Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53
Проаналізовано життєздатність клітин лінії G1 миші при обробці колхіцином у різних концентраціях. Встановлено меншу чутливість до дії колхіцину клітин лінії G1 на 26-му та 114-му пасажах порівняно з ембріональними фібробластами миші лінії BALB/c, що свідчить про ослаблення контрольної точки мітозу в...
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| Опубліковано в: : | Біополімери і клітина |
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| Дата: | 2007 |
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Інститут молекулярної біології і генетики НАН України
2007
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| Цитувати: | Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 / А.П. Яцишина, С.М. Кваша, О.В. Підпала, Т.П. Рубан, І.М. Вагіна, Л.Л. Лукаш // Біополімери і клітина. — 2007. — Т. 23, № 4. — С. 338-346. — Бібліогр.: 46 назв. — укр., англ. |
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Яцишина, А.П. Кваша, С.М. Підпала, О.В. Рубан, Т.П. Вагіна, І.М. Лукаш, Л.Л. 2019-06-20T04:16:41Z 2019-06-20T04:16:41Z 2007 Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 / А.П. Яцишина, С.М. Кваша, О.В. Підпала, Т.П. Рубан, І.М. Вагіна, Л.Л. Лукаш // Біополімери і клітина. — 2007. — Т. 23, № 4. — С. 338-346. — Бібліогр.: 46 назв. — укр., англ. 0233-7657 http://dx.doi.org/10.7124/bc.000772 https://nasplib.isofts.kiev.ua/handle/123456789/157508 576.5+576.35:576.356+577.21 Проаналізовано життєздатність клітин лінії G1 миші при обробці колхіцином у різних концентраціях. Встановлено меншу чутливість до дії колхіцину клітин лінії G1 на 26-му та 114-му пасажах порівняно з ембріональними фібробластами миші лінії BALB/c, що свідчить про ослаблення контрольної точки мітозу в клітинах лінії G1. За допомогою Вестерн-блот аналізу виявлено білок р53 на низькому рівні як у клітинах лінії G1, так і її субліній G1-OA та G1-T. Методом імунопреципітації в клітинах лінії G1 визначено присутність білка р53 обох типів: як дикого, так і мутантного, що може призводити до порушення його функції. Генетична нестабільність клітин лінії G1 миші пов’язана з порушенням функції контрольної точки мітозу і, ймовірно, з функціональною інактивацією р53. The ability of cells of mouse G1 cell line to survive after treatment with different concentrations of colchicine has been analysed. We have shown that G1 cells at 26th and 114th passages are less sensitive to the treatment with colchicine than BALB/c mouse embryonic fibroblasts. These results indicate attenuation of the mitotic checkpoint of G1 cells. The expression of p53 has been detected at low levels in cells of G1 cell line and its sublines G1-OA and G1-T by Western blotting. It has been shown by immunoprecipitation that G1 cells contain wild type and mutant p53 that could lead to the disfunction of p53. Genetic instability of G1 cell line is possibly associated with the failure of the mitotic checkpoint and functional inactivation of p53. Изучена выживаемость мышиных клеток линии G1 при обработке колхицином в разных концентрациях. Установлена меньшая чувствительность к действию колхицина клеток линии G1 на 26-м и 114-м пассажах по сравнению с эмбриональными фибробластами мыши линии BALB/c, что свидетельствует об ослаблении контрольной точки митоза в клетках линии G1. При помощи Вестерн-блот анализа выявлен белок р53 на низком уровне как в клетках линии G1, так и ее сублиний G1-OA и G1-T. Методом иммунопреципитации в клетках линии G1 определено присутствие белка р53 обоих типов: как дикого, так и мутантного, что может вызвать нарушение его функции. Генетическая нестабильность клеток линии G1 мыши связана с нарушением функции контрольной точки митоза и, вероятно, с функциональной инактивацией р53. Роботу частково профінансовано Державним фондомфундаментальних досліджень Міністерства освіти і науки України, проект № Ф18/11-2006. uk Інститут молекулярної біології і генетики НАН України Біополімери і клітина Молекулярна та клітинна біотехнології Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 Генетическая нестабильность эмбриональных герминативных клеток линии G1 мыши и нарушение функций контрольной точки митоза и р53 Genetic instability of mouse embryonic germinative G1 cell line and disfunction of the mitotic checkpoint and p53 Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 |
| spellingShingle |
Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 Яцишина, А.П. Кваша, С.М. Підпала, О.В. Рубан, Т.П. Вагіна, І.М. Лукаш, Л.Л. Молекулярна та клітинна біотехнології |
| title_short |
Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 |
| title_full |
Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 |
| title_fullStr |
Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 |
| title_full_unstemmed |
Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 |
| title_sort |
генетична нестабільність ембріональних гермінативних клітин лінії g1 миші та порушення функцій контрольної точки мітозу і білка р53 |
| author |
Яцишина, А.П. Кваша, С.М. Підпала, О.В. Рубан, Т.П. Вагіна, І.М. Лукаш, Л.Л. |
| author_facet |
Яцишина, А.П. Кваша, С.М. Підпала, О.В. Рубан, Т.П. Вагіна, І.М. Лукаш, Л.Л. |
| topic |
Молекулярна та клітинна біотехнології |
| topic_facet |
Молекулярна та клітинна біотехнології |
| publishDate |
2007 |
| language |
Ukrainian |
| container_title |
Біополімери і клітина |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Генетическая нестабильность эмбриональных герминативных клеток линии G1 мыши и нарушение функций контрольной точки митоза и р53 Genetic instability of mouse embryonic germinative G1 cell line and disfunction of the mitotic checkpoint and p53 |
| description |
Проаналізовано життєздатність клітин лінії G1 миші при обробці колхіцином у різних концентраціях. Встановлено меншу чутливість до дії колхіцину клітин лінії G1 на 26-му та 114-му пасажах порівняно з ембріональними фібробластами миші лінії BALB/c, що свідчить про ослаблення контрольної точки мітозу в клітинах лінії G1. За допомогою Вестерн-блот аналізу виявлено білок р53 на низькому рівні як у клітинах лінії G1, так і її субліній G1-OA та G1-T. Методом імунопреципітації в клітинах лінії G1 визначено присутність білка р53 обох типів: як дикого, так і мутантного, що може призводити до порушення його функції. Генетична нестабільність клітин лінії G1 миші пов’язана з порушенням функції контрольної точки мітозу і, ймовірно, з функціональною інактивацією р53.
The ability of cells of mouse G1 cell line to survive after treatment with different concentrations of colchicine has been analysed. We have shown that G1 cells at 26th and 114th passages are less sensitive to the treatment with colchicine than BALB/c mouse embryonic fibroblasts. These results indicate attenuation of the mitotic checkpoint of G1 cells. The expression of p53 has been detected at low levels in cells of G1 cell line and its sublines G1-OA and G1-T by Western blotting. It has been shown by immunoprecipitation that G1 cells contain wild type and mutant p53 that could lead to the disfunction of p53. Genetic instability of G1 cell line is possibly associated with the failure of the mitotic checkpoint and functional inactivation of p53.
Изучена выживаемость мышиных клеток линии G1 при обработке колхицином в разных концентрациях. Установлена меньшая чувствительность к действию колхицина клеток линии G1 на 26-м и 114-м пассажах по сравнению с эмбриональными фибробластами мыши линии BALB/c, что свидетельствует об ослаблении контрольной точки митоза в клетках линии G1. При помощи Вестерн-блот анализа выявлен белок р53 на низком уровне как в клетках линии G1, так и ее сублиний G1-OA и G1-T. Методом иммунопреципитации в клетках линии G1 определено присутствие белка р53 обоих типов: как дикого, так и мутантного, что может вызвать нарушение его функции. Генетическая нестабильность клеток линии G1 мыши связана с нарушением функции контрольной точки митоза и, вероятно, с функциональной инактивацией р53.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/157508 |
| citation_txt |
Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53 / А.П. Яцишина, С.М. Кваша, О.В. Підпала, Т.П. Рубан, І.М. Вагіна, Л.Л. Лукаш // Біополімери і клітина. — 2007. — Т. 23, № 4. — С. 338-346. — Бібліогр.: 46 назв. — укр., англ. |
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MO LEC U LAR AND CELL BIOTECHNOLOGIES
Ge netic in sta bil ity of mouse em bry onic germinative
G1 cell line and disfunction of the mi totic check point
and ð53
A. P. Iatsyshyna, S. M. Kvasha, O. V. Pidpala, T. P. Ruban, I. M. Va gina, L.L.Lukash
The In sti tute of Mo lec u lar Bi ol ogy and Ge net ics
150 Zabolotny Str., Kyiv 03143, Ukraine
a.p.iatsyshyna@imbg.org.ua
The abil ity of cells of mouse G1 cell line to sur vive af ter treat ment with dif fer ent con cen tra tions of
colchicine has been ana lysed. We have shown that G1 cells at 26th and 114th pas sages are less sen si tive to
the treat ment with colchicine than BALB/c mouse em bry onic fibroblasts. These re sults in di cate at ten u a tion
of the mi totic check point of G1 cells. The ex pres sion of p53 has been de tected at low lev els in cells of G1 cell
line and its sublines G1-OA and G1-T by West ern blot ting. It has been shown by immunoprecipitation that
G1 cells con tain wild type and mu tant p53 that could lead to the disfunction of p53. Ge netic in sta bil ity of G1
cell line is pos si bly as so ci ated with the fail ure of the mi totic check point and func tional in ac ti va tion of p53.
Key words: mouse cell in vi tro, mi totic check point, chro mo somal in sta bil ity, p53
In tro duc tion. Chro mo somal in sta bil ity i.e. loss or gain
of chro mo somes, also aneuploidy, is of ten ob served in
var i ous hu man tu mours and is con sid ered to be one of
the mark ers of solid tu mours [1]. The changes in ploidy
are more sig nif i cant at the later stages of tu mour de vel -
op ment, and thus chro mo somal in sta bil ity cor re lates
with ac qui si tion of ma lig nant phe no types [2, 3]. There
is a con cept that chro mo somal in sta bil ity is a mutator,
in creas ing the num ber of mu ta tions, which re sult in tu -
mour for ma tion [4]. The mo lec u lar bases of chro mo -
somal in sta bil ity have not been stud ied enough, how -
ever, re cently much at ten tion has been paid to the is sue
of ge netic and epigenetic changes of the mi totic check -
point (or the check point of spin dle as sem bly), which
func tions as the pro vider of equal dis tri bu tion of chro -
mo somes be tween two daugh ter cells [5–7]. As the ma -
jor ity of cells are in ca pa ble of ter mi nat ing their di vi sion
as a re sponse of to the pres ence of com pound like
nocodazole [8], the search for mu ta tions re sult ing in
chro mo somal in sta bil ity is nar rowed to the search for
the mu ta tions of genes of mi totic check point. Sim i lar
mu ta tions were de tected in some hu man cell lines,
which are char ac ter ized by chro mo somal in sta bil ity
[9]. How ever, it has been shown that the dam age of mi -
totic check point pri mar ily re sults in the death of cells
[1, 10], and in or der to ob tain the phe no type of the chro -
mo somal in sta bil ity, there should be at least ad di tional
loss of p53 func tion [1]. The cor re la tion be tween chro -
mo somal in sta bil ity (ploidy change) and the loss or
mutational in ac ti va tion of p53 is ob served in nu mer ous
ex per i men tal and clin i cal in ves ti ga tions [11–17].
Tu mour sup pres sor gene p53 (TP53 in hu man and
Trp53 in mice) is mu tated the most in hu man tu mours,
its mu ta tions have been re vealed in more than 50% of
hu man tu mours [18–20]. Mutational in ac ti va tion
and/or de le tion of p53 is com mon for dif fer ent types of
tu mours in the cases of spo radic and fa mil ial can cer dis -
338
ISSN 0233-7657. Biopolymers and cell. 2007. vol. 23. ISS 4. Translated from Ukrainian.
ã A. P. IATSYSHYNA, S. M. KVASHA, O. V. PIDPALA, T. P. RUBAN, I. M. VA GINA,
L.L.LUKASH, 2007
eases [18, 19]. Pro tein p53 is spe cific for the nu mer ous
func tions [21, 22], par tic u larly par tic i pa tion in the in hi -
bi tion of ab nor mal growth of cells [23] and ini ti a tion of
apoptosis [24]. p53 has been called the guard ian of the
ge nome due to the func tion of se cur ing the ge nome in -
teg rity and elim i na tion of dam aged cells [25]. Be sides,
some more im por tant func tions of p53 are the DNA re -
pair [26] and in hi bi tion of angiogenesis [27]; p53 is
know to be the tran scrip tion fac tor of many genes [28].
The ac ti va tion of tar get genes by p53 may re sult in the
growth stop ei ther be fore rep li ca tion of DNA in G1
phase of cell cy cle or prior to mi to sis in G2 phase. The
stop in growth en ables the cells to re pair the dam aged
DNA while due to apoptosis the dam aged cells are
elim i nated, which pre vents the se cur ing the dam ages in
DNA as mu ta tions.
In nor mal cells p53 pro tein is non-func tional, it is
ac ti vated as a re sponse to dif fer ent sig nals only, e.g.
dam age of DNA, ab nor mal proliferative sig nals,
hypoxia, the loss of cell ad he sion, etc [27]. In the ma -
jor ity of hu man tu mours p53 is non-func tional or in cor -
rect func tion ing also [27]. In ac ti va tion of p53 func tion
may be achieved in dif fer ent ways, in some cases not
only through the mu ta tion of p53 gene but also in func -
tional changes of p53 pro tein dur ing its bind ing to vi rus
pro teins, lo cal isa tion of pro tein in cy to plasm, as a re sult
of changes in pro tein en cod ing genes (e.g. Mdm2), as -
so ci ated with p53 etc [27].
Both the stud ies on mor pho log i cal and growth
char ac ter is tics of new em bry onic germ cell line G1
[29, 30] and the re sults de tailed cytogenetic anal y sis
[31] tes tify in fa vour of spon ta ne ous trans for ma tion of
the in ves ti gated cells in vi tro, ac com pa nied by the in -
ten sive evo lu tion of karyotype. Dif fer ent ab er rant
mitoses re vealed [30] as well as high fre quen cies of
aneuploid cells in G1 cell line pop u la tions [31] in di -
cate the de reg u la tion of di vi sion of cells and weak en -
ing of mi totic check point [6]. The pres ence of chro -
mo somal ab er ra tions in the cells un der in ves ti ga tion
re veals the disfunctioning of rep a ra tion [31, 32].
There fore, in or der to iden tify pos si ble fac tors of chro -
mo somal in sta bil ity in mouse cells in vi tro we have
ana lysed the ex pres sion of Trp53 on the level of pro -
tein and in ves ti gated the life ca pac ity of cells of the G1
line af ter treat ment with colchicine at dif fer ent pas -
sages of in vi tro cul ti va tion.
Ma te ri als and Meth ods. Cell cul tures. Mouse G1
cell line, ob tained from em bry onic ma te rial of BALB/c
mouse strain at the De part ment of Hu man Ge net ics of
In sti tute of Mo lec u lar Bi ol ogy and Ge net ics of NAS of
Ukraine, its sublines G1-OA and G1-T, se lected by the
fea tures of trans formed phe no type in vi tro [29, 30], and
12.5-d.p.c. mouse em bryos of BALB/c strain, which
were used to ob tain mouse cell line G1 [29], were used
in the cur rent work.
The cells were cul ti vated in DMEM me dium
(Sigma, USA) with 1–10% of fe tal bo vine se rum
(Sigma), 100 units/ml of pen i cil lin, and 100 mg/ml of
strep to my cin.
Ac tiv ity anal y sis of mi totic check point. To ana lyse
the abil ity of cells to sur vive and to di vide af ter the
treat ment with colchicine, the cells were sown in
96-well plates, 15 thou sand each, and in cu bated at 37°C
and 5% CO
2
in the growth me dium with the cor re spond -
ing con cen tra tion of fe tal bo vine se rum for 24 hours.
The next 24 hours the cells were in cu bated in the
growth me dium with 0.05, 0.5, and 5 mg/ml of
colchicine and with no colchicine ad di tion. The cells
with out any colchicine were used as con trol (con trol 1,
C-1) and with the ad di tion of colchicine sol vent, wa ter,
(con trol 2, C-2) were used as neg a tive con trol. Af ter
the colchicine was washed away, the cells were cul ti -
vated for 0 and 48 hours in the growth me dium. The
sol vent was added to the nu tri tion me dium in the vol -
umes, cor re spond ing the colchicine con cen tra tions of
0.5 and 5 mg/ml. To cal cu late the num bers of sur vived
cells in the mentioned above time, the growth me dium
was added with triazolyl blue tetrazolium bro mide
(Sigma) in the con cen tra tion of 75 mg per 1 ml of me -
dium and in cu bated for 4 hours at 37°C and 5% CO
2
.
The me dium was re moved, added with dimethyl
sulfoxide, 200 ml per well, pipetted gently and de ter -
mined for the spec tro pho to met ric den sity at l=540 nm.
Prep a ra tion of lysates of mam ma lian cells. The
cells were cul ti vated to 70-80% con flu ence and then
were washed twice with phos phate-buf fered sa line
(PBS, pH 7.4) and col lected from the glass us ing 0.02%
EDTA. Cell sus pense was washed twice with PBS (pH
7.4) and cen tri fuged at 15 000 rpm for 10 min. The cell
pel let was added with the cor re spond ing vol ume of
lysis buffer. The pieces of em bry onic tis sues of
BALB/c mouse strain, ob tained in asep tic con di tions,
GE NETIC IN STA BIL ITY OF MOUSE EM BRY ONIC GERMINATIVE G1 CELL LINE
339
were washed in PBS (pH 7.4), pow dered in mor tar with
liq uid ni tro gen and in cu bated with the lysis buffer.
To carry out the immunoprecipitation, lysis of cells
was per formed for 30 min on ice in the buffer of the fol -
low ing con tents: 0.05 M tris-HCl (pH 8.0), 0.15 M
NaCl, 0.005 M EDTA, 1 % NP-40, and 0.002 M
phenylmethylsulfonyl flu o ride.
To carry out the West ern Blot ting, the cell pel let
was added with buffer of: 0.05 M trisHCl (pH 8.0), 0.15
M NaCl, 0.005 M EDTA, 0.003 M 1,4-dithiotreitol,
0.002 M phenylmethylsulfonyl flu o ride and in cu bated
on ice for 30 min. Then the cell lysates were sonicated
on ice by ul tra-sound three times, 10 sec each, with 30
sec in ter val, cen tri fuged for 30 min at 4°C at 10 000 g.
The supernatant was trans ferred to a fresh tube, and the
pel let was dis carded.
West ern Blot anal y sis of tis sue and cell lysates.
Brad ford’s quan ti ta tive method was used to de ter mine
to tal pro tein con cen tra tion in cell lysates [31].
Gel-elec tro pho re sis of cell lysates was per formed in ac -
cor dance to the rec om men da tions [34].
Immunoprecipitation and West ern Blot ting were
car ried out us ing the monoclonal anti-p53 an ti bod ies by
Chemicon In ter na tional, USA: 1) PAb240, ca pa ble of
de tect ing in denaturated con di tions of both forms of
p53: wild type and mu tants, whereas in the na tive con -
di tions – only the mu tant form and 2) an ti bod ies
PAb246, re ac tive to wild type p53 only. Spe cies-spe -
cific an ti bod ies, pro duced by Jack son
ImmunoResearch, USA, con ju gated with horse-rad ish
peroxidase were used as sec ond ary an ti bod ies.
West ern-Blot anal y sis was per formed ac cord ing to
rec om men da tions, pre sented in [34]. The pro teins were
sep a rated by sin gle-level elec tro pho re sis in 10%
SDS-polyacrylamide gels. Sep a rated pro teins were
trans ferred from SDS-PAAG to polyvinylidene flu o -
ride mem brane (Millipore, USA) us ing the method of
semi-dry trans fer in the buffer, which con tained 39 mM
of glycine, 48 mM ba sic tris, 0.037% SDS and 20%
meth a nol, pH 8.3 and us ing the method of tank trans fer
in the buffer of the fol low ing con tents: 192 mM of
glycine, 25 mM tris-HCl, 10% meth a nol, us ing the
trans fer sys tems of BioRad, USA. Af ter the trans fer the
mem branes were in cu bated for 1 hour with gen tle ag i ta -
tion on a plat form shaker to pre vent non-spe cific bind -
ing (0.01 M tris-HCl, pH7.5, 0.15 M NaCl, 5% non fat
dried milk). Then, mem branes were in cu bated for 1
hour at room tem per a ture with gen tle ag i ta tion on a
plat form shaker in the so lu tion of pri mary monoclonal
an ti bod ies (1 ?g/ml of an ti bod ies in the blocking so lu -
tion). The mem brane was washed three times 10 min
each in buffer, con tain ing 0.01 M tris-HCl, pH7.5, 0.15
M NaCl and 0.1% of tween-20. Spe cies-spe cific sec -
ond ary an ti bod ies, con ju gated with horse rad ish
peroxidase, were added as rec om mended by the man u -
fac turer and in cu bated with the mem brane dur ing 1
hour at room tem per a ture with gen tle ag i ta tion on a
plat form shaker.
Chemiluminescent re ac tion of the mem brane was
car ried out for 1 min in the so lu tion of: 100 mM
tris-HCl, pH 8.5, 1.237 mM 3-aminophthalhydrazide
(luminol), 0.673 mM trans-4-hydroxycinnamic acid
(p-coumaric acid), and 0.011% H
2
O
2
. Mem brane was
ex posed to X-ray film, pro duced by Agfa, USA.
Immunoprecipitation. Cell lysates were cen tri fuged
at 10 000 g for 30 min at 4°C. 500 mg of to tal pro tein
were in cu bated with 40 ml of 50% pro tein A-sepharose
dur ing 1 hour, then cen tri fuged at 30 000 g for 2 min.
The lysates was added 15 ml of an ti bod ies (1.5 mg) and
then in cu bated (4°C, 16 hours). Af ter in cu ba tion with
an ti bod ies, 60 ml of 50% protein A-sepharose were
added and in cu bated again (4°C, 1 hour), then cen tri -
fuged at 3 000 g for 2 min. Pro tein A-sepharose was
washed three times in the buffer (0.05 M tris-HCl, pH
8.0, 0.15 M NaCl, 0.005 M EDTA, 1% NP-40, and
0.002 M phenylmethylsulfonyl flu o ride). Pro tein
A-sepharose was added 20 ml 2 x Laemmli buffer and
boiled for 10 min.
Microphotography. Pic tures of microobjects were
taken us ing Jenaval mi cro scope (Carl Zeiss, Aus tria)
with lens of 25x, 40x, and 100x us ing the Canon
Powershot dig i tal cam era (Canon, UK) and Zoom soft -
ware.
The sta tis ti cal anal y sis of the re sult ob tained was
per formed ac cord ing to the method, de scribed in [35].
Re sults and Dis cus sion. The cell pop u la tions of
mouse G1 cell line were re vealed to have dif fer ent ab er -
rant mitoses, namely, cells with dif fer ent num bers of
nu clei, micronuclear cells, bridges, C-mitoses, etc, and
high fre quen cies of aneuploid cells at dif fer ent pas -
sages of in vi tro cul ti va tion [30, 31]. Ab er rant mitoses
re vealed are con se quences of both dam ages of chro mo -
IATSYSHYNA A. P. ET AL.
340
somes and mi totic ap pa ra tus and cytotomy [36]. The
loss or gain of chro mo somes in cells, can cer cells in
par tic u lar, are known to be the re sults of ab er rant
mitoses [37]. Eukaryotic cells are spe cific for the ex is -
tence of a qual i ta tive mech a nism, con trol ling the nor -
mal course of mi to sis i.e. mi totic check point, which
pro vides cor rect dis tri bu tion of chro mo somes dur ing
cell di vi sion [6, 7]. Sup pos edly, mi totic check point in
the cells of the in ves ti gated mouse cell line G1 is ei ther
par tially ac tive or in ac tive at all.
The cells of de fected mi totic check point are not ca -
pa ble of ter mi nat ing its di vi sion if the chromatides are
not at tached to the spin dle fi bers or at tached inproperly
as well as also in the case of dam aged kinetochore or
formed sev eral spin dle poles (so called multi-po lar
mitoses) etc [6, 7]. Thus the cells on set pre ma ture
anaphase. Nor mal cells, treated with so lu tion
depolymerising spin dle microtubule or in hib it ing the
for ma tion of one, stop in the cell cy cle and do not en ter
the mi to sis as their mi totic check point is ac ti vated [38,
39]. The cells with dam aged mi totic check point are not
ca pa ble of ter mi nat ing their di vi sion af ter treat ment
with mi totic poi sons, e.g. nocodazole, colchicine,
colcemide, etc [8]. There fore, we have tested G1 cells
at the 26th and 114th pas sages of in vi tro cul ti va tion for
their ca pa bil ity to sur viv ing and di vid ing af ter treat -
ment with colchicine of dif fer ent con cen tra tions. The
rel a tive num ber of liv ing cells, ob tained test ing the nor -
mal cells – mouse em bry onic fibroblasts (MEFs) iso -
lated from 12.5-d.p.c. BALB/c mouse em bryos, were
used as the con trol.
It was re vealed that the num ber of liv ing cells de -
creased af ter MEFs in cu ba tion with colchicine of dif -
fer ent con cen tra tions in no time af ter the change of the
me dium (Fig.1). The com par i son with the con trol
(cells and sol vent) re vealed the num ber of cells to de -
crease app. 24–33% (Fig.1, b). In 48 hours af ter the
change of the growth me dium, the num ber cells in -
creased in both con trols, at the same time their num ber
de creased in the wells, treated with dif fer ent con cen tra -
tions of colchicine (Fig.1). The cells, treated with 0.05
?g/ml colchicine were the least sen si tive – 63.9% of
cells sur vived; av er age sen si tiv ity of cells to 0.5 ?g/ml
was 45.19% and MEFs were the most sen si tive to 5
?g/ml colchicine per 1 ml of me dium – 27.54% (Fig.1,
b). This fact re veals the cor re la tion be tween the MEFs
sen si tiv ity and colchicine con cen tra tion. Thus, we
have de fined ef fec tive pro lif er a tion block ing and sig -
nif i cant de crease in the num ber of liv ing cells in MEFs
pop u la tion af ter treat ment with colchicine, which also
tes ti fies to the func tional ac tiv ity of mi totic check point
in MEFs.
The num ber of G1 liv ing cells at the 26th pas sage af -
ter treat ment with colchicine in dif fer ent con cen tra tions
de creased in sig nif i cantly (Fig.2, a). The com par i son
with the con trol re vealed app. 13–14% de crease in the
num ber of cells (Fig.2, b). In 48 hours af ter change of
the me dium, the num ber of liv ing cells at 0.05 ?g/ml
colchicine ex ceeded that of MEFs and amounted
76.86% (Fig.1, Fig.2, a, b). The num ber of sur vived G1
cells was also in creased at the 26th pas sage, com pared to
MEFs, af ter treat ment with 0.5 and 5 mg/ml of
GE NETIC IN STA BIL ITY OF MOUSE EM BRY ONIC GERMINATIVE G1 CELL LINE
341
Fig.1 Anal y sis of ac tiv ity of mi totic check point in mouse em bry onic fibroblasts (MEFs, 2nd pas sage in vi tro): a – the num ber of liv ing cells,
ab so lute value of op ti cal den sity (1 – 0 hours; 2 – 48 hours); b – rel a tive num ber of liv ing cells in com par i son to the con trol (1 – 0 hours; 2 –
48 hours); absciss – colchicine con cen tra tion, mg/ml, C-1 – con trol (cells only); C-2 – con trol 2 (cells and colchicine sol vent – wa ter)
colchicine (Fig.2), 65.67 and 68.51%, re spec tively. No
sig nif i cant dif fer ence has been dis cov ered in the sen si -
tiv ity of the in ves ti gated G1 cells to the con cen tra tion
of colchicine of 0.5 and 5 mg/ml. There fore, the ac tiv ity
of mi totic check point at the 26th pas sage of cul ti va tion
of cells of the mouse G1 cell line is lower, com pared
with MEFs.
The anal y sis of the ac tiv ity of the mi totic check -
point of G1 cells at the 114th pas sage of in vi tro cul ti va -
tion re vealed the high est num ber of sur vived cells af ter
the treat ment with colchicine, in com par i son with the
26th pas sage and MEFs. The num ber of sur vived cells in
com par i son with the con trol de creased ~5% only at the
treat ment of colchicine of 0.05 ?g/ml and ~20% at 0.5
and 5 mg/ml (Fig.2, c, d). In 48 hours af ter the change of
the growth me dium, the num ber of sur vived cells at
dif fer ent colchicine con cen tra tions was at a rather high
level (79.82, 79.97, and 76.44% of sur vived cells at the
IATSYSHYNA A. P. ET AL.
342
Fig.2 Anal y sis of ac tiv ity of mi totic check point in the cells of mouse G1 cell
line: a – num ber of liv ing cells at the 26th pas sage, ab so lute val ues of op ti cal
den sity; b – rel a tive num ber of liv ing cells at the 26th pas sage, com pared to
the con trol; c – num ber of liv ing cells at the 114th pas sage, ab so lute value of
op ti cal den sity; d – rel a tive num ber of liv ing cells at the 114th pas sage, com -
pared to the con trol (a–d: 1 – 0 hours, 2 – 48 hours); absciss – colchicine
con cen tra tion, mg/ml, C-1 – con trol (cells only); C-2 – con trol 2 (cells and
colchicine sol vent – wa ter); e – anaphase of G1 line cells at the 104th pas -
sage of in vi tro cul ti va tion with chro mo somal de lay (black ar row) and
bridges be tween groups of anaphase chro mo somes (white ar row); lens 100x,
oc u lar 10x.
afore men tioned colchicine con cen tra tions) (Fig.2).
There fore, the cells of mouse G1 cell line were re vealed
to be in sen si tive to dif fer ent con cen tra tions of
colchicine at the 114th pas sage of in vi tro cul ti va tion,
which tes ti fies about weak ened mi totic con trol.
The anal y sis of mi totic check point in MEFs and in
G1 cell line at the 26th and 114th pas sages on in vi tro cul -
ti va tion also re vealed MEFs to be the most sen si tive to
the ef fect of dif fer ent con cen tra tions of colchicine. G1
cells were shown to be less sen si tive at both early and
late pas sages (Fig.3, a).
In 48 hours of grow ing of cells af ter the change of
the me dium, the sig nif i cant dif fer ence in the num ber of
sur vived cells in MEFs pop u la tion at dif fer ent
colchicine con cen tra tions was ob served as well as in
the num bers of sur vived cells in MEFs and G1 pop u la -
tions (Fig.3, b). G1 cells were re vealed to be in sen si tive
to the ef fect of dif fer ent con cen tra tions of colchicine,
whereas ev ery 10-time in crease in colchicine con cen -
tra tions, MEFs were shown both not to di vide and die.
There fore, the lower sen si tiv ity of G1 cells to the ef fect
of colchicine at the 26th and 114th pas sage of cul ti va tion,
com pared to MEFs, was re vealed, while the cells at the
114th pas sage were spe cific for the low est sen si tiv ity
(Fig.3).
The in ac ti va tion of mi totic check point in cells of
the mouse G1 cell line is ev i denced by the pre ma ture
sep a ra tion of centromeres, com pared with sis ter
chromatides, re vealed at the 15th pas sage of in vi tro cul -
ti va tion [40]. The fac tors, caus ing the loss of chro mo -
some, the for ma tion of micronuclei, and aneuploidy of
the in ves ti gated cell line, may in clude not only the spin -
dle de fects (as multi-nu clear cells in the G1 cell pop u la -
tion and mul ti po lar mitoses in their sublines were
ob served) but also the de fects of kinetochores, par tic u -
larly de lay in anaphase of chro mo somes (Fig.2, e).
There fore, the ob tained re sults re veal that the
disfunction of mi totic check point is re spon si ble for the
chro mo somal in sta bil ity of the cells of mouse G1 cell
line.
Some other fac tors are in volved in the oc cur rence
of phe no type of chro mo somal in sta bil ity in mam ma lian
cells, e.g. the loss of func tion of tumorsupressor p53
[1]. Fibroblasts of p53-/- of mice were shown to re veal
high aneuploidy [11, 12–16]. The cor re la tion be tween
mu ta tion or de le tion of p53 and the oc cur rence of
aneuploidy have been es tab lished in var i ous hu man tu -
mours [17].
The mech a nisms of p53 in ac ti va tion prior to the oc -
cur rence of chro mo somal in sta bil ity have not been clar -
i fied yet. One of the known mech a nisms is pre sented
by the reg u la tion of cy cle of centrosome du pli ca tion
dur ing p53 in ac ti va tion [11, 41, 42]. Thus, loss of p53
was shown to re sult in hy per-am pli fi ca tion of
centrosomes in mouse cells in vi tro, as a re sult high fre -
quency of ab er rant mitoses (mul ti po lar mitoses and
dam aged cytokinesis) was ob served, which in its turn
re sults in the in crease in num ber of er rors in the trans fer
of chro mo somes to the sis ter cells [11, 41]. Other pos -
si ble mech a nisms are re lated to p53 reg u la tion of the
cell cy cle and apoptosis.
There fore, we have ana lysed the ex pres sion of
Trp53 gene in the cells of mouse G1 cell line and its
sublines G1-OA and G1-T at dif fer ent pas sages of in vi -
GE NETIC IN STA BIL ITY OF MOUSE EM BRY ONIC GERMINATIVE G1 CELL LINE
343
Fig.3 Num bers of sur vived cells com pared to the con trol: a – 0 hours and b – 48 hours of cell growth, prior treated with dif fer ent colchicine
con cen tra tions, af ter the change of growth me dium (1 – MEFs, 2nd pas sage; 2 – G1, 26th pas sage; 3 – G1, 114th pas sage); absciss –
colchicine con cen tra tion, mg/ml, C-1 – con trol (cells only); C-2 – con trol 2 (cells and colchicine sol vent – wa ter)
tro cul ti va tion us ing the meth ods of West ern Blot ting
and immunoprecipitation. West ern Blot ting was car -
ried out on monoclonal an ti bod ies PAb240, which in
the denaturated con di tions re cog nised both forms (na -
tive and mu tant) of p53 pro tein [43, 44].
Immuneprecipitation was per formed with an ti bod ies
PAb246, spe cific to wild type p53, and PAb240, which
at the na tive con di tions are bound with mu tant p53 only
[43, 44].
Low level of p53 was re vealed in all in ves ti gated
cell cul tures us ing West ern Blot ting (Fig.4, a). MEFs
lysates were used as the pos i tive con trol of p53 ex pres -
sion.
As PAb240 at denaturated con di tions al lows de tect -
ing p53 of both mu tant and wild types, there fore, to de -
ter mine which of the forms of p53 is pres ent in G1 cells
we have per formed immunoprecipitation of p53 at the
na tive con di tions with PAb240 and pAB246 an ti bod -
ies. At these con di tions PAb240 are bound to mu tant
p53 only and PAb246 with wild type p53. Only wild
type p53 was immunoprecipitated from the MEFs
lysates. At the same time lysates of the G1 cells at the
106th pas sage of cul ti va tion in vi tro re vealed the
immunoprecipitation of both wild and mu tant p53
forms (Fig.4, b). Mu tant p53 were de tected at the 30th
and 106th pas sages of in vi tro cul ti va tion of G1 cells
(Fig.4, b). The pres ence of mu tant and wild p53 forms
may re sult in func tional in ac ti va tion of p53 due to the
for ma tion of p53-tetramere be tween wild type p53 and
mu tant p53, as it has been de scribed in [45, 46].
There fore, the anal y sis of p53 ex pres sion re vealed
the mu tant form of p53 in G1 line cells at the 30th and
106th pas sages of in vi tro cul ti va tion. These data cor re -
late with the re sults of anal y sis of ac tiv ity of mi totic
check point in the in ves ti gated cells. Chro mo somal in -
sta bil ity and de sta bi li sa tion of cell line karyotype are
ex pected to in crease at the con di tions of weak ened mi -
totic con trol and the ab sence of func tional p53. It is
pos si ble that si mul ta neous in ac ti va tion of mi totic
check point and ex pres sion of p53 of both types may
pro vide the ex pla na tion to ex tended stage of es tab lish -
ment in vi tro of mouse G1 cell line with in ten sive evo -
lu tion of karyotype [31].
Thus, hav ing per formed the anal y sis of sur viv abil -
ity of the cells of mouse G1 cell line and divisibility af -
ter treat ment with colchicine, it is pos si ble to con clude
the weak en ing of func tion of mi totic check point at dif -
fer ent pas sages of in vi tro cul ti va tion. West ern-Blot
anal y sis al lowed low-level de tec tion of p53 pro tein in
the cells of G1 line as well as in its sublines G1-OA and
G1-T, whereas the immunoprecipitation of p53 pro tein
at the na tive con di tions re vealed the mu tant p53 form in
the G1 cells at the 30th and 106th pas sages of in vi tro cul -
ti va tion. Both wild type and mu tant p53 were de tected
at the 106th pas sage of G1 cells. Sum ma ris ing the stated
above it has to be said that in creased chro mo somal in -
sta bil ity and in ten sive pro cess of karyotypic evo lu tion
of spon ta ne ously im mor tal ised mouse G1 cell line [29,
31, 32], as well as ab er rant mitoses in the cells of this
line are due to the dam ages in reg u la tion of mi to sis and
pos si bly in ac ti va tion of p53.
The work was par tially sup ported by Ukrai nian
Fun da men tal Re search State Fund of Min is try of Sci -
ence and Ed u ca tion of Ukraine, pro ject
No.Ô18/11-2006.
À. Ï. ßöûøèíà, Ñ. Ì. Êâàøà, Î. Â. Ïèäïàëà, Ò. Ï. Ðóáàí,
È. Í. Âàãèíà, Ë. Ë. Ëóêàø
Ãå íå òè ÷åñ êàÿ íå ñòà áèëü íîñòü ýì áðè î íàëü íûõ ãåð ìè íà òèâ íûõ
êëå òîê ëè íèè G1 ìûøè è íà ðó øå íèå ôóíê öèé êîí òðîëü íîé
òî÷ êè ìè òî çà è ð53
Ðå çþ ìå
Èçó ÷å íà âû æè âà å ìîñòü ìû øè íûõ êëå òîê ëè íèè G1 ïðè îá ðà -
áîò êå êîë õè öè íîì â ðàç íûõ êîí öåí òðà öè ÿõ. Óñòà íîâ ëå íà
ìåíü øàÿ ÷ó âñòâè òåëü íîñòü ê äå éñòâèþ êîë õè öè íà êëå òîê ëè -
íèè G1 íà 26-ì è 114-ì ïàñ ñà æàõ ïî ñðàâ íå íèþ ñ ýì áðè î íàëü -
IATSYSHYNA A. P. ET AL.
344
Fig.4 P53 pro tein de tec tion: a – West ern-Blot anal y sis of p53 with
monoclonal an ti bod ies PAb240 in cells: 1 – MEFs; 2 – G1, 30th
pas sage; 3 – G1, 106th pas sage; 4 – G1-OA, 35th/24th pas sages; 5 –
G1-OA, 35th/65th pas sage; 6 – G1-T, 66th/17th pas sage; 7 – G1-T,
66th/35th pas sage; b – immunoprecipitation of p53 with PAb240
an ti bod ies (1, 2, 4) and PAb246 (3, 5) from pro tein ex tracts of cells:
1 – G1, 30th pas sage; 2, 3 – G1, 106th pas sage; 4, 5 – MEFs
íû ìè ôèá ðîá ëàñ òà ìè ìûøè ëè íèè BALB/c, ÷òî
ñâè äå ò åëüñòâó åò îá îñëàá ëå íèè êîí òðîëü íîé òî÷ êè ìè òî çà â
êëåò êàõ ëè íèè G1. Ïðè ïî ìî ùè Âåñ òåðí-áëîò àíà ëè çà âû ÿâ ëåí
áå ëîê ð53 íà íèç êîì óðîâ íå êàê â êëåò êàõ ëè íèè G1, òàê è åå
ñóá ëè íèé G1-OA è G1-T. Ìå òî äîì èì ìó íîï ðå öè ïè òà öèè â
êëåò êàõ ëè íèè G1 îïðå äå ëå íî ïðè ñó òñòâèå áåë êà ð53 îá îèõ òè -
ïîâ: êàê äè êî ãî, òàê è ìó òàí òíî ãî, ÷òî ìî æåò âûçâàòü íà ðó -
øå íèå åãî ôóíê öèè. Ãå íå òè ÷åñ êàÿ íå ñòà áèëü íîñòü êëå òîê
ëè íèè G1 ìûøè ñâÿ çà íà ñ íà ðó øå íè åì ôóíê öèè êîí òðîëü íîé
òî÷ êè ìè òî çà è, âå ðî ÿò íî, ñ ôóíê öè î íàëü íîé èíàê òè âà öè åé
ð53.
Êëþ ÷å âûå ñëî âà: êëåò êà ìûøè in vitro, êîí òðîëü íàÿ òî÷ êà
ìè òî çà, õðî ìî ñîì íàÿ íå ñòà áèëü íîñòü, ð53.
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ÓÄÊ 576.5+576.35:576.356+577.21
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