Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction
Aim. To investigate correlation between nitric oxide synthase, heat shock protein Hsp70 and apoptosis regulatory gene grim in D. melanogaster. Methods. The heat stress (37 C for 1 hour) induction in third instar larvae of Oregon R strain and transgenic strains, containing additional copies of dNOS1...
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irk-123456789-1538882019-07-06T20:57:10Z Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction Khussainova, E.M. Bulentayeva, Z.A. Bekmanov, B.O. Djansugurova, L.B. Bersimbayev, R.I. Structure and Function of Biopolymers Aim. To investigate correlation between nitric oxide synthase, heat shock protein Hsp70 and apoptosis regulatory gene grim in D. melanogaster. Methods. The heat stress (37 C for 1 hour) induction in third instar larvae of Oregon R strain and transgenic strains, containing additional copies of dNOS1 gene. RT-PCR and Western-blot analysis were used to study the expression of dNOS, Hsp70 and grim genes. Results. It is demonstrated that additional copies of dNOS1 gene in transgenic strains are intensively expressed immediately after heat stress induction. It was revealed that in all Drosophila strains the level of Hsp70 gene expression and its protein synthesis increase with subsequent decline after 2–3 hours; whereas the level of expression of grim increases immediately after heat stress induction in transgenic strains and declines in wild type flies, while the level of Hsp70 expression remains high. Conclusions. The increased level of Hsp70 has negative impact on the expression of grim, whereas additional NO synthesis neutralizes anti-apoptotic effects of Hsp70 and increases the expression level of grim. Thus, we assume the competitive relationships between anti-apoptotic functions of Hsp70 and pro-apoptotic effects of nitric oxide. Мета. Дослідити взаємозв’язок між синтазою оксиду азоту, білком теплового шоку (БТШ70) та апоптоз-регулюючим геном grim у D. melanogaster. Методи. Індукція теплового шоку (37 C, 1 год) у личинок 3-го віку лінії Oregon R і трансгенних ліній з додатковими копіями dNOS1-гена. Для аналізу експресії генів dNOS, hsp70 и grim використано методи ЗТ-ПЛР і Вестерн-блот. Результати. Показано, що в трансгенних лініях додаткові копії гена dNOS1 активно експресуються відразу після теплового стресу. Виявлено, що в усіх використаних лініях рівень експресії hsp70 і його білкового продукту підвищується після індукції стресу і знижується через 2–3 год, у той час як рівень експресії гена grim у трансгенних лініях за таких же умов підвищується, а в контролі зменшується при збереженні високого рівня експресії hsp70. Висновки. Значний рівень БТШ70 негативно впливає на експресію гена grim, додатковий синтез NO нейтралізує антиапоптичну дію БТШ70 і підвищує рівень експресії grim. Таким чином, ми передбачаємо наявність конкурентного взаємозв’зку між антиапоптичною функцією БТШ70 і про-апоптичною дією оксиду азоту. Цель. Исследовать взаимосвязь между синтазой оксида азота, белком теплового шока (БТШ70) и апоптоз-регулирующим геном grim у D. melanogaster. Методы. Индукция теплового шока (37 C, 1 ч) у личинок 3-го возраста линии Oregon R и трансгенных линий с дополнительными копиями dNOS1 гена. Для анализа экспрессии генов dNOS, hsp70 и grim использовали методы ОТ-ПЦР и Вестерн-блот. Результаты. Показано, что в трансгенных линиях дополнительные копии гена dNOS1 активно экспрессируются сразу после теплового стресса. Выявлено, что во всех используемых линиях уровень экспрессии hsp70 и его белкового продукта повышается после индукции стресса и снижается через 2–3 ч, в то время как уровень экспрессии grim в трансгенных линиях в этих же условиях возрастает, а в контроле уменьшается при сохранении высокого уровня экспрессии hsp70. Выводы. Значительный уровень БТШ70 негативно влияет на экспрессию гена grim, дополнительный синтез NO нейтрализует антиапоптическое действие БТШ70 и увеличивает уровень экспрессии grim. Таким образом, мы предполагаем наличие конкурентной взаимосвязи между антиапоптической функцией БТШ70 и про-апоптическим действием оксида азота. 2010 Article Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction / E.M. Khussainova, Z.A. Bulentayeva, B.O. Bekmanov, L.B. Djansugurova, R.I. Bersimbayev // Вiopolymers and Cell. — 2010. — Т. 26, № 2. — С. 194-199. — Бібліогр.: 20 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000156 http://dspace.nbuv.gov.ua/handle/123456789/153888 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Structure and Function of Biopolymers Structure and Function of Biopolymers |
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Structure and Function of Biopolymers Structure and Function of Biopolymers Khussainova, E.M. Bulentayeva, Z.A. Bekmanov, B.O. Djansugurova, L.B. Bersimbayev, R.I. Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction Вiopolymers and Cell |
| description |
Aim. To investigate correlation between nitric oxide synthase, heat shock protein Hsp70 and apoptosis regulatory gene grim in D. melanogaster. Methods. The heat stress (37 C for 1 hour) induction in third instar larvae of Oregon R strain and transgenic strains, containing additional copies of dNOS1 gene. RT-PCR and Western-blot analysis were used to study the expression of dNOS, Hsp70 and grim genes. Results. It is demonstrated that additional copies of dNOS1 gene in transgenic strains are intensively expressed immediately after heat stress induction. It was revealed that in all Drosophila strains the level of Hsp70 gene expression and its protein synthesis increase with subsequent decline after 2–3 hours; whereas the level of expression of grim increases immediately after heat stress induction in transgenic strains and declines in wild type flies, while the level of Hsp70 expression remains high. Conclusions. The increased level of Hsp70 has negative impact on the expression of grim, whereas additional NO synthesis neutralizes anti-apoptotic effects of Hsp70 and increases the expression level of grim. Thus, we assume the competitive relationships between anti-apoptotic functions of Hsp70 and pro-apoptotic effects of nitric oxide. |
| format |
Article |
| author |
Khussainova, E.M. Bulentayeva, Z.A. Bekmanov, B.O. Djansugurova, L.B. Bersimbayev, R.I. |
| author_facet |
Khussainova, E.M. Bulentayeva, Z.A. Bekmanov, B.O. Djansugurova, L.B. Bersimbayev, R.I. |
| author_sort |
Khussainova, E.M. |
| title |
Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction |
| title_short |
Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction |
| title_full |
Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction |
| title_fullStr |
Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction |
| title_full_unstemmed |
Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction |
| title_sort |
relations between nitric oxide synthase dnos1, hsp70 and apoptosis regulatory gene grim in drosophila melanogaster after heat stress induction |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2010 |
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Structure and Function of Biopolymers |
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http://dspace.nbuv.gov.ua/handle/123456789/153888 |
| citation_txt |
Relations between nitric oxide synthase DNOS1, Hsp70 and apoptosis regulatory gene grim in Drosophila melanogaster after heat stress induction / E.M. Khussainova, Z.A. Bulentayeva, B.O. Bekmanov, L.B. Djansugurova, R.I. Bersimbayev // Вiopolymers and Cell. — 2010. — Т. 26, № 2. — С. 194-199. — Бібліогр.: 20 назв. — англ. |
| series |
Вiopolymers and Cell |
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| fulltext |
Relations between nitric oxide synthase DNOS1,
Hsp70 and apoptosis regulatory gene grim in Drosophila
melanogaster after heat stress induction
E. M. Khussainova, Z. A. Bulentayeva, B. O. Bekmanov,
L. B. Djansugurova, R. I. Bersimbayev
Institute of General Genetics and Cytology
75A, al-Farabi Ave., Almaty, Kazakhstan, 050060
khussainova@mail.ru
Aim. To investigate correlation between nitric oxide synthase, heat shock protein Hsp70 and apoptosis re-
gulatory gene grim in D. melanogaster. Methods. The heat stress (37 °C for 1 hour) induction in third instar
larvae of Oregon R strain and transgenic strains, containing additional copies of dNOS1 gene. RT-PCR and
Western-blot analysis were used to study the expression of dNOS, Hsp70 and grim genes. Results. It is
demonstrated that additional copies of dNOS1 gene in transgenic strains are intensively expressed
immediately after heat stress induction. It was revealed that in all Drosophila strains the level of Hsp70
gene expression and its protein synthesis increase with subsequent decline after 2–3 hours; whereas the
level of expression of grim gene increases immediately after heat stress induction in transgenic strains and
declines in wild type flies, while the level of Hsp70 expression remains high. Conclusions. The increased
level of Hsp70 has negative impact on the expression of grim, whereas additional NO synthesis neutralizes
anti-apoptotic effects of Hsp70 and increases the expression level of grim. Thus, we assume the compe-
titive relationships between anti-apoptotic functions of Hsp70 and pro-apoptotic effects of nitric oxide.
Keywords: apoptosis, Drosophila, heat shock proteins, nitric oxide, grim.
Introduction. A large number of data evidence to the
fact that apoptosis in fruit fly Drosophila has many
common characteristics with apoptosis in mammals [1,
2]. Apoptotic signaling mechanisms render complex
sequence of molecular events and a multitude of mole-
cular components is required for realization of apopto-
tic cascades. Recent publications have demonstrated
that different endogenous and exogenous factors and
signaling molecules are either apoptosis inducers or its
inhibitors, being able to play a dual role in particular
conditions [3].
Nitric oxide (NO) is known for a wide spectrum of
characteristics and a range of activities inside the cells,
including physiological and pathological processes.
NO is synthesized during the conversion of L-arginine
to L-citrulline by nitric oxide synthase (NOS) [4]. In
Drosophila NOS gene encodes 10 different transcripts,
which can produce at least seven proteins, including
the only enzymatically active isoform – DNOS1. Other
isoforms are more or less truncated, which results in the
loss of functionally important NOS regions and sub-
sequent loss of activity [5]. The data on NO role in apo-
ptosis development are quite controversial. The bala-
nce between anti- and pro-apoptotic effects of nitric
194
ISSN 0233-7657. Biopolymers and Cell. 2010. Vol. 26. N 3
Ó Institute of Molecular Biology and Genetics NAS of Ukraine, 2010
oxide depends on many factors including the NO tissue
concentration and its interaction with other compo-
nents of apoptosis tools [6].
Many recent studies have shown that heat shock
protein 70 (Hsp70) plays a crucial role in regulating the
apoptotic cascade [7–9]. Hsp70 family is the most con-
served, diverse and best characterized class of Hsps,
which include constitutive and stress-inducible mole-
cules. Hsp70 is believed to interact with a large num-
ber of cellular proteins and therefore is a vital compo-
nent of cellular networks. Despite the fact that Hsp70
was assumed to be mostly anti-apoptotic, its precise
role in well-orchestrated apoptosis machinery is not
clearly defined [10, 11].
Realization of apoptosis program in Drosophila
melanogaster requires activity of specific genes acting
as integrators: reaper (rpr), head involution defective
(hid), and grim [1, 2, 12]. It is believed that these genes
are capable of inducing apoptosis independently and
transcriptionally regulated by different death-inducing
stimuli [13]. However, many aspects of their functions
and protein-protein interactions with other components
of apoptosis network, especially apoptosis regulatory
functions of grim, are yet to be revealed.
Therefore, the aim of our study was to investigate
relationship between nitric oxide synthase, heat shock
protein Hsp70 and apoptosis regulatory gene grim in
D. melanogaster.
Materials and methods. Objects. Experiments
were carried out on D. melanogaster late third instar
larvae, which were kept on standard nutrient medium at
25 oC. Wild strain Oregon R was used as a control. The
following transgenic strains, containing additional co-
pies of cDNAs from full-length DNOS1 transcript
(additional NO production), were used in the expe-
riments:
1. HS dNOS1 – contains a part of dNOS gene under
HS-promoter in X chromosome, which synthesizes
functionally active DNOS1 protein. X chromosome is
marked by mutations y and wa;
2. HS dNOS1 Flag – contains Flag-vector with a
part of dNOS gene under HS-promoter in X chromo-
some. HS dNOS1 Flag synthesizes functionally active
DNOS1 protein. Markers of X chromosome: y and wa;
Heat shock induction. The induction of heat shock
was performed by placing larvae containing tubes into
water bath heated to 37 °C for 1 hour. Afterwards lar-
vae were collected in the following time span: im-
mediately after heat shock induction; 2–3 hours after
heat shock and 5–6 hours after heat shock induction.
Larvae which were not subjected to heat stress served
as a control.
RNA extraction from larvae. Extraction of total
mRNA was performed using the RNA STAT-60 re-
agent (Tel-Test Inc., USA) in accordance with manu-
facturer’s instructions. RNA concentration was mea-
sured spectrophotometrically by determination of opti-
cal density at 260 and 280 nm.
cDNA synthesis. Reverse transcription was perfor-
med using the reverse transcription set (Sileks, Russian
Federation) according to manufacturer’s instructions.
Reaction was performed in 25 ml of reaction mixture,
containing 2 mg of total RNA.
PCR analysis. Reaction was performed in 20 ml of
reaction mixture, 0.3 mM of each primer and 0.5 ml of
cDNA. PCR conditions were as follows: for hsp70
gene (s 5'-CTG CGA GTC GTT GAA GTA CG-3' and
as 5'-TCG GTA TTG ATC TGG GAA CC 3'): 94 °Ñ –
2 min 30 s, and further 35 cycles: 94 °Ñ – 45 s, 56 °Ñ –
45 s, 72 °Ñ – 1 min. Final extinction was conducted at
72 °Ñ for 10 min; for dNOS1gene (s 5'-TTG TTG TGG
CCT CCA CCT TT-3' and as 5'-CAA TCC ATG CTC
GGA AGA CTC-3'): 94 °Ñ – 2 min, and further 40
cycles: 94 °Ñ – 15 s, 60 °Ñ – 1 min. Final extinction was
conducted at 60 °Ñ for 30 s; for grim gene (s 5'-ATG
AGG ACG ACG TTA CC-3' and as 5'-TTC TTG TTG
CTG CGG TTG-3'): 95 °Ñ – 30 s, 53 °Ñ – 30 s, 72 °Ñ –
40 s. Final extinction was conducted at 72 °Ñ for 7 min.
Amplification of b-actin gene fragment (s 5'-cgt cga caa
tgg atc tgg aa-3' and as 5'-cga cca tca cac cct gat ga-3')
was used as internal control. PCR products were visua-
lized in 2 % agarose gel electrophoresis.
Western-blot-analysis. 20 mg of protein extract we-
re separated in 8 % SDS-PAGE gel electrophoresis at
100 V and 4 °C. Then proteins were transferred to
PVDF membrane (Immobilon-P, Millipore Co., USA)
for 1.5 hour at 200 mÀ current at 4 °C. Primary antibo-
dy against Hsp70 (SantaCruz, USA) was used in
1:4000 dilution in the blocking buffer. Data of Wes-
tern-blot-analysis were analyzed with ImageJ software.
Results and discussion. Transgenic Drosophila
strains with additional copies of NOS gene (HS dNOS1
195
RELATIONS BETWEEN NITRIC OXIDE SYNTHASE DNOS1, Hsp70 AND APOPTOSIS REGULATORY GENE grim
and HS dNOS1 Flag) were used to investigate the
relationship between nitric oxide and apoptosis in res-
ponse to heat shock exposure as well as to study the
mechanisms of regulation of full-length DNOS1 pro-
tein activity [14]. As soon as the additional copies of
dNOS1 gene were placed under the heat shock pro-
moter we induced heat shock to «switch on» dNOS
gene. In order to monitor the expression of additional
copies of dNOS gene in dynamics (different time points
after heat shock induction), we performed reverse
transcriptase PCR using the specific primers. We de-
monstrated (Fig. 1) that immediately after heat shock
induction the level of expression of additional copies of
dNOS1 gene in HS dNOS1 and HS dNOS1 Flag (nitric
oxide donors of Drosophila strains increases dramati-
cally and remains on the same level for 2–3 hours, then
the level of expression of the gene decreases, which is
noticeable at 5–6 hours time point after stress induc-
tion. RT-PCR data confirmed the activity of addi-
tional copies of dNOS1 gene in the given fly strains and
demonstrated gene expression dynamics at different ti-
me points.
Many studies demonstrated that heat shock pro-
teins, especially Hsp70, are capable of inhibiting
stress-induced apoptosis and acting as anti-apoptotical
factors, however, exact pathways of this protective me-
chanism are yet to be determined.
To define hsp70 gene expression in third instar,
Drosophila larvae were subjected to heat stress, as
described above. The data obtained demonstrate that
drastic increase of hsp70 gene expression immediately
after heat shock induction is observed in wild type
strain Oregon R, and in transgenic strains HS dNOS1
Flag and HS dNOS1 with subsequent evident decrease
only 5–6 hours after heat stress (Fig. 2).
Results of RT-PCR analysis were proved by Wes-
tern-blot analysis. As it is demonstrated in Fig. 3,
immediately after heat stress the level of Hsp70 in-
creases significantly in control group Oregon R and
transgenic strains, containing additional copies of
dNOS1 gene, which results in generation of full length
functional transcript. Within 2–3 hours after heat
induction the level of expression of the given protein
starts decreasing in the experimental strains HS dNOS1
Flag and HS dNOS1, and only after 5–6 hours it redu-
ces in the control strain.
Very low hsp70 expression was found in intact
animals, though it was undetectable on protein level. It
is notable that intact groups of all studied strains do not
display Hsp70 expression which may be explained by
very insignificant amounts of the protein in samples
that could not be detected. According to Schmitt et al.,
contrary to Hsp90, which is abundantly expressed in
cells in constitutive manner, the expression of Hsp70
and Hsp27 is stimulated by different stress types, and
under normal conditions the expression of these pro-
teins in unstressed cells either does not occur at all or
occurs at a very low level [15].
Results obtained by Lakhotia and Prasanth de-
monstrated that «regulation of synthesis and turnover
of Hsp70 and Hsp64 during and after HS (heat stress
shock) in different cell types of Drosophila is complex,
involving transcriptional, translational, and posttrans-
lational controls». Thus, the authors demonstrated that
in larval malpighian tubule the hsp70 genes are quick-
ly transcribed, however, the protein synthesis requires
196
KHUSSAINOVA E. M. ET AL.
HS dNOS1 Flag HS dNOS1
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Fig. 1. dNOS mRNAs expression in intact and heat shock (37 °Ñ)
treated larvae of dNOS1 transgenic strains (RT-PCR)
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Fig. 2. hsp70 mRNAs expression in intact and heat shock (37 °Ñ)
treated larvae of wild (Oregon R) and dNOS1-transgenic strains
(RT-PCR)
some recovery period. It was revealed that during re-
covery period transport of the hsp70 transcripts from
nucleus to cytoplasm is enabled [16, 17].
According to I. V. Guzhova, mRNA of stress pro-
teins occurs in cell within few minutes after tempe-
rature increase, and the duration of its synthesis takes
several hours depending on heat stress continuance
with subsequent decrease after elevation of synthesis
activity. At 5–6 hours time point after heat induction its
level decreases to initial values. The increase in the
concentration of heat shock proteins starts 1–3 hours
after the heat induction and may remain at the high
level for a long time [18].
It is well known that ectopic expression of at least
one of key apoptotic regulatory genes (rpr, hid or grim)
is enough for realization of apoptosis program. The
activity of each gene is regulated by different upstream
regulatory signals. While the regulation of rpr and hid
expressions was extensively studied under the influ-
ence of a wide range of stimuli, the regulation of grim
expression is still far from elucidation [13]. Therefore,
we have focused our study on peculiarities of this gene
expression.
To determine the effect of excessive nitric oxide
concentrations and heat stress on the regulation of the
expression of apoptosis regulatory gene grim, we con-
ducted PCR amplification using specific primers. Our
results proved (Fig. 4) the expression of grim in intact
samples of both strains, which may testify to apoptosis
induction, associated with natural larval metamor-
phosis.
However, significant difference between wild type
and transgenic strains was observed in samples subjec-
ted to heat stress. In wild type Oregon R strain the exp-
ression level of grim does not change immediately after
heat stress, a slight increase is observed in 2–3 hours,
and a decrease – at 5–6 hours time point. Contrary to
transgenic strain HS dNOS1 Flag, which generates
increased concentrations of nitric oxide after heat stress
induction, the increase in grim gene expression was
observed after heat stress. The expression level of the
gene decreases at 2–3 hours time point after heat stress
with gradual coming to its normal level. In HS dNOS1
transgenic strain there is a significant increase in grim
expression, which decreases slightly at 2–3 hours time
point and remains on the same level at 5–6 hours point.
Recent studies have demonstrated that the expres-
sion of all integrator genes hid/rpr/grim is transcriptio-
nally regulated during organism development. How-
ever, if hid gene is expressed both in living and dying
cells, rpr and grim appear to be specifically expressed
in cells that are doomed to die and their expression pat-
tern is quite similar. At the same time rpr and grim fun-
ction independently of each other, as cell death induced
by grim does not require the expression of rpr. Still,
contrary to other integrator genes, little is known about
the precise regulation of grim expression [13, 19].
Comparison of the results obtained in the analysis
of hsp70 gene expression with the results of grim exp-
ression revealed that in wild type Oregon R strain the
activation of hsp70 gene expression and further protein
synthesis trigger protective mechanisms and suppress
apoptosis. After the level of hsp70 gene expression
reduces, the cell re-enters the apoptotic way.
As the expression of hsp70 in both strains was the
same, the difference in the expression of apoptosis-
197
RELATIONS BETWEEN NITRIC OXIDE SYNTHASE DNOS1, Hsp70 AND APOPTOSIS REGULATORY GENE grim
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b-actin
Oregon R HS dNOS1 Flag HS dNOS1
16
12
8
4
0
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1 2 3 1 2 3 1 2 3
A
B
Fig. 3. Semi-quantitative analysis of Hsp70 protein content in intact
and heat shock (37 °C) treated larvae of wild (Oregon R) and
dNOS1-transgenic strains: A – Western-blot-analysis; B – den-
sitometric scanning of the Western-blot
regulatory gene grim is likely to depend on increased
concentrations of nitric oxide in transgenic strains. As
it was mentioned earlier, the quantity of nitric oxide af-
ter heat stress induction increases drastically, protec-
tive functions of heat shock proteins become uncompe-
titive, and apoptosis program is enabled via apoptosis
regulatory gene grim. However, due to short life span
NO concentration decreases while the expression of
hsp70 gene still continues, which, in turn, results in
apoptosis suppression. Our results demonstrated that in
5–6 hours the response to stress ceased and the process
of programmed cell death re-entered its normal level.
Therefore, taking into consideration the previously
obtained data, it was assumed that nitric oxide can play
a crucial role in cellular signaling and organogenesis. It
is believed that NO regulates cell proliferation in the
imaginal discs of developing larvae and participates in
the development of visual system. One of the mecha-
nisms of regulating visual system development may be
associated with the ability of NO to induce or suppress
apoptosis. Thus, it was demonstrated that exogenous
donors of nitric oxide in LobeRSV-mutants may stimulate
the apoptosis in imaginal discs and therefore contribute
the reduction of ommatidia number in the eyes of flies.
However, many aspects of NO signaling in apoptosis
machinery and its precise role in apoptosis induction
and progression are yet to be revealed [20].
Thus, it is suggested that anti-apoptotic functions
of Hsp70 in D. melanogaster larvae are realized via de-
creased expression of such apoptosis regulatory genes
as, for instance, grim, whereas the increase in nitric
oxide level is present in transgenic strains after heat
stress induction in addition to increased expression of
Hsp70. This increase in NO is likely to suppress anti-
apoptotic function of Hsp70 and to stimulate the
expression of one out of three key apoptosis-inducing
genes – grim.
Despite the fact that a large number of key apo-
ptosis regulatory proteins are well known, the precise
molecular mechanisms of their activities are still far
from elucidation. Detailed study of apoptosis mecha-
nisms on the molecular level will provide better un-
derstanding of the pathology development and there-
fore will facilitate the elaboration of novel therapeu-
tical approaches.
This experimental article is not a complete study,
additional experimental analysis and investigations in
the given direction are to be performed.
Acknowledgements. The authors are thankful to
Dr. Boris Kuzin, Kol’tsov Institute of Developmental
Biology, Moscow, Russian Federation, for providing
the experimental Drosophila strains and Y. Stasiv,
G. Enikolopov, M. Regulski, T. Tully, Cold Spring
Harbor Laboratory, NY, USA, for generating trans-
genic flies.
Å. Ì. Õó ñà¿ íî âà, Ç. À. Áó ëåí òàºâà, Á. Î. Áåê ìà íîâ,
Ë. Á. Äæàí ñó ãó ðî âà, Ð. ². Áåð ñèì áàºâ
Çâ’ÿ çîê ì³æ ñèí òà çîþ îêñè äó àçî òó DNOS1, Hsp70 ³ àïîï òîç-
ðå ãó ëþ þ ÷èì ãå íîì grim ó Drosophila melanogaster
ï³ñëÿ ³íäóêö³¿ òåï ëî âî ãî ñòðå ñó
Ðå çþ ìå
Ìåòà. Äîñë³äèòè âçàºìîç â’ÿ çîê ì³æ ñèí òà çîþ îêñè äó àçî òó,
á³ëêîì òåï ëî âî ãî øîêó (ÁÒØ70) òà àïîï òîç-ðå ãó ëþ þ ÷èì ãå -
íîì grim ó D. melanogaster. Ìå òî äè. ²íäóêö³ÿ òåï ëî âî ãî øîêó
(37 °C, 1 ãîä) ó ëè ÷è íîê 3-ãî â³êó ë³í³¿ Oregon R ³ òðàíñ ãåí íèõ ë³-
í³é ç äî äàò êî âè ìè êîï³ÿìè dNOS1-ãåíà. Äëÿ àíàë³çó åêñïðåñ³¿
ãåí³â dNOS, hsp70 è grim âè êî ðèñ òà íî ìå òî äè ÇÒ-ÏËÐ ³ Âåñ -
òåðí-áëîò. Ðå çóëü òà òè. Ïî êà çà íî, ùî â òðàíñ ãåí íèõ ë³í³ÿõ
äî äàò êîâ³ êîﳿ ãåíà dNOS1 àê òèâ íî åêñïðå ñó þòü ñÿ â³äðàçó ï³-
ñëÿ òåï ëî âî ãî ñòðåñó. Âè ÿâ ëå íî, ùî â óñ³õ âè êî ðèñ òà íèõ ë³í³ÿõ
ð³âåíü åêñïðåñ³¿ hsp70 ³ éîãî á³ëêî âî ãî ïðî äóê òó ï³äâè ùóºòüñÿ
ï³ñëÿ ³íäóêö³¿ ñòðå ñó ³ çíè æóºòüñÿ ÷å ðåç 2–3 ãîä, ó òîé ÷àñ ÿê
ð³âåíü åêñïðåñ³¿ ãåíà grim ó òðàíñ ãåí íèõ ë³í³ÿõ çà òà êèõ æå
óìîâ ï³äâè ùóºòüñÿ, à â êîí òðîë³ çìåí øóºòüñÿ ïðè çáå ðå æåíí³
âè ñî êî ãî ð³âíÿ åêñïðåñ³¿ hsp70. Âèñ íîâ êè. Çíà÷íèé ð³âåíü
ÁÒØ70 íå ãà òèâ íî âïëè âຠíà åêñïðåñ³þ ãåíà grim, äî äàò êî âèé
ñèí òåç NO íå é òðàë³çóº àí òè à ïîï òè÷ íó ä³þ ÁÒØ70 ³ ï³äâè ùóº
ð³âåíü åêñïðåñ³¿ grim. Òà êèì ÷è íîì, ìè ïå ðå äáà ÷àºìî íà ÿâí³ñòü
êîí êó ðåí òíî ãî âçàºìîçâ’çêó ì³æ àí òè à ïîï òè÷ íîþ ôóíêö³ºþ
ÁÒØ70 ³ ïðî-àïîï òè÷ íîþ 䳺þ îêñè äó àçîòó.
Êëþ ÷îâ³ ñëî âà: àïîï òîç, Drosophila, á³ëêè òåï ëî âî ãî øîêó,
îêñèä àçî òó, grim.
198
KHUSSAINOVA E. M. ET AL.
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grim
b-actin
Oregon R HS dNOS1 Flag HS dNOS1
Fig. 4. grim mRNAs expression in intact and heat shock (37 °C)
treated larvae of wild (Oregon R) and dNOS1-transgenic strains
(RT-PCR)
Ý. Ì. Õó ñà è íî âà, Ç. À. Áó ëåí òà å âà, Á. Î. Áåê ìà íîâ,
Ë. Á. Äæàí ñó ãó ðî âà, Ð. È. Áåð ñèì áà åâ
Ñâÿçü ìåæ äó ñèí òà çîé îêñè äà àçî òà DNOS1, Hsp70 è
àïîï òîç-ðå ãó ëè ðó þ ùèì ãå íîì grim ó Drosophila melanogaster
ïî ñëå èí äóê öèè òåï ëî âî ãî ñòðåñ ñà
Ðå çþ ìå
Öåëü. Èññëå äî âàòü âçà è ìîñ âÿçü ìåæ äó ñèí òà çîé îêñè äà àçî -
òà, áåë êîì òåï ëî âî ãî øîêà (ÁÒØ70) è àïîï òîç-ðå ãó ëè ðó þ ùèì
ãå íîì grim ó D. melanogaster. Ìå òî äû. Èíäóê öèÿ òåï ëî âî ãî
øîêà (37 °C, 1 ÷) ó ëè ÷è íîê 3-ãî âîç ðàñ òà ëè íèè Oregon R è
òðàíñ ãåí íûõ ëè íèé ñ äî ïîë íè òåëü íû ìè êî ïè ÿ ìè dNOS1 ãåíà.
Äëÿ àíà ëè çà ýêñ ïðåñ ñèè ãå íîâ dNOS, hsp70 è grim èñ ïîëü çî âà ëè
ìåòîäû ÎÒ-ÏÖÐ è Âåñ òåðí-áëîò. Ðå çóëü òà òû. Ïî êà çà íî,
÷òî â òðàíñ ãåí íûõ ëè íè ÿõ äî ïîë íè òåëü íûå êî ïèè ãåíà dNOS1
àê òèâ íî ýêñ ïðåñ ñè ðó þò ñÿ ñðà çó ïî ñëå òåï ëî âî ãî ñòðåñ ñà. Âû -
ÿâ ëå íî, ÷òî âî âñåõ èñ ïîëü çó å ìûõ ëè íè ÿõ óðî âåíü ýêñ ïðåñ ñèè
hsp70 è åãî áåë êî âî ãî ïðî äóê òà ïî âû øà åò ñÿ ïî ñëå èíäóêöèè
ñòðåñ ñà è ñíè æà åò ñÿ ÷å ðåç 2–3 ÷, â òî âðå ìÿ êàê óðî âåíü ýêñ -
ïðåñ ñèè grim â òðàíñ ãåí íûõ ëè íè ÿõ â ýòèõ æå óñëî âè ÿõ âîç ðàñ -
òà åò, à â êîí òðî ëå óìåíü øà åò ñÿ ïðè ñî õðà íå íèè âû ñî êî ãî
óðîâ íÿ ýêñ ïðåñ ñèè hsp70. Âû âî äû. Çíà ÷è òåëü íûé óðî âåíü
ÁÒØ70 íå ãà òèâ íî âëè ÿ åò íà ýêñ ïðåñ ñèþ ãåíà grim, äî ïîë íè -
òåëü íûé ñèí òåç NO íå é òðà ëè çó åò àí òè à ïîï òè ÷åñ êîå äåéñò-
âèå ÁÒØ70 è óâå ëè ÷è âà åò óðî âåíü ýêñ ïðåñ ñèè grim. Òà êèì îá -
ðà çîì, ìû ïðåä ïî ëà ãà åì íà ëè ÷èå êîí êó ðåí òíîé âçà è ìîñ âÿ çè
ìåæ äó àí òè à ïîï òè ÷åñ êîé ôóíê öè åé ÁÒØ70 è ïðî-àïîï òè ÷åñ -
êèì äå éñòâè åì îêñè äà àçî òà.
Êëþ ÷å âûå ñëî âà: àïîï òîç, Drosophila, áåë êè òåï ëî âî ãî øî-
êà, îêñèä àçî òà, grim.
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RELATIONS BETWEEN NITRIC OXIDE SYNTHASE DNOS1, Hsp70 AND APOPTOSIS REGULATORY GENE grim
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