Synthesis and properties of myelopeptides possessing differentiating activity

Article abstract of DOI:10.1134/S1068162010040035

Bone marrow peptides Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro (MP-4) and Val-Asp-Pro-Pro (MP-6) have been synthesized y the classical and solid-phase methods of peptide chemistry, and their differentiatingactivity has been studied on leukemia cell ines HL-60 and K-562. It has been shown that both peptides induce the terminal differentiation of leukemic blasts; however, their echanisms of action are different. Pleiades Publishing, Ltd., 2010.

Full text of DOI:10.1134/S1068162010040035

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 4, pp. 456–460. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © L.A. Fonina, E.V. Kudryavtseva, Zh.D. Bespalova, M.A. Efremov, A.A. Mikhailova, E. A. Kirilina, 2010, published in Bioorganicheskaya Khimiya, 2010,
Vol. 36, No. 4, pp. 493–497.
Synthesis and Properties of Myelopeptides Possessing
Differentiating Activity
L. A. Foninaa, E. V. Kudryavtsevab, Zh. D. Bespalovab, M. A. Efremova,
1
A. A. Mikhailovaa, and E. A. Kirilinaa,
a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. MiklukhoꢀMaklaya 16/10, Moscow, 117997 Russia
b Institute of Experimental Cardiology, Russian Cardiological Research and Production Association, Russian Agency of Public
Health and Social Development, Tret’ya Cherepkovskaya ul. 15a, Moscow, 121552 Russia
Received October 10, 2009; in final form, December 25, 2009
Abstract—Bone marrow peptides PheꢀArgꢀProꢀArgꢀIleꢀMetꢀThrꢀPro (MPꢀ4) and ValꢀAspꢀProꢀPro (MPꢀ6) have
been synthesized by the classical and solidꢀphase methods of peptide chemistry, and their differentiating
activity has been studied on leukemia cell lines HLꢀ60 and Kꢀ562. It has been shown that both peptides
induce the terminal differentiation of leukemic blasts; however, their mechanisms of action are different.
Key words: myelopeptides MPꢀ4 and MPꢀ6, synthesis, differentiating activity
DOI: 10.1134/S1068162010040035
1
INTRODUCTION
We have found in preliminary experiments that the
tetrapeptide ValꢀAspꢀProꢀPro (MPꢀ6) also exhibits
the differentiating effect, inducing the differentiation
of leukemia cell lines HLꢀ60 and Kꢀ562.
The goal of this work was the synthesis and comꢀ
parative study of the differentiating activity of MPꢀ4
and MPꢀ6.
Immunoregulatory peptides of bone marrow
(myelopeptides) are involved in a complex chain of
immunoregulatory processes occurring in living
2
organisms. They exhibit a wide spectrum of biological
activities (immunocorrecting, macrophageꢀstimulatꢀ
ing, and differentiating) in in vitro and in vivo experiꢀ
ments [1]. At present, it may be considered as estabꢀ
lished that MP are immunoregulatory peptides whose
action is aimed at maintaining the normal state of the
immune status of an organism.
RESULTS AND DISCUSSION
The peptide MPꢀ4 was synthesized using a combiꢀ
nation of solidꢀphase and classical methods of syntheꢀ
It has been shown at earlier steps of the studies of sis. Because the
Cꢀterminal amino acid in MPꢀ4 is
ꢀterminal tripeptide MetꢀThrꢀPro was
the MP that a mixture of MP isolated from bone marꢀ proline, the
C
row contains factors capable of inducing the terminal synthesized by the classical methods of peptide chemꢀ
differentiation of myeloid cells. The addition of a mixꢀ istry in a solution to prevent the formation of dikeꢀ
ture of MP to cells of leukemic lines led to a decrease topiperazine during the elongation of the peptide
in the amount of actively proliferating blasts and the chain. The resulting tripeptide was coupled to a polyꢀ
appearance of more mature cells [2]. After the isolaꢀ meric carrier (chloromethylated copolymer of styrene
tion of individual MP, their identification, and analyꢀ with 1% divinylbenzene; content of chlorine of
sis, it was established that one of these factors is the 1 mmol/g) by the method of cesium salts. The further
octapeptide
(MPꢀ4), which induces the differentiation of leukemic solidꢀphase method using the standard Boc methodolꢀ
cell lines HLꢀ60 (myeloblastic leukemia) and Kꢀ562 ogy.
ꢀAmino groups were blocked by the Boc group,
PheꢀArgꢀProꢀArgꢀIleꢀMetꢀThrꢀPro elongation of the peptide chain was performed by the
α
(erythroblastic leukemia) [3, 4]. It was found that the OH group of threonine was blocked by the Bzl
MPꢀ4 increases the expression of differentiating antiꢀ group, and the guanidino function of arginine was
gens CD14 and CD38 on the surface of HLꢀ60 cells blocked by the Mtr group. After the termination of the
and of antigen CD44 on the surface of Kꢀ562 cells [5]. synthesis, MPꢀ4 was detached from the carrier as
described in [6].
1
Corresponding author; phone: (495) 330ꢀ72ꢀ56; eꢀmail: kiriliꢀ
The MPꢀ6 peptide was synthesized by the classical
methods of peptide chemistry in the solution. The tarꢀ
get peptides MPꢀ4 and MPꢀ6 were isolated and puriꢀ
fied by reversedꢀphase chromatography and characꢀ
naelen@yandex.ru.
2
Abbreviations: Bzl, benzyl; BSA, bis(
mide; Mtr, 4ꢀmethoxyꢀ2,3,6ꢀtrimethylsulfonyl; PMA, phorbol
12ꢀmyristate 13ꢀacetate; MP, myelopeptides.
O,Nꢀtrimethylsilyl)acetaꢀ
456

SYNTHESIS AND PROPERTIES OF MYELOPEPTIDES POSSESSING
457
Table 1. Changes in the proliferationa of HLꢀ60 and Kꢀ562
terized by the data of HPLC, mass spectrometry, and
sequencing. The content of MPꢀ4 and MPꢀ6 was more
than 97%, as indicated by the data of analytical
HPLC.
The differentiating properties of MPꢀ4 and MPꢀ6
were determined from the changes in cell metabolism
typical for the differentiation process, namely, a
decrease in the proliferation of HLꢀ60 and Kꢀ562
cells by the action of MPꢀ4
Proliferation of cells, % of control
Agent added
(concentration)
HLꢀ60
Kꢀ562
– (control
)
100
100
PMA (10 ng/ml
)
53 5.4**
cells. A characteristic feature of these cell lines is that DMSO (0.5%)
67 8.4**
82 14.5*
79 14.8*
42 17.2**
76 19.2*
101 7.5
MPꢀ4 (1
×
×
×
×
×
10–4 M)
10–5 M)
10–6 M)
10–7 M)
10–8 M)
104 11.3
102 8.1
they are capable of spontaneously proliferating; in this
process, only single differentiated (mature) forms
appear in the population of these proliferating cells.
The differentiation of these cells occurs only by the
action of the differentiation factors, which ultimately
results in the appearance of mature cells accompanied
by a decrease in the proliferation level and, hence, in
the rate of DNA synthesis. The known differentiating
substances—PMA, which induces the differentiation
of the cell line HLꢀ60 in the monocytic line, and
DMSO, which has a differentiating effect on the
erythroblastic cell line Kꢀ562—were used as a control.
(1
(1
(1
68 22.2**
77 15.8**
91 11.2
(1
a
3
Note: Incorporation of [ H]thymidine into DNA; * p < 0.05;
** p < 0.01.
Table 2. Changes in the proliferationa of HLꢀ60 and Kꢀ562
cells by the action of MPꢀ6
As is evident from Table 1, MPꢀ4 at concentrations
of
1
×
10–6 and
1
×
10–7 M induced the most proꢀ
Proliferation of cells (% of control)
Agent added
nounced decrease in the DNA synthesis in HLꢀ60
cells, the effect of the peptide being comparable with
the effect of PMA. In Kꢀ562 cells, the effect of MPꢀ4
made itself evident over a broader concentration
(concentration)
HLꢀ60
Kꢀ562
– (control
)
100
100
PMA (10 ng/ml)
43 17.4**
range, both at high (
1
×
10–4 and
1
×
10–5 M) and low
10–6–1
×
10–7 M); in this case, the
DMSO (0.5%)
77 18.9*
82 14.6*
71 11.1*
79 14.7*
73 7.3**
83 14.1*
concentrations (
1
×
MPꢀ6 (1
×
×
×
×
×
10–4 M)
10–5 M)
10–6 M)
10–7 M)
10–8 M)
75 19.1*
76 19.4*
68 4.8**
69 13.2**
111 19.0
effect of MPꢀ4 was comparable in magnitude with the
effect of DMSO.
Thus, the synthetic analogue of the endogenous
immunoregulatory peptide MPꢀ4 induces changes in
cell metabolism that are typical of the differentiation
process, which was shown on two leukemic cell lines
HLꢀ60 and Kꢀ562.
(1
(1
(1
(1
a
3
Note: Incorporation of [ H]thymidine into DNA; * p < 0.05;
** p < 0.01.
The results of the experiments on the effect of
MPꢀ6 on the metabolism of HLꢀ60 and Kꢀ562 cells
are presented in Table 2. As is seen from the table,
the peptide produces the most pronounced effect
10–6 and
10–4
1
×
produces the effect in a broader concentration range
than MPꢀ4.
on HLꢀ60 cells at concentrations of
10–7 M. However, at higher concentrations,
and
10–5 М , the peptide also decreases the
1
×
1
×
1
×
The occurrence of the differentiating activity of
both peptides was also shown by an increase in the
expression of markers CD14 and CD38 (markers of
mature monocytes and macrophages) on HLꢀ60 cells
[5, 7]. The cytometric analysis of HLꢀ60 cells indiꢀ
cated that MPꢀ4 at a maximum effective concentraꢀ
DNA synthesis. Thus, MPꢀ6 affects the metabolism of
HLꢀ60 cells in a broader concentration range than
MPꢀ4.
As for the effect of MPꢀ6 on Kꢀ562 cells, the most
pronounced changes in metabolism occur at the pepꢀ
tide concentrations of
1
×
10–5–1 10–7 M (Table 2),
×
tion of
1
10–6 M increases the expression of the CD14
×
i.e., at the same concentrations as in the case of HLꢀ60
cells. It should be noted that MPꢀ4 at a concentration
and CD38 antigens by eight and seven times as comꢀ
pared with the level of the spontaneous differentiation
of these cells, and MPꢀ6 at the same dose (
1.5 and 1.4 times, respectively. A similar situation was
observed in the case of Kꢀ562 cells. MPꢀ4 (
10–6 M)
of
1
×
10–6 M induces a maximum decrease in DNA
1
10–6 M),
×
synthesis in Kꢀ562 cells (58% of the control).
Based on these data, it can be concluded that both
peptides are capable of affecting the metabolism of the
cells of the two leukemia lines, HLꢀ60 and Kꢀ562, and
their differentiating effects are comparable in magniꢀ
1
×
increased the expression of the marker of mature cells
CD44 by 2.75 times, and MPꢀ6 at the same dose
tude. However, it should be emphasized that MPꢀ6 increased it by 1.9 times [5, 7, 8]. Thus, the effect of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

458
FONINA et al.
MPꢀ4 on the expression of the markers of mature cells
is several times more pronounced than that of MPꢀ6.
Amino acid sequences of peptides were determined
on a gas sequencer (Applied Biosystem 477A, United
States) connected with an automatic analyzer of
amino acid phenylthiohydantoins.
Molecular masses were determined by mass specꢀ
trometry on a Thermo Bioanalysis Vision 2000 device
(England) and were 1017 for MPꢀ4 and 428 for MPꢀ6.
The peptides tested differ by the primary structure;
however, their final effects on leukemic cells are simiꢀ
lar: both peptides induce the terminal differentiation
of leukemic blasts. Despite the similar terminal
effects, the mechanisms of action of the peptides are
different. It has been shown previously that MPꢀ4
labeled at the
Nꢀterminus by fluorescein retains the
Synthesis of ValꢀAspꢀProꢀPro (MPꢀ6)
biological activity inherent in the original peptide; it is
displaced by the unlabeled peptide but is not extruded
by MPꢀ6. In addition, it has been found that labeled
MPꢀ4 is capable of penetrating into the cell and conꢀ
centrating around the nucleus, which presumably govꢀ
erns its differentiating action [7].
BocꢀValꢀAsp(OBzl)ꢀOH. A solution of BocꢀValꢀ
ONSu (2.59 g, 8.24 mmol) in dioxane (15.0 ml) was
added to a solution of ꢀbenzyl ether of aspartic acid
β
(1.84 g, 8.24 mmol) and sodium bicarbonate (1.39 g)
in water (15.0 ml). The reaction mixture was stirred for
12 h at 20
was dissolved in ethyl acetate (70.0 ml) and washed
with a 2% solution of sulfuric acid ( 50 ml) and
°C and evaporated in a vacuum. The residue
The results of the experiments showed that
both peptides are differentiation factors for HLꢀ60
and Kꢀ562 cells. They decrease proliferation and
increase the number of mature cells carrying the difꢀ
ferentiation markers CD14, CD38, and CD44 [5, 7,
8]. Mature, functionally active cells appear under the
action of these two peptides in the populations of leuꢀ
kemia lines.
2
×
water to a neutral reaction. The ethyl acetate solution
was dried over sodium sulfate and evaporated to dryꢀ
ness. The residue was ground with diethyl ether. The
sediment was filtered off, washed with ether, and airꢀ
dried. Yield: 2.44 (70%); mp 130–132°C. Rf 0.31 (C),
0.75 (D).
These peptides may be promising in the developꢀ
ment of drugs based on them that correct hemopoietic
disorders without producing any unfavorable effect on
normal cells.
BocꢀValꢀAsp(OBzl)ꢀProꢀOH.
NꢀHydroxysuccinꢀ
imide (0.34 g, 2.97 mmol) was added to a solution of
BocꢀValꢀAsp(OBzl)ꢀOH (1.0 g, 2.38 mmol) in a mixꢀ
ture of dioxane–methylene chloride (2 : 1), the mixꢀ
ture was cooled to 0°C, and N,Nꢀdicyclohexylcarboꢀ
diimide (0.69 g) was added. The reaction mixture was
stirred for 1 h at 0°C and for 12 h at 20°C. The sediꢀ
ment was separated, and the solvent was removed in a
vacuum. The oily residue was ground with hexane, the
supernatant was decanted, and crystals were used as a
carboxyl component.
EXPERIMENTAL
Commercial amino acids, their derivatives (Reaꢀ
nal, Hungary; Fluka, Switzerland), or derivatives
obtained by standard methods were used. The individꢀ
uality of the compounds obtained at the intermediate
stages of synthesis was checked by TLC on plates of
silica gel (Merck, Germany) using systems of solvents:
chloroform–methanol–32% CH3COOH 60 : 45 : 20
(A); chloroform–methanol–32% CH3COOH 5 : 3 : 1
(B); chloroform–methanol 9.5 : 0.5 (C); chloroform–
methanol 8 : 2 (D); and chloroform–methanol 9 : 1
(E). The substances were detected by ninhydrin.
A solution of the carboxyl component in methylene
chloride (3 ml) was added to a solution of proline
(0.3 g, 3 mmol) in a mixture with BSA (1.4 ml) and
methylene chloride (2.0 ml) at 20°C. The reaction
mixture was stirred for 12 h at 20°C, and the solvent
was removed in a vacuum. The residue was dissolved in
a 5% sodium bicarbonate solution (70.0 ml) and
washed with diethyl ether (
layer was acidified by solid citric acid to pH 3 and
extracted by ethyl acetate ( 20 ml). The organic
2
×
20 ml). The aqueous
Analytical HPLC was performed using an LCꢀ
10ADvp chromatographic system (Shimadzu, Japan)
5
×
on a column of Ultrasphere C18 (4.6 250 mm) using
×
layer was washed with water to the neutral reaction and
dried over sodium sulfate. The solvent was removed in
a vacuum, and the residue was ground with diethyl
ether. The resulting crystals were filtered off, washed
with ether, and airꢀdried. Yield: 0.90 g (70.0%); mp
a gradient of an acetonitrile concentration in 0.1%
TFA (0–80%, 32 min). The elution rate was 1.6
ml/min, and detection was at 214 and 280 nm.
Preparative HPLC of the final peptides was carried
out on a column (50
×
250 mm) of Diasorb C 16ꢀT
74–76°
ValꢀAspꢀProꢀPro (MPꢀ6). The intermediate comꢀ
pound BocꢀValꢀAsp(OBzl)ꢀProꢀProꢀOH was
C, Rf 0.16 (E), 0.58 (D).
using a gradient of a concentration of buffer B in buffer
A. Buffer A: 0.1% TFA; buffer B: 80% acetonitrile in
buffer A. The elution rate was 50 ml/min, and detecꢀ
tion was at 226 nm.
obtained similar to BocꢀValꢀAsp(OBzl)ꢀProꢀOH
starting from the latter compound (0.90 g, 1.73 mmol)
Amino acid analysis was carried out on a Biotronic and proline (0.2 g, 1.73 mmol). The resulting BocꢀValꢀ
5001 automatic analyzer (Germany). Peptides were Asp(OBzl)ꢀProꢀProꢀOH was dissolved in ethanol
hydrolyzed for 27 h at 110
2% thioglycolic acid.
°
C by 6 M HCl containing (50 ml) and hydrated over 5% Pd/C until the starting
compound disappeared (control by TLC). The cataꢀ
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SYNTHESIS AND PROPERTIES OF MYELOPEPTIDES POSSESSING
459
lyzer was filtered off, and the filtrate was evaporated. mixture was evaporated to dryness, and the rest of the
The residue was treated with TFA (50.0 ml) for 1 h at moisture was removed with benzene and evaporated
20 C. The reaction mixture was evaporated, oil was over phosphorus pentoxide to a constant weight in a
°
ground with ether, and the sediment was filtered off vacuum exsiccator. The cesium salt of the tripeptide
and dried over NaOH in a vacuum. The resulting trifꢀ was dissolved in DMF (20 ml), 5 g of the Merrifield
luoroacetate ValꢀAspꢀProꢀPro was dissolved in water polymer (chloromethylated copolymer of styrene with
and purified by preparative HPLC using a gradient of 1% divinylbenzene; chlorine content of 1 mmol/g)
the concentration of buffer C in buffer A (see above) was added, and the mixture was stirred for 12 h at
from 0 to 40% for 80 min. The fractions corresponding 50 C. The peptidyl polymer was filtered off; washed
°
to the major peak were combined, acetonitrile was with DMF, a DMF–H2O mixture, methanol, and
evaporated, and the residue was diluted with water and dichloromethane; and dried in a vacuum exsiccator to
lyophilized. Yield: 0.5 g (67%). Amino acid analysis: a constant weight. Seven grams of a tetrapeptidyl polyꢀ
Asp 0.98 (1), Val 0.93 (1), Pro 1.85 (2). Mass specꢀ mer [BocꢀMetꢀThr(Bzl)ꢀProꢀpolymer] with the
trum: calculated, 425; found, 428. The retention time degree of substitution of 0.46 mmol/g were obtained.
of MPꢀ6 was 6.13 0.6 min. Chromatography was carꢀ The degree of substitution was determined by the
ried out on an Ultrasphere ODS C18 column (4.6
250 mm, 5 m) using a gradient of an acetonitrile conꢀ
centration of 0–100% in 0.1% TFA for 32 min. The
elution rate was 1.5 ml/min. The wavelength was
214 nm.
×
picrin method [9] and by the gain in weight.
μ
PheꢀArgꢀProꢀArgꢀIleꢀMetꢀThrꢀPro (MPꢀ4) The
coupling of the next amino acids was carried out on an
Applied Biosystems model 431 A automatic peptide
synthesizer starting from the BocꢀMetꢀThr(Bzl)ꢀProꢀ
polymer (0.1 mmol, 0.217 g). The Bocꢀderivatives of
amino acids were coupled according to the standard
program of the company for single condensation; all
necessary intermediate washings of the peptidyl polyꢀ
mer were conducted. After the termination of syntheꢀ
sis, the peptidyl polymer was washed with dichloꢀ
romethane and dried in a vacuum. The resulting
octapeptidyl polymer (0.35 g) was suspended in a mixꢀ
ture of thioanisole (0.5 ml), ethane dithiol (0.25 ml),
dimethyl sulfide (0.25 ml), and TFA (5 ml) and stirred
Synthesis of PheꢀArgꢀProꢀArgꢀIleꢀMetꢀThrꢀPro (MPꢀ4)
CF3COOH HꢀThr(Bzl)ꢀProꢀOH. Triton B (7.4 ml)
⋅
was added to proline (1.5 g, 0.013 mol), and the soluꢀ
tion was evaporated in a vacuum of a waterꢀjet pump.
The residue was dissolved in DMF (100 ml), Bocꢀ
Thr(Bzl)ꢀOPcp (6.6 g, 0.012 mol) was added, and the
mixture was stirred for 12 h at 20 C. The solvent was
°
removed from the reaction mixture by evaporation,
and the residue was dissolved in an aqueous 5%
NaHCO3 solution (150 ml) and washed with ethyl aceꢀ
for 10 min at 20 C. Then, trifluoromethanesulfo acid
°
(0.5 ml) was added, and the mixture was stirred for 3 h.
The polymer was filtered off, and the substance was
precipitated by diethyl ether, filtered off, and reprecipꢀ
itated from TFA. The crude product was purified by
HPLC on a Diasorb C 16ꢀT column using a gradient
of a concentration of buffer C in buffer A (0–50%) in
150 min. Yield: 0.07 g (43% as calculated for Bocꢀ
MetꢀThr(Bzl)ꢀProꢀpolymer).
tate (
20% sulfuric acid to pH 3, the main substance was
extracted by ethyl acetate ( 100 ml), the organic
1
×
100 ml). The aqueous phase was acidified by
2
×
layer was washed with water to a neutral reaction, and
ethyl acetate was evaporated. The oily product was disꢀ
solved in TFA (100 ml) and kept for 1 h at 20 C. TFA
°
was evaporated, and dry diethyl ether was added. The
precipitate was filtered off, washed with ether, and
dried over NaOH in a vacuum exsiccator. Yield: 3.85 g
Amino acid analysis: Phe 1.01 (1), Arg 2.16 (2), Pro
1.92 (2), Ile 1.0 (1), Thr 0.81 (1), Met 0.92 (1). Mass
spectrum: calculated 1017.22, found 1017. The retenꢀ
(78%); Rf 0.65 (A) and 0.32 (B).
BocꢀMetꢀThr(Bzl)ꢀProꢀOH. Diisopropylethylꢀ tion time of MPꢀ4 was 11.61 0.5 min. Chromatogꢀ
amine (3.14 ml, 0.018 mol) and BocꢀMetꢀONp (3.4 g, raphy was performed on an Ultrasphere ODS C18 colꢀ
0.009 mol) were added to a solution of CF3COOH
⋅
umn (4.6
HꢀThr(Bzl)ꢀProꢀOH (3.85 g, 0.009 mol) in DMF nitrile concentration 0–100% in 0.1% TFA in 32 min.
(50 ml), and the mixture was stirred for 12 h at 20 C. The elution rate was 1.5 ml/min. The wavelength was
The reaction mixture was evaporated, and the residue 214 nm.
was chromatographed on a column (15 230 mm)
filled with a silica gel suspension (Serva 60, 90–
130 m) in chloroform. The elution was with chloroꢀ
×
250 mm, 5
μ
m) using a gradient of acetoꢀ
°
×
Cell cultivation. HLꢀ60 and Kꢀ562 cells were cultiꢀ
vated in the standard RPMIꢀ1640 medium (ICN,
United States) containing 7% fetal calf serum
(GIBCO, England), a 20ꢀmM HEPES buffer (Flow
μ
form and then stepwise with mixtures of chloroform–
methanol in ratios of 9 : 1; 9 : 2; and 9 : 3. Fractions
containing the target compound were evaporated.
Yield: 4.5 g (91%); Rf 0.83 (A) and 0.52 (B).
Laboratories, England), 2 mM
Laboratories, England), and gentamycin (50
(Bryntsalov Verein, Russia). The cells were grown in
C in a
s2CO3 (1.195 g, 0.0037 mol) in water (6 ml) was humid atmosphere and an atmosphere of 5% CO2.
L
ꢀglutamine (Flow
μ
g/ml)
BocꢀMetꢀThr(Bzl)ꢀProꢀpolymer. A solution of polystyrene flasks in a CO2 incubator at 37
°
C
added to a solution of BocꢀMetꢀThr(Bzl)ꢀProꢀOH Every 48 hours, the cells were examined under a
(4.0 g, 0.0073 mol) in ethyl ether (24 ml). The reaction microscope and, depending on the amount of cells
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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460
FONINA et al.
grown, either the complete incubation medium a package of functions for the statistical processing of
(RPMIꢀ1640) was added or the cells were plated in the Excel program.
new flasks and incubated further until cells accumuꢀ
lated in the amounts required for the experiment. The
number of viable cells after the incubation with pepꢀ
ACKNOWLEDGMENTS
This work was supported by the program of basic
research of the Presidium of the Russian Academy of
Sciences “Molecular and Cellular Biology.”
tides was monitored in each series of experiments.
Incorporation of radioactive label [3H]thymidine.
HLꢀ60 and Kꢀ562 cells were plated in 96ꢀwell plates,
50 000 cells/well (in 150
After 24 h, differentiation factors MPꢀ4, MPꢀ6, PMA,
or DMSO at different concentrations in 50 l of
μl of complete RPMIꢀ1640).
REFERENCES
μ
1. Petrov, R.V., Mikhailova, A.A., Fonina, L.A., and
medium were added to the wells (except for control
wells). An equivalent amount of complete medium
was added to the control wells. For each dose of the
peptide and the control, six replicates (six wells with
cells) were made; at the end of incubation, prior to the
introduction of [3H]thymidine, one of the wells was
stained with trypan blue, and the number of living and
dead cells was counted. The percent of dead cells was,
on average, 5–7% from experiment to experiment (in
all, six experiments were conducted). [3H]Thymidine
Stepanenko, R.N., Mielopeptidy (Myelopeptides),
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μ
⎯
28 C). Before measuring the incorporation of
°
[3H]thymidine, the plates were defrosted, and the
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a scintillation liquid.
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Statistical processing of data was performed using
the STATGRAPH package of computer programs and
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2010