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Blood, 15 August 2007, Vol. 110, No. 4, pp. 1238-1250. Prepublished online as a Blood First Edition Paper on April 9, 2007; DOI 10.1182/blood-2006-02-003772.
NEOPLASIA VEGFR-1 (FLT-1), ß1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome1 Department of Experimental Pathology and Oncology, University of Firenze, Firenze; 2 Department of Internal Medicine, University of Torino; 3 Azienda Ospedaliero Universitaria (A.O.U.). Hematology, Policlinico di Careggi, Firenze; 4 Azienda Ospedaliero Universitaria Careggi, Department of Laboratory Medicine, Firenze; 5 Department of Biotechnology and Biosciences, University of Milano Bicocca, Milano; and 6 Department of Public Health, Firenze, Italy
Leukemia cell motility and transendothelial migration into extramedullary sites are regulated by angiogenic factors and are considered unfavorable prognostic factors in acute leukemias. We have studied cross talk among (1) the vascular endothelial growth factor receptor-1, FLT-1; (2) the human eag-related gene 1 (hERG1) K+ channels; and (3) integrin receptors in acute myeloid leukemia (AML) cells. FLT-1, hERG1, and the ß1 integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for FLT-1 signaling activation and AML cell migration. Both effects were inhibited when hERG1 channels were specifically blocked. A FLT-1/hERG1/ß1 complex was also observed in primary AML blasts, obtained from a population of human patients. The co-expression of FLT-1 and hERG1 conferred a pro-migratory phenotype to AML blasts. Such a phenotype was also observed in vivo. The hERG1-positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, hERG1 expression in leukemia patients correlated with a higher probability of relapse and shorter survival periods. We conclude that in AML, hERG1 channels mediate the FLT-1–dependent cell migration and invasion, and hence confer a greater malignancy.
The persistence of leukemia cells outside the bone marrow (BM) microenvironment and, in particular, their migration from the peripheral blood (PB) into extramedullary organs are considered unfavorable prognostic factors in acute leukemias.1–3 Leukemia cell migration into extramedullary sites may also reduce responsiveness to induction chemotherapy.1–3 Therefore, there is considerable interest in deciphering the molecular mechanisms that regulate leukemia cell motility and transendothelial migration, and, consequently, determining how leukemia cells exit from the BM and infiltrate extramedullary organs. A crucial role is thought to be played by angiogenic factors and angiogenesis-related signals, especially those centered on the vascular endothelial growth factor (VEGF) and its receptors (VEGFRs).4–6 Some lines of evidence suggest that VEGF/VEGFRs binding exerts an autocrine regulatory effect on the leukemic cell population, in addition to the paracrine effect of VEGF on the endothelium.6 Among VEGFRs, both VEGFR-1 (FLT-1) and VEGFR-2 (KDR) are tyrosine kinase receptors. KDR is expressed in endothelial cells. It transduces angiogenic signals5,6 and enhances acute myeloid leukemia (AML) cell survival.7,8 Little is known about the role of FLT-1 in acute leukemia progression. Recent studies suggest that it induces proliferation of AML cell lines and regulates the localization of immature malignant precursors within the BM in myelodysplastic syndromes.9 Studies of multiple myeloma reinforce the idea that FLT-1 regulates the migration of malignant hemopoietic cells.6,10 Fragoso et al11 have recently demonstrated that FLT-1 activation stimulates cell migration of acute lymphoblastic leukemia cells. In addition, FLT-1 neutralization in vivo with specific antibodies impairs leukemia cell exit from the BM and prolongs survival of injected mice.11
A peculiar aspect of VEGFR-mediated signaling is epitomized by their interaction with the integrin family of adhesion receptors.12–14 The For the purposes of the present article, it is important to bear in mind that hERG1 is found in a broad range of human leukemic cell lines and in primary human AML,25 as well as in chronic lymphocytic leukemias.26 Leukemia cells express a truncated form of hERG1 that, although lacking most of the N-terminus, produces functional hERG currents. This isoform is referred to as hERG1B to distinguish it from the full-length hERG1A.27 hERG1 activity is necessary for leukemia cells to progress beyond the G1/S boundary.25 Consistent with these results, the clonogenic potential of AML circulating blasts is reduced by hERG1-specific blockers.25 We sought to determine whether FLT-1, hERG1 channels, and integrins are associated in AML cells, and how this may regulate leukemia cell invasiveness. In addition, by inoculating AML blasts into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, we found that hERG1 channel activity regulates leukemia blast mobilization from BM spaces into extramedullary sites in vivo. Suggested clinical implications of this observation are also reported.
Cell culture and stimulation The human cell lines FLG 29.1, K562, NB4, KG1, HL60, and TF1 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (complete medium; Hyclone, Milan, Italy) at 37°C in 5% CO2. For signaling experiments, cells were deprived of serum for 12 to 14 hours, in RPMI medium, before stimulation with either VEGF165 (100 ng/mL), placental growth factor (PlGF; 50 ng/mL), or TS2/16 antibody (20 µg/mL), or diluted in RPMI medium and applied for 30 minutes at 37°C. Treatment with K+ channel inhibitors We tested the following K+ channel inhibitors: (1) Way 123 398 (Way) and E4031 (these compounds specifically block hERG1 channels when applied to cells maintained in standard extracellular saline solutions, at concentrations of approximately 1 µM22,28); (2) tetra-ethyl-ammonium, a wide-range inhibitor of voltage-dependent K+ channels that does not affect hERG1 at the concentration used (5 mM); and (3) charybdotoxin (C. toxin), an inhibitor of intermediate-conductance Ca2+-activated K+ channels and voltage-dependent Kv 1.3 channels. Way was a kind gift from Dr W. Spinelli (Wyeth-Ayerst Research, Princeton, NJ); E4031 and tetra-ethyl-ammonium were from Sigma (St. Louis, MO); C. toxin was from Alomone Labs (Jerusalem, Israel). All inhibitors were dissolved in phosphate-buffered saline, except C. toxin, which was dissolved in ethanol. Cells were preincubated with the various K+ channels inhibitors, in RPMI medium, for 15 minutes at 37°C, and then stimulated as needed. Migration assay Cell migration was measured by a Boyden chamber assay. The 2 compartments of the chamber (NeuroProbe, Gaithersburg, MD) were separated by a porous polycarbonate membrane (pore diameter 8 µm), previously coated with fibronectin (FN) (50 µg/mL) or bovine serum albumin (BSA; 250 µg/mL). Starved cells (106/mL) were added to the upper well, in the presence or absence of inhibitors, whereas VEGF165 (100 ng/mL) or PlGF (50 ng/mL) were added to the lower well. After 18 hours of incubation, migrated cells were collected from the lower compartment, spun down, and counted using a hemocytometer. Only live cells, as determined by Trypan blue exclusion, were considered in the quantification. Experiments were performed in triplicate, and results are shown as the number of migrating cells/mL. Silencing of Akt by small interfering RNAs FLG 29.1 cells were transiently transfected with purified duplex small interfering (si) RNAs for Akt and with a scrambled control purchased from Qiagen (Valencia, CA), using the Transmessenger kit (Qiagen). After 54 hours, cells were transferred into a Boyden chamber (for cell migration assay) or into 96-well plates for viability (WST) assay, and incubated for further 18 hours. A parallel sample (treated with siRNAs for a total of 72 hours) was processed for the evaluation of Akt expression. Cell viability assay To assess cell viability and proliferation, the cell viability assay (WST; Roche Diagnostics, Mennheim, Germany) was used. Cells were seeded at 105/well in 96-well plates, and incubated for 18 hours. At the end of incubation, the WST reagent was added, and absorbance was measured at 450 nm. VEGF secretion AML cells were seeded into 24-well cell clusters at 2 x 105 cells/mL in serum-free medium. After a 48-hour incubation, the medium was collected and used for VEGF measurement using the DuoSet ELISA Development System (R&D Systems, Wiesbaden, Germany). Cells were subsequently recovered and counted to normalize VEGF secretion data. Immunocytochemistry Immunocytochemistry (ICC) was performed as described.25 Images were acquired on a Leica DM 4000B microscope with a Leica DFC 320 photocamera (Leica Microsystems, Milan, Italy) (PL Fluotar 40x/0.70, PL Fluotar 100x/1.30 OIL objective). Preparation of AML blasts and normal CD34+ cells AML blasts and CD34+ cells were prepared from PB samples and stimulated as described.25 Molecular biologic methods RNA extraction and RT-PCR. Total RNA was extracted, and reverse-transcription-polymerase chain reaction (RT-PCR) for herg1 and gapdh was performed.25 For flt1 and kdr amplification, primers and conditions were as in Dias et al.7 In some samples only the amplification of herg1 and gapdh was performed, due to the amount of RNA extracted from the primary sample. RT Quantitative PCR. Herg1a and herg1b mRNA were quantified by RT quantitative PCR (RQ-PCR), with the ABI PRISM 7700 Sequence Detection System and the SYBR Green Master Mix Kit (Applied Biosystems, Foster City, CA). The ß-glucuronidase (GUS) gene was used as standard reference. The relative expression of herg1a and herg1b was calculated by using a comparative threshold cycle method. The primer sequences were as follows: herg1a sense, 5'-GTGGAAATCGCCTTCTACCG-3'; antisense, 5'-GCCCCATCCTCGTTCTTCA-3'; and herg1b sense, 5'-GCGCATCTCCAGCCTCGTG-3'; antisense, 5'-ACGTCGGCGCCCAGGGACA-3'. Patients PB samples were obtained from 61 patients diagnosed with AML. They represent a group of consecutive patients with high PB blast counts, treated either at the Hematology Unit in Florence or at the Department of Internal Medicine in Turin. In each case, diagnosis was based on morphologic, immunophenotypic, and molecular analysis. All samples were obtained with signed informed consent, in accordance with the Declaration of Helsinki, by using a protocol approved by the Comitato Etico Azienda Ospedaliera Universitaria di Careggi, Firenze, Italy. Patient details are summarized in Table 1. Patients were treated by standard induction chemotherapy with daunorubicin-aracytine. Daunorubicin was administered at 60 mg/m2 on days 1, 2, and 3, in association with aracytine as a continuous infusion for 7 days at 100 or 200 mg/m2, for patients older than or younger than 60 years of age, respectively. Complete remission and relapse were evaluated by standard hematologic parameters.
Additional information is provided in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article).
AML cells express hERG1 and FLT-1 As cellular models, we have used several human AML cell lines of different French-American-British (FAB) phenotypes (Figure 1) and K562 cells (ie, cells derived from the acute phase of a chronic myeloid leukemia). First, we tested the expression of herg1 and assayed hERG1 protein by RT-PCR and Western blot (WB), respectively. All the AML cell lines we examined expressed herg1 (Figure 1A) and contained the corresponding hERG1 protein (Figure 1B). In the WB lanes (Figure 1B), the bands of different molecular weights represent differently glycosylated forms of the hERG1A and hERG1B proteins. In particular, the hERG1A and hERG1B bands with the higher molecular weight represent proteins with the highest level of glycosylation, which is typical of the forms expressed on the plasma membrane.21,27 We conclude that hERG1A and hERG1B are both expressed in AML cell lines, albeit with a prevalence of the latter, and that both isoforms are properly expressed onto the plasma membrane.
We subsequently investigated the expression of transcripts for VEGFRs. All of our cell lines expressed flt1, but not kdr (Figure 1A). The proper translation of flt1 was confirmed by WB (Figure 1C, arrow). To verify that the FLT-1 receptor was functional, we assessed the level of tyrosine phosphorylation (p-Tyr) after VEGF165 treatment. Serum-starved cells were incubated with VEGF165 for 30 minutes, and FLT-1 was subsequently immunoprecipitated and probed with anti p-Tyr antibody. It turned out that VEGF165 always triggered the p-Tyr of FLT-1 (Figure 1D). A variable level of p-Tyr on the FLT-1 receptor was also observed in the absence of VEGF (see below), a finding that can be attributed to autocrine VEGF production by leukemia cells (see below). Nevertheless, p-Tyr was significantly increased by VEGF165 to levels similar to those previously reported in myeloma and chronic lymphocytic leukemia cells.32,33 These data are summarized in Table 2.
Formation of a FLT-1/hERG1/ß1 complex in AML cell lines To test whether FLT-1 associated with hERG1, we performed co-immunoprecipitation experiments on lysates from AML cell lines cultured in the presence of serum. After immunoprecipitating FLT-1, WB tests with an anti pan-hERG1 antibody always revealed co-immunoprecipitation with the ion channel, although the intensity of the effect was different in the different cell lines (Figure 2A). The molecular weight of the hERG1 bands observed after WB suggests that the complex with FLT-1 was predominantly formed by the hERG1B isoform. This was confirmed by treating the blot with a specific anti hERG1B antibody. Subsequent application of an anti ß1 integrin antibody revealed that ß1 also co-immunoprecipitates with FLT-1 and hERG1B. We concluded that FLT-1, hERG1B, and ß1 integrin co-immunoprecipitate in serum-cultured leukemia cells, thereby suggesting the formation of a macromolecular FLT-1/hERG1/ß1 membrane complex.
Effect of VEGF/PlGF and of integrin stimulation on the FLT-1/hERG1/ß1 complex We next studied whether the assembly of the multiprotein complex was affected by VEGF165 addition, concomitant or not with ß1 integrin activation. This point was first assessed in FLG 29.1, a leukemia cell line in which we had previously shown ß1/hERG1 association.34 Co-immunoprecipitation experiments were performed on serum-starved cells, after addition of either BSA (control) or VEGF165. After precipitating FLT-1, the blot was treated with an antibody specific for hERG1B. The hERG1B-specific bands were present on both controls and cells stimulated with VEGF165, and the signal was increased after stimulation with VEGF165 (Figure 2B, top panel). The same effect was observed when leukemia cells were treated with the specific FLT-1 ligand, PlGF6 (Figure 2B, right). The exclusive presence of hERG1B (compared with hERG1A) was confirmed by reprobing the membranes with anti-pan hERG1 antibody. Next, we tested the effects produced on FLT-1/hERG1 assembly by application of a ß1 integrin activating antibody (TS2/16), either in the absence or in the presence of VEGF165 (or BSA for control). As it turned out (Figure 2B, uppermost left panel), addition of TS2/16 scarcely altered the level of FLT-1/hERG1B association obtained after treatment with either BSA or VEGF165. Moreover, probing our blots with anti ß1 integrin antibody showed that ß1 integrin was not recruited to the complex by VEGF165 stimulation in the absence of TS2/16. On the contrary, TS2/16 addition induced the association of the integrin subunit in the FLT-1/hERG1 complex when VEGF165 was absent. Finally, simultaneous stimulation with VEGF165 and TS2/16 produced further increase of the level of integrin association with the FLT-1/hERG1 complex. Our results are better appreciated by observing the corresponding densitometric analyses (Figure 2B, graph). Similar results were found with the other AML cell lines (Figure 2C). VEGF165 addition in the presence of integrin stimulation always induced the formation of a FLT-1/hERG1/ß1 complex. It is particularly noteworthy that ß1 integrin was associated with the FLT-1/hERG1 complex even when cells were cultured in the presence of serum with no supplementary stimulation (Figure 2A). We assume this effect is caused by simultaneous stimulation of both VEGFRs and integrins by serum.13 Functional role of the FLT-1/hERG1/ß1 complex in leukemic cell lines To understand the physiologic importance of these results, we asked: (1) does FLT-1/hERG1/ß1 complex formation affect FLT-1 activation (ie, its p-Tyr); (2) what is the effect of altering hERG1 channel activity on downstream signaling; and (3) does this complex affect AML cell motility, based on the fact that VEGF165 binding to FLT-1 is known to stimulate migration of several normal and leukemic hemopoietic cells.7,32,35–39 Effect of protein complex formation on FLT-1 activation. FLT-1 p-Tyr was increased in FLG 29.1 cells by the addition of either VEGF165, PlGF or the ß1 activating antibody TS2/16, compared with controls (Figure 3A). Densitometric analysis showed that stimulation with VEGF plus TS2/16 increased FLT-1 p-Tyr by 3.7-fold (Figure 3A, graph). Integrin activation thus appears to be necessary for full activation of FLT-1 by VEGF.
Effect of hERG1 activity on FLT-1 activation and downstream signaling. In FLG 29.1 cells, the level of FLT-1 p-Tyr produced in the presence of VEGF165 plus TS2/16 was considerably decreased by inhibiting hERG1. The latter effect was obtained by using either Way or E4031 (Figure 3B). The inhibition of FLT-1 p-Tyr was more than 60% (Figure 3B, inset), whereas no effect was observed by treating the cells with other K+ channel blockers (tetra-ethyl-ammonium and C. toxin)40–44 (Figure 3B). The stimulation of FLT-1 p-Tyr by VEGF plus TS2/16 occurred in all of our AML cell lines (Figure 3C), and the effect was reduced by addition of 1 µM Way. The results obtained with NB4 and KG1 cells are reported in Figure 3C (right panel). Finally, we analyzed the effect of VEGF plus TS2/16 on intracellular signaling pathways switched on by FLT-1 activation.6,12 Figure 3D shows that VEGF plus TS2/16 stimulated phosphorylation of both the mitogen-activated protein kinases (MAPK) and of Akt, the kinase downstream to the phosphatidylinositol-3-kinase (PI3K). Application of 1 µM Way strongly decreased the effect. The levels of total extracellular-signal-related kinases (ERK) 1 and ERK2 and of Akt remained unchanged throughout the experiments (Figure 3D, bottom panels). Role of the FLT-1/hERG1/ß1 complex in leukemia cell migration. By using a Boyden chamber assay, we observed that VEGF165 increased the number of migrating FLG 29.1 cells, and the effect was potentiated by the concomitant stimulation of integrins (VEGF + FN; Figure 4A). The same occurred after addition of the specific FLT-1 ligand PlGF. This result stresses the specific role of the VEGFR-1 in leukemia cell migration. The cell migration induced by VEGF plus FN was inhibited by Way and E4031, but not by tetra-ethyl-ammonium and C. toxin. In this case, however, a full inhibition was only obtained with higher Way concentrations (40 µM), necessary to produce full and sustained channel block when cells are incubated in protein-containing media for prolonged times.28 Leukemia cell migration was also inhibited by applying an antibody against the FN receptor (anti-FN-R), which had been previously shown to block ß1-containing integrins.20 The effect was similar to that produced by 1 µM Way. When the anti-FN-R and 1 µM Way were applied together, their effects cooperated to almost completely block leukemia cell migration, an effect similar to the one exerted by 40 µM Way. We also tested which of the signaling pathways switched on by FLT-1 activation regulated leukemia cell migration. The PI3K inhibitor, LY, strongly reduced FLG 29.1 cell migration. An inhibitory effect was also obtained with Akt-specific small interfering RNAs (Akt-siRNAs). Such Akt-siRNAs decreased Akt expression of FLG 29.1 cells by 50%, in our experimental conditions (Figure 4A, inset). Neither LY nor Akt-siRNAs affected FLG 29.1 cell survival/proliferation, as measured by the WST assay (Figure 4B). However, leukemia cell survival/proliferation was strongly reduced by the MAPK inhibitor, PD.
With regard to the other AML lines, cell migration was stimulated by VEGF plus FN, with an average increment of more than 2-fold, compared with control (BSA). Once again, the effect was significantly reduced in the presence of 1 µM Way (Figure 4C). A positive correlation was found between the number of leukemia cells stimulated to migrate by VEGF plus FN (relative to BSA), and the density of the FLT-1/hERG1/ß1 complex assembled in the same conditions (Figure 4). Expression of hERG1 gene(s), VEGFRs, and FLT-1/hERG1/ß1 complex in primary leukemia blasts We hypothesized that the aforementioned mechanism might operate in primary AML blasts to induce a significant leukemia cell migration. To assess this possibility, we first determined the expression of herg1 and VEGFRs (flt-1 and kdr) transcripts in mononuclear cells obtained from the PB of 61 patients affected by de novo AML. For herg1 detection, we used primers capable of amplifying both the herg1a and the herg1b transcripts. A representative example of the RT-PCR results is reported in Figure 5A. Herg1 mRNA was expressed in the majority of the AML samples (69% of the cases, 42/61), irrespective of the FAB type. In some of the samples, a more quantitative estimate of herg1 transcripts was obtained by applying RQ-PCR, with primers specific for either herg1a or herg1b. Results are shown in Table 3, where data relative to some of the AML cell lines used throughout this study are also reported. The level of herg1a/b transcripts in PB mononuclear cells was taken as 1. When compared with PB mononuclear cells, 81% of our AML cases showed increased transcription of herg1a, and increased transcription of herg1b in 72% of cases. The samples examined by RQ-PCR and reported in Table 3 correspond to some of the samples shown in Figure 5A and analyzed by conventional RT-PCR. Their comparison shows concordance between the level of the transcripts and the intensity of the PCR bands.
Flt1 transcript expression was detected in 71% (38/55) of patients, whereas only 31% (15/47) expressed kdr. These results agree with those reported by others.7,11 Features of AML patients are summarized in Table 1. Statistical analysis showed a significant correlation between herg1 and flt1 gene expression in AML blast (P = .04; Fisher exact test). Such a correlation was also observed at the protein level, because both hERG1 and FLT-1 were detected by ICC in the same AML cases. A representative example relating to case A49 is reported in Figure 5B. Finally, we determined the amount of VEGF secreted by the AML blasts. The amounts of VEGF secreted by some of our AML cell lines are also reported (Table 3). VEGF secretion varied from 59 to 183 pg/mL in primary AML blasts. FLG 29.1 and HL60 stably transfected with the herg1 cDNA (hERG1-HL60) gave the highest secretion of VEGF. In the former, secretion was strongly inhibited by Way. On the whole, a significant correlation emerged between herg1 expression and the amount of secreted VEGF in both primary AML blasts and AML cell lines (Figure 5). Formation of an FLT-1/hERG1/ß1 complex in primary AML blasts and normal hematopoietic precursors We tested whether a FLT-1/hERG1/ß1 complex was formed in primary AML blasts of different FAB as well, by using the approach illustrated in Figure 2A. Here again, the hERG1B protein co-immunoprecipitated with FLT-1 (Figure 6A, top panel) and the complex included the ß1 integrin (Figure 6A, middle panel). The bottom panel of Figure 6A shows the results of reprobing the membrane with the same antibody used to obtain immunoprecipitation. Thus, the results we have obtained with AML blasts match those obtained in AML cell lines.
We had previously shown that when CD34+ collected from PB of normal subjects are stimulated with IL-3, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, herg1 is upregulated and cells enter into the cell cycle.25 Based on this information, we performed co-immunoprecipitation experiments on resting and stimulated normal peripheral CD34+ cells. A strong hERG1 band was observed only in the immunoprecipitate obtained from stimulated CD34+ cells (Figure 6B, top panel), and mostly contained the hERG1B isoform. We conclude that an FLT-1/hERG1/ß1 complex occurs in human hematopoietic immature cells, whether they are activated normal CD34+ or AML blasts. Role of hERG1 channels in leukemia cell migration in vitro As a final step, we investigated whether herg1 expression, in addition to flt-1, might stimulate leukemia blast migration, in vitro. We studied 6 AML samples. All expressed the flt-1 receptor, and 3 of them expressed herg1 as well. Migration was studied in the same conditions applied to AML cell lines (Figure 4A). Cell migration was significantly higher in herg1-positive (hERG1+) compared with herg1-negative (hERG1–) samples (Figure 6C). Interestingly, flt-1-negative leukemias were almost unable to migrate through FN, irrespective of herg1 expression (Figure 6C). Pretreatment with 1 µM Way significantly reduced cell migration of hERG1+, but not of hERG1–, samples. (Figure 6D). Altogether, our results indicate that in vitro, the cell migration mediated by FLT-1 depends on hERG1 channel activity. Overexpression of herg1 confers a higher invasiveness to leukemia cells in vivo To test whether the FLT-1/hERG1/ß1 complex regulates leukemia cell migration in vivo, we used a repopulation assay in NOD-SCID mice. We applied 2 different approaches: (1) inoculation of herg1– and herg1+ leukemia blasts from primary AML; and (2) inoculation of either HL60 or hERG1-HL60 cells that overexpress the herg1 gene (Table 3). The degree of BM engraftment and PB invasion was determined by measuring the amount of human CD45-positive (hCD45+) cells with flow cytometry (Figure 7). In addition, we determined (1) the level of angiogenesis, the state of normal hemopoiesis, and the presence of leukemia blasts in the BM; and (2) the presence and extent of leukemia blast invasion into extramedullary organs (Figure 8 and Figure 7). The BM engraftment was approximately the same irrespective of herg1 expression in the blasts from primary leukemias, as well as in HL60 and hERG1-HL60 (Figure 7). However, the efficiency of bloodstream invasion was significantly higher in herg1+ compared with herg1– blasts from primary AML, as well as in hERG1-HL60 compared with HL60 cells (Figure 7). This result was paralleled by a higher presence of immature cells in the PB of mice inoculated with either herg1+ AML blasts or hERG1-HL60 cells (Figure 7 insets). BM angiogenesis, in particular the total vascular area, was significantly higher in mice inoculated with either herg1+ blasts or hERG1-HL60 cells (Figure 8A). An increased density of undifferentiated leukemia cells and a concomitant decrease of endogenous hemopoiesis were detected in the BM of mice injected with either herg1+ blasts or hERG1-HL60 cells (Figure 8B). The hERG1-positive blasts were often found around new vessels in the BM of herg1+/hERG1-HL60-injected mice (Figure 8B inset). Finally, only mice injected with herg1+/hERG1-HL60 blasts displayed a substantial hepatic (Figure 8C) and splenic (Figure 8D) invasion. A quantitative estimation of these results is also given in Figure 7.
On the whole, the phenotype conferred to AML cells in vitro by herg1 overexpression (enhanced migration through FN after VEGF165 addition, caused by the assembly of a FLT-1/hERG1/ß1 complex) was also observed in vivo and appears to contribute to the exit of leukemia blasts into the bloodstream with subsequent invasion of extramedullary sites. Correlation between herg1 expression and clinical parameters and outcome To follow up these results, we analyzed the correlation between herg1 expression and clinical features and outcome of 42 of the AML patients enrolled in the present study that were undergoing stand | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
vß3 integrin associates with KDR in endothelial cells,
) and on FN-coated filters in the presence of VEGF165 (100 ng/mL), or PlGF (50 ng/mL). Cell migration was carried out at 37°C and 5% CO2 for 18 hours as detailed in "Patients, materials, and methods; Migration assay." Values are reported as number of migrated cells/mL and represent means of 4 experiments, each performed in triplicate. Results shown are means (± SEM). The following inhibitors were used, at the final concentrations reported in parentheses: the hERG1 specific blocker Way (1 and 40 µM); the specific hERG1 blocker E4031 (1µM); tetra-ethyl-ammonium (used at 5 mM, a concentration known to block voltage-dependent K+ channels other than hERG1); C. toxin, a blocker of Ca2+-dependent K+ channels (1 µM); the anti FN-R antibody, known to block all the ß1 containing integrins
) or not (
Evaluated with CD34 staining