Influence of an altered methylation potential on mRNA methylation and gene expression in HepG2 cells

Experimental Cell Research 294 (2004) 325 – 334 www.elsevier.com/locate/yexcr

Influence of an altered methylation potential on mRNA methylation and gene expression in HepG2 cells Marina Hermes, Hartmut Osswald, Julia Mattar, and Doris Kloor * Department of Pharmacology and Toxicology, Faculty of Medicine, University of Tu¨bingen, D-72074 Tu¨bingen, Germany Received 28 July 2003, revised version received 24 November 2003

Abstract S-adenosylhomocysteine (AdoHcy), a by-product and inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions, is removed by AdoHcy hydrolase. The ratio of AdoMet and AdoHcy, also termed methylation potential (MP), is a metabolic indicator for cellular methylation status. In the present study, we have investigated the influence of hypoxia and inhibition of AdoHcy hydrolase on MP in HepG2 cells. Furthermore, we studied the impact of deviations in MP on mRNA and DNA methylation and the expression of selected genes: erythropoietin, VEGF-A, AdoHcy hydrolase, cyclophilin, and HIF-1a. Under hypoxic conditions, the MP raised from 53.4 F 3.3 to 239.4 F 24.8, which is the result of increased AdoMet and decreased AdoHcy levels. Inhibition of AdoHcy hydrolase by adenosine-2V,3V-dialdehyde leads to a 40-fold reduction of the MP under both normoxic and hypoxic conditions. Hypoxia increases erythropoietin (2.7-fold) and VEGF-A (5-fold) mRNA expression. During a reduced MP erythropoietin mRNA expression is lowered under normoxia and hypoxia by 70%, whereas VEGF-A mRNA expression is only reduced under hypoxic conditions by 60%. The mRNA expression of AdoHcy hydrolase, HIF-1a, and cyclophilin is insensitive to an altered MP. Furthermore, decreased MP leads to a highly significant decrease in overall mRNA methylation. Our results show that the mRNA levels of the studied genes respond differentially to changes in MP. This implies that genes with a slower transcription rate and mRNAs with a slower turnover are insensitive to short-term changes in MP. D 2004 Elsevier Inc. All rights reserved. Keywords: S-adenosylhomocysteine hydrolase; mRNA methylation; DNA methylation; Methylation potential; Gene expression; HepG2 cells; Erythropoietin

Introduction Biological methylation reactions, which utilize Sadenosylmethionine (AdoMet) as a methyl donor, encompass several important processes, including the methylation of phospholipids, proteins, DNA and RNA [1]. After the transfer of the methyl group, AdoMet is converted to S-adenosylhomocysteine (AdoHcy) within the active site of the methyltransferase enzyme (Fig. 1). Since most methyltransferases bind AdoHcy with a higher affinity than AdoMet, AdoHcy is a potent product inhibitor of most AdoMet-dependent transmethylations [2]. Therefore, a fast removal of AdoHcy is required for efficient methyltransferase reactions. S-adenosylhomocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1) is the only * Corresponding author. Department of Pharmacology and Toxicology, Faculty of Medicine, University of Tu¨bingen, Wilhelmstrasse 56, D-72074 Tu¨bingen, Germany. Fax: +49-7071-294942. E-mail address: [email protected] (D. Kloor). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2003.12.001

enzyme known in vertebrates that catalyses the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) [3]. Although the equilibrium of the reaction favors the synthesis of AdoHcy, the hydrolysis of AdoHcy to Ado and Hcy prevails under physiological conditions [4]. AdoHcy hydrolase appears to act near methyltransferases, even in the nucleus [5]. The crystal structure analysis of AdoHcy hydrolase has revealed that the catalytic domain is almost identical to that reported for methyltransferases, suggesting that AdoHcy can travel between the two enzymes [6]. The observation that deletion of AdoHcy hydrolase gene is associated with embryonic lethality in mice shows the biological importance of this enzyme [7]. The ratio AdoMet/AdoHcy is an important metabolic indicator for cellular methylation status and also termed methylation potential (MP). A decrease in AdoMet/AdoHcy ratio in presence of significant increase in AdoHcy is associated with DNA hypomethylation [8,9] and decreased

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internal standard. Since the erythropoietin and VEGF gene expression are stimulated by hypoxia in HepG2 cells [19 – 21], the influence of a decreased MP on gene expression was also studied under hypoxia.

Materials and methods Cell line and reagents

Fig. 1. Overview of AdoMet, AdoHcy, Hcy and Ado metabolism. Since AdoHcy is a product-inhibitor of AdoMet-dependent transmethylations, fast removal by AdoHcy hydrolase is required for efficient methylation reactions. AdoHcy: S-adenosylhomocysteine; AdoMet: S-adenosylmethionine; THF: tetrahydrofolate; METH: 5-methyltetrahydrofolate-homocysteine S-methyltransferase; MAT: methionine adenosyl transferase; BHMT: betaine-homocysteine S-methyltransferase; DMG: dimethylglycine [34].

methyltransferase activity [10]. Furthermore, an altered AdoMet/AdoHcy ratio by inhibition of AdoHcy hydrolase results in inhibition of lipid methylation, protein carboxymethylation, and RNA methylation [11,12]. Therefore, specific inhibitors of AdoHcy hydrolase can serve as a pharmacological target to influence methylation reactions and are of great medical interest because of their antiviral, immunosuppressive, and anti-inflammatory effects [13]. The antiviral effect seems to be, at least in part, due to lowered methylation of 5V-cap structure of viral mRNA [14]. This cap structure plays a significant role in the interaction of mRNA with the ribosomes during initiation of protein synthesis. Moreover, the 5V-terminal cap structure may be resistant to the action of 5V-3V-exoribonuclease, yielding the stability of mRNA within the cell [15]. The identification of AdoHcy hydrolase as a specific target for the design of immunosuppressive and anti-inflammatory agents [16] is based on the observation that the activation of lymphocytes is more dependent on methylation than the activation of other cell types [17], but the precise mechanism by which inhibition of AdoHcy hydrolase leads to inhibition of T-cell proliferation remains unclear [18]. In the present study, we have investigated the impact of changes in methylation potential (achieved by AdoHcy hydrolase inhibition and changes of equilibrium of the reaction catalyzed by AdoHcy hydrolase after addition of Ado and Hcy) on mRNA and DNA methylation and the expression of following genes: erythropoietin and vascular endothelial growth factor A (VEGF-A, as examples for differentially regulated genes), hypoxia inducible factor 1a (HIF-1a, as important factor under hypoxia), cyclophilin (as classical housekeeping gene), and AdoHcy hydrolase (as part of the methylation pathway) by using 18S rRNA as

The human hepatoblastoma cell line HepG2 [22,23] was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ Braunschweig, Germany). RPMI 1640 medium, newborn calf serum, 0.05% trypsin: 0.02% EDTA, Dulbecco’s phosphate buffered saline (PBS) and penicillin/streptomycin were provided by Invitrogen. AdoMet, AdoHcy, Ado, Hcy, methyladenosine, adenosine-2V,3Vdialdehyde (Ado-2V,3V-dial), and ammonium phosphate were obtained from Sigma. HPLC grade methanol and acetonitril were from Merck. The radiolabeled precursors were purchased from the source indicated: L-[methyl-3H]-methionine (71 Ci/mmol) Perkin Elmer and [14C]-uridine (55 Ci/mol) Bio Trend. Cell culture Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated newborn calf serum, 100 U/ml penicillin and 100 Ag/ml streptomycin in a humidified atmosphere containing 5% CO 2 at 37jC. For hypoxia experiments, the oxygen partial pressure was lowered to 0.9 kPa (1% O2 by vol.) for 24 h. To change the AdoMet/ AdoHcy ratio, cells were rinsed with PBS and then incubated with Ado-2V,3V-dial (AdoHcy hydrolase inhibitor) or Ado + Hcy in serum-free RPMI 1640 for 24 h. At the end of each incubation period, the culture medium was harvested, clarified by centrifugation and stored frozen at 20jC until assayed. The cells were removed with trypsin/EDTA, and cell counts were determined with a hemocytometer. Metabolite measurement AdoMet and AdoHcy concentrations in cultured HepG2 cells were measured in perchloric acid cell extracts. Cells were harvested by incubation with trypsin/EDTA and then collected by centrifugation at 200  g for 10 min. The cell pellet was washed with PBS and extracted with 0.6N perchloric acid. The protein precipitate was removed by centrifugation (10 min, 20,000  g) and the supernatant was adjusted to a pH between 5.5 and 6.0 by adding 2 M K2CO3/1M KH2PO4. All samples and standards were supplemented with 1 AM N6-methyladenosine as internal standard. The precipitated potassium perchlorate was discarded after centrifugation at 20,000  g and the supernatant was applied onto solid-phase extraction column (BondElut, ICT, Germany). Elution of the compounds was performed with

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HCl (0,1 M) and the eluate was analyzed by high performance liquid chromatography (HPLC) according to Delabar et al. [24]. mRNA labeling RNA methylation was determined by the incorporation of radioactivity from L-[methyl-3H]-methionine into RNA and RNA synthesis by incorporation of [14C]-uridine. The inhibitor was added to the cells and incubated for 2 h before 50 ACi L-[methyl-3H]-methionine/ml and 0.2 ACi [14C]uridine/ml were added. The cells were incubated with the isotopes for 22 h. Radioactivity incorporated in mRNA was determined by liquid scintillation counting. Isolation of total RNA and mRNA Total RNA was isolated with Tri-Reagent/Trizol as described previously [25]. Polyadenylated mRNA was isolated using Oligotex mRNA mini kit (Qiagen).

327

following LightCycler experimental run protocol was used: denaturation program (95jC for 10 min), amplification, and quantification program repeated 45 times (95jC for 10 s, ‘‘touch down PCR’’ 68jC–58jC with a step size of 0.5jC/ cycle for 10 s, 72jC for 16 s with a single fluorescence measurement), melting curve program (65jC – 95jC with a heating rate of 0.1jC/s and a continuous fluorescence measurement) and finally, a cooling step to 37jC. ‘‘Fit point method’’ was performed in the LightCycler software 3.5.3 (Roche). Gene expression was calculated according to a mathematical model for relative quantification established by Pfaffl [26]. Erythropoietin assay Quantitative detection of erythropoietin in the supernatant was performed with a commercially available monoclonal enzyme immunoassay (Epo-ELISA, medac) according to the manufacturer’s instructions. Twenty-five microliters of each medium sample was used without any prior dilution.

cDNA synthesis Preparation of total protein and protein quantification Reverse transcription was performed with 300 ng of total RNA as template and 0.25 Ag random hexamers (Promega) as primer using 12.5 U AMV reverse transcriptase (peqLab) in a total volume of 20 Al (20jC for 10 min, 42jC for 1 h and 95jC for 5 min). Real-time-PCR analysis For LightCycler reaction, a master mix of the following reaction components was prepared to the indicated endconcentration: 12.6 Al H2O, 2.4 Al MgCl2 (4 mM), 0.5 Al forward primer (0.5 AM), 0.5 Al reverse primer (0.5 AM), and 2.0 Al Fast Start DNA Master SYBR Green I (Roche Diagnostics). Two-microliter cDNA was added to 18 Al LightCycler master mix as PCR template. The primer sequences used in this study are given in Table 1. The

Table 1 The primer sequences used in this study Gene

Primer sequences (F, forward; R, reverse)

Product size bp

Erythropoietin

F: GCC AGA GGA ACT GTC CAG AG R: ATG GTA GGT GCG AAA ACA GG F: GGG GAG AAA GGA TTT GGC TA R: ACA TGC TTG CCA TCC AGC C F: GGT ATC GGT TGA AGA ATG G R: GGT ACT TGT CTG GAT GGG TC F: GCA GAA TCA TCA CGA AGT GG R: GCA TGG TGA TGT TGG ACT CC F: TCA TCC ATG TGA CCA TGA GG R: TTC ATA TCC AGG CTG TGT CG F: CGG CTA CCA CAT CCA AGG AA R: GCT GGA ATT ACC GCG GCT

206

Cyclophilin AdoHcy hydrolase VEGF-A [54] HIF-1a [54] 18S rRNA [55]

Cells were harvested by incubation with trypsin/EDTA and then collected by centrifugation at 200  g for 10 min. The cell pellet was washed with PBS and cells were homogenized in Tris –HCl (20 mM, pH 7.2) by sonification. The homogenate was centrifuged at 20,000  g for 15 min and supernatant was processed as described below. The protein concentration was determined according to the method of Bradford using a protein assay kit (BioRad) with bovine serum albumin (BSA) as standard. Isoelectric focusing Isoelectric focusing was performed in a 0.5-mm polyacrylamide gel with a final concentration of T5C3 acrylamide and N,N-methylene-bisacrylamide and 6.5% carrier ampholytes divided in six parts pH 4 –6; one part pH 5 – 6 and three parts pH 3 – 10. Anolyte and catholyte were 1 M H3PO4 and 1 M NaOH, respectively. Focusing was performed at 1500 V, 25 mA, and 5 W for 4500 Vh, 6 W for 750 Vh, 7 W for 500 Vh, and 8 W for 250 Vh. Western blotting analysis

256 159 212 345 187

After isoelectric focusing, proteins were transferred from the gel onto a nitrocellulose membrane by upward blotting for 1 h. The membrane was blocked in PBS and 5% defatted milk powder overnight, then washed three times with Tween/PBS for 10 min. The first antibody (rabbit antiAdoHcy hydrolase; [27]) was carried out for 3 h in PBS + 0.5% BSA at a dilution of 1:500. The membrane was washed and the second antibody (goat –anti-rabbit coupled

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to horseradish peroxidase, Dako) reaction was performed for 2 h in PBS + 0.5% BSA at a dilution of 1:1500. The membrane was washed three times and the staining of the membrane was performed using 4-chloro-1-naphthol, H2O2, and ortho-toluidine. When color appeared on the membrane, the reaction was stopped by washing with water. Global DNA-methylation using cytosine extension assay Global DNA methylation was determined as described by Pogribny et al. [28]. Genomic DNA was isolated using DNeasy Tissue Kit (Qiagen) and 2 Ag were digested for 15 h with 20 units HpaII (Fermentas) according to the manufacturer’s instructions. A second DNA aliquot was incubated without enzyme and served as background control. For cytosine extension assay 0.5 Ag DNA were incubated with 1  NH4-reaction buffer for PANScript Polymerase, 1 mM MgCl2, 0.25 units PANScript DNA Polymerase (PAN) and 0.1 Al [3H]dCTP (40 – 60 Ci/mmol, NEN Life Science Products) in a total volume of 25 Al for 1 h at 56jC. Duplicate 10-Al aliquots from each reaction were filtered through Whatman DE-81 ion exchange filters and filters were washed three times with 3 ml 0.5 M sodium phosphate buffer (pH 7.0). Radioactivity incorporated in DNA and adsorbed on the filters was determined by liquid scintillation counting. Calculation and statistics Results are expressed as mean F SEM. Data were analyzed by Student’s t test or Alternate t test (InStat). A difference between groups was considered to be significant when P value was <0.05.

Results Changes in methylation potential by inhibition of AdoHcy hydrolase Under control conditions, the intracellular concentration of AdoMet and AdoHcy is 1.76 F 0.1 nmol/107 cells

and 0.034 F 0.002 nmol/107 cells, respectively, resulting in a AdoMet/AdoHcy ratio of 53.4 F 3.3 (Table 2). Inhibition of AdoHcy hydrolase by Ado-2V,3V-dial was found to decrease AdoMet/AdoHcy ratio in a concentration-dependent manner. Inhibition of AdoHcy hydrolase by 1 AM Ado-2V,3V-dial results in a decrease of the AdoMet/AdoHcy ratio to 7.7 F 1.3. The maximal inhibitory effect was observed with 30 AM Ado-2V,3V-dial (Fig. 2A). Ado + Hcy were also found to lower the methylation potential in a dose-dependent manner, with maximal effect occurring at 1 mM Ado + Hcy (Fig. 2B). Therefore for all following experiments, Ado-2V,3V-dial was chosen in a concentration of 30 AM and Ado + Hcy 1 mM. Inhibition of AdoHcy hydrolase by Ado-2V,3V-dial 30 AM results in an enhanced accumulation of intracellular AdoHcy to 2.25 F 0.16 nmol/107 cells, which is about 70 times the amount in control cells (Table 2). Furthermore, Ado-2V,3V-dial leads to a 1.8-fold increased AdoMet concentration. Therefore, the MP decreased to 1.4 in Ado-2V,3V-dial-treated cells. Highest intracellular AdoHcy and AdoMet concentrations were measured after the simultaneous addition of the AdoHcy hydrolase substrates (Ado + Hcy) to the culture medium: a 1000fold increased AdoHcy level is accompanied by an 8fold increased AdoMet concentration (compared to control), which means that the MP is reduced by 99% (Table 2). Immunological detection of native AdoHcy hydrolase after isoelectric focusing on ampholine-PAG reveals one band under control conditions and after incubation with Ado + Hcy or hypoxia. After inhibition of AdoHcy hydrolase with Ado-2V,3V-dial, no band can be detected (Fig. 3) because binding of Ado-2V,3V-dial to AdoHcy hydrolase leads to changes in the conformation of the enzyme structure [6]. Effect of hypoxia on the methylation potential Hypoxia results in a 4-fold increase of MP to 239.4 F 24.8, which is due to a 2-fold increased AdoMet concentration and a decreased AdoHcy concentration

Table 2 Influence of hypoxia on intracellular AdoMet and AdoHcy concentration under control conditions and inhibition of AdoHcy hydrolase Experimental condition

n

AdoMet nmol/107 cells

AdoHcy nmol/107 cells

AdoMet/AdoHcy

Control Hypoxia 30 AM Ado-2V,3V-dial 30 AM Ado-2V,3V-dial + hypoxia 1 mM Ado + Hcy 1 mM Ado + Hcy + hypoxia

25 13 6 5 10 5

1.76 4.24 3.16 8.29 15.12 8.72

0.034 0.019 2.25 1.56 40.22 3.75

53.42 239.44 1.40 5.24 0.44 5.85

F F F F F F

0.10 0.55* 0.28* 1.86** 2.09* 1.27

F F F F F F

0.002 0.003* 0.16* 0.27** 6.20* 1.58

F F F F F F

3.25 24.82* 0.06* 0.45** 0.07* 2.39**

Cells were incubated with the indicated substances for 24 h under normoxic (21%) and hypoxic (1%) conditions in serum-free RPMI 1640. The cells were extracted with perchloric acid and the metabolites were separated by HPLC as described in Materials and methods. Data are averages F SEM for n determinations. * P < 0.01 versus control. ** P < 0.01 versus hypoxia.

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Hcy 5.9), but the MP is always higher than under normoxic conditions after administration of the same substances. This is due to a reduced endogenous AdoHcy concentration. Effect of hypoxia on mRNA expression Real-time-PCR analysis showed that erythropoietin mRNA levels are three times increased in HepG2 cells grown in an atmosphere containing 1% oxygen and 5% CO2 for 24 h compared to levels in comparable cells grown in an atmosphere containing 21% oxygen (Fig. 4A). A 5fold up regulation of gene expression induced by hypoxia is found in VEGF-A (Fig. 4B). In contrast, AdoHcy hydrolase gene expression is significantly lowered by hypoxia (30% versus control; Fig. 5A). The expression

Fig. 2. Effects of increasing concentrations of Ado-2V,3V-dial (A) and Ado + Hcy (B) on the AdoMet/AdoHcy ratio in HepG2 cells. The Hcy concentration was 1 mM when cells were incubated with different Ado concentrations. After incubation with the substances for 24 h in serum-free RPMI 1640, cells were harvested, extracted with perchloric acid and the metabolites were separated by HPLC as described in Materials and methods. Data are mean F SEM for 5 – 25 determinations.

(Table 2). Like under normoxic conditions, the inhibition of AdoHcy hydrolase results in a decreased MP versus hypoxic control (Ado-2V,3V-dial 5.2 and Ado +

Fig. 3. Isoelectric focusing of total cellular protein. Cells were grown under control conditions (1) or incubated with 30 AM Ado-2V,3V-dial (2, 3) and 1 mM Ado + Hcy (4, 5) or grown under hypoxia (6, 7) for 24 h in serum-free RPMI. Proteins were separated by isoelectric focusing, blotted onto nitrocellulose membrane. AdoHcy hydrolase was detected immunologically by a specific antibody.

Fig. 4. Changes in relative gene expression of erythropoietin (A) and VEGF-A (B) estimated by real-time-RT-PCR. HepG2 cells were cultured for 24 h under the indicated conditions. The relative expression level of each gene was calculated according to Pfaffl [26]. The expression of 18S rRNA served as internal standard. Data represent mean F SEM for 3 – 18 experiments; *P < 0.05 versus control; §P < 0.05 versus hypoxia.

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of cyclophilin and HIF-1a is independent of the O2concentration (Figs. 5B, C). Influence of an altered methylation potential on gene expression Inhibition of AdoHcy hydrolase with Ado-2V,3V-dial leads to a decreased erythropoietin mRNA expression under both normoxic and hypoxic conditions. Changes in AdoMet/ AdoHcy ratio by the substrates of AdoHcy hydrolase (Ado + Hcy) also lower erythropoietin mRNA levels (Fig. 4A). In contrast, the VEGF-A expression is only reduced by Ado-2V,3V-dial under normoxic conditions. Incubation with Ado + Hcy leads to a 2-fold induction of VEGF-A expression compared to control (Fig. 4B). However, in the presence of Ado-2V,3V-dial or Ado + Hcy, hypoxia causes only a 2-fold increase in VEGF-A mRNA level. This means that reduction of methylation potential decreases VEGF-A expression about 60%, but only under hypoxia. The expression of the housekeeping genes cyclophilin and AdoHcy hydrolase as well as the expression of HIF-1a are not or only slightly affected by an altered methylation potential (Fig. 5). Inhibition of mRNA methylation and stimulation of mRNA synthesis To investigate whether AdoHcy hydrolase activity is required for efficient mRNA methylation and synthesis, HepG2 cells were simultaneously labeled with [14C]uridine and L-[methyl-3H]-methionine in the presence or absence of inhibitors of AdoHcy hydrolase. After cells were incubated under conditions to increase AdoHcy, the amount of [3H]-CH3 incorporated in poly(A)-RNA was reduced about 40% compared to control (Table 3). In contrast, [14C]-uridine incorporation into mRNA was increased after adding Ado-2V,3V-dial or Ado + Hcy 1.3-fold.

Table 3 Effects of AdoHcy hydrolase inhibition on mRNA methylation

Fig. 5. Relative amounts of AdoHcy hydrolase (A), HIF-1a (B) and cyclophilin (C) mRNA levels determined by real-time-RT-PCR. HepG2 cells were cultured for 24 h under the indicated conditions. The relative expression level of each gene was calculated according to Pfaffl [26]. The expression of 18S rRNA served as internal standard. Data are mean F SEM for 3 – 20 determinations. *P < 0.05 versus control; §P < 0.05 versus hypoxia.

Treatment

n

3 H incorporated %

14 C incorporated %

3

H/14C

Control 30 AM Ado-2V,3V-dial 1 mM Ado + Hcy

6 2

96.7 F 1.6 61.9 F 1.4*

63.0 F 3.6 84.2 F 7.5*

1.56 F 0.08 0.63 F 0.01*

4

58.6 F 5.8*

80.3 F 6.8*

0.73 F 0.05*

Cells were incubated with the indicated substances and labeled with L[methyl-3H]-methionine and [14C]-uridine as described under Materials and methods. The polyadenylated mRNA was isolated and the amount of 3H and 14C incorporated in mRNA was determined by liquid scintillation counting. The numbers are percentages with respect to the highest value observed in one experiment. Data represent averages F SEM for n determinations. * P < 0.05 versus control.

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The effect on mRNA methylation was determined by the changes in the ratio 3H/14C in mRNA. Ado-2V,3V-dial decreases 3H/14C ratio about 60%, Ado + Hcy about 55% (Table 3). Furthermore, we were able to show that Ado-2V,3Vdial inhibits mRNA methylation and stimulates mRNA synthesis in a dose-dependent manner (data not shown): even application of 1 AM decreases the 3H/14C ratio. The maximal inhibition of mRNA methylation was reached at concentrations of 100 AM, which correlates with the dosedependent modification of AdoMet/AdoHcy ratio by this inhibitor (Fig. 2A). Effect on erythropoietin expression Under control conditions, HepG2 cells show a constitutive release of erythropoietin into the culture medium of 5.33 F 0.32 mIU/106 cells (Fig. 6). The exposure of HepG2 cells to hypoxia for 24 h results in a 2-fold increase in erythropoietin production. Treatment with Ado-2V,3V-dial decreases expression about 40% under both normoxic and hypoxic conditions. The lowest erythropoietin level is detected after administration of Ado + Hcy. Under normoxic as well as under hypoxic conditions, the erythropoietin level is only about 2 mIU/106 cells.

Fig. 7. Comparison of the global DNA methylation in Ado-2V,3V-dial- and Ado + Hcy-treated HepG2 cells. Cells were grown under the different conditions for 24 h. The final concentrations of Ado-2V,3V-dial and Ado + Hcy were 100 AM and 1 mM each, respectively. Global DNA methylation was determined using cytosine extension assay as described in Materials and methods.

Ado + Hcy for 24 h does not change the amount of radioactivity incorporated into DNA compared to control (Fig. 7).

Influence of an altered MP on global DNA methylation The methyl-sensitive restriction enzyme HpaII cuts at non-methylated recognition sites leaving a guanosine overhang, where [3H]dCTP is incorporated by DNA polymerase. Therefore, the amount of radioactivity incorporated into the DNA reflects the number of unmethylated cytosine residues. The administration of Ado-2V,3V-dial or

Fig. 6. Comparison of erythropoietin concentrations in the culture medium of cells exposed to Ado + Hcy or Ado-2V,3V-dial under normoxia and hypoxia. Cells were grown under the different conditions for 24 h. Data represent averages F SEM for 3 – 6 experiments. Erythropoietin released into the medium was determined with an Epo-ELISA. *P < 0.05 versus control; §P < 0.05 versus hypoxia.

Discussion The ratio of AdoMet/AdoHcy concentrations, also termed methylation potential (MP), is considered as an indicator of the flow of methyl groups transferred from AdoMet to an acceptor [4]. In this study, the impact of MP on transmethylation reactions was analyzed on mRNA and DNA level. To determine MP, a sensitive HPLC method for simultaneous measurement of AdoMet and AdoHcy was used [24]. To alter MP, we chose two different methods to increase AdoHcy concentration in HepG2 cell line. First, AdoHcy level was increased by inhibition of the AdoHcy metabolizing enzyme AdoHcy hydrolase with Ado-2V,3V-dial, which appears to be selective for AdoHcy hydrolase [29]. Cory and Mansell [30] showed that 630 AM Ado-2V,3V-dial lowers the ribonucleotide reductase activity in treated tumor cells by 57%. Since the inhibitor concentrations used in our experiments is about 30 AM, we do not expect any effect on ribonucleotide reductase activity. In contrast to other potent AdoHcy hydrolase inhibitors, for example, 3-deazaadenosine [31], Ado-2V,3V-dial does not serve as substrate for AdoHcy hydrolase and is therefore a useful probe to study AdoMet-dependent transmethylations. Moreover, this inhibitor does not interfere with the HPLC measurement of Ado and AdoHcy, since Ado-2V,3V-dial has another retention time (data not shown). The second method to decrease the MP is the induction of AdoHcy synthesis by administration of Ado + Hcy [32,33]. Both, Ado-2V,3V-

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dial and Ado + Hcy, lead to a dose-dependent decrease in MP from 53.4 (control) to 1.4 and 0.44, respectively (Table 2). The MP of 53.4 in HepG2 cells is within the physiological range obtained by in vivo experiments [34]. In our experiments, hypoxia leads to increased AdoMet and decreased AdoHcy levels resulting in a 4-fold increased MP (Table 2). Since AdoHcy is a potent inhibitor of AdoMet-dependent methyltransferases [4,35], a low AdoHcy level leads to a high MP and enables a high transmethylation activity. Hypoxia arises in early development of rapidly proliferating tumor cells [36,37] and leads to the expression of several genes, for example, erythropoietin, VEGF [38,20], glycolytic enzymes [39], and inducible nitric oxide synthase [40]. An increased MP could therefore be an advantage for transcriptional active and rapidly proliferating tumor cells because of an increased methylation capacity. Real-time PCR analysis revealed that erythropoietin expression was enhanced by hypoxia in HepG2 cells confirming previous data [19,21]. Decreasing the MP by Ado2V,3V-dial or Ado + Hcy was associated with lowered erythropoietin mRNA levels under both normoxic and hypoxic conditions. The erythropoietin release into the medium goes parallel to mRNA expression. To compare erythropoietin expression with another factor induced by hypoxia, VEGFA mRNA expression was included in the experiments. VEGF-A mRNA expression was 5-fold increased by 24 h hypoxia and was decreased by Ado-2V,3V-dial under normoxia and hypoxia (Fig. 4). In contrast, reduction of MP by Ado + Hcy decreased VEGF-A expression only under hypoxia (compared to hypoxic control cells). Under normoxia, VEGF-A expression was 2-fold enhanced when cells were incubated with Ado + Hcy. This could be explained by the observation that Ado stimulates or inhibits the release of VEGF, dependent on the cell type examined [41 –43], because of differential expression of Ado receptor subtypes [44]. The expression of the housekeeping genes, AdoHcy hydrolase and cyclophilin, was not affected by an altered MP. Also HIF-1a expression was neither stimulated by hypoxia nor by an altered MP, which is consistent with the observation that HIF-1a is regulated by posttranscriptional modification [45 –47]. We also found that a decreased MP reduces overall mRNA methylation, probably by AdoHcy product inhibition of mRNA-methyltransferases. Similar results were also obtained by Backlund et al. [11] using 3-deazaadenosine and 3-deazaaristeromycin as AdoHcy hydrolase inhibitors. Radomski et al. [5] showed that inhibitors of AdoHcy hydrolase interfere with mRNA methylation and elongation of poly(A)+ RNA in Xenopus laevis. Moreover, they suggest a close association of AdoHcy hydrolase to the transcriptional elongation complex to prevent product inhibition of RNA guanine-7-methyltransferase [5,48]. Interestingly, in our experiments, mRNA synthesis was

increased by Ado-2V,3V-dial and Ado + Hcy, whereas mRNA methylation was reduced. These results obtained in human HepG2 cell line are comparable to observations by Furuichi [49], who showed that AdoHcy functions in two different ways in cytoplasmic polyhedrosis virus. First, as a strong stimulator of RNA synthesis and second, as a potent methylation inhibitor, resulting in the synthesis of viral mRNA containing blocked (GpppA) and unblocked (ppA) rather than capped (m7 GpppA) 5V-termini. This leads to the assumption that in our experiments, the increased total mRNA synthesis could also be the result of increased AdoHcy levels. Although the effects of AdoHcy hydrolase inhibition on mRNA-methylation have been studied extensively, the effects on mRNA and protein levels of distinct genes have not been investigated until now. Our results show that a decreased methylation potential is associated with reduced erythropoietin and VEGF-A mRNA levels (Fig. 4). One explanation for the lowered erythropoietin and VEGF-A mRNA levels could be decreased stability of the respective mRNAs, which could be the result of unblocked 5V-termini in the presence of elevated AdoHcy levels [49,50]. The cap structure protects mRNA from degradation and thus increases its stability, uncapped mRNAs is degraded by 5V-3V-exoribonuclease before translation takes place [50,51]. This assumption is supported by erythropoietin protein levels, which are also lowered under a decreased MP (Fig. 6). Another explanation for decreased erythropoietin and VEGF-A mRNA levels could be an altered active transcription rate of both genes, which can be studied by nuclear run-on assays [52]. Whether reduced mRNA levels are associated with a decreased mRNA stability or with a reduced transcription rate remains to be determined. In contrast, AdoHcy hydrolase, cyclophilin and HIF-1a mRNA levels are not affected by an altered MP within 24 h. This observation could be caused by a longer half-life time of the respective mRNAs which might be insensitive to short-term changes in MP. The fact that a reduced MP does not change the global DNA methylation suggests that the differential gene expression is not associated with changes of methylation pattern of the DNA. Moreover, these experiments show that DNA methylation is not influenced by short-term alterations in MP in our model although the MP is reduced about 98%. This observation is not amazing since DNA methylation occurs concurrently with replication during S-phase of cell cycle [53]. Yi et al. [9] observed that even a 2-fold increased AdoHcy-level results in DNA hypomethylation in human lymphocytes and Caudill et al. [8] showed that a decreased AdoMet/AdoHcy ratio correlates with global DNA hypomethylation in tissues of methyl-deficient cystathionine ßsynthase heterozygous mice. In summary, our results show that the mRNA levels of the studied genes respond differentially to short-term changes in MP. Thus, the results do not support the concept of a general importance of MP for gene expression.

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Acknowledgments The authors wish to thank Dr. Ch. Ko¨hle and Dr. S. Harsch for valuable suggestions concerning LightCycler analysis and primer sequences. They are grateful to Dr. U. Delabar for help with HPLC measurements, R. Riehle for technical assistance and T. Rieg for computational assistance. This work was supported by a grant from the DFG Graduiertenkolleg 686-1.

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