Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers
Aim. Earlier, we have shown that antibody (AT) preparations obtained by precipitation with 50 % ammonium sulfate from milk of some healthy mothers possess pro-proliferative activity toward transformed and tumor cells in vitro (Kit et al., 2008). We hypothesized that this effect is associated with th...
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Starykovych, M.O. Stoika, R.S. Kit, Yu.Ya. 2019-06-11T19:18:42Z 2019-06-11T19:18:42Z 2013 Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers / M.O. Starykovych, R.S. Stoika, Yu.Ya. Kit // Вiopolymers and Cell. — 2013. — Т. 29, №. 2. — С. 117-123. — Бібліогр.: 33 назв. — англ, укр. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00080E https://nasplib.isofts.kiev.ua/handle/123456789/152484 577.155.2 Aim. Earlier, we have shown that antibody (AT) preparations obtained by precipitation with 50 % ammonium sulfate from milk of some healthy mothers possess pro-proliferative activity toward transformed and tumor cells in vitro (Kit et al., 2008). We hypothesized that this effect is associated with the presence of the anti-histone H1 sIgAs in AT preparations. Methods. To check this hypothesis, we obtained electrophore- tically homogeneous anti-histone H1 sIgAs from milk of healthy mothers by sequential chromatography on protein A-Agarose, protein G- Sepharose and histone H1-Sepharose respectively. These Ab were tested on a proliferative activity toward human T-leukemia Jurkat and human melanoma SK-MEL cells. Results. It was found that anti-histone H1 sIgAs are able to stimulate proliferation of both tumor cell lines. Mitotic effect of these AB was confirmed with an increase of signal proteins involved in cell proliferation (c-Myc, MAP-and cdc2-protein kinases), detected by Western-blot analysis. We also studied the antigenic reactivity of anti-histone H1 sIgAs toward SK-MEL cell proteins. It was observed that these AB possessed an affinity for a number of melanoma cell proteins with molecular masses of 60, 55, 48 and 38 kDa. Conclusions. It has been found that anti-histone H1 sIgA antibodies can stimulate proliferation of human T-leukemia Jurkat and human melanoma SK-MEL cells in vitro. The cross reactivity of these AB could serve as an explanation of their mitotic activity toward the target cells. Мета. Раніше нами показано, що препарати антитіл (АТ), отримані з молока деяких клінічно здорових породіль осадженням 50 %-розчином сульфату амонію, володіють проліферативною активністю щодо трансформованих і пухлинних клітин in vitro (Кіт Ю. та ін., 2008). Ми припустили, що мітотична активність цих препаратів АТ може бути пов’язана з присутністю у них анти-гістон Н1 sIgA-антитіл. Методи. Для перевірки цієї гіпотези з молока клінічно здорових породіль методом постадійних хроматографій білків на білок А-агарозі, білок G-сефарозі і гістон Н1-сефарозі одержано електрофоретично гомогенні анти-гістон Н1 sIgA і досліджено їхній вплив на життєздатність Т-клітин лінії Jurkat лейкозу людини та клітин лінії SK-MEL меланоми людини. Результати. Встановлено, що анти-гістон Н1 sIgA стимулюють проліферацію клітин лінії Jurkat, а також клітин лінії SK-MEL in vitro. Мітотичний ефект цих АТ підтверджено імуноблотингом за зростанням у клітинах рівня деяких сигнальних білків, залучених до проліферації (c-Myc, MAP- і cdc2-протеїнкінази). Нами також досліджено антигенну реактивність анти-гістон Н1 sIgA щодо білків лізатів клітин SK-MEL. Визначено, що ці АТ виявляють спорідненість до низки білків з молекулярною масою 60, 55, 48 і 38 кДа. Висновки. Показано, що анти-гістон Н1 sIgA-антитіла здатні стимулювати проліферацію Т-клітин ліній лейкозу Jurkat та меланоми SK-MEL людини in vitro. Мітотична дія цих АТ може бути пов’язана з їхньою перехресною імунореактивністю. Цель. Ранее нами показано, что препараты антител (АТ), полученные из молока некоторых клинически здоровых рожениц осаждением 50 %-м раствором сульфата аммония, обладают пролиферативной активностью в отношении трансформированных и опухолевых клеток in vitro (Кот Ю. др., 2008). Мы предположили, что митотическая активность этих препаратов может быть связана с присутствием у них анти-гистон Н1 sIgA-антител. Методы. Для проверки этой гипотезы из молока клинически здоровых рожениц методом постадийной хроматографии белков на белок А-агарозе, белок G-сефарозе и гистон Н1-сефарозе получены электрофоретически гомогенные анти-гистон Н1 sIgA и исследовано их влияние на жизнеспособность Т-клеток лейкоза человека линии Jurkat и клеток меланомы человека линии SK-MEL. Результаты. Установлено, что анти-гистон Н1 sIgA стимулируют пролиферацию клеток линии Jurkat, а также клеток линии SK-MEL in vitro. Митотический эффект этих АТ подтвержден иммуноблоттингом по возрастанию в клетках уровня некоторых сигнальных белков, вовлеченных в пролиферацию (c-Myc, MAP- и cdc2-протеинкиназы). Нами также исследована антигенная реактивность анти-гистон Н1 sIgA к белкам лизатов клеток SK- MEL. Определено, что эти АТ проявляют сродство к ряду белков с молекулярной массой 60, 55, 48 и 38 кДа. Выводы. Показано, что анти-гистон Н1 sIgA-антитела способны стимулировать пролиферацию Т-клеток лейкоза линий Jurkat и меланомы SK-MEL человека in vitro. Митотическая активность этих АТ может быть связан с их перекрестной иммунореактивностью. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers Мітотична активність анти-гістон Н1 sIgA-антитіл молока клінічно здорових породіль Митотическая активность анти-гистон Н1 sIgA-антител молока клинически здоровых рожениц Article published earlier |
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| title |
Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers |
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Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers Starykovych, M.O. Stoika, R.S. Kit, Yu.Ya. Structure and Function of Biopolymers |
| title_short |
Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers |
| title_full |
Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers |
| title_fullStr |
Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers |
| title_full_unstemmed |
Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers |
| title_sort |
mitotic activity of anti-histone h1 siga-antibodies from milk of healthy mothers |
| author |
Starykovych, M.O. Stoika, R.S. Kit, Yu.Ya. |
| author_facet |
Starykovych, M.O. Stoika, R.S. Kit, Yu.Ya. |
| topic |
Structure and Function of Biopolymers |
| topic_facet |
Structure and Function of Biopolymers |
| publishDate |
2013 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Мітотична активність анти-гістон Н1 sIgA-антитіл молока клінічно здорових породіль Митотическая активность анти-гистон Н1 sIgA-антител молока клинически здоровых рожениц |
| description |
Aim. Earlier, we have shown that antibody (AT) preparations obtained by precipitation with 50 % ammonium sulfate from milk of some healthy mothers possess pro-proliferative activity toward transformed and tumor cells in vitro (Kit et al., 2008). We hypothesized that this effect is associated with the presence of the anti-histone H1 sIgAs in AT preparations. Methods. To check this hypothesis, we obtained electrophore- tically homogeneous anti-histone H1 sIgAs from milk of healthy mothers by sequential chromatography on protein A-Agarose, protein G- Sepharose and histone H1-Sepharose respectively. These Ab were tested on a proliferative activity toward human T-leukemia Jurkat and human melanoma SK-MEL cells. Results. It was found that anti-histone H1 sIgAs are able to stimulate proliferation of both tumor cell lines. Mitotic effect of these AB was confirmed with an increase of signal proteins involved in cell proliferation (c-Myc, MAP-and cdc2-protein kinases), detected by Western-blot analysis. We also studied the antigenic reactivity of anti-histone H1 sIgAs toward SK-MEL cell proteins. It was observed that these AB possessed an affinity for a number of melanoma cell proteins with molecular masses of 60, 55, 48 and 38 kDa. Conclusions. It has been found that anti-histone H1 sIgA antibodies can stimulate proliferation of human T-leukemia Jurkat and human melanoma SK-MEL cells in vitro. The cross reactivity of these AB could serve as an explanation of their mitotic activity toward the target cells.
Мета. Раніше нами показано, що препарати антитіл (АТ), отримані з молока деяких клінічно здорових породіль осадженням 50 %-розчином сульфату амонію, володіють проліферативною активністю щодо трансформованих і пухлинних клітин in vitro (Кіт Ю. та ін., 2008). Ми припустили, що мітотична активність цих препаратів АТ може бути пов’язана з присутністю у них анти-гістон Н1 sIgA-антитіл. Методи. Для перевірки цієї гіпотези з молока клінічно здорових породіль методом постадійних хроматографій білків на білок А-агарозі, білок G-сефарозі і гістон Н1-сефарозі одержано електрофоретично гомогенні анти-гістон Н1 sIgA і досліджено їхній вплив на життєздатність Т-клітин лінії Jurkat лейкозу людини та клітин лінії SK-MEL меланоми людини. Результати. Встановлено, що анти-гістон Н1 sIgA стимулюють проліферацію клітин лінії Jurkat, а також клітин лінії SK-MEL in vitro. Мітотичний ефект цих АТ підтверджено імуноблотингом за зростанням у клітинах рівня деяких сигнальних білків, залучених до проліферації (c-Myc, MAP- і cdc2-протеїнкінази). Нами також досліджено антигенну реактивність анти-гістон Н1 sIgA щодо білків лізатів клітин SK-MEL. Визначено, що ці АТ виявляють спорідненість до низки білків з молекулярною масою 60, 55, 48 і 38 кДа. Висновки. Показано, що анти-гістон Н1 sIgA-антитіла здатні стимулювати проліферацію Т-клітин ліній лейкозу Jurkat та меланоми SK-MEL людини in vitro. Мітотична дія цих АТ може бути пов’язана з їхньою перехресною імунореактивністю.
Цель. Ранее нами показано, что препараты антител (АТ), полученные из молока некоторых клинически здоровых рожениц осаждением 50 %-м раствором сульфата аммония, обладают пролиферативной активностью в отношении трансформированных и опухолевых клеток in vitro (Кот Ю. др., 2008). Мы предположили, что митотическая активность этих препаратов может быть связана с присутствием у них анти-гистон Н1 sIgA-антител. Методы. Для проверки этой гипотезы из молока клинически здоровых рожениц методом постадийной хроматографии белков на белок А-агарозе, белок G-сефарозе и гистон Н1-сефарозе получены электрофоретически гомогенные анти-гистон Н1 sIgA и исследовано их влияние на жизнеспособность Т-клеток лейкоза человека линии Jurkat и клеток меланомы человека линии SK-MEL. Результаты. Установлено, что анти-гистон Н1 sIgA стимулируют пролиферацию клеток линии Jurkat, а также клеток линии SK-MEL in vitro. Митотический эффект этих АТ подтвержден иммуноблоттингом по возрастанию в клетках уровня некоторых сигнальных белков, вовлеченных в пролиферацию (c-Myc, MAP- и cdc2-протеинкиназы). Нами также исследована антигенная реактивность анти-гистон Н1 sIgA к белкам лизатов клеток SK- MEL. Определено, что эти АТ проявляют сродство к ряду белков с молекулярной массой 60, 55, 48 и 38 кДа. Выводы. Показано, что анти-гистон Н1 sIgA-антитела способны стимулировать пролиферацию Т-клеток лейкоза линий Jurkat и меланомы SK-MEL человека in vitro. Митотическая активность этих АТ может быть связан с их перекрестной иммунореактивностью.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152484 |
| citation_txt |
Mitotic activity of anti-histone H1 sIgA-antibodies from milk of healthy mothers / M.O. Starykovych, R.S. Stoika, Yu.Ya. Kit // Вiopolymers and Cell. — 2013. — Т. 29, №. 2. — С. 117-123. — Бібліогр.: 33 назв. — англ, укр. |
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| fulltext |
117
STRUCTURE AND FUNCTION OF BIOPOLYMERS
UDC 577.155.2
Mitotic activity of anti-histone H1 sIgA-antibodies from
milk of healthy mothers
M. O. Starykovych, R. S. Stoika, Yu. Ya. Kit
Institute of Cell Biology, NAS of Ukraine
14/16, Drahomanov Str., Lviv, Ukraine, 79005
kit@сellbiol.lviv.ua
Aim. Earlier, we have shown that antibody (AT) preparations obtained by precipitation with 50 % ammonium
sulfate from milk of some healthy mothers possess pro-proliferative activity toward transformed and tumor cells
in vitro (Kit et al., 2008). We hypothesized that this effect is associated with the presence of the anti-histone H1
sIgAs in AT preparations. Methods. To check this hypothesis, we obtained electrophoretically homogeneous
anti-histone H1 sIgAs from milk of healthy mothers by sequential chromatography on protein A-Аgarose,
protein G-Sepharose and histone H1-Sepharose respectively. These Ab were tested on a proliferative activity
toward human T-leukemia Jurkat and human melanoma SK-MEL cells. Results. It was found that anti-histone
H1 sIgAs are able to stimulate proliferation of both tumor cell lines. Mitotic effect of these AB was confirmed
with an increase of signal proteins involved in cell proliferation (c-Myc, MAP-and cdc2-protein kinases),
detected by Western-blot analysis. We also studied the antigenic reactivity of anti-histone H1 sIgAs toward
SK-MEL cell proteins. It was observed that these AB possessed an affinity for a number of melanoma cell
proteins with molecular masses of 60, 55, 48 and 38 kDa. Conclusions. It has been found that anti-histone H1
sIgA antibodies can stimulate proliferation of human T-leukemia Jurkat and human melanoma SK-MEL cells in
vitro. The cross reactivity of these AB could serve as an explanation of their mitotic activity toward the target
cells.
Keywords: human milk, autoantibodies, anti-histone H1 sIgA, tumor cell, proliferation.
Introduction. Immune system of mammals ensures the
protection of the organism from harmful environmental
agents and is involved into the regulation of biological
functions, defining its homeostasis [1]. An important
role in maintaining homeostasis play antibodies (AT),
directed to foreign and self-antigens (auto-Ab) [1, 2].
Auto-ATs were found in the organisms of patients with
autoimmune and oncological diseases as well as in cli-
nically healthy people [3]. In healthy people, auto-ATs
are mainly presented by polyspecific low-affinity IgM
or high-affinity IgGs (anti-idiotypic AT), involved into
the regulation of immune response [3, 4]. Humans with
autoimmune diseases were revealed to have high-affi-
nity IgG auto-AT, which may participate in the deve-
lopment of autoimmune processes [5]. The determina-
tion of the level of these auto-ATs in human blood se-
rum is a new approach, widely used in the diagnostics
of various autoimmune diseases as well as in predicting
the clinical course in patients [6].
Auto-ATs of various specificity were also found in
human secretory fluids. A significant level of secretory
immunoglobulin A (sIgA) with affinity to human acti-
ne, myosin, tubulin and spectrin was detected in saliva
and colostrum of clinically healthy women [7]. These
immunoglobulins were called polyspecific sIgA-anti-
bodies (poly-sIgA).
Poly-sIgAs with broad antigenic specificity are be-
lieved to be produced by B1-lymphocytes and to ensure
ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 2. P. 117–123 (Translated from Ukrainian) doi: 10.7124/bc.00080E
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013
118
STARYKOVYCH M. O., STOIKA R. S., KIT Yu. Ya.
the protection of mother and child from pathogenic
microflora [8].
Another type of auto-AT, revealed in human
secretions, is low-affinity anti-alpha-galactosyl AT
(anti-Gal AT). These AB interact with carbohydrate
residues Gal-α-1,3-Gal-β-1,4-GalcNAc-R (alpha-gala-
ctosyl epitopes) [9, 10]. They are synthesized in about 1
% B-lymphocytes in response to the effect of antigens
of gastric bacteria. Anti-Gal ATs were found in human
milk, saliva, vaginal smears and bile. They are capable
of causing agglutination of rabbit erythrocytes
(REA-AT) are binding bovine thyroglobulins, contain-
ing alpha-galactosyl epitopes [11].
Contrary to the secretory fluids where the sIgA le-
vel is high (3–5 mg/ml), the concentration of sIgA in
blood serum of clinically healthy people is low
(0.01–0.02 mg/ml). It was shown that the sIgA level in
human blood serum essentially increases due to some
chronic diseases of liver and gastrointestinal system,
autoimmune, oncological and a number of infectious
diseases of viral and bacterial origin. The functional
activity of secretory immunoglobulins in blood plasma
is poorly studied; although there are the data about their
influence on immune cells [11–19].
Our previous studies have demonstrated that the
preparations of antibodies, isolated from colostrum and
milk of healthy mothers have different effect on
T-leukemia Jurkat cells. Depending on donors, they are
capable of either induce apoptosis of Jurkat T-cells or
stimulate proliferation of these cells in vitro [20]. These
data demonstrate that secretory antibodies may also
influence the growth and viability of human tumor
cells. Our further studies showed that [the] cytotoxic
activity of secretory AT preparations towards tumor
cells could be linked with anti-DNA sIgA-antibodies
[21], though the mitogenic effect of secretory anti-
bodies remained unstudied.
Previously we have found that blood serum of
patients with multiple myeloma contains IgG-anti-
bodies with affinity to histon H1 (anti-histon H1 sIgA-
antibodies), capable of stimulating in vitro proliferation
of T-leukemia CEM-T4 cells [22].
We assumed that the milk of some mothers might
contain anti-histon H1 sIgA-antibodies possessing
mitogenic activity similar to the anti-histon H1
IgG-antibodies from blood serum of patients with
multiple myeloma.
The aim of this work is purification of electro-
phoretically homogeneous preparations of anti-histon
H1 sIgA-antibodies from human milk and study of their
effect on tumor cells in vitro.
Materials and Methods. Mother's milk was pro-
vided by the Lviv Regional Perinatal Center according
to the cooperation agreement.
The isolation of antibodies with affinity to histon
H1 (anti-histon H1 AT) from human milk. Anti-H1
sIgA was purified from milk of clinically healthy
mothers by sequential chromatography according to the
scheme, described in [23]. As the first step, the milk
was centrifuged at 5,000 rpm with subsequent purifica-
tion of the fraction of total AT (IgG and sIgA) on
chromatography column with protein A-agarose ("Sig-
ma", USA) according to [26]. Further IgG and sIgA-
antibodies were separated on chromatography column
with protein G-Sepharose. The fraction of sIgA (1–3
mg of protein) that did not bind the sorbent, was dia-
lyzed against 20 mM tris-HCl-buffer, pH 7.5, contain-
ing 140 mM NaCl (tris-saline buffer, TSB), for 18 h,
and applied to column with histon H1-Sepharose (1
ml), previously balanced by the same buffer. The co-
lumn was washed with TBS and anti-histon H1 AT was
eluted with 0.1 M glycine-HCl, pH 2.6. The eluate was
neutralized with 1.5 M tris-HCl, pH 7.5, and dialyzed
against TBS for 18 h. Protein concentration in the pre-
parations of anti-histon H1 AT was measured at Nano-
Drop ND-1000 spectrophotometer (NanoDrop Techno-
logies, USA) at 280 nm wavelength.
The effect of anti-histon H1 AT preparations of hu-
man milk on tumor cells in vitro. Human T-leukemia
Jurkat cells, and SK-MEL human melanoma cells were
used to study the effect of sIgA preparations. Cell lines
were obtained from the collection of R. E. Kavetsky In-
stitute of Experimental Pathology, Oncology and Ra-
diology, NAS of Ukraine. The cells were cultivated in
Carrel flasks in RPMI-1640 and DMEM mediums
("Sigma Chem. Co.", USA) with addition of 10 % bo-
vine embryonic blood serum ("Sigma Chem. Co."), 50
µg/ml gentamicin ("Sigma Chem. Co.") until sub con-
fluent state of the cells. Jurkat cells in the concentration
of 1.5·106 cells/ml were seeded into 96-well cultural
119
MITOTIC ACTIVITY OF ANTI-HISTONE H1 sIgA-ANTIBODIES FROM MILK OF HEALTHY MOTHERS
plastic vial, and SK-MEL cells – into 24-well vials at
starting concentration of 2.7·104 cells/ml.
After 2 h incubation to the suspension of cells was
added the preparation of anti-H1 sIgA AT (final con-
centration of 0.04 mg/ml) and incubated for 24, 48 and
72 h. Dead cells were stained by 0.1 % trypan blue
solution. The number of unstained living and stained
dead cells was calculated in the hemocytometry cham-
ber under light microscope Biolam R (LOMO, Russian
Federation).
Western-blotting of cell lysates. To the preliminary
washed in phosphate-buffer saline (PBS) cells was
added lysing buffer (1 % triton X-100, 20 mM tris-HCl,
pH 7.4 in the presence of the mixture of protease
inhibitors (CompleteTM, "Roche", France)) in ratio 50 µl
of lysing buffer per 1 million cells. Cells were perfor-
med for 30 min on ice and cell lysates were centrifuged
for 15 min at 12,000 g. The supernatant was dissolved
in 4-fold Laemmli buffer and heated at 900C for 5 min.
Samples were kept at – 20°C until used. Proteins were
separated by denaturing electrophoresis in 12 % poly-
acrylamide gel (PAAG) at 0.1 % SDS according to La-
emmli [24]. Proteins from PAAG were transferred to
nitrocellulose membrane ("Hybond", USA) with sub-
sequent processing of obtained blots using specific
antibodies. Membranes were blocked at room tempe-
rature for 1 h by 5 % solution of skimmed milk powder
in PBS, containing 0.05 % twin-20. Then the membra-
ne was incubated in the presence of monospecific anti-
c-Myc rabbit antibodies, phospho-p44/42 MAP Kinase
(Thr 202/Tyr204), cdc2 (Tyr15) ("Cell Signaling",
USA) and β-actine ("Sigma") for 12 h at 4 °C.
Immunoreactive proteins on the membrane were re-
vealed using conjugates of horseradish peroxidase and
goat antibodies (Sigma), specific to rabbit IgG. For esti-
mation of proteins amount on the membrane the β-acti-
ne-specific rabbit antibodies were used.
To determine the cross-reactivity of anti-histon H1
sIgA we used Western-blotting analysis with two alter-
native methods of detection. In one case the cell lysates
were separated by SDS-electrophoresis in 12 % PAAG
with following electroblotting proteins on nitrocellu-
lose membrane. The membranes were blocked by 3 %
albumin solution in PBS with 0.05 % twin-20 for 18 h at
40 °C, and then incubated with previously biotinylated
anti-histon H1 sIgA-antibodies (60 µg/ml) in the
blocking buffer. Immunoreactive proteins were detec-
ted after treatment of membranes with avidin-horsera-
dish peroxidase solution (1:10,000). In the second case,
the membranes after blocking were incubated with na-
tive anti-histon H1 sIgA-antibodies (60 µg/ml) dissol-
ved in buffer for blocking. Immunoreactive proteins
were detected after treatment of membranes by rabbit
IgG antibodies, conjugated with horseradish peroxi-
dase, mono specific towards heavy chains of human
IgA ("Sigma-Aldrich", USA) in 1:6,000 dilution.
Statistical processing of investigation results. All
experiments were repeated 3–5 times. Average values
and standard deviations (M ± m) are presented in the
work. Statistical analysis was performed by Student's
criterion (t). The data at p ≤ 0.05 were considered re-
liable. The charts and statistical processing of the data
were performed using Origin 4.0 and Excel 97 soft-
ware.
Results and Discussion. It was previously found
that sIgA-antibodies possess the affinity to protein
A-agarose, but contrary to IgG-antibodies, they are not
capable of binding to protein G-Sepharose [25]. Ac-
cording to these data, anti-histon H1 sIgA was isolated
from milk by sequential affinity chromatography,
including: obtaining total fraction of milk AT by
chromatography on the column with protein A-agarose
(Fig. 1, path 1), purification of sIgA antibodies from
IgG by chromatography on the column with protein
G-Sepharose (Fig. 1, path 2); isolation of anti-histon H1
sIgA-antibodies by chromatography on the column
with histon H1-Sepharose. Electrophoretic analysis of
these purified proteins demonstrated their similarity to
polypeptides corresponding to the secretory component
(SC), heavy (H) and light (L) chains of sIgA [26].
The purified preparations of anti-histon H1 sIgA-
antibodies were studied for their ability to affect the
growth and survival of cells in vitro. Target cells were
Jurkat T-cells (human leukemia) and SK-MEL (human
melanoma). As seen from the data, presented in Fig. 2,
a, growth-stimulating effect of anti-histon H1 sIgA on
Jurkat line cells was observed at the 24th and 48th h with
maximal growth (1.4-fold) at the 72 h of incubation.
The similar effect was also observed for the antibody-
treated SK-MEL melanoma cells (Fig. 2, b). Here the
maximum of cell growth was detected at the 48th h of in-
cubation.
120
STARYKOVYCH M. O., STOIKA R. S., KIT Yu. Ya.
These data demonstrate that anti-histon H1 sIgA-
antibodies, isolated from milk of clinically healthy
mothers, are capable of stimulating proliferation of
both types of tumor cells. To prove this assumption we
studied the level of some signaling proteins, directly
involved in the regulation of cell proliferation. For this
we used Western-blotting of proteins of Jurkat and
SK-MEL cells, treated and untreated by anti-histon H1
sIgA-antibodies (Fig. 3). It was established that the
level of MAP-kinase (p42/p44 Erk 1/2) (42, 44 kDa) in
the cells, incubated with AT, was essentially increased
comparing with the control cells. Additionally, in
treated by anti-histon H1 sIgA-antibodies cells was
revealed increased level of the transcriptional factor
c-Myc (57 kDa) which is an intracellular molecular
target for MAP-kinase, participates in the regulation of
cellular growth and may initiate cell proliferation
[27–29].
We also detected considerable increase of the phos-
phorylated form of cyclin-dependent kinase p-cdc2
(Tyr15) (34 kDa) in the Jurkat and SK-MEL cells in the
presence of anti-histone H1 sIgA. These data suggest
that the AT treated cells are in phase G1/S of the mitotic
cycle [30].
Thus, we determined that the anti-histon H1 sIgA-
antibodies, isolated from milk of healthy mothers, are
capable of stimulating proliferation of both human
T-leukemia Jurkat cells and human melanoma SK-
MEL cells in vitro (Fig. 3, a, b).
Mechanism involved in the stimulation of cell pro-
liferation by the anti-histon H1 sIgA-antibodies re-
mains unknown. We assume that pro-proliferative ef-
fect of these secretory antibodies could be linked with
their cross reactivity to positively charged proteins. We
revealed also the antibodies with similar antigenic spe-
cificity and pro-proliferative activity in the blood serum
of patients with multiple myeloma and systemic lupus
erythematosus [22–31]. This property might provide
the interaction of anti-histon H1 sIgA-antibodies with
varied protein antigens of target cells.
To check this assumption, the SK-MEL cells were
lysed in hypotonic buffer in the presence of 1 % triton
X-100, then antigen specificity of the anti-histone H1
sIgA-antibodies towards lysate proteins was deter-
mined by Western-blotting using two independent
ways. In one case, immune reactive proteins were
detected using the previously biotinylated anti-histon
H1 sIgA with their subsequent identification on the
membrane by conjugate avidin-horseradish peroxidase
(Fig. 4, path 1). In the second case, for detection of the
immune reactive proteins on the membrane we used
← H
← L
← SC
11 →
17 →
26 →
32 →
43 →
55 →
72 →
95 →
130 →
170 →
M 1 2 3kD
Fig.1. Electrophoresis in 12% PAAG in the presence of sodium
dodecylsulfate of sIgA preparations, purified from human milk by
consequential chromatography on columns with protein A-agarose
(1), protein G-agarose (2) and histon H1-sepharose (3) M – markers of
molecular mass of proteins. Arrows on the right hand indicate the
position of sIgA polypeptides on the gel (SC – secretory component; H
– heavy and L – light chains)
b
a
0
0,5
1
1,5
2
2,5
3
3,5
0 24 48 72 hour
0
20
40
60
80
100
120
0 24 48 72 hour
1
2
1
2
Fig.2. The effect of anti-histon H1 sIgA-antibodies on the growth of
human T-leukemia Jurkat (a) and human melanoma SK-MEL cells (b)
121
MITOTIC ACTIVITY OF ANTI-HISTONE H1 sIgA-ANTIBODIES FROM MILK OF HEALTHY MOTHERS
rabbit antibodies, monospecific to alpha-chains of
human IgA (Fig. 4, path 2). The data obtained
demonstrated that the anti-histon H1 sIgA-antibodies
possess an affinity to different proteins of melanoma
cell preferentially binding with p60, p55, p48 and p38
kDa. Although the nature of these proteins is yet to be
determined, one may suggest that these protein antigens
include receptors of the plasmatic membrane, involved
in the induction of cell proliferation (for instance,
receptors of polypeptide growth factors [32]).
Since it is well-known that colostrum and milk of
mothers contain a broad spectrum of biologically active
compounds, possessing growth-stimulating as well as
growth-inhibiting activity towards different types of
cells [20, 21, 25], the data obtained do not allow us to
claim that pro-proliferative activity is a function of only
antibodies. The steroid hormones and growth factors,
previously found in human milk, could also be engaged
in this effect [33].
At present, the effect of anti-histon H1 sIgA-
antibodies toward normal human blood lymphoid cells
needs to be clarified. Our previous data demonstrate
that these antibodies do not have considerable effect on
the human blood lymphocytes in vitro. Meanwhile they
are capable of stimulating the differentiation of isolated
monocytes into macrophages in the presence of phorbol
myristate acetate (unpublished results).
Conclusions. It was established that the sIgA-anti-
bodies from healthy mothers' milk, isolated by chro-
matography on the column with histon H1-Sepharose
(anti-histon H1-sIgA), are capable of stimulating the
proliferation of human T-leukemia Jurkat and human
melanoma SK-MEL cells in vitro. This effect may be a
result of cross reactivity of these antibodies to
membrane protein receptors of target cells.
a bJurkat
SK-MEL
c-myc
p-p44/42
MAP
p-cdc 2
β-actine
Control
Control
К
sIgA
sIgA
c-myc
c-myc p-p44/42
MAP
p-cdc 2 β-actine
p-p44/42
MAP
p-cdc 2 β-actine
c-myc
p-p44/42
MAP
p-cdc 2
β-actine
К
100
75
25
50
0
100
75
25
50
0
Fig.3. The level of some proteins,
involved in the regulation of cell
proliferation in Jurkat and SK-
MEL cells after their incubation
with the anti-histon H1 sIgA-
antibodies: a – immunoblotting
of proteins of cell lysates; b –
comparative content of regula-
tory proteins in cells
← 38
← 48
← 55
← 60
17 →
24 →
34 →
43 →
55 →
72 →
95 →
130 →
170 →
kD 1 2 k D
Fig.4. The affinity of anti-histon H1 sIgA-antibodies towards proteins
of melanoma SK-MEL cells: 1 – membranes were treated with pre-
viously biotinylated anti-histon H1 sIgA-antibodies, avidin-horse-
radish peroxidase, were used for detection of antigens; 2 – membranes
were treated with anti-histon H1 sIgA-antibodies, immune complexes
were determined using rabbit IgG-antibodies, monospecific to heavy
chains of human IgA, conjugated with horseradish peroxidase.
122
STARYKOVYCH M. O., STOIKA R. S., KIT Yu. Ya.
The authors express their sincere gratitude to the Head
doctor of Lviv prenatal center, L. B. Yaniv for provided
samples of mothers' milk.
М. О. Ста ри ко вич, Р. С. Стой ка, Ю. Я. Кіт
Міто тич на ак тивність анти-гістон Н1 sIgA-ан титіл мо ло ка
клінічно здо ро вих по роділь
Інсти тут біології клітини НАН Украї ни
Вул. Дра го ма но ва, 14/16, Львів, Украї на, 79005
Summary
Мета. Раніше нами по ка за но, що пре па ра ти ан титіл (АТ), от ри -
мані з мо ло ка де я ких клінічно здо ро вих по роділь осад жен ням 50
%-роз чи ном суль фа ту амонію, во лодіють проліфе ра тив ною ак -
тивністю щодо транс фор мо ва них і пух лин них клітин in vitro
(Кіт Ю. та ін., 2008). Ми при пус ти ли, що міто тич на ак тивність
цих пре па ратів АТ може бути по в’я за на з при сутністю у них
анти-гістон Н1 sIgA-ан титіл. Ме то ди. Для пе ревірки цієї гіпо -
те зи з мо ло ка клінічно здо ро вих по роділь ме то дом по стадійних
хро ма- тог рафій білків на білок А-ага розі, білок G-се фа розі і
гістон Н1-се фа розі одер жа но елек тро фо ре тич но го мо генні
анти-гістон Н1 sIgA і дослідже но їхній вплив на життєздатність
Т-клітин лінії Jurkat лей ко зу лю ди ни та клітин лінії SK-MEL ме ла -
но ми лю ди ни. Ре зуль та ти. Вста нов ле но, що анти-гістон Н1 sIgA
сти му лю ють проліфе рацію клітин лінії Jurkat, а та кож клітин
лінії SK-MEL in vitro. Міто тич ний ефект цих АТ підтвер дже но
іму ноб ло тин гом за зрос тан ням у кліти нах рівня де я ких сиг наль -
них білків, за лу че них до проліфе рації (c-Myc, MAP- і cdc2-про -
теїнкінази). Нами та кож дослідже но ан ти ген ну ре ак тив- ність
анти-гістон Н1 sIgA щодо білків лізатів клітин SK-MEL. Виз на -
че но, що ці АТ ви яв ля ють спорідне- ність до низ ки білків з мо ле ку -
ляр ною ма сою 60, 55, 48 і 38 кДа. Вис нов ки. По ка за но, що
анти-гістон Н1 sIgA-ан титіла здатні сти му лю ва ти проліфе -
рацію Т-клітин ліній лей ко зу Jurkat та ме ла но ми SK-MEL лю ди ни
in vitro. Міто тич на дія цих АТ може бути по в’я за на з їхньою пе -
ре хрес ною іму но ре ак тивністю.
Клю чові сло ва: мо ло ко по роділь, ав то ан титіла, анти-гістон
Н1 sIgA, пух линні клітини, проліфе рація.
М. А. Ста ри ко вич, Р. С. Стой ка, Ю. Я. Кит
Ми то ти чес кая ак тив ность анти-гис тон Н1 sIgA-ан ти тел мо ло ка
кли ни чес ки здо ро вых ро же ниц
Ре зю ме
Цель. Ра нее нами по ка за но, что пре па ра ты ан ти тел (АТ), полу-
чен ные из мо ло ка не ко то рых кли ни чес ки здо ро вых ро же ниц осаж-
де ни ем 50 %-м рас тво ром суль фа та ам мо ния, об ла да ют про ли -
фе ра тив ной ак тив нос тью в от но ше нии транс фор ми ро ван ных и
опу хо ле вых кле ток in vitro (Кот Ю. др., 2008). Мы пред по ло жи ли,
что ми то ти чес кая ак тив ность этих пре па ра тов мо жет быть
свя за на с при су тстви ем у них анти-гис тон Н1 sIgA-ан ти тел.
Ме то ды. Для про вер ки этой ги по те зы из мо ло ка кли ни чес ки здо -
ро вых ро же ниц ме то дом по ста дий ной хро ма тог ра фии бел ков на
бе лок А-ага ро зе, бе лок G-се фа ро зе и гис тон Н1-се фа ро зе по лу че -
ны элек тро фо ре ти чес ки го мо ген ные анти-гис тон Н1 sIgA и ис -
сле до ва но их вли я ние на жиз нес по соб ность Т-кле ток лей ко за че-
ло ве ка ли нии Jurkat и кле ток ме ла но мы че ло ве ка ли нии SK-MEL.
Резуль та ты. Уста нов ле но, что анти-гис тон Н1 sIgA сти му ли ру-
ют про ли фе ра цию кле ток ли нии Jurkat, а так же кле ток ли нии
SK-MEL in vitro. Ми то ти чес кий эф фект этих АТ под твер жден
им му ноб лот тин гом по воз рас та нию в клет ках уров ня не ко то рых
сиг наль ных бел ков, вов ле чен ных в про ли фе ра цию (c-Myc, MAP- и
cdc2-про те ин ки на зы). Нами так же ис сле до ва на ан ти ген ная ре-
ак тив ность анти-гис тон Н1 sIgA к бел кам ли за тов кле ток SK-
MEL. Опре де ле но, что эти АТ про яв ля ют сро дство к ряду бел ков
с мо ле ку ляр ной мас сой 60, 55, 48 и 38 кДа. Вы во ды. По ка за но,
что анти-гис тон Н1 sIgA-ан ти те ла спо соб ны сти му ли ро вать
про ли фе ра цию Т-кле ток лей ко за ли ний Jurkat и ме ла но мы SK-MEL
че ло ве ка in vitro. Ми то ти чес кая ак тив ность этих АТ мо жет быть
свя зан с их пе ре крес тной им му но ре ак тив нос тью.
Клю че вые сло ва: мо ло ко ро же ниц, ау то ан ти те ла, анти-гис -
тон Н1 sIgA, опу хо ле вые клет ки, про ли фе ра ция.
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