Plant and animal lectines as modulators of mgmt and marp gene expression in vitro
Aims. Previously for the first time we have studied the ability of lectins to influence the processes of mutagenesis and antimutagenesis in different test systems. The aim of present study was to examine the effect of panel of lectins on the MGMT and MARP expression levels in tumor and non-tumor mam...
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| Cite this: | Plant and animal lectines as modulators of mgmt and marp gene expression in vitro / L.L. Macewicz, V.V. Lylo, I.S. Karpova, K.V. Kotsarenko, T.A. Ruban, L.L. Lukash // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2014. — Т. 15. — С. 260-264. — Бібліогр.: 26 назв. — англ. |
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Macewicz, L.L. Lylo, V.V. Karpova, I.S. Kotsarenko, K.V. Ruban, T.A. Lukash, L.L. 2021-02-18T15:22:28Z 2021-02-18T15:22:28Z 2014 Plant and animal lectines as modulators of mgmt and marp gene expression in vitro / L.L. Macewicz, V.V. Lylo, I.S. Karpova, K.V. Kotsarenko, T.A. Ruban, L.L. Lukash // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2014. — Т. 15. — С. 260-264. — Бібліогр.: 26 назв. — англ. 2219-3782 https://nasplib.isofts.kiev.ua/handle/123456789/178342 575.224 Aims. Previously for the first time we have studied the ability of lectins to influence the processes of mutagenesis and antimutagenesis in different test systems. The aim of present study was to examine the effect of panel of lectins on the MGMT and MARP expression levels in tumor and non-tumor mammalian cells in vitro. Methods. Standard cell cultivation methods and Western blot analysis were used. Results. The influence of plant and animal lectins (perk egg lectin, lentil seeds lectin and elderberry bark lectin) on expressiom of proteins recognized by anti-MGMT monoclonal antibodies (MGMT and MARP) on stable and destabilized human non-tumor and tumor-derived cell lines was studied. Conclusions. Studied lectins are able to modulate the expression of MGMT and MARP. The influence of SNA-I on MARP and MGMT expression levels depends on origin and genomic stability of cell line. SNA-I is perspective for further study as potential drug in anti-tumor therapy optimization schemes. Key words: MGMT expression, MARP expression, lectins, NiCl2, cell lines. en Інститут молекулярної біології і генетики НАН України Фактори експериментальної еволюції організмів Прикладна генетика і селекція Plant and animal lectines as modulators of mgmt and marp gene expression in vitro Article published earlier |
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Plant and animal lectines as modulators of mgmt and marp gene expression in vitro |
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Plant and animal lectines as modulators of mgmt and marp gene expression in vitro Macewicz, L.L. Lylo, V.V. Karpova, I.S. Kotsarenko, K.V. Ruban, T.A. Lukash, L.L. Прикладна генетика і селекція |
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Plant and animal lectines as modulators of mgmt and marp gene expression in vitro |
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Plant and animal lectines as modulators of mgmt and marp gene expression in vitro |
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Plant and animal lectines as modulators of mgmt and marp gene expression in vitro |
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Plant and animal lectines as modulators of mgmt and marp gene expression in vitro |
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plant and animal lectines as modulators of mgmt and marp gene expression in vitro |
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Macewicz, L.L. Lylo, V.V. Karpova, I.S. Kotsarenko, K.V. Ruban, T.A. Lukash, L.L. |
| author_facet |
Macewicz, L.L. Lylo, V.V. Karpova, I.S. Kotsarenko, K.V. Ruban, T.A. Lukash, L.L. |
| topic |
Прикладна генетика і селекція |
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Прикладна генетика і селекція |
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2014 |
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English |
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Фактори експериментальної еволюції організмів |
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Інститут молекулярної біології і генетики НАН України |
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Aims. Previously for the first time we have studied the ability of lectins to influence the processes of mutagenesis and antimutagenesis in different test systems. The aim of present study was to examine the effect of panel of lectins on the MGMT and MARP expression levels in tumor and non-tumor mammalian cells in vitro. Methods. Standard cell cultivation methods and Western blot analysis were used. Results. The influence of plant and animal lectins (perk egg lectin, lentil seeds lectin and elderberry bark lectin) on expressiom of proteins recognized by anti-MGMT monoclonal antibodies (MGMT and MARP) on stable and destabilized human non-tumor and tumor-derived cell lines was studied. Conclusions. Studied lectins are able to modulate the expression of MGMT and MARP. The influence of SNA-I on MARP and MGMT expression levels depends on origin and genomic stability of cell line. SNA-I is perspective for further study as potential drug in anti-tumor therapy optimization schemes.
Key words: MGMT expression, MARP expression, lectins, NiCl2, cell lines.
|
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2219-3782 |
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https://nasplib.isofts.kiev.ua/handle/123456789/178342 |
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Plant and animal lectines as modulators of mgmt and marp gene expression in vitro / L.L. Macewicz, V.V. Lylo, I.S. Karpova, K.V. Kotsarenko, T.A. Ruban, L.L. Lukash // Фактори експериментальної еволюції організмів: Зб. наук. пр. — 2014. — Т. 15. — С. 260-264. — Бібліогр.: 26 назв. — англ. |
| work_keys_str_mv |
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| first_indexed |
2025-11-26T01:39:48Z |
| last_indexed |
2025-11-26T01:39:48Z |
| _version_ |
1850603842985525248 |
| fulltext |
260
UDC 575.224
MACEWICZ L.L., LYLO V.V., KARPOVA I.S., KOTSARENKO K.V., RUBAN T.A., LUKASH L.L.
Institute of Molecular Biology and Genetics of Natl. Acad. Sci. of Ukraine,
Ukraine, 03680, Kyiv, Akad. Zabolotnogo str., 150, е-mail: lukash@imbg.org.ua
PLANT AND ANIMAL LECTINES AS MODULATORS OF MGMT AND MARP GENE
EXPRESSION IN VITRO
Repair enzyme O6-methylguanine-DNA
methyltransferase (MGMT) is the major defence
factor against the mutagenic, carcinogenic and
cytotoxic effects of alkylating agents [1, 2]. It can
transfer an alkyl group from the O6-position of
guanine to its own cysteine residue with further
degradation of MGMT molecule [3, 4]. Thus,
MGMT protects normal cells from endogenous and
exogenous mutagenes and carcinogens. But
alkylating agents are widely used in cancer
chemotherapy schemes. The MGMT expression and
activity in malignant cells are often high, and need
to be inhibited. Besides the secondary
tumorigenesis after the treatment with alkylating
agents is possible. Thus, the protection of non-
tumor cells from genotoxic anti-cancer drugs is an
issue. Therefore, this enzyme is considered to be
one of the targets to regulate antitumor efficacy of
alkylating agents [1, 5, 6].
The expression level of MGMT gene can
differ widely in individuals, in tissues and organs of
the same organism, and in normal and tumor cells
of the same organ [7, 8]. In our previous
investigations we found and described the unknown
protein with M.w. ≈ 50 kDa [9, 10] named as
MARP (anti-Methyltransferase Antibody
Recognizable Protein), shown to be highly
inducible, and presumed to be the part of DNA
repair system in mammalian cells. It can be
regulated by some cytokines and growth factors
[11].
Therefore the search for effective modulators
of MGMT activity and expression is actual for
improving anti-tumor efficacy of alkylating agents
and preventing of their side effects in normal cells.
To date only a small number of DNA repair genes
have been shown to be inducible in mammalian
cells and MGMT is presumably one of the best
studied ones. And the inducibility of MARP have
not been studied yet. According to the literature data
the MGMT gene expression may be affected by
various factors: the alkylating agents, single-strand
DNA breaks, transcription factors, activators of
protein kinase C, MGMT gene promoter
hypermethylation, etc. [12]. Regulation of
expression can also be indirect through different
intracellular signaling pathways [13]. Some
cytokines are known to be able to influence on the
MGMT and MARP gene expression as well [1, 13–
15].
Plant and animal lectines are recognized as
regulating proteins due to their broad spectrum of
biological activity in all living systems. Biological
functions of these proteins are based on their
carbohydrate specificity. Exogenous lectins can
influence through glycosylated surface of the cell
membrane, and can up- and downregulate adhesion,
migration, apoptosis, proliferation etc in pro- and
eukaryotic systems [16]. Previously for the first
time we have studied the ability of such a lectins to
influence the processes of mutagenesis and
antimutagenesis in prokaryotic test systems [19] and
mammalian cell cultures [18–20]. The elderberry
bark lectins were shown to have both mutagenic and
toxic activity (at high concentrations) and protective
properties (at low concentrations) [18, 21]. In our
studies it has also been shown that plant lectins are
able to modify DNA damage repair process [22].
Thus, we have assumed that repair enzyme
MGMT could also be a target of lectin regulatory
impact on mutation process. The aim of our study
was to examine the effect of panel of lectins on the
MGMT and MARP expression levels in tumor and
non-tumor mammalian cells in vitro.
Materials and methods
The following cell cultures were used in the
experiments: 4BL – fibroblast-like cell line,
received in our laboratory from blood of an adult
donor; the standard line of Hep2 cells. The cells
were cultivated in a standard DMEM growth
medium with an addition of 10 % Fetal bovine
serum (PAA) and antibiotics (penicillin and
streptomycin) at 37oC with 4 % CO2. As biological
factors commercial preparations of elderberry
(Sambucus nigra) bark lectins, perch eggs lectins,
lentil seed lectins (Lectinotest, Lviv, Ukraine) were
used and as a chemical mutagen – nickel chloride.
Conditions of cell treatment with lectins and NiCl2
were described previously [22]. Protein extracts
were obtained according to a previously described
method [15].
SDS electrophoresis of the proteins was
performed in 12 % polyacrylamide gel by the
Laemmli method [23]. The total protein
261
concentration was determined by the Bradford
method [24] in every sample in order to an equal
quantity of the protein was loaded into every lane.
Monoclonal antibodies against human MGMT
(clone 23.2, isotype IgG2b) were obtained from
Novus Biologicals, USA. Secondary antibodies
peroxidase conjugated with horseradish were from
Sigma. The procedure of MGMT identification in
the samples was performed according to the
recommendation of the manufacturer of monoclonal
antibodies [http://www.novusbio.com/]. For even
loading control the densitometry of the stained
hybridized membrane was used (Scion Image
program) as there are some literature data that total
protein stains is an acceptable alternative to single-
protein loading controls [25].
Results and discussion
4BL cell culture was treated by panel of
lectins including perch egg lectin (PEL), lentil seed
lectin (LSL) and elderberry bark lectin (SNA-I) in
concentrations 20 and 80 mkg/ml during 4h. With
Western-blot analysis it was shown absence of
MGMT expression both in control and in treated
cells (fig. 1, a), but MARP protein was detected in
all the samples. Under the SNA-I treatment MARP
expression was increased in cells treated by 20
mkg/ml, while 80 mkg/ml concentration had no
effect. The similar tendency was shown in the case
of animal lectin (PEL) treated cells but MARP
expression level was higher. The effect of LSL
treatment has another dose dependence: 20 mkl/ml
concentration have no affect MARP expression, but
80 mkg/ml extremely increased the amount of
protein. Thus, all lectins studied were shown to
influence the MARP expression on protein level,
depending on the origin of lectin.
Further we have performed the more detailed
study of the dose dependence of PEL treatment
effect in 4BL cells (fig. 1, b). PEL was shown to
induce MARP expression with all treatment
concentrations (0.2, 2 and 20 mkg/ml), but level of
expression was the same in all treated samples .
Thus, PEL active dose is rather low.
The ability of of SNA-I to induce MARP
expression were the lowest in studied lectin panel,
and the in some cases were undetectable (fig. 1, c).
But, newertheless, such small induction was
observed even in samples treated by low
0.2 mkg/ml lectin concentration.
Though the low influence of SNA-I-induced
MARP expression level changes in non-tumor intact
cells, in our earlier studies we exhibited the DNA
repair induction by SNA-I in nickel-damaged cells
[21]. And this was the reason to study the joint
effect of this lectin and NiCl2 (fig. 2).
So, the moderate up-regulation of MARP
expression level in cells treated by 0.2 mkg/ml of
lectin, and downregulation of this level in nickel-
treated cells was shown. But in cells treated both by
nickel chloride and lectin the MARP expression
level was the same as in untreated cells. Thus, we
can conclude that in normal cell cultures SNA-I
lectin modulates the MARP expression rather
slightly but we suppose that it can cooperate with
some components of repair system after mutagen
action.
a b c
Fig. 1. The MARP expression in 4BL cells treated by perch eggs lectin (PEL), lentil seed lectin (LSL)
and elderberry bark lectin (SNA-I). a) 1 – control, 2 – SNA-I, 20 mkg/ml, 3 – SNA-I, 80 mkg/ml, 4 – PEL,
20 mkg/ml, 5 – PEL, 80 mkg/ml, 6 – LSL, 20 mkg/ml, 7 – LSL, 80 mkg/ml. b) 1 – PEL, control, 2 – PEL, 20
mkg/ml, 3 – PEL, 2 mkg/ml, 4 – PEL, 0.2 mkg/ml. c) 1 – SNA-I, control, 2 – SNA-I, 20 mkg/ml, 3 – SNA-I,
2 mkg/ml, 4 – SNA-I, 0.2 mkg/ml, 5 – SNA-I, 20 mkg/ml 72h postincubation, 6 – SNA-I, 2 mkg/ml 72h
postincubation, 7 – SNA-I, 0.2 mkg/ml 72h postincubation
262
Fig. 2. The MARP expression in 4BL cells
treated by elderberry bark lectin (SNA-I) and NiCl2
(500 mkg/ml): 1 – control; 2 – SNA-I, 2 mkg/ml,
3 – SNA-I, 0,2 mkg/ml; 4 – NiCl2; 5 – NiCl2 +
SNA-I, 0,2 mkg/ml
Moreover we were interested if SNA-I can
change its action in other stress conditions. And in
our futher experiments we did demonstrate that in
unstable cell culture the SNA-I effect is much more
manifested. Cell culture destabilization was induced
by cultural medium with high ionic strength, and
was accompanied by lost of genomic stability [26].
Enhancing MARP expression in these conditions
under the treatment with SNA-I was substantial and
persisted at least up to 5 days (fig. 3, a).
a b
Fig. 3. The MARP expression in destabilized
4BL cells (a) and Hep-2 tumor cells (b) treated by
elderberry bark lectin (SNA-I): a) 1 – control,
2 – SNA-I (20 mkg/ml), 3 – 5 days postincubation
after SNA-I (20 mkg/ml) treatment; b) 1 – control,
2 – SNA-I (80 mkg/ml), 3 – SNA-I (20 mkg/ml),
4 – SNA-I (2 mkg/ml), 5 – SNA-I (0.2 mkg/ml)
Substantial influence of SNA-I both on
MARP and MGMT expression was demonstrated in
cells of tumor origih (Hep-2) (fig. 3, b). But the
character of such influence was completely different
from the one found in 4Bl. The lowest
concentration 0,2 mkg/ml which was shown to be
genoprotective in non-malignant cell lines and
primary human cells, causes near two-fold
decreasing MGMT expression. MARP expression
was not detected at all, whilst this enzyme is present
in intact Hep-2 cells. In cell culture treated with 2
mkg/ml of lectin the expression levels of both
MGMT and MARP are close to the control level,
and in 20 mkg/ml treated cells are substantially
enhanced. But further increasing lectin
concentration leads to decrease of MGMT and
MARP expression levels.
As result of this work SNA-I lectin was
shown to discriminate non-tumor, destabilizated
non-tumor and tumor cells in vitro according to its
influence on the MGMT and MARP expression
levels. Elderberry bark lectin SNA-I had only slight
effect on MARP expression in stable non-tumor cell
line 4BL. But in cell line on the stage of induced
secondary genomic instability SNA-I was shown to
substantionaly increase MARP expression level.
And in malignant cell line Hep-2 we demonstrated
both supress and enhance of MARP and MGMT
expression levels under the SNA-I treatment.
In early works lectins were assumed to be
only carbohydrate-binding proteins, but further they
were shown to affect the different kinds of enzyme
activities. For the first time we have shown that
MARP and repair enzyme MGMT expression can
be regulated by these biologically active substances
as well.
Conclusions
Lectins of plant and animal origin (perk egg
lectin, lentil seeds lectin and elderberry bark lectin)
are able to modulate the expression of proteins
recognized by anti-MGMT monoclonal antibodies
(MGMT and MARP).
The influence of SNA-I on MARP and
MGMT expression levels depends on origin and
genomic stability of cell line. Destabilized 4BL cell
line was shown to be more sensitive to SNA-I
modulating action. In malignant cells both
expression up- and downregulating effect of SNA-I
lectin was described, so SNA-I is perspective for
futrther study as potential drug in anti-tumor
therapy optimization schemes.
263
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264
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MACEWICZ L.L., LYLO V.V., KARPOVA I.S., KOTSARENKO K.V., RUBAN T.A., LUKASH L.L.
Institute of Molecular Biology and Genetics of Natl. Acad. Sci. of Ukraine,
Ukraine, 03680, Kyiv, Akad. Zabolotnogo str., 150, е-mail: lukash@imbg.org.ua
PLANT AND ANIMAL LECTINES AS MODULATORS OF MGMT AND MARP GENE
EXPRESSION IN VITRO
Aims. Previously for the first time we have studied the ability of lectins to influence the processes of
mutagenesis and antimutagenesis in different test systems. The aim of present study was to examine the
effect of panel of lectins on the MGMT and MARP expression levels in tumor and non-tumor mammalian
cells in vitro. Methods. Standard cell cultivation methods and Western blot analysis were used. Results. The
influence of plant and animal lectins (perk egg lectin, lentil seeds lectin and elderberry bark lectin) on
expressiom of proteins recognized by anti-MGMT monoclonal antibodies (MGMT and MARP) on stable
and destabilized human non-tumor and tumor-derived cell lines was studied. Conclusions. Studied lectins
are able to modulate the expression of MGMT and MARP. The influence of SNA-I on MARP and MGMT
expression levels depends on origin and genomic stability of cell line. SNA-I is perspective for further study
as potential drug in anti-tumor therapy optimization schemes.
Key words: MGMT expression, MARP expression, lectins, NiCl2, cell lines.
UDC 633.11:631.523:633.11
MOTSNY I.I. 1, SUDARCHUK L.V. 1, GALAEV A.V. 1, CHEBOTAR S.V. 1, 2
1 Plant Breeding and Genetics Institute – National Center of Seed and Cultivar investigations,
Ukraine, 65036, Odessa, Ovidiopolskaya dor., 3, e-mail: motsnyyii@gmail.com
2 Odessa National Mechnikov University, Department of Genetics and Molecular Biology,
Ukraine, 65026, Odessa, Dvoryanskaya str., 2
APPLICATION OF PCR MARKERS FOR DETECTING 1BL.1RS WHEAT-RYE CHROMOSOME
TRANSLOCATIONS AND (1B)1R SUBSTITUTIONS
Nikolai Vavilov was the first to recognize the
utilization of wheat relatives is a promising source
for wheat improvement [1]. As an development of
Vavilov`s ideas a number of wheat introgression
stocks with a high resistance to powdery mildew,
leaf and stem rusts, frost tolerance, high protein
content and some morphological characters has
been obtained as a result of wide crosses [2, 3]. For
a successful practical application the stocks require
an identification of the alien introgressions. DNA
markers become a useful tool for gene or
chromosome identification, especially being
valuable in respect of new for wheat an alien
genetic material.
This paper deals with PCR marker assisted
detection of (1B)1R wheat-rye chromosome
substitution and 1BL.1RS translocation, their meiotic
behavior and genetic analysis of certain alien
characters, incorporated into wheat. The
investigation was carried out within a program for
the development of a genetic collection of bread
wheat lines with qualitative characters.
Material and methods
A set of original primitive introgression
stocks (2n = 42): Erythrospermum 200_97-2 (in
further E200_97-2), Erythrospermum 217_97
(E217_97), Hostianum 242_97-1 (H242_97-1),
Hostianum 242_97-2 (H242_97-2), Hostianum
273_97 (H273_97), Hostianum 274_97 (H274_97)
and ОН232_03, collection sib-strains H74_90-245
and H74_90-258, winter bread wheat cv. Odesskaya
267 (Od267) and F1 hybrids between Od267 and all
the lines have been investigated. The majority of the
stocks were developed from a cross: triticale (8x)
cv. AD825/T. durum Desf. cv. Chernomor and
spontaneous hybridization of the F3 hybrids with the
strain H74_90-245 or H74_90-258, or without it.
Triticale AD825 is a primary amphidiploid (T.
aestivum L. cv. Hostianum 237/S. cereale L. cv.
Voronezhskaya SHI) [4]. The strains H74_90-245
and H74_90-258 were derived in Dobroudja
Agricultural Institute (General Toshevo, Bulgaria)
from the step cross: Dr. Savov`s synthetic (T.
timopheevii Zhuk./Ae. tauschii Coss.)/Tom Pouce
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