Influence of EMAP II , IFN-2 b and its medicinal preparations on the MGMT protein amount in human cells in vitro

Aim. To study the effect of EMAP II, IFN2b and its medicinal preparations on the amount of O 6 -methylguanine-DNA methyltransferase (MGMT) protein in human cells in vitro. Methods. The human cells of 4BL and Hep-2 lines were treated with the purified recombinant proteins EMAP II, IFN2b and its commercial me dicinal preparations. Changes in the MGMT gene expression were studied at a protein level by Western blot analysis. Results. Treatment of Hep-2 and 4BL cells with EMAP II at the concentrations of 0.02 g/ml and 2 g/ml respectively led to induction of the MGMT gene expression. EMAP II at the concentrations of 0.2–20 g/ml caused decrease of the MGMT protein amount in Hep-2 cells. The regulating activity of EMAP II was also observed for MARP (anti-Methyltransferase Antibody Recognizable Protein). IFN2b and Laferon-PharmBiotek with the activity of 200 and 2000 IU/ml were shown to cause an increase of the MGMT protein amount in Hep-2 cells. Conclusions. The purified recombinant proteins EMAP II and IFN2b which are substrates for the medicinal preparations influenced on the amount of MGMT protein in the human cell cultures in a concentration-dependent manner. At the same time the effect of medicinal preparations differs from that of the purified protein IFN2b. Possibly it depends on the presence of stabilizing components in their compositions.

Introduction.The repair enzyme O 6 -methylguanine-DNA methyltransferase (MGMT) eliminates O 6 -methylguanin adducts in DNA and protects normal cells from damaging effects of alkylating agents.At the same time MGMT makes tumor cells resistant to alkylating drugs such as temozolomide [1,2].
Therefore, in medicine MGMT is considered as a target which needs to be regulated.Various regulators of the MGMT enzyme activity and expression are known but they are often toxic not only for tumor but also for normal human cells [3].Cytokines are natural factors and some of them were shown to be promising in the MGMT gene expression regulation.For example, IFN-b downregulated the MGMT transcription and sensitized the re-sistant glioma and neuroblastoma cells to temozolomide in vitro [4,5].One of the members of interleukin family -IL-24 down-regulated the MGMT gene expression via activation of p53 and therefore helped to overcome the melanoma cells resistance to temozolomide [6].
Interferons elicit pleiotropic biological effects, so they are widely used either alone or in combination with other antitumor agents, in particular with nitrosoureas.Preadministration of interferons to patients might be a part of a novel biochemotherapy approach that may help to overcome the resistance to alkylating drugs.The combined therapy with IFN-b and temozolomide was shown to provide better clinical outcomes in the patients whose tumor cells had an unmethylated MGMT promoter [7].For the patients with progressive malignant glioma, the complex therapy with BCNU and IFN-a2b (Intron A, «Schering Corporation», USA) appeared to be a feasible and promising treatment strategy.The effect of IFN-a2b on the cell sensitivity to BCNU was proposed to be implemented by inhibition of the MGMT gene expression [8].However, this suggestion needs to be confirmed.
EMAP II (endothelial monocyte-activating polypeptide II) is a multifunctional cytokine, which is formed in malignant tumors of mammals due to the alternative splicing and posttranslational processing of its precursor -the p43 protein [9].EMAP II suppresses the endothelial cell migration; stimulates their apoptosis and influences the activity of monocytes, neutrophils and macrophages, facilitating inflammatory processes in tumors [10].The antitumor activity of this cytokine was evidenced in the experimental models of glioma, sarcoma, stomach and pancreas cancer [11].
In our previous works different exogenous cytokines, growth factors and plant components (extracts, lectins) were shown to be able to change the level of the MGMT gene expression in some human cell cultures [12,13].To our opinion IFN-a2b and EMAP II which possess antitumor activity were the most promising agents for this purpose.One of the important questions in this study was determination of the dependence of IFN-a2b and EMAP II action on their concentration.Moreover the another problem has arrived: does the effect of cytokine medicinal preparation differ from that of their purified substances?
Additionally it should be noted that the human MGMT protein has a molecular weight of 22-24 kDa [14].However, in our previous works the Western blot analysis with monoclonal anti-MGMT antibodies, clone 23.2, revealed two highly specific immunoreactive bands: 24 kDa (classic MGMT protein) and 48 kDa (anti-Methyltransferase Antibody Recognizable Protein or MARP) [12,15].In those works the MARP nature and its induction by exogenous cytokines in human cells in vitro have been discussed.
The aim of the present work was to compare the effect of different concentrations of purified IFN-a2b and EMAP II and medicinal preparations of IFN-a2b on the MGMT and MARP protein amount in human cell cultures.
For cytokine treatments, 8 × 10 5 cells were plated and allowed attaching during 24 h.The next day, the cells were treated with cytokines and cytokine-containing preparations in the serum-free DMEM growth medium.After 8-h exposure the medium was removed, the cells were rewashed with PBS buffer and harvested in DMEM with 10 % of serum during 16 h.The cells were then trypsinized, washed, centrifuged, and stored at -20 o C. Control intact cells were subjected to the similar procedure but without adding cytokines.
Protein extracts were obtained from cell pellets in lysis buffer (50 mM Tris HCl; 0.1 mM EDTA; 5 mM DTT; pH 7.5).The suspensions were incubated on ice, exposed to three 10-s pulses of sonication with 30-s intervals and 50 mM of PMSF in ethanol were added.Sonicates were then centrifuged at 13,000 g for 30 min at 4 o C. The supernatants were collected and frozen at -80 o C for later use.SDS-PAGE (12 % gel) was performed by Laemmli method [21].
The concentration of total protein in cell lysates was measured colorimetrically according to Bradford method [22].
The following antibodies were used: anti-MGMT monoclonal antibodies, clone 23.2, isotype IgG2b («Novus Biologicals», USA), secondary antibodies conjugated with horseradish peroxidase («Sigma», USA).The procedure of MGMT identification in the samples was performed by Western blot analysis according to the methodological instructions of the manufacturer of mAbs [23].Densitometry of stained membranes was used for loading control according to [24] by ScionImage 4.0.2 and Origin 8.1 programs.
Results and discussion.It was shown by Western blot analysis that cytokine EMAP II at a concentration of 0.02 mg/ml induced the MGMT gene expression (Fig. 1, lane 2) in a clone of Hep-2 cells which did not express the MGMT gene under normal conditions (Fig. 1, lane 1).However, an increase of EMAP II doses to 0.2-20 mg/ml did not lead to any changes of the MGMT protein amount in these cells (Fig. 1, lanes 3-5).After the treatment of Hep-2 cells, that normally expressed the MGMT gene, with cytokine EMAP II at a concentration of 2 mg/ml we observed decrease of the MGMT protein amount (Fig. 1, lanes 6 and 7).Thus, the EMAP II effect on the MGMT protein amount in Hep-2 cells differs depending on concentrations.
In our previous work, 4BL cells were shown to lose the possibility to express the MGMT gene during more than 130 passages cultivation and cell line stabilization [26].However, the treatment of these cells (137 passage) with cytokine EMAP II led to induction of the MGMT gene expression (Fig. 2), which allowed us to suggest reversibility of the MGMT gene silencing.Induction of the MGMT gene expression in 4BL cells was detected after 8-h incubation with EMAP II at a concentration 2 mg/ml.However this effect almost disappeared during longer incubation time -16 and 32 h (Fig. 2, lanes 3 and 4).A reduction of the MGMT protein amount may be the result of increasing time of serum-free conditions.
The regulating activity of EMAP II was observed not only for the MGMT protein but also for MARP.The MARP protein is stably expressed in both Hep-2 and 4BL cells in contrast to MGMT.An inverse concentration dependence was observed after treatment of Hep-2 cells with recombinant protein EMAP II: at concentrations of 0.02 and 0.2 mg/ml it induced the MARP expression, but at higher concentrations (2 and 20 mg/ml) it inhibited the MARP expression (Fig. 3, lanes 2-5).EMAP II caused a slight increase of the MARP amount in 4BL cells (Fig. 3, lanes 7-9).
In our earlier work IFN-a2b-containing preparation Laferobion («Biofarma») at concentrations of 200 and 2000 IU/ml was shown to cause a dramatic decrease in the MGMT protein amount in Hep-2 cells [12].However, in our current work it was shown that IFN-a2b at concentrations of 200 and 2000 IU/ml increases the MGMT protein amount in Hep-2 cells (Fig. 4, lanes 2 and 3).The similar tendency was observed after treatment of Hep-2 cells with Laferon-PharmBiotek preparation («Interpharmbiotech») (Fig. 4, lanes 6 and 7).
At the same time no changes in the MGMT protein amount were observed after treatment of 4BL cells with IFN-a2b and Laferon (data not shown).
Laferon-PharmBiotek and Laferobion preparations have similar composition (recombinant human IFN-a2b, salts, dext ran) but they showed an opposite effect on the MGMT protein amount.According to the literature data, a preparation Intron A (recombinant IFN-a2b, EDTA, NaCl, m-Kresol, Polysorbate 80, Na 2 HPO 4 , NaH 2 PO 4 ) decreased the MGMT gene expression in patients, who had progressive malignant glioma [8].Thus Laferobion and Intron A showed similar regu lating effects although they have different composition of stabilizing components [8,12].According to these data we may suggest that regulating effect of the IFN-a2b-containing preparations depends not only on the composition but also on their purification degree.Therefore these results could be useful for planning the experiments with the IFN-a2b-containing preparations and for IFN-a2b therapy.
There are several hypotheses about the mechanisms of the MGMT gene expression regulation under the influence of cytokines.According to one of them IFN-b regulates the MGMT transcription via p53 protein and specificity protein 1 (Sp1) [4,5].Another one suggests that IFN-b activates transcriptional factor NF-kB and thus affects the transcription of MGMT gene, which belongs to the NF-kB target genes [26].According to the literature data, all IFNs of type I (IFN-a, IFN-b, IFN-e, IFN-w) bind to the specific cell surface receptor complex known as the IFN-b receptor (IFNAR) [27].Therefore we suppose that regulation of the MGMT gene expression by IFN-b and IFN-a2b may involve the similar pathways (Fig. 5).
The way of EMAP II impact on the MGMT gene expression remains unclear.In some works it was shown that EMAP II at low and high concentrations activates various signaling pathways in cells [28,29].For instance, in a blood-tumor barrier (BTB) model, EMAP II at low concentration (0.05 nM) induced three isoforms of protein kinase C (PKC): PKC-a, b, and z, and, through them, caused functional, biochemical, and morphological alterations in BTB [29].PKC is known to regulate the MGMT gene expression [30].Its regulating effect is mediated by binding to an activator protein 1 (AP-1) and further binding to the AP-1 site in the MGMT gene promotor, affecting the MGMT gene transcription.Moreover, EMAP II can also activate the NF-kB transcription factor [31].Therefore we suppose that regulation of the MGMT gene expression by IFN-a2b and EMAP II occurs with participation of transcription factors NF-kB, Sp1 and AP-1.A scheme of possible cell signaling pathways involved in these processes is shown in Fig. 5.We plan further study of the mechanisms of the MGMT gene expression regulation under the influence of cytokines to establish a connection between the action of cytokines and the repair processes in human cells.
Conclusions.The EMAP II influence on the MGMT protein amount depends on the concentration.The treatments with cytokine IFN-a2b at concentrations of 200 and 2000 IU/ml lead to the increase of MGMT protein amount in Hep-2 cells.The effect of the purified protein IFN-a2b on the MGMT and MARP protein amounts differs from that of medicinal preparations.Possibly it depends on the presence of stabilizing components in their compositions.