Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells

Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all- trans - retinoic acid (ATRA) or 1,25 dihydroxyvitamin D 3 (1,25D), or the use of both these differentiation - inducing agents. However, the effect of combination treatment varies in different AML cell lines , and this is due to ATRA either down - or up - regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated . Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein , the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down regulation of the level of RARα leads to increased expression of VDR . We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D .


Introduction
Acute myeloid leukemia (AML) is the predominant acute leukemia among adults. This disease is difficult to treat due to variable underlying causes and most patients are elderly and often excluded from aggressive chemotherapy trials [1]. A very attractive and gentler way to treat patients with AML is so called differentiation therapy [2,3]. The most successful differentiation therapy agent is all-trans-retinoic acid (ATRA). This is used routinely to treat a very rare form of AML called acute promyelocytic leukemia (APL), in which a PML-RARα fusion protein is generated by a t(15;17)(q22;q12) chromosomal translocation [4]. However, the success of ATRA-based differentiation therapy has not been extended to other forms of AML [5]. 1,25-dihydroxyvitamin D 3 (1,25D) is capable of inducing in vitro differentiation of AML cell lines [6], but was not found to be very effective in early clinical trials of AML [7]. Previous work has shown that a combination of 1,25D and ATRA can produce a synergistic differentiation effect [8]. However, our recent research has shown that the effect of this combination treatment varies in AML cell lines [9]. This is due to either down-or up-regulation of vitamin D receptor (VDR) transcription in response to ATRA in the AML cell lines examined. HL60 cells have a high constitutive level of VDR mRNA and VDR expression is downregulated by ATRA, whereas KG1 cells have a very low basal level of VDR mRNA, and ATRA upregulates VDR expression [9].
The gene encoding human VDR is located on chromosome 12, it covers about 100 kb of genomic DNA [10] and its composition is complex. The gene is composed of 14 exons, and translation of VDR protein spans from the exon 2 to the exon 9 [11,12]. Due to T to C polymorphism, which eliminates the most 5'-located ATG codon in the exon 2, translation starts from the second in-frame ATG codon in some individuals. Thus, two variants of VDR protein exist, one three amino-acids shorter (aa and 424) than the other (427 aa) [13]. The 5' region is very complex, and consists of the six exons 1a-1f, which together with the corresponding promoter regions are alternatively used in transcription regulation in various tissues [11]. Only three promoter regions have been identified in the region that codes for exons 1a-1f. Transcripts originating from exon 1a and from exon 1d are regulated by the promoter upstream to exon 1a, and exons 1f and 1c have their own upstream promoters ( Figure 1A). The regulation of remaining exons remains to be elucidated [11][12][13].
Transcripts which originate from exon 1a and 1d are expressed in most of the 1,25Dresponsive tissues, while a transcript originating in exon 1f is selectively expressed in tissues that play a role in calcium-phosphate homeostasis [11]. Moreover, it has been shown that the transcripts starting from exon 1d give rise to a longer VDR protein, named VDR B1 [11,14]. ATRA-mediated upregulation of VDR transcription has been reported in the past. However, due to the lack of retinoic acid response elements (RARE) in VDR promoter region, the mechanism of the influence of ATRA on VDR transcription remains unclear. Non-classical ATRA-responsive regions have been reported to be present in a regulatory element localized downstream of exon 1c (as detected in reporter assays using transfected HeLa cells) [13] and in the promoter region upstream of exon 1c (detected in similar assays using breast cancer cells) [15]. It is thus tempting to speculate that the mechanism of regulation of VDR by ATRA, and whether the outcome is up-or down-regulation of VDR expression, are cell-context dependent.
ATRA is a non-selective agonist of the three distinct isoforms of retinoic acid receptors (RAR) α, β and γ, and these occur as numerous splicing variants [16][17][18]. RARs, similarly to VDR, form heterodimers with retinoid X receptors (RXR) and act as ligand-regulated transcription factors via binding to specific RAREs. However, it should be remembered that un-ligated RARα, and to much lesser extent RARβ and γ, may act as transcriptional repressors to certain genes [19,20]. Due to the differences in the ligand binding domains of distinct RAR isoforms, it has been possible to synthesize a number of selective RAR agonists and antagonists [21]. The means to selectively activate or inhibit distinct isoforms of RARs have led to discovery of their diverse functions, which often relate to various aspects of cell differentiation [22].
To gain a better understanding of the molecular mechanisms of regulation of VDR transcription by RARs in AML cells we have examined the transcriptional variants produced by these cells. We have used a rapid amplification of cDNA ends (5'-RACE) method to investigate this problem.
The use of selective RAR agonists has allowed us to identify the isoform of RAR that regulates a transcriptional variant of VDR in AML cells. Through luciferase reporter assays, we localized the region in the VDR promoter which is involved in the regulation of VDR expression in response to ATRA. Finally, we have addressed the role of un-ligated RARα in AML cells by using shRNA gene silencing technology.

Cell lines and cultures
HL60 cells were obtained from the local cell bank at the Institute of Immunology and Experimental Therapy in Wrocław, and KG1, U973, MV4-11, MOLM-13 and NB-4 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 (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin (Sigma, St Louis, MO) and maintained at standard cell culture conditions.

Chemicals and antibodies
1,25D was purchased from Cayman Europe (Tallinn, Estonia) and ATRA was from Sigma. The compounds were dissolved in an absolute ethanol to 1000x final concentrations, and subsequently diluted in the culture medium to the required concentration. AGN191183 (pan RAR agonist), AGN195183 (RARα agonist) and AGN205327 (RARγ agonist) have been described previously [23] and were synthesized at the Shangai Institute of Materia Medica. Tazarotene (RARβγ agonist) [24] and BMS453 (RARβ agonist) [25] were purchased from Tocris Bioscience (Bristol, UK). These compounds were stored at 10 mM concentrations in 50% methanol/50% dimethylsulphoxide at -20°C and subsequently diluted in the culture medium to the required concentration. Rabbit polyclonal anti-RARα (sc-550), anti-actin (sc-1616), anti-Histone H1 (sc-10806) and mouse monoclonal anti-VDR (sc-13133) antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-lamin C2 custom made antibody was a kind gift from Prof. Ryszard Rzepecki (Faculty of Biotechnology; University of Wrocław). Goat anti-rabbit IgG and a goat anti-mouse IgG conjugated to peroxidase were obtained from Jackson ImmunoResearch (West Grove, PA).

cDNA synthesis and Real-time PCR
Isolation of total RNA, reverse transcription into cDNA and Real-time PCR reactions were performed as published before [9], using CFX Real-time PCR System (Bio-Rad Laboratories Inc., CA).
Quantification of gene expression was analyzed with either the ∆Cq or ∆∆Cq method using GAPDH as the endogenous control. Primers efficiencies were measured in all cell lines using a Real-time PCR reaction based on the slope of the standard curve. The results were normalized to primer efficiencies to compare gene expression in different cell lines. Real-time PCR assays were performed at least in triplicate.

Flow cytometry
The expression of cell surface markers of differentiation was determined by flow cytometry.
The cells were incubated with 10 nM 1,25D or/and 1 µM ATRA for 96 h, then washed and stained with 1 µl of fluorescently labeled antibodies CD11b/FITC and CD14/PE or with the appropriate control immunoglobulins (all from ImmunoTools; Friesoythe, Germany) for 1 hour on ice. Next, they were washed with ice-cold PBS and suspended in 0.5 ml PBS supplemented with 0.1% BSA prior to analysis on FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA). The acquisition parameters were set for an isotype control. The experiments were repeated at least three times and data analysis was performed using WinMDI 2.8 software (freeware by Joseph Trotter).

Identification of transcriptional start sites of VDR transcripts
To identify the transcriptional start sites of VDR transcripts, 5'-RACE (rapid amplification of cDNA ends) was used [27].  Terminator Cycle Sequencing Kit (Life Technologies). In total, 67 clones were sequenced. The sequencing reaction was analyzed using ABI Prism 310 Genetic Analyzer. The sequences of VDR transcripts obtained were aligned with the genomic sequence of VDR gene using Spidey software to identify exons and transcriptional start sites.

Reporter assays
Two forms of the VDR 1a promoter: 1a long (-1935/+71) and 1a short (-464/+71) were cloned upstream of the firefly luciferase gene in the pGL3 Basic vector (Promega) using SLIC cloning. First, promoter fragments were obtained by amplification of the genomic DNA isolated from KG1 cells in a PCR reaction using Phusion Green High-Fidelity Polymerase (Thermo Scientific) and primer pairs: VDRp1aDF-pGL3, VDRp1aR and VDRp1aKF-pGL3, VDRp1aR-pGL3 for the long and the short form respectively. The SLIC reaction was performed as following: 0.038 pmol of BglII/HindIII-digested pGL3Basic, 0.08 pmol of VDR 1a promoter fragment (long or short form), 1 x BSA, 1 x NEB buffer 2, 0.5 µl T4 polymerase were incubated for 2.5 minutes at room temperature in a final volume of 10 µl.
Then 20 ng of RecA protein were added and the reaction was stored on ice. Chemically competent DH5α cells were transformed with 5 µl of the reaction product using thermal shock method (45 For the reporter assays, HL60 cells were co-transfected with 2.5 μg of the reporter pGL3 plasmids and with the 1 μg of the control pRL-TK plasmid, using Neon®Transfection System

Gene silencing reagents and procedure
The gene silencing was performed using shRNA lentiviral particles: the RARA shRNA lentiviral particles (sc-29465-V) containing three target-specific 19-25nt shRNAs designed to specifically knockdown RARA gene expression, and the control shRNA lentiviral particles (sc-108080) containing scrambled shRNA sequences which were used as a negative control (both Santa Cruz Biotechnology, Inc.). KG1 cells were seeded on 24-well plates (2 × 10 4 cells per well) and after 24 h the cells were infected with 20 μl of lentivirus particles in medium containing 1 μg/ml polybrene (Santa Cruz Biotechnology, Inc.) for 8 h. The medium was changed and the cells were grown for two more days.
After that time, the medium was replaced with selection medium containing 1 μg/ml puromycin (Santa Cruz Biotechnology, Inc.).

Western blots
In order to obtain cytosolic and nuclear extracts 5x10 6 cells/sample were washed and lysed using Pierce NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the user's manual.
Lysates were denatured by adding 5x sample buffer (1/4 volume of the lysate) and boiled for 5 min.
25 µl of each lysate were separated in SDS-PAGE and electroblotted to PVDF membrane. The membranes were then dried, and incubated sequentially with primary and a horseradish peroxidaseconjugated secondary antibody. The protein bands were visualized by chemiluminescence. Then the membranes were stripped, dried again and probed with subsequent antibodies. Western blots were repeated 3 times.

Statistical analysis
For statistical analysis one-way ANOVA was used to test the null hypothesis that samples in two or more groups are drawn from populations with the same mean values. When the ANOVA test had shown that the null hypothesis is not true, Student's t-test for independent samples was used to analyze the differences between the pairs of groups (Excel, Microsoft Office and free ANOVA Calculator: http://www.danielsoper.com/statcalc3/calc.aspx?id=43).

Regulation of VDR in response to ATRA in six AML cell lines
Our previous work has shown that transcription of VDR gene is regulated in an opposite manner in response to ATRA in two of the commonly used cell lines that typify AML cells.  Table 1. HL60, KG1, U973, MOLM-13, NB-4 and NOMO-1 cells were exposed for 24, 48, 72 and 96 h to 1 µM ATRA, and the expression of VDR was measured by Real-time PCR using GAPDH as a reference gene. The highest level of constitutive expression of VDR was observed in NB-4 cells ( Figure 2A). However, it should be remembered that NB-4 cells harbor one copy of the PML-RARA fusion gene, which encodes the fusion protein PML-RARα. Previous studies have shown that PML/RARα impairs the localization of VDR in the nucleus by binding to VDR and by this means inhibits its transcriptional activity [28]. VDR expression was up-regulated by ATRA to a significant extent in NB-4 cells (2.4 times), but to a lesser extent than observed for KG1 cells (more than 7 times). In contrast, VDR expression was down-regulated in response to ATRA in HL60 cells (3.2 times), MOLM-13 (2.9 times) and NOMO-1 (5.2 times) cell lines ( Figure 2B). The absolute level of VDR expression was very low in U937 cells, and it was further down-regulated in response to ATRA (1.8 times). However, this decrease was not statistically significant. Additionally, constitutive VDR expression is about 10 times higher in HL60 than in KG1 cells.
The differentiating effects of 1,25D or/and ATRA were examined by flow cytometry. Figures 2C-2H show that for all the cell lines studied the combined effect of ATRA and 1,25D was to increase the level of granulocytic differentiation (increased CD11b expression). The situation was different in regard to expression of monocytic marker CD14. For the cell lines in which ATRA up-regulated VDR expression (KG1 and NB-4) combined treatment also up-regulated CD14, when compared to single treatment. For the cell lines in which ATRA down-regulated VDR expression it also down-regulated CD14, when compared to 1,25D alone. These results point to increased VDR expression shifts the differentiation option from the granulocytic to the monocytic pathway.

Effects of 1,25D and ATRA on regulation of CYP24A1 expression
The CYP24A1 gene encodes the enzyme 24-hydroxylase of 1,25D, which is the key enzyme in the degradation of 1,25D to calcitroic acid. It is well documented that CYP24A1 is the most strongly regulated of all the 1,25D-target genes [29], thus 1,25D-dependent up-regulation of CYP24A1 confirms that VDR protein is expressed and active in cells. For the AML cell lines that responded well to 1,25D, the expression of CYP24A1 was slowly, but significantly, up-regulated up to a thousandfold, as compared to untreated cells [26]. As shown in Figure 3A and 3C, measurements of CYP24A1 levels reflected ATRA-driven changes in VDR expression and activation levels in KG1 and in HL60 cells.
Similarly in MOLM-13 ( Figure 3D) and U937 ( Figure 3F) cells ATRA-driven changes in VDR expression were further reflected in expression of CYP24A1. In the case of NB-4 ( Figure 3B) and in NOMO-1 cells ( Figure 3E) 1,25D-induced CYP24A1 expression was not altered to a significant degree by ATRA. The data presented in Figure 3 show that ATRA alone does not influence CYP24A1 expression. Moreover, ATRA-induced changes to CYP24A1 expression confirmed that increased VDR mRNA levels lead to increased translation of VDR protein, which is activated by ligand. It is noteworthy that the observed 1,25D-induced CYP24A1 expression in HL60 cells is one order of magnitude higher than in MOLM-13 and NOMO-1 cells, two orders of magnitude higher than in NB-4 cells and three to four orders of magnitude higher than in KG1 and U937 cells.

Transcriptional variants of VDR in HL60 and KG1 cells as identified by 5'-RACE
For these studies we chose to use the HL60 and KG1 cell lines as the most sensitive and least sensitive to 1,25D, respectively. We considered that variable regulation of VDR gene in these cell lines might result from the usage of different VDR promoters. Therefore, we decided to identify the promoter regions that regulate VDR transcription in un-stimulated and ATRA-stimulated HL60 and KG1 cells, and examined the transcript variants occurring in those cells. We used the 5'-RACE method to identify 5' portions of the VDR transcripts -upstream of the VDR exon 2. The results of our experiments are presented in Figure 1B and aligned to the published sequence. We found that in both un-stimulated and ATRA-stimulated HL60 cells, VDR transcripts originated from exon 1a which was spliced to exon 2 either directly or through exon 1c. The same transcript variants were the most frequently detected transcripts in un-stimulated and ATRA-stimulated KG1 cells. In addition, we observed that KG1 cells express a more diverse set of VDR transcripts than HL60 cells. In unstimulated KG1 cells, we detected transcript variants originating from exon 1d and, from a so-farunidentified exon, depicted in Figure 1B as exon 1g, and localized downstream of exon 1d. In contrast to HL60, in KG1 cells we detected also exon 1b present in transcripts originating either from exon 1a or 1g. Aside from the differences between HL60 and KG1 cells, the majority of transcripts expressed in both cell types, regardless of the presence or absence of ATRA, originated from exons controlled by the 1a promoter. This strongly suggests a predominant role of the 1a promoter in regulating VDR expression in these cells.

Regulation of VDR variants in response to ATRA and to selective RAR agonists
The  Table 2. As shown in Figure 4C (HL60) and 4D (KG1), the pan-RAR agonist, the selective RARα agonist and the mixed RARβγ agonist (tazarotene) significantly regulated transcription of VDR 1a. Among the transcript variants of VDR gene in KG1 cells we identified rare transcripts, which originated from a newly discovered exon 1g. We tested whether these transcripts are also regulated by the RAR agonists. In HL60 cells such regulation did not occur (not shown), but VDR1g transcript appeared to be regulated by RAR agonists in KG1 cells ( Figure 4E). The pattern of regulation of VDR1g and VDR1a in KG1 cells is similar, which suggests that the promoter region used to regulate both transcripts is the same. The data presented in Figure 4 indicate that the most important isoform of RAR involved in VDR transcription is RARα. Significant up-regulation in KG1 cells, and significant down-regulation in HL60 cells of VDR1a by tazarotene suggests that RARβ and γ also have limited regulatory effects, which is further supported by the observation that combination treatment using RARβ and RARγ agonists mimics the effect of tazarotene (not shown here).

Reporter assays in HL60 cells treated with ATRA
In order to determine whether the promoter of exon 1a responds directly to ATRA stimulation, two reporter vectors were constructed that contained different portions of the 1a promoter region, no effects of ATRA were observed, but when the 1a long promoter was used luciferase activity was significantly down-regulated by ATRA treatment, in keeping with the regulation of the VDR 1a variant in response to ATRA in HL60 cells ( Figure 5A).

Expression of VDR in the absence of RARα
Having shown that RARα ligated by its selective agonist regulates transcription of the VDR gene in HL60 and KG1 cells, we investigated the role of un-ligated RARα in these cells. Our experiments had shown that constitutive expression of RARA mRNA in KG1 cells is much higher than in HL60 cells ( Figure 5B). Also the protein level of RARα in the nucleus was higher in KG1 than in HL60 cells ( Figure 5C). Even though RARB mRNA levels were high in both cell lines, RARβ protein was undetectable in our experiments. RARG expression was low in both cell lines, and RARγ protein was not detectable.
Therefore, we silenced the expression of RARA in KG1 cells. The gene silencing was performed using RARA shRNA lentiviral particles and the scrambled shRNA lentiviral particles as a control. After selecting transduced cells in puromycin containing medium, we obtained two KG1 sublines; KG1-RARα(-), from the use of RARA shRNA, and KG1-CTR, from the use of scrambled shRNA.
The level of gene silencing was examined by Real-time PCR ( Figure 6A) and by Western blotting ( Figure 6B, top panel). Whilst RARα silencing was not complete in KG1-RARα(-) cells the level was significant at around 85%. Next, we investigated whether the loss of RARα influenced the basal level of transcription of VDR. The level of VDR mRNA in KG1-RARα(-) cells was observed to be almost 5 times higher than in the cells transfected with the control plasmid ( Figure 6C). This level was less than the basal level of VDR mRNA in HL60 cells, but more than the level in wild-type KG1 cells. In order to confirm that increased expression of VDR gene in KG1-RARα(-) cells resulted in the translation of functional VDR protein, we measured VDR protein levels in both sublines. It has been documented earlier that VDR protein is stabilized by 1,25D, and that a high level of VDR in the nucleus can be seen only after addition of 1,25D to AML cells (presented in Figure 5D) [30]. Thus, we exposed KG1-CTR cells and KG1-RARα(-) cells to 10 nM 1,25D for 24 h, or left untreated. As presented in Figure 6D, VDR protein level was observed to be higher in nuclear fractions from KG1-RARα(-) cells as compared to KG1-CTR cells, especially after exposure to 1,25D.
We analyzed the influence of the selective RAR agonists on VDR expression levels in both KG1 sublines. KG1-CTR cells were similarly responsive to wild-type KG1 cells, while KG1-RARα(-) had lost responsiveness to RAR agonists. The selective RARα agonist induced a significantly higher VDR expression in KG1-CTR cells than any other agonist. The mixed RARβγ agonist (tazarotene) induced significant up-regulation of VDR, but to a much lower extent than when the RARα agonist was used alone ( Figure 6E).

Discussion
The fact that retinoids regulate the expression of the VDR gene in bone and mammary cells is well described, and this effect is known to be cell-type dependent [12,15]. Also, it is well established that RARα acts as transcriptional repressor when un-ligated [19], and after binding its physiological agonist ATRA then functions to activate transcription [20]. However, precise details are lacking of the molecular events that occur in AML cell lines. The transcriptional regulation of VDR gene is very complex because of the use of several alternative promoter regions in the large regulatory region encompassing 65 kb upstream of the coding region (exons 2-9) [13]. In our studies we have identified transcripts starting from exon 1a within KG1 and HL60 cells. These are the transcripts that are regulated when cells are treated either with ATRA or a RARα specific agonist. From our studies, we have also identified a new non-coding exon, here termed 1g, which either can be used as a transcript start, or alternatively spliced into the transcripts starting from exon 1a.
A more complete understanding of the mechanism whereby expression of the VDR gene is regulated in response to retinoids is confounded by the fact that a classical RARE does not exist within the entire VDR promoter region. However, it should be remembered that the location of RAREs is highly variable, and ranges from 10,145 bases upstream to 8,141 bases downstream of the 5' end of known transcription start site [31], so the regulatory element does not need to be located in close proximity to the VDR gene. It has also been documented that RAREs are often located in intronic regions [32]. Moreover, recent studies have revealed a high level of diversity in the topology and spacing of RAREs. Classical RAREs are composed of two direct repeats of a core hexameric nucleotide sequence, spaced by 1, 2 or 5 random nucleotides (named DR1, DR2 and DR5). New data have revealed that RAREs do not have to be spaced at all (DR0), they can be spaced by 8 nucleotides (DR8) or composed of the inverted repeats (IR) [33]. To circumvent the, as yet, lack of a RARE involved in VDR regulation, it has been suggested that retinoids regulate VDR transcription in a secondary manner, by using cis-regulatory elements which cooperate with the promoter [13]. Until now, these elements have been localized in the vicinity of exon 1c, either downstream [13] or upstream [15], depending on the cell type. Here we have shown for AML cells that a putative cisregulatory element, which is used by RARs, is located in the promoter region of exon 1a, between nucleotides -1935 and -464 relative to the transcriptional start site of 1a. The attempts to specify the exact location of the regulatory element are underway in our laboratories.
As mentioned above, whether ATRA treatment of AML cell lines leads to up-or downregulation of expression of VDR depends on the cell line tested. It is important to bear in mind that several kinase signal transduction pathways are rapidly activated when cells are treated with ATRA.
[34]. This, in turn, might influence the phosphorylation status of particular RAR isoforms [35].      The cells were exposed to 10 nM 1,25D or to 1 µM ATRA and after 96 h the levels of VDR 1a and VDR 1d mRNA were measured relative to GAPDH mRNA levels.
Results that differ significantly (p<0.01) from respective controls are marked with asterisks. The influence of non-selective and selective RAR agonists towards VDR 1a expression was examined in HL60 (C) and in KG1 (D) cells. The cells were exposed to 10 nM 1,25D or 100 nM RAR agonists and after 96 h the levels of VDR 1a mRNA were measured relative to GAPDH mRNA levels by Real-time PCR. The bar charts show the mean values (±SEM) of the fold changes. Results that differ significantly (p<0.05) from the respective control are marked with asterisks. The influence of non-selective and selective RAR agonists towards VDR 1g expression was examined in KG1 (E) cells. The cells were exposed to 10 nM 1,25D or 100 nM RAR agonists and after 96 h the levels of VDR 1g mRNA were measured relative to GAPDH mRNA levels by Real-time PCR. The bar charts show the mean values (±SEM) of the fold changes. Results that differ significantly (p<0.05) from the respective control are marked with asterisks. The levels of RARα (C) and VDR (D) proteins were determined in the cytosol and nuclei of HL60 and KG1 cells by Western blots. In order to visualize VDR protein, the cells were exposed to 10 nM 1,25D for 24 h. The cytosolic (C) and nuclear (N) extracts were separated by SDS-PAGE, transferred to PVDF membranes and the proteins were revealed using anti-RARα, anti-VDR and anti-actin. The OD ratio of each receptor band was calculated versus the OD of the respective actin band and the means (±SEM) are presented below the blots. In order to visualize VDR protein, the cells were exposed to 10 nM 1,25D for 24 h. The cytosolic (C) and nuclear (N) extracts were separated by SDS-PAGE, transferred to PVDF membranes and the proteins were revealed using anti-RARα, anti-VDR and antiactin. Anti-Lamin C antibody was used to show the purity of cell fractionation. The OD ratio of each receptor band was calculated versus the OD of a respective actin band and the means (±SEM) are presented below the blots.