Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro
Aim. To investigate an effect of biologically active compounds IFN-α2b, EMAPII, Card medium, fibronectin on the amount of MGMT (O6-methylguanine-DNA methyltransferase) and MARP (anti-Methyltransferase Antibody Recognizable Protein) proteins in human cells in vitro. Methods. The human c...
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Інститут молекулярної біології і генетики НАН України
2014
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| Zitieren: | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro / K.V. Kotsarenko, V.V. Lylo, L.L. Macewicz, T.P. Ruban, Yu.S. Luchakivska, M.V. Kuchuk, L.L. Lukash // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 203-208. — Бібліогр.: 29 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860112270055440384 |
|---|---|
| author | Kotsarenko, K.V. Lylo, V.V. Macewicz, L.L. Ruban, T.P. Luchakivska, Yu.S. Kuchuk, M.V. Lukash, L.L. |
| author_facet | Kotsarenko, K.V. Lylo, V.V. Macewicz, L.L. Ruban, T.P. Luchakivska, Yu.S. Kuchuk, M.V. Lukash, L.L. |
| citation_txt | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro / K.V. Kotsarenko, V.V. Lylo, L.L. Macewicz, T.P. Ruban, Yu.S. Luchakivska, M.V. Kuchuk, L.L. Lukash // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 203-208. — Бібліогр.: 29 назв. — англ. |
| collection | DSpace DC |
| container_title | Вiopolymers and Cell |
| description | Aim. To investigate an effect of biologically active compounds IFN-α2b, EMAPII, Card medium, fibronectin on the amount of MGMT (O6-methylguanine-DNA methyltransferase) and MARP (anti-Methyltransferase Antibody Recognizable Protein) proteins in human cells in vitro. Methods. The human cells of 4BL, Hep-2 and A102 lines were treated with growth factors and cytokines. Changes in the amount of MGMT and MARP proteins were studied by Western blot analysis with anti-MGMT mAbs. Results. The treatment of A102 cells with EMAPII, fibronectin, Laferon and Card medium led to a decreased level of the MGMT protein, whereas the amount of MARP was highly increased in these cells. The treatment with the recombinant protein IFN-α2b increased the amount of MGMT and MARP proteins in Hep-2 cells. The treatment with extracts of transgenic plants,containing human IFN-α2b, caused a significant decrease in the content of both proteins in Hep-2 cells and MARP in 4BL cells. Conclusions. Both MGMT and MARP are highly inducible proteins. Their amount in cells can be changed by some growth factors (Card medium, fibronectin), cytokine (IFN-α2b), cytokine-like (EMAPII) or cytokine-containing substances (Laferon and IFN-α2b in plant extracts). This regulation depended not only on the type of biologically active substances but on the cell line used in this study as well.
Мета. Дослідження впливу біологічно активних сполук IFN-α2b, EMAPII, середовища Card і фібронектину на вміст білків MGMT (О6-метилгуанін-ДНК метилтрансфераза) і MARP (білок, що розпізнається анти-MGMT антитілами) у клітинах людини in vitro. Методи. Клітини людини ліній 4BL, Hep-2 і A102 обробляли ростовими факторами і цитокінами. Зміни в кількості білків MGMT і MARP досліджували з використанням Вестерн блот аналізу і моноклональних анти-MGMT антитіл. Результати. Обробка клітин A102 препаратами EMAPII, фібронектину і Лаферону призводить до зниження кількості білка MGMT на фоні значного зростання кількості білка MARP у цих клітинах. Обробка рекомбінантним білком IFN-α2b підвищує кількість білків MGMT і MARP у клітинах Hep-2, а екстрактами трансгенних рослин, які містять IFN-α2b людини, – зменшує кількість обох білків у клітинах Hep-2 та білка MARP у клітинах 4BL. Висновки. MGMT і MARP є високо-індуцибельними білками. Їхня кількість може варіювати під дією деяких ростових факторів (середовище Card і фібронектин), цитокіну (IFN-α2b), цитокіноподібного (EMAPII) та цитокіновмісних (Лаферон і IFN-α2b у композиції з рослинними екстрактами) препаратів. Виявлена регуляція залежить не лише від типу біологічно активних речовин, але й від клітинних ліній, використаних в експериментах.
Цель. Исследовать влияние биологически активных соединений IFN-α2b, EMAP II, среды Card и фибронектина на содержание белков MGMT (О6-метилгуанин-ДНК метилтрансфераза) и MARP (белок, распознаваемый анти-MGMT антителами) в клетках человека in vitro. Методы. Клетки человека линий 4BL, Hep-2 и A102 обрабатывали ростовыми факторами и цитокинами. Изменения в количестве белков MGMT и MARP исследовали с использованием Вестерн блот анализа и моноклональных анти-MGMT антител. Результаты. Обработка клеток А102 препаратами EMAPII, фибронектина, Лаферона и среды Card снижает количество белка MGMT на фоне значительного возрастания количества белка MARP в этих клетках. Обработка рекомбинантным белком IFN-α2b увеличивает количество белков MGMT и MARP в клетках Hep-2, а экстрактами трансгенных растений, содержащих человеческий IFN-α2b, – существенно снижает количество обоих белков в клетках Hep-2 и MARP в клетках 4BL. Выводы. MGMT и MARP являются высокоиндуцибельными белками. Их количество может варьировать под действием некоторых ростовых факторов (среда Card и фибронектин), цитокина (IFN-α2b) цитокиноподобного (EMAP II) и цитокинсодержащих (Лаферон и IFN-α2b в композиции с растительными экстрактами) препаратов. Выявленная регуляция зависит не только от типа биологически активных веществ, но и от клеточных линий, использованных в экспериментах.
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UDC 575.224 + 577.218
Influence of some biologically active substances
on amount of MGMT and MARP proteins in
human cells in vitro
K. V. Kotsarenko1, V. V. Lylo1, L. L. Macewicz1, T. P. Ruban1,
Yu. S. Luchakivska 2, M. V. Kuchuk2, L. L. Lukash1
1Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
2Institute of Cell Biology and Genetic Engineering, NAS of Ukraine
148, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
lukash@imbg.org.ua
Aim. To investigate an effect of biologically active compounds IFN-�2b, EMAPII, Card medium, fibronectin on
the amount of MGMT (O6-methylguanine-DNA methyltransferase) and MARP (anti-Methyltransferase Antibo-
dy Recognizable Protein) proteins in human cells in vitro. Methods. The human cells of 4BL, Hep-2 and A102 li-
nes were treated with growth factors and cytokines. Changes in the amount of MGMT and MARP proteins were
studied by Western blot analysis with anti-MGMT mAbs. Results. The treatment of A102 cells with EMAPII, fib-
ronectin, Laferon and Card medium led to a decreased level of the MGMT protein, whereas the amount of MARP
was highly increased in these cells. The treatment with the recombinant protein IFN-�2b increased the amount
of MGMT and MARP proteins in Hep-2 cells. The treatment with extracts of transgenic plants,containing human
IFN-�2b, caused a significant decrease in the content of both proteins in Hep-2 cells and MARP in 4BL cells.
Conclusions. Both MGMT and MARP are highly inducible proteins. Their amount in cells can be changed by
some growth factors (Card medium, fibronectin), cytokine (IFN-�2b), cytokine-like (EMAPII) or cytokine-con-
taining substances (Laferon and IFN-�2b in plant extracts). This regulation depended not only on the type of
biologically active substances but on the cell line used in this study as well.
Keywords: human cell lines, repair enzyme MGMT, MARP, cytokines, growth factors, Western blot analysis.
Introduction. The repair enzyme O6-methylguanine-
DNA methyltransferase (MGMT) removes alkyl ad-
ducts from the O6-position of guanine in DNA. It is the
major defense factor against the mutagenic, carcino-
genic and cytotoxic effects of alkylating agents [1, 2].
MGMT protects both normal cells from endogenous
and exogenous carcinogens and tumor cells from che-
motherapeutic alkylating compounds. Therefore, this
enzyme is considered to be one of the targets to regulate
antitumor efficacy of the alkylating agents [3].
According to the literature data the expression of
MGMT gene may be affected by various factors: the al-
kylating agents, single-strand DNA breaks, transcrip-
tion factors, activators of protein kinase C, the MGMT
gene promoter hypermethylation, p53 protein etc. [4,
5]. There are some works where cytokines have been
also shown to be able to influence the MGMT gene ex-
pression. For example, IFN-� decreased the MGMT ge-
ne expression in human glioma and neuroblastoma cells
in vitro and increased the sensitivity of these cells to the
antitumor alkylating agent temozolomide [6, 7]. IL-24
also reduced the level of the MGMT gene expression in
the human melanoma cells in dose-dependent manner
[8]. A combined action of two cytokines IL-1� and IFN-
� increased the level of this gene expression in the rat �-
cells in vitro [9]. Nowadays, application of regulation
203
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 3. P. 203–208 doi: http://dx.doi.org/10.7124/bc.000897
� Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
204
of the MGMT gene expression with some cytokines is
launched in clinical practice. Complex treatment of the
patients, who had newly diagnosed primary glioblasto-
ma multiforme, with cytokine IFN-� and temozolomide
has resulted in favorable outcome, particularly in cases
when the tumor cells contained the unmethylated MGMT
promoter [10]. So the combined action of cytokines,
which reduced the level of MGMT gene expression, and
antitumor alkylating agents improves their efficiency
in both cell lines and humans.
The aim of our study was to investigate the effect of
biologically active compounds such as IFN-�2b, EMAP
II, Card medium, fibronectin, on the amount of MGMT
and MARP proteins in the human cells in vitro.
Recombinant interferon �2b (IFN-�2b) is widely
used in oncology due to its well-known antitumor ac-
tivity [11]. Endothelial-monocyte activating polypep-
tide II (EMAP II), whose properties are not completely
understood, also exhibits the anticancer activity [12].
Since the MGMT gene expression is known to change
during cell differentiation [13], such factors as Card me-
dium, ATP and fibronectin, which take part in the pro-
cess of cell differentiation, were also investigated in
this study.
Biological properties of the substances mentioned
above have been known for a long time, but their pos-
sible role in regulation of the MGMT gene expression
has not been studied yet.
Materials and methods. The following human
cells were used in this study: 4BL – fibroblast-like cell
line derived in our laboratory [14], standard Hep-2 cell
line (laryngeal cancer) and A102 cell line (skin fib-
roblasts), kindly provided by Prof. McCormick (Michi-
gan State University, The United States).
The cells were cultivated in standard DMEM
(«PAA», USA) with 10 % FBS («Sigma», USA) and
antibiotics penicillin (0.02 %) and streptomycin (0.02 %)
at 37 oC with 4 % CO2.
Different commercial and non-commercial biolo-
gically active substances were used. IFN-�2b was used
as purified recombinant protein («Interpharmbiotek»,
Ukraine), in the form of commercial Laferon prepara-
tion («Interpharmbiotek») and as a component of the
crude extract of transgenic carrot plants (Daucus caro-
ta L., Nantskaya and Perfektsiya varieties). The transge-
nic carrot plants expressing IFN-�2b were obtained via
Agrobacterium tumefaciens-mediated transformation
using two vector constructs containing: i) the sequen-
ces encoding interferon gene fused with Nicotiana plum-
bagenifolia calreticulin apoplast targeting signal driven
by 35S CaMV promoter and selective neomycin phos-
photransferase II (nptII) gene in order to obtain the trans-
genic carrot plants able to accumulate human IFN-�2b
protein; ii) the sequence encoding gusA gene driven by
35S CaMV promoter and the selective nptII gene for ob-
taining the transgenic carrot plants that we used as cont-
rol ones in our study – so-called «empty» vector. Accor-
ding to the previous studies the transgenic carrot plants
were characterized by high level of the recombinant hu-
man IFN-�2b protein accumulation. The protein extracts
of these plants were characterized by a high antiviral
activity [15].
The purified recombinant protein EMAPII was
kindly provided by Prof. A. I. Korneluk (IMBG, Ukrai-
ne). «Card differentiating» medium supplemented with
growth factors of different origin was developed and
used in our laboratory for the differentiation of stem cells
into cardiomyocytes [16]. The commercial preparations
Fibronectin and ATP were received from «Sigma».
We used the chemical agents 5-azacytidine and mi-
tomycin C. The conditions of cell treatment with the
biologically active substances in the serum-free culture
medium were described previously [17].
Cell lysates were prepared according to [18]. SDS-
PAGE (12 % gel) was performed using Laemmli me-
thod [19]. The concentration of total protein in cell ly-
sates was measured colorimetrically according to Brad-
ford method [20].
Monoclonal anti-MGMT antibodies (clone 23.2,
isotype Ig G2b) were obtained from Novus Biologicals
(USA), secondary antibodies conjugated with horsera-
dish peroxidase were obtained from «Sigma». The pro-
cedure of MGMT identification in the samples was
performed by Western blot analysis according to the me-
thodological instructions of the manufacturer of mono-
clonal antibodies (http://www.novusbio.com/ support/
protocols/protocol-specific-for-mgmt-antibody-nb100-
168.html). Densitometry of stained membranes by Scion-
Image 4.0.2 and Origin 8.1 programs was used as loading
control [21, 22].
Results and discussion. The human MGMT pro-
tein was shown to have molecular weight of 22–24 kDa
KOTSARENKO K. V. ET AL.
[1]. However, the Western blot analysis with mo-
noclonal anti-MGMT antibodies (clone 23.2) revealed
two highly specific immunoreactive bands – 24 kDa
(classic MGMT protein) and 48 kDa (anti-Methyl-
transferase Antibody Recognizable Protein or MARP)
(Fig. 1). It should be noted that in our previous works
MARP was named as a modified form of MGMT, whi-
le the classic 24 kDa protein was named as an unmodi-
fied form of MGMT [17, 23]. We proposed several hy-
potheses about the nature of MARP, namely, post-trans-
lation modifications, dimerization of MGMT, etc.,
which have been discussed in the mentioned articles.
So far there is an open question regarding the nature of
the 48 kDa protein.
In this work we have compared the effect of various
exogenous biologically active substances on the amount
of both MGMT and MARP proteins in the human cell
lines. The results of the A102 cells treatment with Card
medium, EMAPII, Laferon, fibronectin and ATP are
shown in the Fig. 2. The treatment of A102 cells with
different exogenous factors led to the dramatic changes
of the amount of MGMT and MARP proteins. Hep-2
cells were used as positive control in Western blots, be-
cause they express both types of proteins (Fig. 1).
Almost all of the studied factors (except ATP) de-
creased the level of MGMT protein amount in the A102
cells (lanes 4–7) compared with the control A102 cells
(lane 2). The treatment with ATP and to a greater extent
with Card medium (lane 4), EMAP II (lane 5), Laferon
(lane 6) and fibronectin (lane 7) led to an increased
amount of MARP.
In this work we studied the influence of purified
recombinant protein IFN-�2b and the extract of transge-
nic carrot tissues, expressing human IFN-�2b, on the
levels of MGMT and MARP proteins in Hep-2 and 4BL
cell lines.
As shown in Fig. 3, the treatment of Hep-2 cells
with the human recombinant protein IFN-�2b led to
slight increase of the amount of MGMT and MARP
proteins (lanes 2 and 3). No significant changes in the
amount of MARP were detected in 4BL cells. In cont-
rast to these data, the results presented in Fig. 4 show a
significant decrease of the MGMT and MARP proteins
amount in Hep-2 cells as well as the MARP amount in
4BL cells after their treatment with the extracts of trans-
genic plant tissues, expressing human IFN-�2b. On the
other hand the amount of MGMT in Hep-2 cells and
MARP in both cell lines did not significantly change
after the treatment with the extract of plants without in-
terferon gene.
We suppose that different effects of the purified re-
combinant protein IFN-�2b and the interferon-con-
taining plant extract on the MGMT and MARP proteins
amount may be caused by the influence of some plant
extract components on the action of interferon.
Thus, the results presented in Fig. 2–4 show that the
effect of interferon preparations may differ depending
on the composition of these preparations and the type of
cell lines. There are several hypotheses about the mecha-
nisms of modulating the MGMT gene expression by cy-
tokines. According to one of them the p53 protein is in-
volved in regulation of the MGMT transcription by cy-
205
INFLUENCE OF ACTIVE SUBSTANCES ON AMOUNT OF THE MGMT AND MARP PROTEINS IN VITRO
1 2
kDa
48 �
24 �
Fig. 1. Western blot analysis of MGMT gene expression in cells Hep-2
(1) and 4BL (2)
24 �
48 �
kDa
a
b
0
10
20
30
1 2 3 4 5 6 7
C
o
n
v
.
d
e
n
s.
,
u
n
it
s
MARP
MGMT
Fig. 2. Effect of different biologically active substances on the amount
of MGMT and MARP proteins in A102 cells: a – Western blot analysis
(1 – Hep-2, positive control; 2 – A102– control; 3 – A102 + ATP (20
�g/ml); 4 – A102 + Card medium; 5 – A102 + EMAP II (10�g/ml); 6 –
A102 + Laferon (20 IU/ml); 7 – A102 + fibronectin (10 �g/ml)); b –
results of densitometry. The vertical line represents the level of MGMT
and MARP proteins amount in conventional densitometry units
tokines [6, 8]. Another one suggests that the transcrip-
tional factor NF-�B is activated by cytokines and affects
the transcription of target genes including MGMT [24,
25]. We plan to continue our research of involving the-
se transcriptional factors in regulation of the MGMT gene
expression under the influence of the studied cytokines.
We presume that the gene encoding MARP protein
does not belong to the housekeeping genes because its ex-
pression can be regulated by various exogenous factors.
The Hep-2 cells usually have both proteins (Fig. 1, lane
1) as well as the 4BL cells in early passages [26]. How-
ever, after prolonged cultivation (more than 130 passa-
ges) the 4BL cells have lost the conventional MGMT
protein (Fig. 1, lane 2). According to the literature data,
hypermethylation of the MGMT gene promoter com-
monly occurs causing a gene silencing in both different
cell lines and human cells in vivo [1, 5, 10]. Therefore
in our experiments 5-azacytidine was selected as a de-
methylating agent that can affect the MGMT gene ex-
pression in the MGMT-deficient 4BL cells (Fig. 5). 5-
azacytidine was the first identified demethylating agent
that inhibits DNA methyltransferases and reverses DNA
hypermethylation, restoring the expression of silenced
genes [27]. The mechanisms involved in the 5-azacyti-
206
KOTSARENKO K. V. ET AL.
1 2 3 4 5 6 7 8 9
24 �
48 �
kDa
5 6 7 8 9
MARP
MGMT
0
2
4
6
Variant
a
b
1 2 3 4
0
20
40
Fig. 3. The effect of purified recombinant protein IFN-a2b on the
MGMT and MARP proteins amount in the human cell lines: a –
Western blot analysis (1 – Hep-2, control; 2 – Hep-2 + IFN-a2b (2000
IU/ml); 3 – Hep-2 + IFN-a2b (200 IU/ml); 4 – Hep-2 + IFN-a2b (2
IU/ml); 5 – 4BL, 182 p., control; 6 – 4BL + IFN-a2b (2000 IU/ml); 7 –
4BL + IFN-a2b (200 IU/ml); 8 – 4BL + IFN-a2b (20 IU/ml); 9 – 4BL +
IFN-a2b (2 IU/ml)); b – results of densitometry. The vertical line
represents the amount of MGMT and MARP proteins in conventional
densitometry units
1 2 3 4 5 6
0
2
4
6
4 5 6
Variant
MARP
MGMT
24 �
48 �
kDa
a
b
0
5
10
15
20
1 2 3
C
o
n
v
.
d
e
n
s.
,
u
n
it
s
Fig. 4. The effect of extract of transgenic carrot on MGMT and MARP
proteins amount in the human cell lines: a – Western blot analysis (Hep-
2: 1 – control; 2 – extract of transgenic carrot plants, containing human
IFN-a2b (600 IU/ml); 3 – extract of transgenic carrot cells transfected
with an «empty» vector; 4BL: 4 – 182 p., control; 5 – extract of trans-
genic carrot cells transfected with an «empty» vector; 6 – extract of
transgenic carrot plants, containing human IFN-a2b (600 IU/ml)); b –
results of densitometry. The vertical line represents the level of MGMT
and MARP proteins in conventional densitometry units
Variant
24 �
48 �
kDa
a
b
1 2 3 4 5 6 7
0
1
2
3
4
1 2 3 4 5 6 7
Fig. 5. The effect of 5-azacytidine on the MARP protein amount in the
4BL cells (180 p.): a – Western blot analysis (1 – control; 2 – mitomycin
C + 5-aza (10 �g/ml), 8 h; 3 – 5-aza (1 �g/ml), 11 days; 4 – 5-aza (10
�g/ml), 6 days; 5 – 5-aza (10�g/ml), 11 days; 6 – mitomycin C + 5-aza (1
�g/ml), 6 days; 7 – mitomycin C + 5-aza (10�g/ml), 6 days); b – results
of densitometry. The vertical line represents the MGMT and MARP
proteins amount in conventional densitometry units
dine cytotoxic effects include inhibiting DNA, RNA
and protein synthesis, drug incorporation into DNA and
RNA, as well as the activation of DNA damage path-
ways. Since the non-proliferating cells are practically
insensitive to azacytidine [28], mitomycin C was used in
this study as a cytostatic agent for the inhibition of cell
proliferation [29].
As shown in Fig. 5, the treatment of 4BL cells with
5-azacytidine alone or in the combination with mito-
mycin C did not influence the MGMT protein amount.
These results may indicate that the absence of the con-
ventional MGMT protein in the 4BL cells is not asso-
ciated with the promotor hypermethylation. However,
the MARP amount significantly changed under the 5-
azacytidine alone or in combination with mitomycin C
treatment. This fact requires further investigation.
Conclusions. Monoclonal anti-MGMT antibodies
(clone 23.2) recognize both the MGMT protein (M. w.
~ 24 kDa) and the unknown protein (M. w. ~ 48 kDa),
named as MARP, in Western blot analysis. These pro-
teins are highly inducible and their amount can be chan-
ged by some growth factors (Card medium, fibronec-
tin), cytokine (IFN-�2b), cytokine-like (EMAP II) or
cytokine-containing substances (Laferon and IFN-�2b
in plant extracts). However, this regulation depends not
only on the type of biologically active substances but on
the type of cell lines. The absence of MGMT protein in
4BL cells is unlikely due to the MGMT gene promotor
hypermethylation. The mechanisms of regulation of
the MGMT gene expression by growth factors and cy-
tokines require further studies.
Ê. Â. Êîöàðåíêî, Â. Â. Ëèëî, Ë. Ë. Ìàöåâè÷, Ò. Ï. Ðóáàí,
Þ. Ñ. Ëó÷àê³âñüêà, Ì. Â. Êó÷óê, Ë. Ë. Ëóêàø
Âïëèâ äåÿêèõ á³îëîã³÷íî àêòèâíèõ ðå÷îâèí íà âì³ñò á³ëê³â MGMT
³ MARP ó êë³òèíàõ ëþäèíè in vitro
Ðåçþìå
Ìåòà. Äîñë³äæåííÿ âïëèâó á³îëîã³÷íî àêòèâíèõ ñïîëóê IFN-�2b,
EMAPII, ñåðåäîâèùà Card ³ ô³áðîíåêòèíó íà âì³ñò á³ëê³â MGMT
(Î6-ìåòèëãóàí³í-ÄÍÊ ìåòèëòðàíñôåðàçà) ³ MARP (á³ëîê, ùî ðîç-
ï³çíàºòüñÿ àíòè-MGMT àíòèò³ëàìè) ó êë³òèíàõ ëþäèíè in vitro.
Ìåòîäè. Êë³òèíè ëþäèíè ë³í³é 4BL, Hep-2 ³ A102 îáðîáëÿëè ðîñ-
òîâèìè ôàêòîðàìè ³ öèòîê³íàìè. Çì³íè â ê³ëüêîñò³ á³ëê³â MGMT
³ MARP äîñë³äæóâàëè ç âèêîðèñòàííÿì Âåñòåðí áëîò àíàë³çó ³ ìî-
íîêëîíàëüíèõ àíòè-MGMT àíòèò³ë. Ðåçóëüòàòè. Îáðîáêà êë³-
òèí A102 ïðåïàðàòàìè EMAPII, ô³áðîíåêòèíó ³ Ëàôåðîíó ïðèçâî-
äèòü äî çíèæåííÿ ê³ëüêîñò³ á³ëêà MGMT íà ôîí³ çíà÷íîãî çðî-
ñòàííÿ ê³ëüêîñò³ á³ëêà MARP ó öèõ êë³òèíàõ. Îáðîáêà ðåêîìá³íàí-
òíèì á³ëêîì IFN-�2b ï³äâèùóº ê³ëüê³ñòü á³ëê³â MGMT ³ MARP ó
êë³òèíàõ Hep-2, à åêñòðàêòàìè òðàíñãåííèõ ðîñëèí, ÿê³ ì³ñòÿòü
IFN-�2b ëþäèíè, – çìåíøóº ê³ëüê³ñòü îáîõ á³ëê³â ó êë³òèíàõ Hep-2
òà á³ëêà MARP ó êë³òèíàõ 4BL. Âèñíîâêè. MGMT ³ MARP º âèñîêî-
³íäóöèáåëüíèìè á³ëêàìè. ¯õíÿ ê³ëüê³ñòü ìîæå âàð³þâàòè ï³ä 䳺þ
äåÿêèõ ðîñòîâèõ ôàêòîð³â (ñåðåäîâèùå Card ³ ô³áðîíåêòèí), öè-
òîê³íó (IFN-�2b), öèòîê³íîïîä³áíîãî (EMAPII) òà öèòîê³íîâì³ñ-
íèõ (Ëàôåðîí ³ IFN-�2b ó êîìïîçèö³¿ ç ðîñëèííèìè åêñòðàêòàìè)
ïðåïàðàò³â. Âèÿâëåíà ðåãóëÿö³ÿ çàëåæèòü íå ëèøå â³ä òèïó á³îëî-
ã³÷íî àêòèâíèõ ðå÷îâèí, àëå é â³ä êë³òèííèõ ë³í³é, âèêîðèñòàíèõ â
åêñïåðèìåíòàõ.
Êëþ÷îâ³ ñëîâà: ë³í³¿ êë³òèí ëþäèíè, ðåïàðàòèâíèé ôåðìåíò
MGMT, MARP, öèòîê³íè, ðîñòîâ³ ôàêòîðè, Âåñòåðí-áëîò àíàë³ç.
Å. Â. Êîöàðåíêî, Â. Â. Ëûëî, Ë. Ë. Ìàöåâè÷, Ò. À. Ðóáàí,
Þ. Ñ. Ëó÷àêèâñêàÿ, Í. Â. Êó÷óê, Ë. Ë. Ëóêàø
Âëèÿíèå íåêîòîðûõ áèîëîãè÷åñêè àêòèâíûõ âåùåñòâ íà
ñîäåðæàíèå áåëêîâ MGMT è MARP â êëåòêàõ ÷åëîâåêà in vitro
Ðåçþìå
Öåëü. Èññëåäîâàòü âëèÿíèå áèîëîãè÷åñêè àêòèâíûõ ñîåäèíåíèé
IFN-�2b, EMAP II, ñðåäû Card è ôèáðîíåêòèíà íà ñîäåðæàíèå
áåëêîâ MGMT (Î6-ìåòèëãóàíèí-ÄÍÊ ìåòèëòðàíñôåðàçà) è
MARP (áåëîê, ðàñïîçíàâàåìûé àíòè-MGMT àíòèòåëàìè) â êëåò-
êàõ ÷åëîâåêà in vitro. Ìåòîäû. Êëåòêè ÷åëîâåêà ëèíèé 4BL, Hep-2
è A102 îáðàáàòûâàëè ðîñòîâûìè ôàêòîðàìè è öèòîêèíàìè.
Èçìåíåíèÿ â êîëè÷åñòâå áåëêîâ MGMT è MARP èññëåäîâàëè ñ èñ-
ïîëüçîâàíèåì Âåñòåðí áëîò àíàëèçà è ìîíîêëîíàëüíûõ àíòè-
MGMT àíòèòåë. Ðåçóëüòàòû. Îáðàáîòêà êëåòîê À102 ïðåïàðà-
òàìè EMAPII, ôèáðîíåêòèíà, Ëàôåðîíà è ñðåäû Card ñíèæàåò
êîëè÷åñòâî áåëêà MGMT íà ôîíå çíà÷èòåëüíîãî âîçðàñòàíèÿ êî-
ëè÷åñòâà áåëêà MARP â ýòèõ êëåòêàõ. Îáðàáîòêà ðåêîìáèíàíò-
íûì áåëêîì IFN-�2b óâåëè÷èâàåò êîëè÷åñòâî áåëêîâ MGMT è
MARP â êëåòêàõ Hep-2, à ýêñòðàêòàìè òðàíñãåííûõ ðàñòåíèé,
ñîäåðæàùèõ ÷åëîâå÷åñêèé IFN-�2b, – ñóùåñòâåííî ñíèæàåò êî-
ëè÷åñòâî îáîèõ áåëêîâ â êëåòêàõ Hep-2 è MARP â êëåòêàõ 4BL.
Âûâîäû. MGMT è MARP ÿâëÿþòñÿ âûñîêîèíäóöèáåëüíûìè áåëêà-
ìè. Èõ êîëè÷åñòâî ìîæåò âàðüèðîâàòü ïîä äåéñòâèåì íåêîòî-
ðûõ ðîñòîâûõ ôàêòîðîâ (ñðåäà Card è ôèáðîíåêòèí), öèòîêèíà
(IFN-�2b) öèòîêèíîïîäîáíîãî (EMAP II) è öèòîêèíñîäåðæàùèõ
(Ëàôåðîí è IFN-�2b â êîìïîçèöèè ñ ðàñòèòåëüíûìè ýêñòðàêòà-
ìè) ïðåïàðàòîâ. Âûÿâëåííàÿ ðåãóëÿöèÿ çàâèñèò íå òîëüêî îò òè-
ïà áèîëîãè÷åñêè àêòèâíûõ âåùåñòâ, íî è îò êëåòî÷íûõ ëèíèé, èñ-
ïîëüçîâàííûõ â ýêñïåðèìåíòàõ.
Êëþ÷åâûå ñëîâà: ëèíèè êëåòîê ÷åëîâåêà, ðåïàðàòèâíûé ôåð-
ìåíò MGMT, MARP, öèòîêèíû, ðîñòîâûå ôàêòîðû, Âåñòåðí-
áëîò àíàëèç.
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KOTSARENKO K. V. ET AL.
|
| id | nasplib_isofts_kiev_ua-123456789-154307 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T17:34:32Z |
| publishDate | 2014 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Kotsarenko, K.V. Lylo, V.V. Macewicz, L.L. Ruban, T.P. Luchakivska, Yu.S. Kuchuk, M.V. Lukash, L.L. 2019-06-15T12:35:35Z 2019-06-15T12:35:35Z 2014 Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro / K.V. Kotsarenko, V.V. Lylo, L.L. Macewicz, T.P. Ruban, Yu.S. Luchakivska, M.V. Kuchuk, L.L. Lukash // Вiopolymers and Cell. — 2014. — Т. 30, № 3. — С. 203-208. — Бібліогр.: 29 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000897 https://nasplib.isofts.kiev.ua/handle/123456789/154307 575.224 + 577.218 Aim. To investigate an effect of biologically active compounds IFN-α2b, EMAPII, Card medium, fibronectin on the amount of MGMT (O6-methylguanine-DNA methyltransferase) and MARP (anti-Methyltransferase Antibody Recognizable Protein) proteins in human cells in vitro. Methods. The human cells of 4BL, Hep-2 and A102 lines were treated with growth factors and cytokines. Changes in the amount of MGMT and MARP proteins were studied by Western blot analysis with anti-MGMT mAbs. Results. The treatment of A102 cells with EMAPII, fibronectin, Laferon and Card medium led to a decreased level of the MGMT protein, whereas the amount of MARP was highly increased in these cells. The treatment with the recombinant protein IFN-α2b increased the amount of MGMT and MARP proteins in Hep-2 cells. The treatment with extracts of transgenic plants,containing human IFN-α2b, caused a significant decrease in the content of both proteins in Hep-2 cells and MARP in 4BL cells. Conclusions. Both MGMT and MARP are highly inducible proteins. Their amount in cells can be changed by some growth factors (Card medium, fibronectin), cytokine (IFN-α2b), cytokine-like (EMAPII) or cytokine-containing substances (Laferon and IFN-α2b in plant extracts). This regulation depended not only on the type of biologically active substances but on the cell line used in this study as well. Мета. Дослідження впливу біологічно активних сполук IFN-α2b, EMAPII, середовища Card і фібронектину на вміст білків MGMT (О6-метилгуанін-ДНК метилтрансфераза) і MARP (білок, що розпізнається анти-MGMT антитілами) у клітинах людини in vitro. Методи. Клітини людини ліній 4BL, Hep-2 і A102 обробляли ростовими факторами і цитокінами. Зміни в кількості білків MGMT і MARP досліджували з використанням Вестерн блот аналізу і моноклональних анти-MGMT антитіл. Результати. Обробка клітин A102 препаратами EMAPII, фібронектину і Лаферону призводить до зниження кількості білка MGMT на фоні значного зростання кількості білка MARP у цих клітинах. Обробка рекомбінантним білком IFN-α2b підвищує кількість білків MGMT і MARP у клітинах Hep-2, а екстрактами трансгенних рослин, які містять IFN-α2b людини, – зменшує кількість обох білків у клітинах Hep-2 та білка MARP у клітинах 4BL. Висновки. MGMT і MARP є високо-індуцибельними білками. Їхня кількість може варіювати під дією деяких ростових факторів (середовище Card і фібронектин), цитокіну (IFN-α2b), цитокіноподібного (EMAPII) та цитокіновмісних (Лаферон і IFN-α2b у композиції з рослинними екстрактами) препаратів. Виявлена регуляція залежить не лише від типу біологічно активних речовин, але й від клітинних ліній, використаних в експериментах. Цель. Исследовать влияние биологически активных соединений IFN-α2b, EMAP II, среды Card и фибронектина на содержание белков MGMT (О6-метилгуанин-ДНК метилтрансфераза) и MARP (белок, распознаваемый анти-MGMT антителами) в клетках человека in vitro. Методы. Клетки человека линий 4BL, Hep-2 и A102 обрабатывали ростовыми факторами и цитокинами. Изменения в количестве белков MGMT и MARP исследовали с использованием Вестерн блот анализа и моноклональных анти-MGMT антител. Результаты. Обработка клеток А102 препаратами EMAPII, фибронектина, Лаферона и среды Card снижает количество белка MGMT на фоне значительного возрастания количества белка MARP в этих клетках. Обработка рекомбинантным белком IFN-α2b увеличивает количество белков MGMT и MARP в клетках Hep-2, а экстрактами трансгенных растений, содержащих человеческий IFN-α2b, – существенно снижает количество обоих белков в клетках Hep-2 и MARP в клетках 4BL. Выводы. MGMT и MARP являются высокоиндуцибельными белками. Их количество может варьировать под действием некоторых ростовых факторов (среда Card и фибронектин), цитокина (IFN-α2b) цитокиноподобного (EMAP II) и цитокинсодержащих (Лаферон и IFN-α2b в композиции с растительными экстрактами) препаратов. Выявленная регуляция зависит не только от типа биологически активных веществ, но и от клеточных линий, использованных в экспериментах. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro Вплив деяких біологічно активних речовин на вміст білків MGMT і MARP у клітинах людини in vitro Влияние некоторых биологически активных веществ на содержание белков MGMT и MARP в клетках человека in vitro Article published earlier |
| spellingShingle | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro Kotsarenko, K.V. Lylo, V.V. Macewicz, L.L. Ruban, T.P. Luchakivska, Yu.S. Kuchuk, M.V. Lukash, L.L. Structure and Function of Biopolymers |
| title | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro |
| title_alt | Вплив деяких біологічно активних речовин на вміст білків MGMT і MARP у клітинах людини in vitro Влияние некоторых биологически активных веществ на содержание белков MGMT и MARP в клетках человека in vitro |
| title_full | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro |
| title_fullStr | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro |
| title_full_unstemmed | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro |
| title_short | Influence of some biologically active substances on amount of MGMT and MARP proteins in human cells in vitro |
| title_sort | influence of some biologically active substances on amount of mgmt and marp proteins in human cells in vitro |
| topic | Structure and Function of Biopolymers |
| topic_facet | Structure and Function of Biopolymers |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/154307 |
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