ISSN 00954527, Cytology and Genetics, 2013, Vol. 47, No. 4, pp. 202–209. © Allerton Press, Inc., 2013. Original Russian Text © K.V. Kotsarenko, V.V. Lylo, L.L. Macewicz, L.A. Babenko, A.I. Kornelyuk, T.A. Ruban, L.L. Lukash, 2013, published in Tsitologiya i Genetika, 2013, Vol. 47, No. 4, pp. 9–18.
Change in the MGMT Gene Expression under the Influence of Exogenous Cytokines in Human Cells In Vitro K. V. Kotsarenko, V. V. Lylo, L. L. Macewicz, L. A. Babenko, A. I. Kornelyuk, T. A. Ruban, and L. L. Lukash Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine email:
[email protected] Received May 14, 2012
Abstract—The influence of cytokines LIF, SCF, IL3, and EMAP II and the Laferobion (IFNa2b) drug on the MGMT gene expression in human cell cultures has been studied. It was shown that exogenous cytokines can modulate the MGMT gene expression at the protein level. EMAP II is able to increase or decrease the MGMT level, depending on the experimental conditions. Cytokines LIF, SCF, IL3 and Laferobion decreased the MGMT expression level in human cells in vitro. Some conditions leading to the destruction of the MGMT protein complex were identified. Keywords: human cell culture, reparative enzyme MGMT, expression regulation, cytokines, protein dimers DOI: 10.3103/S0095452713040087
INTRODUCTION In the repair of primary DNA lesions caused by alkylating compounds, reparative enzyme O6meth ylguanineDNA methyl transferase (MGMT), encoded by the MGMT gene, plays a key role [1]. Although DNA can be alkylated at different sites, the introduction of alkyl groups in the O6 position of gua nine has the strongest carcinogenic, mutagenic, and cytotoxic potential [2]. Methyl transferase transfers the alkyl group from O6methylguanine to its own cysteine residue at the active site, which leads to DNA repair and irreversibly inactivates MGMT [1, 3]. Since the action of enzyme MGMT is one of the key mechanisms in protecting an organism from alky lating agents, it is important to identify the factors that influence its expression. Studying the regulation of the expression of the gene encoding reparative enzyme MGMT has both fundamental (understanding the regulation pathways of gene expression and the struc ture (function) of the protein) and practical (develop ment of effective inhibitors (activators of MGMT) importance [3]. For example, a decrease in the MGMT gene expression in tumor cells is a relevant problem in oncology because this increases their sensitivity to chemotherapy based on alkylating compounds [4]. At the same time, an increase in the level of expression is important for the protection of normal cells from the action of alkylating agents. According to published data [5–7], the level of MGMT gene expression is influenced by various fac tors, such as alkylating agents, singlestranded DNA breaks, and transcription factors. A special role is
played by the hypermethylation of the MGMT gene promoter. The expression level can also be regulated indirectly through various intracellular signaling path ways (for example, those involving the p53 protein) [6]. Some published data indicate that certain cytok ines can also affect the MGMT gene expression level (see table). Recently, the study of the effect of cytokines on the MGMT gene expression level has transited from cell cultures to the bodily level. For example, in the treat ment of patients with newly diagnosed primary glioblas tomas, the administration of IFNβ simultaneously with the chemotherapeutic drug temozolomide con tributed to a favorable outcome, especially in patients with an unmethylated MGMT gene promoter [12]. For our study, we have selected a number of cytok ines with different directions of biological action, which are involved in various regulatory signaling pathways. Recombinant interferon α2b (IFNα2b) is widely used in medicine due to its antiviral and anti cancer activity, particularly in oncology. Therefore, of great interest is the possibility to use this cytokine to regulate the MGMT gene expression level both in nor mal and neoplastic human cells. According to some published data, the expression of the gene encoding this reparative enzyme in cells can be modified during their differentiation [13]. With this in mind, to study the possibilities of influencing its expression, we selected cytokines, such as LIF, IL3, and SCF, which are directly involved in cell division, growth, and dif ferentiation. In addition to these cytokines, we contin ued to study cytokinelike protein EMAP II, which fulfills a multitude of functions in cells. For example,
202
CHANGE IN THE MGMT GENE EXPRESSION UNDER THE INFLUENCE
203
Cytokines affecting the MGMT gene expression level Cytokines
Literature source
Result
IFNβ (100 U/mL)
Reduces the level of MGMT gene expression and sensitizes human glioma cells to temozolomide Recombinant human IFNβ (50 U/mL) Reduces the level of MGMT gene expression in human neuroblastoma cells IL24 (from 0 to 39 ng/mL) Reduces the level of MGMT gene expression in human melanoma cells in a dosedependent manner IL1β (50 U/mL) + IFNγ (1000 U/mL) Combination of cytokines IL1β + IFNγ increases the MGMT gene expression level in rat pancreatic βcells
it can inhibit the migration and stimulate apoptosis of endothelial cells and affect the activity of monocytes, neutrophils, and macrophages, promoting inflamma tion in tumors [14, 15]. In addition, according to our previous data [16], it stimulates the MGMT gene expression in human cells. The biological properties of these cytokines have long been known; however, their possible role in the regulation of the MGMT gene expression level has not been studied yet. The purpose of this study was to investigate the effect of a number of exogenous cytokines on the level of MGMT gene expression in cultured human cells. MATERIALS AND METHODS In this study, we used human cell cultures derived in our laboratory (fibroblastlike cells 4BL from adult donors' blood, fibroblastlike cells CB1 from umbilical cord blood), standard cell line Hep2 (cancer of lar ynx), and A102 cells (skin fibroblasts), which were kindly provided by Professor McCormick. Cells were cultured in standard growth medium DMEM (Sigma, United States) supplemented with 10% fetal bovine serum (Sigma, United States) and antibiotics penicil lin and streptomycin. We used commercially produced cytokines Lafero bion (Biopharma, Ukraine), LIF, IL3, and SCF (Sigma, United States). Cytokinelike protein EMAP II was obtained in the bacterial system Escherichia coli BL21 (DE3) pLysE/rETZOa EMAPII as described previously [17]. The conditions for the treatment of cells with cytokines in a serumfree culture medium were described in [16]. Cell lysates were prepared as described previously [18]. SDSPAGE of protein extracts was performed in a 12% polyacrylamide gel according to LaemmLi [19]. To destroy dimers, protein extracts prior to SDS PAGE were treated in several ways in a 2X standard sample buffer (250 mM TrisHCl (pH 6.8), 2% SDS, 5% (βmercaptoethanol, 0.5% bromophenol blue, and 20% glycerol) by heating for 5 min in a boiling water bath. To uniformly load proteins in wells, the total protein concentration in each sample was deter mined according to Bradford [20] and 50 μg of protein CYTOLOGY AND GENETICS
Vol. 47
No. 4
2013
[8] [9] [10] [11]
extracts was added to each well. The changes in the MGMT gene expression level were estimated at the protein level by Western blot analysis according to the methodical instructions of the manufacturer of mono clonal antibodies against human MGMT (clone 23.2) Novus Biologicals (Littleton Co., United States) [21]. Speciesspecific antibodies conjugated to horseradish peroxidase (Sigma, United States) were used as sec ondary antibodies. To control the application of anti bodies, we performed a densitometrical analysis of a stained membrane [22]. An image analysis was per formed using the ScionImage 4.0.2 and Origin 8.1 software. The densitometrical analysis results were corrected for the control of loading. RESULTS Our previous studies showed that monoclonal anti MGMT antibodies (clone 23.2), used in Western blot analysis, recognize not only a protein with a molecular weight of 24 kDa, which corresponds to a molecular weight of the MGMT protein according to published data, but also a 48kDa protein [23]. According to our hypothesis, the socalled modified form of the protein (48 kDa) is a homodimer of the classical MGMT pro tein (24 kDa). In this study, we tested this hypothesis using heating (boiling for 1 h) or chemical treatment (0.5 M dithiothreitol (DTT) or 8 M urea) of protein extracts. The results of experiments presented in Fig. 1 show that the exposure of protein extracts to the above mentioned factors resulted in a complete (lanes 2 and 7) or partial (lanes 3–5, 8) destruction of the modified form (48 kDa) of the protein and an increase in the content of the MGMT protein in the unmodified form (24 kDa). Thus, we have obtained evidence that the signal at 48 kDa in Western blot analysis corresponds to the dimeric form of the studied protein and the sig nal at 24 kDa corresponds to its monomers. In further studies, before SDSPAGE, we treated protein extracts under conventional conditions and assayed both the unmodified and modified forms of the protein. Before conducting experiments with cytokines, we tested some experimental conditions. For example, an
204
KOTSARENKO et al. 1
2
3
4
83
80 79 86
5
6
7
8
47 kDa
26 kDa 20 kDa Control of loading, %
90 93 90 88
150 100 50 0
1
2
3
4
5
6
7
8
Fig. 1. Changes in the ratio of the modified and unmodi fied forms of the MGMT protein in extracts under the influence of thermal and chemical treatment (the histo gram shows the densitometry results of signals): (1) Hep2, boiling for 5 min (pH 6.8); (2) Hep2, boiling for 60 min (pH 6.8); (3) Hep2, incubation with 0.5 M DTT for 4 h (pH 6.8) and boiling for 5 min; (4) Hep2, incubation with 0.5 M DTT for 4 h (pH 8.0) and boiling for 5 min; (5) Hep2, incubation with 8 M urea for 60 min (pH 6.8) and boiling for 5 min; (6) 4BL, boiling for 5 min (pH 6.8); (7) 4BL, incubation with 0.5 M DTT for 4 h (pH 6.8) and boiling for 5 min; and (8) 4BL, incubation with 0.5 M DTT for 4 h (pH 8.0) and boiling for 5 min. Here and in Figs. 2–7, the unmodified and modified forms of the enzyme are shown in dark and light gray, respectively.
1
2
69
67
3
4
5
6
7
8
92
90 100 77
47 kDa 26 kDa 20 kDa Control of loading, % 250
59 77
200 150 100 50 0
1
2
3
4
5
6
7
8
Fig. 2. Western blot analysis of the level of MGMT gene expression depending on the duration of cultivation of Hep2 and 4BL cells: (1) 4BL (24 h), (2) 4BL (48 h), (3) 4BL (72 h), (4) 4BL (96 h), (5) Hep2 (24 h), (6) Hep2 (48 h), (7) Hep2 (72 h), and (8) Hep2 (96 h).
important task of the study was to select optimal con ditions for culturing cells and their subsequent treat ment with exogenous cytokines. For this reason, we at first performed experiments to determine at which stage after the inoculation of a culture medium with various cell lines the synthesis of the MGMT protein was maximum, because it was important to adequately analyze the results. In one of the experiments, we compared the changes in the MGMT gene expression at the protein level in 4BL and Hep2 cells cultured for 24–96 h during one passage (Fig. 2). The maximum level of MGMT gene expression in 4BL and Hep2 cells was observed after 48 and 72 h of culturing, respectively. It should be noted that, in 4BL cells, the MGMT protein at all stages of the study was detected only in the modified form. Conversely, in Hep2 cells, it was detected in the unmodified form at nearly all stages (the first, second, and fourth days). Only after 72 h of cultivation, the protein synthesized in Hep2 cells was detected in the modified form. Thus, the optimal time to detect MGMT gene expres sion is the second to third day of cell cultivation after inoculation. In our experiments, we also established the impor tance of proper selection of culture media. In DMEM with a high glucose content (4.5 g/L), cells not only grew better, but the level of MGMT expression was higher than in a medium with a low glucose content (1 g/L). This fact was taken into account in our subsequent experiments. Of great importance is the duration of incubation of cells with exogenous cytokines. The results shown in Fig. 3 indicate that the stimulation of gene expres sion at the level of the MGMT protein by the cytok inelike EMAP II protein was observed during 8–32 h of incubation of cells with this agent. Treatment of cells with EMAP II not only affected the overall level of MGMT expression but also led to the appearance of the protein in the classical unmodified form, which practically was not detected in the control variant under these conditions. Note that the incubation of cells for 8 h gave a more pronounced effect compared to longer incubation periods (16 and 32 h). Probably, this phenomenon was due to a decrease in the gene expression level in cells that were incubated for a long period of time in a depleted serumfree medium. At the next stage of the study, we compared the effect of EMAP II on the level of MGMT gene expres sion in tumor cells and in cells obtained from healthy donors [16]. Hep2 cells were treated with EMAP II at a concentration of 2 μg/mL for 8 h. As seen in Fig. 4, this treatment significantly decreased the level of MGMT gene expression and only small amounts of the protein in the unmodified form were detected. We have shown previously [16] that EMAP II caused an increase in the MGMT gene expression level in human 4BL cells at concentrations ranging from 0.2 to 20.0 μg/mL and that this effect was concentration dependent. A similar effect of EMAP II was also CYTOLOGY AND GENETICS
Vol. 47
No. 4
2013
CHANGE IN THE MGMT GENE EXPRESSION UNDER THE INFLUENCE
detected in other human cell lines. These data indicate that the effect of EMAP II depends both on its con centration and on the nature of cells, as well as, prob ably, that of the initial level of MGMT expression in cells. The effect of cytokines IL3 and SCF was studied using 4BL cells. As shown in Fig. 5, after treatment with these cytokines, the protein in the unmodified form disap peared completely and the amount of the protein in the modified form decreased significantly. Similar results were observed when primary human skin fibro blast and Hep2 cancer cells were treated with cytok ine LIF [24]. The results of one experiment are shown in Fig. 6. This result suggests that cytokine LIF is also appar ently able to inhibit MGMT gene expression. As a result of treatment with this cytokine, the protein in the unmodified form disappeared completely and the amount of the protein in the modified form was com parable with the control level. As was shown earlier, cytokine IFNα2b, which is the active substance in the Laferobion formulation, is also probably able to modulate MGMT gene expres sion at the level of proteins in cultured human cells [25]. In this study, after the treatment of 4BL cells with Laferobion in a wide concentration range, a signifi cant decrease in the amount of the MGMT protein in the modified form was observed at concentrations of 20 and 2000 U/mL (Fig. 7). After the treatment of Hep2 cells with Laferobion, a decrease in the amount of the MGMT protein in the unmodified form was observed at nearly all concentra tions of the drug (2–2000 U/mL), except for a con centration of 20 U/mL (Fig. 7, lane 8), and the amount of the protein in the modified form was at approximately the same level as in the control. Thus, Laferobion can apparently affect the MGMT gene expression level, although no direct dependence of its effect on the concentration was observed. This com plex effect is probably determined by the fact that cytokine IFNα2b at different concentrations affects different regulatory signaling pathways. Experiments with CB1 cells showed that the amount of the MGMT protein in the modified form decreased at a Laferobion concentration of 200 U/mL [25]. However, in the case of A102 cells, treatment with this drug at a concentration of 20 U/mL led to an increase in the amount of the MGMT protein in the modified form. In our opinion, the effect of Lafero bion should be studied more thoroughly, because this medicine is used in clinical practice for the treatment of cancer patients, and it is important to understand the consequences of its influence on the reparative enzyme, which determines the success of chemother apy with alkylating agents. Thus, we confirmed the data of other authors that exogenous cytokines can affect the amount of the CYTOLOGY AND GENETICS
Vol. 47
No. 4
2013
205
1
2
3
4
66
80
79
61
47 kDa 26 kDa 20 kDa Control of loading, % 150 100 50 0
1
2
3
3
Fig. 3. Western blot analysis of the level of MGMT gene expression depending on the time of incubation of 4BL6 cells with the drug EMAP II: (1) 4BL6, control; (2) 4BL6 + EMAP II (2 μg/mL), 8 h; (3) 4BL6 + EMAP II (2 μg/mL), 16 h; and (4) 4BL6 + EMAP II (2 μg/mL), 32 h.
1
2
92
88
1
2
47 kDa
26 kDa
20 kDa Control of loading, % 80 60 40 20 0
Fig. 4. Western blot analysis of the effect of the EMAP II drug on the MGMT gene expression level in Hep2 tumor cells: (1) Hep2 (control) and (2) Hep2 + EMAP II (2 μg/mL).
206
KOTSARENKO et al. 1
2
3
1
2
77
100
1
2
47 kDa
47 kDa
26 kDa
26 kDa
20 kDa Control of loading, %
54 57 51
20 kDa Control of loading, %
500
300
400 300
200
200 100
100 0
1
2
3
0
Fig. 5. Western blot analysis of the effect of cytokines IL3 and SCF on the MGMT gene expression level in immortal ized human cells in vitro: (1) 4BL, control; (2) 4BL + IL3 (20 ng/mL); and (3) 4BL + SCF (20 ng/mL).
Fig. 6. Western blot analysis of the effect of cytokine LIF on the level of MGMT gene expression in Hep2 cells (the histogram shows the densitometry results of signals): (1) Hep2 (control) and (2) Hep2 + LIF (20 ng/mL).
reparative enzyme of interest in human cells [8–11] and expanded the range of cytokines that are potential regulators of the MGMT gene expression level. At the next stages of our research, we plan to determine the level at which MGMT expression is regulated (protein or RNA) and study the regulatory pathways involved in this process.
It is known that cytokines in high doses can cause DNA lesions, which are in turn among the main trig gers that stimulate DNA repair systems.
DISCUSSION
In studies performed with human melanoma cells, a positive effect of IFNβ on the potentiation of the sensitivity of melanoma cells to temozolomide was explained by the involvement of this cytokine in the recovery of procaspase8, which triggers an apoptotic cascade [28]. However, we incline to believe that the regulation of MGMT under the influence of cytokines is quite possible and that whole cascades of regulatory signaling pathways that are triggered by various cytok ines are involved in this process. For example, it is known that interferons exhibit antiviral and antitumor activities. The antiviral activity of these cytokines is manifested in the suppression of protein synthesis. In response to interferon, cells produce high quantities of protein kinase R, as a result of which the level of pro tein synthesis in a cell decreases. The stimulation of the synthesis of protein kinase R is followed by the stimulation of the synthesis of ribonuclease L, which cleaves cellular RNA and further reduces the level of protein synthesis. In general, the IFNdependent
In literature, hundreds of articles devoted to study ing the reparative MGMT enzyme appear each year. Since the discovery of this unique protein and estab lishment of its major function—the removal of alkyl groups from the DNA molecule—researchers have made significant progress in the understanding of this repair system [26]. However, some issues concerning the structure and function of the protein itself are still to be elucidated. In this paper, we consider one important aspect— the possibility to regulate the MGMT gene expression level using exogenous biologically active substances. Before this work, it was known that cytokines IFNβ, IFNγ, IL1β, and IL24 can influence the regulation of MGMT gene expression [8–11]. The authors of the abovementioned studies assumed that the factors directly involved in the regulation process can be DNA breaks or the p53 protein.
According to published data, the expression level of the p53 protein is inversely proportional to the expres sion level of MGMT [27]. However, some authors deny the possibility of regulation of the MGMT gene expression level by cytokine IFNβ.
CYTOLOGY AND GENETICS
Vol. 47
No. 4
2013
CHANGE IN THE MGMT GENE EXPRESSION UNDER THE INFLUENCE 1
2
3
4
5
6
7
8
9
207
10
47 kDa
26 kDa 20 kDa Control of loading, % 100 80 60 40 20 0
1
86 89
2
3
87 96 86 94 80 96 100 95
4
5
6
7
8
9
10
Fig. 7. Effect of the Laferobion drug on the level of MGMT gene expression in 4BL6 and Hep2 cells (incubation time, 8 h). (1–5) 4BL Cells: (1), control; (2), IFNα2b (2 U/mL); (3), IFNα2b (20 U/mL); (4), IFNα2b (200 U/mL); and (5), IFNα2b (2000 U/mL). (6–10) Hep2 Cells: (6) control, (7) IFNα2b (2 U/mL), (8) IFNα2b (20 U/mL), (9) IFNα2b (200 U/mL), and (10) IFNα2b (2000 U/mL).
inhibition of translation is detrimental for both virus and host cells. In addition to the effect on translation, interferons can activate hundreds of other genes involved in the protection of cells from viruses [29]. Furthermore, interferon limits the distribution of viral particles by activating the p53 protein, which results in the apop tosis of infected cells [30]. With allowance for the published data, which showed an inverse relationship between the level of expression of the p53 protein and MGMT, it can be assumed that these signaling pathways are involved in the regulation of the expression of the gene encoding the reparative enzyme. The second aspect covered in this work was the presence of modifications of the MGMT protein. According to published data, the selfassociation of proteins with the formation of dimers and higher oli gomers is a very common phenomenon. Oligomers can be represented by homodimers, which are com prised of identical subunits, and heterodimers, which consist of different subunits [31]. Recent structural and biophysical studies have demonstrated that dimerization and/or oligomerization are one of the key factors in the regulation of various proteins, such as enzymes, ion channels, receptors, and transcription factors. It should be noted that selfassociation in some way helps to minimize the size of the genome, with retaining the advantages of the formation of mod ular complexes [32]. Despite the fact that electrophoresis of proteins under denaturing conditions (such as βmercaptoeth anol, SDS, and heating) usually destroys intermolec ular and intramolecular disulfide bonds, there exist CYTOLOGY AND GENETICS
Vol. 47
No. 4
2013
stable protein complexes that can be destroyed only with the use of additional destabilizing factors (pH, denaturing agents, high pressure, high ionic strength, temperature changes, etc.) [33, 34]. Some of these factors were used in our study to destroy the 48kDa protein, because the protein complex was not destroyed by standard SDSPAGE. Thus, our data on the destruction of the MGMT protein complex with a molecular weight of 48 kDa by the thermal and chemical treatment of protein extracts, as a result of which the protein with a molec ular weight of 24 kDa is formed, confirm the hypothe sis regarding the possible formation of a stable homodimer (i.e., the protein–protein interaction between two molecules of the MGMT protein). A the oretical confirmation of our results is the implementa tion of MGMT polymerization in a cellfree system [35], as well as the fact that structural elements of pro teins, such as cysteine residues and zinc ions, which are present in the MGMT molecule, play an impor tant role in the formation of dimers [36]. We plan to further study both forms of the protein at the molecu lar–genetic level. CONCLUSIONS Our data suggest that a number of exogenous cytokines—SCF, LIF, IL3, EMAP II, and IFNα2b (drug Laferobion)—can affect the expression of the gene encoding the reparative MGMT enzyme in human cells in vitro. It is shown that the pattern of the effect of these cytokines may vary depending on the concentration of cytokines, the cell type, and the level of expression of the relevant gene in test cells. How ever, the mechanisms by which this regulation is
208
KOTSARENKO et al.
implemented remain obscure and require further studies. It is assumed that the modified form of MGMT with a molecular weight of 48 kDa is a protein complex. ACKNOWLEDGMENTS We are grateful to Professor J. McCormick (Mich igan State University, United States) for kindly provid ing the A102 cell line. REFERENCES 1. Margison, G., Povey, A., Kaina, B., and Santibáñez Koref, M., Variability and regulation of O6alkylgua nineDNA alkyltransferase, Carcinogenesis, 2003, vol. 24, no. 4, pp. 625–635. 2. Fang, Q., Noronha, A.M., Murphy, S.P., et al., Repair of O6GalkylO6G interstrand crosslinks by human O6alkylguanhinedna alkyltransferase, Biochemistry, 2008, vol. 47, no. 41, pp. 10892–10903. 3. Kaina, B., Christmann, M., Naumann, S., and Roos, W., MGMT: key node in the battle against geno toxicity, carcinogenicity and apoptosis induced by alky lating agents, DNA Rep., 2007, vol. 6, pp. 1079–1099. 4. Sabharwal, A. and Middleton, M.R., Exploiting the role of O6methylguanineDNAmethyltransferase (MGMT) in cancer therapy, Curr. Opin. Pharm., 2006, vol. 6, pp. 355–363. 5. Sharma, S., Salehi, F., Scheithauer, B.W., et al., Role of MGMT in tumor development, progression, diagnosis, treatment and prognosis, Anticancer Res., 2009, vol. 29, no. 10, pp. 3759–3768. 6. Verbeek, B., Southgate, T.D., Gilham, D.E., and Mar gison, G.P., O6methylguanineDNA methyltrans ferase inactivation and chemotherapy, Brit. Med. Bull., 2008, vol. 85, pp. 17–33. 7. Niture, S.K., Doneanu, C.E., Velu, C.S., et al., Pro teomic analysis of human O6methylguanineDNA methyltransferase by affinity chromatography and tan dem mass spectrometry, Biochem. Biophys. Res. Com mun., 2005, vol. 337, pp. 1176–1184. 8. Natsume, A., Ishii, D., Wakabayashi, T., et al., IFNβ downregulates the expression of DNA repair gene MGMT and sensitizes resistant glioma cells to temozo lomide, Cancer Res., 2005, vol. 65, no. 17, pp. 7573– 7579. 9. Rosati, S.F., Williams, R.F., Nunnally, L.C., et al., IFNbeta sensitizes neuroblastoma to the antitumor activity of temozolomide by modulating O6meth ylguanine DNA methyltransferase expression, Mol. Cancer Ther., 2008, vol. 7, no. 12, pp. 3852–3858. 10. Zheng, M., Bocangel, D., Ramesh, R., et al., Interleu kin24 overcomes temozolomide resistance and enhances cell death by downregulation of O6meth ylguanineDNA methylransferase in human melanoma cells, Mol. Cancer Ther., 2008, vol. 7, no. 12, pp. 3842– 3851. 11. Cardozo, A.K., Kruhoffer, M., Leeman, R., et al., Identification of novel cytokineinduced genes in pan creatic βcells by highdensity oligonucleotide arrays, Diabetes, 2001, vol. 50, pp. 909–920.
12. Motomura, K., Natsume, A., Kishida, Y., et al., Bene fits of interferonβ and temozolomide combination therapy for newly diagnosed primary glioblastoma with the unmethylated MGMT promoter, Cancer, 2011, vol. 117, no. 8, pp. 1721–1730. 13. Briegert, M., Enk, A.H., and Kaina, B., Change in expression of MGMT during maturation of human monocytes into dendritic cells, DNA Rep., 2007, vol. 6, pp. 1255–1263. 14. Ivakhno, S.S. and Kornelyuk, A.I., Cytokinelike activities of some aminoacyltRNA synthetases and auxiliary p43 cofactor of aminoacylation reaction and their role in oncogenesis, Exp. Oncol., 2004, vol. 26, no. 4, pp. 250–255. 15. Schwarz, M.A., Kandel, J., Brett, J., et al., Endothe lialmonocyte activating polypeptide II, a novel antitu mour cytokine that suppresses primary and metastatic tumor growth and induces apoptosis in growing endot helial cells, J. Exp. Mes., 1999, vol. 190, no. 3, pp. 341– 354. 16. Lylo, V.V., Matsevich, L.L., Kotsarenko, E.V., et al., Activation of gene expression of the O6methylgua nineDNA transferase repair enzyme upon the influ ence of EMAP II cytokine in human cells in vitro, Cytol. Genet., 2011, vol. 45, no. 6, pp. 373–378. 17. Dubrovsky, A.L., Brown, J.N., Kornelyuk, A.I., et al., Bacterial expression of fulllength and truncated forms of cytokine EMAP2 and cytokinelike domain of mammalian tyrosyltRNA synthetase, Biopolym. Cell, 2000, vol. 16, no. 3, pp. 229–235. 18. Morton, E.N. and Margison, G.P., Increased O6alky lguanineDNA alkyltransferase activity in Chinese hamster V79 cells following selection with chloroethy lating agents, Carcinogenesis, 1988, vol. 9, no. 1, pp. 45–49. 19. Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage t4, Nature, 1970, vol. 227, no. 5259, pp. 680–685. 20. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein–dye binding, Anal. Biochem., 1976, vol. 72, no. 1/2, pp. 248–254. 21. www.novusbio.com/MGMTAntibodyMT232_NB100 168.html 22. Aldridge, G.M., Podrebarac, D.M., Greenough, W.T., and Weiler, I.J., The use of total protein stains as load ing controls: an alternative to highabundance single protein controls in semiquantitative immunoblotting, J. Neurosci. Meth., 2008, vol. 172, no. 2, pp. 250–254. 23. Lylo, V.V., Identification of a modified form of the repair enzyme O6alkylguanineDNA alkyltransferase, Aktual. Probl. Akush. Ginekol. Klet. Biol. Med. Genet., 2010, vol. 19, pp. 299–305. 24. Kotsarenko, E.V., Lylo, V.V., Matseevich, L.L., et al., Cytokine LIF as a modulator MGMT gene expression in human cells in vitro, in Factors of Experimental Evo lution of Organisms: Collected Scientific Papers, 2011, vol. 11, pp. 489–493. 25. Kotsarenko, E.V., Shaposhnik, L.A., Lylo, V.V., et al., Interferons as possible regulators of MGMT gene expression, Ukr. Biokhim. Zh., 2010, vol. 82, p. 35. CYTOLOGY AND GENETICS
Vol. 47
No. 4
2013
CHANGE IN THE MGMT GENE EXPRESSION UNDER THE INFLUENCE 26. Pegg, A.E., Multifaceted roles of alkyltransferase and related proteins in DNA repair, DNA damage, resis tance to chemotherapy, and research tools, Chem. Res. Toxicol., 2011, vol. 24, no. 5, pp. 618–639. 27. Blough, M.D., Zlatescu, M.C., and Cairncross, J.G., O6methylguanineDNA methyltransferase regulation by p53 in astrocytic cells, Cancer Res., 2007, vol. 67, no. 2, pp. 580–584. 28. Roos, W.P., Jost, E., Belohlavek, C., et al., Intrinsic anticancer drug resistance of malignant melanoma cells is abrogated by IFNβ and valproic acid, Cancer Res., 2011, vol. 71, no. 12, pp. 4150–4160. 29. De Veer, M.J., Holko, M., Frevel, M., et al., Func tional classification of interferonstimulated genes identified using microarrays, J. Leuk. Biol., 2001, vol. 69, no. 6, pp. 912–920. 30. Takaoka, A., Hayakawa, S., Yanai, H., et al., Integra tion of interferonα/β signaling to p53 responses in tumour suppression and antiviral defense, Nature, 2003, vol. 424, pp. 516–523. 31. Traut, T.W., Dissociation of enzyme oligomers: a mech anism for allosteric regulation, Crit. Rev. Biochem. Mol. Biol., 1994, vol. 29, no. 2, pp. 135–163.
CYTOLOGY AND GENETICS
Vol. 47
No. 4
2013
209
32. Marianayagam, N.J., Sunde, M., and Matthews, J.M., The power of two: protein dimerization in biology, Trends Biochem. Sci., 2004, vol. 29, no. 11, pp. 618– 625. 33. Henriksen, U., Fog, J.U., Litman, T., and Gether, U., Identification of intra and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2, J. Biol. Chem., 2005, vol. 280, no. 44, pp. 36926–36934. 34. Kolodziejski, P.J., Rashid, M.B., and Eissa, N.T., Intracellular formation of “undisruptable” dimmers of inducible nitric oxide synthase, Proc. Natl. Acad. Sci. U.S.A., 2003, vol. 100, no. 24, pp. 14263–14268. 35. Adams, C.A., Melikishvili, M., Rodgers, D.W., et al., Topologies of complexes containing O6alkylguanine DNA alkyltransferase and DNA, J. Mol. Biol., 2009, vol. 389, pp. 248–263. 36. Duguid, E.M., Rice, P.A., and He, C., The structure of the human AGT protein bound to DNA and its impli cations for damage detection, J. Mol. Biol., 2005, vol. 350, pp. 657–666.
Translated by M. Batrukova
