Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro
Aim. To study the effect of EMAP II, IFN-α2b and its medicinal preparations on the amount of O6-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, IFN-α2b and its com...
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| Опубліковано в: : | Вiopolymers and Cell |
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| Дата: | 2014 |
| Автори: | , , , , , , |
| Формат: | Стаття |
| Мова: | Англійська |
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Інститут молекулярної біології і генетики НАН України
2014
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro / K.V. Kotsarenko, V.V. Lylo, T.P. Ruban, L.L. Macewicz, A.I. Kornelyuk, S.I. Chernykh, L.L. Lukash // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 448-453. — Бібліогр.: 31 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859655451901165568 |
|---|---|
| author | Kotsarenko, K.V. Lylo, V.V. Ruban, T.P. Macewicz, L.L. Kornelyuk, A.I. Chernykh, S.I. Lukash, L.L. |
| author_facet | Kotsarenko, K.V. Lylo, V.V. Ruban, T.P. Macewicz, L.L. Kornelyuk, A.I. Chernykh, S.I. Lukash, L.L. |
| citation_txt | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro / K.V. Kotsarenko, V.V. Lylo, T.P. Ruban, L.L. Macewicz, A.I. Kornelyuk, S.I. Chernykh, L.L. Lukash // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 448-453. — Бібліогр.: 31 назв. — англ. |
| collection | DSpace DC |
| container_title | Вiopolymers and Cell |
| description | Aim. To study the effect of EMAP II, IFN-α2b and its medicinal preparations on the amount of O6-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, IFN-α2b 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 mg/ml and 2 mg/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). IFN-α2b 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 IFN-α2b 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 IFN-α2b. Possibly it depends on the presence of stabilizing components in their compositions.
Мета. Дослідити вплив EMAP II, IFN-α2b та його медичних препаратів на кількість білка MGMT у клітинах людини in vitro. Методи. Клітини людини 4BL і Hep-2 обробляли EMAP II, IFN-α2b і його комерційними препаратами. Зміни в експресії гена MGMT на рівні білка досліджували за використання Вестерн-блот аналізу. Результати. Обробка клітин Hep-2 і 4BL цитокіном EMAP II в концентрації 0,02 і 2 мкг/мл відповідно призводить до індукції експресії гена MGMT. EMAP II в концентраціях 0,2–20 мкг/мл знижує кількість білка MGMT у клітинах Hep-2. Регулювальну активність EMAP II спостерігали також і відносно MARP (білка, який розпізнається моноклональними анти-MGMT антитілами). Показано, що IFN-α2b і Лаферон-ФармБіотек з активністю 200 і 2000 МО/мл підвищують кількість білка MGMT у клітинах Hep-2. Висновки. Очищені рекомбінантні білки EMAP II і IFN-α2b, які є субстратами для медичних препаратів, впливають на кількість білка MGMT у клітинах людини in vitro залежно від концентрації. У той же час дія медичних препаратів відрізняється від ефекту очищеного білка IFN-α2b, що, можливо, пов’язано з присутністю стабілізувальних компонентів у його складі.
Цель. Исследовать влияние EMAP II, IFN-α2b и его медицинских препаратов на количество белка MGMT в клетках человека in vitro. Методы. Клетки человека 4BL и Hep-2 обрабатывали EMAP II, IFN-α2b и его коммерческими препаратами. Изменения в экспрессии гена MGMT исследовали с использованием Вестерн-блот анализа. Результаты. Обработка клеток Hep-2 и 4BL цитокином EMAP II в концентрации 0,02 и 2 мкг/мл соответственно приводит к индукции экспрессии гена MGMT. EMAP II в концентрациях 0,2–20 мкг/мл снижает уровень экспрессии гена MGMT в клетках Hep-2. Регулирующую активность EMAP II наблюдали также и относительно MARP (белка, распознаваемого моноклональными анти-MGMT антителами). Показано, что IFN-α2b и Лаферон-ФармБиотек с активностью 200 и 2000 МЕ/мл повышают количество белка MGMT в клетках Hep-2. Выводы. Очищенные рекомбинантные белки EMAP II и IFN-α2b, являющиеся субстратами для медицинских препаратов, влияют на количество белка MGMT в клетках человека in vitro зависимым от концентрации образом. В то же время действие медицинских препаратов отличается от влияния очищенного белка IFN-α2b, что, возможно, связано с присутствием стабилизирующих компонентов в его составе.
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GENOMICS, TRANSCRIPTOMICS AND PROTEOMICS
UDC 575.224 + 577.218
Influence of EMAP II, IFN-�2b and its
medicinal preparations on the MGMT protein
amount in human cells in vitro
K. V. Kotsarenko, V. V. Lylo, T. P. Ruban, L. L. Macewicz, A. I. Kornelyuk,
S. I. Chernykh, L. L. Lukash
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str.,Kyiv, Ukraine, 03680
lukash@imbg.org.ua
Aim. To study the effect of EMAP II, IFN-�2b and its medicinal preparations on the amount of O
6
-methylgua-
nine-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, IFN-�2b and its commercial me dicinal
preparations. Changes in the MGMT gene expression were studied at a protein level by Western blot analysis. Re-
sults. Treatment of Hep-2 and 4BL cells with EMAP II at the concentrations of 0.02 �g/ml and 2 �g/ml respecti-
vely led to induction of the MGMT gene expression. EMAP II at the concentrations of 0.2–20 �g/ml caused dec-
rease 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). IFN-�2b 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 IFN-�2b which are substrates for the medicinal preparations in-
fluenced 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 IFN-�2b. Possibly it depends
on the presence of stabilizing components in their compositions.
Keywords: MGMT, MARP, IFN-�2b, EMAP II, human cell cultures.
Introduction. The repair enzyme O6-methylguanine-
DNA methyltransferase (MGMT) eliminates O6-me-
thylguanin adducts in DNA and protects normal cells
from damaging effects of alkylating agents. At the sa-
me time MGMT makes tumor cells resistant to alkyla-
ting 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 nor-
mal 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-� down-
regulated the MGMT transcription and sensitized the re-
sistant glioma and neuroblastoma cells to temozolomi-
de in vitro [4, 5]. One of the members of interleukin fa-
mily – IL-24 down-regulated the MGMT gene expres-
sion via activation of p53 and therefore helped to over-
come 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 com-
bined therapy with IFN-� and temozolomide was shown
to provide better clinical outcomes in the patients who-
se tumor cells had an unmethylated MGMT promoter
[7]. For the patients with progressive malignant glioma,
the complex therapy with BCNU and IFN-�2b (Intron
448
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 6. P. 448–453 doi: http://dx.doi.org/10.7124/bc.0008BF
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
A, «Schering Corporation», USA) appeared to be a fea-
sible and promising treatment strategy. The effect of
IFN-�2b 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 poly-
peptide II) is a multifunctional cytokine, which is for-
med in malignant tumors of mammals due to the alterna-
tive splicing and posttranslational processing of its pre-
cursor – the p43 protein [9]. EMAP II suppresses the en-
dothelial 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, sarco-
ma, stomach and pancreas cancer [11].
In our previous works different exogenous cytoki-
nes, growth factors and plant components (extracts, lec-
tins) 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-�2b and EMAP II which pos-
sess 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-�2b
and EMAP II action on their concentration. Moreover
the another problem has arrived: does the effect of cyto-
kine medicinal preparation differ from that of their pu-
rified 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, clo-
ne 23.2, revealed two highly specific immunoreactive
bands: 24 kDa (classic MGMT protein) and 48 kDa (an-
ti-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 ef-
fect of different concentrations of purified IFN-�2b and
EMAP II and medicinal preparations of IFN-�2b on the
MGMT and MARP protein amount in human cell
cultures.
Materials and methods. The following human cell
lines were used: a standard line Hep-2 (laryngeal can-
cer) and 4BL line (fibroblast-like cells) derived in our la-
boratory [16]. Cells were cultivated in standard DMEM
(PAA) with 10 % FBS («Sigma») and antibiotics peni-
cillin (0.02 %) and streptomycin (0.02 %) at 37 oC with
4 % CO2.
The following interferon agents were used: human
recombinant protein IFN-�2b («Interpharmbiotek»,
Ukraine) in 0.1 M NaCl; Laferon-PharmBiotek («In-
terpharmbiotek») containing the human recombinant
protein IFN- �2b and additional components: NaCl,
Dextran 70, KH2PO4, Na2HPO4; Laferobion («Biofar-
ma», Ukraine) containing the human recombinant pro-
tein IFN-�2b and additional components: NaCl, Dext-
ran 70, KH2PO4, Na3PO4 � 12H2O.
Recombinant EMAP II protein was expressed in E. co-
li, purified and studied as described previously [17–20].
For cytokine treatments, 8 �105 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 oC.
Control intact cells were subjected to the similar proce-
dure 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 in-
tervals and 50 mM of PMSF in ethanol were added.
Sonicates were then centrifuged at 13,000 g for 30 min
at 4 oC. The supernatants were collected and frozen at
–80 oC for later use. SDS-PAGE (12 % gel) was perfor-
med by Laemmli method [21].
The concentration of total protein in cell lysates was
measured colorimetrically according to Bradford me-
thod [22].
The following antibodies were used: anti-MGMT
monoclonal antibodies, clone 23.2, isotype IgG2b («No-
vus Biologicals», USA), secondary antibodies conjuga-
ted with horseradish peroxidase («Sigma», USA). The
procedure of MGMT identification in the samples was
performed by Western blot analysis according to the me-
thodological instructions of the manufacturer of mAbs
[23]. Densitometry of stained membranes was used for
449
INFLUENCE OF IFN-�2b, EMAP II AND THEIR MEDICINAL PREPARATIONS ON THE MGMT PROTEIN
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 �g/ml induced the MGMT gene expression (Fig.
1, lane 2) in a clone of Hep-2 cells which did not ex-
press the MGMT gene under normal conditions (Fig. 1,
lane 1). However, an increase of EMAP II doses to 0.2–
20�g/ml did not lead to any changes of the MGMT pro-
tein 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 �g/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 de-
pending 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 re-
versibility 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 �g/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 re-
sult 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 concentra-
tion dependence was observed after treatment of Hep-2
cells with recombinant protein EMAP II: at concentra-
tions of 0.02 and 0.2�g/ml it induced the MARP expres-
sion, but at higher concentrations (2 and 20 �g/ml) it in-
hibited 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-�2b-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]. How-
ever, in our current work it was shown that IFN-�2b 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 treat-
ment of Hep-2 cells with Laferon-PharmBiotek prepa-
ration («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-�2b and Laferon (data not shown).
Laferon-PharmBiotek and Laferobion preparations
have similar composition (recombinant human IFN-�2b,
salts, dext ran) but they showed an opposite effect on the
MGMT protein amount. According to the literature da-
ta, a preparation Intron A (recombinant IFN-�2b, EDTA,
NaCl, m-Kresol, Polysorbate 80, Na2HPO4, NaH2PO4)
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 compo-
nents [8, 12].
450
KOTSARENKO K. V. ET AL.
A
B
1 2 3 4 5
0
5
10
15
6 7
0
1
2
3
1 2 3 4 5 6 7
Fig. 1. Effect of cytokine EMAP II at different concentrations on the
amount of MGMT protein in MGMT non-expressing (A) and MGMT-
expressing (B) Hep-2 cells: A – Western blot analysis (1 – 0 �g/ml; 2 –
0.02 �g/ml; 3 – 0.2 �g/ml; 4 – 2 �g/ml; 5 – 20 �g/ml); B – Western
blot analysis (1 – 0 �g/ml; 2 – 2 �g/ml); C, D – results of densitometry
(the vertical bar represents the level of MGMT protein amount, conven-
tional densitometry units)
24 kDa
A B
0
1
2
3
4
5
1 2 3 4
1 2 3 4
Fig. 2. Time-dependent effect of cytokine EMAP II on the amount of
MGMT protein in 4BL cells: A – Western blot analysis; 1–4 – 4BL,
137 passages + EMAP II (1 – 0�g/ml, 8 h; 2 – 2�g/ml, 8 h; 3 – 2�g/ml,
16 h; 4 – 2 �g/ml, 32 h); B – results of densitometry (the vertical bar re-
presents the level of MGMT protein amount, conventional densitomet-
ry units)
According to these data we may suggest that regula-
ting effect of the IFN-�2b-containing preparations de-
pends not only on the composition but also on their puri-
fication degree. Therefore these results could be useful
for planning the experiments with the IFN-�2b-contai-
ning preparations and for IFN-�2b therapy.
There are several hypotheses about the mechanisms
of the MGMT gene expression regulation under the in-
fluence of cytokines. According to one of them IFN-�
regulates the MGMT transcription via p53 protein and
specificity protein 1 (Sp1) [4, 5]. Another one suggests
that IFN-� activates transcriptional factor NF-�B and
thus affects the transcription of MGMT gene, which be-
longs to the NF-�B target genes [26]. According to the
literature data, all IFNs of type I (IFN-�, IFN-�, IFN- ,
IFN-
) bind to the specific cell surface receptor comp-
lex known as the IFN-� receptor (IFNAR) [27]. There-
fore we suppose that regulation of the MGMT gene ex-
pression by IFN-� and IFN-�2b may involve the similar
pathways (Fig. 5).
The way of EMAP II impact on the MGMT gene ex-
pression 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 ins-
tance, in a blood–tumor barrier (BTB) model, EMAP II
at low concentration (0.05 nM) induced three isoforms
of protein kinase C (PKC): PKC-�, �, and �, and, through
them, caused functional, biochemical, and morpholo-
gical 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. Mo-
reover, EMAP II can also activate the NF-�B trans-
cription factor [31]. Therefore we suppose that regula-
tion of the MGMT gene expression by IFN-�2b and
EMAP II occurs with participation of transcription fac-
tors NF-�B, Sp1 and AP-1. A scheme of possible cell sig-
naling pathways involved in these processes is shown
in Fig. 5.
451
INFLUENCE OF IFN-�2b, EMAP II AND THEIR MEDICINAL PREPARATIONS ON THE MGMT PROTEIN
A B
48 kDa
C D
1 2 3 4 5
0
2
4
6
8
10
6 7 8 9
0
2
4
6
1 2 3 4 5 6 7 8 9
Fig. 3. Effect of recombinant protein EMAP II on the amount of MARP
in human cell cultures: A – 1–5 – Hep-2 + EMAP II (1 – 0 �g/ml; 2 –
0.02 �g/ml; 3 – 0.2 �g/ ml; 4 – 2 �g/ml; 5 – 20 �g/ml); 6–9 – 4BL, 137
p. + EMAP II (6 – 0�g/ml, 8 h; 7 – 2�g/ml, 8 h; 8 – 2�g/ml, 16 h; 9 – 2
�g/ml, 32 h); B – results of densitometry (the vertical bar represents the
level of MARP amount, conventional densitometry units)
24 kDa
A
B
1 2 3 4
0
2
4
6
5 6 7
0
5
10
15
1 2 3 4 5 6 7
Fig. 4. Effect of IFN-�2b and Laferon-PharmBiotek on the MGMT
protein amount in Hep-2 cells: A – Western blot analysis (1 – IFN-�2b,
0 IU/ml; 2 – IFN-�2b, 2000 IU/ml; 3 – IFN-�2b, 200 IU/ml; 4 –
IFN-�2b, 2 IU/ml; 5 – Laferon, 0 IU/ml; 6 – Laferon, 2000 IU/ml; 7 –
Laferon-PharmBiotek, 200 IU /ml); B – results of densitometry (the ver-
tical bar represents the level of MGMT protein amount, conventional
densitometry units)
STAT3
ISGF3 NF-�B
AP-1
PKC
p53
Sp1
EMAP IIIFN-�2�
NF-�B
Cell
membrane
Nuclear
membrane
MGMT gene
promoter
Fig. 5. Possible cell signaling pathways involved in regulation of the
MGMT gene expression under the influence of IFN-�2b and EMAP II;
STAT3 – Signal transducer and activator of transcription 3; ISGF3 –
Interferon-stimulated gene factor 3
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 treat-
ments with cytokine IFN-�2b 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-�2b 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.
Âïëèâ EMAP II, IFN-�2b òà éîãî ìåäè÷íèõ ïðåïàðàò³â
íà ê³ëüê³ñòü á³ëêà MGMT ó êë³òèíàõ ëþäèíè in vitro
Ê. Â. Êîöàðåíêî, Â. Â. Ëèëî, Ò. Ï. Ðóáàí, Ë. Ë. Ìàöåâè÷,
Î. ². Êîðíåëþê, Ñ. ². ×åðíèõ, Ë. Ë. Ëóêàø
Ðåçþìå
Ìåòà. Äîñë³äèòè âïëèâ EMAP II, IFN-�2b òà éîãî ìåäè÷íèõ ïðå-
ïàðàò³â íà ê³ëüê³ñòü á³ëêà MGMT ó êë³òèíàõ ëþäèíè in vitro. Ìå-
òîäè. Êë³òèíè ëþäèíè 4BL ³ Hep-2 îáðîáëÿëè EMAP II, IFN-�2b ³
éîãî êîìåðö³éíèìè ïðåïàðàòàìè. Çì³íè â åêñïðåñ³¿ ãåíà MGMT íà
ð³âí³ á³ëêà äîñë³äæóâàëè çà âèêîðèñòàííÿ Âåñòåðí-áëîò àíàë³çó.
Ðåçóëüòàòè. Îáðîáêà êë³òèí Hep-2 ³ 4BL öèòîê³íîì EMAP II â
êîíöåíòðàö³¿ 0,02 ³ 2 ìêã/ìë â³äïîâ³äíî ïðèçâîäèòü äî ³íäóêö³¿ åêñ-
ïðåñ³¿ ãåíà MGMT. EMAP II â êîíöåíòðàö³ÿõ 0,2–20 ìêã/ìë çíè-
æóº ê³ëüê³ñòü á³ëêà MGMT ó êë³òèíàõ Hep-2. Ðåãóëþâàëüíó àê-
òèâí³ñòü EMAP II ñïîñòåð³ãàëè òàêîæ ³ â³äíîñíî MARP (á³ëêà,
ÿêèé ðîçï³çíàºòüñÿ ìîíîêëîíàëüíèìè àíòè-MGMT àíòèò³ëàìè).
Ïîêàçàíî, ùî IFN-�2b ³ Ëàôåðîí-ÔàðìÁ³îòåê ç àêòèâí³ñòþ 200 ³
2000 ÌÎ/ìë ï³äâèùóþòü ê³ëüê³ñòü á³ëêà MGMT ó êë³òèíàõ Hep-2.
Âèñíîâêè. Î÷èùåí³ ðåêîìá³íàíòí³ á³ëêè EMAP II ³ IFN-�2b, ÿê³ º
ñóáñòðàòàìè äëÿ ìåäè÷íèõ ïðåïàðàò³â, âïëèâàþòü íà ê³ëüê³ñòü
á³ëêà MGMT ó êë³òèíàõ ëþäèíè in vitro çàëåæíî â³ä êîíöåíòðàö³¿.
Ó òîé æå ÷àñ ä³ÿ ìåäè÷íèõ ïðåïàðàò³â â³äð³çíÿºòüñÿ â³ä åôåêòó
î÷èùåíîãî á³ëêà IFN-�2b, ùî, ìîæëèâî, ïîâ’ÿçàíî ç ïðèñóòí³ñòþ
ñòàá³ë³çóâàëüíèõ êîìïîíåíò³â ó éîãî ñêëàä³.
Êëþ÷îâ³ ñëîâà: MGMT, MARP, IFN-�2b, EMAP II, êóëüòóðè êë³-
òèí ëþäèíè.
Âëèÿíèå EMAP II, IFN-�2b è åãî ìåäèöèíñêèõ ïðåïàðàòîâ
íà êîëè÷åñòâî áåëêà MGMT â êëåòêàõ ÷åëîâåêà in vitro
Å. Â. Êîöàðåíêî, Â. Â. Ëûëî, Ò. À. Ðóáàí, Ë. Ë. Ìàöåâè÷,
À. È. Êîðíåëþê, Ñ. È. ×åðíûõ, Ë. Ë. Ëóêàø
Ðåçþìå
Öåëü. Èññëåäîâàòü âëèÿíèå EMAP II, IFN-�2b è åãî ìåäèöèíñêèõ
ïðåïàðàòîâ íà êîëè÷åñòâî áåëêà MGMT â êëåòêàõ ÷åëîâåêà in vit-
ro. Ìåòîäû. Êëåòêè ÷åëîâåêà 4BL è Hep-2 îáðàáàòûâàëè EMAP
II, IFN-�2b è åãî êîììåð÷åñêèìè ïðåïàðàòàìè. Èçìåíåíèÿ â ýêñ-
ïðåññèè ãåíà MGMT èññëåäîâàëè ñ èñïîëüçîâàíèåì Âåñòåðí-áëîò
àíàëèçà. Ðåçóëüòàòû. Îáðàáîòêà êëåòîê Hep-2 è 4BL öèòîêèíîì
EMAP II â êîíöåíòðàöèè 0,02 è 2 ìêã/ìë ñîîòâåòñòâåííî ïðèâîäèò
ê èíäóêöèè ýêñïðåññèè ãåíà MGMT. EMAP II â êîíöåíòðàöèÿõ 0,2–
20 ìêã/ìë ñíèæàåò óðîâåíü ýêñïðåññèè ãåíà MGMT â êëåòêàõ
Hep-2. Ðåãóëèðóþùóþ àêòèâíîñòü EMAP II íàáëþäàëè òàêæå è
îòíîñèòåëüíî MARP (áåëêà, ðàñïîçíàâàåìîãî ìîíîêëîíàëüíûìè
àíòè-MGMT àíòèòåëàìè). Ïîêàçàíî, ÷òî IFN-�2b è Ëàôåðîí-
ÔàðìÁèîòåê ñ àêòèâíîñòüþ 200 è 2000 ÌÅ/ìë ïîâûøàþò êîëè-
÷åñòâî áåëêà MGMT â êëåòêàõ Hep-2. Âûâîäû. Î÷èùåííûå ðåêîì-
áèíàíòíûå áåëêè EMAP II è IFN-�2b, ÿâëÿþùèåñÿ ñóáñòðàòàìè
äëÿ ìåäèöèíñêèõ ïðåïàðàòîâ, âëèÿþò íà êîëè÷åñòâî áåëêà MGMT
â êëåòêàõ ÷åëîâåêà in vitro çàâèñèìûì îò êîíöåíòðàöèè îáðàçîì.
 òî æå âðåìÿ äåéñòâèå ìåäèöèíñêèõ ïðåïàðàòîâ îòëè÷àåòñÿ
îò âëèÿíèÿ î÷èùåííîãî áåëêà IFN-�2b, ÷òî, âîçìîæíî, ñâÿçàíî ñ
ïðèñóòñòâèåì ñòàáèëèçèðóþùèõ êîìïîíåíòîâ â åãî ñîñòàâå.
Êëþ÷åâûå ñëîâà: MGMT, MARP, IFN-�2b, EMAP II, êóëüòóðû
êëåòîê ÷åëîâåêà.
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INFLUENCE OF IFN-�2b, EMAP II AND THEIR MEDICINAL PREPARATIONS ON THE MGMT PROTEIN
|
| id | nasplib_isofts_kiev_ua-123456789-154591 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T13:38:46Z |
| publishDate | 2014 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Kotsarenko, K.V. Lylo, V.V. Ruban, T.P. Macewicz, L.L. Kornelyuk, A.I. Chernykh, S.I. Lukash, L.L. 2019-06-15T16:37:45Z 2019-06-15T16:37:45Z 2014 Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro / K.V. Kotsarenko, V.V. Lylo, T.P. Ruban, L.L. Macewicz, A.I. Kornelyuk, S.I. Chernykh, L.L. Lukash // Вiopolymers and Cell. — 2014. — Т. 30, № 6. — С. 448-453. — Бібліогр.: 31 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008BF https://nasplib.isofts.kiev.ua/handle/123456789/154591 575.224 + 577.218 Aim. To study the effect of EMAP II, IFN-α2b and its medicinal preparations on the amount of O6-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, IFN-α2b 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 mg/ml and 2 mg/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). IFN-α2b 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 IFN-α2b 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 IFN-α2b. Possibly it depends on the presence of stabilizing components in their compositions. Мета. Дослідити вплив EMAP II, IFN-α2b та його медичних препаратів на кількість білка MGMT у клітинах людини in vitro. Методи. Клітини людини 4BL і Hep-2 обробляли EMAP II, IFN-α2b і його комерційними препаратами. Зміни в експресії гена MGMT на рівні білка досліджували за використання Вестерн-блот аналізу. Результати. Обробка клітин Hep-2 і 4BL цитокіном EMAP II в концентрації 0,02 і 2 мкг/мл відповідно призводить до індукції експресії гена MGMT. EMAP II в концентраціях 0,2–20 мкг/мл знижує кількість білка MGMT у клітинах Hep-2. Регулювальну активність EMAP II спостерігали також і відносно MARP (білка, який розпізнається моноклональними анти-MGMT антитілами). Показано, що IFN-α2b і Лаферон-ФармБіотек з активністю 200 і 2000 МО/мл підвищують кількість білка MGMT у клітинах Hep-2. Висновки. Очищені рекомбінантні білки EMAP II і IFN-α2b, які є субстратами для медичних препаратів, впливають на кількість білка MGMT у клітинах людини in vitro залежно від концентрації. У той же час дія медичних препаратів відрізняється від ефекту очищеного білка IFN-α2b, що, можливо, пов’язано з присутністю стабілізувальних компонентів у його складі. Цель. Исследовать влияние EMAP II, IFN-α2b и его медицинских препаратов на количество белка MGMT в клетках человека in vitro. Методы. Клетки человека 4BL и Hep-2 обрабатывали EMAP II, IFN-α2b и его коммерческими препаратами. Изменения в экспрессии гена MGMT исследовали с использованием Вестерн-блот анализа. Результаты. Обработка клеток Hep-2 и 4BL цитокином EMAP II в концентрации 0,02 и 2 мкг/мл соответственно приводит к индукции экспрессии гена MGMT. EMAP II в концентрациях 0,2–20 мкг/мл снижает уровень экспрессии гена MGMT в клетках Hep-2. Регулирующую активность EMAP II наблюдали также и относительно MARP (белка, распознаваемого моноклональными анти-MGMT антителами). Показано, что IFN-α2b и Лаферон-ФармБиотек с активностью 200 и 2000 МЕ/мл повышают количество белка MGMT в клетках Hep-2. Выводы. Очищенные рекомбинантные белки EMAP II и IFN-α2b, являющиеся субстратами для медицинских препаратов, влияют на количество белка MGMT в клетках человека in vitro зависимым от концентрации образом. В то же время действие медицинских препаратов отличается от влияния очищенного белка IFN-α2b, что, возможно, связано с присутствием стабилизирующих компонентов в его составе. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Genomics, Transcriptomics and Proteomics Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro Вплив EMAP II, IFN-α2b та його медичних препаратів на кількість білка MGMT у клітинах людини in vitro Влияние EMAP II, IFN-α2b и его медицинских препаратов на количество белка MGMT в клетках человека in vitro Article published earlier |
| spellingShingle | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro Kotsarenko, K.V. Lylo, V.V. Ruban, T.P. Macewicz, L.L. Kornelyuk, A.I. Chernykh, S.I. Lukash, L.L. Genomics, Transcriptomics and Proteomics |
| title | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro |
| title_alt | Вплив EMAP II, IFN-α2b та його медичних препаратів на кількість білка MGMT у клітинах людини in vitro Влияние EMAP II, IFN-α2b и его медицинских препаратов на количество белка MGMT в клетках человека in vitro |
| title_full | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro |
| title_fullStr | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro |
| title_full_unstemmed | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro |
| title_short | Influence of EMAP II, IFN-α2b and its medicinal preparations on the MGMT protein amount in human cells in vitro |
| title_sort | influence of emap ii, ifn-α2b and its medicinal preparations on the mgmt protein amount in human cells in vitro |
| topic | Genomics, Transcriptomics and Proteomics |
| topic_facet | Genomics, Transcriptomics and Proteomics |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/154591 |
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