Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation
- Alejandra B Ochoa-Hernández1, 2,
- Moisés Ramos-Solano3,
- Ivan D Meza-Canales3,
- Beatriz García-Castro3,
- Mónica A Rosales-Reynoso4,
- Judith A Rosales-Aviña3,
- Esperanza Barrera-Chairez5,
- Pablo C Ortíz-Lazareno3,
- Georgina Hernández-Flores3,
- Alejandro Bravo-Cuellar3,
- Luis F Jave-Suarez3,
- Patricio Barros-Núñez1 and
- Adriana Aguilar-Lemarroy3, 6Email author
© Ochoa-Hernández et al; BioMed Central Ltd. 2012
Received: 28 July 2011
Accepted: 7 February 2012
Published: 7 February 2012
WNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the WNT7A gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation.
We analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL), and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR) analysis were performed to determine relative WNT7A expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures.
WNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (p ≤0.001). By restoring WNT7A expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of WNT7A expression in Jurkat cells did not activate the canonical Wnt/β-catenin pathway.
To our knowledge, this is the first report evidencing quantitatively decreased WNT7A levels in leukemia-derived cells and that WNT7A restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible function of WNT7A as a tumor suppressor gene as well as a therapeutic tool.
KeywordsWNT7A Wnt signaling Leukemia Anti-proliferative Non-canonical pathway
The Wnt signaling pathway describes a complex network of proteins involved in differentiation, proliferation, migration, and cell polarity, which play important roles during embryonic development, tissue regeneration, and in homeostatic mechanisms [1, 2]. Wnt molecules are a highly conserved group of secreted cysteine-rich lipoglycoproteins that work as signaling molecules. Nineteen different Wnt family members have been described in humans to date. The binding of these ligands to its receptor complex (Frizzled/LRP-5/6) leads to activation of the pathway [1, 3]. Distinct sets of Wnt and Frizzled ligand-receptor pairs can activate different pathways and lead to unique cellular response [3, 4]. Wnt signals are transduced through at least three different intracellular pathways: Wnt/β-catenin, also known as canonical pathway; Wnt/Ca++, and the Planar cell polarity (Wnt/JNK) pathway [1, 3, 5]. In the canonical pathway, receptor activation leads to stabilization of β-catenin by inhibiting the phosphorylation activity of the Glycogen synthase kinase (GSK)-3β. Unphosphorylated β-catenin accumulates in the cytoplasm and then translocates into the nucleus, activating target gene expression through a complex network of co-receptors (TCF/LEF transcription factors) and repressors (Groucho) [6–8]. The Wnt/β-catenin pathway is involved in the self-renewal and proliferation of hematopoietic stem cells and has been also implicated in numerous types of cancers [7, 9, 10]. Dysregulation of this pathway is a hallmark of several types of tumors [7, 11–13].
Leukemic cells are highly heterogeneous, and their mechanisms of tumorigenesis are poorly understood. Recently, dysregulation of the Wnt signaling pathway has been implicated in the pathogenesis of some leukemia types [14–17]. Moreover, different expression profiles of some WNT genes and their related signaling molecules have been reported in hematological cancers [12, 18–22]. However, there are limited numbers of studies regarding the role of WNT7a, both in normal and in leukemia-derived cells [19, 23, 24].
Because our group has observed strongly decreased expression of WNT7A in different tumor-derived cell lines (unpublished data), we have focused our attention on the expression of WNT7A in normal peripheral blood cells, in leukemia-derived cell lines, and in patients with Acute lymphoblastic leukemia (ALL).
Fourteen peripheral blood samples from patients with leukemia were collected from the Hospital Civil Fray Antonio Alcalde and blood samples from healthy volunteers at the Instituto Mexicano del Seguro Social (IMSS) Blood Bank after approval by the Ethical and Research Committee No. 1305 of the Centro de Investigación Biomédica de Occidente (CIBO) - IMSS (project approval numbers 1305-2006-07 and 1305-2010-2). Written informed consent from patients and healthy volunteers (following local Ethics Committee guidelines and international norms) was also required prior to blood sample collection.
Cell line culture
The human leukemia-derived cell lines Jurkat, K562, CEM, HL60, and the BJAB cells (lymphoma-derived B cells) were used as study model. Jurkat and CEM possess a lymphoblastic phenotype, whereas K562 and HL60 have a myeloid origin. Cells were cultured in RPMI-1640 medium supplemented with 10% Fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere of 5% CO2. All products mentioned previously were obtained from the GIBCO™ Invitrogen Corporation.
Sorting of CD3- and CD19-positive cells
Peripheral blood mononuclear cells (PBMC) obtained from five healthy volunteers (12 mL of peripheral blood) were isolated by density-gradient centrifugation with Ficoll-Paque™ PLUS (GE Healthcare). PBMC included lymphocytes, monocytes, macrophages, NK cells, and also basophils and dendritic cells. These cells can be extracted from whole blood using Ficoll, which separates the blood into a top layer of plasma, followed by a layer of PBMC and a bottom fraction of polymorphonuclear cells and erythrocytes. The PBMC were resuspended in PBS and stained with an anti-CD3 antibody (sc-1179-FITC, Santa Cruz Biotechnology) to select T-lymphocytes and with an anti-CD19 antibody (sc-19650-PE, Santa Cruz Biotechnology) to select B-lymphocytes. After incubation with both antibodies, cells were washed and positive cells for CD3 or CD19 were sorted on a FACSAria (Becton Dickinson).
Leukemia-derived cell lines were seeded at a density of 5 × 106 in 75-cm3 flasks and harvested after 24 h for Total RNA extraction by using the PureLink™ Micro-to-Midi Total RNA Purification System (cat. no. 12183-018, Invitrogen) as described by the manufacturers. RNA from PBMC, CD3+ and CD19+cells was also extracted from five healthy volunteers by this method and put together to create a representative sample group of each cell type for the qRT-PCR analysis.
Gender and Age of Control and Patients
For cell lines, cDNA synthesis was performed from 5 μg of total RNA. For patients or healthy volunteers, we used the maximum volume of mRNA permitted in the kit (8 μL). cDNA was obtained by using the SuperScript™ III First-Strand Synthesis System primed with oligo(dT) (cat. no. 18080051, Invitrogen). The protocol was carried out as recommended by the manufacturers.
Primer design and qRT-PCR assays
Information of the oligonucleotides used for the qRT-PCR analysis
Sequence Accession Number
Primer Location (Exon
Wingless-type MMTV integration site family, member 7A
V-myc myelocytomatosis viral oncogene homolog (avian)
FOS-like antigen 1
Ribosomal protein L32
Ribosomal protein S18
Gene expression analysis was achieved by qRT-PCR on the 1.5 LightCycler® (Roche Diagnostics) using the LightCycler-FastStart DNA MasterPLUS SYBR Green I Kit (cat. no. 03515885001, Roche Applied Science) as recommended by the manufacturers. A standard curve with four serial dilution points and a negative control were included in each run. Relative expression was calculated with LightCycler software version 4.1 by taking GAPDH, RPL32, or RPS18 as reference genes.
Analysis in cell lines was performed by taking the values obtained from two independent RNA extractions in duplicate. In patients and healthy volunteers, experiments were performed three times in each individual sample.
In order to demonstrate that the reference genes selected and used in our cell model were appropriate, some samples were also tested with additional reference genes, as shown in Additional File 1.
Δ CP analysis
For analysis of WNT7A expression in patients and in healthy volunteers, we used ΔCP to facilitate analysis by taking only intrinsic references from each sample. We compared the ΔCP values obtained for both groups, i.e., the WNT7A CP minus the reference gene CP from the same sample. GAPDH and RPL32 were used for normalization in this analysis. It is very important to point out that ΔCP is inversely proportional to the expression of the target gene.
Isolation and culture of mononuclear cells
Peripheral blood mononuclear cells (PBMC) of healthy volunteers were isolated by density gradient using Ficoll-Paque PLUS (GE Healthcare, Sweden). Blood with anticoagulant was diluted 1:1 in PBS without MgCl2 and subsequently 1:1 in Ficoll-Paque PLUS. After 30-min centrifugation at 1,500 rpm, the cells' pellet was washed with PBS and resuspended in RPMI medium complemented with 10% FBS and antibiotics. Cells were led to growth at 37°C in an atmosphere of 5% CO2 in the presence or absence of phytohemagglutinin (PHA - 2 μg/mL).
Treatment with WNT7a recombinant protein
Jurkat and PBMC were seeded at a density of 2 × 104 cells in a 96-well microtiter plate in 200 μL of RPMI medium. Recombinant human WNT7a (cat. no. 3008-WN/CF, R&D Systems) was resuspended first in sterile PBS and was afterward diluted 1:100 in the cell culture medium. Every 24 h the recombinant protein was added fresh to each well at a final concentration of 3 μg/mL; incubations at 37°C were performed for 24 and 48 h.
Measurement of cell survival
After WNT7A overexpression or treatment with WNT7a recombinant protein, cell survival was determined by cleavage of tetrazolium salt WST-1 to formazan (cat. no. 11 644 807 001, Roche Applied Science) by reading the absorbance of treated and untreated cells at 440 nm on a microtiter plate reader (Synergy™ HT Multi-Mode Microplate Reader. Biotek. Winooski, VT, USA). The value of untreated cells was used as 100% cell survival.
WNT7A Open reading frame (ORF) (GeneID: 7476; NM _004625) was amplified from human non-tumorigenic keratinocytes utilizing the Expand High Fidelity PCR System (cat. no. 11 732 650 001, Roche Applied Science) with the following set of primers: forward 5'-GGG ACT ATG AAC CGG AAA GC -3'; reverse: 5'- CGG GGC TCA CTT GCA CGT GTA C -3'. Afterward, the PCR product was cloned into the pCR2.1TOPO vector (Invitrogen). Construction was sequenced employing M13 Forward and Reverse primers (Invitrogen) with the BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems). After corroborating the WNT7A sequence with that reported in GenBank, WNT7A ORF was isolated from pCR2.1TOPO vector by EcoRI restriction and subcloned into the EcoRI site of the lentiviral expression vector pLVX-Puro or pLVX-Tight-Puro (Clontech Laboratories, USA).
Lentivirus production and infection
To produce infectious viral particles, Lenti-X 293 T cells were transient-transfected by the Lentiphos HT/Lenti-X HT Packaging Systems with the lentiviral vectors pLVX-Puro or pLVX-WNT7A-Puro as described by the manufacturers (Clontech Laboratories. USA). Tet-Express Inducible Expression Systems were also used (pLVX-Tet-On Advanced and pLVX-Tight-WNT7A-Puro). After 48 h, supernatants were checked with Lenti-X GoStix (Clontech Laboratories. USA) to determine whether sufficient viral particles were produced before transducing target cells. Supernatants were filtered through a 0.45-μm PES filter to eliminate detached cells, were aliquoted, and subsequently stored at -80°C until use. Jurkat cells were transduced with 200 μL of supernatants obtained with pLVX-Puro or pLVX-WNT7A-Puro. RNA and protein extractions were obtained after 2 days of transduction and 1 week of puromycin selection (1 μg/mL). Cell proliferation was also measured by adding WST-1 to the culture cells at this point.
For inducible WNT7A expression in the leukemia-derived cell lines, cells were first transduced with the pLVX-Tet-On (regulator vector) and selected with G418 (cat. no. 631307, Clontech Laboratories, USA). Afterward, cells were transduced with the pLVX-Tight-WNT7A-Puro and selected with Puromycin for 2 weeks (1.5 μg/mL). After selection, cells were grown in the absence or presence of Doxycycline (Doxy) (750 ng/mL) to overexpress WNT7A.
Western blot assays
Cells were harvested by scraping and were lysed with RIPA buffer by sonication (15 pulses, 50% amp). Extracts were incubated for 30 min at 4°C and obtained by centrifugation (14,000 rpm for 5 min at 4°C). Protein concentrations were determined using the Bio-Rad DC Protein Kit (cat. no. 500-0114 Protein DC - BioRad, Hercules, CA, USA) and 50 μg of whole-cell extracts were electrophoresed in 12% SDS-PAGE. Proteins were then transferred onto a PVDF membrane (Millipore) and incubated with 1% Western blocking reagent (cat no. 11921681001, Roche, Germany) to block nonspecific binding. Primary antibodies were incubated over night at 4°C and the secondary antibodies were incubated with the membrane for 2 h at RT, followed by chemiluminescent detection using Immobilon Western substrate (Millipore Corporation, USA) with the ChemiDoc XRS (Biorad Laboratories, USA). The primary antibodies used were: anti-β-Catenin (cat. no. SC-7199, Santa Cruz Biotechnology, USA), anti-WNT7A (cat. no. AF3008 and cat. no. K-15, from R&D and Santa Cruz Biotechnology, respectively), or anti-β-Actin (cat. no. SC-26361, Santa Cruz, Biothechnology).
Cell death was measured by flow cytometry using propidium iodide (cat. no. P4864, Sigma-Aldrich) and Annexin-V-FLUOS (cat. no. 1828681, Roche Applied Science) as recommended by these manufacturers. Cells were seeded at a density of 2.5 × 105 cells per flask in 10 mL RPMI medium with or without Doxycycline (750 ng/mL). After a 72 h incubation, cells were washed with PBS and incubated with Annexin and propidium iodide for 15 min; 10,000 events from each sample were analyzed in a FACS Aria cytometer (BD Biosciences).
Statistical analysis was performed with SPSS Statistics software version 17.0. Post-hoc tests (Tukey HSD, Bonferroni, and Dunnett 3 T) were utilized for multiple comparisons between groups, and one-way ANOVA was employed to compare the means among more than two different groups. Only p values < 0.05 were considered as significant.
T-lymphocytes are the main cell population in peripheral blood that expresses WNT7A
WNT7Aexpression is downregulated upon T-lymphocytes activation
Reduced WNT7Agene expression levels in leukemia-derived cell lines
Because WNT7A was expressed in resting peripheral T-lymphocytes, but severely reduced in activated T-lymphocytes, we assumed that leukemia-derived cell lines (which are undifferentiated and have a high grade of proliferation) should express low levels of this ligand. To test this hypothesis, we selected five different leukemia-derived cell lines: two of lymphoid origin (Jurkat and CEM); two of myeloid origin (K562 and HL60), and a lymphoma-derived B cell line (BJAB). We included also myeloid immature cell lines to determine whether this myeloid lineage indeed (as previously calculated by the analysis obtained from peripheral blood volunteer's samples) expresses very low levels of WNT7A. Therefore, we determined the quantitative expression of WNT7A by qRT-PCR assays. As a reference for comparison, we also included in this analysis a mix of cDNA obtained from total peripheral blood cells of five clinically healthy volunteers (control group). As can be observed in Figure 2B, relative expression utilizing GAPDH, RPL32, and RPS18 as reference genes exhibited very low expression levels of WNT7A in all leukemia-derived cell lines when compared with the control group. We observed relative values ranging from 0.026 in Jurkat cells to 0.002 in K562 cells when normalized to the control group (set as 1). After qRT-PCR assays, all amplified products were resolved in 1.5% agarose gels and visualized with Ultraviolet (UV) light for photo-documentation (data not shown).
In conclusion, the experiments performed previously allow us to demonstrate quantitatively that leukemia-derived cell lines of both myeloid and lymphoid origins express very low levels of WNT7A.
Peripheral blood cells of patients with leukemia also showed a significant decrease in WNT7Agene expression
Taking the median of the ΔCP values of controls and patients with ALL obtained with both reference genes, we calculated the average of WNT7A relative expression. As can be observed in Figure 3B, the average of WNT7A expression in patients in comparison to the control group (set as 1) diminished to 0.043 (GAPDH) or to 0.135 (RPL32).
In summary, there is a statistically significant decreased expression of WNT7A, not only in leukemia-derived cell lines in comparison with the control group, but also in patients with ALL when compared with healthy volunteers. Peripheral blood cells from four Acute myeloid leukemias (AML), three Chronic myeloid leukemia (CML), and five Chronic lymphocytic leukemia (CLL) also revealed a tendency to exhibit reduced WNT7A expression when compared with the control group (data not shown).
Restoration of WNT7Ainhibits cell growth in Jurkat cells
Restoration of WNT7Adoes not induce the canonical β-catenin pathway in Jurkat cells
Restoration of WNT7Ainhibits cell growth in K562, BJAB, and CEM cells
Wnt signaling is conserved from invertebrates to vertebrates and regulates early embryonic development, as well as the homeostasis of adult tissues; as a central pathway in both physiological processes, dysregulation of Wnt signaling is associated with many human diseases, and particularly with cancer . Recently, Wnt signaling has also been implicated in hematopoiesis, both in self-renewal and in differentiation [1, 10, 18]. Based on these observations, it is hypothesized that dysregulation of the WNT pathway might contribute to the pathogenesis of lymphoproliferative diseases .
Despite the modest number of reports on the potential roles of Wnt signaling in leukemia, it is increasingly clear that Wnt signaling is dysregulated in several types of leukemia [12, 18, 27]. Some of these findings involve over- or underexpression of several Wnt ligands or Frizzled receptors [16, 19, 28, 29], hypermethylation of natural WNT inhibitors , and overexpression of β-catenin .
Despite this knowledge, there are a very limited number of publications on the expression of WNT7A and its role in the biology of blood cells. One of the first observations of the implication of WNT7A in hematological disorders was the frequent deletion of the 3p25 chromosome band observed in patients with AML, CML, and ALL . As is known, WNT7A is also localized at this chromosomal region [32, 33] and its deletion could be an important step during the neoplastic transformation.
In this paper, we report the expression of WNT7A in normal peripheral T-lymphocytes and strongly reduced WNT7A expression, not only in leukemia-derived cell lines, but also in the peripheral blood cells of patients with leukemia.
We were able to demonstrate that T-lymphocytes, but not B-lymphocytes, express WNT7A (ΔCP 11.47 ± 1.2). In agreement with this observation, Lu et al. also found expression of this ligand in peripheral blood lymphocytes (ΔCP 11.81 ± 0.99), but do not determined that this expression was afforded mainly from T-cells . In contrast, Sercan et al. found WNT7A expression in both T- and B-cells obtained from healthy volunteers . Discrepancies in these data could be due to the different method employed for quantification. The previously mentioned research group quantified WNT7A expression by comparing the densities of amplified WNT7A and β-actin PCR products visualized on agarose gels, while our group did this by performing qRT-PCR assays, which afford very precise data for quantification analysis.
We found from 38- to 500-fold lower expression in leukemia-derived cell lines than in healthy control cells (see Figure 2B). These results are in agreement as reported recently by Sercan et al., in which they did not find WNT7A expression in leukemia-derived cell lines K562, HL60, Jurkat, and Namalwa . However, the authors measured qualitatively, while we determined WNT7A expression quantitatively.
On the other hand, expression of WNT7A in hematological diseases has been only determined in patients with CLL and AML. Memarian et al. observed reduced expression of WNT7A in Iranian patients with AML compared with normal subjects ; however, the authors did not find this difference in patients with CLL . It is noteworthy that in both of these previously mentioned reports, WNT7A expression was calculated using the band densities of WNT7A and β-actin after conventional PCR. In agreement with the results of Memarian et al. we also observed reduced expression of WNT7A also in patients with AML, but statistical significance was not reached, probably due to the low number of patients with AML whom we analyzed (data not shown). Regarding expression of this ligand in patients with CLL; Lu et al. also observed lower WNT7A expression in patients with CLL (ΔCP 15.43 ± 2.94) when compared with healthy peripheral blood lymphocytes (11.81 ± 0.99). In this sense, we also observed this behavior in 4 out of 5 CLL patients (ΔCP 16.3 ± 1.5). Despite this low number of CLL patients, we found a statistical significance of p ≤0.02 when compared with healthy control cells (ΔCP 11.47 ± 1.2) (data not shown).
Interestingly, when we analyzed peripheral blood cells from 14 patients with ALL, these also expressed reduced WNT7A expression (ΔCP 15.19 ± 2.5) and we found a statistically significant difference of p ≤0.001 (GAPDH) and p ≤0.003 (RPL32) when compared with the control group (Figure 3).
Another important observation that we discerned is that WNT7A decreases acutely after PHA activation. To our knowledge, this is the first report evidencing that lymphocytes require reduction of their WNT7A levels in order to proliferate and suggests that dysregulation in the expression of this ligand needs to occur during oncogenesis to lose control of cell proliferation. Interestingly, it has been reported that T-cell activation by phytohemagglutinin results in a strong increase of phosphorylated GSK3β , which in turn targets beta-catenin for ubiquitylation and proteasomal degradation .
With respect to the reference genes used in the qRT-PCR assays, it is important to mention that there are no perfect reference genes for every treatment in every cell line. Thus, we used at least two reference genes in each assay and also evaluated some samples with a total of five reference genes (please see Additional Files 1 and 2). It has been determined that one of the reference genes that we used (RPS18) is useful as internal control for quantitative PCR in human lymphoblastoid cells, because constant levels of expression across the cell lines used were found following exposure to ionizing radiation as well as to PHA . However, it could be that some, but not all, of the changes in WNT7A expression may be caused by changes in reference-gene expression when cells were treated with PHA.
To our knowledge, no other papers relating WNT7A and leukemia have been published; however, reduced or absent expression of WNT7A has also been observed in lung cancer when compared with normal lung and mortal, short-term bronchial epithelial culture by qRT-PCR assay [36, 37].
Furthermore, it has been reported that WNT7a activates E-cadherin expression in lung cancer cells and that WNT7A loss may be important in lung cancer development or in progression due to its effects on E-cadherin, because E-cadherin in cancer has been associated with dedifferentiation, invasion, and metastasis . In addition to the role of WNT7A observed in leukemia and lung cancer, disruptions or alterations of the WNT7A gene have also been found in oral premalignant lesions  and in esophageal squamous cells .
We were also able to demonstrate, in the Jurkat leukemia-derived cell line, that restoration of WNT7A (by lentiviral overexpression or the addition of human recombinant protein) inhibits cell proliferation (Figure 5). Moreover, with the inducible-lentiviral overexpression system, we also confirmed this observation in K562, BJAB, and CEM cells after WNT7a expression; however, induction of cell death was not observed (Figure 8). Spinsanti et al. also reported an anti-proliferative action of WNT7a expression in undifferentiated PC12 cells . Additionally, recent studies have demonstrated that the combined expression of WNT7A and Frizzled 9 (Fzd9) in Non-small cell lung cancer (NSCLC) cell lines inhibits transformed growth by activating ERK5 and increasing PPARgamma activity, representing a novel tumor suppressor pathway in lung cancer [36, 42, 43]. However, the biological role of WNT7A action in cancer is controversial at present; some evidence supports its activity as an oncogene, but there is also evidence of its tumor suppressor action [36, 44, 45]. This dual role can be explained by the FZD proteins that bind WNT7a. It has been reported that the binding of WTN7a and FZD5 induces the canonical pathway, which has been related with cancer development [46, 47]. On the other hand, WTN7a can also bind FZD-10 and -9, which in turn activated the c-Jun NH2-terminal kinase pathway (JNK). Activation of JNK has been shown to antagonize the canonical pathway .
Due to the reported dual behavior of WNT7A as a cell-proliferation inducer or blocker, it is reasonable to think that Jurkat cells preferentially express anti-proliferative FZD partners of WNT7A. To address this question, we analyzed the presence of FZD mRNAs in Jurkat cells compared with T-lymphocytes from healthy controls and found overexpression of FZD-3 and -6 and downmodulation of FZD-5 and -10 in Jurkat cells (data not shown). On analyzing hematopoietic cells and leukemia-derived cells, Sercan et al. also found expression of FZD-3 and -6 in leukemia-derived T-lymphocytes . The presence of FZD-6 in lymphocytes is interesting, because it has been shown that FZD-6 can act as a negative regulator of the canonical pathway . Whether FZD-6 can interact with WTN7a in lymphocytes and what the biological consequences of this interaction would be are questions that remain open.
It has been observed that increased expression of some WNT ligands such as WNT3a, induces activation of the canonical pathway, accompanied by an increase in the proliferation and survival of leukemia cells . In addition, it has been reported that β-catenin comprises an integral part of AML cell proliferation and cell cycle progression . Because we observed downmodulation of WNT7A in leukemia-derived cells, it appears that WNT7a in Jurkat cells does not activate the WNT/β-catenin pathway. Evidence that supports this notion is the finding that mRNA levels of the putative canonical target genes AXIN2, MYC, JUN, and FRA-1 were not increased after WNT7a restoration (see Figure 6). In contrast, mRNA from JUN and FRA-1 were strongly downregulated in WNT7A-expressing cells, but again restored (FRA-1) or even upregulated (JUN) when cells were treated with LiCl (Figures 6A & 6B). Concerning this point, it has been reported that the β-catenin - T cell-factor/lymphoid-enhancer-factor complex directly interacts with the promoter region of JUN and FRA-1 . Because we observed restoration of the expression levels of JUN and FRA-1 after LiCl treatment, it is very probable that LiCl antagonize WNT7a activity in these cells. An additional observation that supports the idea that WNT7a is working in this model in a non-canonical pathway is that expression of β-catenin was not increased after WNT7a restoration (as seen in Figure 6C).
In conclusion, we are demonstrating, by qRT-PCR analysis, that WNT7A is significantly reduced in leukemia-derived cell lines as well as in patients with leukemia when compared with clinically healthy volunteers. The finding that WNT7A restoration inhibits proliferation of leukemia-derived Jurkat cells, but not of PBMC, allows us to assume that WNT7A can be acting as a modulator of cell proliferation, especially in T-cells that are producing this protein. In this regard, impairment of this anti-proliferative function could be an important event in leukemia and in some cancer-cell types. If this is true, therapeutic tools directed toward restoring WNT7A expression in patients with leukemia might increase the probabilities of their overcoming this disease.
We thank our technicians María de Jesús Delgado and Leticia Ramos for their efficient support in the laboratory. We are grateful with Maggie Brunner and José David Ramos Solano for proofreading of the manuscript. ABOH is grateful for the scholarship obtained from CONACYT-Mexico. This work was supported by grants CONACYT-CB-2008-01-105705 and FIS/IMSS/PROT/G10/817 (both to AAL).
- Chen X, Yang J, Evans PM, Liu C: Wnt signaling: the good and the bad. Acta Biochim Biophys Sin (Shanghai). 2008, 40: 577-594. 10.1111/j.1745-7270.2008.00440.x.View ArticleGoogle Scholar
- Nusse R, Fuerer C, Ching W, Harnish K, Logan C, Zeng A, et al: Wnt signaling and stem cell control. Cold Spring Harb Symp Quant Biol. 2008, 73: 59-66. 10.1101/sqb.2008.73.035.View ArticlePubMedGoogle Scholar
- Gordon MD, Nusse R: Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem. 2006, 281: 22429-22433. 10.1074/jbc.R600015200.View ArticlePubMedGoogle Scholar
- Miller JR: The Wnts. Genome Biol. 2002, 3: REVIEWS3001-PubMedGoogle Scholar
- van AR, Mikels A, Nusse R: Alternative wnt signaling is initiated by distinct receptors. Sci Signal. 2008, 1: re9-10.1126/scisignal.135re9.Google Scholar
- Willert K, Jones KA: Wnt signaling: is the party in the nucleus?. Genes Dev. 2006, 20: 1394-1404. 10.1101/gad.1424006.View ArticlePubMedGoogle Scholar
- Fuerer C, Nusse R, ten BD: Wnt signalling in development and disease. Max Delbruck Center for Molecular Medicine meeting on Wnt signaling in Development and Disease. EMBO Rep. 2008, 9: 134-138. 10.1038/sj.embor.7401159.View ArticlePubMedPubMed CentralGoogle Scholar
- MacDonald BT, Tamai K, He X: Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009, 17: 9-26. 10.1016/j.devcel.2009.06.016.View ArticlePubMedPubMed CentralGoogle Scholar
- Duncan AW, Rattis FM, DiMascio LN, Congdon KL, Pazianos G, Zhao C, et al: Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nat Immunol. 2005, 6: 314-322. 10.1038/ni1164.View ArticlePubMedGoogle Scholar
- Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K, et al: A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature. 2003, 423: 409-414. 10.1038/nature01593.View ArticlePubMedGoogle Scholar
- Logan CY, Nusse R: The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004, 20: 781-810. 10.1146/annurev.cellbio.20.010403.113126.View ArticlePubMedGoogle Scholar
- Groen RW, Oud ME, Schilder-Tol EJ, Overdijk MB: ten BD, Nusse R et a.: Illegitimate WNT pathway activation by beta-catenin mutation or autocrine stimulation in T-cell malignancies. Cancer Res. 2008, 68: 6969-6977. 10.1158/0008-5472.CAN-08-1322.View ArticlePubMedGoogle Scholar
- Pongracz JE, Stockley RA: Wnt signalling in lung development and diseases. Respir Res. 2006, 7: 15-10.1186/1465-9921-7-15.View ArticlePubMedPubMed CentralGoogle Scholar
- Majeti R, Becker MW, Tian Q, Lee TL, Yan X, Liu R, et al: Dysregulated gene expression networks in human acute myelogenous leukemia stem cells. Proc Natl Acad Sci USA. 2009, 106: 3396-3401. 10.1073/pnas.0900089106.View ArticlePubMedPubMed CentralGoogle Scholar
- Valencia A, Roman-Gomez J, Cervera J, Such E, Barragan E, Bolufer P, et al: Wnt signaling pathway is epigenetically regulated by methylation of Wnt antagonists in acute myeloid leukemia. Leukemia. 2009, 23: 1658-1666. 10.1038/leu.2009.86.View ArticlePubMedGoogle Scholar
- Nygren MK, Dosen G, Hystad ME, Stubberud H, Funderud S, Rian E: Wnt3A activates canonical Wnt signalling in acute lymphoblastic leukaemia (ALL) cells and inhibits the proliferation of B-ALL cell lines. Br J Haematol. 2007, 136: 400-413. 10.1111/j.1365-2141.2006.06442.x.View ArticlePubMedGoogle Scholar
- Wu QL, Zierold C, Ranheim EA: Dysregulation of Frizzled 6 is a critical component of B-cell leukemogenesis in a mouse model of chronic lymphocytic leukemia. Blood. 2009, 113: 3031-3039. 10.1182/blood-2008-06-163303.View ArticlePubMedPubMed CentralGoogle Scholar
- Khan NI, Bendall LJ: Role of WNT signaling in normal and malignant hematopoiesis. Histol Histopathol. 2006, 21: 761-774.PubMedGoogle Scholar
- Lu D, Zhao Y, Tawatao R, Cottam HB, Sen M, Leoni LM, et al: Activation of the Wnt signaling pathway in chronic lymphocytic leukemia. Proc Natl Acad Sci USA. 2004, 101: 3118-3123. 10.1073/pnas.0308648100.View ArticlePubMedPubMed CentralGoogle Scholar
- Tickenbrock L, Hehn S, Sargin B, Choudhary C, Baumer N, Buerger H, et al: Activation of Wnt signalling in acute myeloid leukemia by induction of Frizzled-4. Int J Oncol. 2008, 33: 1215-1221.PubMedGoogle Scholar
- Mai YJ, Qiu LG, Li ZJ, Yu Z, Li CH, Wang YF, et al: The expression of beta-catenin and its significance in leukemia cells. Zhonghua Xue Ye Xue Za Zhi. 2007, 28: 541-544.PubMedGoogle Scholar
- Ge X, Wang X: Role of Wnt canonical pathway in hematological malignancies. J Hematol Oncol. 2010, 3: 33-10.1186/1756-8722-3-33.View ArticlePubMedPubMed CentralGoogle Scholar
- Sercan Z, Pehlivan M, Sercan HO: Expression profile of WNT, FZD and sFRP genes in human hematopoietic cells. Leuk Res. 2010, 34: 946-949. 10.1016/j.leukres.2010.02.009.View ArticlePubMedGoogle Scholar
- Memarian A, Jeddi TM, Vossough P, Sharifian RA, Rabbani H, Shokri F: Expression profile of Wnt molecules in leukemic cells from Iranian patients with acute myeloblastic leukemia. Iran J Immunol. 2007, 4: 145-154.PubMedGoogle Scholar
- Rao AS, Kremenevskaja N, Resch J, Brabant G: Lithium stimulates proliferation in cultured thyrocytes by activating Wnt/beta-catenin signalling. Eur J Endocrinol. 2005, 153: 929-938. 10.1530/eje.1.02038.View ArticlePubMedGoogle Scholar
- Silva AK, Yi H, Hayes SH, Seigel GM, Hackam AS: Lithium chloride regulates the proliferation of stem-like cells in retinoblastoma cell lines: a potential role for the canonical Wnt signaling pathway. Mol Vis. 2010, 16: 36-45.PubMedPubMed CentralGoogle Scholar
- Weerkamp F, van Dongen JJ, Staal FJ: Notch and Wnt signaling in T-lymphocyte development and acute lymphoblastic leukemia. Leukemia. 2006, 20: 1197-1205. 10.1038/sj.leu.2404255.View ArticlePubMedGoogle Scholar
- Martin V, Valencia A, Agirre X, Cervera J, Jose-Eneriz ES, Vilas-Zornoza A, et al: Epigenetic regulation of the non-canonical Wnt pathway in acute myeloid leukemia. Cancer Sci. 2010, 101: 425-432. 10.1111/j.1349-7006.2009.01413.x.View ArticlePubMedGoogle Scholar
- Memarian A, Hojjat-Farsangi M, Asgarian-Omran H, Younesi V, Jeddi-Tehrani M, Sharifian RA, et al: Variation in WNT genes expression in different subtypes of chronic lymphocytic leukemia. Leuk Lymphoma. 2009, 50: 2061-2070. 10.3109/10428190903331082.View ArticlePubMedGoogle Scholar
- Roman-Gomez J, Cordeu L, Agirre X, Jimenez-Velasco A, San Jose-Eneriz E, Garate L, et al: Epigenetic regulation of Wnt-signaling pathway in acute lymphoblastic leukemia. Blood. 2007, 109: 3462-3469. 10.1182/blood-2006-09-047043.View ArticlePubMedGoogle Scholar
- Johansson B, Billstrom R, Kristoffersson U, Akerman M, Garwicz S, Ahlgren T, et al: Deletion of chromosome arm 3p in hematologic malignancies. Leukemia. 1997, 11: 1207-1213. 10.1038/sj.leu.2400718.View ArticlePubMedGoogle Scholar
- Bui TD, Lako M, Lejeune S, Curtis AR, Strachan T, Lindsay S, et al: Isolation of a full-length human WNT7A gene implicated in limb development and cell transformation, and mapping to chromosome 3p25. Gene. 1997, 189: 25-29. 10.1016/S0378-1119(96)00808-6.View ArticlePubMedGoogle Scholar
- Ikegawa S, Kumano Y, Okui K, Fujiwara T, Takahashi E, Nakamura Y: Isolation, characterization and chromosomal assignment of the human WNT7A gene. Cytogenet Cell Genet. 1996, 74: 149-152. 10.1159/000134404.View ArticlePubMedGoogle Scholar
- Lendeckel U, Scholz B, Arndt M, Frank K, Spiess A, Chen H, et al: Inhibition of alanyl-aminopeptidase suppresses the activation-dependent induction of glycogen synthase kinase-3beta (GSK-3beta) in human T cells. Biochem Biophys Res Commun. 2000, 273: 62-65. 10.1006/bbrc.2000.2883.View ArticlePubMedGoogle Scholar
- Metcalfe C, Bienz M: Inhibition of GSK3 by Wnt signalling - two contrasting models. J Cell Sci. 2011, 124: 3537-3544. 10.1242/jcs.091991.View ArticlePubMedGoogle Scholar
- Winn RA, Marek L, Han SY, Rodriguez K, Rodriguez N, Hammond M, et al: Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiation. J Biol Chem. 2005, 280: 19625-19634. 10.1074/jbc.M409392200.View ArticlePubMedGoogle Scholar
- Calvo R, West J, Franklin W, Erickson P, Bemis L, Li E, et al: Altered HOX and WNT7A expression in human lung cancer. Proc Natl Acad Sci USA. 2000, 97: 12776-12781. 10.1073/pnas.97.23.12776.View ArticlePubMedPubMed CentralGoogle Scholar
- Ohira T, Gemmill RM, Ferguson K, Kusy S, Roche J, Brambilla E, et al: WNT7a induces E-cadherin in lung cancer cells. Proc Natl Acad Sci USA. 2003, 100: 10429-10434. 10.1073/pnas.1734137100.View ArticlePubMedPubMed CentralGoogle Scholar
- Tsui IF, Rosin MP, Zhang L, Ng RT, Lam WL: Multiple aberrations of chromosome 3p detected in oral premalignant lesions. Cancer Prev Res (Phila Pa). 2008, 1: 424-429. 10.1158/1940-6207.CAPR-08-0123.View ArticleGoogle Scholar
- Chattopadhyay I, Singh A, Phukan R, Purkayastha J, Kataki A, Mahanta J, et al: Genome-wide analysis of chromosomal alterations in patients with esophageal squamous cell carcinoma exposed to tobacco and betel quid from high-risk area in India. Mutat Res. 2010, 696: 130-138.View ArticlePubMedGoogle Scholar
- Spinsanti P, De VT, Caruso A, Melchiorri D, Misasi R, Caricasole A, et al: Differential activation of the calcium/protein kinase C and the canonical beta-catenin pathway by Wnt1 and Wnt7a produces opposite effects on cell proliferation in PC12 cells. J Neurochem. 2008, 104: 1588-1598. 10.1111/j.1471-4159.2007.05111.x.View ArticlePubMedGoogle Scholar
- Winn RA, Van SM, Hammond M, Rodriguez K, Crossno JT, Heasley LE, et al: Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferator-activated receptor gamma. J Biol Chem. 2006, 281: 26943-26950. 10.1074/jbc.M604145200.View ArticlePubMedGoogle Scholar
- Tennis MA, Van Scoyk MM, Freeman SV, Vandervest KM, Nemenoff RA, Winn RA: Sprouty-4 inhibits transformed cell growth, migration and invasion, and epithelial-mesenchymal transition, and is regulated by Wnt7A through PPARgamma in non-small cell lung cancer. Mol Cancer Res. 2010, 8: 833-843. 10.1158/1541-7786.MCR-09-0400.View ArticlePubMedPubMed CentralGoogle Scholar
- Lindberg D, Akerstrom G, Westin G: Mutational analyses of WNT7A and HDAC11 as candidate tumour suppressor genes in sporadic malignant pancreatic endocrine tumours. Clin Endocrinol (Oxf). 2007, 66: 110-114.Google Scholar
- Kirikoshi H, Katoh M: Expression of WNT7A in human normal tissues and cancer, and regulation of WNT7A and WNT7B in human cancer. Int J Oncol. 2002, 21: 895-900.PubMedGoogle Scholar
- Caricasole A, Ferraro T, Iacovelli L, Barletta E, Caruso A, Melchiorri D, et al: Functional characterization of WNT7A signaling in PC12 cells: interaction with A FZD5 x LRP6 receptor complex and modulation by Dickkopf proteins. J Biol Chem. 2003, 278: 37024-37031. 10.1074/jbc.M300191200.View ArticlePubMedGoogle Scholar
- Carmon KS, Loose DS: Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells. Mol Cancer Res. 2008, 6: 1017-1028. 10.1158/1541-7786.MCR-08-0039.View ArticlePubMedGoogle Scholar
- Golan T, Yaniv A, Bafico A, Liu G, Gazit A: The human Frizzled 6 (HFz6) acts as a negative regulator of the canonical Wnt. beta-catenin signaling cascade. J Biol Chem. 2004, 279: 14879-14888. 10.1074/jbc.M306421200.View ArticlePubMedGoogle Scholar
- Khan NI, Bradstock KF, Bendall LJ: Activation of Wnt/beta-catenin pathway mediates growth and survival in B-cell progenitor acute lymphoblastic leukaemia. Br J Haematol. 2007, 138: 338-348. 10.1111/j.1365-2141.2007.06667.x.View ArticlePubMedGoogle Scholar
- Siapati EK, Papadaki M, Kozaou Z, Rouka E, Michali E, Savvidou I, et al: Proliferation and bone marrow engraftment of AML blasts is dependent on beta-catenin signalling. Br J Haematol. 2011, 152: 164-174. 10.1111/j.1365-2141.2010.08471.x.View ArticlePubMedGoogle Scholar
- Mann B, Gelos M, Siedow A, Hanski ML, Gratchev A, Ilyas M, et al: Target genes of beta-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas. Proc Natl Acad Sci USA. 1999, 96: 1603-1608. 10.1073/pnas.96.4.1603.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/60/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.