Restored expression of vitamin D receptor and sensitivity to 1,25-dihydroxyvitamin D3 in response to disrupted fusion FOP2–FGFR1 gene in acute myeloid leukemia cells
© Marchwicka et al. 2016
Received: 25 July 2015
Accepted: 25 January 2016
Published: 2 February 2016
Acute myeloid leukemia (AML) cells can be induced to undergo terminal differentiation with subsequent loss of tumorigenicity using 1,25-dihydroxyvitamin D3 (1,25D) alone or in combination with hematopoietic cytokines. KG1 cells are resistant to 1,25D-induced cell differentiation. These cells have the aberrant signal transduction resulting from a constitutively active fusion protein FOP2-FGFR1, a constitutively active STAT1 and a high level of interferon (IFN) stimulated genes (ISGs).
In this paper we report that in KG1 cells with constitutively activated protein FOP2-FGFR1 delivery of plasmid DNA disrupted FOP2-FGFR1 fusion gene.
As a consequence, STAT1 signal transduction pathway became switched off, the expression of vitamin D receptor (VDR) gene was increased and sensitivity to 1,25D-induced differentiation was restored. The activation of ISGs in KG1 cells resulted in resistance to externally added IFNs, and also this effect was reversed in cells with disrupted FOP2-FGFR1 fusion gene.
In this paper we have documented for the first time a link between constitutively active STAT1 signal transduction pathway, high level of ISGs and low expression of VDR gene.
We show in this paper that delivery of plasmid DNA to the cells may disrupt fusion gene FOP2-FGFR1 which occurs in a disease entity called 8p11 myeloproliferative syndrome. Inhibition of the FOP2-FGFR1 signal transduction pathway restored sensitivity of the cells to 1,25D-induced cell differentiation.
KeywordsDNA delivery Leukemia Differentiation Vitamin D receptor Fusion protein Transcription factor Interferon signaling
Chromosomal translocations are characteristic features of lymphoma and leukemia. A number of malignancies are driven by chromosomal translocations which involve the gene for fibroblast growth factor receptor 1 (FGFR1) and fuse it to the distant aminoterminal partners. In blood cells, these translocations are associated with the disease entity called 8p11 myeloproliferative syndrome, which rapidly transforms to acute myeloid leukemia (AML) . The only available cell line model for this disease is the KG1 cell line, where FGFR1 oncogene partner 2 (FOP2)–FGFR1 fusion gene was identified, which results in the generation of a constitutively active fusion protein FOP2–FGFR1 . KG1 cells have been characterized by a constitutive activation of signal transducer and activator of transcription (STAT) 5  and STAT1 . Under physiological conditions interferons (IFNs) activate STAT signal transduction pathways, leading to transcription of IFN-stimulated genes (ISGs) . This is the basic immune mechanism which controls the spread of viral infections. OAS proteins which activate degradation of viral RNA by 2′,5′-oligoadenylate-dependent ribonuclease L (RNAse L) are among ISGs [5, 6]. Other ISGs include the one that encodes protein MX1, which inhibits the replication cycle of influenza virus . G1P2 encodes a ubiquitin-like protein which binds to target proteins in response to IFNα or IFNβ stimulation and has chemotactic activity of neutrophils , while IFIT1 gene encodes a protein which may inhibit viral replication and translational initiation .
AML is characterized by the accumulation of primitive hematopoietic blast cells, which lose their ability of normal differentiation . AML cells can be induced to undergo terminal differentiation with subsequent loss of tumorigenicity. However, at present the clinical success of differentiation therapy for AML is limited to one rare subtype, which can be cured using all-trans retinoic acid (ATRA) . There is a need to develop differentiation therapies to other subtypes, for example, using 1,25-dihydroxyvitamin D3 (1,25D) alone or in combination with hematopoietic cytokines or phytonutrients . KG1 cells have been reported to be resistant to 1,25D-induced differentiation , and our earlier experiments revealed that this was caused by a very low expression level of vitamin D receptor (VDR) gene and protein . There are hundreds of VDR-controlled genes, many of them responsible for maintaining the calcium-phosphate homeostasis , however, there are also many involved in blood cell functions, exemplified by CD14, a macrophage co-receptor for bacterial LPS . VDR is not essential for blood cells development, but is important for their proper function [17, 18], thus low VDR level and low VDR activity in leukemic cells may contribute to their malignant phenotype.
In this study we have addressed the possible reasons of KG1 cells’ resistance to 1,25D-induced differentiation. In our search for the role of interactions between various nuclear receptors, we wanted to generate genetically modified KG1 subline with retinoic acid receptor α (RARA) gene silenced. Using electroporation DNA delivery method we have obtained two sublines: KG1-CtrA (transfected with a plasmid containing scrambled DNA sequence) and KG1-RARA (transfected with the plasmid coding short hairpin (sh) RNA against RARA gene). In both transfected cell lines VDR gene and protein expression levels increased and 1,25D-resistance was reversed, however this was not due to the gene silencing. We have therefore addressed the molecular events that have led to the reversal of 1,25D resistance. We found that the high level of FOP2–FGFR1 and ISGs transcription, constitutively present in KG1 cells, were suppressed in KG1-CtrA and KG1-RARA cells. Similarly, constitutive activity of STAT1 in KG1 cells, was not longer present in transfected cells. In contrast, in KG1-CtrA and KG1-RARA cells the expression and activity of VDR were much higher than in KG1 cells. The high activation of ISGs in KG1 cells resulted in resistance to externally added IFNs, and also this effect was reversed in transfected cells. The low level of VDR expression in KG1 cells wasn’t caused by the repressed transcription, but at least in part by degradation of VDR mRNA. Addition of curcumin, an inhibitor of RNAse L, to KG1 cells partly restored 1,25D-induced cell differentiation.
Differentiation of KG1, HL60, KG1-CtrA and KG1-RARA
In order to validate whether the expression of RARA gene was indeed efficiently knocked down in KG1-RARA cells, the RARα mRNA (Fig. 1c) and protein levels (Fig. 1d) were compared in KG1-CtrA and KG1-RARA cells. The mRNA expression was reduced to approximately 40 % of initial level, and was followed by reduced RARα protein content in the nuclei of KG1-RARA cells.
The plasmids that were used in our experiments confer the resistance to puromycin, an antibiotic which is toxic to eukaryotic cells. Transfected KG1 cells were selected from untransfected in the culture using this antibiotic. Since puromycin inhibits protein translation, it seemed unlikely that the effect of 1,25D-induced differentiation was caused by the exposure of the cells to puromycin. However, in order to verify that, we cultured KG1 cells at sub-lethal concentrations of puromycin (250 nM) and exposed them to 10 nM 1,25D and we detected that KG1 still did not differentiate (not shown). These experiments confirmed that cell differentiation of KG1-CtrA and KG1-RARA cells wasn’t caused by puromycin.
VDR in HL60, KG1, KG1-CtrA and KG1-RARA
VDR mRNA in HL60, KG1 and KG1-CtrA and its regulation in response to ATRA
Chromatin accessibility assay in HL60, KG1 and KG1-CtrA
FGFR1-FOP signaling in KG1, KG1-CtrA and KG1-RARA cells
As it has been presented in the past, there is a constitutive activation of STAT1 transcription factor in KG1 cells . In our experiments we tested, if this transcription factor is also constitutively active in KG1-CtrA and KG1-RARA cells. We tested the presence of Tyr-701 phospho-STAT1 in these cells, relative to the total amount of STAT1 and to the actin content. The results presented in Fig. 5c document constitutive activation of STAT1 in KG1 cells, but neither in KG1-CtrA nor in KG1-RARA cells. However, not only the levels of phosphorylated STAT1 were higher in wild type, than in transfected cells. The total amount of STAT1 was also higher in KG1 than in both transfected sublines, when compared to the actin content.
IFN stimulated genes in HL60, KG1, KG1-CtrA and KG1-RARA cells
Differentiation of KG1, KG1-CtrA and KG1-RARA cells after externally added IFNs
Differentiation of KG1 after externally added curcumin
Delivery of DNA into cancer cells in vitro and in vivo has become a standard protocol worldwide. It has been believed that delivery of control plasmid DNA has no significant effect to the cells, however some data were published which show that this is not always the truth. It has been shown that in some tumor cell lines delivery of control plasmid DNA caused significant increase in the expression of following ISGs: IRF7, STAT1, MIG, MICA and ITGAL . The phenomenon of activation of IFN signaling in transfected cells was attributed predominantly to delivery of short interfering (si) RNAs to the cells . In this paper we show that plasmid DNA delivered to acute myeloid leukemia cells may integrate into genomic DNA and disrupt FOP2–FGFR1 fusion gene. Our results show that this “side effect” of DNA delivery might have positive influence towards cell phenotype.
The above phenomenon has encouraged us to address the question of low VDR expression level in KG1 cells. Disruption of FOP2–FGFR1 fusion gene unexpectedly restored sensitivity of KG1 cells to 1,25D. This was caused by increased levels of VDR mRNA, followed by translation of VDR protein. VDR protein in genetically modified KG1-CtrA and KG1-RARA cells was transcriptionally functional and these cells resembled 1,25D-sensitive HL60 cells. In transfected KG1 sublines 1,25D induced expression of CD14 cell surface marker, which is a characteristic feature of mature monocytes and necessary for phagocytosis . Moreover, the regulation of VDR expression by ATRA, which in KG1 cells is opposite from that in HL60 cells, showed to be identical in both HL60, KG1-CtrA and KG1-RARA cells. We then hypothesized that the transcription of VDR gene was silenced in KG1 cells in comparison with KG1-CtrA or HL60 cells. Since the epigenetic mechanisms of gene silencing are variable, we took advantage of EPIQ chromatin analysis test. The results of this test show the accessibility of chromatin in the promoter region of studied gene, regardless the epigenetic modifications. These results showed that promoter region of VDR has a moderate degree of accessibility within all HL60, KG1 and KG1-CtrA cell lines, thus epigenetic gene silencing was not the reason of various VDR expression levels in HL60, KG1 and KG1-CtrA cells.
Subsequently, we looked at the aberrant signal transduction in KG1 cells. It was reported before that transcription factor STAT1 is constitutively active in KG1 . Indeed, we have found high levels of STAT1 protein, and its constitutive activation in KG1 cells, but not longer in KG1-CtrA and in KG1-RARA cells. In response to disruption of FOP2–FGFR1, STAT1 signal transduction became switched off, which pointed out that constitutively active fusion FOP2–FGFR1 kinase is an upstream activator of this pathway.
Among the ISG genes constitutively activated in KG1 cells were the ones that code for OAS proteins, which in turn activate RNase L, highly regulated, latent endoribonuclease . Since OAS genes were overexpressed in KG1, but neither in HL60, KG1-CtrA nor in KG1-RARA cells, the degradation of mRNA could be one of the reasons of VDR low level. In order to verify whether RNAse L activation contributes to the resistance of KG1 cells to 1,25D, we used curcumin, which has been reported as the only cell permeable RNAse L inhibitor. Our experiments revealed, that curcumin itself is a differentiation-inducing factor towards KG1 cells, and in combination with 1,25D it had synergistic pro-differentiation effect. It should be noted however, that degradation of VDR RNA may in part contribute to resistance of KG1 cells to 1,25D, but it is not the only reason.
In summary, we show in this paper that delivery of plasmid DNA to the cells may disrupt fusion gene FOP2–FGFR1 which occurs in a disease entity called 8p11 myeloproliferative syndrome. Whether this is limited to the cell line or present also in blast cells from patients needs to be studied in future, even though this disease is very rare. More importantly, inhibition of the FOP2–FGFR1 signal transduction pathway restored sensitivity of the cells with fusion kinase to 1,25D-induced cell differentiation. We suppose that this finding needs to be explored in more detail as it can be important for therapeutic purposes.
Cell lines and cultures
HL60 cells were a from a local cell bank at the Institute of Immunology and Experimental Therapy in Wroclaw (Poland), while KG1 cells were purchased from the German Resource Center for Biological Material (DSMZ GmbH, Braunschweig, Germany). The cells were grown in RPMI-1640 medium with 10 % fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin (Sigma, St Louis, MO) and kept at standard cell culture conditions.
Chemicals and antibodies
1,25D was purchased from Cayman Europe (Tallinn, Estonia), ATRA and curcumin were from Sigma. The compounds were dissolved in an absolute ethanol to 1000 × final concentrations, and subsequently diluted in the culture medium to the required concentration. IFNα (cat. no. 11343506), IFNγ (cat. no. 11343536) and antibodies CD11b-FITC (cat. no. 21279113) and CD14-PE (cat. no. 21270144), as well as appropriately labeled isotype controls were from ImmunoTools (Friesoythe, Germany). Mouse monoclonal anti-VDR (sc-13133), anti-Stat1 p84/p91 (sc-464) and anti-p-STAT1 (sc-8394), rabbit polyclonal anti-actin (sc-1616), anti-HDAC1 (sc-7872) and anti-Histone H1 (sc-10806) were from Santa Cruz Biotechnology Inc. (Santa Cruz Biotechnology Inc., CA). Goat anti-rabbit IgG, anti-mouse IgG conjugated to peroxidase, anti-mouse conjugated to biotin and streptavidin conjugated to peroxidase were from Jackson ImmunoResearch (West Grove, PA).
Transfection reagents and procedure
Electrotransfection by Neon® Transfection System (Invitrogen™, Carlsbad, CA) was performed as before  using control shRNA plasmid-A (sc-108060) or RARA shRNA plasmid (sc-29465-SH; both Santa Cruz). In order to obtain additional control cells, KG1 cells were seeded on 24-well plates (2 × 104 cells per well) and after 24 h the cells were infected with 20 μl of lentiviral particles containing scrambled shRNA sequences (sc-108080; Santa Cruz) in medium containing 1 μg/ml polybrene (Santa Cruz) for 8 h. The medium was changed and the cells were grown for 2 more days. After transfection the cells were grown in a medium supplemented with 1 µg/ml puromycin (Santa Cruz). Medium and selective antibiotic were changed every 2 days and puromycin non-resistant cells were cleared from the culture.
The expression of cell surface markers of differentiation was determined by flow cytometry. The cells were incubated with 1,25D ± IFNs or curcumin for 96 h, then washed and stained with 1 µl of fluorescently labeled antibody (or the appropriate control immunoglobulins) for 1 h on ice. Next, they were washed with ice-cold PBS and suspended in 0.5 ml of PBS supplemented with 0.1 % BSA prior to analysis on FACS Calibur flow cytometer (Becton–Dickinson, San Jose, CA). Experiments were repeated at least three times. The acquisition parameters were set for an isotype control. Data analysis was performed with use of WinMDI 2.8 software (freeware by Joseph Trotter).
Isolation of total RNA, reverse transcription into cDNA and Real-time PCR reactions were performed as published before , using CFX Real-time PCR System (Bio-Rad Laboratories Inc., CA). The sequences of VDR and GAPDH primers together with reaction conditions were described previously . The FOP2–FGFR1, FOP2 and FGFR1 primers were as published before . The interferon response was evaluated by IFNr qRT-Primers (Invivogen) which allow to quantify the mRNA expression of well characterized IFN-induced genes: IFNB, OAS1, MX1, G1P2, IFIT1. Relative quantification (RQ) of gene expression was analyzed with ∆∆Cq method using GAPDH as the endogenous control. Experiments were repeated at least three times.
In order to prepare cytosolic, nucleosolic and chromatin fractions 5 × 106 cells/sample (equivalent of 15 μl packed cell volume) were washed with PBS and lysed using either Pierce Subcellular Protein Fractionation Kit or NE-PER Nuclear and Cytoplasmic Extraction Reagents (both from Thermo Fisher Scientific Inc., Worcester, MA) according to the user’s manual. Obtained lysates were denatured by adding 5× sample buffer and boiling for 5 min. For western blotting 25 μl of lysates were separated on 10 % SDS-PAGE gels and transferred to PVDF membranes. The membranes were then dried, and incubated sequentially with primary (3 h) and a horseradish peroxidase-conjugated secondary antibody (1 h) at room temperature. In case of STAT1 detection biotin-conjugated secondary antibody and peroxidase-conjugated streptavidin were used. The protein bands were visualized with a chemiluminescence (Santa Cruz). Then the membranes were stripped, dried again and probed with subsequent antibodies. These experiments were repeated 2–5 times.
Digestion of chromatin was carried out using EpiQ chromatin Analysis Kit according to manufacturer’s guidelines (Bio-Rad). All cells were viable and actively growing in culture at the time of experiment, approximately 2.5 × 105 cells per sample were harvested. Cells were pelleted and resuspended in 100 µl of chromatin buffer. Digested (D) samples were treated with 2 µl of EpiQ nuclease, undigested (U) samples were not treated with nuclease. Both D and U samples were incubated at 37 °C for 1 h. Stop buffer was added to the samples for 10 min at 37 °C to stop chromatin digestion and thereafter the genomic DNA was extracted and purified, chromatin accessibility was then assessed by real-time quantitative PCR using CFX Real-time PCR system. For each cell type three digested samples and three undigested samples were analyzed using EpiQ chromatin Kit Data Analysis Tool (http://www.bio-rad.com/epiq) and normalized against the RHO (Rhodopsin) gene as a negative reference to a closed chromatin structure. The GAPDH gene was a positive reference to an open chromatin structure. Primers to analyze the proximal promoter of the human VDR gene were designed as recommended by the manufacturer using Primer3 software. The sequences for VDR were forward: 5′-GGCTGAAGCGGGTATCCGCACCTAT-3′, and reverse: 5′-TTTGACAAGCAGAGACAGCCCAGCA-3′. Experiments were repeated three times.
Genomic DNA from 5 × 106 of cells was isolated using GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma). FOP2 forward (AGATGATCCGGGTATAATAA) and FGFR1 reverse (AGAAGAACCCCAGAGTTCAT) primers were used to amplify the ~ 5 kb genomic fusion sequence . A PCR reaction was performed with 1 μl of Marathon DNA polymerase (A&A Biotechnology, Gdansk, Poland), 250 μM of each dNTP, 200 ng of each primer and 500 ng of genomic DNA in 50 μl of reaction mixture. The PCR reaction conditions were according to the polymerase protocol with annealing temperature of 53 °C, 5 min of elongation step and with 35 cycles. PCR products were visualized on a 1 % agarose gel stained with ethidium bromide and HyperLadder™ 1 kb and 25 bp (Bioline, London, UK).
The Student’s t test for independent samples was used to analyze the results obtained in experiments (Excel, Microsoft Office).
acute myeloid leukemia
all-trans retinoic acid
fibroblast growth factor receptor 1
Fms-related tyrosine kinase 3
FGFR1 oncogene partner 2
IFN stimulated genes
mean channel of fluorescence
retinoic acid receptor α gene
- RNAse L:
signal transducer and activator of transcription
vitamin D receptor
AM performed transfections of KG1 cells, selected stable transfectants, performed and analyzed all Real-time PCR experiments, performed and analyzed some flow cytometry experiments, some western blot experiments and described the methods used. AC performed and analyzed the experiment presented in Fig. 4, described the method and the results of this experiment and participated in edition of the paper text. KB designed and performed experiments presented in Fig. 5b which allowed for the analysis of fusion gene. EM designed conception of the study, performed and analyzed some flow cytometry experiments and some western blot experiments, wrote the article, revised it critically. All authors read and approved the final manuscript.
The research was supported by Wroclaw Research Center EIT + under the Project “Biotechnologies and advanced medical Technologies–BioMed” (POIG 01.01.02-02-003/08-00) financed by the European Regional Development Fund (Operational Program Innovative Economy, 1.1.2). AC gratefully acknowledges receipt of a Marie Curie Research Associate post funded by European Union’s Seventh Framework Programme FP7/2007–2013/under REA Grant Agreement No. 315902. Publication cost was supported by Wroclaw Center of Biotechnology Program, The Leading National Research Center (KNOW) for years 2014–2018.
The authors declare that they have no competing interests.
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