Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells
To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis p...
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Інститут клітинної біології та генетичної інженерії НАН України
2012
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| Цитувати: | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells / Ya.A. Sheremet, A.I. Yemets, A. Azmi, K. VissenBerg, J.P. Verbelen, Ya.B. Blume // Цитология и генетика. — 2012. — Т. 46, № 5. — С. 3-11. — Бібліогр.: 44 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860246094650277888 |
|---|---|
| author | Sheremet, Ya.A. Yemets, A.I. Azmi, A. VissenBerg, K. Verbelen, J.P. Blume, Ya.B. |
| author_facet | Sheremet, Ya.A. Yemets, A.I. Azmi, A. VissenBerg, K. Verbelen, J.P. Blume, Ya.B. |
| citation_txt | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells / Ya.A. Sheremet, A.I. Yemets, A. Azmi, K. VissenBerg, J.P. Verbelen, Ya.B. Blume // Цитология и генетика. — 2012. — Т. 46, № 5. — С. 3-11. — Бібліогр.: 44 назв. — англ. |
| collection | DSpace DC |
| container_title | Цитология и генетика |
| description | To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.
Для изучения участия обратимого фосфорилирования белков в прохождении митоза растительной клеткой исследовано влияние на этот процесс и организацию микротрубочек в синхронизированной культуре BY-2 ингибиторов тирозинкиназ – гербимицина А, генистеина и тирфостина AG18, а также ингибитора тирозинфосфатаз – ортованадата натрия. Обнаружено, что обработка клеток BY-2 ингибиторами тирозинкиназ при прохождении G2/M фазы не приводила к видимым нарушениям митотических структур микротрубочек, однако вызывала уменьшение их количества. Возможно, снижение уровня фосфорилирования белков микротрубочек по остаткам тирозина может нарушать динамические свойства микротрубочек при прохождении интерфазы/профазы. Все использованные ингибиторы тирозинкиназ приводили к задержке вступления клеток в профазу: гербимицин А и генистеин – на 2 ч, тирфостин AG18 – на 1 ч. После обработки гербимицином А или генистеином отмечали задержку в появлении пика митоза на 1 ч по сравнению с контролем, а после обработки тирфостином AG18 изменений не было. Обработка культуры клеток ингибитором тирозинфосфатаз ортованадатом натрия на протяжении 1 ч оказывала противоположное воздействие на прохождение митоза: вступление клеток в профазу ускорялось, но без изменений во времени формирования митотического пика по сравнению с контролем. Можно предположить, что фосфорилирование белков по остаткам тирозина является важным звеном в регуляции перехода клеток из интерфазы в М-фазу посредством регуляции динамических свойств микротрубочек в растительных клетках.
Щоб дослідити участь зворотного фосфорилювання білків у проходженні мітозу рослиною клітини, вивчено вплив на цей процес і організацію мікротрубочок в синхронізованій культурі BY-2 інгібіторів тирозинкіназ – гербіміцину А, геністеїну та тирфостину AG18, а також інгібітора тирозинфосфатаз – ортованадату натрію. Виявлено, що обробка клітин BY-2 інгібіторами тирозинкіназ при проходженні G2/M не призводила до очевидних порушень мітотичних структур, проте викликала зменшення їхньої кількості. Можливо, що зниження рівня тирозинфосфорилювання білків мікротрубочок по залишках тирозину може порушувати динамічні властивості мікротрубочок впродовж інтерфази/профази. Всі використані нами інгібітори тирозинкіназ призводили до затримки входження клітин у профазу: гербіміцин А і геністеїн – на 2 год, тирфостин AG18 – на 1 год. Після обробки гербіміцином А або геністеїном виявлено затримку в прояві піку мітозу на 1 год у порівнянні з контролем, а після обробки тирфостином AG18 змін не було. Обробка культури клітин інгібітором тирозинфосфатаз, ортованадатом натрію впродовж 1 год призводила до протилежного впливу на проходження мітозу: вступ клітин в профазу прискорювався, проте без змін в часі формування мітотичного піку в порівнянні із контролем. Можна зробити припущення про те, що зворотне тирозинфосфорилювання білків по залишках тирозину є важливою ланкою в регуляції переходу із інтерфази в М-фазу за рахунок регуляції динамічних властивостей мікротрубочок в рослинних клітинах.
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Îðèãèíàëüíûå ðàáîòû
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
© Ya.A. SHEREMET, A.I. YEMETS, A. AZMI, K. VISSENBERG,
J.-P. VERBELEN, Ya.B. BLUME, 2012
Ya.A. SHEREMET 1, A.I. YEMETS 1, A. AZMI 2,
K. VISSENBERG 2, J.-P. VERBELEN 2, Ya.B. BLUME 1
1 Institute of Food Biotechnology and Genomics, National Academy
of Sciences of Ukraine, Kiev
2 University of Antwerp, Plant Growth and Development,
Biology Department, Belgium
Е-mail: yarasheremet@gmail.com
EFFECTS OF TYROSINE KINASE
AND PHOSPHATASE INHIBITORS
ON MITOSIS PROGRESSION IN
SYNCHRONIZED TOBACCO BY-2
CELLS
To test whether reversible tubulin phosphorylation
plays any role in the process of plant mitosis the effects
of inhibitors of tyrosine kinases, herbimycin A, genistein
and tyrphostin AG 18, and of an inhibitor of tyrosine
phosphatases, sodium orthovanadate, on microtubule
organization and mitosis progression in a synchronized
BY-2 culture has been investigated. It was found that
treatment with inhibitors of tyrosine kinases of BY-2 cells
at the G2/M transition did not lead to visible disturbances
of mitotic microtubule structures, while it did reduce
the frequency of their appearance. We assume that a
decreased tyrosine phosphorylation level could alter
the microtubule dynamic instability parameters during
interphase/prophase transition. All types of tyrosine kinase
inhibitors used caused a prophase delay: herbimycin A and
genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter
the peak of mitosis was displaced for 1 h by herbimycin A
or genistein exposure, but after tyrphostin AG18 treatment
the timing of the mitosis-peak was comparable to that in
control cells. Enhancement of tyrosine phosphorylation
induced by the tyrosine phosphatase inhibitor resulted in
the opposite effect on BY-2 mitosis transition. Culture
treatment with sodium orthovanadate during 1 h resulted
in an accelerated start of the prophase and did not
lead to the alteration in time of the mitotic index peak
formation, as compared to control cells. We suppose that
the reversible tyrosine phosphorylation can be involved in
the regulation of interphase to M phase transition possibly
through regulation of microtubule dynamics in plant cells.
Introduction. Reversible phosphorylation is
one of the most common post-translational
protein modifications, which regulates numerous
biological processes in eukaryotic cells, namely it
plays an important role in cellular proliferation,
growth and development [1]. It is known that
combined activity of protein kinases, which ca-
talyze the phosphorylation of serine/threonine or
tyrosine residues, and protein phosphatases, that
remove the phosphate groups from appropriate
residues, determines the level of substrate protein
phosphorylation [2, 3].
Tyrosine phosphorylation, despite its overwhel-
ming importance in animals, has been largely ne-
glected in plants simply because typical tyrosine
kinases were not found in higher plants. Recently
with the development of mass spectrometry, it
has been reported that tyrosine phosphorylation
is as important in plants as in animals [4]. By
searching for tyrosine kinase-specific sequence
motifs, a bioinformatic screen for tyrosine ki-
nases in plants predicted two tyrosine kinases in
the Arabidopsis thaliana genome [5], 34 putative
tyrosine kinases and five dual-specificity proteins,
indicating that nearly 4 % of all A. thaliana ki-
nases can phosphorylate tyrosine residues [6].
Moreover putative tyrosine kinases are found in
several plants: six tyrosine kinases in Oryza sa-
tiva ssp. Indica and seven in Oryza sativa ssp.
Japonica [5]. Using bioinformatics methods on
the basis of homology to the Mus musculus Zap70
tyrosine kinase catalytic domain, the existence of
494 protein kinase genes in A. thaliana was pre-
dicted. Moreover non-receptor Syk-like tyrosine
kinase was identified in the green algal species,
which could have a critical role in algal growth
and development [7].
A. thaliana has few protein tyrosine-specific
phosphatases. Recent studies have shown that
specific tyrosine phosphatases exist in higher
plants [3] and play a key role in some physiologi-
cal processes, such as the bending of touch-sen-
sitive petioles in Mimosa pudica [8], regulation
of stomata movement in Commelina communis
leaves [9], in pollen germination and pollen tube
growth [10].
Tyrosine phosphorylated proteins have been
detected in different plant species [8, 11–13]. It
is known that protein tyrosine phosphorylation is
involved in the control of specific steps in plant
development [11, 14] and in plant signalling pro-
cesses [15–17].
4
Ya.A. Sheremet, A.I. Yemets, A. Azmi et al.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
As it was revealed in higher plants one of
the cytoskeleton components, microtubules, can
be intensively regulated by post-translational
modifications of tubulin, including phosphory-
lation [18, 19]. Recently it was established that
both - and -tubulins undergo phosphorylation
on tyrosine residues [19]. Immunofluorescence
microscopy revealed that tyrosine phosphorylation
of -tubulin could be one of the targets for
tyrosine kinases [20]. In our previous paper we
have provided data that tyrosine kinase inhibitors
as herbimycin A, genistein and tyrphostin AG18
as well as an inhibitor of tyrosine phosphatases,
sodium orthovanadate, affect microtubule organi-
zation in Arabidopsis thaliana root cells. As a
result root growth and development are altered,
leading to the interpretation that tubulin phos-
phorylation/dephosphorylation events are invo-
lved in these regulating cascades [21]. However,
at present no evidences exist for possible roles of
these modifications in the plant’s microtubular
organization, nor for its functional impact on the
cell-cycle progression.
To investigate the potential involvement of
tyrosine phosphorylation of plant proteins in
regulation of mitosis, the effects of tyrosine
kinase inhibitors (herbimycin A, genistein and
tyrphostin AG18) and a tyrosine phosphatase
inhibitor (sodium orthovanadate) on microtu-
bule organization and mitotic progression in
synchronized suspension Nicotiana tabacum
BY-2 cell culture were investigated. It was found
that at G2/M transition treatment of BY-2 cells
with inhibitors of tyrosine kinases caused no vis-
ible disturbances of microtubular organization,
but that the frequency of the mitotic figures
was reduced. It is suggested that the decrease of
tyrosine phosphorylation level could cause the
slowing down of the interphase/prophase tran-
sition. Probably this is the result of alterations
in dynamic parameters of plant microtubules in
dividing cells via regulation of phosphorylation
levels of microtubular proteins during cell cycle-
specific stages transition.
Materials and methods. Cell culture and syn-
chronization. The tobacco BY-2 (Nicotiana taba-
cum L. cv. Bright Yellow-2) suspension cell line
expressed GFP-MBD [22] was maintained as
described by Nagata et al. [23] with modifications.
The culture samples was refreshed weekly by
transfer of 2,5 ml of 7-day-old culture into 50 ml
of fresh Murashige and Skoog medium [24],
(«Duchefa», Netherlands) pH 5.8, containing 3 %
(w/v) sucrose («Duchefa», Netherlands), 0,2 g/L
KH2PO4 («Merck», Germany), 10 mg/L myo-
inositol («Sigma», USA), 1 mg/L thiamin hydro-
chloride («Sigma», USA), and 0,2 mg/L 2,4-D
(«Serva», Germany). The culture was kept at
+28 °C at constant darkness and 130 rpm.
Cell culture synchronization protocol was ba-
sed on the method of Nagata et al. [23]. The
stationary culture was transferred in a proportion
15:100 to the fresh Murashige and Skoog me-
dium, supplied with 5 g/ml aphidicolin (ICN
Biomedicals, Belgium). After 20 h incubation,
cells were released by extensive washing (5 L per
115 ml of blocked culture). After the aphidicolin
release the main population of cells was at the
early S-phase [23]. Afterwards, cells were trans-
ferred into fresh medium and divided equally into
five subcultures, which were subsequently treated
with inhibitors (Fig. 1, see color plate). Untreated
culture was used as a control. For experiments we
used previously defined effective concentrations
of herbimycin A, genistein, tyrphostin AG18 and
sodium orthovanadate [21]. The synchronization
of BY-2 culture by aphidicolin at the beginning
of S-phase, a drug that inhibits DNA polymerase
[23], enables to investigate effects of tyrosine
kinase and tyrosine phosphatase inhibititors on
both G2/M and mitosis transitions.
Confocal laser scanning microscopy. Effects of
tyrosine kinase and tyrosine phosphatase inhibi-
tors on microtubule organization in synchronized
BY-2 cells were visualized after 1–8 h of treat-
ment. GFP-labeled microtubules in tobacco BY-2
(GFP-MBD) cells were visualized in vivo using
confocal laser scanning microscopes C1 («Nikon»,
Japan) and LSM 510 META («Carl Zeiss»,
Germany) with 60× water-immersion and 63×
oil-immersion objectives respectively, using the
488 line of the Ar-lazer.
Mitotic index calculation. For calculation of the
mitotic index cells were sampled during 10 h at
1 h intervals (0,5 ml per sample), and fixed in a
ethanol/acetic acid 3:1 (v/v) solution. Cells were
washed in phosphate-buffered saline, stained with
4’,6-diamino-phenylindole (DAPI, 5 mg/ml) and
counted (Nikon fluorescent microscope, Nikon
Europe, Badhoevedorp, The Netherlands). The
5
Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
mitotic index was established as the sum of the
percentages of cells in pro-, meta-, ana- and
telophases. The percentage of pro-, meta-, ana-
and telophases was assessed as a number of
cells in correspondence phase of mitosis to the
percentage of the total number of cells. For each
determination more than one thousand cells were
examined. All results were presented as a mean ±
standard error (SE) of at least three replicates.
Chemicals. The inhibitor of non-receptor tyro-
sine kinase, herbimycin A («Sigma», USA) [25],
was dissolved in DMSO as 1 mM stock solution
and used in 30 M concentration. The inhibitor
of receptor tyrosine kinases, genistein («Sigma»,
USA) [26] was dissolved in DMSO at 10 mM stock
solution and used in 10 M concentration. The
inhibitor of receptor tyrosine kinase, tyrphostin
AG18 («Calbiochem», Germany) [27] was dis-
solved in DMSO at 5 mM stock solution and
used in 50 M concentration. Stock solutions of
tyrosine kinase inhibitors were stored at –20 °C.
The concentration of DMSO in all experiments did
not exceed 0,5 %. The inhibitor of protein tyrosine
phosphatases, sodium orthovanadate («Sigma»,
USA) [28] was dissolved immediately before use
in H2O at 250 M concentration.
Results. Effects of tyrosine kinase and tyro-
sine phosphatase inhibitors on the mitotic microtu-
bules organization in synchronized BY-2 cells. In
control BY-2 cells all microtubule arrays typical
for higher plant cells were observed. The inter-
phase microtubule network in non-treated cells
consists of cortical microtubules oriented mostly
transverse to the cell elongation axis (Fig. 2, a,
see color plate). In prometaphase, cortical micro-
tubules were substituted by the preprophase band
(Fig. 2, b). Dissolution of the preprophase band
and nuclear envelope coincides with formation
of the mitotic spindle (Fig. 2, c). As mitosis pro-
ceeds, the phragmoplast (Fig. 2, d) forms in the
midzone of the late anaphase spindle, precisely
at the site landmarked by the preprophase band.
The phragmoplast, like the preprophase band, is
a structure unique to plant cells. In conclusion,
in higher-plant cells, successful cell division re-
quires the temporal and spatial coordination of
the assembly of highly organized microtubule
arrays, where these microtubule reorganizations
are closely linked to the acquisition of specialized
properties and functions [29].
Control synchronized BY-2 culture entered
into mitosis after 4 h following aphidicolin re-
lease as far as at this time first PPBs were ob-
served. It was estimated that all used tyrosine ki-
nase inhibitors reduced the frequency of mitotic
figure appearance in treated synchronized BY-2
cells but that it did not cause the alteration of
microtubular organization. It was found that after
3 h treatment with inhibitors of tyrosine kina-
ses in synchronized BY-2 culture all typical mi-
totic microtubular arrays were present without any
visible disturbances (Fig. 3, see color plate).
The interphase microtubules also maintain the
normal organization (Fig. 3, a, e, i). Long-term
(6 h) incubation of cells with tyrosine kinase in-
hibitors did not lead to any dramatic alteration of
mitotic microtubules.
The same results were obtained after tyrosine
phosphatase inhibitor treatment. It was shown that
BY-2 treatment with sodium orthovanadate did
not lead to the alteration in organization of both
mitotic and cortical microtubular arrays (Fig. 4,
a–d, see color plate), as compared to non-treated
cells (Fig. 2, a–d). Short- as well as long-term
incubation of cells with sodium orthovanadate did
not disturb organization, nor orientation of the
preprophase band, spindle and phragmoplast in
treated cells.
Inhibitors of tyrosine kinases and tyrosine phos-
phatases influence the mitotic index of BY-2 cells.
In a second set of experiments the effects of
treatment with tyrosine kinase and tyrosine phos-
phatase inhibitors on the mitotic index of the
synchronized BY-2 culture were investigated. It
was established that treatment with all tested ty-
rosine kinase inhibitors as well as with one ty-
rosine phophatase inhibitor caused alterations in
mitotic progression as compared to control cells
(Fig. 5). The peak of mitotic index was reached
at 35 ± 1,9 % in control culture after 7 h follow-
ing aphidicolin release (Fig. 5). The most obvi-
ous alterations in mitotic progression were found
after application of herbimycin A and genistein.
In the synchronized BY-2 culture treated with
30 M herbimycin A and 10 M genistein the
cells reached a maximum mitotic index of 16 ±
± 2,2 and 17 ± 1,4 %, correspondingly (1 h later
than in control cells) (Fig. 5). Both inhibitors
caused similar changes in mitotic progression of
synchronized BY-2 cells, namely, dividing cells
6
Ya.A. Sheremet, A.I. Yemets, A. Azmi et al.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
entered mitosis 1 h later and finish the process
with 2 h delay. On the other hand, BY-2 cell treat-
ment with another inhibitor, tyrphostin AG18,
did not cause changes in cells exiting mitosis. Be-
sides the reduction of the mitotic index level to
24,8 ± 1,4 %, there were no differences at the
time of mitotic index peak after tyrphostin AG18
treatment in comparison to control cells (Fig. 5).
Culture treatment with the tyrosine phospha-
tase inhibitor sodium orthovanadate resulted in a
reduction of the mitotic index peak (20 ± 1,9 %),
but it did not lead to the alteration in the timing
of the mitotic index peak formation, as compared
to control cells (Fig. 5).
Effects of tyrosine kinases and tyrosine phospha-
tase inhibitors on the progression of mitotic phases
in synchronized BY-2 cells. In the next step the
effects of tyrosine kinase and tyrosine phospha-
tase inhibitors on different mitotic phases’ pro-
gression of synchronized BY-2 culture were de-
termined. In the control culture the percentage
of cells in pro-/metaphases was maximal at 7 h
after aphidicolin release, in ana- and telophases
this occurred at 8 h (Fig. 6). Treatment with ty-
rosine kinase inhibitors of a synchronized BY-2
cell culture led to obvious changes in prophase
progression. Both herbimycin A (Fig. 7) and ge-
nistein (Fig. 8) treatments led to a 2 h delay in
prophase entry; tyrphostin AG18 caused a 1 h
lagging into prophase entry (Fig. 9), as compared
to the control (Fig. 6). Moreover, the amount of
inhibitor-treated cells entering the prophase was
significantly lower, probably due to the further
decrease in the level of mitotic index (Fig. 7–9).
It was found that the observed delay into mito-
sis entry after herbimycin A and genistein treat-
ment led to the lagging of mitotic index peak and
a subsequent delayed exit of cells from mitosis
(Fig. 5), as compared to control cells.
On the other hand, BY-2 treatment with an
inhibitor of tyrosine phosphatases caused the op-
posite effect on mitotic phase’s progression, as
compared to influence of tyrosine kinases inhibi-
tors. It was found that 1 h treatment with sodium
orthovanadate causes an acceleration of cells
entering prophase (Fig. 10). However the total
amount of dividing cells after sodium orthovana-
date treatment was significantly lower as a result
of the obvious reduction of the number of cells
entering prophase (Fig. 10), as compared to the
control.
Discussion. It was found that BY-2 treat-
ment with inhibitors of tyrosine kinases at the
point of G2/M transition did not lead to visible
disturbances of mitotic microtubular structures,
while it significantly reduced the frequency of
the typical mitotic figure appearances. The to-
tal amount of the cells with a preprophase band
after 1–2 h tyrosine kinases inhibitors treatment
was significantly reduced without disruption in
preprophase band architecture, in comparison
with control. Moreover cell treatment with any
tyrosine kinases inhibitor did not affect mitotic
microtubules (spindle and phragmoplast), nor did
Fig. 5. The effects of different types of tyrosine kinase
and tyrosine phosphatase inhibitors on mitotic activity
in synchronized BY-2 cells. Inhibitors were applied 4 h
after aphidicolin release (arrow)
Fig. 6. The mitotic index and percentage of cells in
different phases of mitosis in control synchronized
BY-2 culture. The peak of cells in pro-, meta-, ana-
and telophases are indicated an arrow
7
Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
it affect the cortical microtubule network. Thus
it was demonstrated that all used tyrosine kinase
inhibitors at tested concentrations did not cause
mitotic arrest in G2/M, but that they did lead to
slower prophase entry. As argument for this find-
ing we can note that all types of used tyrosine
kinases inhibitors caused prophase delay: herbi-
mycin A and genistein – for 2 h, and tyrphostin
AG18 – for 1 h. Thereafter the peak of mitosis
was displaced 1 h later after herbimycin A or ge-
nistein exposure, whereas after tyrphostin AG18
treatment it was comparable to this in control
cells. Moreover the total amount of mitotic cells
was significantly reduced.
These results demonstrate that herbimycin A
and genistein have a more potent action on mi-
totic progression in synchronized BY-2 cell cul-
ture than tyrphostin AG18. It is known that her-
bimycin A is a benzoquinoid ansamycin antibiotic
that acts as a very specific inhibitor of non-re-
ceptor tyrosine kinases of the src family, binding
directly to the kinase domain (probably to thiol
groups) and preventing ATP binding [30]. Other
types of tyrosine kinase inhibitors, like genistein
and tyrphostin, are potent receptor-type broad
range tyrosine kinase inhibitors that interact with
ATP binding sites [26] or act through binding
the substrate binding site [31], subsequently. Pos-
sibly, the observed distinction in the effects of
tyrosine kinase inhibitors could be a result of the
different substrate specificity and mechanisms of
action of the tested inhibitors.
Previously, effects of different tyrosine kinase
inhibitors on cell cycle progression were inves-
tigated only on animal cells. It was shown that
herbimycin A and genistein were able to inhibit
the proliferation of some cancer cells lines in a
dose-dependent manner [32, 33]. Several cancer
cell studies have shown that genistein can trigger
G2/M cell cycle arrest and inhibit cell growth
[34, 35]. Our data show for the first time that
inhibition of receptor and non-receptor tyrosine
kinases lead to delay of G2/M cell cycle transi-
tion in synchronized BY-2 cells as a result of
2 h prophase lagging after herbimycin A and ge-
nistein treatment, as compared to mitotic pro-
gression in control cells.
Enhancement of tyrosine phosphorylation in-
duced by the tyrosine phosphatase inhibitor did
not cause the alteration in organization of both
Fig. 7. The mitotic index and percentage of cells in
different phases of mitosis in synchronized BY-2 cul-
ture after herbimycin A treatment. Inhibitor applica-
tion is indicated by the arrow
Fig. 8. The mitotic index and percentage of cells in
different phases of mitosis in synchronized BY-2 cul-
ture after genestein treatment. Inhibitor application is
indicated by the arrow
Fig. 9. The mitotic index and percentage of cells in
different phases of mitosis in synchronized BY-2 cul-
ture after tyrphostin AG18 treatment. Inhibitor appli-
cation is indicated by the arrow
8
Ya.A. Sheremet, A.I. Yemets, A. Azmi et al.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
cortical and mitotic microtubule arrays but re-
sulted in the opposite effect on BY-2 mitotic tran-
sition in comparison with effects of tyrosine ki-
nases inhibitors. One hour treatment with sodium
orthovanadate slightly promotes cells entering into
prophase; the total amount of the cells that entered
mitosis was higher than in the control. Though,
in cells incubated with sodium orthovanadate the
mitotic index was reduced, which is in accordance
with results obtained for animal cells [36]. It was
shown for different animal cells that sodium or-
thovanatade protects cells from apoptosis but that
in certain situations it can indeed induce growth
inhibition and apoptosis [37, 38]. Downregula-
tion of several tyrosine phosphatases resulted in
a significantly reduced mitotic index [39]. Up to
now, effects of sodium orthovanadate on mitotic
microtubule organization as well as mitosis transi-
tion in higher plant cells were not investigated.
Only in animal cells it was shown that sodium or-
thovanadate increases phosphorylation on tyrosine
residues of cellular proteins, including -tubulin
[40]. Sodium orthovanadate plus H2O2 decreases
the degree of microtubule polymerization in hepa-
tocyte cultures and antagonizes the effect of her-
bimycin A. The authors proposed that the degree
of tyrosine phosphorylation of -tubulin after the
cells’ exposure to inhibitors of tyrosine kinases and
tyrosine phosphatases is an important factor to de-
termine the state of assembly of microtubules in
animal cells [40].
Recently using double-label staining experi-
ments on A. thaliana microtubules with a poly-
clonal anti-phosphotyrosine antibody (P-Tyr),
a monoclonal anti- -tubulin (TU-01) and an
anti- -tubulin antibody (TUB 2.1) it was shown
that phosphotyrosine labeling correlated with
cortical microtubules in interphase cells as well
as with all types of mitotic microtubules [20]. It
was also estimated that the signal detected with
the anti-phosphotyrosine antibody was markedly
enhanced after pretreating the cells with sodium
orthovanadate, as compared with control (un-
treated) cells. Thereby immunofluorescence mi-
croscopy revealed tyrosine phosphorylation on
polymerized microtubules in plant cells, imply-
ing that - and -tubulins could be targets for
tyrosine kinases [20].
Taken together, our observations revealed that
inhibitors of tyrosine kinases could slow down
the interphase/prophase transition, probably as
a result of alterations in dynamic parameters of
plant microtubules in dividing cells by regulating
phosphorylation levels of microtubule proteins
during specific stages of cell cycle transition. Dy-
namic instability of microtubules is characterized
by four parameters: growth rate, shrinkage rate,
frequency of transition from growth to shrinkage
(catastrophe frequency), and frequency of transi-
tion from shrinkage to growth (rescue frequency)
[41]. It is known that the dynamic instability
parameters of plant microtubules are important
determinants for the transition from interphase
microtubules to the preprophase band [42]. Du-
ring the transition from interphase to prepro-
phase band formation the microtubule growth
rate and catastrophe frequency doubles, but the
shrinkage rate and rescue frequency remains
constant, which makes microtubules shorter and
more dynamic [43]. Earlier it was estimated in
animal cells that -tubulin phosphorylation on
C-terminal tyrosine residues by non-receptor ty-
rosine kinases effects microtubules polymeriza-
tion [44] since phosphorylated -tubulin fails to
polymerize into microtubules [44].
Comparison of our results with previously
published data [21] suggests that the role of ty-
rosine phosphorylation/dephosphorylation pro-
cesses in dividing and differentiated plant cells
can be different. Earlier we found that the most
sensitive microtubules to the action of tyrosine
kinases in A. thaliana primary roots were those in
differentiated cells. Cortical microtubules in cells
Fig. 10. The mitotic index and percentage of cells in
different phases of mitosis in synchronized BY-2 cul-
ture after sodium orthovanadate treatment. Inhibitor
application is indicated by the arrow
9
Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
of the roots differentiation zone (trichoblasts and
atrichoblasts) and root hairs are more sensitive
to tyrosine kinases inhibitors than microtubules
in meristematic cells [21]. We suggested that the
tyrosine phosphorylation/dephosphorylation pro-
cess is involved in regulating the development
and differentiation of Arabidopsis root cells. This
assumption is in accordance with previous data
on carrot and A. thaliana cells were protein tyro-
sine phosphorylation involvement in the control
of specific steps in plant development was esti-
mated [11].
Using specific inhibitors of non-receptor and
receptor tyrosine kinases and inhibitors of tyro-
sine phosphatases we found that tyrosine phos-
phorylation/dephosphorylation processes partici-
pate in mitosis progression in synchronized BY-2
culture. We suppose that inhibition of tyrosine
phosphorylation causes the slow down in the
transition from the interphase to the prophase in
mitotic cells through regulation of phosphoryla-
tion levels of microtubule proteins. We proposed
that cells respond to stimuli, and that the regu-
lation of cellular morphogenesis and control of
cell division can be partly interpreted in terms
of the microtubule protein phosphorylation/de-
phosphorylation status.
This work was supported by grants from the
Research Foundation – Flanders (FWO), the Uni-
versity of Antwerp and INTAS ¹ 03-51-6459.
ß.À. Øåðåìåò, À.È. Åìåö, À. Àçìè,
Ê. Âèññåíáåðã, Æ.-Ï. Âåðáåëåí, ß.Á. Áëþì
ÂËÈßÍÈÅ ÈÍÃÈÁÈÒÎÐΠÒÈÐÎÇÈÍÊÈÍÀÇ
È ÒÎÐÎÇÈÍÔÎÑÔÀÒÀÇ ÍÀ ÏÐÎÕÎÆÄÅÍÈÅ
ÌÈÒÎÇÀ Â ÑÈÍÕÐÎÍÈÇÈÐÎÂÀÍÍÎÉ
ÊÓËÜÒÓÐÅ ÒÀÁÀÊÀ BY-2
Äëÿ èçó÷åíèÿ ó÷àñòèÿ îáðàòèìîãî ôîñôîðèëè-
ðîâàíèÿ áåëêîâ â ïðîõîæäåíèè ìèòîçà ðàñòèòåëü-
íîé êëåòêîé èññëåäîâàíî âëèÿíèå íà ýòîò ïðî-
öåññ è îðãàíèçàöèþ ìèêðîòðóáî÷åê â ñèíõðîíèçè-
ðîâàííîé êóëüòóðå BY-2 èíãèáèòîðîâ òèðîçèíêè-
íàç – ãåðáèìèöèíà À, ãåíèñòåèíà è òèðôîñòèíà
AG18, à òàêæå èíãèáèòîðà òèðîçèíôîñôàòàç –
îðòîâàíàäàòà íàòðèÿ. Îáíàðóæåíî, ÷òî îáðàáîòêà
êëåòîê BY-2 èíãèáèòîðàìè òèðîçèíêèíàç ïðè
ïðîõîæäåíèè G2/M ôàçû íå ïðèâîäèëà ê âèäèìûì
íàðóøåíèÿì ìèòîòè÷åñêèõ ñòðóêòóð ìèêðîòðóáî-
÷åê, îäíàêî âûçûâàëà óìåíüøåíèå èõ êîëè÷åñòâà.
Âîçìîæíî, ñíèæåíèå óðîâíÿ ôîñôîðèëèðîâàíèÿ
áåëêîâ ìèêðîòðóáî÷åê ïî îñòàòêàì òèðîçèíà ìî-
æåò íàðóøàòü äèíàìè÷åñêèå ñâîéñòâà ìèêðîòðó-
áî÷åê ïðè ïðîõîæäåíèè èíòåðôàçû/ïðîôàçû. Âñå
èñïîëüçîâàííûå èíãèáèòîðû òèðîçèíêèíàç ïðè-
âîäèëè ê çàäåðæêå âñòóïëåíèÿ êëåòîê â ïðîôàçó:
ãåðáèìèöèí À è ãåíèñòåèí – íà 2 ÷, òèðôîñòèí
AG18 – íà 1 ÷. Ïîñëå îáðàáîòêè ãåðáèìèöèíîì À
èëè ãåíèñòåèíîì îòìå÷àëè çàäåðæêó â ïîÿâëåíèè
ïèêà ìèòîçà íà 1 ÷ ïî ñðàâíåíèþ ñ êîíòðîëåì,
à ïîñëå îáðàáîòêè òèðôîñòèíîì AG18 èçìåíåíèé
íå áûëî. Îáðàáîòêà êóëüòóðû êëåòîê èíãèáèòîðîì
òèðîçèíôîñôàòàç îðòîâàíàäàòîì íàòðèÿ íà ïðî-
òÿæåíèè 1 ÷ îêàçûâàëà ïðîòèâîïîëîæíîå âîçäåé-
ñòâèå íà ïðîõîæäåíèå ìèòîçà: âñòóïëåíèå êëåòîê â
ïðîôàçó óñêîðÿëîñü, íî áåç èçìåíåíèé âî âðåìåíè
ôîðìèðîâàíèÿ ìèòîòè÷åñêîãî ïèêà ïî ñðàâíåíèþ
ñ êîíòðîëåì. Ìîæíî ïðåäïîëîæèòü, ÷òî ôîñôîðè-
ëèðîâàíèå áåëêîâ ïî îñòàòêàì òèðîçèíà ÿâëÿåòñÿ
âàæíûì çâåíîì â ðåãóëÿöèè ïåðåõîäà êëåòîê èç
èíòåðôàçû â Ì-ôàçó ïîñðåäñòâîì ðåãóëÿöèè äè-
íàìè÷åñêèõ ñâîéñòâ ìèêðîòðóáî÷åê â ðàñòèòåëü-
íûõ êëåòêàõ.
ß.À. Øåðåìåò, À.². ªìåöü, À. Àçì³,
Ê. ³ññåíáåðã, Æ.-Ï. Âåðáåëåí, ß.Á. Áëþì
ÂÏËÈ ²ÍòÁ²ÒÎв ÒÈÐÎÇÈÍʲÍÀÇ
² ÒÈÐÎÇÈÍÔÎÑÔÀÒÀÇ ÍÀ ÏÐÎÕÎÄÆÅÍÍß
̲ÒÎÇÓ Â ÑÈÍÕÐÎͲÇÎÂÀͲÉ
ÊÓËÜÒÓв ÒÞÒÞÍÓ BY-2
Ùîá äîñë³äèòè ó÷àñòü çâîðîòíîãî ôîñôîðèëþ-
âàííÿ á³ëê³â ó ïðîõîäæåíí³ ì³òîçó ðîñëèíîþ êë³-
òèíè, âèâ÷åíî âïëèâ íà öåé ïðîöåñ ³ îðãàí³çàö³þ
ì³êðîòðóáî÷îê â ñèíõðîí³çîâàí³é êóëüòóð³ BY-2
³íã³á³òîð³â òèðîçèíê³íàç – ãåðá³ì³öèíó À, ãåí³ñ-
òå¿íó òà òèðôîñòèíó AG18, à òàêîæ ³íã³á³òîðà
òèðîçèíôîñôàòàç – îðòîâàíàäàòó íàòð³þ. Âèÿâ-
ëåíî, ùî îáðîáêà êë³òèí BY-2 ³íã³á³òîðàìè
òèðîçèíê³íàç ïðè ïðîõîäæåíí³ G2/M íå ïðèçâî-
äèëà äî î÷åâèäíèõ ïîðóøåíü ì³òîòè÷íèõ ñòðóê-
òóð, ïðîòå âèêëèêàëà çìåíøåííÿ ¿õíüî¿ ê³ëüêîñ-
ò³. Ìîæëèâî, ùî çíèæåííÿ ð³âíÿ òèðîçèíôîñôî-
ðèëþâàííÿ á³ëê³â ì³êðîòðóáî÷îê ïî çàëèøêàõ
òèðîçèíó ìîæå ïîðóøóâàòè äèíàì³÷í³ âëàñòèâîñ-
ò³ ì³êðîòðóáî÷îê âïðîäîâæ ³íòåðôàçè/ïðîôàçè.
Âñ³ âèêîðèñòàí³ íàìè ³íã³á³òîðè òèðîçèíê³íàç ïðè-
çâîäèëè äî çàòðèìêè âõîäæåííÿ êë³òèí ó ïðî-
ôàçó: ãåðá³ì³öèí À ³ ãåí³ñòå¿í – íà 2 ãîä,
òèðôîñòèí AG18 – íà 1 ãîä. ϳñëÿ îáðîáêè ãåðá³-
ì³öèíîì À àáî ãåí³ñòå¿íîì âèÿâëåíî çàòðèìêó
â ïðîÿâ³ ï³êó ì³òîçó íà 1 ãîä ó ïîð³âíÿíí³ ç
êîíòðîëåì, à ï³ñëÿ îáðîáêè òèðôîñòèíîì AG18
çì³í íå áóëî. Îáðîáêà êóëüòóðè êë³òèí ³íã³á³-
òîðîì òèðîçèíôîñôàòàç, îðòîâàíàäàòîì íàòð³þ
âïðîäîâæ 1 ãîä ïðèçâîäèëà äî ïðîòèëåæíîãî
âïëèâó íà ïðîõîäæåííÿ ì³òîçó: âñòóï êë³òèí â
ïðîôàçó ïðèñêîðþâàâñÿ, ïðîòå áåç çì³í â ÷àñ³
10
Ya.A. Sheremet, A.I. Yemets, A. Azmi et al.
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
ôîðìóâàííÿ ì³òîòè÷íîãî ï³êó â ïîð³âíÿíí³ ³ç
êîíòðîëåì. Ìîæíà çðîáèòè ïðèïóùåííÿ ïðî òå,
ùî çâîðîòíå òèðîçèíôîñôîðèëþâàííÿ á³ëê³â ïî
çàëèøêàõ òèðîçèíó º âàæëèâîþ ëàíêîþ â ðåãóëÿ-
ö³¿ ïåðåõîäó ³ç ³íòåðôàçè â Ì-ôàçó çà ðàõóíîê ðå-
ãóëÿö³¿ äèíàì³÷íèõ âëàñòèâîñòåé ì³êðîòðóáî÷îê â
ðîñëèííèõ êë³òèíàõ.
REFERENCES
1. Hunter T. Porotein kinases and phosphatases:
the yin and yang of protein phosphorylation and
signaling // Cell. – 1995. – 80. – P. 225–236.
2. Hubbard S., Till J. Protein tyrosine kinase structure
and function // Annu. Rev. Biochem. – 2000. –
69. – P. 373–378.
3. Wang H., Chevalier D., Larue C., Ki Cho S.,
Walker J.C. The protein phosphatases and protein
kinases of Arabidopsis thaliana // The Arabidopsis
book / Eds C.R. Somerville, E.M. Meyerowitz. –
Rockville MD Amer. Soc. Plant Biol., 2007. –
P. 1–38.
4. De la Fuente van Bentem S., Hirt H. Protein tyrosine
phosphorylation in plants : More abundant than
expected? // Trends Plant Sci. – 2009. – 14, ¹ 2. –
P. 71–76.
5. Miranda-Saavedra D., Barton G.J. Classification
and functional annotation of eukaryotic protein
kinases // Proteins. – 2007. – 68. – P. 893–914.
6. Carpi A., Di Maria G., Vedovato M., Rossi V., Nac-
cari T., Floridus M., Terzi M., Filippini F. Com-
parative proteome bioinformatics: identification
of a whole complement of putative protein tyro-
sine kinases in the model flowering plant Arabi-
dopsis thaliana // Proteomics. – 2002. – 2, ¹ 11. –
P. 1494–1503.
7. Dash A. Evidence for the presence of non-receptor
protein tyrosine kinases in algal cells // Acta
Physiol. Plant. – 2010. – 32, ¹ 1. – P. 177–182.
8. Kameyama K., Kishi Y., Yoshimura M., Kanzawa N.,
Sameshima M., Tsuciya T. Tyrosine phosphorylation
in plant bending // Nature. – 2000. – 407. –
P. 37.
9. MacRobbie E. Evidence of a role of protein
tyrosine phosphatase in the control of ion release
from the guard cell vacuole in stomatal closure //
Proc. Nat. Acad. Sci. USA. – 2002. – 99. –
P. 11963–11968.
10. Zi H., Xiang Y., Wang T., Ren H. Reversible protein
tyrosine phosphorylation affects pollen germination
and pollen tube growth via the actin cytoskele-
ton // Protoplasma. – 2007. – 230. – P. 183–191.
11. Barizza E., Schiavo F., Terzi M., Filippini F. Evi-
dence suggesting protein tyrosine phosphorylation
in plants depends on the developmental condi-
tions // FEBS Lett. – 1999. – 447. – P. 191–194.
12. Huang H.J., Lin Y.M., Huang D.D., Takahashi T.,
Sugiyama M. Protein tyrosine phosphorylation
during phytohormone-stimulated cell proliferation
in Arabidopsis hypocotyls // Plant Cell. Physiol. –
2003. – 44. – P. 770–775.
13. Sugiyama N., Nakagami H., Mochida K., Daudi A.,
Tomita M., Shirasu K., Ishihama Y. Large-scale
phosphorylation mapping reveals the extent of
tyrosine phosphorylation in Arabidopsis // Mol.
Syst. Biol. – 2008. – 4. – P. 193.
14. Islas-Flores I., Oropeza C., Hernandez-Sotomayor S.M.
Protein phosphorylation during coconut zygotic
embryo development // Plant Physiol. – 1998. –
118. – P. 257–263.
15. Bretz J.R., Mock N.M., Charity J.C., Zeyad S.,
Baker C.J., Hutcheson S.W. A translocated protein
tyrosine phosphatase of Pseudomonas syringae pv.
tomato DC3000 modulates plant defence response
to infection // Mol. Microbiol. – 2003. – 49. –
P. 389–400.
16. Ghelis T., Bolbach G., Clodic G., Habricot Y.,
Miginiac E., Sotta B., Jeannette E. Protein tyrosine
kinases and protein tyrosine phosphatases are
involved in abscisic acid-dependent processes in
Arabidopsis seeds and suspension cells // Plant
Physiol. – 2008. – 148, ¹ 3. – P. 1668–1680.
17. Jaillais Y., Hothorn M., Belkhadir Y., Dabi T.,
Nimchuk Z.L., Meyerowitz E.M., Chory J. Tyrosine
phosphorylation controls brassinosteroid receptor
activation by triggering membrane release of its
kinase inhibitor // Genes Dev. – 2011. – 25, ¹ 3. –
P. 232–237.
18. Blume Ya., Smertenko A., Ostapets N., Viklick V.,
Draber P. Post-translational modifications of plant
tubulin // Cell Biol. Int. – 1997. – 21. – P. 918–
920.
19. Blume Ya.B., Yemets A., Sulimenko V., Sulimenko T.,
Chan J., Lloyd C., Dráber P. Evidence of tyrosine
phosphorylation of plant tubulin // Planta. – 2008. –
229. – P. 143–150.
20. Blume Ya., Yemets A., Sheremet Ya., Nyporko A.,
Sulimenko V., Sulimenko T., Dráber P. Exposure of
beta-tubulin regions defined by antibodies on an
Arabidopsis thaliana microtubule protofilament model
and in the cells // BMC Plant Biol. – 2010. – 10,
¹ 29. – P. 1–10. – doi: 10.1186/1471-2229-10-29.
21. Yemets A., Sheremet Ya., Vissenberg K., Van Orden J.,
Verbelen J.-P., Blume Ya. Effects of tyrosine kinase
and phosphatase inhibitors on microtubules in
Arabidopsis root cells // Cell Biol. Int. – 2008. –
32. – P. 630–637.
22. Granger C., Cyr R. Microtubule reorganization in
tobacco BY-2 cells stably expressing GFP-MBD //
Planta. – 2000. – 210. – P. 502–509.
23. Nagata T., Nemoto Y., Hasezawa S. Tobacco BY-2
11
Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2012. ¹ 5
cell line as the HeLa in the cell biology of higher
plants // Int. Rev. Cyt. – 1992. – 132. – P. 1–30.
24. Murashige T., Skoog F. A revisied medium for rapid
growth and bioassays with tobacco tissue culture //
Physiol. Plant. – 1962. – 15. – Ð. 473–497.
25. Nagaishi K., Adachi R., Matsui S., Yamaguchi T.,
Kasahara T., Suzuki K. Herbimycin A inhibits both
dephosphorylation and translocation of cofilin
induced by opsonized zymosan in macrophagelike
U937 cells // J. Cell Physiol. – 1999. – 180, ¹ 3. –
P. 345–354.
26. Akiyama T., Ishida J., Nakagawa S., Ogawara H.,
Watanabe S., Itoh N., Shibuya M., Fukami Y. Ge-
nistein, a specific inhibitor of tyrosine-specific pro-
tein kinases // J. Biol. Chem. – 1987. – 262. –
P. 5592–5595.
27. Wolbring G., Hollenbergf M.G., Schnetkamp P. Inhi-
bition of GTP-utilizing enzymes by tyrphostins //
J. Biol. Chem. – 1994. – 269, ¹ 36. – P. 22470–
22472.
28. Gordon J.A. Use of vanadate as protein-phospho-
tyrosine phosphatase inhibitor // Methods Enzy-
mol. – 1991. – 201. – P. 477–482.
29. Vantard M., Cowling R., Delichére C. Cell cycle
regulation of the microtubular cytoskeleton //
Plant Mol. Biol. – 2000. – 43. – Ð. 691–703.
30. Fukazawa H., Uehara Y., Murakami Y., Mizuno S., Ha-
mada M., Takeuchi T. Labeling of v-src and bcr-abl
tyrosine kinases with (14C) herbimycin A and its use in
the elucidation of the kinase inactivation mechanism //
FEBS Lett. – 1994. – 340. – P. 155–158.
31. Nowak F., Jacquemin-Sablon A., Pierre J. Epidermal
growth factor receptor signalling cascade as target
for thyrphostin (RG50864) in epithelial cells //
Biochem. Pharmacol. – 1997. – 53. – P. 287–298.
32. Farival R., Gardner-Thorpe J., Ito H., Archad H.,
Zinner M., Ashley S., Whang E. The efficacy of
tyrosine kinase inhibitors on human pancreatic can-
cer cell lines // J. Surg. Res. – 2003. – 115, ¹ 2. –
P. 219–225.
33. Hunáková L., Sedlák J., Klobušickšá M., Durai J.,
Chorváth B. Tyrosine kinase inhibitor-induced
differentiation of K-562 cells: alterations of cell
cycle and cell surface phenotype // Cancer Lett. –
1994. – 81, ¹ 1. – P. 81–87.
34. Oki T., Sowa Y., Hirose T., Tokagaki N., Horinaka M.,
Nakanishi R., Yasuda C., Yoshida T., Kanazawa M.,
Satomi Y., Nishino H., Miki T., Sakai T. Genistein
induces Gadd45 gene and G2/M cell cycle arrest
in the DU145 human peostate cancer cell line //
FEBS Lett. – 2004. – 577. – P. 55–59.
35. Li Z., Li J., Mo B., Hu C., Liu H., Qi H., Wang X.,
Xu J. Genistein induces G2/M cell cycle arrest via
stable activation of ERK1/2 pathway in MDA –
MB -231 breast cancer cells // Cell Biol. Toxicol. –
2008. – 24, ¹ 5. – P. 401–409.
36. Humaguchi T., Sudo T., Osada H. RK-682, a
potent inhibitor of tyrosine phosphatase, arrested
the mammalian cell cycle progression at G1 phase
// FEBS Lett. – 1995. – 132, ¹ 1. – P. 54–58.
37. Cruz T., Morgan A., Min W. In vitro and in vivo
antineoplastic effects of orthovanadate // Mol. Cell
Biochem. – 1995. – 153. – P. 161–166.
38. Figiel I., Kaczmarek L. Orthovanadate induces cell
death in rat dentate gyrus primary culture // Neu-
roreport. – 1997. – 8. – P. 2465–2470.
39. Chen F., Archambault V., Kar A., Lio P., D’Avino
P., Sinka R., Lilley K., Laue E., Deak P., Capalbo
L., Glover D. Multiple protein phosphatases are
required for mitosis in Drosophila // Curr. Biol. –
2007. – 17. – P. 293–303.
40. Ishibashi K., Fujioka T., Ui M. Decreases in cAMP
phosphodiesterase activity in hepatocytes cultured
with herbimycin A due to cellular microtubule
polymerization related to inhibition of tyrosine
phosphorylation of -tubulin // Eur. J. Biochem. –
1999. – 260. – P. 398–408.
41. Desai A., Mitchison T. Microtubule polymerization
dynamics // Annu. Rev. Cell Dev. Biol. – 1997. –
10. – P. 153–180.
42. Vassileva V., Fujii Y., Ridge R. Microtubule dyna-
mics in plants // Plant Biotech. – 2005. – 22,
¹ 3. – P. 171–178.
43. Dhonukshe P., Gadella T. Alteration of microtubule
dynamic instability during preprophase band
formation revealed by yellow fluorescent protein–
CLIP170 microtubule plus-end labeling // Plant
Cell. – 2003. – 15. – P. 597–611.
44. MacRae T. Tubulin post-translational modifications.
Enzymes and their mechanisms of actions // Eur.
J. Biochem. – 1997. – 244. – P. 265–278.
Received 10.02.12
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| id | nasplib_isofts_kiev_ua-123456789-126487 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0564-3783 |
| language | English |
| last_indexed | 2025-12-07T18:36:53Z |
| publishDate | 2012 |
| publisher | Інститут клітинної біології та генетичної інженерії НАН України |
| record_format | dspace |
| spelling | Sheremet, Ya.A. Yemets, A.I. Azmi, A. VissenBerg, K. Verbelen, J.P. Blume, Ya.B. 2017-11-24T21:01:18Z 2017-11-24T21:01:18Z 2012 Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells / Ya.A. Sheremet, A.I. Yemets, A. Azmi, K. VissenBerg, J.P. Verbelen, Ya.B. Blume // Цитология и генетика. — 2012. — Т. 46, № 5. — С. 3-11. — Бібліогр.: 44 назв. — англ. 0564-3783 DOI: 10.3103/S0095452712050088 https://nasplib.isofts.kiev.ua/handle/123456789/126487 To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells. Для изучения участия обратимого фосфорилирования белков в прохождении митоза растительной клеткой исследовано влияние на этот процесс и организацию микротрубочек в синхронизированной культуре BY-2 ингибиторов тирозинкиназ – гербимицина А, генистеина и тирфостина AG18, а также ингибитора тирозинфосфатаз – ортованадата натрия. Обнаружено, что обработка клеток BY-2 ингибиторами тирозинкиназ при прохождении G2/M фазы не приводила к видимым нарушениям митотических структур микротрубочек, однако вызывала уменьшение их количества. Возможно, снижение уровня фосфорилирования белков микротрубочек по остаткам тирозина может нарушать динамические свойства микротрубочек при прохождении интерфазы/профазы. Все использованные ингибиторы тирозинкиназ приводили к задержке вступления клеток в профазу: гербимицин А и генистеин – на 2 ч, тирфостин AG18 – на 1 ч. После обработки гербимицином А или генистеином отмечали задержку в появлении пика митоза на 1 ч по сравнению с контролем, а после обработки тирфостином AG18 изменений не было. Обработка культуры клеток ингибитором тирозинфосфатаз ортованадатом натрия на протяжении 1 ч оказывала противоположное воздействие на прохождение митоза: вступление клеток в профазу ускорялось, но без изменений во времени формирования митотического пика по сравнению с контролем. Можно предположить, что фосфорилирование белков по остаткам тирозина является важным звеном в регуляции перехода клеток из интерфазы в М-фазу посредством регуляции динамических свойств микротрубочек в растительных клетках. Щоб дослідити участь зворотного фосфорилювання білків у проходженні мітозу рослиною клітини, вивчено вплив на цей процес і організацію мікротрубочок в синхронізованій культурі BY-2 інгібіторів тирозинкіназ – гербіміцину А, геністеїну та тирфостину AG18, а також інгібітора тирозинфосфатаз – ортованадату натрію. Виявлено, що обробка клітин BY-2 інгібіторами тирозинкіназ при проходженні G2/M не призводила до очевидних порушень мітотичних структур, проте викликала зменшення їхньої кількості. Можливо, що зниження рівня тирозинфосфорилювання білків мікротрубочок по залишках тирозину може порушувати динамічні властивості мікротрубочок впродовж інтерфази/профази. Всі використані нами інгібітори тирозинкіназ призводили до затримки входження клітин у профазу: гербіміцин А і геністеїн – на 2 год, тирфостин AG18 – на 1 год. Після обробки гербіміцином А або геністеїном виявлено затримку в прояві піку мітозу на 1 год у порівнянні з контролем, а після обробки тирфостином AG18 змін не було. Обробка культури клітин інгібітором тирозинфосфатаз, ортованадатом натрію впродовж 1 год призводила до протилежного впливу на проходження мітозу: вступ клітин в профазу прискорювався, проте без змін в часі формування мітотичного піку в порівнянні із контролем. Можна зробити припущення про те, що зворотне тирозинфосфорилювання білків по залишках тирозину є важливою ланкою в регуляції переходу із інтерфази в М-фазу за рахунок регуляції динамічних властивостей мікротрубочок в рослинних клітинах. This work was supported by grants from the Research Foundation – Flanders (FWO), the University of Antwerp and INTAS № 03-51-6459. en Інститут клітинної біології та генетичної інженерії НАН України Цитология и генетика Оригинальные работы Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells Влияние ингибиторов тирозинкиназ и торозинфосфатаз на прохождение митоза в синхронизированной культуре табака BY-2 Вплив інгібіторів тирозинкіназ і тирозинфосфатаз на проходження мітозу в синхронізованій культурі тютюну BY-2 Article published earlier |
| spellingShingle | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells Sheremet, Ya.A. Yemets, A.I. Azmi, A. VissenBerg, K. Verbelen, J.P. Blume, Ya.B. Оригинальные работы |
| title | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells |
| title_alt | Влияние ингибиторов тирозинкиназ и торозинфосфатаз на прохождение митоза в синхронизированной культуре табака BY-2 Вплив інгібіторів тирозинкіназ і тирозинфосфатаз на проходження мітозу в синхронізованій культурі тютюну BY-2 |
| title_full | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells |
| title_fullStr | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells |
| title_full_unstemmed | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells |
| title_short | Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells |
| title_sort | effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco by-2 cells |
| topic | Оригинальные работы |
| topic_facet | Оригинальные работы |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/126487 |
| work_keys_str_mv | AT sheremetyaa effectsoftyrosinekinaseandphosphataseinhibitorsonmitosisprogressioninsynchronizedtobaccoby2cells AT yemetsai effectsoftyrosinekinaseandphosphataseinhibitorsonmitosisprogressioninsynchronizedtobaccoby2cells AT azmia effectsoftyrosinekinaseandphosphataseinhibitorsonmitosisprogressioninsynchronizedtobaccoby2cells AT vissenbergk effectsoftyrosinekinaseandphosphataseinhibitorsonmitosisprogressioninsynchronizedtobaccoby2cells AT verbelenjp effectsoftyrosinekinaseandphosphataseinhibitorsonmitosisprogressioninsynchronizedtobaccoby2cells AT blumeyab effectsoftyrosinekinaseandphosphataseinhibitorsonmitosisprogressioninsynchronizedtobaccoby2cells AT sheremetyaa vliânieingibitorovtirozinkinazitorozinfosfataznaprohoždeniemitozavsinhronizirovannoikulʹturetabakaby2 AT yemetsai vliânieingibitorovtirozinkinazitorozinfosfataznaprohoždeniemitozavsinhronizirovannoikulʹturetabakaby2 AT azmia vliânieingibitorovtirozinkinazitorozinfosfataznaprohoždeniemitozavsinhronizirovannoikulʹturetabakaby2 AT vissenbergk vliânieingibitorovtirozinkinazitorozinfosfataznaprohoždeniemitozavsinhronizirovannoikulʹturetabakaby2 AT verbelenjp vliânieingibitorovtirozinkinazitorozinfosfataznaprohoždeniemitozavsinhronizirovannoikulʹturetabakaby2 AT blumeyab vliânieingibitorovtirozinkinazitorozinfosfataznaprohoždeniemitozavsinhronizirovannoikulʹturetabakaby2 AT sheremetyaa vplivíngíbítorívtirozinkínazítirozinfosfataznaprohodžennâmítozuvsinhronízovaníikulʹturítûtûnuby2 AT yemetsai vplivíngíbítorívtirozinkínazítirozinfosfataznaprohodžennâmítozuvsinhronízovaníikulʹturítûtûnuby2 AT azmia vplivíngíbítorívtirozinkínazítirozinfosfataznaprohodžennâmítozuvsinhronízovaníikulʹturítûtûnuby2 AT vissenbergk vplivíngíbítorívtirozinkínazítirozinfosfataznaprohodžennâmítozuvsinhronízovaníikulʹturítûtûnuby2 AT verbelenjp vplivíngíbítorívtirozinkínazítirozinfosfataznaprohodžennâmítozuvsinhronízovaníikulʹturítûtûnuby2 AT blumeyab vplivíngíbítorívtirozinkínazítirozinfosfataznaprohodžennâmítozuvsinhronízovaníikulʹturítûtûnuby2 |