HDAC INHIBITORS SODIUM BUTYRATE AND SODIUM VALPROATE DO NOT AFFECT HUMAN NCOR 1 AND NCOR 2 GENE EXPRESSION IN HL-60 CELLS

Aim. This study was designed to examine whether the class I and class IIa histone deacetylase (HDAC) inhibitors, sodium butyrate and sodium valproate alter the expression of human NCOR1 and/or NCOR2 genes coding for N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors), respectively. Methods. Human leukemia HL-60 cells were treated for 24 h with 0.5 and 1 mM sodium butyrate, 1 to 3 mM sodium valproate, 1 mcM all-trans retinoic acid (ATRA) or cotreated with 1 mcM ATRA and 0.5 mM sodium butyrate. The acetylation of histones H3 and H4 was analysed by western blotting. The levels of NCOR1 and NCOR2 mRNA were determined by quantitative real-time PCR. Expression of NCF2 gene coding for the NADPH oxidase subunit p67phox was evaluated as a marker of myeloid differentiation. Results. Both butyrate and valproate increased the acetylation of histone H3 at Lys9 and/or Lys14 as well as histone H4 at Lys12. Both HDAC inhibitors caused a significant increase in NCF2 mRNA levels without affecting NCOR1 or NCOR2 mRNA levels. Similarly, ATRA alone or in combination with butyrate induced NCF2 gene expression without any significant influence on the expression of NCOR1 or NCOR2 genes. Conclusion. We conclude that inhibitors of class I and class IIa HDACs do not alter the expression of human NCOR1 or NCOR2 genes and that the onset of myeloid differentiation is not accompanied by induction or repression of these genes in HL-60 cells.


INTRODUCTION
N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone receptors) are ubiquitously expressed corepressor proteins.They interact with unliganded nuclear receptors and form large corepressor complexes that mediate transcriptional repression through their association with histone deacetylases (HDACs).The relative hypoacetylation restores a positive charge in histone proteins and causes closer association of the histones with DNA that becomes less accessible to transcription factors 1,2 .HDACs that have been shown to interact with N-CoR or SMRT complexes include class I HDACs 1, 2 and 3 and class IIa HDACs 4, 5 and 7 (ref. 3).N-CoR and SMRT have been recognized to play important roles, among others, in development and cell differentiation 1 .For instance, neutrophilic differentiation is regulated through the nuclear retinoic acid receptor α (RARα).Unliganded RARα interacts with N-CoR or SMRT which associate through the corepressor mSin3 (mammalian switch independent 3 protein) with HDAC1 or HDAC2.The corepressor complexes, however, dissociate from RARα at physiological concentrations of all-trans retinoic acid (ATRA) and subsequent recruitment of coactivators with histone acetylase activity relieves the transcriptional repression of differentiationrelated genes 4 .On the other hand, abnormal transcriptional repression resulting from aberrant recruitment of the N-CoR/SMRT-HDAC complexes appears to underlie the molecular pathogenesis of cancers such as acute promyelocytic leukemia 5 .In accordance with the crucial role of HDACs in corepressor-dependent transcriptional repression, inhibitors of HDAC activity have emerged as a new class of anticancer agents 6 .
Human N-CoR and SMRT are encoded by NCOR1 (nuclear receptor corepressor 1) and NCOR2 (nuclear receptor corepressor 2) genes, respectively.To date, little is known about their transcriptional regulation.It has been shown that the steroid hormone, 17β-estradiol decreases N-CoR protein levels in estrogen receptor-positive breast cancer cells without affecting NCOR1 mRNA levels 7 .NCOR1 mRNA levels were also unchanged in rat pituitary GH 3 cells exposed to 17β-estradiol or to the thyroid hormone triiodothyronine 8 .The steroid hormone, 1α,25-dihydroxyvitamin D 3 has been found to induce a moderate and transient upregulation of NCOR2 but not NCOR1 mRNA levels in MCF-7 breast cancer cells 9 .Both NCOR1 and NCOR2 mRNA levels remained unaf-fected in rat hepatocytes treated with ATRA or colchicine, a microtubule interfering agent 10 .
Since HDAC inhibitors are considered to modulate the expression of 7-10% of genes in human cancer cells 11 , we examined whether the expression of human NCOR1 and/or NCOR2 genes could be altered by two HDAC inhibitors, sodium butyrate and sodium valproate.

Chemicals
Sodium butyrate (B5887), sodium valproate (P4543) and ATRA (R2625) were obtained from Sigma-Aldrich (St. Louis, MO, USA).Stock solutions of sodium butyrate and sodium valproate (1 M) were prepared in water for molecular biology and stored at -20 °C.Stock solution of ATRA (1 mM) in ethanol was prepared freshly before use with all manipulations performed in subdued light.

Reverse transcription and quantitative real-time PCR
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.The concentration of RNA was determined by spectrophotometry at 260 nm.RNA samples (2 μg) were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's recommendations.Real-time PCR was performed on a LightCycler 480 II system (Roche Diagnostics) using the TaqMan Universal PCR Master Mix and the TaqMan Gene Expression Assays consisting of specific primers and FAM dye-labelled TaqMan minor groove binder probes (Applied Biosystems).The assay ID for NCOR1 was Hs00196920_m1; for NCOR2, Hs00196955_m1; for NCF2, Hs00166416_m1; and for 18S, Hs99999901_s1, respectively.Amplification conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles with 95 °C for 15 s and 60 °C for 1 min.Crossing point values, equivalent to C T , were determined automatically using the second derivative maximum analysis.Relative changes in gene expression were calculated by the comparative C T method using the 2 -∆∆C T equation with results normalized to 18S rRNA levels.

Statistical analysis
Results were expressed as means ± S.D. of three independent experiments.The differences in mean values were analysed by Student's t-test.A p value of less than 0.05 was considered as statistically significant.

Hyperacetylation of histones H3 and H4 by butyrate and valproate
We chose for our study non-cytotoxic concentrations of butyrate and valproate on the basis of our previous results 12 .To confirm efficient inhibition of HDAC activity, human leukemia HL-60 cells were treated with 0.5 and 1 mM butyrate or 1 to 3 mM valproate and the whole cell lysates were analysed by western blotting using antibodies detecting specific acetylations of histones H3 and H4.After 24 h treatment, both butyrate and valproate increased the acetylation of histone H3 at Lys9 and/or Lys14 as well as the acetylation of histone H4 at Lys12 (Fig. 1).

Effect of butyrate and valproate on NCOR1, NCOR2 and NCF2 gene expression
The exposure of HL-60 cells to butyrate or valproate is associated with multiple changes in gene expression leading to monocytic differentiation 13,14 .We examined the expression of human NCOR1 and NCOR2 genes encoding HDAC inhibitors sodium butyrate and sodium valproate do not affect human NCOR1 and NCOR2 gene expression in HL-60 cells N-CoR and SMRT, respectively, concurrently with the expression of NCF2 (neutrophil cytosolic factor 2) gene coding for the NADPH oxidase subunit p67phox, a marker of myeloid differentiation 12,15 .After 24 h treatment of HL-60 cells, both butyrate and valproate induced a dosedependent increase in NCF2 gene expression as shown by the real-time PCR technique (Fig. 2A).In contrast, neither butyrate nor valproate caused any substantial changes in the expression of NCOR1 or NCOR2 genes under the same conditions.The NCOR1 mRNA levels induced by butyrate or valproate reached (0.83 ± 0.09)-fold to (1.08 ± 0.04)-fold values, while the NCOR2 mRNA levels varied between (0.80 ± 0.11)-fold and (1.25 ± 0.22)-fold values compared to control cells (Fig. 2A).We also examined the effect of the HDAC inhibitors in the presence of ATRA, an inducer of neutrophilic differentiation 13 .Our preliminary experiments (data not shown) demonstrated that only 0.5 mM butyrate synergized with ATRA in the induction of the NCF2 gene expression without parallel induction of the cytotoxic effect 12 .While the NCF2 mRNA levels in HL-60 cells exposed for 24 h to 0.5 mM butyrate or 1 μM ATRA reached 2.9-fold (Fig. 2A) and 7.2-fold values (Fig. 2B), respectively, the coexposure to ATRA and butyrate caused 17.3-fold increase in NCF2 mRNA level compared to control cells (Fig. 2B).On the other hand, ATRA either alone or in combination with butyrate did not substantially affect the expression of either NCOR1 or NCOR2 genes (Fig. 2B).

DISCUSSION
N-CoR and SMRT are the most extensively studied corepressor proteins, but little is known about their transcriptional regulation.This study was designed to examine whether the expression of human NCOR1 and NCOR2 genes coding for N-CoR and SMRT, respectively, could be altered by two HDAC inhibitors, sodium butyrate and sodium valproate.Human HDAC enzymes are classified on the basis of their homology to yeast HDACs.Classical HDACs are Zn 2+ -dependent enzymes and in-clude class I (HDAC1, 2, 3, 8), class IIa (HDAC4, 5, 7, 9), class IIb (HDAC6, 10) and class IV (HDAC11).Class III enzymes require NAD + for their activity and comprise seven members known as sirtuins 6 .Inhibitors of HDAC activity are able to cause both induction and repression of gene expression 16 .These effects may result from the increased acetylation of histones and/or nonhistone HDAC substrates that include a number of transcription factors and other important proteins 11 .Butyrate and valproate inhibit at millimolar concentrations class I and class IIa HDACs 11,16,17 , i.e. enzymes known to associate with N-CoR and SMRT (ref. 3 ).The HL-60 cell line used in our study was derived from a patient with acute myeloblastic leukemia type M2 according to the French-American-British classification 18 .HL-60 cells may be induced to differentiate along the neutrophilic pathway, e.g. by ATRA, or along the monocytic pathway by butyrate 13 , valproate 14 and other compounds.ATRA activates the transcription of differentiation-related genes through its binding to RARα leading to the release of the N-CoR/ SMRT-HDAC complexes 4 .Butyrate and valproate may induce expression of the same genes as ATRA by inhibiting HDACs in the corepressor complexes 5 .Moreover, a combination of HDAC inhibitors with ATRA has been shown to synergize in differentiation of ATRA-sensitive myeloid leukemic cells 19 and to overcome the block in differentiation of ATRA-resistant cells 20 .In this study, we found that neither butyrate nor valproate, like ATRA, induced any significant changes in the expression of NCOR1 and NCOR2 genes under conditions of efficient HDAC inhibition and/or induction of myeloid gene expression.We conclude that the inhibitors of class I and class IIa HDACs do not alter the expression of human NCOR1 and NCOR2 genes and that the onset of myeloid differentiation is not accompanied by induction or repression of these genes in HL-60 cells.