Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells
Aim: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its phenotypic and growth patterns. Materials and Methods: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T line, stable T-rex HEK-hB...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2006
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| Цитувати: | Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells / E. Zhuravel, D. Lytvyn, M. Soldatkina, S. Zeleniy, T. Shestakova, P. Pogrebnoy // Experimental Oncology. — 2006. — Т. 28, № 4. — С. 288-292. — Бібліогр.: 21 назв. — англ. |
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irk-123456789-1379292018-06-18T03:06:57Z Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells Zhuravel, E. Lytvyn, D. Soldatkina, M. Zeleniy, S. Shestakova, T. Pogrebnoy, P. Original contributions Aim: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its phenotypic and growth patterns. Materials and Methods: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T line, stable T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in culture medium was generated. The morphological patterns, growth characteristics and colony forming activity of these cells were studied using routine techniques. Results: T-rex HEK-HBD2-m cell subline was shown to express both mRNA and hBD-2m protein upon the presence of 1 µg/ml tetracycline in culture medium as it was demonstrated by RT-PCR and immunocytochemical approach. Upon prolonged expression of hBD-2, the cells acquired special features: they lost ability to grow in monolayer in vitro and to form colonies in soft agar, characteristic to parental HEK293T cells, but possess higher growth rate and longer survival in FBS-free medium than wild type cells. Conclusion: Expression of hBD-2 in T-rex HEK-HBD2-m cell subline results in specific biological consequences that favor cell survival. Цель: создать линию клеток с регулируемой экспрессией бета-дефенсина-2 человека (hBD-2) и проанализировать влияние экспрессии этого пептида на особенности фенотипа и рост клеток. Материалы и методы: клеточная сублиния T-rex HEK hBD2-m, экспрессирующая биологически активную зрелую форму hBD-2 при индукции клеток тетрациклином, получена путем клонирования на основе эмбриональных клеток почки человека линии HEK293T. Морфологические особенности, характеристики роста и показатели колониеобразования в полужидкой среде исследовали стандартными методами. Результаты: с помощью методов РТ-ПЦР и иммуноцитохимии показано, что сублиния клеток T-rex HEK-HBD2- экспрессирует hBD2 в присутствии 1 µг/мл тетрациклина в среде инкубации. Продолжительная экспрессия hBD-2 приводила к тому, что клетки утрачивали способность образовывать монослой при культивировании in vitro и образовывать колонии в полужидкой среде, но характеризовались более высокой скоростью роста и способностью к более продолжительному выживанию в бессывороточной среде, чем исходная линия клеток HEK293T. Выводы: экспрессия hBD-2 в сублинии клеток T-rex HEK-HBD2- cell обусловливает специфические биологические эфеекты, способствующие выживанию клеток. 2006 Article Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells / E. Zhuravel, D. Lytvyn, M. Soldatkina, S. Zeleniy, T. Shestakova, P. Pogrebnoy // Experimental Oncology. — 2006. — Т. 28, № 4. — С. 288-292. — Бібліогр.: 21 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137929 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Zhuravel, E. Lytvyn, D. Soldatkina, M. Zeleniy, S. Shestakova, T. Pogrebnoy, P. Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells Experimental Oncology |
| description |
Aim: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its phenotypic and growth patterns. Materials and Methods: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T line, stable T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in culture medium was generated. The morphological patterns, growth characteristics and colony forming activity of these cells were studied using routine techniques. Results: T-rex HEK-HBD2-m cell subline was shown to express both mRNA and hBD-2m protein upon the presence of 1 µg/ml tetracycline in culture medium as it was demonstrated by RT-PCR and immunocytochemical approach. Upon prolonged expression of hBD-2, the cells acquired special features: they lost ability to grow in monolayer in vitro and to form colonies in soft agar, characteristic to parental HEK293T cells, but possess higher growth rate and longer survival in FBS-free medium than wild type cells. Conclusion: Expression of hBD-2 in T-rex HEK-HBD2-m cell subline results in specific biological consequences that favor cell survival. |
| format |
Article |
| author |
Zhuravel, E. Lytvyn, D. Soldatkina, M. Zeleniy, S. Shestakova, T. Pogrebnoy, P. |
| author_facet |
Zhuravel, E. Lytvyn, D. Soldatkina, M. Zeleniy, S. Shestakova, T. Pogrebnoy, P. |
| author_sort |
Zhuravel, E. |
| title |
Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells |
| title_short |
Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells |
| title_full |
Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells |
| title_fullStr |
Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells |
| title_full_unstemmed |
Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells |
| title_sort |
regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2006 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/137929 |
| citation_txt |
Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells / E. Zhuravel, D. Lytvyn, M. Soldatkina, S. Zeleniy, T. Shestakova, P. Pogrebnoy // Experimental Oncology. — 2006. — Т. 28, № 4. — С. 288-292. — Бібліогр.: 21 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T04:45:22Z |
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2025-07-10T04:45:22Z |
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| fulltext |
288 Experimental Oncology 28, 288–292, 2006 (December)
It is accepted now that immune system plays an impor-
tant role in the control of tumor growth. Numerous experi-
mental studies carried out in recent years have revealed
that the character of interaction between tumor and host
organism is much more complex and multifactorial than
it has been postulated by the theory of immune control.
Among the factors of such interaction one may hypo-
thetically mention defensins — small cationic antimicrobal
peptides that are the components of innate immunity.
In the last decade it has been shown that a number
of human tumors are characterized by hyperexpression
or down-regulation of certain defensin genes and in
some cases serum levels of defensins may serve as ad-
ditional diagnostic markers of neoplastic process [1–7].
However, it remains unknown yet what is a functional role
of defensin expression in tumor cells — whether these
antimicrobials may protect the host from tumor develop-
ment by killing tumor cells, or benefit malignization and
metastasis. It has been revealed that defensins may exert
oppositely directed influence on cultured cells dependent
on their local concentration [8, 9]. Experiments carried
in vitro demonstrated that defensins may cause a strong
proliferative response in cultured cells in a concentra-
tion-dependent fashion, stimulating cell proliferation in
micromolar concentration range [10] and promote motili-
ty and invasiveness of cancer cells in vitro [11]. However,
in higher concentrations defensins are usually cytotoxic
and cause lysis of tumor cells [8] or may induce cell
death via mitochondrial injury and intracellular signalling
pathways [12]. In vivo conditions are more complicated,
because in tumor microenvironment defensins produced
by malignant cells or by blood cells infiltrating tumor may
interact with various cell receptors or be bound by HMW
proteins of blood plasma. Up to date, just few reports are
discussing possible local concentrations of defensins in
oral squamous cell carcinoma [13].
To answer the question on the potential role of
defensins in cancer it looks reasonable to create an
appropriate model system to study various effects
of defensins in vitro and in vivo. Such system should
represent the regulated expression of antimicrobial
peptide with possibility to evaluate the exact level of
peptide’s production per cell and the one where such
production may be stopped in a given moment.
In present study we aimed to create cell line with regu-
lated expression of human beta-defensin-2 (hBD-2) on
the base of human embryonic kidney cells of HEK293T
line and evaluate the influence of endogeneously pro-
duced hBD-2 on its phenotypic and growth patterns..
MATERIALS AND METHODS
Cell lines. HEK293T (human embryonic kidney)
cells were purchased from the Europian Collection of
Animal Cell Cultures. Cells were cultivated in DMEM
with high glucose content supplemented with 10% fetal
bovine serum (FBS), 100 units/mL penicillin G sodium
salt, 100 µg/ml streptomycin sulfate and 0.25 µg/ml
amphotericin B as fungizone.
Gene cloning and plasmid constructions. The
genes for mature β-defensin-2 were cloned from total
RNA isolated from A431 cells treated with 1 µg/ml EGF [14]
by RT-PCR using specific primers. To hBD-2m (mature
molecule): forward — 5´-ACT-TAA-GCT-TGC-CAT-GGG-
TAT-AGG-CGA-TCC-TGT-TAC-3´, and reverse — 5´-TCT-
TGG-AAT-TCT-CAT-GGC-TTT-TTG-CAG-CATTTT-G-3´)
(GenBank accession no. AF040153). PCR products were
digested with HindIII and EcoRI and cloned into the same
sites of inducible expression vector, pcDNA4/TO/neo
modified by us as follow: neomycin phosphotransferase
gene was cut from the pcDNA3 plasmid (Invitrogen Life
Technologies, USA) and ligated in the pcDNA4/TO (Invit-
rogen Life Technologies, USA). The obtained construct
REGULATED EXPRESSION OF HUMAN BETA-DEFENSIN-2 LEADS
TO ALTERED PHENOTYPE AND GROWTH PATTERNS OF CULTURED
HUMAN EMBRYONAL KIDNEY CELLS
E. Zhuravel*, D. Lytvyn, M. Soldatkina, S. Zeleniy, T. Shestakova, P. Pogrebnoy
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv, Ukraine
Aim: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its
phenotypic and growth patterns. Materials and Methods: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T
line, stable T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in cul-T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in cul- cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in cul-
ture medium was generated. The morphological patterns, growth characteristics and colony forming activity of these cells were studied using
routine techniques. Results: T-rex HEK-HBD2-m cell subline was shown to express both m���� and hBD-2m protein upon the presence ofT-rex HEK-HBD2-m cell subline was shown to express both m���� and hBD-2m protein upon the presence of cell subline was shown to express both m���� and hBD-2m protein upon the presence of
1 µg/ml tetracycline in culture medium as it was demonstrated by �T-PC� and immunocytochemical approach. Upon prolonged expression
of hBD-2, the cells acquired special features: they lost ability to grow in monolayer in vitro and to form colonies in soft agar, characteristic to
parental HEK293T cells, but possess higher growth rate and longer survival in FBS-free medium than wild type cells. Conclusion: Expression
of hBD-2 in T-rex HEK-HBD2-m cell subline results in specific biological consequences that favor cell survival.T-rex HEK-HBD2-m cell subline results in specific biological consequences that favor cell survival. cell subline results in specific biological consequences that favor cell survival.
Key Words: human beta-defensin-2, regulated expression, colony formation, HEK293 cells.
Received: November 17, 2006.
*Correspondence: E-mail: po�rebno��oncone�.�iev.�apo�rebno��oncone�.�iev.�a
Abbreviations used: ���-2 �� ��man be�a-de�ensin-2�� ���-2m �����-2 �� ��man be�a-de�ensin-2�� ���-2m ��
ma��re molec�le o� ��man be�a-de�ensin-2�� P�K1 �� p�os-
p�oinosi�ide-dependen� �inase 1�� RT-PCR �� reverse �ranscrip�ion
pol�merase c�ain reac�ion.
Exp Oncol 2006
28, 4, 288–292
Experimental Oncology 28, 288–292, 2006 (December) 28928, 288–292, 2006 (December) 289December) 289) 289 289
pcDNA4/TO/neo was verified by the restriction analysis
using endonucleases NruI and XhoI.
The correctness of insert in abovementioned
construct carrying hBD-2m gene was verified by the
DNA sequencing and plasmids were purified using a
standard protocol [15].
Transfection HEK293T cells. The resulting
constructs were cotransfected with the regulatory
plasmid, pcDNA6/TR (Invitrogen Life Technologies,
USA), which contained a selective marker — the blas-
ticidin S deaminase gene, into HEK293T cells using
FuGENE 6 Reagent (Roche Molecular Biochemicals,
USA) according to the instructions of manufacturer.
After transfection cells were selected in medium with
5 µg/ml blasicidin (Invitrogen Life Technologies, USA)
and 600 µg/ml G418 (Gibco, USA) for 2 weeks. Resis-
tant cells were treated with tetracycline to derepress
the hybrid CMV/TetO2 promoter in pcDNA4/TO/neo
and to induce expression of hBD-2 mature form.
Expression of mature hBD2 mRNA. After 24 h
incubation of obtained cell line T-rex-HEK-hBD-2m
with tetracycline, mRNA was isolated and the level of
expression of hBD-2 was analysed by RT-PCR with the
use of abovementioned primers.
Immunocytochemical analysis of hBD�2 expres�hBD�2 expres�expres�
sion. Cells were sedimented by centrifugation, and cell were sedimented by centrifugation, and cellwere sedimented by centrifugation, and cell sedimented by centrifugation, and cellsedimented by centrifugation, and cell, and celland cell
pellet was fixed with 4% formaline for 4 h, washed with4% formaline for 4 h, washed withh, washed with, washed withwashed with
PBS and stored in 70 70° ethanol. Later the paraffin blocksethanol. Later the paraffin blocks. Later the paraffin blocks
were prepared, 4 µm slides were cut and analysed im-µm slides were cut and analysed im-m slides were cut and analysed im-
munocytochemically using anti-hBD-2-MoAbs [16]MoAbs [16][16]
at the dilution of 1 : 10 and 1 : 25. For the part of the of 1 : 10 and 1 : 25. For the part of theand 1 : 25. For the part of the 1 : 25. For the part of the
samples, renewal of antigenic properties using Target
Retrieval Solution (DAKO, USA) according to protocolUSA) according to protocol) according to protocolaccording to protocol
of the manufacturer was applied. To block nonspecific. To block nonspecificTo block nonspecific
binding, biotin-blocking system (DAKO, USA) was used. (DAKO, USA) was used.USA) was used.) was used.
Anti-mouse antibodies labeled with biotin (Sigma, USA) (Sigma, USA)USA))
were used at the dilution of 1 : 400�� Vectastain ABC Kit of 1 : 400�� Vectastain ABC Kit
(Vecor Lab., USA) with peroxidase probe was used as theUSA) with peroxidase probe was used as the) with peroxidase probe was used as thewith peroxidase probe was used as the
third coating. As negative control, 1% BSA (Dia�, Rus-. As negative control, 1% BSA (Dia�, Rus-As negative control, 1% BSA (Dia�, Rus- 1% BSA (Dia�, Rus-BSA (Dia�, Rus- (Dia�, Rus-Dia�, Rus-, Rus-Rus-
sia) prepared on 0.02 � phosphate buffer (��� 7.2) was) prepared on 0.02 � phosphate buffer (��� 7.2) was prepared on 0.02 � phosphate buffer (��� 7.2) was 0.02 � phosphate buffer (��� 7.2) was� phosphate buffer (��� 7.2) was phosphate buffer (��� 7.2) wasphosphate buffer (��� 7.2) was (��� 7.2) was��� 7.2) was7.2) was was
used instead of specific antibodies. As positive control,. As positive control,As positive control,
gastric tumor tissue samples expressinghBD-2[16]were hBD-2 [16] were
used. Peroxidase conjugate was developed by 0.05%Peroxidase conjugate was developed by 0.05%0.05%
3’3’-diaminobenzidinetetrahydrochloride (Sigma, USA)diaminobenzidinetetrahydrochloride (Sigma, USA)(Sigma, USA)USA))
solution containing 0.01% hydrogen peroxide. Nucleihydrogen peroxide. Nuclei. NucleiNuclei
were counterstained with Mayer’s hematoxyline.
The study was performed using microscope Axio-
plan (Zeiss, Germany) equipped with the videocamera
Sony DXC-151AP, EZ Capture, computer Pentium IV,
and program for analysis of images (Media Cybernetic,
USA). Ten fields of view were studied per sample.
Colony formation analysis. Analysis of colony
forming ability of the cells was carried out by routine
technique as described elsewhere [17] in DMEM me-
dium supplemented with 15% FBS and 0.4% methylcel-
lulose without/with 1 µg/ml tetracycline. The cells were
dispensed in 6-well plates (1 х 103 cells per 1 ml of me-
dium) and incubated in СО2-incubator for 14–16 days.
The colonies were visualized using staining procedure
with 0.2% p-iodonitrotetrazolium violet dye.
RESULTS AND DISCUSSION
Generation of T-REX-HEK-hBD-2 cell sublines. For
cloning and expression of hBD-2 gene in mammalian
cells two-vector T-REx system was choosed. Such sys-
tem contains expressing vector pcDNA4/TO designed
for cloning of the gene of interest, and pcDNA6/TR
vector coding Tet repressor. Co-transfection of these
two vectors allows to perform tetracycline-regulated
expression of the gene of interest in mammalian cells.
Plasmid pcDNA4/TO contains hybrid promoter provid-
ing high level of tetracycline-regulated expression in
different types of cells. Promoter is composed from
two parts — early promoter of human cytomegalo-
virus (CMV) and region of two tetracycline operators
(TetII O2). For selection of stable cell lines, the gene of
resistance to zeocin is introduced in this plasmid. Two
TetII O2 sequences present the region of binding of
4 Tet repressor molecules (two Tet homodimers). Tet
repressor is expressed by pcDNA6/TR plasmid. In the
absence of tetracycline expression of the cloned gene
is suppressed through the binding of Tet repressor
homodimers with TetII O2 sequences. Addition of tet-
racycline to the cells derepresses hybrid CMV/ TetII O2
promoter in pcDNA4/TO plasmid via its binding with Tet
repressor and allows expression of the cloned gene.
T-REx system responded to all our requirements —
cloning of hBD-2 gene, except the site of resistance
to zeocin — this antibiotic is too expensive and rare��
that’s why we decided to replace it by another selective
marker — Neomycin: the gene of zeocin resistance
has been removed from pcDNA4/TO by restriction at
NruI/XhoI sites, and neomycin gene cut from the plas-
mide pcDNA3.1+ (Invitrogen, USA) was inserted. The
generated vector was named pcDNA4/TO/neo.
After this 151 bp fragment received by HindIII/EcoRI
restriction was cloned into pcDNA4neo plasmid by
HindIII/EcoRI sites. Selection of recombinant clones
was performed by restriction analysis allowed to
choose clone pcDNA4/TO/neo/hBD2m, containing
the sequence coding mature hBD-2 molecule.
Next, HEK293T cells were cotransfected with
pcDNA6/TR plasmid and pcDNA4/TO/neo/hBD2m by
standard procedure. Selection of clones was based on
blasticidin/G418 resistance. As the positive control,
pcDNA4/TO/lacZ vector was used. Co-transfection of
pcDNA4/TO/lacZ and pcDNA6/TR resulted in induction
of β-galactosidase gene upon addition of tetracycline,
and may be registered by staining using X-Gal.
Expression of hBD�2 in T�REX�HEK�hBD�2 cell
sublines. As we have shown earlier, the highest level
of defensin expression in engineered cell line could be
achieved in 20 h after induction with 1 µg/ml tetracycline.
RT-PCR analysis has demonstrated that the highest
level of transcription of gene coding mature molecule
of hBD-2 is registered in cell subline T-rex HEK-hBD2- T-rex HEK-hBD2-
m, induced with 1 µg/ml tetracycline, at lower level — in1 µg/ml tetracycline, at lower level — intetracycline, at lower level — in
cell lines T-rex-HEK-hBD2-m grown in the absence of T-rex-HEK-hBD2-m grown in the absence of
antibiotic, whilst in parental HEK293T cells expression expressionexpression
of hBD-2 mRNA wasn’t registered by the applied tech-
nique (Fig. 1). These data indicated that to some extent(Fig. 1). These data indicated that to some extent
290 Experimental Oncology 28, 288–292, 2006 (December)
the tet-regulated promoter is leaking (the most possible
explanation is that there are trace amounts of tetracycline
in the components of culture medium). Unfortunately, we
were unable to analyze the expression of defensin mol-
ecules using Western blot analysis because anti-hBD-2
MoAbs generated in our lab earlier [16] fail to recognize
native antigen in this assay. For this reason production
of hBD-2 peptide in cell subline has been studied using hBD-2 peptide in cell subline has been studied usingpeptide in cell subline has been studied using
immunocytochemical approach with the use of anti-hBD-
2-mAbs (that method was appropriate for detection of
hBD-2, as it was estimated using the samples of human
gastric tumors characterized by hyperexpression of hBD-
2 [16] or A431 cells induced by EGF as a positive control
(data not presented)). As one may see from Fig. 2, the. As one may see from Fig. 2, theAs one may see from Fig. 2, the one may see from Fig. 2, theone may see from Fig. 2, the may see from Fig. 2, themay see from Fig. 2, the see from Fig. 2, thesee from Fig. 2, the from Fig. 2, thefrom Fig. 2, the Fig. 2, theFig. 2, the. 2, the
distribution of cells by hBD-2 content looks as follow:looks as follow:
parental HEK293T cells < T-rex HEK-HBD2-m Tetr- <
T-rex HEK-HBD2-m Tetr+.
Fig. 1. RT-PCR analysis of hBD-2 expression. � — DNA lad- — DNA lad-DNA lad-
der�� 1, 2 — HEK293T cells without/with tetracycline incubation���� 1, 2 — HEK293T cells without/with tetracycline incubation��cells without/with tetracycline incubation����
3, 4 — T-rex HEK-hBD2m cells without induction with tetracy-hBD2m cells without induction with tetracy-BD2m cells without induction with tetracy-cells without induction with tetracy- without induction with tetracy-without induction with tetracy- induction with tetracy-induction with tetracy- with tetracy-with tetracy- tetracy-tetracy-
cline�� and after induction with tetracycline respectively�� and after induction with tetracycline respectivelyafter induction with tetracycline respectively induction with tetracycline respectivelyinduction with tetracycline respectively with tetracycline respectivelywith tetracycline respectively tetracycline respectivelytetracycline respectively
Expression of hBD�2 leads to altered cell growth
patterns and ability to form colonies in methylcel�
lulose clonal culture. The study of growth patterns of
cell subline T-rex HEK-hBD2-m compared to these of
parental ��ЕК293Т cells was performed by methods of
light microscopy and colony formation assay. We have
registered the phenomenon that T-rex HEK-hBD2-m
cells (contrary to parental strain) acquire ability for pro-
longed growth in culture (up to 148 h) even in the absence
of FBS, i. e. on “starving” medium (upon such conditions,
��ЕК293T cells die in 48–62 h). Using direct cell counting,
it was found out that the number of T-rex HEK-hBD2-m
cells cultivated in the presence of tetracycline in the
“starving” medium, increased nearly 1.7 fold during the
1st day, and nearly 3-fold — till the end of the 2nd day
versus 1.5- and 2-fold increase in T-rex HEK-hBD2-mT-rex HEK-hBD2-m
cultivated without tetracycline (the number of parentalthe number of parental
cells did not alter significantly) (Table).
Table. T�e n�mber o� cells eval�a�ed b� direc� cell co�n�in�
Cell line Cell n�mber (106 cells) (mean sq�are devia�ion)
Ini�ial 18 � 26 � 52 �
HEK293T Te�– 0.50 1.0 ± 0.25 0.40 ± 0.1* 0.50 ± 0.13*
HEK293T Te�+ 0.50 0.52 ± 0.16* 0.34 ± 0.01* 0.40 ± 0.05*
T-rex HEK-���2-m Te�– 0.30 0.52 ± 0.09* 0.50 ± 0.12* 0.65 ± 0.05*
T-rex HEK-���2-m Te�+ 0.30 0.56 ± 0.06* 0.60 ± 0.22 0.95 ± 0.05*
*S�a�is�icall� si�ni�ican�, p < 0.05.
The growth-stimulatory patterns of hBD-2 expres-
sion in the cells are in agreement with our previous
results obtained with the use of recombinant hBD-2
expressed in bacterial cells [10] that showed the pro-
moting effect of rec-hBD-2 at the concentrations of
0.1–2 µg/ml on proliferation and viability of eukaryotic
cells in vitro. Yet we can’t evaluate the exact level of
hBD-2 production per cell in generated cell sublines,
but one may speculate that according to the observed
biologic effect, its production may be close to the
mentioned above concentration range.
Another specific pattern that has been noticed
is the phenomenon of deprivation of ability to form
confluent monolayer by ��ЕК/hBD-2m cells cultured
in the presence of 1 µg/ml tetracycline (Fig. 3, a–d).
Whilst parental HEK293 cells are growing in typical
monolayer, where the cells possess flattened or spread
morphology, tetracycline-induced ��ЕК/hBD-2m cells
seem to loose this property forming multilayer clusters
and possessing mostly rounded morphology. It looks
like that normal cell-to-cell contacts are disturbed, as
Fig. 2.. 2. Immunocytochemical analysis of hBD-2 expression in wild type ��ЕК cells (wild type ��ЕК cells ( type ��ЕК cells (type ��ЕК cells ( ��ЕК cells (��ЕК cells ( cells (cells ( (a), T-rex HEK-hBD2m TetrhBD2m TetrBD2m Tetr_ (b), T-rex HEK-hBD2mhBD2mBD2m
Tetr+(c). x 100x 100
Experimental Oncology 28, 288–292, 2006 (December) 29128, 288–292, 2006 (December) 291December) 291) 291 291
well as adhesion properties of the cells. We speculate
that such effect may be explained in part by possible
alteration of cell adherence in hBD-2-producing cells.
The morphological features of tetracycline-induced
T-rex��ЕК-hBD-2m cells closely resembled these of
HEK293T cells expressing chymeric constructs of
ADAM9 (a desintegrin and metalloprotease 9), mol-
ecule interacting with β-integrins in renal cells and
affecting their adherence properties [19]. There are
some reports evidencing the possible link between
alpha-defensins and adhesion molecules. It has been
shown that alpha-defensins may specifically inhibit
alpha5beta1-integrin-dependent migration of bovine
retinal endothelial cells to FN in vitro [20]. Another
research demonstrated that human neutrophil pep-
tides (HNPs) promote the binding of fibronectin to
alpha5beta1 integrin and may affect endothelial cell
adhesion and migration in FN-dependent manner [21].
So, there are some grounds to speculate that specific
morphological and growth patterns of T-rexHEK-hBD-
2m cells could be related to hBD-2-dependent altera-
tion of adhesive properties of the cells.
Finally we have performed colony-formation as-
say using 0.4% methylcellulose without/with 1 µg/ml
tetracycline, and found out that after 2 weeks of
growth HEK293T cells formed multiple colonies, but
T-rexHEK-hBD-2m cells seeded at the same density
of 1 x 103 cells/ml did not and form only single colonies
in tetracycline free medium. So, one may conclude
that expression of hBD-2 in T-rexHEK-hBD-2m cells
may result in the loss of the pattern of malignization
(namely, ability to form colonies in semiliquid medium)
characteristic for parental HEK293T cells.
In conclusion, we may assume that stable cell subline
T-rexHEK-hBD-2m expressing mature hBD-2 molecule
in tetracycline-dependent manner has been created��
whilst wild type cells do not express and produce hBD-2,
T-rexHEK-hBD-2 cells express hBD-2 upon incubation
with 1 µM tetracycline in incubation medium, as it has
been shown by RT-PCR and immunocytochemical
analysis. Our data have demonstrated that prolonged
expression (> 48 h) of endogeneous hBD-2 resulted in
a number of biological effects: hBD-2-producing cells
lost normal adhesive properties as well as the ability to
form colonies in agar, characteristic to parental HEK
cells, but acquire some features favoring the acceler-
ated growth and survival. Our preliminary data allow to
suppose that some signal cascades in these cells (in
particular, PDK1-Akt, and p70S6 kinase) are involved
in mediating abovementioned events�� the research of
these enzymes and expression of adhesion molecules
is currently under study.
ACKNOWLEDGEMENT
The work was supported by grant № 2.2.5.260/2004,
the Program “Newest Medico-Biological Problems and
Fig. 3. The morphological features of T-rex-HEK-hBD-2m cells Tet+ (b–d) and of parental HEK293T cells (a) cultured on “starving”
medium for 48 h
292 Experimental Oncology 28, 288–292, 2006 (December)
Environment”, Part 2. “Biologically Active Compounds
for Human Health” (Ukraine). Authors wish to express
their gratitude to Dr. Liudmila Drobot (Ukraine) and
Dr. Tatyana Klymenko (Germany) for their help and
advices in solving gene-engineering tasks.
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Cop�ri��� © Experimen�al Oncolo��, 2006
ИЗМЕНЕНИЕ ФЕНОТИПА И ОСОБЕННОСТЕЙ РОСТА
В КУЛЬТУРЕ ЛИНИИ ЭМБРИОНАЛЬНЫХ КЛЕТОК ПОЧКИ
ПРИ РЕГУЛИРУЕМОЙ ЭКСПРЕССИИ БЕТА-ДЕФЕНСИНА-2
Цель: создать линию клеток с регулируемой экспрессией бета-дефенсина-2 человека (hBD-2) и проанализировать влияние
экспрессии этого пептида на особенности фенотипа и рост клеток. Материалы и методы: клеточная сублиния T-rex HEK-T-rex HEK--rex HEK-rex HEK- HEK-HEK--
hBD2-m, экспрессирую�ая биологически активную зрелую форму hBD-2 при индук�ии клеток тетра�иклином, получена2-m, экспрессирую�ая биологически активную зрелую форму hBD-2 при индук�ии клеток тетра�иклином, полученаm, экспрессирую�ая биологически активную зрелую форму hBD-2 при индук�ии клеток тетра�иклином, получена, экспрессирую�ая биологически активную зрелую форму hBD-2 при индук�ии клеток тетра�иклином, получена
путем клонирования на основе эмбриональных клеток почки человека линии HEK293T. Морфологические особенности,
характеристики роста и показатели колониеобразования в полужидкой среде исследовали стандартными методами. Результаты:
с помо�ью методов РТ-ПЦР и иммуно�итохимии показано, что сублиния клеток T-rex HEK-HBD2-m экспрессирует hBD2T-rex HEK-HBD2-m экспрессирует hBD2-rex HEK-HBD2-m экспрессирует hBD2rex HEK-HBD2-m экспрессирует hBD2 HEK-HBD2-m экспрессирует hBD2HEK-HBD2-m экспрессирует hBD2-HBD2-m экспрессирует hBD2HBD2-m экспрессирует hBD22-m экспрессирует hBD2m экспрессирует hBD2 экспрессирует hBD2
в присутствии 1 µг/мл тетра�иклина в среде инкуба�ии. Продолжительная экспрессия hBD-2 приводила к тому, что клетки
утрачивали способность образовывать монослой при культивировании in vitro и образовывать колонии в полужидкой среде, но
характеризовались более высокой скоростью роста и способностью к более продолжительному выживанию в бессывороточной
среде, чем исходная линия клеток HEK293T. Выводы: экспрессия hBD-2 в сублинии клеток T-rex HEK-HBD2-m cellT-rex HEK-HBD2-m cell-rex HEK-HBD2-m cellrex HEK-HBD2-m cell HEK-HBD2-m cellHEK-HBD2-m cell-HBD2-m cellHBD2-m cell2-m cellm cell cell
обусловливает спе�ифические биологические эфеекты, способствую�ие выживанию клеток.
Ключевые слова: бета-дефенсин-2 человека, регулируемая экспрессия, колониеобразование, клетки линии HEK293.
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