Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2008 Feb 21;134(8):861–872. doi: 10.1007/s00432-008-0356-8

Additive effects of PI3-kinase and MAPK activities on NB4 cell granulocyte differentiation: potential role of phosphatidylinositol 3-kinase γ

Sebastian Scholl 1,✉, Tzvetanka Bondeva 2, Yuantao Liu 2, Joachim H Clement 1, Klaus Höffken 1, Reinhard Wetzker 2
PMCID: PMC12160749  PMID: 18288489

Abstract

Purpose

In acute promyelocytic leukemia (APL) the chromosome translocation t(15;17) resulting in the PML–RARα fusion protein is responsible for a blockage of myeloid differentiation. In this study we investigated the expression of different Phosphatidylinositol 3-kinase (PI3K) isoforms during granulocyte differentiation of NB4 cells induced by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9cisRA) or retinoic acid receptor (RAR) agonists.

Methods

NB4 cells were analysed for their ability to differentiate into granulocytic lineage by the use of ATRA, 9cisRA or RAR agonists. Expression of signalling proteins was investigated by western blot and real-time PCR. PI3K activity was determined by in vitro kinase assays.

Results

Co-treatment of NB4 cells with either LY294002 to inhibit PI3Ks or PD98059 in order to suppress MEK activity led to significant reduction of CD11b surface expression during ATRA, 9cisRA or the RARα agonist Ro40-6055 dependent NB4 cells granulocyte differentiation. We also show that only the G-protein coupled receptor activated PI3Kγ isoform demonstrates up-regulated protein and mRNA expression during myeloid differentiation of NB4 cells via RARα and RARβ—dependent mechanism. Furthermore, activation of MAPK cascade including phosphorylation of MEK increases during retinoid induced differentiation of NB4 cells. Interestingly, protein kinase assays of immunoprecipitated PI3Kγ revealed a protein of about 50 kDa that is phosphorylated when NB4 cells were treated with the RARα agonist Ro40-6055.

Conclusion

Collectively, our data suggest additive effects of PI3K and MAPK activity on ATRA-dependent NB4 cells granulocyte differentiation.

Keywords: AML, APL, NB4, MAPK, PI3-kinase

Introduction

Acute myeloid leukemia is characterised by a blockage of differentiation of the malignant clone resulting in uncontrolled growth of leukemic blasts. Beside the possibility of cytotoxic and/or immune therapies (e.g. chemotherapy, allogeneic stem cell transplantation) there is another therapeutic strategy: to overcome the differentiation stop by differentiating agents. Among these substances only all-trans-retinoic acid (ATRA) is regularly used in the treatment of acute promyelocytic leukemia (APL or AML M3 according to FAB classification) (Tallman et al. 1997, 2002; Huang et al. 1988; Warrell et al. 1993; Fenaux et al. 1997). This subset of acute myeloid leukemia is characterised by the PML–RARα fusion protein due to the translocation t(15;17) (Benoit et al. 2001; Melnick and Licht 1999).

The expression of PML–RARα leads to loss of nuclear structures called PML bodies that represent important loci in terms of gene transcription (Koken et al. 1997; Grande et al. 1996; Mu et al. 1996). ATRA that is limited to the treatment of the APL subset of FAB AML subtypes can overcome the impaired expression of a wide range of genes involved in granulocyte differentiation by reassembling of PML bodies (Wang et al. 2004; Yang et al. 2003). NB4 cells provide the best-investigated model to analyse ATRA induced differentiation of APL cells (Lanotte et al. 1991).

Several signalling pathways are attributed to be important in the differentiation of hematopoietic cells. In this intracellular network the mitogen activated protein kinase (MAPK) pathway plays a pivotal role in terms of the critical balance between proliferation and differentiation (Lewis et al. 1994; Marshall 1994; Seger and Krebs 1995). Drug induced stimulation of endogenous MAPK or overexpression of MAPK can result in growth arrest and induction of differentiation in hematopoietic cell lines (Miranda et al. 2002; Kang et al. 1993). In detail, the activation of Erk2 in retinoic acid mediated differentiation including an involvement of Raf phosphorylation has been demonstrated (Battle et al. 2001; Yen and Varvayanis 2000; Yen et al. 1999).

PI3Ks are intracellular signalling proteins that regulate cellular functions like proliferation, differentiation, and apoptosis. The PI3K isoforms α, β, and δ are expressed as heterodimeric proteins consisting of a p110 catalytic subunit and a p85 adapter subunit and are mostly regulated by the receptor tyrosine kinases (Carpenter and Cantley 1996; Vanhaesebroeck et al. 1997; Vanhaesebroeck and Waterfield 1999; Hiles et al. 1992; Hu et al. 1993). In contrast, PI3Kγ isoform is found to be activated by βγ subunits of heterotrimeric G proteins (Stoyanov et al. 1995). It is tightly associated with a protein called p101 with no homology to other known proteins (Stephens et al. 1997).

Bertagnolo et al. (1999) could demonstrate a functional role of PI3K activity in granulocyte differentiation of HL-60 cells. Antisense strategies to suppress the expression of p85 lead to a strong inhibition of ATRA-mediated differentiation.

The involvement of PI3Kγ in the differentiation process of erythroid progenitor cells was recently demonstrated (Schmidt et al. 2004).

We could previously demonstrate that PI3Kγ can be up-regulated by ATRA in U937 cells while the expression of the other PI3K isoforms was not affected (Baier et al. 1999). The isoform specific up-regulation of PI3Kγ did not only lead to an increase of endogenous PI3K activity but also resulted in an enhanced MAP kinase activity (Erk2). Thus, it was hypothesised that PI3Kγ is involved in MAP kinase activation during ATRA induced U937 cell differentiation.

Based on these findings in this study we especially investigated the expression of different PI3K isoforms during granulocyte differentiation of NB4 cells induced by ATRA or different RAR agonists. We also analysed the activation of the MAPK pathway (Raf, MEK, Erk) by western blot analyses in these experiments. Furthermore, we addressed the question whether the inhibition of PI3K by LY294002 or the inhibition of MEK by PD 98059 influences myeloid differentiation after treatment with the above mentioned substances. In this context the addition of ATRA, cis-RA or RAR alfa agonist to the NB4 cells induced PI3K and MAPK paths dependent myeloid differentiation. In between PI3Ks only the PI3Kγ expression on mRNA and protein levels was increased and this finding correlates as well as with an increased lipid and protein kinase activities of the enzyme. Neither LY 294002 nor PD98059 were able to completely block the myeloid differentiation of NB4 cells when used alone, therefore we suggest a co-operative mechanism regulating this process involving in a concert PI3K and MAPK pathways.

Material and methods

Reagents and antibodies

All-trans-retinoic acid (ATRA) and 9-cis-retintoic acid (9cisRA) were purchased from Sigma. PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 were obtained from Calbiochem (Vlahos et al. 1994; Alessi et al. 1995). RARα agonist (Ro40-6055), RARα inhibitor (Ro41-5253), RARβ agonist (Ro48-2249) and RARγ agonist (Ro44-4753) were kindly provided by Dr. Mohr (Roche, Germany). All substances were solved in DMSO. Antibody conjugates used for flow cytometry (anti-CD11b-APC, anti-CD14-PE and the corresponding isotype control) were from Becton Dickinson. Antibodies used in western blots were purchased from Upstate Biotechnology (anti-p85), Santa Cruz (anti-actin, anti-mouse-HRP, anti-rabbit-HRP), Cell Signalling Technology (anti-phospho-Ser217/221-MEK1/2, anti-phospho-Thr202/Tyr204-Erk1/2, anti-phospho-Ser338-Raf, anti-MEK1/2, anti-Erk1/2) or Transduction Laboratories (anti-Raf). Anti-vinculin mouse monoclonal antibody was obtained from Serotec (United Kingdom). Anti-PI3Kγ monoclonal antibody, directed against the N-terminal fragment of PI3Kγ was used for Western blot. Anti-PI3Kγmonoclonal antibody, clone 641, was used for immunoprecipitation (both PI3Kγ antibodies are produced in FSU Jena facilities). Anti-PI3Kγ polyclonal antibody (s-19) was purchased from Santa Cruz (Germany).

Cell culture and cell lysis

The human promyelocytic cell line NB4 was kindly provided by Dr. M. Lanotte (Paris, France). NB4 cells were maintained in DMEM (Gibco) containing 10% (v/v) fetal calf serum (Biochrom), 2 mM l-glutamine, and 0,125% gentamicin. For differentiation experiments cells were splitted and adjusted to 5 × 105 cells/ ml in DMEM. Cell culture flasks were incubated using different combinations of reagents for 48 h. After this, cells were harvested, washed three times with cold PBS and lysed on ice with lysis buffer containing 10 mM Tris pH 7.5, 130 mM NaCl, 1% NP-40, 10 mM NaPPi, 1 mM PMSF, 0.1 mM Na3VaO4. Lysates were transferred to microcentrifuge tubes and then precleared by centrifugation at 11,900g for 15 min at 4°C. Protein concentrations were determined using Bradford reagent (Sigma) according to the manufacturer’s instructions.

SDS-PAGE electrophoresis and immunoblotting

After determination of protein concentration lysates of each experiment were adjusted to equal protein concentration and 50 μg protein per lane was fractionated by 7.5 or 10% SDS-PAGE, followed by western blot analysis.

FACS analysis

For surface staining of differentiation marker cells were washed two times with PBS (5 × 105 cells per tube) and stained with anti-CD11b-APC or anti-CD14-PE (20 μl per tube) or stained with IgG-APC or IgG-PE as isotype controls. After washing two times samples were measured using a FACS Calibur cytometer (acquisition of 10,000 cells per test).

Cell cycle analysis was performed using CycleTestR (Becton Dickinson) according to the manufacturers instructions. In short, cells were washed in PBS, pelleted and incubated with a trypsin containing solution followed by trypsin inhibitor solution to stop cell digestion. Nuclei were stained with propidium iodide while co-treatment with RNase. Annexin V assay kit was purchased from Pharmingen. After surface staining (APC-conjugated isotype control vs. anti-CD11b-APC) 5 × 105 cells were washed twice in PBS and once in Ca2+ free annexin binding buffer. After resuspension in 185 μl annexin binding buffer 5 μl FITC conjugated annexin V and 10 μl propidium iodide solution supplied by the manufacturer were added or cells left unstained (autofluorescence as control of cells prior stained with isotype control) and incubated for 30 min at room temperature in the dark. Before FACS analyses 300 μl annexin binding buffer was added to each sample. Furthermore, two separate samples (annexin-V-FITC or PI alone) were prepared to adjust the compensation of the cytometer.

RNA preparation and real-time PCR

Total RNA was routinely isolated from 5 to 10 × 106 NB4 cells by the use of RNA easy Qiagen kit. After this, 1 μg RNA was used for synthesis of cDNA according to a standard protocol. The following primer sets were used for real-time PCR amplifications: human p101: forward primer 5′-gacatgcacggaggaccgcatccag-3′ and reverse primer 5′-acagcaaggaactcggcctg-3′; human PI3-K γ: forward primer 5′-catattgacttcgggcacattcttg-3′ and reverse primer 5′-gtctctgcaaacttcgatctgatc-3′; human PBGD: forward primer 5′-ggcaatgcggctgcaa-3′and reverse primer 5′-gggtacccacgcgaatcac-3′. Real-time PCR based on SYBRGreen was performed using the LightCycler system (Roche). The PCR set up was as follows: 1 μl cDNA, 0.5 μM of each primer, 4 μl of five fold master mix and sterile water (up to 20 μl). Amplification of p101 and PI3Kγ was performed with denaturation at 95°C for 5 s, annealing at 62°C at 10 s and elongation at 72°C for 10 s for 45 cycles while the amplification cycles of the housekeeping gene PBGD contained consisted of denaturation at 95°C for 5 s, annealing at 66°C at 10 s and elongation at 72°C for 5 s. Melting curve analysis of each PCR demonstrated the product specificity.

PI3K protein kinase activity assay

Cells were lysed and subjected to immunoprecipitation using PI3Kγ antibody clone 641. Lysates were incubated with the antibody overnight at 4°C and then the immunecomplexes were collected by the aid of 10 μl Gamma-bind Sepharose beads (Amersham) for an additional 2 h at 4°C. The immunoprecipitations were washed three times with lysis buffer and ones with a protein kinase buffer containing 12.5 mM MOPS pH 7.5, 12.5 mM β-glycerophosphate, 7.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM NaF. As an in vitro protein substrate was used 2 μg MBP (Sigma)/reaction. The beads were then re-suspended in 50 μl of protein kinase buffer, incubated at the presence or absence of 100 nM Wortmannin for 30 min at 37°C. The reactions were initiated by the addition of 20 μM cold ATP and 10 μCi [32P]γ-ATP to the protein kinase buffer and the assay was performed for 30 min at 37°C on a termomixer. Reactions were terminated with 12.5 μl of 5× SDS-PAGE loading buffer. Samples were denatured at 95°C for 10 min. and resolved on 12% SDS-PAGE. After silver or Coomassie staining, the gel was dried on a Biorad vacuum gel-dryer. The phosphorylated bands were visualised using a PhosphorImager or by exposure on a BioMax MR-1 Film.

PI3K lipid kinase assay

PI3Kγ or p85 were immunoprecipitated as described above and the complexes were resuspended in 25 μl dH20. To each sample 25 μl of 3× lipid kinase buffer (20 mM Tris–HCl pH 7.4, 4 mM MgCl2, 100 mM NaCl), 10 μg phosphatidylinositol (sonicated in water), 10 μM cold ATP and 5 μCi [32P]γ-ATP were added. The reaction was incubated at 37°C for 15 min and was terminated with 100 μl 1 M HCl. The lipids were extracted by addition of 400 μl chloroform: methanol (1:1) and the organic phase was washed twice with 200 μl 1 M HCl. Samples were loaded on a silicagel 60 thin layer chromatography (TLC) plates using Linomat IV (Camag). After the run was completed the plate was dried and phosphorylated lipids were visualised with the PhosphorImager system.

Statistics

Western blots represent one of two or three independent experiments as indicated. Histograms show the mean of three to four different experiments as indicated. Statistical significance was calculated using Student’s t test for independent variables by Excel software (Microsoft). P values < 0.05 were considered statistically significant.

Results

Involvement of PI3K in ATRA or Ro40–6055 (a RARα specific agonist) dependent granulocyte differentiation of NB4 cells

The differentiation block of promyelocytic leukemia cells is characterised by the PML–RARα fusion protein and it can be overcome by treatment with retinoids. Nevertheless, little is known about the genes and proteins involved in the regulation of this process. Recently, we have demonstrated that in U937 cells addition of ATRA induced macrophage like lineage differentiation in a Wortmannin sensitive manner. Testing the activity and the expression of the PI3K isoforms from class I we have observed, that only PI3Kγ has shown an increased protein concentration and as well treatment with ATRA led to an increased lipid kinase activity of PI3Kγ (Schmidt et al. 2004). Based on this, we now investigated the ability of NB4 cells to differentiate to granulocyte lineage and the involvement of PI3K in this process. The differentiation status of NB4 cells was determined by flow cytometry analysing the surface expression of CD11b indicating granulocyte differentiation. In order to evaluate what isoforms of RARs (RARα, β and γ) are involved in this process, we used different substances able to stimulate or to inhibit the corresponding RAR isoforms (Table 1). We treated NB4 cells for 2 days with ATRA, a pan-RAR agonist, or with Ro40–6055 that represents a RARα specific agonist to test whether LY294002, a commonly used inhibitor of PI3K, could affect NB4 granulocyte differentiation by ATRA or the RARα agonist Ro40–6055. To approach this, NB4 cells were incubated for 2 days with LY294002 alone or in a combination with ATRA or Ro40–6055 (Fig. 1a), followed by FACS analysis. Similar to ATRA, treatment of NB4 cells with Ro40–6055 for 2 days led to CD11b expression as a marker for granulocyte differentiation of more than 90% of cells while no expression of CD14 (monocyte differentiation) was observed. The treatment of NB4 cells with LY294002 alone did not affect the differentiation status of these cells but inhibition of PI3-K could significantly inhibit granulocyte differentiation of NB4 cells as induced by ATRA (Fig. 1a, plus LY294002 mean 57%, range 48–64%, P 0.001) or the RARα agonist Ro40–6055 (Fig. 1a, plus LY294002 mean 57%, range 51–64%, P 0.001).

Table 1.

Specificity of applied modulators of retinoid signalling

RAR
α Β γ
ATRA + + + 1 μM (and 0.1 μM)
9cisRA + + + 1 μM (and 0.1 μM)
Ro40–6055 + w/o w/o 10nM
Ro41–5253 − w/o w/o 1 μM
Ro48–2249 w/o + w/o 10 nM
Ro44–4753 w/o w/o + 1 μM
Ro61–8431 − − − 1 μM
Open in a new tab

Characteristics of each substance interfering with RAR signalling as well as its concentration used in this study are shown

+ agonist, − inhibitor, w/o without effect

Fig. 1.

Fig. 1

Open in a new tab

Influence of inhibition of PI3Ks and influence of inhibition of RARα receptor on differentiation of NB4 cells induced by ATRA or the RARα agonist Ro40–6055. a NB4 cells were treated with 1 μM all-trans-retinoic acid (ATRA) or 10 nM RARα agonist (Ro40–6055) for 48 h at 37°C to induce granulocyte differentiation measured by CD11b surface expression (a). Expression of CD11b under these conditions was set 100%, respectively. NB4 cells treated with 0.2% (v/v) DMSO was used as control as well as NB4 cells were incubated with 20 μM of the PI3K inhibitor LY294002 alone as indicated. The influence of this inhibitor is shown by co-treatment of both differentiating agents and LY294002. Percentage of CD 11b expression indicates the inhibitory effects compared to single treatment with ATRA or RARα agonist (a) respectively. The diagrams show mean and p values of four independent experiments; b NB4 cells were pretreated with 1 μM of the RARα antagonist Ro41–5253 for 4 h at 37°C. After this, cells were treated with 1 μM all-trans-retinoic acid (ATRA) or 10nM RARα agonist (Ro40–6055) for 48 h at 37°C to induce granulocyte differentiation measured by CD11b surface expression. Figure 1a shows the primary histograms of flow cytometry when cells were stained with anti-CD11b-APC. Figure 1b shows expression of CD11b after treatment with ATRA was set 100%. DMSO 0.2% (v/v) was used as control beginning during the pre-treatment period. Percentage of CD11b expression indicates the inhibitory effect of Ro41–5253 when NB4 cells are treated with ATRA or RARα agonist, respectively. The diagram shows mean and P value and standard deviation of three independent experiments

In addition, we measured the potential induction of apoptosis as well as changes of cell cycle distribution during myeloid differentiation dependent on inhibition of PI3K using LY294002 (see Table 2). Our data demonstrate that induction of granulocyte differentiation by ATRA or Ro40–6055 is not associated with increased apoptosis of CD11b expressing cells. Single treatment of NB4 cells with LY294002 lead to an increase not only of Annexin V-FITC positive cells but also to an increased amount of cells within the sub-G1 fraction of the cell cycle. Both observations can be interpreted as an induction of cell suicide in these cells and reflect an enhanced sensitivity towards pro-apoptotic stimuli. These cellular effects are markedly reduced during co-treatment with one of the differentiation inducing substances, ATRA or the RARα agonist Ro40–6055. In addition, CD11b expression after incubation with ATRA or the RARα agonist Ro40–6055 is associated with a decrease of the proliferative index represented by the S+G2/M fraction of the cell cycle.

Table 2.

Analyses of apoptosis with Annexin V assay and cell cycle distribution

AnnexinV-FITC positive AnnexinV- and CD11b positive sub-G1 G0/G1 S+G2/M
DMSO 0.2% (v/v) 21 1 6 51 43
ATRA 1 μM 11 2 8 60 33
Ro40–6055 10nM 8 5 6 63 31
LY 20 μM 42 0 21 49 30
ATRA/LY 23 1 13 61 27
Ro40–6055/LY 20 1 10 64 27
Open in a new tab

Percentage of total apoptotic cells and apoptotic cells with CD11b expression as well as fractions of cell cycle are indicated for each experimental condition. NB4 cells were treated with 1 μM all-trans-retinoic acid (ATRA) or 10 nM RARα agonist (Ro40–6055) for 48 h at 37°C to induce granulocyte differentiation measured by CD11b surface expression. Expression of CD11b under these conditions was set 100% respectively. NB4 cells treated with 0.2% (v/v) DMSO were used as control. Furthermore, NB4 cells were incubated with 20 μM of the PI3K inhibitor LY294004 alone as indicated. The influence of this inhibitor is demonstrated by co-treatment of ATRA or the RARα agonist and LY294002. Histograms show CD11b surface expression using anti-CD11b-APC conjugate compared with isotype controls conjugated with APC while NB4 cells treated with DMSO 0.2% (v/v) were used as control. For cell cycle analysis of NB4 cells we used isolated nuclei as described above. Histograms of FL-2A were used to discriminate cells being in G0/G1-, S- or G2/M phase while cells of S- and G2/M-phase reflect the mitotic index. Data show one representative of three independent experiments

ATRA induced granulocyte differentiation of NB4 cells is not restricted to the RARα receptor

As ATRA, a pan-RAR agonist, and the RARα agonist Ro40–6055 were equally potent in inducing granulocyte differentiation of NB4 cells, we further addressed the question whether retinoid mediated differentiation is restricted to RARα signalling. For this purpose, we tested the ability of Ro41–5253, a RARα specific antagonist, to affect this process. Figure 1b demonstrates that pre-treatment of NB4 cells with the RARα antagonist Ro41–5253 can nearly prevent differentiation as induced by the RARα agonist Ro40–6055 (mean 7%, range 3–13% vs. 130%, 106–156%, P 0.005). Interestingly, pre-incubation of NB4 cells with Ro41–5253 before treatment with ATRA had almost no inhibitory effect on granulocyte differentiation (90%, 75–100%, P 0.17). We also did not observed any influence of Ro41–5253 pre-treatment on ATRA mediated NB4 differentiation when higher concentrations of the inhibitor (up to 10 μM) were used (data not shown). Nevertheless, this inhibitor can nearly completely abolish the Ro40–6055 dependent granulocyte differentiation.

Involvement of MAPK activity in NB4 cells granulocyte differentiation

The activation of MAPK is well documented to be involved in the balance between the proliferation and differentiation process in hematopoietic cells. Therefore, we have addressed the question whether MAPK activation is related to the NB4 cells differentiation by ATRA or Ro40–6055. To test that, we have applied to the cells the PD98059, an inhibitor of MEK1/2 kinases, and analysed its effect on the expression of CD11b. We observed that addition of 10 μM PD98059 was not effective in changing the differentiation status of NB4 cells but was demonstrating an ability to inhibit granulocyte differentiation induced by ATRA (Fig. 2a, plus PD98059 mean 46%, range 23–64%, P 0.006) or the RARα agonist Ro40–6055 (Fig. 2a, plus PD98059 mean 51%, range 44–63%, P 0.001). Furthermore, we have analysed the activation of the proteins in the MAPK signalling path and their dependence on ATRA or the RARα agonist Ro40–6055 treatment. As it is present in Fig. 2b, both ATRA and Ro40–6055 led to an activation of Raf-1, MEK1/2 and Erk1/2 kinases. To further investigate the role of the RARα in the stimulation of the MAPK signalling cascade, we have applied an inhibitor to the RARα, the substance Ro41–5253, prior to the treatment of NB4 cells with ATRA or R40–5253. We observed that the inhibitor had no effect on ATRA induced Raf-1, MEK1/2 or Erk1/2 phosphorylation, but significantly reduced Ro40–6055 mediated activation of the MAPK pathway (Fig. 2b). In addition, when applied alone, the RARα inhibitor Ro41–5253 was not affecting the expression levels of c-Raf, MEK1/2, and Erk1/2 proteins (data not shown).

Fig. 2.

Fig. 2

Open in a new tab

Influence of inhibition of MAPK kinase by PD98059 on granulocyte differentiation and phosphorylation status of Raf, MEK and Erk1/2 in NB4 cell differentiation. a NB4 cells were treated with 1 μM all-trans-retinoic acid (ATRA) or 10 nM RARα agonist (Ro40–6055) for 48 h at 37°C to induce granulocyte differentiation measured by CD11b surface expression. Expression of CD11b under these conditions was set 100%, respectively. NB4 cells treated with 0.2 % (v/v) DMSO was used as control as well as NB4 cells were incubated with 10 μM PD98059 alone as indicated. Percentage of CD11b expression indicates the inhibitory effect of PD98059 compared to single treatment with ATRA or RARα agonist, respectively. The diagram shows mean and p values and standard deviation of four independent experiments as indicated; b NB4 cells were pretreated with 1 μM of RARα antagonist (Ro41–5253) for 4 h at 37°C. After this, cells were treated with 1 μM all-trans-retinoic acid (ATRA) or 10 nM RARα agonist (Ro40–6055) for 48 h at 37°C to induce granulocyte differentiation while DMSO at 0.2% (v/v) was used as control beginning during the pre-treatment period. Cells were lysed and protein concentrations were determined. After SDS electrophoresis and western blot membranes were incubated with primary and secondary antibodies for 1 h, respectively. Blots were developed with ECL. One representative of three independent experiments is shown. In these experiments, Western blot of vinculin was used to demonstrate equal amount of protein loading

Since we could demonstrate that the induction of NB4 cell granulocyte differentiation is inhibited by blocking either PI3Ks or MEK1/2 kinases, we also applied a combination of both inhibitors LY294002 and PD98059 to NB4 cells. We could observe a total inhibition of CD11b expression when NB4 cells were treated with ATRA in combination with both inhibitors. In contrast to our experiments with either LY294002 or PD98059, there was a strong induction of apoptosis when NB4 cells were treated with both inhibitors (data not shown).

Activation of MAPK preceeds the up-regulation of PI3Kγ subunits on mRNA and protein level during ATRA induced differentiation

We were next interested in the kinetic of the ATRA induced increasing of PI3Kγ and the time dependent activation of MAPK. For this reason, a quantitative real-time PCR was established in order to quantify mRNA levels of both, the regulatory and the catalytic subunit of PI3Kγ. The “housekeeping gene” PBGD was used as an internal control. After confirmation of equal PCR efficiencies for all three PCR’s, the ΔΔC T method was used for quantitative analysis (Pfaffl 2001). Time dependent induction of the regulatory and catalytic subunit of PI3Kγ after treatment with ATRA is demonstrated in Fig. 3a and b, respectively. We can show a significant increase of p101 mRNA level after 14 h (6.21 fold increase, P 0.03) while after 9 h the indicated up-regulation is not yet significant (P 0.06, Fig. 3a). A similar kinetic can be observed in terms of the catalytic subunit of PI3Kγ (Fig. 3b). In detail, a significant increase of the mRNA level of PI3Kγ catalytic subunit can be detected beginning at 14 h (5.24-fold increase, P 0.008). Furthermore, we investigated the time course of ATRA induced up-regulation of PI3Kγ catalytic subunit on protein level and MAPK activation by western blot analysis. Figure 3c demonstrates that an increase of the protein amount of the catalytic subunit of PI3Kγ is detectable after 24 h of NB4 cell treatment with ATRA. This observation is correlating with the data we obtained from quantitative analysis of PI3Kγ mRNA levels. Surprisingly, a significant difference in MAPK activation measured by phosphorylation of MEK1/2 kinases can already be observed after treatment of NB4 cells for 6 h.

Fig. 3.

Fig. 3

Open in a new tab

Time course of mRNA expression of the regulatory and catalytic PI3Kγ subunits and kinetic of PI3Kγ protein expression and phosphorylation of MEK during ATRA induced differentiation of NB4 cells. a NB4 cells were treated with 1 μM all-trans-retinoic acid (ATRA) or 0.2% (v/v) DMS0 for 24 h at 37°C. At different time points (after 0, 3, 6, 9, 14 and 24 h) cells were harvested and RNA as well as protein lysates were isolated. a The kinetic of mRNA expression of the regulatory PI3Kγ subunits p101 was determined by quantitative realtime PCR with the LightCycler system using SYBRGreen while product specificity was confirmed by melting curve analysis. Quantification was performed by the ΔΔC T method using the PBGD expression as the control after similar efficiencies for all realtime PCR’s were demonstrated. The error bars represent two fold of standard deviation. The pictogram shows that p101 expression did not changed significantly when NB4 cells were treated with DMSO alone; b Time course of PI3Kγ catalytic subunit expression following ATRA treatment. Expression analysis and quantification was carried out as described for p101 expression. The pictogram reflects the equal expression of PI3Kγ catalytic subunit during DMSO treatment while two fold of standard deviation are indicated as well; c Protein lysates that were collected at each time point were separated by SDS-PAGE. Protein expression of PI3Kγ, phosphorylated MEK and total MEK were determined by western blot. The blots show a representative of three independent experiments

NB4 cell granulocyte differentiation by 9-cis-retinoid-acid can also be inhibited by LY294002 or PD98059

Since its is impossible to exclude that ATRA is converted to its isomer 9-cis-retinoid-acid (9cisRA), we continued to investigate whether 9cisRA is able to induce granulocyte differentiation of NB4 cells as well. We can demonstrate that ATRA and 9cisRA are able to induce CD11b expression at different concentrations (Fig. 4a). Furthermore, we were interested in the functional role of the PI3Ks and of the MAPK pathway in 9cisRA induced NB4 cell differentiation. Interestingly, when 9cisRA was combined with the PI3Ks inhibitor LY294002, suppression of 9cisRA induced differentiation was comparable with the treatment of ATRA plus LY294002 or PD98059, (at 1 μM 13.8% vs. 13.9%, P 0.007 vs. 0.002, Fig. 4a). Similar results were obtained when the MEK1/2 kinase inhibitor PD98059 was combined with 9cisRA or ATRA (at 1 μM 17.5% vs. 19.0%, P 0.002 vs. 0.001, data not shown). Therefore, we next investigated the expression of PI3Kγ as well as the phosphorylation of MEK dependent of 9cisRA treatments of NB4 cells. We can show (Fig. 4b) that not only the induction of PI3Kγ on protein level but also the enhanced phosphorylation of MEK is nearly identical after treatment with ATRA or 9cisRA and there is no influence of LY294002 on phosphorylation of MEK1/2 at Ser 217/221. Figure 4c and d shows the concentration dependent mRNA up-regulation of p101 (Fig. 4c) and of the catalytic subunit of PI3Kγ (Fig. 4d) after treatment with ATRA or 9cisRA. There is no significant difference for both subunits between ATRA and 9cisRA. Furthermore, at concentrations 0.1 and 1 μM the effect of ATRA or 9cisRA on PI3Kγ mRNA expression did not differ significantly.

Fig. 4.

Fig. 4

Open in a new tab

Influence of inhibition of PI3Ks and expression of PI3Kγ (protein vs. mRNA) and phsophorylation of MEK during differentiation of NB4 cells induced by ATRA or the isomer 9-cis-retonid-acid (9cisRA). a NB4 cells were treated with 0.1 versus 1 μM all-trans-retinoic acid (ATRA) or 0.1 versus 1 μM of its isomer 9-cis-retonid-acid (9cisRA) alone or in combination with the PI3K inhibitor LY294002 (20 μM) for 48 h at 37°C to induce granulocyte differentiation measured by CD11b surface expression. Differentiation of NB4 cells treated with 0.2 % (v/v) DMSO (used as control) and 20 μM of the PI3K inhibitor LY294002 are shown. The influence of this inhibitor is demonstrated by co-treatment of both differentiating agents and LY294002. Percentage of CD11b expression indicates the inhibitory effects compared to single treatment with ATRA or 9cisRA, respectively. The diagrams show mean and p values as well as standard deviations of three independent experiments; b NB4 cells were treated under the same conditions as described for a. Protein lysates were separated by SDS-PAGE followed by western blot analysis for protein expression of PI3Kγ, phosphorylated MEK and total MEK. The blots show a representative of three independent experiments; c, d) NB4 cells treated with either DMSO or different concentrations of ATRA or 9cisRA for 48 h at 37°C were collected for RNA isolation. Quantitative PCR analysis for mRNA expression of the regulatory subunit of PI3Kγ (p101, c) as well quantification of the PI3Kγ catalytic subunit mRNA (d) was performed as described above. Mean values and standard deviation of three independent experiments are indicated

Influence of RARβ agonist Ro48–2249 and RARγ agonist Ro44–4753 on granulocyte differentiation of NB4 cells

In addition to the RARα agonist (Ro40–6055) we tested the ability of RARβ and RARγ agonist (Ro48–2249 and Ro44–4753, respectively) to induce granulocyte differentiation in NB4 cells. We found that treatment of NB4 cells with the RARβ agonist (Fig. 5a) led to differentiation of a minor fraction of the cells (mean 33%, range 29–38%, P 0.001) while the RARγ agonist had no effect on this process (mean 7%). As the stimulation of the NB4 cells with the RARα specific inhibitor Ro41–5253 was not demonstrating an effect on ATRA induced granulocyte differentiation, we next investigated the possibility of ATRA to induce granulocyte differentiation of NB4 cells via a RAR-independent pathway. To asses that we have treated the cells with the pan-RAR’s antagonist Ro61–8431 alone or in combination with ATRA. Surprisingly, as it is shown in Fig. 5a the addition of this inhibitor did not affect the ATRA dependent differentiation of NB4 cells (mean 94%, range 88–99%, P 0.11). Furthermore, we have analysed as well whether Ro48–2249 or Ro44–4753 agonists are capable of inducing changes in the protein expression level of PI3K isoforms (Fig. 5b). Treatment of NB4 cells with the above substances unveil that RARβ agonist can induce an increase of PI3Kγ, which was weaker than this observed by ATRA, but it has no effect on PI3Kα (not shown) or its regulatory subunit p85 (Fig. 5b). In addition, we did not observe differences in the protein expression or phosphorylation properties of the analysed proteins when RARγ agonist was added to the cells. We found as well that Ro48–2249 was equally potent as ATRA in inducing MEK1/2 phosphorylation (Fig. 5b). Nevertheless, pre-treatment of the cells with the pan-RAR´s antagonist Ro61–8431 was unable to suppress ATRA dependent PI3Kγ protein expression and had only a minor effect on MEK1/2 phosphorylation driven by ATRA.

Fig. 5.

Fig. 5

Open in a new tab

Effect of RARβ (Ro48–2249) and RARγ agonist (Ro44–4753) on differentiation of NB4 cells and expression of PI3Kγ and MEK1/2 activation. a NB4 cells were left untreated or were incubated with 1 μM of pan-RAR-inhibitor Ro61–8431 for 4 h at 37°C prior to treatment with 1 μM all-trans-retinoic acid (ATRA), the RARβ agonist Ro48–2249 or the RARγ agonist Ro44–4753 for 48 h at 37°C. Granulocyte differentiation measured by CD11b surface expression is shown. Expression of CD11b after treatment with ATRA was set 100%. DMSO with 0.2% (v/v) was used as control. The diagram shows mean and p value of three independent experiments (* DMSO vs. Ro48–2249 and ** ATRA vs. Ro61–8431 plus ATRA, respectively); b cells were treated for 48 h as described above, lysed and equal amounts of protein were separated by SDS-PAGE. Expression of PI3Kγ was monitored by western blot. One representative of two independent experiments is shown

Expression of PI3K isoforms during ATRA and RARα agonist Ro40–6055 mediated differentiation of NB4 cells

Recently, we have demonstrated that PI3Kγ protein expression level was augmented during ATRA driven macrophage differentiation of U937 cells (Baier et al. 1999). Therefore, we have addressed as well the question whether there are detectable changes in the protein expression levels of the PI3K when ATRA or RARα agonist Ro40–6055 were applied to NB4 cells. For this approach we have analysed the expression pattern of the PI3Kα, PI3Kγ and the class IA PI3K adaptor protein p85. As it is demonstrated in Fig. 6a the adaptor molecule p85 was unchanged during induced differentiation, as well no changes were detected in the expression of PI3Kα catalytic subunit. When the protein expression of the PI3Kγ catalytic subunit was analysed a significant induction was observed.

Fig. 6.

Fig. 6

Open in a new tab

Expression of PI3K isoforms on protein level and lipid vs. protein kinase activity of PI3Kγ during NB4 cell differentiation. a NB4 cells were pretreated with 1 μM of RARα antagonist (Ro41–5253) for 4 h at 37°C. After this, cells were treated with 1 μM all-trans-retinoic acid (ATRA) or 10 nM RARα agonist (Ro40–6055) for 48 h at 37°C to induce granulocyte differentiation while DMSO at 0.2% (v/v) was used as control beginning during the pre-treatment period. Cells were lysed and protein concentrations were determined; b lipid kinase activity of PI3Kγ after treatment of NB4 cells with DMSO (1), ATRA (2) or Ro40–6055 (3): after immunoprecipitation of PI3Kγ phosphatidlyinositol was used as substrate in the presence of γ32P-ATP. After separation of remaining γ32P-ATP the product was measured by liquid scintillation counting (see histogram) and an aliquot of the hydrophobic phase was separated by thin layer chromatography (upper left). The western blot demonstrates nearly equal amount of immunoprecipitates per reaction. Lane 4 of the chromatography represents a positive control for phosphatidylinositol 3-phosphate; c similar to the lipid kinase assay after identical treatment of NB4 cells protein kinase activity was determined using MBP as substrate. One additional protein that binds to PI3Kγ shows an enhanced phosphorylation especially after treatment with Ro40–6055. The molecular weight of this protein is approximately 50 kDa. One representative of three independent experiments for each subset is shown

Furthermore, we have studied the effect of the RARα antagonist Ro41–5253 on ATRA and RARα dependent increase of PI3Kγ protein expression. Addition of the RARα inhibitor had almost no effect on ATRA mediated PI3Kγ protein expression, but it can specifically affect the RARα stimulated effects on PI3Kγ protein levels (Fig. 6a). We next analysed lipid as well as protein kinase activity of PI3Kγ after immuoprecipitation from cell lysates when NB4 cells were treated with ATRA or Ro40–6055 (Fig. 6b, c, respectively). We can demonstrate about two folds increase of both lipid and protein kinase activities of PI3Kγ when NB4 cells underwent granulocyte differentiation by ATRA or Ro40–6055. In addition, we observed a protein of about 50 kDa that co-immunoprecipitates with PI3Kγ and shows an enhanced phosphorylation after treatment with Ro40–6055 (Fig. 6c).

Discussion

We can demonstrate that the NB4 cell line can be differentiated into granulocyte lineage not only by ATRA treatment, but as well by the RARα specific agonist Ro40–6055. Both mechanisms we have found to be sensitive to LY294002, an inhibitor of PI3-K isoforms, and to PD98059, which inhibits MAPK pathway. These data suggest PI3K and MAPK cascades as important elements in the process of NB4 granulocyte differentiation.

LY294002 can principally inhibit all known PI3K isoforms. In consideration of the demonstrated specific up-regulation of the PI3Kγ isoform by ATRA or RARα agonist Ro40–6055—while receptor tyrosine kinases dependent isoforms like PI3Kα or their adaptor protein p85 were completely unaffected—we hypothesise a direct or indirect role of PI3Kγ in the NB4 cells granulocyte differentiation. Analysis of the MAPK cascade path demonstrates a simultaneous and concomitant phosphorylation of these proteins after treatment with ATRA or Ro40–6055. Moreover, the addition of the MEK1/2 inhibitor PD98059 was significantly reducing the granulocyte differentiation as tested by the CD11b expression. These data indicate a functional role of the MAPK pathway.

Analysis of apoptosis as well as cell cycle analysis using flow cytometry lead to complementary results. We can show that granulocyte differentiation of NB4 cells does not lead to a significant induction of cellular suicide as observed in other hematopoietic systems. In contrast, increased ratio of apoptotic cells when NB4 cells are treated with LY294002 can be reduced by co-treatment with ATRA or Ro40–6055. A differentiation specific process after treatment with ATRA or Ro40–6055 is represented by a decrease of the proliferative index (S+G2/M) of NB4 cells under these conditions.

Zheng et al. (2005) published comprehensive analysis of transcriptome and proteome in retinoic acid and/or arsenic trioxide induced NB4 cell differentiation. The methodological approaches applied here allow insights into putative molecular networks during granulocyte differentiation. The authors found that treatment of NB4 cells with ATRA can activate an antiapoptotic program, which enables terminal differentiation of granulocytes. Since we can demonstrate a higher percentage of apoptotic cells when NB4 cells were treated with LY294002 alone compared with the combined treatment with either ATRA or Ro40–6055, our observations correlate with these data (Zheng et al. 2005).

Recently, Matkovic could demonstrate a role for Akt activation in ATRA-induced granulocyte differentiation of NB4 cells. In consideration of the linkage between PI3K and Akt, these data support the involvement of PI3K in this process (Matkovic et al. 2006).

The inhibition of RARα signalling by Ro41–5253 can prevent the cellular effects as mediated by the RARα agonist Ro40–6055 including the up-regulation of PI3Kγ. Furthermore, we could show that the induction of phosphorylation of proteins that belong to the MAP kinase pathway is not inhibited when Ro41–5253 is co-incubated with ATRA. These results suggest that ATRA can not only induce differentiation via RARα. Thus, the presumed key role of the PML–RARα fusion protein as generated by the translocation t(15;17) in promyelocytic leukemia has to be considered critically.

We propose two possible alternative mechanisms how ATRA can induce myeloid differentiation of NB4 cells: First, ATRA could act via RARβ and/or RARγ signalling. This hypothesis can be supported by the experiments showing granulocyte differentiation of NB4 cells by RARβ agonist Ro48–2249. Stimulation of NB4 cells with Ro48–2249 is also associated with up-regulation of PI3Kγ and induction of MEK1/2 phosphorylation. We therefore suggest that RARβ also plays a functional role in ATRA mediated differentiation of NB4 cells. Second, the cellular effects of ATRA could also be mediated independent on RAR signalling in a receptor independent manner. This model is supported by the experiments with blockage of RAR signalling by the pan-RAR antagonist Ro61–8431 that can neither prevent ATRA mediated MEK1/2 phosphorylation nor ATRA induced increase of PI3Kγ protein expression. In contrast, 0.1 μM of 9cisRA were also able to induce NB4 cell granulocyte differentiation. Thus, we can not exclude that ATRA is partially converted to 9cisRA that can activate NB4 cell differentiation via RXR signalling (Benoit et al. 1999).

We have shown previously that PI3Kγ is able to induce MAPK activity when over-expressed in COS7 cells in a protein kinase activity dependent manner (Bondeva et al. 1998; Lopez et al. 1997). On the other side, treatment with ATRA in U937 cells was inducing macrophage like differentiation and demonstrated an increase of the lipid kinase activity in this cell system (Baier et al. 1999). Therefore, we have investigated the protein and lipid kinase activity of PI3Kγ and p85 associated PI3Ks. Our data show that PI3Kγ lipid and protein kinase activity was increased upon addition of ATRA and RARα agonist about two fold. Furthermore, an approximately 50 kDa protein was found to co-immunoprecipitate with PI3Kγ being significantly phosphorylated during NB4 cell differentiation induced by Ro40–6055. In the meanwhile, we determined the site on MEK1 being phosphorylated by PI3Kγ with the used of MALDI–MS analysis (T. Bondeva et al., unpublished observation) being different from the one phosphorylated by Raf1 (Ser 221). Therefore, we can not measure the PI3Kγ mediated phosphorylation of MEK with the antibody used in the present study. Further experiments need to be performed to identify the phosphorylated 50 kDa protein that was observed to co-immunoprecipitate with PI3Kγ.

In contrast to our previous results from U937 cells, the data we obtained from NB4 cells do not propagate a direct involvement of PI3Kγ activity in the activation of the MAPK cascade.

Furthermore, our data let us hypothesise that in NB4 cells MAPK and PI3K pathway represent separate signalling cascades that both contribute to retinoid induced myeloid differentiation. The involvement of the MAPK and the PI3K pathway in the differentiation of myeloid precursors might be considered as cell type specific.

In contrast to Bertagnolo et al., we provide biochemical and morphological evidence for a specific involvement of PI3Kγ but not p85 associated PI3Ks in NB4 cell granulocyte differentiation (Bertagnolo et al. 1999).

In summary, the up-regulation of PI3Kγ protein expression leading to activation of its both activities—lipid and protein kinase acitivity—as well as MAPK induction by ATRA are both important for retinoid induced NB4 cells differentiation. Therefore we suggest that additive effects of PI3K and MAPK activities are necessary to promote granulocyte differentiation.

Acknowledgments

We thank Prof. M. Lanotte for sending us NB4 cells. Furthermore, we are especially grateful to Dr Mohr (Roche, Germany) who provided us several RAR modulating substances.

Footnotes

S. Scholl and T. Bondeva have contributed equally to this work.

References

  1. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270:27489–27494 [DOI] [PubMed] [Google Scholar]
  2. Baier R, Bondeva T, Klinger R, Bondev A, Wetzker R (1999) Retinoic acid induces selective expression of phosphoinositide 3-kinase gamma in myelomonocytic U937 cells. Cell Growth Differ 10:447–456 [PubMed] [Google Scholar]
  3. Battle TE, Roberson MS, Zhang T, Varvayanis S, Yen A (2001) Retinoic acid-induced blr1 expression requires RARalpha, RXR, and MAPK activation and uses ERK2 but not JNK/SAPK to accelerate cell differentiation. Eur J Cell Biol 80:59–67 [DOI] [PubMed] [Google Scholar]
  4. Benoit G, Altucci L, Flexor M, Ruchaud S, Lillehaug J, Raffelsberger W, Gronemeyer H, Lanotte M (1999) RAR-independent RXR signaling induces t(15;17) leukemia cell maturation. EMBO J 18:7011–7018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benoit G, Roussel M, Pendino F, Segal-Bendirdjian E, Lanotte M (2001) Orchestration of multiple arrays of signal cross-talk and combinatorial interactions for maturation and cell death: another vision of t(15;17) preleukemic blast and APL-cell maturation. Oncogene 20:7161–7177 [DOI] [PubMed] [Google Scholar]
  6. Bertagnolo V, Neri LM, Marchisio M, Mischiati C, Capitani S (1999) Phosphoinositide 3-kinase activity is essential for all-trans-retinoic acid-induced granulocytic differentiation of HL-60 cells. Cancer Res 59:542–546 [PubMed] [Google Scholar]
  7. Bondeva T, Pirola L, Bulgarelli-Leva G, Rubio I, Wetzker R, Wymann MP (1998) Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science 282:293–296 [DOI] [PubMed] [Google Scholar]
  8. Carpenter CL, Cantley LC (1996) Phosphoinositide kinases. Curr Opin Cell Biol 8:153–158 [DOI] [PubMed] [Google Scholar]
  9. Fenaux P, Chomienne C, Degos L (1997) Acute promyelocytic leukemia: biology and treatment. Semin Oncol 24:92–102 [PubMed] [Google Scholar]
  10. Grande MA, van der Kraan I, van Steensel B, Schul W, de The H, van der Voort HT, de Jong L, van Driel R (1996) PML-containing nuclear bodies: their spatial distribution in relation to other nuclear components. J Cell Biochem 63:280–291 [DOI] [PubMed] [Google Scholar]
  11. Hiles I, Otsu M, Volinia S, Gout I, Dhand R, Panayotou G, Ruiz-Larrea F, Tompson A, Totty N, Courtneidge S, Parker PJ, Waterfield MD (1992) Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell 70:419–429 [DOI] [PubMed] [Google Scholar]
  12. Hu P, Mondino A, Skolnik EY, Schlessinger J (1993) Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase, identification of its binding site on p85. Mol Cell Biol 3:7677–7688 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, Gu LJ, Wang ZY (1988) Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 72: 567–572 [PubMed] [Google Scholar]
  14. Kang CD, Do IR, Kim KW, Ahn BK, Kim SH, Chung BS, Jhun BH, Yoo MA (1993) Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells. Exp Mol Med 31:76–82 [DOI] [PubMed] [Google Scholar]
  15. Koken MH, Reid A, Quignon F, Chelbi-Alix MK, Davies JM, Kabarowski JH, Zhu J, Dong S, Chen S, Chen Z, Tan CC, Licht J, Waxman S, de The H, Zelent A (1997) Leukemia-associated retinoic acid receptor alpha fusion partners, PML and PLZF, heterodimerize and colocalize to nuclear bodies. Proc Natl Acad Sci USA 94:10255–10260 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R (1991) NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 77:1080–1086 [PubMed] [Google Scholar]
  17. Lewis TS, Shapiro PS, Ahn NG (1994) Signal transduction through MAP kinase cascades. Adv Cancer Res 74:49–139 [DOI] [PubMed] [Google Scholar]
  18. Lopez IM, Crespo P, Pellici PG, Gutkind JS, Wetzker R (1997) Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase gamma. Science 275:394–397 [DOI] [PubMed] [Google Scholar]
  19. Marshall CJ (1994) MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev 4:82–89 [DOI] [PubMed] [Google Scholar]
  20. Matkovic K, Brugnoli F, Bertagnolo V, Banfic H, Visnjic D (2006) The role of the nuclear Akt activation and Akt inhibitors in all-trans-retinoic acid-differentiated HL-60 cells. Leukemia 20:941-951 [DOI] [PubMed] [Google Scholar]
  21. Melnick A, Licht JD (1999) Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 93:3167–3215 [PubMed] [Google Scholar]
  22. Miranda MB, McGuire TF, Johnson DE (2002) Importance of MEK-1/-2 signaling in monocytic and granulocytic differentiation of myeloid cell lines. Leukemia 16:683-692 [DOI] [PubMed] [Google Scholar]
  23. Mu ZM, Le XF, Glassman AB, Chang KS (1996) The biologic function of PML and its role in acute promyelocytic leukemia. Leuk Lymphoma 23:277–285 [DOI] [PubMed] [Google Scholar]
  24. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schmidt EK, Fichelson S, Feller SM (2004) PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors. BMC Biol 2:7–18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Seger R, Krebs EG (1995) The MAPK signaling cascade. FASEB J 9:726–735 [PubMed] [Google Scholar]
  27. Stephens LR, Eguinoa A, Erdjument BH, Lui M, Cooke F, Coadwell J, Smrcka AS, Thelen M, Cadwallader K, Tempst P, Hawkins PT (1997) The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101. Cell 89:105–114 [DOI] [PubMed] [Google Scholar]
  28. Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkoy M, Malek D, Stoyanova S, Vanhaesebroeck B, Dhand R, Nürnberg B, Gierschil P, Seedorf K, Hsuan JJ, Waterfield MD, Wetzker R (1995) Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science 269:690–693 [DOI] [PubMed] [Google Scholar]
  29. Tallman MS, Andersen JW, Schiffer CA, Appelbaum FR, Feusner JH, Ogden A, Shepherd L, Willman C, Bloomfield CD, Rowe JM, Wiernik PH (1997) All-trans-retinoic acid in acute promyelocytic leukemia. N Engl J Med 337:1021–1028 [DOI] [PubMed] [Google Scholar]
  30. Tallman MS, Andersen JW, Schiffer CA, Appelbaum FR, Feusner JH, Woods WG, Ogden A, Weinstein H, Shepherd L, Willman C, Bloomfield CD, Rowe JM, Wiernik PH (2002) All-trans retinoic acid in acute promyelocytic leukemia: long-term outcome and prognostic factor analysis from the North American Intergroup protocol. Blood 100:4298–4302 [DOI] [PubMed] [Google Scholar]
  31. Vanhaesebroeck B, Waterfield MD (1999) Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253:239–254 [DOI] [PubMed] [Google Scholar]
  32. Vanhaesebroeck B, Welham MJ, Kotani K, Stein R, Warne PH, Zvelebil MJ, Higashi K, Volinia S, Waterfield MD (1997) P110deta, a novel phosphoinositide 3-kinase in leukocytes. Proc Natl Acad Sci USA 94:4330–4335 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vlahos CJ, Matter WF, Hui KY, Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269:5241–5248 [PubMed] [Google Scholar]
  34. Wang D, Jensen R, Gendeh G, Williams K, Pallavicini MG 2004) Proteome and transcriptome analysis of retinoic acid-induced differentiation of human acute promyelocytic leukemia cells, NB4. J Proteome Res 3:627–635 [DOI] [PubMed] [Google Scholar]
  35. Warrell RP, Jr de The H, Wang ZY, Degos L (1993) Acute promyelocytic leukemia. N Engl J Med 329:177–189 [DOI] [PubMed] [Google Scholar]
  36. Yang L, Zhao H, Li SW, Ahrens K, Collins C, Eckenrode S, Ruan QG, McIndoe RA, She JX (2003) Gene expression profiling during all-trans retinoic acid-induced cell differentiation of acute promyelocytic leukemia cells. J Mol Diagn 5:212–221 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yen A, Varvayanis S (2000) Retinoic acid increases amount of phosphorylated RAF; ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced GO arrest is. In Vitro Cell Dev Biol Anim 36:249–255 [DOI] [PubMed] [Google Scholar]
  38. Yen A, Roberson MS, Varvayanis S (1999) Retinoic acid selectively activates the ERK2 but not JNK/SAPK or p38 MAP kinases when inducing myeloid differentiation. In Vitro Cell Dev Biol Anim 35:527–532 [DOI] [PubMed] [Google Scholar]
  39. Zheng PZ, Wang KW, Zhang QY, Huang QH, Du YZ, Zhang QH, Xiao DK, Shen SH, Imbeaud S, Eveno E, Zhao CJ, Chen YL, Fan HY, Waxman S, Auffray C, Jin G, Chen SJ, Chen Z, Zhang J (2005) Systems analysis of transcriptome and proteome in retinoic acid/arsenic trioxide-induced cell differentiation/apoptosis of promyelocytic leukemia. Proc Natl Acad Sci USA 102:7653–7658 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES