Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури
За умов моношарової культури тиреоцитів щура відбувається внутрішньоклітинний перерозподіл S6K1/2, які поряд з цитоплазмою детектуються у ядрах клітин. Метою роботи було дослідити зв’язок між активацією процесів міграції, проліферації, втрати фолікулярної організації тиреоцитів за різних умов культи...
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Хоруженко, А.І. Чередник, О.В. Філоненко, В.В. 2019-06-21T05:35:42Z 2019-06-21T05:35:42Z 2008 Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури / А.І. Хоруженко, О.В. Чередник, В.В. Філоненко // Біополімери і клітина. — 2008. — Т. 24, № 6. — С. 470-475. — Бібліогр.: 20 назв. — укр., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0007BF https://nasplib.isofts.kiev.ua/handle/123456789/157885 577.25, 591.87, 611.44 За умов моношарової культури тиреоцитів щура відбувається внутрішньоклітинний перерозподіл S6K1/2, які поряд з цитоплазмою детектуються у ядрах клітин. Метою роботи було дослідити зв’язок між активацією процесів міграції, проліферації, втрати фолікулярної організації тиреоцитів за різних умов культивування та субклітинною локалізацією S6K1/2. Виявлено загальне зростання вмісту S6K1/2 у проліферуючих клітинах. У тиреоцитах, культивованих у вигляді фолікулів, внутрішньоклітинна локалізація S6K1/2 не змінюється відносно нормальної тканини. Субклітинну релокалізацію S6K1/2 відзначено лише для окремої популяції клітин, що за певних причин втратили фолікулярну організацію і відповідно функціональну активність. Отже, зміна субклітинної локалізації S6K1/2 у культивованих тиреоцитах безпосередньо пов’язана зі зміною рівня диференціації на відміну від проліферації та міграції цих клітин. The aim of this work was to study the link between activation of processes of migration, proliferation, loss of thyrocyte follicle organization at different culture conditions and subcellular localization of S6K1/2. The subcellular redistribution of S6K1/2 takes place in the rat thyrocyte monolayer culture where S6K1/2 was detected not only in the cytoplasm but in the nuclei of cells as well|. In addition, the content of S6K1/2 in proliferating cells was increased. The subcellular| localization of S6K1/2 in thyrocytes cultivated as follicles was similar to that observed in normal thyroid tissue. Subcellular relocalization of S6K1/2 was detected only in certain cellular population, which for some reasons lost follicle organization and, consequently, functional activity. Thus, the changes in subcellular localization of S6K1/2 in cultivated thyrocytes are directly related to the level of differentiation, unlike proliferation and migration of these cells. В условиях монослойной культуры тиреоцитов крысы происходит внутриклеточное перераспределение S6K1/2, которые наряду с цитоплазмой детектируются в ядрах клеток. Цель работы состояла в изучении связи между активацией процессов миграции, пролиферации, потери фолликулярной организации тиреоцитов при разных условиях культивирования и субклеточной локализацией S6K1/2. Обнаружено общее повышение содержания S6K1/2 в пролиферирующих клетках. В тиреоцитах, культивируемых в виде фолликулов, внутриклеточная локализация S6K1/2 не изменялась по сравнению с нормальной тканью. Субклеточная релокализация S6K1/2 отмечена лишь для отдельной популяции клеток, которые по различным причинам теряли фолликулярную организацию и соответственно функциональную активность. Таким образом, изменение субклеточной локализации S6K1/2 в культивируемых тиреоцитах непосредственно связано с изменением уровня дифференциации в отличие от пролиферации и миграции этих клеток. uk Інститут молекулярної біології і генетики НАН України Біополімери і клітина Клітинна біологія Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури Cубклеточная локализация S6K1 и S6K2 форм киназы рибосомного белка S6 в тиреоцитах крыс в условиях двух- и трехмерной культуры Subcellular localization of S6K1 and S6K2 forms of ribosomal protein S6 kinase in rat thyrocytes under conditions of two- and three-dimensional culture Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури |
| spellingShingle |
Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури Хоруженко, А.І. Чередник, О.В. Філоненко, В.В. Клітинна біологія |
| title_short |
Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури |
| title_full |
Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури |
| title_fullStr |
Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури |
| title_full_unstemmed |
Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури |
| title_sort |
субклітинна локалізація s6k1 і s6k2 форм кінази рибосомного білка s6 у тиреоцитах щурів за умов дво- та тривимірної культури |
| author |
Хоруженко, А.І. Чередник, О.В. Філоненко, В.В. |
| author_facet |
Хоруженко, А.І. Чередник, О.В. Філоненко, В.В. |
| topic |
Клітинна біологія |
| topic_facet |
Клітинна біологія |
| publishDate |
2008 |
| language |
Ukrainian |
| container_title |
Біополімери і клітина |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Cубклеточная локализация S6K1 и S6K2 форм киназы рибосомного белка S6 в тиреоцитах крыс в условиях двух- и трехмерной культуры Subcellular localization of S6K1 and S6K2 forms of ribosomal protein S6 kinase in rat thyrocytes under conditions of two- and three-dimensional culture |
| description |
За умов моношарової культури тиреоцитів щура відбувається внутрішньоклітинний перерозподіл S6K1/2, які поряд з цитоплазмою детектуються у ядрах клітин. Метою роботи було дослідити зв’язок між активацією процесів міграції, проліферації, втрати фолікулярної організації тиреоцитів за різних умов культивування та субклітинною локалізацією S6K1/2. Виявлено загальне зростання вмісту S6K1/2 у проліферуючих клітинах. У тиреоцитах, культивованих у вигляді фолікулів, внутрішньоклітинна локалізація S6K1/2 не змінюється відносно нормальної тканини. Субклітинну релокалізацію S6K1/2 відзначено лише для окремої популяції клітин, що за певних причин втратили фолікулярну організацію і відповідно функціональну активність. Отже, зміна субклітинної локалізації S6K1/2 у культивованих тиреоцитах безпосередньо пов’язана зі зміною рівня диференціації на відміну від проліферації та міграції цих клітин.
The aim of this work was to study the link between activation of processes of migration, proliferation, loss of thyrocyte follicle organization at different culture conditions and subcellular localization of S6K1/2. The subcellular redistribution of S6K1/2 takes place in the rat thyrocyte monolayer culture where S6K1/2 was detected not only in the cytoplasm but in the nuclei of cells as well|. In addition, the content of S6K1/2 in proliferating cells was increased. The subcellular| localization of S6K1/2 in thyrocytes cultivated as follicles was similar to that observed in normal thyroid tissue. Subcellular relocalization of S6K1/2 was detected only in certain cellular population, which for some reasons lost follicle organization and, consequently, functional activity. Thus, the changes in subcellular localization of S6K1/2 in cultivated thyrocytes are directly related to the level of differentiation, unlike proliferation and migration of these cells.
В условиях монослойной культуры тиреоцитов крысы происходит внутриклеточное перераспределение S6K1/2, которые наряду с цитоплазмой детектируются в ядрах клеток. Цель работы состояла в изучении связи между активацией процессов миграции, пролиферации, потери фолликулярной организации тиреоцитов при разных условиях культивирования и субклеточной локализацией S6K1/2. Обнаружено общее повышение содержания S6K1/2 в пролиферирующих клетках. В тиреоцитах, культивируемых в виде фолликулов, внутриклеточная локализация S6K1/2 не изменялась по сравнению с нормальной тканью. Субклеточная релокализация S6K1/2 отмечена лишь для отдельной популяции клеток, которые по различным причинам теряли фолликулярную организацию и соответственно функциональную активность. Таким образом, изменение субклеточной локализации S6K1/2 в культивируемых тиреоцитах непосредственно связано с изменением уровня дифференциации в отличие от пролиферации и миграции этих клеток.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/157885 |
| citation_txt |
Субклітинна локалізація S6K1 і S6K2 форм кінази рибосомного білка S6 у тиреоцитах щурів за умов дво- та тривимірної культури / А.І. Хоруженко, О.В. Чередник, В.В. Філоненко // Біополімери і клітина. — 2008. — Т. 24, № 6. — С. 470-475. — Бібліогр.: 20 назв. — укр., англ. |
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2025-11-25T16:08:49Z |
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| fulltext |
CELL BIOLOGY
Subcellular localization of S6K1 and S6K2 forms of
ribosomal protein S6 kinase in rat thyrocytes under
conditions of two- and three-dimensional culture
A. I. Khoruzhenko, O. V. Cherednyk, V. V. Filonenko
Institute of molecular biology and genetics NAS of Ukraine
150, Zabolotnogo Str, Kyiv Ukraine, 03680
a.i.khoruzhenko@imbg.org.ua
The aim of this work was to study the link between activation of processes of migration, proliferation, loss of
thyrocyte follicle organization at different culture conditions and subcellular localization of S6K1/2. The
subcellular redistribution of S6K1/2 takes place in the rat thyrocyte monolayer culture where S6K1/2 was
detected not only in the cytoplasm but in the nuclei of cells as well|. In addition, the content of S6K1/2 in
proliferating cells was increased. The subcellular| localization of S6K1/2 in thyrocytes cultivated as
follicles was similar to that observed in normal thyroid tissue. Subcellular relocalization of S6K1/2 was
detected only in certain cellular population, which for some reasons lost follicle organization and,
consequently, functional activity. Thus, the changes in subcellular localization of S6K1/2 in cultivated
thyrocytes are directly related to the level of differentiation, unlike proliferation and migration of these
cells.
Keywords: ribosomal protein S6 kinase, thyrocytes, level of differentiation.
Introduction. The main function of thyroid, as an
endocrine organ, consists in the production of thyroid
hormones which play an important role in the
metabolism of human and mammalians. (A) Basic
structural and functional unit of thyroid is a follicle,
which consists of closed prismatic or cuboidal
epithelium monolayer with an apical part forming a
lumen. The latter is filled with a colloid which
contains a glycoprotein thyroglobulin. Follicle
organization of thyrocytes ensures the multistep
process of thyroid hormones synthesis. It was shown
on cultivated thyrocytes that the loss of follicle
organization and depolarization lead to
dedifferentiation of these cells and to a decrease in
thyroglobulin content [1, 2].
The functions of thyroid are regulated by a few
factors with the main role of pituitary thyroid
stimulating hormone (TSH). It was shown, that TSH
influences the thyroid cells through the
Phosphatidylinositol 3-Kinase (PI3K) signal
transduction pathway [3]. The link between the
receptor of TSH and regulatory ð85a subunit of PI3K
was demonstrated on cultivated thyrocytes. It was
noted that TSH stimulates the interaction between the
receptor of TSH and PI3K, which results in PI3K- and
protein kinase A (PKA) dependent translocation of
470
ISSN 1993-6842. Biopolymers and cell. 2008. vol. 24. N 6. Translated from Ukrainian
© A. I. KHORUZHENKO, O. V. CHEREDNYK, V. V. FILONENKO, 2008
phospholipid dependent kinase 1 (PDK1). TSH
stimulates ribosomal protein kinase S6K through
PI3K-, PDK1- and PKA dependent pathway and as a
consequence affects in vitro proliferation and
functional activity of follicular cells [4].
S6Ê belongs to (an) AGC family of
serine/threonine protein kinases which includes protein
kinase C, protein kinase B, SGKs, and 90 kDa
ribosomal S6 protein kinase [5]. There are two forms of
S6 kinase - S6Ê1 and S6Ê2 and each (of them) has
cytoplasmic (S6Ê1II and S6Ê2 II) and nuclear (S6Ê1 I
and S6Ê2 I) izoforms [6]. It was demonstrated that
activity of S6Ê1/2 is regulated by
phosphorylating/dephosphorylating in response to
different extracellular stimuli, which include growth
factors, cytokines and hormones. Nowadays no highly
specific inhibitors of S6Ê are known. Numerous data
demonstrate that PI3K/mTOR signalling pathway
play(s) a key role in complete activating S6Ê.
Earlier an analysis of S6K expression was
performed on the tumours of human thyroid, normal
tissue and cell lines [7, 8, and 9]. Overexpression of
these kinases was detected in papillary carcinoma in
comparison to normal tissue. Studies carried out by
Western-blot analysis and immunohistochemical
technique revealed cytoplasmic localization of S6K1 in
normal tissue and in papillary carcinoma of human
thyroid.
We have shown that in rat thyroid gland S6Ê1 and
S6Ê2 are localized mainly in the cytoplasm of
thyrocytes [10]. At the cultivation of the isolated
follicles of thyroid under the conditions of monolayer
culture during spreading of follicles and loss of
follicle organization of thyrocytes, a positive
immunocytochemical reaction was detected not only in
the cytoplasm but in the cell nuclei as well. Initially
(the) S6K1/2 positive nuclei were detected on the edge
of cell colonies, later a positive reaction was
determined in the nuclei of all other cells [10]. These
data led to an assumption about the link between
activation of cell migration and subcellular
redistribution of (the) indicated kinases. On the other
side, it is known that S6Ê is involved in control of cell
proliferation [11]. In the previous studies we didn’t
found a correlation between the expression of Ki-67
antigen and the appearance of S6Ê in the nuclei of
thyrocytes [10]. That is why a possible role of S6K1/2
in regulation of thyrocyte proliferation has to be
studied further. Besides, it was shown that after
10-days of the cultivation of follicles under conditions
of two-dimensional culture the content of
thyroglobulin in thyrocytes is substantially diminished
[10]. It points out a possible connection between the
decline of functional activity of thyrocytes in vitro and
the changes in subcellular localization of S6Ê1 and
S6Ê2. Consequently, the process of thyrocyte
cultivation is accompanied by activating the migration
processes, cell proliferation and changes in tissue
structural organization as well as the functional
activity. However, it concerns to a lesser degree the
follicles cultivation at under three-dimensional
conditions [2].
The aim of this work was to study the relation
between these processes and the content and
subcellular localization of S6K1/2 in (the) thyrocytes
cultivated under different culture conditions. The
research included generation of the monolayer
cultures and three-dimensional cultures with the
maintenance of follicle organization of thyrocytes. The
cell migration was studied as well using polycarbonate
filters [2, 12] with a subsequent immunocytochemical
analysis. The detection of proliferating cells and
thyroglobulin content was carried out by the
immunoperoxidase and immunofluorescence methods.
Materials and methods. Reagents. Reagents for
the cell culture and chemiluminiscent reagent were
obtained from “Sigma” (USA). Antibodies to the
thyroglobulin and Ki-67 were from “Dako”
(Denmark); antibodies to mitogen-activated protein
kinases (Phospho-p42-44 MAPK) were obtained from
“BioLabs” (USA). Polyclonal and monoclonal
antibodies to S6K1/2 were generated in our laboratory.
Transwell with the pore diameter of polycarbonate
membrane 8 mm were obtained from “Costar” (USA).
Generation of thyrocyte cultures. The suspension
of thyroid follicles from rats of Wistar line was
obtained by the method described earlier [10]. Thyroid
tissue was cut into small fragments, incubated with
(the) mixture of collagenase/dispase with addition of
trypsin inhibitor during 2 hours at 370C. Thereafter
(the) growth medium (RPMI-1640, 17 % FCS, 4 mM
471
SUBCELLULAR LOCALIZATION OF S6K1 AND S6K2 FORMS OF RIBOSOMAL PROTEIN S6 KINASE IN RAT THYROCYTES
glutamine, 50 u/ml of penicillin, 50 mg/ml
streptomycin, 1 mg/ml amphotericin B) was added. The
disaggregated material was filtered through a nylon
mesh with (the) pore diameter (of) 180 mm and
collected on a nylon filter with (the) pore diameter (of)
30 mm. The isolated follicles were washed with (the)
growth medium and centrifuged. To obtain the
monolayer cultures the follicles were cultivated in
Petry dishes. The cultures of aggregates of follicles
were obtained on dishes, preliminary coated with 1 %
agarose. Cultivation was carried out at 37 0 C, in a
humidified gas phase 7 % CO2/air.
Migration of thyrocytes through Transwell. The
suspension of follicles was transferred into the upper
chamber of transwell, in RPMI-1640 supplemented
with 1 % human serum albumin, 4 mM glutamine and
(the) mixture of antibiotics. The lower chamber
contained RPMI-1640 with 20 % FCS to stimulate the
chemotaxis of thyrocytes. (The) Cultivation was
carried out for 4 days; the medium in lower and upper
chambers was changed daily. (The) Filters were fixed
with methanol for 5 min, cells in the upper chamber
were removed by a cotton tampon, retaining the cells
that migrated to the opposite filter surface.
Immunocytochemical and immunohistochemical
analysis. Detections of thyroglobulin (TG), S6K1 and
S6K2 were performed by the indirect
immunoperoxidase method on the paraffin sections of
the cultivated aggregates of follicles, monolayer
cultures and in the thyrocytes, cultivated in transwells.
Histological sections were deparaffinised and
rehydrated. Endogenous peroxidase was quenched
with 3 % H2O2 for 30 min. After blocking non-specific
binding by 10 % FCS, (the) sections were incubated
with primary monoclonal antibodies (S6K1, 1:50;
S6K2, 1:200; TG, 1:100) for 90 min at 370C.
Incubation with the secondary antibodies, labelled with
peroxidase, was carried out for 1 h at 370C. The
reaction proceeded in 3, 3’-diaminobenzidine
tetrachloride solution. For double immunochemical
reaction the first antigen was detected by the
immunoenzymatic method using corresponding first
antibodies (anti-thyroglobulin 1: 100, anti-MARK 1:
50, anti- Ki-67 1: 50) and secondary antibodies labelled
with horse- radishperoxidase. S6K1 and S6K2 were
determined by the immunofluorescent method, using
the secondary antibodies labelled with FITC. To
decrease the autofluorescence background the
preparations were incubated for 30 min in 10 mM
CuSO4 and 50 mM of CH3COONH4 at pH 5.0. Control
preparations were incubated without primary
antibodies.
All experiments were repeated no less than 3 times.
Microscopic studies were carried out using Leica
DM1000 microscope (Germany).
Results and discussion. To detect S6K1/2 in
proliferating thyrocytes in monolayer cultures a double
immunocytochemical staining was applied. This
approach was initially tested using the MCF-7 cell line
which is characterized by a high level of the ribosomal
protein S6 kinases expression. The data obtained
demonstrate that after three days of incubation of
follicles suspension at the conditions of (the)
monolayer culture an amount of proliferating cells
positive for Ki-67 antigen was insufficient. Starting
from (the) sixth day of cultivation the number of Ki-67
positive cells increased. In these thyrocytes the
increased reaction on S6K1 and S6K2 was revealed as
well (Fig. 1, b, d). In addition, double
immunocytochemical staining of activated MAP
kinases and S6K1 or S6K2 in thyrocytes was carried
out. Like in the case of Ki-67 antigen in the cells
positive on phosphorylated MAP kinases (Fig. 1, e, g),
an increased reaction on S6K1/S6K2 (Fig. 1, f, h) was
detected. Thus, on a primary culture of rat thyrocytes
an increased content of S6K1/S6K2 in proliferating
cells was demonstrated.
The obtained results correlate with the literature
data on the involvement of S6K in regulation of cell
proliferation. (An) Activation of S6K1 and MARK was
revealed during stimulation of human endothelial cells
proliferation by angiopoetin [13]. In addition, the
activation of protein synthesis, mediated by S6K in the
human vascular endothelium, was determined as a key
stage in passage through the cell cycle [14]. In the
inducible mice tumours producing TSH, the activation
of S6K was detected at aberrant pituitary growth [15].
However, as we reported earlier (,) the alterations in
subcellular localization of S6K1/2 kinases are not
connected directly with proliferation of thyrocytes
[10].
472
KHORUZHENKO A. I , CHEREDNYK O. V., FILONENKO V. V.
For the detection of S6K1 and S6K2 localization in
migrating rat thyrocytes the transwells were used.
Since the initiating of thyrocytes migration through a
porous membrane requires follicles attachment to the
plastic surface and their spreading out, the cultivation
of follicles was carried out for 4 days. This term of
cultivation was chosen experimentally.
In the control experiments to avoid the stimulation
of chemotaxis, the contents of FCS in upper and lower
chambers were identical. The cells were fixed on
membranes with methanol with subsequent
immunocytochemical determination of a subcellular
localization of S6K1 and S6K2. On the opposite side of
the membrane the colonies of migrating cells of the
regular shape or lacking central part were found. This
fact leads to the assumption that the boundary cells,
unlike those located in a centre of the colony have
higher mobility potential. In thyrocytes migrating to
the opposite surface of the membrane, and also in those
which remained on topside, the localization of S6K1
and S6K2 was predominantly cytoplasmic (Fig. 2).
There are no literature data concerning subcellular
localization of S6K1/2 in migrating thyrocytes.
However, there are some data relating to the
participation of PI3K signal pathway and S6K in
migration. Recently it was shown that suppression of
mTOR by rapamycin leads to inactivation of this
pathway that in turn inhibits (in dose-dependent
manner) the migration of the NUGC4 line cells, which
originate from the carcinoma of stomach [16]. The
above-mentioned data are in accordance with the work
of Zhou H.Y. performed on the cells of ovarian
carcinoma. It was shown that the constitutively active
form of S6K1 caused induction of invasive and
migratory phenotype of cells. In addition, activating
S6K1 predetermined an increase in the expression level
and proteolytic activity of matrix metalloproteinases,
in particular MMP-9 [17]. Our data do not exclude a
possibility of activation of indicated kinases in a
cytoplasm of thyrocytes, but point out that appearance
of S6K1 and S6K2 in the nuclei does not relate to the
cell migration.
Earlier we have shown that dedifferentiation of
thyrocytes is accompanied by subcellular
relocalization of S6K1 and S6K2 in the monolayer
culture [10]. That is why (in this work) we studied
subcellular localization of S6K1/2 in thyrocytes of the
follicles cultivated on the agarose layer at conditions
when the follicles form aggregates and keep tissue
organization of the thyroid cells. However, because of
partial damage during isolation the follicles collapsed
which resulted in aggregate areas with lost follicle
organization. The aggregates of follicles were fixed
and then (the) localization of indicated kinases was
determined immunohistochemically on 3d, 6th and 10th
days of cultivation (Fig. 3). It was revealed that in the
areas with the follicle structure organization S6K1 and
S6K2 were localized mainly in a cytoplasm of
thyrocytes.
It should be noted that the maintenance or loss of
follicle organization at cultivation under
three-dimensional conditions correlated with the
functional state of thyrocytes that was determined by
thyroglobulin content in the cells (Fig. 4 a, c). It was
detected that cells in follicles were positive on a
thyroglobulin, however, the cells with lost primary
tissue organization did not contain a thyroglobulin
which indicated the decreased level of their functional
activity. Double immunohistochemical detection of
thyroglobulin and S6K1/S6K2 in the cells was
performed. In the cells containing thyroglobulin S6K1
and S6K2 had mainly cytoplasm localization, as well as
those in the initial rat thyroid tissue. After 10 days of
cultivation in the population of cells which did not
save the follicle organization and, accordingly, did not
contain thyroglobulin the positive reaction on S6K1/2
was detected in thyrocyte nuclei as well as in the case
of prolongated thyrocyte cultivation in the monolayer
culture (Fig. 4). In addition, the immunostaining of
S6K1/2 in the thyroglobulin negative cells was greater
than in the cells containing thyroglobulin. These
findings enable to suggest that ribosomal protein S6
kinases – S6K1 and S6K2 - are involved into the
process of thyrocytes differentiation. At condition of
artificially initiated dedifferentiation there is a
relocalization of these kinases in comparison with the
normal thyroid tissue. The role of S6K in
differentiation was studied previously on neural cells,
neutrophiles and other types of cells [18, 19]. It was
shown that suppression of S6K1 delays the
differentiation of neural cells. It should be noted that
(earlier in our laboratory was demonstrated) the
473
SUBCELLULAR LOCALIZATION OF S6K1 AND S6K2 FORMS OF RIBOSOMAL PROTEIN S6 KINASE IN RAT THYROCYTES
presence of positive reaction on S6K2 in the nuclei of
human breast carcinoma cells, which was not observed
in normal tissue. Besides, it was revealed that the
amount of S6K1/2 detected in nuclei of malignant cells
increased considerably in the tumours of low
differentiation grade [20].
Thus, our data demonstrate that the proliferation
and migration of thyroid cells in vitro are not associated
directly with the changes in S6K1 and S6K2
localization. Alternatively, the connection between cell
proliferation and increase of S6 kinases cell content has
been shown. As a result of the performed research, the
dependence between subcellular localization of
S6K1/2 and functional state of thyrocytes has been
found that can be useful for an analysis of different
compounds modulating the thyroid function at
pathology.
À. ². Õî ðó æåí êî, Î. Â. ×å ðåä íèê, Â. Â. Ô³ëî íåí êî
Ñóáêë³òèí íà ëî êàë³çàö³ÿ S6K1 ³ S6K2 ôîðì ê³íàçè ðè áî ñîì íî ãî
á³ëêà S6 ó òè ðå î öè òàõ ùóð³â çà óìîâ äâî- òà òðè âèì³ðíî¿ êóëü -
òó ðè
Ðå çþ ìå
Çà óìîâ ìî íî øà ðî âî¿ êóëü òó ðè òè ðå î öèò³â ùóðà â³äáó âàºòüñÿ
âíóòð³øíüîêë³òèí íèé ïå ðå ðîç ïîä³ë S6K1/2, ÿê³ ïî ðÿä ç öè òîï -
ëàç ìîþ äå òåê òó þòü ñÿ ó ÿä ðàõ êë³òèí. Ìå òîþ ðî áî òè áóëî
äîñë³äèòè çâ’ÿ çîê ì³æ àê òè âàö³ºþ ïðî öåñ³â ì³ãðàö³¿, ïðîë³ôå -
ðàö³¿, âòðà òè ôîë³êó ëÿð íî¿ îðãàí³çàö³¿ òè ðå î öèò³â çà ð³çíèõ
óìîâ êóëü òè âó âàí íÿ òà ñóá êë³òèí íîþ ëî êàë³çàö³ºþ S6K1/2. Âè -
ÿâ ëå íî çà ãàëü íå çðîñ òàí íÿ âì³ñòó S6K1/2 ó ïðîë³ôå ðó þ ÷èõ
êë³òè íàõ. Ó òè ðå î öè òàõ, êóëü òè âî âà íèõ ó âèã ëÿä³ ôîë³êóë³â,
âíóòð³øíüîêë³òèí íà ëî êàë³çàö³ÿ S6K1/2 íå çì³íþºòüñÿ â³äíîñ -
íî íîð ìàëü íî¿ òêà íè íè. Ñóáêë³òèí íó ðå ëî êàë³çàö³þ S6K1/2
â³äçíà ÷å íî ëèøå äëÿ îêðå ìî¿ ïî ïó ëÿö³¿ êë³òèí, ùî çà ïåâ íèõ ïðè -
÷èí âòðà òè ëè ôîë³êó ëÿð íó îðãàí³çàö³þ ³ â³äïîâ³äíî
ôóíêö³îíàëü íó àê òèâí³ñòü. Îòæå, çì³íà ñóá êë³òèí íî¿ ëî -
êàë³çàö³¿ S6K1/2 ó êóëü òè âî âà íèõ òè ðå î öè òàõ áåç ïî ñå ðåä íüî
ïî â’ÿ çà íà ç³ çì³íîþ ð³âíÿ äè ôå ðåíö³àö³¿ íà â³äì³íó â³ä ïðîë³ôå -
ðàö³¿ òà ì³ãðàö³¿ öèõ êë³òèí.
Êëþ ÷îâ³ ñëî âà: ê³íàçà ðè áî ñîì íî ãî á³ëêà S6, òè ðå î öè òè,
ð³âåíü äè ôå ðåíö³àö³¿.
À. È. Õî ðó æåí êî, Î. Â. ×å ðåä íèê, Â. Â. Ôè ëî íåí êî
Cóá êëå òî÷íàÿ ëî êà ëè çà öèÿ S6K1 è S6K2 ôîðì êè íà çû
ðè áî ñîì íî ãî áåë êà S6 â òè ðå î öè òàõ êðûñ â óñëî âè ÿõ äâóõ-
è òðåõ ìåð íîé êóëü òó ðû
Ðå çþ ìå
 óñëî âè ÿõ ìî íîñ ëîé íîé êóëü òó ðû òè ðå î öè òîâ êðû ñû ïðî èñ õî -
äèò âíóò ðèê ëå òî÷ íîå ïå ðåðàñ ïðå äå ëå íèå S6K1/2, êî òî ðûå íà -
ðÿ äó ñ öè òîï ëàç ìîé äå òåê òè ðó þò ñÿ â ÿä ðàõ êëå òîê. Öåëü
ðà áî òû ñî ñòî ÿ ëà â èç ó÷å íèè ñâÿ çè ìåæ äó àê òè âà öè åé
ïðî öåñ ñîâ ìèã ðà öèè, ïðî ëè ôå ðà öèè, ïî òå ðè ôîë ëè êó ëÿð íîé
îðãà íè çà öèè òè ðå î öè òîâ ïðè ðàç íûõ óñëî âè ÿõ êóëü òè âè ðî âà -
íèÿ è ñóá êëå òî÷ íîé ëî êà ëè çà öè åé S6K1/2. Îáíà ðó æå íî îá ùåå
ïî âû øå íèå ñî äåð æà íèÿ S6K1/2 â ïðî ëè ôå ðè ðó þ ùèõ êëåò êàõ. Â
òè ðå î öè òàõ, êóëü òè âè ðó å ìûõ â âèäå ôîë ëè êó ëîâ, âíóò ðèê ëå -
òî÷ íàÿ ëî êà ëè çà öèÿ S6K1/2 íå èç ìå íÿ ëàñü ïî ñðàâ íå íèþ ñ íîð -
ìàëü íîé òêàíüþ. Ñóá êëå òî÷ íàÿ ðå ëî êà ëè çà öèÿ S6K1/2
îò ìå ÷å íà ëèøü äëÿ îò äåëü íîé ïî ïó ëÿ öèè êëå òîê, êî òî ðûå ïî
ðàç ëè÷ íûì ïðè ÷è íàì òå ðÿ ëè ôîë ëè êó ëÿð íóþ îðãà íè çà öèþ è ñî -
îò âå òñòâåí íî ôóíê öè î íàëü íóþ àê òèâ íîñòü. Òà êèì îá ðà çîì,
èç ìå íå íèå ñóá êëå òî÷ íîé ëî êà ëè çà öèè S6K1/2 â êóëü òè âè ðó å -
ìûõ òè ðå î öè òàõ íå ïîñ ðå äñòâåí íî ñâÿ çà íî ñ èç ìå íå íè åì óðîâ -
íÿ äèô ôå ðåí öè à öèè â îò ëè ÷èå îò ïðî ëè ôå ðà öèè è ìèã ðà öèè
ýòèõ êëå òîê.
Êëþ ÷å âûå ñëî âà: êè íà çà ðè áî ñîì íî ãî áåë êà S6, òè ðå î öè òû,
óðî âåíü äèô ôå ðåí öè à öèè.
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Received 15.05.08
ISSN 1993-6842. Biopolymers and cell. 2008. vol. 24. N 6. Translated from Ukrainian
Fig. 1. Dou ble im mu -
no chemi cal de tec tion of
Ki-67, MAPK, S6K1
and S6K2 in rat
thyrocytes un der con di -
tions of monolayer cul -
ture. a, c, e, g –
immunoperoxidase re -
ac tion; b, d, f , h –
immunofluorescent re -
ac tion. a, c – de tec tion
of an an ti gen Ki-67, b, d
- de tec tion of S6K1 and
S6K2 cor re spond ingly
in the same thyrocytes,
both ar rows show the
same cell. e, g – de tec -
tion of MAPK, Oc u lar
x10, objective x40.
a b
c
e
g
d
f
h
ISSN 1993-6842. Biopolymers and cell. 2008. vol. 24. N 6. Translated from Ukrainian
Fig. 2. Imunoperoxidase detection of localization of S6K1 (a, c) and S6K2 (b, d) in thyrocytes on the topside of transwell membrane (a, b) and
on a lower surface after migration (c, d). There is cytoplasmic localization of S6K1/2 in all cases. During migration of thyrocytes from the
spreaded follicle, the cells located at an edge of the colonies migrate faster than the cells of central area (Fig. 2Â.) Ocular x10, objective x5.
a b
c d
ISSN 1993-6842. Biopolymers and cell. 2008. vol. 24. N 6. Translated from Ukrainian
Fig. 3. Detection of subcellular localization of S6K1 (a, c, e) and S6K2 (b, d, f) in the cells of aggregates of follicles cultivated under
three-dimensional conditions for 3 days (a, b), 6 days (c, d) and 10 days (e, f). There is mainly cytoplasmic localization of indicated kinases
in thyrocytes. Ocular x10, objective x40.
a b
c d
e f
S6K1 S6K2
3
ya
D
6
ya
D
9
ya
D
ISSN 1993-6842. Biopolymers and cell. 2008. vol. 24. N 6. Translated from Ukrainian
Fig. 4. Double
immunocytochemical detection of
colocalization of thyroglobulin (a,
c – immunoperoxidase method)
and S6K1/2 (accordingly, b, d –
immunofluorescent method) in the
aggregates of follicles on the 10th
day of cultivation. Arrows show
areas where a follicle structure and
thyroglobulin are absent. In
thyrocytes without thyroglobulin
S6K1/2 positive nuclei are
revealed. Ocular x 10, objective x
40.
a b
c d
|