precursors to Notch signaling. Hes1 was upregulated within 1 day, while other T lineage differentiation gene expression was delayed until about Day 3 of DLL1 signaling (GATA-3 and TCF-1, CD25) when CD44CD25 DN2 thymocytes appeared. Stable induction of a T cell development was dependent on the duration of the contact, density of ligands, and modulating proteins (such as Fringe) (21). A role for Notch in T cell leukemias was demonstrated in animal models, as well as in 50% of human T-ALL with activating mutations in Notch1 (22). Schmitt et al. (3) established a cell line that supports T cell development in vitro by transfecting the OP9 stromal cells with Delta1. The OP9-DL1 culture system stimulates the differentiation of T lymphocytes from hematopoietic stem cells from bone marrow or umbilical cord (23). However, deregulation of Notch signaling was found to have a role in the progression of chronic myeloid leukemia (24). The role of Notch in myelopoiesis and in myeloid leukemia has been studied in the recent years (4–6,25). These studies investigated Notch effects in different model systems and perhaps as a consequence of the four closely related receptors the conclusions drawn were often conflicting as to whether Notch promotes or blocks myeloid differentiation. Our results show that Notch/Delta interaction can influence myelocytic differentiation. We investigated the regulation of the CD11b (integrin) gene in the promyeloblastic leukemic cell line, HL60, following differentiation along the monocytic pathway with PMA and ATRA. The steady-state level of CD11b increased markedly over 48 hr from the undetectable level present before differentiation. Our results indicate that OP9DL1 inhibits PMA induced CD11b expression on HL-60 cells as a marker of HL-60 differentiation. We show here a protective role of Notch activation in the differentiation process. As an additional control we also followed the expressionof CD4 on differentiated cells. Our results indicate that rapid downregulation of CD4 upon PMA stimulation may be independent of Notch/DLL1 interaction. On the other hand, ATRA alone does not influence the expression of CD4, but interestingly this was achieved in the presence of DLL1. CD4 expression in regard to Notch1/DLL1 interaction is a matter of further investigation. A reversion of a blockage stage in certain leukemia types is a base for treatment with different stimulators of differentiation. ATRA is a potent inducer of differentiation and inducer of proliferation and has been widely used in the treatment of acute promyelocytic leukemia (APL). ATRA induces complete remission in patients with APL, but cannot completely eliminate the leukemic clone and to be effective is Cytometry Part A 00A: 0000, 2014
combined with chemotherapy. Since, ATRA alone is not sufficient to eliminate the leukemic clone it is possible that some cells remain protected from differentiation induction if interacted with a Delta expressing tissue. Our findings suggest that the cells which do not differentiate after ATRA stimulation, and therefore cannot be deleted are protected through the Notch pathway. While this paper was in revision, additional evidence for Notch involvement in acute myelogenous leukemia has been published (26,27). The results on primary leukemia cell samples confirm our findings and validate the conclusions on cell lines. The authors propose the development of Notch agonists as a potential therapeutic approach in AML. Our study focused on the effects of Notch on ATRA or PMA treated cells and we suggest that HL-60 cells can be a useful tool for analysis of the mechanisms involved in Notch/ Delta1 interactions in cell differentiation.
ACKNOWLEDGMENTS Authors would like to thank Professor J. C. ZunigaPfl€ ucker (Department of Immunology, University of Toronto, Canada) for his generous gift of the transfected OP9 and OP9-DL1 cells used in our experiments and Professor A. Boyd (Queensland Institute of Medical Research Brisbane, Australia) for his generous gift of the NALM-1 and MOLT-4 cells. Authors also thank M. Paradzik for providing technical assistance in the preparation of the manuscript, Prof. H. Sill and Prof. L. Cicin-Sain for critically reading the manuscript.
AUTHOR CONTRIBUTIONS M.A. designed and performed research and wrote the manuscript; M. M., and J. S. performed research, D. R.-K., I. K-S., D. G. and M. A. contributed to experimental design, analyzing data and writing of the manuscript.
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Notch Affects Leukemic Cell Differentiation
