Petr Mlejnek
email: mlejnek_petr@volny.cz

Petr Dolezel

Edgar Faber

Petr Kosztyu

Interactions of N-desmethyl imatinib, an active metabolite of imatinib, with P-glycoprotein in human leukemia cells

Keywords: MDR1, ABCB1, CGP 74588, intracellular drug level, cytotxicity

We measured intracellular accumulation of N-desmethyl imatinib (CGP 74588), the main pharmacologically active metabolite of imatinib (Gleevec or STI-571), in Bcr-Abl positive cells. Using a sensitive and robust non-radioactive in vitro assay, we observed that CGP74588 accumulates in significantly higher amount than imatinib in sensitive K562 cells.

In contrast, the intracellular level of CGP74588 was significantly lower than that of imatinib in K562/Dox cells, which represent a multidrug resistant variant of K562 cells due to P-glycoprotein (ABCB1, MDR1) overexpression. An in vitro enzyme based assay provided evidence that CGP74588 might serve as an excellent substrate for P-gp. Accordingly, we found that CGP74588 up to 20 M concentration neither induced apoptosis nor inhibited substantially cell proliferation in resistant K562/Dox cells. In contrast, CGP74588 was capable to inhibit cell proliferation and induced apoptosis in sensitive K562 cells, although its effect was approximately 3 to 4 times lower than that of imatinib in the same cell line. Our results indicate that CGP74588 could hardly positively contribute to the treatment of CML where ABCB1 gene overexpression represents a possible mechanism of resistance to imatinib in vivo.

Introduction

Imatinib mesylate (Gleevec or STI-571), a rationally developed inhibitor of the Bcr-Abl tyrosine kinase, is capable of blocking proliferation and inducing apoptosis in chronic myeloid leukemia (CML) cells both in vitro and in vivo [START_REF] Buchdunger | Inhibition of the Abl protein-kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative[END_REF][START_REF] Druker | Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells[END_REF]. In addition to Bcr-Abl tyrosine kinase, imatinib serves as an efficient inhibitor of other tyrosine kinases, including plateletderived growth-factor receptor (PDGFR) tyrosine kinases [START_REF] Buchdunger | Inhibition of the Abl protein-kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative[END_REF], and c-Kit, a receptor tyrosine kinase structurally similar to PDGFR [START_REF] Heinrich | Inhibition of ckit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor[END_REF]. Later on, it was also approved for treatment of c-Kit positive gastrointestinal stromal tumors [START_REF] Dagher | Approval summary: imatinib mesylate in the treatment of metastatic and/or unresectable malignant gastrointestinal stromal tumors[END_REF].

The pharmacokinetic properties revealed that imatinib is well absorbed after oral administration with high bioavailability exceeding 90% [START_REF] Peng | Absolute bioavailability of imatinib (Glivec) orally versus intravenous infusion[END_REF]. Imatinib becomes metabolized by cytochrome P450 (CYP), particularly by CYP3A4 and CYP3A5, to form several metabolites.

The most abundant metabolite is N-desmethyl imatinib (CGP 74588), which represents 10-15% of administrated imatinib [START_REF] Gschwind | Metabolism and disposition of imatinib mesylate in healthy volunteers[END_REF]. Although CGP74588 has been reported to be pharmacologically active [START_REF] Gschwind | Metabolism and disposition of imatinib mesylate in healthy volunteers[END_REF], little is known about its cellular distribution and possible interactions with ABC transporters, including P-glycoprotein (P-gp, ABCB1). To address this issue, we measured intracellular accumulation of imatinib and CGP74588 in Bcr-Abl positive cells which overexpressed P-glycoprotein.

Materials and methods

Patients

Only patients with chronic phase CML were included in the study that was conducted in accordance with the Declaration of Helsinki. All patients have signed informed consent approved by the local Ethics committee before sampling. 18 men and 7 women at median age 56 years (range 29-81) have already achieved complete cytogenetic response at the time of blood collection. All but four of them have achieved also major molecular response.

Drug administration

Imatinib was given to 7 patients as a front-line treatment, while in 18 patients was imatinib indicated due to the failure of interferon. Average duration of imatinib therapy was 54.5 and median 60 months (range 4-94). Imatinib was applied in a single daily dose of 400mg in all patiens. Blood sample was collected in median 22 hours after drug ingestion (range 18-24).

Cell lines

A suspension of human chronic myelogenous leukaemia K562 cells, obtained from ECACC, was cultured in RPMI-1640 medium supplemented with a 10% calf foetal serum and antibiotics in 5% CO 2 atmosphere at 37 o C. K562/Dox cells, which represent a MDR variant of K562 cells with overexpressed P-gp (ABCB1, MDR1), were kindly provided by prof. J.P. Marie (University of Paris 6, France). K562/Dox cells were cultured under the same conditions.

Chemicals

Imatinib mesylate (STI571; purity 98%) and N-desmethyl imatinib (CGP 74588; purity 98%) were kindly provided by Novartis (Basel, Switzerland).

Detection of P-gp mediated rhodamine 123 efflux

Cells at the density of 0.2 x 10 6 /ml were loaded with rhodamine 123 (Rh123; 2.0µM) in a growth medium containing 2µM Cyclosporin A (CsA) in the dark. After 30minute incubation cells were transferred into Rh123-free medium with or without 2µMCsA. Cell fluorescence, which reflects the Rh123 content and thus P-gp mediated dye efflux was analyzed in Rh123free medium using FACSscan as described previously [START_REF] Ludescher | Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123[END_REF]. P-gp expression analysis P-gp expression in K562 and K562/Dox cells was measured using UIC2 (Pgp, Beckman Coulter, USA) monoclonal antibody conjugated with phycoerythrin according to the manufacturer's instruction. The fluorescence of the cells was measured and analyzed by flow cytometry. Protein expression was quantified as the median fluorescence intensity shift.

Extraction of CGP 74588 and imatinib from plasma

Peripheral blood samples were taken by direct venipuncture. Plasma was separated by centrifugation (5000rpm/10min/4°C) and resulting supernatant was deproteinised with 4% formic acid (w/v) in 80% methanol/water (v/v)). CGP 74588 and imatinib contents in clarified samples were quantified using HPLC/MS/MS.

Extraction of CGP 74588 and imatinib from cells

Cells at density 5x10 5 cells/ml were incubated in growth medium with various concentration of CGP 74588 or imatinib for 5 hours at 37°C. Afterwards cells were centrifugated through silicone oil and resulting pellet was extracted with 1% formic acid (w/v) in 50% methanol/water (v/v)). CGP 74588 and/or imatinib contents in cell extract were quantified using HPLC/MS/MS.

Quantitation of CGP 74588 and imatinib by LC/MS/MS analysis

The HPLC system consisted of UltiMate 3000 RS pump, degasser, autosampler and column compartment (Dionex, Germering, Germany). Separations were performed at ambient temperature on a Polaris C 18-A 250 x 2.0mm (i.d.), 5µm particle size column (Varian Inc., Lake Forest, CA, USA) connected with a guard C 18 4.0 x 2.0mm (i.d.) precolumn (Phenomenex, Torrance, CA, USA). The mobile phase was composed of methanol-water Ion spray probe parameters were set to the following values: needle voltage 5500 V, declustering potential 71 V for imatinib and 66 V for CGP 74588, temperature 400ºC, curtain gas (nitrogen) 1.38bar, nebulizer gas (zero air quality) 3.45 bar, turbo V-gas (zero air quality) 3.45 bar. The nitrogen pressure in the second quadrupole was measured at 3.7x10 -8 bar, the collision energy and entrance potential was set at 33.0 eV and 5.0 V, respectively. The instrument was operated in unit resolution.

Measurement of drug-stimulated membrane ATPase

Principle of this assay: transport of substrates out of the cell by P-gp is coupled with hydrolysis of ATP, which serves as an energy source. Owing to the fact that these processes are stoichiometrically coupled, the detection of ATPase activity of P-gp is a measure of transporter activity. The assay is performed using purified membrane vesicles from Sf9 (Spodoptera frugiperda) cells, expressing high level of P-gp (Sigma, Saint Louis, Missouri, USA). The ATPase activity was measured as described elsewhere [START_REF] Sarkadi | Expression of the human multidrug resistance cDNA in insect cells generates a high activity drugstimulated membrane ATPase[END_REF].

Determination of cell survival and proliferation

The MTT assay was used for estimation of cell viability and growth as originally described by [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF]. Cells were incubated in the presence of 0.5mg MTT/ml (final concentration) for 1 hour at 37°C, then pelleted by centrifugation and extracted by isopropanol acidified with 0.01M HCl.

Absorbance was read at 570 nm in extracts clarified by centrifugation. The amount of formazane produced is proportional to the number of live and metabolically active cells.

Apoptosis assessment by flow cytometry

We adopted protocol published by [10]. Briefly, cells were washed in PBS and then fixed in ice-cold 70% ethanol in PBS. Fixed cells were washed in PBS and then stained for at least 30 minutes in PBS containing PI (10µg/ml) and RNase A (100µg/ml) prior to flow-cytometric analysis on a Cytomics FC 500 System (Beckman Coulter). The apoptotic population was identified within 10 000 of analysed cells which DNA content was lower than in G1/G0 (hypoploid population) cell cycle phase [10].

Results and Discussion

We measured plasma levels of CGP74588, a main metabolite of imatinib, in 25 patients after administration of imatinib mesylate at daily dosage 400mg. Similarly to other researchers we found that conversion of imatinib into CGP74588 varied from 8 to 24% with the mean value approximately 16% of (Fig. 1). Our data are in a good agreement with those reported in the literature. Indeed, Gschwind and co-workers recently reported that 10 to 15% of imatinib become converted into CGP74588 in healthy volunteers [START_REF] Gschwind | Metabolism and disposition of imatinib mesylate in healthy volunteers[END_REF].

Considering the fact that CGP74588 might represent even more than 20% of imatinib (Fig. 1), and it is pharmacologically active [START_REF] Gschwind | Metabolism and disposition of imatinib mesylate in healthy volunteers[END_REF], we addressed the question whether it could efficiently affect cell survival of Bcr-Abl positive cells which expressed P-gp. Indeed, P-gp was identified as important factor that could confer resistance to imatinib [START_REF] Hegedus | Interaction of tyrosine kinase inhibitors with the human multidrug transporter proteins, MDR1 and MRP1[END_REF][START_REF] Mahon | MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models[END_REF]. However, to our knowledge nothing was reported about the cytotoxic properties of CGP74588 in sensitive, and more importantly in resistant, Bcr-Abl positive cells.

To address this issue, we measured the intracellular levels of CGP74588 and imatinib in K562 cells and their multidrug resistant (MDR) variant K562/Dox cells, which overexpress P-gp [START_REF] Tang | Zosuquidar restores drug sensitivity in P-glycoprotein expressing acute myeloid leukemia (AML)[END_REF]. Expression of P-gp was verified in both cell lines using specific monoclonal antibody (Fig. 2). Function of P-gp, i.e., its ability to transport substrates across the cytoplasmic membrane, was confirmed using the rhodamine 123 efflux assay (Fig. 3).

Analysis of cell extracts revealed that CGP74588 accumulated cells in significantly higher amount than imatinib in K562 (Fig. 4a). In contrast, the intracellular level of CGP74588 was significantly lower than that of imatinib in K562/Dox cells, which overexpress P-g (Fig. 4b).

The finding that CGP74588 accumulated approximately two times more in sensitive K562 cells than imatinib was rather surprising as there is only a little structural difference between imatinib and its N-desmethyl metabolite. We can speculate that demethylation of the piperazine ring might lead to its higher sequestration within lysosomes due to the changed protonation. On the other hand, 3 to 4 fold decrease in content of CGP74588 in comparison to imatinib in resistant K562/Dox cells indicated that CGP74588 was more efficiently pumped out of the cells than imatinib. These results suggested that CGP74588 could serve as a "better substrate" for P-gp than imatinib.

To confirm this hypothesis we investigated whether CGP74588 could serve as a substrate for P-gp in an independent experiment. An in vitro enzyme based assay provided evidence that CGP74588 might serve as a substrate for P-gp (Fig. 5). Interestingly, CGP74588 seemed to be a more powerful stimulator of ATPase than imatinib (Fig. 5). Indeed, the stimulatory effect of CGP74588 on the ATPase activity was comparable to that found for verapamil (Fig. 5), which serves as a model substrate for P-gp in in vitro assays [START_REF] Sarkadi | Expression of the human multidrug resistance cDNA in insect cells generates a high activity drugstimulated membrane ATPase[END_REF].

We further studied cytotoxic effects of CGP74588 in sensitive K562 and resistant K562/Dox cells. We found that CGP74588 inhibited cell proliferation and induced apoptosis in K562 cells although its antiproliferative and proapoptotic effect was 3 to 4 times lower than that of imatinib (Figs. 6, 7, Tab. 1). Not surprisingly, CGP74588 and also imatinib exhibited only weak antiproliferative and proapoptotic effects in resistant K562/Dox cells (Figs. 6,7, Tab. 1). However, while cytotoxicity of imatinib was reduced in resistant K562/Dox cells approximately 10 times, cytotoxicity of CGP74588 was diminished more than 30 times (Fig. 6, Tab 1). Similar results were found for proapoptotic effects of CGP74588 in K562/Dox cells (Fig. 7). These results are consistent with the data found for intracellular levels of CGP74588 in K562 and K562/Dox cells (Fig. 4).

Our results indicated that CGP74588 exhibited only weak antiproliferative and proapoptotic effects in sensitive K562 cells. In contrast, CGP74588 could hardly influence cell proliferation and cell survival in Bcr-Abl positive cells that overexpress P-glycoprotein.

Legends to Figures

Fig. 1. Analysis of plasma levels of CGP74588 and imatinib. Blood samples collected from 25 patients after a single oral dose of imatinib were assayed for imatinib and CGP74588 contents. P a t i e n t 1 P a t i e n t 2 P a t i e n t 3 P a t i e n t 4 P a t i e n t 5 P a t i e n t 6 P a t i e n t 7 P a t i e n t 8 P a t i e n t 9 P a t i e n t 1 0 P a t i e n t 1 1 P a t i e n t 1 2 P a t i e n t 1 3 P a t i e n t 1 4 P a t i e n t 1 5 P a t i e n t 1 6 P a t i e n t 1 7 P a t i e n t 1 8 P a t i e n t 1 9 P a t i e n t 2 0 P a t i e n t 2 1 P a t i e n t 2 2 P a t i e n t 2 3 P a t i e n t 2 4 P a t i e n t

(

  65:35, v/v) containing 7mM ammonium acetate. The HPLC flow rate was 300µl min -1 and sample injection volume was set at 10µl. An API 3200 triple quadrupole mass spectrometer (MDS SCIEX, Ontario, Canada) with electrospray ionization in positive ion mode was used for detection. The mass spectrometer was operated in the multiple-reaction monitoring (MRM) mode. Imatinib and CGP 74588 were monitored by MRM transitions 494.2 > 394.0 and 480.2 > 394.0 (dwelltime = 300 miliseconds), respectively.

Fig. 2 .

 2 Fig. 2. Analysis of P-glycoprotein expression. Protein expression is quantified as the median fluorescence intensity shift. K562 cells (dotted line); K562/Dox (solid line).

Fig. 3 .

 3 Fig. 3. Detection of P-gp function. Cells were loaded with Rh123 and then transferred into a fresh medium with or without CsA. After 30min incubation fluorescence was measured by flow cytometry. Panel (a) K562 cells incubated with (solid line) or without (dotted line) CsA. Panel (b) K562/Dox cells incubated with (solid line) or without (dotted line) CsA.

Fig. 4 .

 4 Fig. 4. Determination of intracellular levels of CGP74588 and imatinib in sensitive K562 and resistant K562/Dox cells. Cells were incubated in growth medium with equal concentrations of CGP74588 and imatinib, as indicated. After 4h incubation cells were harvested and intracellular contents of both drugs were determined using LC/MS/MS. CGP74588 (triangles); imatinib (circles). Panel a) K562 cells; panel b) K562/Dox cells. The experimental points represent mean values from three replicate experiments, with standard deviations.

Fig. 5 .

 5 Fig. 5. Effect of CGP74588 and imatinib on ATPase activity in isolated membranes containing P-gp. No drug added (vehicle) -white circles; imatinib -black triangles; CGP74588 -black squares; verapamil -white hexagons. The experimental points represent mean values from three replicate experiments, with standard deviations.

Fig. 6 .

 6 Fig. 6. Effect of CGP74588 and imatinib on cell survival and cell proliferation in sensitive K562 and resistant K562/Dox cells. Cells were treated with CGP74588 (white triangles) or imatinib (black circles), as indicated. After 48hour incubation cells were subjected to the MTT assay. Cells cultured in standard medium without CGP74588 or imatinib were taken as a control. Panel a) K562 cells; panel b) K562/Dox cells. The experimental points represent mean values from three replicate experiments, with standard deviations.

Fig. 7 .

 7 Fig. 7. Effect of CGP74588 and imatinib on apoptosis induction in K562 and K562/Dox cells. Cells were cultured in a standard medium containing CGP74588 and imatinib, as indicated. After 48hour incubation cells were fixed in ice-cold 70% ethanol in PBS, stained in PI, and further processed for flow cytometric analysis using Cytomics FC 500 System (see Materials and Methods). Panel a, b, and c -K562 cells; panel d, e, and f, -K562/Dox cells. Untreated cells were taken as a control. Histograms represent typical results.

Fig. 1 .

 1 Fig. 1.

  Fig.2

Fig. 3 .Fig

 3 Fig. 3.

Fig

  Fig. 4b.

Table 1

 1 Effect of CGP74588 and imatinib on proliferation of K562 and K562/Dox cells

	Compound	K562 cells	K562/Dox cells
		IC 50 (μM)	IC 50 (μM)
	Imatinib	0.17	1.9
	CGP74588	0.58	> 20

Acknowledgement

This work was supported by grant NS/9627-3 (Ministry of Health) and partly by grant MSM 6198959216 (Ministry of Education, Youth and Sports).


Lidia Sobkow

F Philipp Seib

Ljupco Prodanov

Ina Kurth

Juliane Drichel

Martin Bornhäuser

Carsten Werner
email: werner@ipfdd.de

Prof Werner Carsten

Prolonged transendothelial migration of human haematopoietic stem and progenitor cells (HSPCs) towards hydrogel-released SDF-1

Keywords: HSPC, SDF1, Heparin, collagen, migration, gradient

INTRODUCTION

In healthy adults haematopoietic stem cells/progenitors (HSPCs) reside in the bone marrow microenvironment [START_REF] Lane | The leukemic stem cell niche: current concepts and therapeutic opportunities[END_REF] and are able to rapidly respond to the specific demand of their progeny. Current concept defining stem cell niche identifies specific niches (and close interactions between them)for normal hematopoietic stem cell renewal and maintenance: one associated closely with the endosteal surface with self-renewal and quiescence related function, and the other one in close proximity to the vasculature that provides signals for HSPC motility/migration and differentiation [START_REF] Lymperi | The HSC niche concept has turned 31. Has our knowledge matured?[END_REF][START_REF] Xie | Detection of functional haematopoietic stem cell niche using real-time imaging[END_REF][START_REF] Nie | CXCR4 is required for the quiescence of primitive hematopoietic cells[END_REF] .

Accumulating evidence suggests that there is a close cross talk between those compartments and HSPC migration/location is in part dependent on the pre-conditioning regime the bone marrow compartment has been subjected to [START_REF] Xie | Detection of functional haematopoietic stem cell niche using real-time imaging[END_REF]. In spite of the still limited knowledge about molecular pathways governing HSPC homing to the bone marrow, there are attempts to manipulate the involved processes [START_REF] Dar | Mutual, reciprocal SDF-1/CXCR4 interactions between hematopoietic and bone marrow stromal cells regulate human stem cell migration and development in NOD/SCID chimeric mice[END_REF][START_REF] Lapidot | How do stem cells find their way home?[END_REF][START_REF] Lapidot | The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice[END_REF][START_REF] Peled | The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice[END_REF][START_REF] Waskow | Hematopoietic stem cell transplantation without irradiation[END_REF]. Indeed, the therapeutic success of HSPC transplantation is critically dependent on HSPC engraftment in the bone marrow [START_REF] Tyndall | Autologous haematopoietic stem cell transplants for autoimmune disease--feasibility and transplant-related mortality. Autoimmune Disease and Lymphoma Working Parties of the European Group for Blood and Marrow Transplantation, the European League Against Rheumatism and the International Stem Cell Project for Autoimmune Disease[END_REF][START_REF] Almeida-Porada | Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero[END_REF] One of the key factors known to influence HSPC migration (in and out of the bone marrow) is stromal cell derived factor 1 (SDF1) [START_REF] Hattori | The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1[END_REF].

As nicely reviewed by Liekens and colleagues [START_REF] Liekens | CXCL12-CXCR4 Axis in Angiogenesis, Metastasis and Stem Cell Mobilization[END_REF], current classification system for chemokines places SDF-1 in the group of CXC class and the protein has been assigned the name CXCL12.

CXCL12 interaction with CXCR4 receptor is extensively studied and it has been implicated in many biological events from embryonic and adult stem cell trafficking, through angiogenic processes and endothelial cells migration in ischemic tissue repair and tumors, to cancer cells migration and metastasis [START_REF] Liekens | CXCL12-CXCR4 Axis in Angiogenesis, Metastasis and Stem Cell Mobilization[END_REF][START_REF] Sun | CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression[END_REF].

Several earlier studies described the adsorption of SDF1 to tissue culture plastic [START_REF] Divietro | Immobilized stromal cellderived factor-1alpha triggers rapid VLA-4 affinity increases to stabilize lymphocyte tethers on VCAM-1 and subsequently initiate firm adhesion[END_REF], heparin coated dishes [START_REF] Netelenbos | Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells[END_REF][START_REF] Netelenbos | Proteoglycans guide SDF-1induced migration of hematopoietic progenitor cells[END_REF], or fibronectin layers [START_REF] Pelletier | Presentation of chemokine SDF-1 alpha by fibronectin mediates directed migration of T cells[END_REF] with the aim to manipulate the migratory response of HSPC and/or the adhesion of T cells to immobilized SDF1. All of those studies used two-dimensional culture systems with limited SDF1 loading capacity that is not amenable for in vivo studies. None of these studies specifically aimed at designing release system that allowed the formation of SDF1 gradients in vivo.

In the current study we used Collagen1-Heparin based matrix [START_REF] Salchert | Fibrillar collagen assembled in the presence of glycosaminoglycans to constitute bioartificial stem cell niches in vitro[END_REF] as a SDF1 delivery system. Collagen1-Heparin matrix was functionalized with SDF1 and tested for its ability to induce HSPC migration in vitro.

MATERIALS AND METHODS

Preparation of Collagen1 -Heparin.

For the preparation of reconstituted Collagen type I based matrices, annealed PEMA (poly-ethylene maleic anhydride) thin films were used as the substrate for fibrillogenesis [START_REF] Salchert | Fibrillar collagen assembled in the presence of glycosaminoglycans to constitute bioartificial stem cell niches in vitro[END_REF][START_REF] Salchert | In vitro reconstitution of fibrillar collagen type I assemblies at reactive polymer surfaces[END_REF][START_REF] Pompe | Maleic anhydride copolymers--a versatile platform for molecular biosurface engineering[END_REF]. Collagen type I (1.2mg/ml) (PureCol®, Inamed Biomaterials, Nutacon, Netherlands) supplemented with heparin (0.4mg/ml) (14 kDa, sodium salt from porcine intestinal mucosa, Calbiochem, USA) was prepared as detailed previously [START_REF] Salchert | In vitro reconstitution of fibrillar collagen type I assemblies at reactive polymer surfaces[END_REF]. For cell culture experiments the surface-bound matrices were UV sterilized for 30 min. The thickness of swollen collagen heparin layer was determined by confocal microscopy to be  1µm.

Hydrogel functionalisation and release of SDF1

All functionalisation studies were performed with substrate that was fixed in custom made immobilisation chambers with a nominal surface area of 1 cm 2 . To determine the amount of SDF1 in the system 125 I labelled SDF1 was used. SDF1 (Strathman, Germany) was labelled with radioactive iodine 125 I (CHELATEC SAS, Nantes, France) by employing IodoBeads according to the manufacturer's instructions (IodoBeads, Pierce Biotechnology, USA). During the optimization we determined that SDF1 saturated substrate within 3 h) thus for all subsequent experiments a 3 h immobilization time was used.

The selected solution concentration of 10µg/ml and 1µg/ml SDF1 were applied in volume of 266µl (i.e. 2600ng and 260ng of total protein respectively) to the respective substrate, immobilised for 3h and subsequently washed three times with PBS or CellGro SCGM cell culture Medium (CellGenix, Germany). Surface associated radioactivity was quantified using a beta counter (Berthold Technologies, Germany) and the amount of protein immobilized was calculated. For release studies, samples were incubated with either PBS or CellGro SCGM Medium (CellGenix, Germany) and surface associated radioactivity was determined at the indicated time point.

Isolation of primary human cells

The study was approved by the local institutional review board of the University Hospital, Technical University Dresden. Samples from granulocyte colony-stimulating factor (G-CSF) mobilised leukapheresis products were obtained from healthy donors after informed consent. CD133+ HSPC were isolated using immunomagnetic assisted selection (MACS, Miltenyi Biotech, Bergisch Gladbach, Germany) as previously described [START_REF] Freund | Comparative analysis of proliferative potential and clonogenicity of MACS-immunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor[END_REF]. HSPC were placed in CellGro SCGM Medium (CellGenix, Germany) and allowed to recover over night (37°C, 5%CO 2 ) before commencing migration studies.

Human primary endothelial cells (HUVECs) were isolated in house from umbilical cord veins as previously described [START_REF] Kohler | Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels[END_REF]. HUVEC were cultured in EC Medium containing supplement mix (Promo Cell, Germany) and 2% serum on transwell filters until confluency, and then used in the transendothelial migration experiment (detailed below). Labelling of HUVECs with the SDF1 polyclonal antibody (Abcam) showed that the protein is expressed internally in HUVECs (data no shown) which is consistent with previous reports [START_REF] Salvucci | Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1[END_REF]. However, we were not able to detect the presence of SDF1 in the conditioned culture medium by ELISA (detection limit ~ 40 pg/ml).

Flow cytometry (FACS) of HSPC

Migrating and non migrating cells were collected at 1h, 4h, 24h, 3days and 7days, counted and stained with anti-CXCR4-phycoerythrin-cy5 (PE-Cy5; BD Biosciences), anti-CD133-phycoerythrin (PE), anti-CD38-fluoresceinisothiocyanat (FITC), anti-CD34-allophycocyanin (APC) and/or anti-CD34-FITC (all Miltenyi) according to manufacturers protocol and subjected to FACS analysis using a FACS Calibur with CELL Quest Software (Becton Dickinson, San Jose, USA). Data Analysis was done with the FlowJo (TreeStar Inc. USA) software. Analysis of expression profile of HSPC showed that all isolated cells expressed CD38 initially and population of CD133+CD34+CD38-cells is very small, often beyond abilities of standard MACS isolation kits [START_REF] Freund | Comparative analysis of proliferative potential and clonogenicity of MACS-immunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor[END_REF].

Transwell migration assays of HSPCs

All migration experiments were performed in 24well transwell plates (Corning, USA) with a 5µm pore size filter. To generate a SDF1 gradient respective SDF1 loaded substrate was placed in the lower chamber. 15mm ø glass slide with SDF1-loaded biopolymer (prepared according to immobilization protocol detailed above) was placed in one well of 24-well-plate with surface facing up. 1ml of CellGROW medium was placed in each well containing SDF1-loaded biopolymer slide. HSPCs suspension (100µl) was placed on the filter and cells were allowed to migrate at 37°C, 5%CO 2 conditions.

Cultures were maintained in CellGro SCGM Medium (CellGenix, Germany) without cytokines and cultured at 37°C in a 5%CO 2 atmosphere. For migration experiment 3x10 5 HSPCs (in 100µl) were used per insert. Migration was determined after 1h, 4h, 24h, 3days and 7days. The medium from the lower chamber (1ml) was collected and migrated HSPC cells were counted. The medium in the lower chamber was immediately replaced with the fresh medium and HSPC cells were allowed to migrate until the next time point. The total number of HSPC migrated at each time point is calculated as cumulative value integrating all previous time points.

As a control 1 ml of 500ng/ml SDF1 solution was placed in one well of 24-well-plate and HSPC were allowed to migrate. Furthermore, CellGrow Medium only was used to determined spontaneous HSPC migration through the membrane. To ensure that in our set up migration was indeed SDF1-induced, HSPC were incubated for 2 hours with 500ng/ml SDF1 with the CXCR4 antagonist AMD3100 prior to SDF1 migration assay [START_REF] Rosenkilde | Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor[END_REF].

Colony forming unit assay

HSPCs that migrated towards the SDF1 gradient were collected and CFU assay was performed according to standard protocol using a complete semi-solid CFU medium with cytokines/EPO (MethoCult, Stem Cell Technologies, Germany). After 14 days, the percentage of granulocyte (CFU-G), granulocyte-macrophage (CF-GM), erythroid burst-forming units (BFU-E) and mixed colonies (CFU-GEMM) was determined. Two independent experiments with cells from different donors were performed.

RESULTS

SDF-1gradient and HSPC migration.

The Collagen-1 -Heparin matrix showed loading efficiency at 26% for 10g /ml of loaded SDF1 and 32% for 1g/ml (Fig1). Samples were washed with cell culture medium to measure SDF1 protein release in our culture setup. The initial release of SDF1 to the medium was 470ng from gel loaded with 10g/ml of SDF1. This resulted in the amount of the protein in the medium to be in the same range (on the same level) as in the soluble control (500ng/ml).

The next release step was 10 times smaller, and each subsequent was minimal. In the gel loaded with 1g/ml the initial protein release was approximately 60ng with each subsequent medium change resulting in minimal SDF1 release.

Next, basal HSPC migration levels at 1,4, 24h and 3days were determined that to be ~ 1, 2, 7 and 8 % respectively in the absence of stimulation. Migration of HSPCs towards soluble SDF1 (500ng/ml) reached 9% after 3 days. Cells were allowed to migrate through the filter alone or through the confluent layer of HUVECs plated on the filter. (Fig2)

The specificity of this response was verified by blocking of the SDF1 receptors CXCR4 with AMD 3100 [START_REF] Rosenkilde | Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor[END_REF] that reduced migration to basal levels (data not shown).

HSPCs migration was subsequently studied up to 7 days. Collagen1-Heparin system was compared to control that employed diffusible SDF1 only (500ng/ml). In the absence of endothelial lining (i.e. no HUVECS, sample SDF1(10) and SDF1(1)), samples reached more than 8% and over 12% of the cells migrating towards SDF1 gradient from gel loaded with 10g/ml and 1g/ml respectively after 7 days.

Migration rate increasedto over 23% of migrating cells for SDF1 gradients from hydrogel loaded with both, 10g/ml and 1g/ml respectively in samples where HUVEC layer was present. (Fig2) Substantial differences between the hydrogel system and the controls were evident from 24 h onwards.

At early time points (1 and 4 h) there were no differences between the systems. Sample with 1g/ml immobilized SDF1 and HUVECS (SDF1(1)-H on Fig2) however, showed migration in the range of 8% already after 1h, and increased to 23% of migrated cells in total after 7 days. Control cultures that contained no SDF1 also showed basal migration levels that were less than for cultures that contained diffusible SDF1. At time points 24h and 3d, migration levels for the controls were comparable for cultures containing diffusible SDF1, whereas we observed boost of cell migration towards SDF1 released from hydrogel layers. ANOVA statistics was applied to test the significance of observed differences and it resulted in p values below 0.002.

Clonogenicity and phenotype.To test whether long term culture of hematopoietic stem cells subjected to SDF1 gradient will have an effect on their clonogenicity and differentiation potential we performed colony forming unit assay. We used Collagen1-Heparin hydrogel loaded with 1g/ml SDF1 for the long term profiling of migrating HSPCs upon exposure to SDF1 gradient (Fig. 3.B). The non-migrating (NM) control tended to contain more CFU-M and CFU-GM colonies than those that migrated within first 4h. Furthermore, the contribution of erythroid colonies in the control group was only 2 % whereas hydrogel samples at 4h, 24h and 72h contained 13, 10 and 7% erythroid colonies, respectively. Even after 7 days of exposure to the SDF1 gradient cells were able to generate CFU-GM and CFU-GEMM, suggesting that our culture set-up was able to maintain clonogenic hematopoietic progenitor cells for at least one week. At all time points FACS analysis confirmed the expression of CD34, CD133 and CXCR4 (CD184) on the HSPC in vitro suggesting that the progeny generated during the observation period was able to follow the persistent chemokine gradient. (Fig3.A)

DISCUSSION

Previous studies by us and others could show that hydrogel systems are applicable for a number of tissue engineering applications including strategies aimed at neuronal and cardiac tissue repair [START_REF] Freudenberg | A star-PEG-heparin hydrogel platform to aid cell replacement therapies for neurodegenerative diseases[END_REF][START_REF] Tabesh | The role of biodegradable engineered scaffolds seeded with Schwann cells for spinal cord regeneration[END_REF][START_REF] Jawad | Myocardial tissue engineering[END_REF][START_REF] Spadaccio | Drug releasing systems in cardiovascular tissue engineering[END_REF] In particular the (controlled) release of growth factors and morphogens are critical parameters in this process as they (in part) are attributed to regulate the migration and mobilisation of resident stem/progenitor cells. Sulphated GAGs, in particular heparan sulphate, exhibit a particularly high affinity to SDF1 and have been reported to support presentation of SDF1 to the vascular lumen in the stem cell niche [START_REF] Netelenbos | Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells[END_REF][START_REF] Netelenbos | Proteoglycans guide SDF-1induced migration of hematopoietic progenitor cells[END_REF][START_REF] Mbemba | Glycan and glycosaminoglycan binding properties of stromal cell-derived factor (SDF)-1alpha[END_REF].

It is also known that heparin is a natural protector of SDF1 [START_REF] Sadir | Heparan sulfate/heparin oligosaccharides protect stromal cell-derived factor-1 (SDF-1)/CXCL12 against proteolysis induced by CD26/dipeptidyl peptidase IV[END_REF] that prevents the protein from degradation. Therefore, sulphated polysaccacharides define a promising starting point for the design of biomimetic matrices capable of modulating SDF1 gradients. The migration experiments performed with the matrix-bound SDF1 samples described above showed the highest migratory response of HSPC to SDF1 gradients in vitro reported so far. The control samples with pure medium and soluble control show migration below 10%, which is consistent with previous reports describing similar assays [START_REF] Mohle | Transendothelial migration of CD34+ and mature hematopoietic cells: an in vitro study using a human bone marrow endothelial cell line[END_REF].

However, heparin containing matrix influences the SDF1 retention in ways resulting in longer lasting gradient formation and successful chemotactic control of HSPC. Presence of HUVEC on the transwell filter, with their own ECM and its ability to bind and present SDF1 [START_REF] Netelenbos | Proteoglycans on bone marrow endothelial cells bind and present SDF-1 towards hematopoietic progenitor cells[END_REF] and the ability to process and re-secrete functional SDF1 [START_REF] Dar | Chemokine receptor CXCR4-dependent internalization and resecretion of functional chemokine SDF-1 by bone marrow endothelial and stromal cells[END_REF] was found to further enhance the migratory response of HSPC in the investigated systems. This could explain the effect that we observe for sample SDF1(1)-H on Fig2, where small amounts of released SDF1 in the presence of HUVEC supporting layer cause increased HSPC migration. Referring to the current understanding of the hematopoietic microenvironment, we consider the transendothelial migration experiment as a representative in-vitro system modelling some aspects of the bone marrow 'niche': HUVEC growing on the filter mimic may represent the endothelial lining of bone marrow sinusoids through which HSPC have to pass to reach the peri-vascular niche. By modulating the immobilized amount of SDF1 we show that the migratory responses of HSPC could be maintained over time periods of at least 7 days. Presence of clonogenic progenitors with minimal changes in phenotype after 7 days in culture validates the supportive role of HUVEC in our system.

Another interesting option of the reported SDF1 releasing polymer matrices concerns possible in vivo applications where local accumulation of hematopoietic progenitors would be desirable. Here, long term SDF1 release combined with additional biomolecular functionalization may open up new therapeutic avenues.

Hematopietic progenitor cells migrate selectively to the SDF-1 gradient [START_REF] Wright | Hematopoietic stem cells are uniquely selective in their migratory response to chemokines[END_REF]. It has been already described that functional CXCL12-CXCR4 axis is essential for proper mobilisation vs quiescence balance of HSCs in the bone marrow [START_REF] Petit | G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4[END_REF][START_REF] Broxmeyer | Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist[END_REF]. This phenomenon can be used as a main regulatory mechanism in stem cell transplantations and therapies. Since many stem and progenitor cells express CXCR4 receptor, and are known to respond to SDF-1 gradient [START_REF] Bleul | A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1)[END_REF], site-targeted, artificially elevated levels of SDF-1 (as shown here) are attractive tool to direct stem cells migration and homing to the required site e.g. bone marrow.

The field on a fast track for bioengineered matrices containing biologically relevant molecules is tissue repair and regeneration. Hypoxia and tissue damage causes elevation of HIF-1 in endothelial cells, which in turn causes increase of SDF1 expression chemo-attracting CXCR4+ tissue specific stem cells to participate in the regeneration process [START_REF] Ceradini | Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1[END_REF][START_REF] Ratajczak | Stem cell plasticity revisited: CXCR4-positive cells expressing mRNA for early muscle, liver and neural cells 'hide out' in the bone marrow[END_REF]. Again, artificially elevated levels of SDF1 limited to the site of interest can enhance therapy and limit potentially damaging preparatory treatments (e.g. radiation, invasive surgery). The tissue repair process can be enhanced by angiogenetic properties of CXCL12-CXCR4 axis ( [START_REF] Chen | Stromal cell-derived factor-1/CXCR4 signaling modifies the capillary-like organization of human embryonic stem cell-derived endothelium in vitro[END_REF] and reviewed in [START_REF] Liekens | CXCL12-CXCR4 Axis in Angiogenesis, Metastasis and Stem Cell Mobilization[END_REF]) in case of the hindlimb ischemia [START_REF] Zemani | Ex vivo priming of endothelial progenitor cells with SDF-1 before transplantation could increase their proangiogenic potential[END_REF], stroke and heart myocardial infarction [START_REF] Yu | SDF-1/CXCR4-mediated migration of transplanted bone marrow stromal cells toward areas of heart myocardial infarction through activation of PI3K/Akt[END_REF][START_REF] Paczkowska | Clinical evidence that very small embryonic-like stem cells are mobilized into peripheral blood in patients after stroke[END_REF] or liver damage [START_REF] Kollet | HGF, SDF-1, and MMP-9 are involved in stressinduced human CD34+ stem cell recruitment to the liver[END_REF]. Collagen-1-Heparin matrix immobilisation of SDF-1 can provide such a tool for site-targeted and localized elevation of SDF-1.

In summary, we describe SDF1 gradients from heparin-based polymer matrix which is biologically natural and permits the production of long lasting SDF1 gradients in vitro. The results provide a base for the use of chemokine gradients to control HSPC in tissue engineering strategies in vitro and in vivo.

We have been able to show in vitro, that SDF1 functionalised hydrogel have the potential to support HSPC migration well above levels seen for diffusible SDF1. It remains to be seen if such hydrogel based system applied in vivo could also support an increased HSPC homing into bone marrow following stem cell transplantation.

Support and Financial Disclosure Declaration: Authors declare no competing financial interests. Enriched HSPCs were added to the upper chamber and allowed to migrate into the receiver chamber in the presence and absence of SDF1. Cells were migrating through the filter alone or endothelial lining of HUVECs monolayer cultivated on the filter. Specificity of SDF1 dependant migration was measured by addition of AMD3100 to the medium (data not shown). Either diffusible soluble SDF1 (500ng/ml) or SDF1 functionalised hydrogels were as a SDF-1 source. Abbreviations: SDF1 [START_REF] Tyndall | Autologous haematopoietic stem cell transplants for autoimmune disease--feasibility and transplant-related mortality. Autoimmune Disease and Lymphoma Working Parties of the European Group for Blood and Marrow Transplantation, the European League Against Rheumatism and the International Stem Cell Project for Autoimmune Disease[END_REF] or SDF1(1)surface loaded with SDF1 solution of 10µg/ml or 1µg/ml respectively; H -HUVECs monolayer on the filter; 500ng/mlconcentration of soluble SDF1 (500ng/ml) in CellGROW medium; ctrlcontrol, culture medium without SDF1. * -ANOVA statistical test was applied resulting in p≤<0.002.

Figure1

Click here to download high resolution image

Figure3

Click here to download high resolution image

Figure2

Click here to download high resolution image

Figure 1 .

 1 Figure 1. SDF1 functionalisation of Collagen1 -Heparin substrate. Two different solution concentrations were used (10µg/ml and 1µg/ml) to functionalise substrate with SDF1. Bars represent the amount of SDF1 immobilized initially on the surface (Initial) and after 1, 2, and 3 washing steps respectively. Samples were washed with cell culture medium and the amount of surface associated 125 I SDF1 radioactivity was measured. Each condition was tested in at least 5 separate experiments in triplicate.

Figure 2 .

 2 Figure 2. Migration of human haematopoetic stem and progenitor cells (HSPCs) in response to SDF1.

Figure 3 .

 3 Figure 3 . Characterisation of progenitor cells from migration assay . FACS and CFU analysis of migrating cells. A. Cells from the lower compartment in the migration assay were collected at 1h, 4h, 24, 3d, 7d, stained against the CD133, CD34, CD38 and CD184(CXCR4). Non-migrating cells were also subjected to the FACS analysis showing the same expression profile (not shown). B. The same cells were analysed for clonogenicity and potential to differentiate to different hematopoietic lineages. Graph shows differentiation potential of migrated cells at different time points and non-migrated (N-M) cells after 7 days. Assay was performed for two donor samples in triplicate for each condition.

ACKNOWLEDGEMENTS

This work was supported by the Leibniz Association (Senatswettbewerb LS, MB and CW), the Deutsche Forschungsgemeinschaft, Collaborative Research Centre: Cells into tissues-stem cell and progenitor commitment and interactions during tissue formation (SFB 655, Dresden, FPS, MB and CW) and the Centre for Regenerative Therapies Dresden (DFG Research Centre and Cluster of Excellence).

ABSTRACT

The therapeutic success of haematopoetic stem and progenitor cell (HSPC) transplantation is critically dependent on HSPC engraftment in the bone marrow. Gradients of stromal cell derived factor 1 (SDF1) direct HSPC homing both in vitro and vivo. Potentially, regulating delivery levels of exogenous SDF1 applied to the bone marrow could augment HSPC engraftment. Thus the aim of the present study was to revise the ability of biocompatible hydrogels to direct HSPC migration in vitro.

The delivery system of choice is based on heparin cross-linked with Collagen1. We confirm that hydrogel is capable to trap and release SDF1 and used it to generate protein gradient in transendothelial (Human Umbilical Vein Endothelial Cell -HUVEC) migration experiments. The use of SDF1 functionalised hydrogel to produce chemokine gradient revealed sustained and increased HSPC migration when compared to diffusible SDF1 controls.

In conclusion, regulating SDF1 gradients with heparin-containing hydrogels may offer valuable options to direct site-specific migration of HSPC.


Enriqueta Albizua

Miguel Gallardo

Santiago Barrio

Inmaculada Rapado

Ana Jimenez

Rosa Ayala

Daniel Rueda

Beatriz Sanchez-Espiridion

Eulalia Puigdecanet

Blanca Espinet

Lourdes Florensa

Carles Besses

Joaquin Martinez-Lopez

Joaquin Martínez-López

Differential expression of JAK2 and Src kinase genes in response to hydroxyurea

Keywords: polycythemia vera, essential thrombocythemia, gene expression profile, RT-PCR, response to hydroxyurea, JAK2, Src family kinase (SFK)

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The discovery of the JAK2 V617F point mutation has become a hallmark in the diagnosis of the classic chronic Philadelphia-negative myeloproliferative neoplasms (MPN) namely, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). [START_REF] James | A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera[END_REF] As a result, WHO diagnostic criteria were revised in 2007, and JAK2 mutations and MPL mutations were included as major criteria for the diagnosis of PV, ET and PMF. [START_REF] Tefferi | Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel[END_REF] Moreover, the JAK2 V617F allele burden has been included in response criteria for PV and ET by a recent European Leukaemia Net (ELN) consensus conference. [START_REF] Barosi | Response criteria for essential thrombocythemia and polycythemia vera: result of a European LeukemiaNet consensus conference[END_REF] Despite the fact that JAK2 V617F has facilitated the diagnosis of MPN, and improved our knowledge on their pathogenesis, there are still a number of unanswered questions. [START_REF] Goldman | Chronic myeloproliferative diseases with and without the Ph chromosome: some unresolved issues[END_REF] Although formerly believed to be a single-step process, recent evidence has shown that the JAK2 V617F mutation is neither the sole, nor sufficient molecular event, responsible for MPN. It may also not be the initial one, and other somatic mutations may precede JAK2 V617F . [START_REF] Levine | X-inactivation-based clonality analysis and quantitative JAK2V617F assessment reveal a strong association between clonality and JAK2V617F in PV but not ET/MMM, and identifies a subset of JAK2V617F-negative ET and MMM patients with clonal hematopoiesis[END_REF][START_REF] Kralovics | Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders[END_REF][START_REF] Lambert | In essential thrombocythemia, multiple JAK2-V617F clones are present in most mutant-positive patients: a new disease paradigm[END_REF] There is evidence of clonal heterogeneity and presence of different mutations in the same patient. [START_REF] Li | Clonal heterogeneity in polycythemia vera patients with JAK2 exon12 and JAK2-V617F mutations[END_REF][START_REF] Delhommeau | Mutation in TET2 in myeloid cancers[END_REF] The current first line of treatment for PV and ET patients with a high risk of thrombosis is still hydroxyurea (HU). [START_REF] Vannucchi | Advances in understanding and management of myeloproliferative neoplasms. CA: a cancer journal for clinicians[END_REF] The cytoreductive activity of HU is based on its anti-metabolic role as a cell cycle-specific analog of urea, with activity in the S-phase. HU inhibits the enzyme, ribonucleotide reductase, which converts ribonucleotides to deoxiribonucleotides (critical precursors for de novo DNA biosynthesis and repair). Up to 15 to 25% of patients show resistance or intolerance to HU. [START_REF] Harrison | Hydroxyurea compared with anagrelide in high-risk essential thrombocythemia[END_REF][START_REF] Barosi | A unified definition of clinical resistance/intolerance to hydroxyurea in essential thrombocythemia: results of a consensus process by an international working group[END_REF] The mechanism of resistance is believed to be the increased expression of ribonucleotide reductase due to gene amplification, increased transcription, and post-transcriptional mechanisms. [START_REF] Akerblom | Overproduction of the free radical of ribonucleotide reductase in hydroxyurea-resistant mouse fibroblast 3T6 cells[END_REF] Despite this knowledge, there is still no clear understanding of the underlying molecular mechanism and consequences of HU activity.

We aimed to analyze the differential gene expression profile of JAK2 V617F positive MPN patients with or without response to HU treatment, to determine possible mechanisms for a poor response. As a consequence, we hoped to identify alternative routes for targeted therapy. In addition, we aimed to correlate these results with clinical and laboratory features.

Design and methods

Patients

Forty-nine patients, diagnosed with MPN (21 PV and 28 ET) with the JAK2 V617F mutation were included in the study, as well as eight patients with secondary erythrocytosis (SE) to hypoxemia, and 30 healthy control donors. A MPN diagnosis was established on the basis of World Health Organization criteria (WHO) 2001/2008, [START_REF] Tefferi | Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel[END_REF] or the Polycythemia Vera Southern Study Group (according to the standard criteria at the moment of diagnosis). Twenty MPN patients were studied at diagnosis (as control population) and 29 after treatment.

Clinical and laboratory features at diagnosis and follow-up features of the JAK2 V617F PV and ET patients were assessed and are shown in Table 1.

Response to treatment with HU in PV and ET was defined according to update recommendations [START_REF] Vannucchi | Advances in understanding and management of myeloproliferative neoplasms. CA: a cancer journal for clinicians[END_REF] and to clinico-hematological response criteria for PV and ET recently published by the European Leukaemia Net (ELN) consensus conference [START_REF] Barosi | Response criteria for essential thrombocythemia and polycythemia vera: result of a European LeukemiaNet consensus conference[END_REF].

The study was approved by the local ethics committee and written informed consent was obtained from all patients, according to the Declaration of Helsinki.

Nucleic acids extraction and JAK2 V617F quantification

Peripheral venous blood was collected in EDTA and immediately processed. Granulocytes were isolated by Ficoll-Paque (Sigma-Aldrich, Saint Louis, Mi USA). density gradient centrifugation, as previously described. Erythrocytes were eliminated by lysis with a commercial buffer (Red Blood Lysis Buffer, from Roche Applied Sciences, Mannheim, Germany). DNA was extracted using the Maxwell 16 SEV automated extraction system (Promega co., Madison, Wi, USA). mRNA was extracted from 1×10 6 cells, with TRIzol(R) Reagent (Invitrogen Life Technologies, Inc., Paisley, UK) according to the manufacturer's instructions, with more than 90% granulocytes. Lymphocyte contamination was assessed in five samples by flow cytometry, and was less than 2% of the total cell count. DNA and RNA quantity and quality were assessed on a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) for low density array experiments, and by nanoelectrophoresis using the Nano lab-on-a-chip assay for total eukaryotic RNA (Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA) for microarrays. Retrotranscription was performed with a MMLV-modified enzyme, according to manufacturer's instructions (Applied Biosystems, Palo Alto, CA, USA). Mutational screening for JAK2 V617F was performed with real-time (RT) PCR using DNA from whole peripheral blood from the study sample. [START_REF] Rapado | Validity test study of JAK2 V617F and allele burden quantification in the diagnosis of myeloproliferative diseases[END_REF][START_REF] Rapado | High resolution melting analysis for JAK2 Exon 14 and Exon 12 mutations: a diagnostic tool for myeloproliferative neoplasms[END_REF]

Microarray analyses

As screening proceeding fourteen samples from JAK2 V617F positive patients diagnosed with MPN (three PV and 11 ET) were included in the study, as well as 20 samples from healthy control donors (in four pools from five samples); 11 MPN were under treatment and 3 were under no treatment. Microarray expression profiles were obtained using the Whole Human Genome Oligo Microarray Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Hybridization was performed with Stratagene Universal Human Reference RNA, and a mix of tumor cell lines (Agilent Technologies Stratagene, Inc., Santa Clara, CA, USA). Briefly, for each sample 2 µg of total RNA was mixed with 2 µL of a 5,000-fold dilution of Agilent's Two-Color Spike-in RNA control. The mixture was amplified using the low-input RNA amplification kit (Agilent Technologies Inc., Santa Clara, CA, USA). Following amplification and labeling with Cy3, each sample was assessed on the Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA) to measure yield and specific activity. The amplified and labeled samples were hybridized in a rotating oven to Agilent 44K human whole genome microarrays, according to the manufacturer's instructions. The images were scanned with a G2565BA Microarray Scanner System (Agilent Technologies Inc., Santa Clara, CA, USA). The data were processed and normalized with the use of Feature Extraction (v.9.0) software. Each time point was additionally normalized to time zero. Genes with more than 10% missing values were discarded. Normalized log2 ratios were scaled between arrays to make all data comparable, and processed again with pre-processor of GEPAS 3.1 (http://gepas3.bioinfo.cipf.es/) to purify the data.

The student´s test was employed as statistical tool in the gene expression pattern analysis suite (GEPAS 3.1), Pomelo II (http://pomelo2.bioinfo.cnio.es/) and gene set enrichment analysis (GSEA) programs, used to interpret gene expression data and to determine statistical significance of differential gene expression, as described. [START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles[END_REF] The supervised classification of healthy control donors, ET and PV samples into categories was performed.

Low-density quantitative array (LDA)

To reassess microarray data, forty JAK2 V617F positive patients, diagnosed with MPN were included in the study: 20 MPN patients receiving HU treatment (10 PV, and 10 ET) and 20 MPN newly diagnosed patients (10 PV, and 10 ET), used as control population to assay the influence of phenotype. Eight secondary erythrocytosis (SE) to hypoxemia as well as 10 healthy control donors (in two pools from five samples) were also included in the study. Related to the 20 MPN patients with HU treatment, nine of them showed response to HU (four PV and five ET) and 11 were nonresponders (six PV and five ET). Among the MPN patients with HU treatment, two PV and three ET patients had been previously included in the microarray assay Eighty-four genes were selected for their differential expression from several different dicothomous analyses in the microarray study. Another nine genes, though without differential expression in microarray analysis,were selected for their key role in erythropoiesis, thrombopoiesis, or signalling pathways in MPN; and three housekeeping genes (GADPH, 18s rRNA, and ABL1) were also included (Supplementary Table 1: Supplementary data). All these genes were assayed by quantitative PCR (using the 96 TaqMan Low Density Arrays platform LDA). Besides, three individual TaqMan assays were performed with three additional genes, of interest according to other MPN studies, in the same samples of patients.

Custom-made TaqMan low-density arrays with a setup of 96 different genes were applied for relative mRNA quantification. cDNA samples were subjected to RT-PCR in duplicate in an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Palo Alto, CA, USA). Relative expression of each gene was quantified by the comparative cycle threshold (Ct) method, using the Abelson murine leukemia viral oncogene homolog 1 (ABL1) as an endogenous control. Gene expression quantification was achieved using the comparative Ct method for relative quantification, in which the amount of target is expressed as 2 -ΔCt . [START_REF] Rapado | High resolution melting analysis for JAK2 Exon 14 and Exon 12 mutations: a diagnostic tool for myeloproliferative neoplasms[END_REF]

Statistical analyses

Statistical analyses of gene expression results in LDA, and of the clinical and biological data, and their relationship with the gene expression data, were performed using the SPSS15 software (SPSS Inc., Chicago, Illinois, USA).

Differences between non-normally distributed group data were analyzed using the non-parametric Wilcoxon analysis.

Statistical significance was considered when P was less than 0.05.

Results

Microarray results based on biological and functional pathways analyses

Microarray analysis, used as screening test, showed 84 genes with significant differential expression by GSEA (conditions required in GSEA analysis: core enrichment: YES), related to biological and functional pathways of GSEA tool (significant different expression by GSEA was considered when P was < 0.05 and/or the false discovery rate (FDR) was < 0.25).

Genes was selected from different analyses performed in a dichotomic manner: ET versus PV, ET versus Controls, PV versus Controls and MPN with response to HU against those without response to HU.

Low density array analysis

Gene expression profile and response to HU treatment

A set of 34 genes were found to be differentially expressed in MPN patients who either responded or showed no response to HU treatment, and were significantly over-expressed in those patients with no response (Table 2; Figure 1). Interestingly, the following genes were significantly over-expressed in patients who showed no response to HU treatment: JAK2 (ER = 2.482 vs 1.096; P = 0.008), IGF1R (ER = 3.076 vs 2.259; P = 0.026), MAPK14 (ER = 4.885 vs 3.834; P = 0.007), FCRE1G (ER = 4.760 vs 3.295; P = 0.004), LYN (ER = 5.372 vs 4.386; P = 0.017), BTK (ER = 1.121 vs 0.082; P = 0.016) and PIK3CA (ER = 4.826 vs 3.731; P = 0.011) (Figure 1). JAK2 was over-expressed in patients who did not respond to treatment; and in patients who did respond to treatment, JAK2 expression was lowered to equivalent levels to those of SE patients and healthy controls. A similar gene expression pattern was observed for MAPK14, FCRE1G, LYN, BTK and PIK3CA (Figure 1). These differences were not due to disease duration because when we compared patients with a short disease duration (bellow two years) and patients with a long disease duration (over two years) no differences in gene expression profile were found (Supplementary Table 2, supplementary data).

The gene expression profile of the control group was similar to that of patients who responded to HU treatment.

JAK2 allele burden quantification

In order to assay if the lowering expression of JAK2 gene was due to a decrease of the JAK2 V617F allele burden, it was measured in patients at diagnosis and under HU treatment. When measured after HU treatment, JAK2 V617F allele burden diminished in PV, but only in a significant manner in ET (P = 0.003). However, the allele burden differences between patients who responded, or those who did not respond to HU, were not statistically significant (Figure 1).

Gene expression profile associated with response is not influenced by phenotypic diversity

To assay whether the different gene expression profile between responders to HU treatment and the non responder group could be due to phenotypical differences, gene expression of patients under HU treatment was compared with a balanced population of patients at diagnosis. Upon separate analysis of samples from PV and ET patients receiving HU treatment, and those patients at diagnosis, only one gene, MMP14, was over-expressed in PV newly diagnosed patients compared to ET at diagnosis. Moreover, this over-expression was maintained when comparison was established between treated an untreated ET patients against PV treated and untreated (ER: expression ratios = -4.442 vs -2.794; P = 0.000) (Table 2; Figure 2).

Correlation between clinical and laboratory features and gene expression profile in PV and ET

Clinical and laboratory variables, referred in Table 1 were analyzed and compared in a dichotomic manner with gene expression profile, to search correlates: (1) at diagnosis: gender, age, splenomegaly, hepatomegaly, hemoglobin, hematocrit, white blood cells, platelets, lactate dehydrogenase, thrombotic events, hemorrhagic events, JAK2 V617F allele burden; (2) and in the follow up: disease duration, treatment duration, thrombotic events after diagnosis, hemorrhagic events after diagnosis, and response to HU treatment (Supplementary Table 2: Supplementary data).

Discussion

Microarray analysis looking for a differential gene fingerprint profile between JAK2 V617F MPN different groups treated with HU resulted in the identification of 84 genes with significant differential expression. These genes play key roles as transcription factors (LYN, PIK3CA), in MAP kinase (MAPK) signaling pathways (MAPK14, RAF1, RASA1, YWHAH), as tyrosine kinases (JAK2, BTK), in development and function of the hematological system (CD44, MAP1B, JARID2, HMGB2, IKZF1), and in other signaling and apoptotic pathways (SKAP2, PLAUR, LRMP). Some of these genes have been previously outlined in other microarray analyses or functional studies. [START_REF] Pellagatti | Gene expression profiling in polycythemia vera using cDNA microarray technology[END_REF][START_REF] Kralovics | Altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of Jak2[END_REF] When we looked at this gene signature with LDA (a real time PCR approach), we observed a differential gene expression profile in MPN patients according to response to HU. We found 34 genes that were over-expressed in patients who did not respond to HU treatment. Interestingly, among these 34 over-expressed genes, some participate in four important proliferative pathways: MAPK14, MAP2K1, RAF1 (MAPK pathway), PIK3CA, FRMD4B (AKT pathway), LYN, FCER1G, BTK, SKAP2 (Src family kinase, SFK), and JAK2, IGFR1 (JAK2 pathway). Gene expression did not change with disease evolution but it did with the response to treatment.

A number of genes in the MAPK pathway were differentially expressed in MPN patients relative to their response to HU. An over-expression of genes implicated in this pathway was shown in the group who did not respond to HU, notably RAF1, MAP2K1 and MAPK14. In the group of patients who did respond to HU treatment, MAPK genes expression was lowered to levels equivalent to those observed in SE patients and controls. The activation and over-expression of MAPK14 has been described in PV pathogenesis. [START_REF] James | A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera[END_REF][START_REF] Pellagatti | Gene expression profiling in polycythemia vera using cDNA microarray technology[END_REF][START_REF] Schwemmers | JAK2V617F-negative ET patients do not display constitutively active JAK/STAT signaling[END_REF] Over-expression of genes in the MAPK pathway may be involved in the lack of response to HU treatment, although by yet unknown mechanisms, and probably related to JAK2 gene expression.

The AKT gene, with a crucial role in the AKT pathway, has been widely implicated in the pathogenesis of MPN (PV and ET), by over-activation and over-expression. [START_REF] James | A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera[END_REF][START_REF] Schwemmers | JAK2V617F-negative ET patients do not display constitutively active JAK/STAT signaling[END_REF][START_REF] Shide | Development of ET, primary myelofibrosis and PV in mice expressing JAK2 V617F[END_REF] We found that the PIK3CA gene, an AKT regulator, was also over-expressed in patients who did not respond to HU treatment. This over-expression may also be involved in the lack of response to treatment, and correlate with JAK2 over-expression.

The SFK are also suggested to contribute to signalling by EpoR, [START_REF] Chin | Lyn physically associates with the erythropoietin receptor and may play a role in activation of the Stat5 pathway[END_REF] although they seem not to be indispensable for the PV phenotype induced by JAK2 V617F , as shown by bone marrow reconstitution studies in mice deficient for LYN, FYN or Fgr kinases. [START_REF] Zaleskas | Molecular pathogenesis and therapy of polycythemia induced in mice by JAK2 V617F[END_REF] Our results showed that BTK and other SFK members, such as LYN, FCER1G and SKAP2, were over-expressed in patients who did not respond to HU treatment.. Thus, an over-expression of these genes may be related to a lack of response to HU, while decreased levels, similar to those observed in SE patients and controls, may be related to a good response to HU treatment. These findings could be of great interest, since those patients with over-expressed SFK genes may benefit from treatment with SFK inhibitors (such as Dasatinib). In fact, some groups are testing Dasatinib in cell cultures from PV patients with some success. [START_REF] Zaleskas | Molecular pathogenesis and therapy of polycythemia induced in mice by JAK2 V617F[END_REF][START_REF] Wappl | Dasatinib inhibits progenitor cell proliferation from polycythaemia vera[END_REF] In addition, HU treatment in MPN patients showed a resulting decrease in, not only the JAK2 V617F allele burden, but also in response to treatment in JAK2 gene expression. Interestingly, while JAK2 was over-expressed in patients who did not respond to treatment, in those patients who responded well to HU, JAK2 gene expression was lowered to levels equivalent to those of SE patients and healthy controls. We also showed that, although the JAK2 V617F allele burden diminished after HU treatment, as reported by others, [START_REF] Girodon | Frequent reduction or absence of detection of the JAK2-mutated clone in JAK2V617F-positive patients within the first years of hydroxyurea therapy[END_REF] there was no statistical difference between the levels of the JAK2 V617F allele burden in our different patient groups, with or without a response to HU. However, JAK2 gene differential expression between the same groups of patients with or without a response to HU treatment was statistically significant. Thus, JAK2 over-expression may reflect of lack of response to HU, although the mechanism for this is unknown. The criteria for a lack of response to HU treatment could thus be expressed, either by the mutant allele burden, and/or by JAK2 over-expression. To our knowledge, this is the first study to show these results, and could be useful as an additional parameter of response.

Finally we found that this different pattern of expression was not due to phenotypic differences, as demonstrated when compared with a control group of patients without treatment.

Only one gene, MMP14, remained significant between PV and ET patients at diagnosis. MMP14 is a matrix metalloprotease involved, among other things, in tumor metastasis. It has an indirect connection with some proliferative pathways, such as the MAPK pathway. Some authors have made reference to the importance of the MAPK pathway in PV pathogenesis, and its connection with JAK2. [START_REF] James | A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera[END_REF][START_REF] Pellagatti | Gene expression profiling in polycythemia vera using cDNA microarray technology[END_REF] On the other hand, MMP14 has also been demonstrated to be over-expressed in PMF, and is modulated by TIMP2, which has erythroid-potentiating activity. [START_REF] Bock | Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status[END_REF][START_REF] Stetler-Stevenson | Tissue inhibitor of metalloproteinase-2 (TIMP-2) has erythroid-potentiating activity[END_REF] Thus, the over-expression of MMP14 in PV, compared with ET, may contribute to the final myeloproliferative phenotype of PV by enhancing the commitment to erythropoiesis via MAPK pathway, either by JAK/STAT, TIMP2 or by alternative routes.

To conclude, our results suggest that a differential molecular fingerprint of genes distinguishes MPN patients who either do or do not respond to HU treatment. JAK2, MAPK14, PIK3CA and SFK genes are over-expressed in patients who do not respond to HU. In our group of patients the JAK2 V617F allele burden decreased with HU treatment, however a significant diminution was not observed in response to treatment. On the other hand, patients with no response to treatment over-expressed JAK2. These results need to be confirmed with further functional studies.

Figure 1

 1 Figure 1 The differential gene expression profile in polycythemia vera (PV) and

Figure 2 MMP14

 2 Figure 2 MMP14 gene is differentially expressed in JAK2 V617F positive

Table 1 . The clinical and laboratory features of JAK2 V617F positive PV and ET patients at diagnosis and follow-up

 1 . PV, polycythemia vera; ET, essential thrombocythemia; HU, hydroxyurea; M, male; F, female; WBC, white blood cells, LDH, lactate dehydrogenase NA: not applicable; a. Median value (range) is reported.

Table 2 . Differential gene expression of JAK2 V617F positive polycythemia vera (PV) and essential thrombocythemia (ET) patients.

 2 Results of the low density array (LDA) analysis (P < 0.05) and results of Wilcoxon analyses between clinical and laboratory features relative to the gene expression profile are shown.

Table 2 . Differential gene expression of JAK2 V617F positive polycythemia vera (PV) and essential thrombocythemia (ET) patients.

 2

	Genes		ET vs PV		ET vs PV (with HU treatment)	ET vs PV (at diagnosis)	Response to HU Treatment
		(P-value)		(ratio)	(P-value)		(ratio)	(P-value)		(ratio)	(P-value)		(ratio)
			ET	PV		ET	PV		ET	PV		Yes	No
	Number of patients	20/20.	20	20	10/10.	10	10	10/10.	10	10	11/9.	11	9
	ACSL	0.044*	4..072	4..609	0.000*	3..611	5..602	0.598	4..534	3..617	0.017*	4..235	5..060
	ANXA1	0.040*	2.282	2.756	0.000*	1.953	3.275	0.975	2.610	2.209	0.020*	2.320	2.973
	BTK	0.0032*	0.467	1.636	0.000*	-0.707	1.807	0.729	1.642	1.466	0.016*	0.082	1.121
	CASP8	0.003*	2.838	3.459	0.000*	2.210	3.775	1.000	3.466	3.144	0.039*	2.667	3.391
	CD44	0.008*	2.422	3.229	0.000*	1.796	3.961	0.424	3.049	2.497	0.005*	2.348	3.527
	CXCL1	0.054	2.359	3.156	0.000*	1.214	3.168	0.940	3.504	3.144	0.014*	1.713	2.775
	EDN1	0.218	-1.292	-0.679	0.002*	-1.990	-0.433	0.598	-0.593	-0.926	0.009*	-1.818	-0.471
	F2R	0.02*	-2.409	-2.308	0.091	-2.404	-1.632	0.452	-2.414	-3.019	0.019*	-2.420	-1.526
	FCER1G	0.0021*	3.511	4.682	0.000*	2.494	5.415	0.497	4.527	3.950	0.004*	3.295	4.760
	FRMD4B	0.001*	1.148	2.579	0.000*	0.104	2.931	0.845	2.191	2.228	0.001*	0.735	2.473
	GAPDH	0.011*	2.464	3.167	0.000*	1.719	4.164	0.577	3.208	2.170	0.037*	2.520	3.457
	GSK3B	0.094	1.550	2.191	0.000*	0.567	2.650	0.220	2.533	1.733	0.024*	1.176	2.137
	HMGB2	0.014*	3.188	3.732	0.000*	2.185	4.375	0.869	4.190	3.089	0.014*	2.754	3.924
	IGF1R	0.052	2.417	2.703	0.000*	1.715	3.538	0.798	3.120	1.867	0.026*	2.259	3.076
	IKZF1	0.022*	1.102	2.187	0.000*	0.503	2.190	0.752	1.700	2.184	0.024*	0.970	1.807
	ITGAM	0.065	2.718	3.384	0.000*	1.905	3.912	0.283	3.532	2.857	0.004*	2.400	3.530
	JAK2	0.002*	1.527	2.862	0.000*	0.444	2.995	0.892	2.611	2.729	0.008*	1.096	2.482
	LITAF	0.002*	4.366	5.142	0.000*	4.146	6.122	0.497	4.586	4.161	0.016*	4.693	5.673
	LRMP	0.007*	2.496	3.532	0.000*	1.558	3.740	0.752	3.434	3.324	0.001*	2.134	3.280
	LYN	0.002*	4.535	5.686	0.000*	0.648	1.931	0.616	5.321	5.450	0.017*	4.386	5.372
	MAP2K1	0.053	1.070	1.683	0.001*	1.931	0.647	0.598	1.493	1.435	0.045*	0.972	1.677
	MAPK14	0.006*	3.606	4.314	0.000*	3.200	5.414	0.460	4.012	3.215	0.007*	3.834	4.885
	MMP14	0.000*	-4.422	-2.794	0.001*	-4.430	-2.759	0.003*	-4.414	-2.829	0.106	-3.995	-3.105
	PECAM1	0.021	2.641	3.562	0.000*	1.676	3.782	0.869	3.605	3.342	0.030*	2.414	3.114
	PIK3CA	0.015*	-0.490	0.217	0.000*	-1.424	0.434	0.752	0.445	0.000	0.020*	-0.881	-0.024
	PLA2G4A	0.082	-1.904	-1.446	0.002*	-2.609	-1.301	0.798	-1.199	-1.590	0.022*	-2.289	-1.547
	RAF1	0.005*	2.039	2.954	0.000*	1.352	3.082	0.845	2.726	2.826	0.003*	1.811	2.713
	S100A4	0.059	3.779	4.334	0.000*	3.072	4.845	0.478	4.486	3.823	0.037*	3.679	4.301
	SEC31B	0.114	-0.260	0.194	0.002*	-0.463	0.750	0.346	-0.058	-0.391	0.026*	-0.184	0.544
	SELL	0.005*	3.681	4.666	0.000*	2.765	5.148	0.892	4.596	4.184	0.014*	3.527	4.482
	SELP	0.010*	-1.000	0.040	0.002*	-1.635	0.166	0.641	-0.365	-0.086	0.019*	-1.067	-0.327
	SKAP2	0.008*	2.230	3.254	0.000*	1.330	3.675	0.845	3.131	2.832	0.013*	2.053	3.052
	SPON1	0.469	-5.401	-5.314	0.030*	-5.398	-4.389	0.459	-5.403	-6.239	0.027*	-5.269	-4.435
	TAP1	0.072	2.677	3.522	0.000*	2.441	3.923	0.684	2.913	3.121	0.042*	2.921	3.500
	YWHAH	0.025*	1.650	2.436	0.000*	1.188	2.937	0.660	2.137	1.880	0.034*	1.672	2.538