,
* Signal Transduction Unit/Laboratory of Cell Biology, Section of Human Anatomy, Department of Morphology and Embryology, and
# MIUR ICSI (Interdisciplinary Center for the Study of Inflammation), University of Ferrara, Italy;
Department of Biomorphology, University "G.D’Annunzio", Chieti, Italy;
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland;
L.A. Seragnoli Institute of Haematology, University of Bologna, Italy; and
|| Department of Normal Human Morphology, University of Trieste, Italy
Correspondence: Silvano Capitani, 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. E-mail: cps{at}dns.unife.it
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Key Words: neutrophils • cellular differentiation • hematopoiesis • signal transduction • lipid mediators
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(RAR) and the PML genes, resulting in the formation of a chimeric gene encoding the PML/RAR fusion protein. The presence of PML/RAR confers to leukemic cells a unique sensitivity to all trans-retinoic acid (ATRA), which induces complete remission in more than 90% of APL patients [1
, 2
]. However, patients treated with ATRA invariably relapse, so ATRA is now used in association with chemotherapy in the induction and consolidation/maintenance phases of antileukemic therapy [3
4
5
6
]. In addition, many APL patients fail to respond or relapse, even when treated with ATRA plus chemotherapy. This has prompted different groups of investigators to search innovative methods for easily monitoring the responsiveness of APL patients to ATRA treatment and/or to other drugs to be used cooperatively with ATRA or as an alternative therapy when ATRA fails. The mechanism by which ATRA induces granulocytic differentiation of APL cells involves the retinoid receptors (RARs and RXRs), a family of transcription factors that play a key role in the differentiation of myeloid cells (reviewed in ref. [7 ]), although their downstream target genes are still largely unknown.
Several studies addressing the mechanism underlying the response to ATRA were performed using the HL-60 myeloid cell line, which shows biological and morphological modifications typical of granulocytic differentiation upon ATRA treatment [8
]. Much evidence provided by our and other groups indicate that the ATRA-induced differentiation of the HL-60 cell line involves enzymes related to the inositide metabolism, such as phospholipase C (PLC), protein kinase C, and phosphoinositide 3 kinase [9
10
11
12
13
14
15
]. In particular, we have shown that the ß and 
subfamilies of PLC increase their nuclear amount and activity in an ATRA-dependent manner [12
].
Concerning the role of PLC in granulocytes, recent works demonstrated that in mature neutrophils, ß2 and ß3 are the sole PLC isoforms that are activated by chemoattractants and that appear involved in pathways leading to superoxide production and in the chemoattractant-induced reduction of c-jun NH2 (JNK) and activation of mitogen-activated protein kinase (MAPK) [16 , 17 ].
As these studies indicate that ß PLCs have a relevant role in neutrophil functions, we therefore sought here to investigate their expression during the course of leukemic and normal granulocytic differentiation from pluripotent hematopoietic progenitors. For this purpose, we have chosen the APL NB4 cell line [18 ] containing the t(15;17) translocation as a model system, which represents the most reliable in vitro model to investigate the mechanisms underlying the induction of granulocytic differentiation by ATRA. The expression of ß PLC subfamily members during granulocytic differentiation induced by ATRA was also analyzed in primary APL blasts, as well as in the cytokine-induced granulocytic differentiation of normal CD34+ hematopoietic progenitors. Our data demonstrate that granulocytic differentiation of malignant and normal cells is accompanied by striking modifications of the ß2 isoform of PLC, which can be considered as a marker for granulocytic differentiation and is of potential use to in vitro predict the in vivo response of APL patients to ATRA therapy.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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NB4 cell line was obtained from German Collection of Microorganisms and Cell Cultures (Braunschweing, Germany). The NB4-R clone (NB4-R) was kindly provided by Dr. Carlo Gambacorti-Passerini (Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy). Both cell lines were grown in RPMI 1640 (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Gibco-BRL) in a 94/6% air/CO2 atmosphere, at an optimal cell density between 5 x 105/ml and 1.5 x 106/ml.
Primary blast cells were obtained from 10 patients with AML (seven M3 and three M2) at diagnosis before any therapy and after obtaining informed consent according to the Helsinki Declaration of 1975. AML blasts were washed twice in phospate-buffered saline (PBS) and then cultured in Iscove’s modified Dulbecco’s medium (Gibco-BRL) supplemented with 10% FCS in a 94/6% air/CO2 atmosphere at an optimal cell density of 1.5 x 106/ml. NB4 cell lines and primary AML blasts were treated with ATRA at the indicated concentrations and times.
In experiments in which the PLC inhibitor U-73122 (Calbiochem, La Jolla, CA) was used, NB4 cells, growing in control conditions or in the presence of ATRA, were treated with 2 µM inhibitor, supplemented to the medium after 48 and 72 h of cell culture.
For the isolation of primary CD34+ hematopoietic cells, informed consent to the study was obtained according to the Helsinki Declaration of 1975 from seven normal blood adult donors. Mononuclear cells were isolated from leukopheresis units by Ficoll-Hypaque (d=1.077 g/mL; Pharmacia, Uppsala, Sweden) and were adherence-depleted overnight. After removal of adherent cells, CD34+ cells were isolated using a magnetic cell-sorting program (Mini-MACS, Milteny Biotech, Sunnyvale, CA) and the CD34+ isolation kit in accordance with the manufacturer’s instructions and as described previously [19 ]. Purity of CD34-selected cells was determined for each isolation by FACScan (Becton-Dickinson, San Jose, CA) using a monoclonal antibody (mAb), which recognizes a separate epitope of the CD34 molecule (HPCA-2, Becton-Dickinson) conjugated directly to fluorescein as demonstrated previously [20 ]. CD34+ cell purity ranged from 85 to 97%. CD34+ cells were then seeded in the serum-free ExVivo-20 medium (BioWittaker, Walkersville, MD). Cells were adjusted to the optimal cell density of 5 x 104/ml and cultured in the presence of self-consistent field (SCF; 50 ng/ml) + interleukin (IL)-3 (2 ng/ml) and granulocyte-colony stimulating factor (G-CSF) (20 ng/ml) to induce granulocytic differentiation. All cytokines were purchased from PeproTech Inc. (Rocky Hill, NJ). Every 3–4 days, cultures were demi-populated by removing a half-volume of the medium, which was substituted with fresh medium supplemented with SCF + IL-3 + G-CSF. At these time points, the cells removed were counted, stained, and analyzed by flow cytometry, and the cell density was readjusted to 0.5 x 105/ml.
Mature granulocytic neutrophils with a purity greater than 95% were isolated from healthy adult volunteers, as previously described, using dextran precipitation followed by a Ficoll-Hypaque gradient separation for removal of mononuclear cells and by hypotonic lysis to eliminate contaminating erythrocytes [21 ]. Neutrophils were used within 2 h from the isolation.
Characterization of granulocytic differentiation
The degree of granulocytic differentiation of NB4 and NB4-R cell lines, as well as of primary AML blasts and primary normal CD34-derived cells, was monitored by measuring the nuclear morphology after staining with 4'-6-diamidino-2-phenylindole (DAPI), as previously reported [12
], and by evaluating the phenotypic expression of CD11b and CD15 myeloid surface markers by direct staining with phycoerythrin- (PE) or fluorescein isothiocyanate (FITC)-conjugated anti-CD11b and anti-CD15 mAb (all from Cymbus Biotechnology, Hants, England). Briefly, aliquots of 1.5 x 105 cells were stained with 5 µl of each mAb in 200 µl PBS containing 2% fetal bovine serum at 4°C for 30 min. Nonspecific fluorescence was assessed by using isotype-matched controls. After staining procedures, samples were analyzed by flow cytometry. Data collected from 10,000 cells are presented as % of positive cells or mean fluorescence intensity (MFI) values.
Preparation of cell homogenates and immunochemical analysis
Cell homogenates were prepared as previously described [12
]. Briefly, NB4 and NB4-R cell lines, AML blasts, and primary normal CD34-derived cells, were harvested at different culture times, washed twice with cold PBS containing 1 mM Na3VO4, and resuspended in a lysis buffer containing 50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40 (NP-40); 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid; 1 mM phenylmethylsulfonyl fluoride; 1 µg/ml each aprotinin and leupeptin; 1 mM Na3VO4; and 1 mM NaF (all from Calbiochem).
Rabbit peptide-specific Ab against PLC-ß1, -ß2, -ß3, and -ß4 isoforms were from Santa Cruz Biotechnology (Santa Cruz, CA). Total lysates (50 µg protein) were separated on 7.5% polyacrylamide denaturing gels and blotted to nitrocellulose membranes (Amersham Life Science, Little Chalfont, UK). The blots were then saturated in Tris-buffered saline solution, containing 5% milk and 0.05% Tween-20 (blocking buffer) for 60 min at room temperature and incubated overnight at 4°C with the same solution containing the primary antibodies. After washes in blocking buffer, they were incubated for 45 min at room temperature with peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; Sigma Chemical Co.), diluted 1:2000 in blocking buffer, and washed as above. The final detection was performed using the enhanced chemiluminescence system (DuPont, NEN Research Products, Boston, MA), according to the manufacturer’s instructions.
Densitometric analysis was performed on the Molecular Analyst GS670 (Bio-Rad, Hercules, CA).
Immunocytochemical analysis
After two washes in PBS, cells were cytocentrifuged onto glass slides, fixed with freshly prepared 4% paraformaldehyde (10 min at room temperature), washed in PBS (5 min), and reacted with a polyclonal antibody (diluted 1:60; Santa Cruz Biotechnology) directed against PLC-ß2 in NET gel (150 mM NaCl, 5 mM ethylenediaminetetraacetate, 50 mM Tris-HCl, pH 7.4, 0.05% NP-40, 0.25% carragenin, 0.02% NaN3) for 2 h at room temperature. Samples were then reacted with a secondary antibody (FITC-coniugated anti-rabbit IgG, diluted 1:150) in NET gel for 45 min at room temperature. After one wash with NET gel and one wash with PBS, samples were incubated (for 30 s–5 min) with 0.5 µg/mL DAPI (in PBS), then washed in PBS, dried with ethanol, mounted in glycerol containing 1,4-diazabicyclo [2.2.2] octane to retard fading, and analyzed with a fluorescence microscope (Carl Zeiss Axiophot 100), as previously reported [12
].
PLC activity
Control and ATRA-treated cells, cultured or not in the presence of the PLC inhibitor U-73122, were subjected to in vitro assay of PLC activity. For this purpose, total homogenates (100 µg protein), after two washes in PBS to remove culture medium, were incubated in a final volume of 100 µl for 15 min at 37°C in the presence of 3 nmoles phosphatidylinositol 4, 5-bisphosphate (PIP2), 30,000 dpm [3H] PIP2, 0.06% taurodeoxycholate, 0.6% NaCl, 0.1 M Tris-HCl, pH 6.8, 0.1 mM CaCl2. The reaction was stopped by addition of the lipid extraction mixture, and the hydrosoluble products were counted as described previously [12
].
Statistical analysis
The results were expressed as means ± SD of three or more experiments performed in duplicate. Statistical analysis was performed using the two-tailed Student’s t-test for unpaired data.
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Figure 1. Analysis of PLC isoforms in parental NB4 and NB4-R cells. Cell lysates from control (-) or differentiated (+; 4 days of ATRA treatment) NB4 (A) and NB4-R (B) were subjected to Westen blot and were analyzed with the antibodies against the different isoforms of the PLC-ß subfamily. Densitometric analysis of autoradiograms was performed and is shown as a.u. (C) Immunocytochemical analysis, performed with an anti-PLC-ß2 antibody, of parental NB4 in control conditions and after treatment with ATRA for 4 days and NB4-R cells treated with ATRA for 4 days. The nuclear morphology was detected by specific staining of DNA with- DAPI. The data are representative of three separate experiments performed in duplicate. Original magnification, 250x. (D) Effect of different concentrations of ATRA (10 nM–100 µM) on the amount of PLC-ß2 expressed in NB4 cells at various time points of treatment. Western blot followed by densitometric analysis of autoradiograms. The data are representative of four separate experiments performed in duplicate.
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As the most striking up-regulation involved the PLC-ß2 isozyme, the expression of this protein was also evaluated by immunocytochemical analysis (Fig. 1C) , which confirmed a marked increase of the level of this enzyme in parental NB4 cells upon ATRA treatment. The increase in the levels of PLC-ß2 paralleled the granulocytic differentiation of NB4 cells, evaluated by nuclear staining with DAPI and analysis of the surface expression of CD11b and CD15 Ag. Again, the amount of PLC-ß2 in NB4-R cells, as well as their degree of granulocytic differentiation, was significantly lower than in ATRA-responsive NB4 cells (Fig. 1C ; Table 1 ).
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Table 1. Mean Fluorescence Intensity Values of the Indicated Surface Antigens
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Granulocytic differentiation of primary APL blasts obtained from ATRA-responsive patients is characterized by the up-regulation of PLC-ß2 expression
To better analyze the involvement of PLC-ß2 in the process of ATRA-induced differentiation of APL, the amount of PLC-ß2 was evaluated in primary APL and non-APL blasts, purified at diagnosis from the bone marrow of patients with AML. As shown in Figure 2 A
, when APL blasts were purified from patients showing a good in vivo response to the ATRA treatment, only a small amount of PLC-ß2 was present in freshly isolated blasts, and after 4 days of in vitro ATRA treatment, PLC-ß2 expression increased drastically. The immunocytochemical analysis (Fig. 2B)
confirmed these findings and also showed that the nuclear morphology of ATRA-treated APL blasts was typical of mature granulocytes. Of note, analysis of PLC-ß2 expression in APL blasts obtained from one patient who rapidly developed resistance to ATRA therapy (Resistant M3 patient 1, Fig. 2A
) revealed that this enzyme was not affected by in vitro treatment of blasts with ATRA. The evaluation of morphology of ATRA-treated blasts confirmed the failure of ATRA in inducing granulocytic maturation of these primary cells. In addition, the analysis of PLC-ß2 expression in APL blasts obtained from one patient who developed a resistance after several cycles of treatment with ATRA (Resistant M3 patient 2, Fig. 2A
) revealed a mixed population of morphologically differentiated and undifferentiated cells (Fig. 2B)
. It is interesting that in this particular case, PLC-ß2 was only expressed in cells with a nuclear granulocyte-like morphology (Fig. 2A
and 2B
). Treatment of these blasts with ATRA for 4 days increased the number of differentiated cells expressing high levels of PLC-ß2. However, a significant number of undifferentiated cells with a low level of enzyme persisted (Fig. 2B)
, indicating that only a fraction of the cells was responsive to ATRA. Figure 2
also demonstrates the analysis of PLC-ß2 expression in blasts purified from one patient affected by an M2 AML. As shown by immunochemical (Fig. 2A)
and immunocytochemical (Fig. 2B)
analyses, these blasts displayed a low expression level of PLC-ß2 that was unaffected by ATRA treatment. On the whole, the analysis of PLC-ß2 expression was extended to primary blasts obtained from 10 AML patients (seven diagnosed as APL and three as non-APL; Table 2
). By semiquantitative Western blot analysis of PLC-ß2 in AML blasts treated or not with ATRA for 4 days in vitro, a clear-cut correlation was observed between the appearance of a differentiated phenotype and a significant (P<0.05) increase of PLC-ß2 expression in five out of seven APL cases. More importantly, the up-regulation of PLC-ß2 observed upon treatment with ATRA in vitro, except for patient number 7, correlated fully with the in vivo response to ATRA. On the contrary, in blasts from all patients with M2 acute leukemia, the level of PLC-ß2 remained very low (Table 2)
.
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Figure 2. Analysis of PLC-ß2 in primary APL and non-APL AML blasts. Blasts from APL patients, responsive (Responsive M3 patient) or with a developed resistance (Resistant M3 patients 1 and 2) to ATRA treatment, and from a not responsive patient with an M2 myeloid leukemia (Not responsive M2 patient) were subjected to immunochemical (A) and immunocytochemical (B) analysis with an anti-PLC-ß2 antibody in control conditions (-) and after 4 days of treatment with ATRA (+). The data are representative of five ATRA-responsive cases, of two ATRA-resistant cases, and of three ATRA-nonresponsive M2 samples. (B) Original magnification, 250x.
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Table 2. Analysis of PLC-ß2 Expression and Blast Differentiation
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Figure 3. Analysis of PLC-ß2 in normal granulocytic cells. CD34-derived granulocytic cells, obtained from CD34+ primary progenitors seeded in liquid cultures supplemented with IL-3 + G-CSF for the indicated times, were subjected to immunochemical (A) and immunocytochemical (B) analysis with an anti-PLC-ß2 antibody. The expression of PLC-ß2 in mature neutrophils obtained from the peripheral blood of adult donors is also shown. Data from densitometric analysis of autoradiograms are expressed in a.u. (A); (B) original magnification, 250x. (C) The progression through granulocytic differentiation of primary CD34+ progenitors was monitored by evaluation of specific surface markers. The data are representative of three separate experiments peformed in duplicate.
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Figure 4. Analysis of PLC activity in NB4 cells. NB4 cells were cultured in the presence of ATRA and/or the PLC inhibitor U-73122 for 4 days, with the inhibitor added after 48 and 72 h of culture. Cell lysates were then subjected to in vitro assay of phosphodiesterase activity by using tritiated PIP2 as substrate (A) and analysis of the progression through granulocytic differentiation by flow cytometric determination of the expression levels of CD11b (B). The amount of PLC-ß2 and PLC-ß3 was evaluated by means of immunochemical analyses on lysates from cells under the different conditions (C). Densitometric analysis of autoradiograms are expressed as a.u. The data are representative of three separate experiments performed in duplicate.
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The effect of the reduced PLC activity on the expression of the different PLC isoforms was also analyzed. As shown in Figure 4C , neither the ATRA-induced strong increase of PLC-ß2 nor the less-evident increase of PLC-ß3 was affected by the presence of U-73122 in cell-culture medium. Concerning other PLC isoforms present in NB4, we also found the 1 and 2 isozymes, which decreased in amount during the ATRA treatment (unpublished results).
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Recent studies [16 , 17 ] have shown that PLC-ß2 and PLC-ß3, the only isoforms of the ß subfamily of PLC expressed in mature neutrophils, are activated by chemoattractants in these cells. PLC-ß2 and PLC-ß3 appear to be involved in pathways leading to superoxide production in response to the formyl peptide N-formyl-Met-Leu-Phe (fMLP) as well as in the chemoattractant-mediated decrease of JNK and MAPK activation. These studies, performed with PLC-deficient mouse lines, indicated a relevant role for PLCs in granulocytic functions and in particular, in the down-modulation of hyperinflammatory conditions [16 ].
In this paper, we have demonstrated that, of the ß subfamily, only PLC-ß3 was present in significant amounts in the APL-derived NB4 cell line, and ß2 was almost absent. However, when parental NB4 cells were treated with ATRA, the expression of PLC-ß2 increased strongly. Thus, the deficiency of PLC-ß2 isoform was one of the features characterizing the NB4 cell line, which is unable to progress through the granulocytic pathway. Conversely, treatment with ATRA abolished the differentiation block by inducing a set of events that included the increase of expression of PLC-ß2. The analogy between PLC-ß2 expression and ATRA-dependent granulocytic differentiation of NB4 was confirmed by using an ATRA-resistant NB4 cell line, in which, at variance with the parental cells, the levels of PLC-ß2 remained low after treatment with ATRA.
More importantly, low levels of PLC-ß2 were also present in primary APL blasts purified from the bone marrow of patients bearing APL, and the level of PLC-ß2 expression after ATRA treatment in vitro correlated strikingly with the in vivo ATRA responsiveness of the APL patients. Moreover, the analysis of blasts purified from patients affected by M2 AML, who do not respond to the in vivo treatment with ATRA, failed to show significant modifications of PLC-ß2 after in vitro ATRA treatment. We could also demonstrate that the up-regulation of PLC-ß2 characterizes not only ATRA-induced APL blast differentiation but also normal granulocytic differentiation of primary CD34+ hematopoietic progenitors.
Taken togheter, these data suggest that up-regulation of PLC-ß2 is a marker for ATRA-induced APL blast differentiation and normal granulocytic differentiation.
We demonstrate that PLC activity may be important for the ATRA-induced granulocytic differentiation of NB4. Pharmacological data with the PLC inhibitor U-73122 showed a correlation between inhibition of PLC activity and inhibition of NB4 neutrophil-like maturation. Although the U-73122 inhibitor does not show specificity toward a particular PLC isoform, the inhibition of PIP2 phosphodiesterase activity suggests the involvement of PLC-ß2, because PIP2 is the preferred substrate for the PLC-ß isoforms [27 ].
That the levels of PLC- isoforms decrease during the ATRA treatment makes it unlikely that these isoforms contribute to the increased PLC activity observed during differentiation or to the differentiation process. However, additional experiments will be required to elucidate the role of PLC isoforms, particularly PLC-ß2, in granulocytopoiesis.
It should be noted that the therapeutic treatment with ATRA of APL patients has radically modified the prognosis of this kind of AML. Unfortunately, a significant number of APL patients relapse after one or more cycles of ATRA administration, used alone or in combination with chemotherapy. The fate of these patients is hardly predictable, but it has been shown that they invariably relapse if not retreated [28
29
30
31
]. A current major problem is the identification of relapsed patients who maintain responsiveness to ATRA versus patients who have lost the ability to respond to ATRA. In fact, the former group can be treated again with ATRA, and the latter group would benefit from alternative therapies such as arsenic administration or intensive chemotherapy. At the moment, the only method to predict resistance or future relapse of the disease is the analysis of the transcripts of PML-RAR gene. However, molecular positivity is not closely related to prognostic significance. For these reasons, an in vitro test able to determine the in vivo responsiveness of APL patients to ATRA appears of great interest, especially after relapse.
The data presented in this paper indicate that PLC-ß2 is a sensitive marker to monitor granulocytic differentiation of normal and malignant myeloid progenitors. The analysis of PLC-ß2 expression may represent an easy and relatively rapid in vitro method for predicting the response of APL patients to ATRA treatment in vivo. In particular, the evaluation of the levels of PLC-ß2 in leukemic blasts after in vitro treatment with ATRA may allow a rapid identification of the best therapeutic strategy for the treatment of each APL patient at diagnosis or in case of relapse.
Received October 25, 2001; revised January 24, 2002; accepted January 24, 2002.
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locus due to a variant t (11;17) translocation associated with acute promyelocytic leukemia EMBO J 12,1161-1167[Medline], its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia Blood 93,3167-3215 in chemoattractant-mediated signal transduction Science 287,1046-1049 through proteasome pathway mediates retinoic acid resistance Blood 5,1477-1481This article has been cited by other articles:
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