24-Epibrassinolide at subnanomolar concentrations modulates growth and production characteristics of a mouse hybridoma

Article abstract of DOI:10.1135/cccc20032190

Brassinosteroids are known to stimulate plant growth and to possess antistress activities in plants. This work was aimed at exploring possible beneficial effects of 24-epibrassinolide on cultured mammalian cells. A mouse hybridoma was cultured either in standard serum-free medium, or in medium diluted to 30%, in which the cells underwent nutritional stress. Steady-state parameters of semicontinuous cultures conducted at 24-epibrassinolide concentrations from 10^-16 to 10^-9 mol l^-1 were evaluated. Typical effects of the agent found both in standard and in diluted media were (i) increase in the value of mitochondrial membrane potential, (ii), drop of intracellular antibody level, (iii) increase in the fraction of the cells in the G₀/G¹ phase, and (iv) decrease in the fraction of the cells in the S phase. Alleviation of nutritional stress manifested itself in cultures conducted in diluted media. Viable cell density was significantly higher (relative to control) at 24-epibrassinolide concentrations 10^-13 and 10^-12 mol l^-1. The results of this exploratory study show that the plant hormone 24-epibrassinolide may induce perturbations in the cell division mechanism, in mitochondria performance, and in secreted protein synthesis in a mammalian cell line. At the lowest brassinosteroid concentrations, the number of steroid molecules in the culture was of the same order of magnitude as the number of viable cells in the culture. This implies involvement of a complex cascade mechanism, through which the steroid molecule induces alterations in gene expression leading finally to significant changes in cell culture parameters.

Full text of DOI:10.1135/cccc20032190

2
190
Franěk, Eckschlager, Kohout:
2
4-EPIBRASSINOLIDE AT SUBNANOMOLAR CONCENTRATIONS
MODULATES GROWTH AND PRODUCTION CHARACTERISTICS
OF A MOUSE HYBRIDOMA+
a,
b
c
František FRANĚK *, Tomáš ECKSCHLAGER and Ladislav KOHOUT
a
Laboratory of Growth Regulators, Institute of Experimental Botany,
Academy of Sciences of the Czech Republic, Radiová 1, 102 27 Prague 10, Czech Republic;
e-mail: franek@biomed.cas.cz
Department of Pediatric Oncology, 2nd Faculty of Medicine, Charles University, V Úvalu 84,
b
c
1
50 06 Prague 5, Czech Republic; e-mail: tomas.eckschlager@lfmotol.cuni.cz
Department of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6,
Czech Republic; e-mail: kohout@uochb.cas.cz
Received Jun e 26, 2003
Accepted Septem ber 30, 2003
Brassin osteroids are kn own to stim ulate plan t growth an d to possess an tistress activities in
plan ts. Th is work was aim ed at explorin g possible ben eficial effects of 24-epibrassin olide on
cultured m am m alian cells. A m ouse h ybridom a was cultured eith er in stan dard serum -free
m edium , or in m edium diluted to 30%, in wh ich th e cells un derwen t n utrition al stress.
Steady-state param eters of sem icon tin uous cultures con ducted at 24-epibrassin olide con cen -
–
16
–9
–1
tration s from 10
to 10 m ol l were evaluated. Typical effects of th e agen t foun d both in
stan dard an d in diluted m edia were (i) in crease in th e value of m itoch on drial m em bran e po-
ten tial, (ii), drop of in tracellular an tibody level, (iii) in crease in th e fraction of th e cells in
th e G /G ph ase, an d (iv) decrease in th e fraction of th e cells in th e S ph ase. Alleviation of
0
1
n utrition al stress m an ifested itself in cultures con ducted in diluted m edia. Viable cell den -
–
13
sity was sign ifican tly h igh er (relative to con trol) at 24-epibrassin olide con cen tration s 10
an d 10
–
12
–1
m ol l . Th e results of th is exploratory study sh ow th at th e plan t h orm on e
2
4-epibrassin olide m ay in duce perturbation s in th e cell division m ech an ism , in m itoch on -
dria perform an ce, an d in secreted protein syn th esis in a m am m alian cell lin e. At th e lowest
brassin osteroid con cen tration s, th e n um ber of steroid m olecules in th e culture was of th e
sam e order of m agn itude as th e n um ber of viable cells in th e culture. Th is im plies in volve-
m en t of a com plex cascade m ech an ism , th rough wh ich th e steroid m olecule in duces alter-
ation s in gen e expression leadin g fin ally to sign ifican t ch an ges in cell culture param eters.
Keyw ord s: Steroids; Brassin osteroids; 24-Epibrassin olides; Hybridom a cells; Mitoch on drial
m em bran e poten tial; Cell cycle; Mon oclon al an tibodies; Plan t growth regulators.
+
Part CDXVI in th e Series On Steroids; Part CDXV see ref.1
Collect. Czech. Chem. Commun. (Vol. 68) (2003)
doi:10.1135/cccc20032190

2
4-Epibrassinolide
2191
Brassin osteroids are a group of ph ytoh orm on es, like auxin s, cytokin in s, gib-
berellin s an d abscisic acid. Th ese com poun ds are widespread in n ature, an d
can be foun d in h igh er an d lower plan ts, gym n osperm s, m on ocotyledon s,
dicotyledon s an d algae. Bein g presen t in alm ost every part of th e plan t,
th eir h igh est con cen tration is in pollen (10–5 or less per cen t), an d h igh con -
cen tration in seeds. Th e term brassin olide is derived from Brassica napus L.,
2
from th e pollen of wh ich th ey were isolated for th e first tim e . Brassin olide,
i.e. (22R,23R,24S)-2α,3α,22,23-tetrahydroxy-24-methyl-7-oxa-7a-homo-α-cholestan-
6
-on e, is th e m ain represen tative.
As th e distribution of brassin osteroids in plan t m aterial is very low an d
th eir isolation from th is source affords am oun ts, in sufficien t for practical
use, m an y brassin osteroids with h igh er or lower brassin olide activity were
syn th esized. Som e of th e syn th etically prepared brassin osteroids were later
foun d to occur in n ature. An exam ple of such com poun d, syn th etically
m ore easily available in com parison with brassin olide, is its 24-epim er,
2
4-epibrassin olide, (22R,23R,24R)-2α,3α,22,23-tetrah ydroxy-24-m eth yl-7-
oxa-7a-h om o-α-ch olestan -6-on e (h ereafter epibrassin olide or com poun d 6).
3
4
Its syn th esis was described in 1979, an d isolation from green algae in
987. Due to a difficult availability of brassin olide, m ost studies an d practi-
1
cal application s utilize 24-epibrassin olide, wh ich h as alm ost th e sam e bio-
logical activity as brassin olide. 24-Epibrassin olide was also used in th is
work. Th e term s brassin olide an d epibrassin olide are som etim es in ter-
ch an ged in literature, wh ich can lead to serious m isun derstan din g. Th ere-
5
fore, it is n ecessary to follow exactly th e correct use of th ese n am es .
Th e best kn own ph ysiological activity of brassin osteroids is th eir ability
6
to stim ulate plan t growth . Th ey cause elon gation of all parts of plan ts, of
stem s, sh oots, roots, th e quan tity of buds, quan tity an d weigh t of pods,
th ey also act in cell division , in fluen ce seed germ in ation , etc. Elon gation ,
swellin g an d splittin g of youn g bean s were observed wh en brassin osteroids
7
were applied at th e begin n in g of th e secon d in tern ode growth . Especially
in terestin g are th eir an tistress activities, i.e., again st tem perature stress,
water stress, salt stress an d ch illin g stress, wh ich are n ow very in ten sely
studied. For exam ple, th e protection of cucum ber plan ts again st h eat stress
(
40 °C) after treatm en t with brassin olide was foun d to result in an in crease
8
of germ in ation ability, better growth an d h igh er dry weigh t . A decrease of
th e effect of water stress in m un g bean s treated with brassin olide was
sh own to be associated with a h igh er ability of plan ts to assim ilate water .
9
Th e use of brassin osteroids for th e elim in ation of stress in plan t develop-
1
0
m en t was also reported in paten t literature .
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

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Franěk, Eckschlager, Kohout:
Som e effects of brassin osteroids were observed in vertebrates as well.
Treatm en t of Russian sturgeon Acipenser gueldenstaedti fin gerlin gs with
–
4
–1
1
0
m g l epibrassin olide before exposure sh owed a sign ifican t decrease of
th e n egative in fluen ce of stress factors, such as pollutan ts, oxygen defi-
1
1
–5
cien cy an d h igh salin ity . Treatm en t of several Acipenser species at 10 to
–
9
–1
1
0
m g l with epibrassin olide in creased fecun dity, h atch in g an d larvae
survival. Th e auth ors also reported a sign ifican tly in creased viability of
–
6
–1
sperm atozoa in sperm treated with epibrassin olide at 10 m g l .
Th e aim of th is work was to exam in e th e activity of epibrassin olide in
m am m alian cells tran sform ed to perm an en t growth in vitro. A m odel of
lym ph ocyte h ybridom as growin g in ch em ically defin ed m edia free of ste-
roids an d an y n atural m acrom olecules offered a possibility to study th e ef-
fect of epibrassin olide with out in terferen ce caused by ill-defin ed n atural
m aterials. To our kn owledge, th is is th e first study devoted to explorin g th e
effects of brassin osteroids in a m am m alian cell system .
1
2
HO
O
4
3
H
O
O
OH
OH
OH
OH
HO
HO
HO
HO
5
O
6
H
H
O
O
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2
4-Epibrassinolide
2193
EXPERIMENTAL
2
4-Epibrassin olide Syn th esis
Th e syn th esis of 24-epibrassin olide used in th is work is a m odification of previously de-
scribed syn th eses, resultin g in a h igh er yield of th e substan ce (Sch em e 1). Th e startin g m ate-
rial for th e syn th esis was ergosterol (Fluka, purum ; >97% (HPLC)), i.e. 3-h ydroxy-24-m eth yl-
ch olest-5,7,22-trien e (1). In 3α,5-cyclo-6-oxo-7-en e (2), available from ergosterol via i-steroid
rearran gem en t an d oxidation , th e 7-double bon d was reduced with Na S O in th e presen ce
2
2
4
1
2,13
of a ph ase tran sfer catalyst . Th e obtain ed 3,5-cyclo-6-oxo-24-m eth ylch olest-22-en e (3)
was th en con verted to 6-oxo-2-olefin e (4). Com poun d 3 was isom erized in dim eth ylacet-
am ide with lith ium brom ide an d pyridin ium 4-toluen esulfon ate . Im provem en t of h ydro-
xylation led to th e m ore desired 22R,23R-isom er 5 upon asym m etric dih ydroxylation with
O-(4-ch loroben zyl)h ydroquin idin e (Fluka; >99% (HPLC)). In th is step, we obtain ed 24-epi-
castasteron e [(22R,23R,24R)-2α,3α,22,23-tetrah ydroxy-24-m eth yl-5α-ch olestan -6-on e; (5)].
Th e con version of 24-epicastasteron e 5 in to 24-epibrassin olide 6 was effected by Baeyer–
Villiger oxidation . We im proved th is part of th e syn th esis by excludin g th e steps of pro-
tection an d deprotection of th e h ydroxyl groups in position s 2,3,22,23. Hen ce, we obtain ed
1
4
1
5
1
6
2
4-epibrassin olide [(22R,23R,24R)-2α,3α,22,23-tetrah ydroxy-24-m eth yl-7-oxa-7a-h om o-5α-
ch olestan -6-on e; (6)] directly from 24-epicastasteron e on treatm en t with trifluoroperoxy-
acetic acid. Th e product 6 was obtain ed as fin e wh ite n eedles with m .p. 257–258 °C, with
3
,17
IR, NMR an d an alytical data iden tical with th ose in th e literature
.
Cell Culture
Mouse h ybridom a cell lin e ME-750, producin g an IgG m on oclon al an tibody, was cultured in
DMEM/F12/RPMI 1640 (2:1:1) m edium supplem en ted with am in o acids an d with th e iron -
1
8,19
rich protein -free growth -prom otin g m ixture as described in detail previously
. Cell cul-
ture m edia an d supplem en ts were from Life Tech n ologies. Th e m edia for cell starvation were
prepared by dilutin g th e above defin ed m edia with ph osph ate-buffered salin e supplem en ted
with th e iron -rich growth -prom otin g m ixture. Th e cultures were kept at 37 °C in a h um idi-
fied atm osph ere con tain in g 5% CO2.
Com poun d 6 was dissolved in itially in dim eth yl sulfoxide to 10 m m ol l–1 con cen tration .
Furth er dilution s were prepared in th e culture m edium .
Assays of th e action of 6 were set up in a total volum e of 6.0 m l in 25 cm 2 T-flasks in th e
sem icon tin uous culture m ode. In sem icon tin uous cultures, a stan dard fraction of th e culture
volum e was with drawn in a stepwise m ode on ce a day, an d replaced by m edium con tain in g
1
9
th e respective con cen tration of 6 (ref. ). Viable an d dead cells were coun ted in a h em ocyto-
m eter usin g th e Trypan Blue exclusion test. Th e con cen tration of m on oclon al an tibody (fur-
th er abbreviated MAb) in th e culture supern atan t was determ in ed by im m un oturbidim etry
2
0
in th ree parallels for each sam ple
.
Th e values of viable an d dead cell con cen tration s, as well as th e values of th e m on oclon al
an tibody (MAb) con cen tration were determ in ed daily. Four to five values of cell con cen tra-
tion s an d of MAb con cen tration s, obtain ed on in dividual days of culture, were averaged to
obtain steady-state m ean values.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2
194
Franěk, Eckschlager, Kohout:
Distribution of Cell Cycle Ph ases
6
For th e determ in ation of cell kin etics, th e cells (5–10 × 10 ) were perm eabilized an d stain ed
usin g DNA Prep kit (Beckm an Coulter, Hialeah , U.S.A.) followin g th e producer’s protocol.
After 30-m in in cubation , th e sam ples were m easured with a FACSCalibur flow cytom eter
(
3
Beckton Dickin son , Fran klin Lakes (NJ), U.S.A.), an d th e data were an alysed by ModFit LT
.1 DNA an alysis software (Verity Software House, In c., Topsh am (ME), U.S.A.). Percen tages
of cells in G /G , S an d G /M ph ases were evaluated.
0
1
2
Mitoch on drial Mem bran e Poten tial
Th e flow cytom eter was used to an alyze cells, stain ed for m itoch on drial poten tial usin g
Rh odam in e 123 (Sigm a–Aldrich , Prague). Th e cells were diluted with th e m edium to a con -
5
–1
–1
cen tration of 5–6 × 10 cells m l , stain ed with a fin al con cen tration of 2 µg m l
Rh odam in e 123, an d in cubated at 37 °C for 10 m in . Th e cells were cen trifuged, resuspen ded
in ph osph ate buffered salin e, an d stored on ice in th e dark. Th e stain ed cell were an alysed
with in 15–30 m in at th e excitation wawelen gth 488 n m an d th e em ission wawelen gth
5
30 n m (ref.21). Th e m itoch on drial poten tial was evaluated as m ean fluorescen ce.
In tracellular An tibody
5
In tracellular level of MAb’s was m easured by flow cytom etry. Cells (2–5 × 10 ) were perm e-
abilized usin g FIX&PERM Cell Perm eabilization Kit (Kaum berg, Austria) accordin g to th e
m an ufacturer’s recom m en dation s, an d stain ed by ph ycoeryth rin -labelled Goat An ti-Mouse
IgG polyclon al an tibody F(ab′)2 fragm en t (Beckm an Coulter, Nyon , Switzerlan d). Th e MAb
con ten t in in dividual cells was evaluated as m ean fluorescen ce at 585 n m .
RESULTS
In th e presen t experim en ts, we em ployed th e m eth odical approach of sem i-
2
2
con tin uous culture, based on th e ch em ostat m ode of cell culture . Th is
m ode of cell culture allows to suppress possible tran sien t effects appearin g
upon adm in istration of a sin gle dose of th e tested substan ce, an d to obtain
reliable sets of data for statistical evaluation from a steady-state culture.
Exploratory batch experim en ts carried out with con cen tration s of 6 in
th e ran ge 10–12–10 m ol l sh owed culture param eters sign ifican tly differ-
en t from th ose of th e con trol on ly at th e lowest con cen tration s tested.
Th erefore, in th e fin al sem icon tin uous culture experim en ts, a lower con -
–6
–1
cen tration ran ge was selected, i.e. 10– –10 m ol l .
16
–9
–1
Th e con trol culture was carried out in a stan dard protein -free m edium . In
order to guaran tee a steady excess of n utrien ts, th e dilution rate was ad-
–
1
justed to 0.5 day . Th e resultin g viable cell den sity was approxim ately
6
–1
1
.9 × 10 cells m l , an d th e culture viability was m ore th an 90% (Table I).
As expected, un der th ese con dition s, 6 did n ot in duce an y ch an ge in th e vi-
able cell den sity or viability. Sim ilarly, n o sign ifican t effect on th e MAb
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2
4-Epibrassinolide
2195
con cen tration in th e m edium was foun d. However, even un der th e con di-
tion s of an excess of n utrien ts, sign ifican t ch an ges in th e level of in tra-
cellular MAb, an d in th e distribution of cell cycle ph ases could be observed
even at th e lowest con cen tration of 6 (Table II). At all con cen tration s of 6
tested, th e proportion of th e G /G ph ase was h igh er, an d th e proportion
0
1
of th e S ph ase was lower th an in th e con trol culture. An elevated m ito-
ch on drial m em bran e poten tial, an d a lower value of th e in tracellular MAb
–
15
–1
con cen tration , relative to th e con trol, were foun d at 1 × 10
m ol l an d
h igh er con cen tration s of 6.
Th e task of culturin g cells un der a severe n utrition al stress was fulfilled
by suppressin g th e con cen tration s of all m edia com pon en ts to 30%, an d,
–
1
m oreover, by decreasin g th e dilution rate to 0.25 day . Con sequen tly, th e
6
–1
steady-state viable cell den sity was on ly ca 0.3 × 10 cells m l , an d th e cul-
ture viability between 62 an d 68% (Table III). Th e an ti-stress activity of 6
–
12
–13
–1
m an ifested itself at con cen tration s in th e ran ge 10 –10
m ol l . In th is
con cen tration ran ge, th e steady-state viable cell den sity was h igh er by m ore
th an 15% in com parison with th e con trol experim en t. Th e viability was
TABLE I
Sem icon tin uous cultures in stan dard m edium at th e dilution rate D = 0.5 day–1. Cell culture
param eters
Con cen tration
Viable cell
den sity/total
cell den sity
MAb
in
m edium
Mitoch on drial
m em bran e
poten tial
Viable cell
den sity
MAb
in tracellular
of 6
a
a
–
1
a
a
m ol l
0
1
1
1
1
1
1
1
1
(Con trol)
× 10–16
× 10–15
× 10–14
× 10–13
× 10–12
× 10–11
× 10–10
× 10–9
1.00 ± 0.04
1.00 ± 0.04
0.98 ± 0.01
1.00 ± 0.04
0.99 ± 0.04
0.99 ± 0.04
1.06 ± 0.10
1.01 ± 0.07
1.02 ± 0.05
0.93
0.91
0.90
0.95
0.92
0.96
0.93
0.95
0.92
1.00 ± 0.08 1.00 ± 0.03
0.86 ± 0.07 0.91 ± 0.04
1.00 ± 0.04
1.01 ± 0.01
0.90 ± 0.05 0.66 ± 0.08*** 1.05 ± 0.01*
0.95 ± 0.09 0.80 ± 0.05*** 1.07 ± 0.03*
1.06 ± 0.08 0.71 ± 0.09*** 1.13 ± 0.02**
1.11 ± 0.05 0.79 ± 0.05*** 1.06 ± 0.04*
0.97 ± 0.04 0.69 ± 0.03*** 1.09 ± 0.02**
1.07 ± 0.12 0.84 ± 0.04*** 1.09 ± 0.04**
0.98 ± 0.10 0.76 ± 0.10*** 1.08 ± 0.03**
a
Values relative to th ose in th e con trol culture with out com poun d 6. Mean ± stan dard devi-
ation values are given . Mean viable cell den sity in th e con trol culture was (1890 ± 76)
3
–1
×
1
10 cell m l . Mean MAb con cen tration in th e supern atan t of th e con trol culture was
–
1
6.9 ± 1.4 m g l . Sign ifican tly differen t from con trol: * p < 0.1, ** p < 0.05, *** p < 0.01.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2
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Franěk, Eckschlager, Kohout:
TABLE II
Sem icon tin uous cultures in stan dard m edium at th e dilution rate D = 0.5 day–1. Distribution
of cell cycle ph ases
G /G
S
G /M
2
0
1
Con cen tra-
tion of 6
m ol l
relative
to
relative
to
relative
to
–
1
%
%
%
con trol
con trol
con trol
0
1
1
1
1
1
1
1
1
(Con trol)
× 10–16
× 10–15
× 10–14
× 10–13
× 10–12
× 10–11
× 10–10
× 10–9
49.1 ± 1.0
1.00
1.03
1.04
1.08
1.12
1.16
1.10
1.10
1.10
41.0 ± 2.1
1.00
0.94
0.91
0.82
0.76
0.77
0.84
0.84
0.80
9.9 ± 1.1
1.00
1.11
1.17
1.35
1.40
1.17
1.18
1.16
1.34
50.5 ± 1.1*
38.5 ± 0.6*
11.0 ± 1.2*
11.6 ± 0.9*
13.4 ± 0.7***
13.9 ± 0.7***
11.6 ± 1.5
51.1 ± 0.8**
53.0 ± 1.0***
54.8 ± 1.3***
56.9 ± 2.2***
53.9 ± 0.4***
54.0 ± 0.8***
53.9 ± 0.9***
37.3 ± 0.4***
33.6 ± 0.4***
31.3 ± 0.9***
31.5 ± 1.2***
34.4 ± 0.7***
34.5 ± 1.8***
32.8 ± 1.9***
11.7 ± 0.8*
11.5 ± 1.6
13.3 ± 1.2**
Mean ± stan dard deviation values are given . Sign ifican tly differen t from con trol: * p < 0.1,
* p < 0.05, *** p < 0.01.
*
TABLE III
Sem icon tin uous cultures in 30% m edium at th e dilution rate D = 0.25 day–1. Cell culture pa-
ram eters
Con cen tration
Viable cell
den sity/total
cell den sity
MAb
in
m edium
Mitoch on drial
m em bran e
poten tial
Viable cell
den sity
MAb
in tracellular
of 6
a
a
–
1
a
a
m ol l
0
1
1
1
1
1
1
1
1
(Con trol)
× 10–16
× 10–15
× 10–14
× 10–13
× 10–12
× 10–11
× 10–10
× 10–9
1.00 ± 0.04
1.00 ± 0.04
1.04 ± 0.04
0.98 ± 0.05
1.16 ± 0.10**
1.19 ± 0.10**
1.01 ± 0.04
1.00 ± 0.07
1.04 ± 0.06
0.62 ± 0.06
0.65 ± 0.04
0.63 ± 0.04
0.62 ± 0.05
0.68 ± 0.07
0.67 ± 0.05
0.63 ± 0.04
0.64 ± 0.05
0.65 ± 0.04
1.00 ± 0.03 1.00 ± 0.03
1.00 ± 0.03 0.90 ± 0.05
1.00 ± 0.03
1.01 ± 0.06
0.98 ± 0.05 0.76 ± 0.05*** 1.14 ± 0.07**
0.97 ± 0.06 0.67 ± 0.05*** 1.10 ± 0.06**
0.98 ± 0.09 0.69 ± 0.09*** 1.07 ± 0.04*
0.93 ± 0.07 0.66 ± 0.05*** 1.11 ± 0.04**
0.96 ± 0.09 0.75 ± 0.03*** 1.10 ± 0.06**
0.92 ± 0.08 0.68 ± 0.04*** 1.16 ± 0.05**
0.95 ± 0.09 0.58 ± 0.10*** 1.18 ± 0.08**
a
Values relative to th ose in th e con trol culture with out com poun d 6. Mean ± stan dard
deviation values are given . Mean viable cell den sity in th e con trol culture was (330 ± 16) ×
3
–1
1
1
0 cells m l . Mean MAb con cen tration in th e supern atan t of th e con trol culture was 9.7 ±
–
1
.1 m g l . Sign ifican tly differen t from con trol: * p < 0.1, ** p < 0.05, *** p < 0.01.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2
4-Epibrassinolide
2197
sligh tly h igh er, but th e differen ce was n ot statistically sign ifican t. Th e in -
crease in viable cell den sity was n ot accom pan ied by an in crease in MAb
con cen tration s in th e m edium . Th e effect of 6 on th e level of in tracellular
MAb an d on th e m itoch on drial m em bran e poten tial was an alogous to th at
observed in th e stan dard m edium . Th e cultures in th e diluted m edium were
ch aracterized by a strikin g accum ulation of cells in th e S ph ase (Table IV).
Th e gen eral ch aracter of th e sh ift of proportion s between G /G an d S ph a-
0
1
ses, wh ich occurred even at th e lowest con cen tration of 6, was th e sam e as
in th e stan dard m edium , i.e., in crease of G /G ph ase with a sim ultan eous
0
1
decrease of S ph ase cells.
DISCUSSION
Recen t studies on th e m ech an ism of action of brassin osteroids in plan ts in -
dicate th at m ore th an on e m olecular m ech an ism of brassin osteroid action
exists: biosyn th esis of en zym es an d oth er ph ysiological factors via an effect
on gen om e expression , an d an effect on m em bran es. Th ese m odes of action
6
are obviously in tercon n ected . Brassin osteroids are kn own to act on th e ge-
n om e in th e form of a h orm on e-receptor com plex. Th e action of th ese sub-
stan ces results in gen om e activity regulation s, an d could be respon sible for
TABLE IV
Sem icon tin uous cultures in 30% m edium at th e dilution rate D = 0.25 day–1. Distribution of
cell cycle ph ases
G /G
S
G /M
0
1
2
Con cen tra-
tion of 6
m ol l
–
1
relative to
con trol
relative to
con trol
%
%
%a
0
1
1
1
1
1
1
1
1
(Con trol)
× 10–16
× 10–15
× 10–14
× 10–13
× 10–12
× 10–11
× 10–10
× 10–9
29.6 ± 1.8
29.5 ± 1.2
1.00
1.00
1.15
1.15
1.18
1.11
1.12
1.20
1.14
70.2 ± 2.8
69.4 ± 2.1
65.9 ± 2.1*
65.2 ± 2.9*
64.6 ± 2.5*
66.8 ± 1.5*
66.0 ± 2.2*
64.5 ± 2.8*
65.3 ± 3.4*
1.00
0.99
0.94
0.93
0.92
0.95
0.94
0.92
0.93
0.2 ± 0.2
1.1 ± 0.3
0.1 ± 0.2
0.7 ± 0.5
0.4 ± 0.3
0.2 ± 0.2
0.8 ± 0.5
0.1 ± 0.2
1.0 ± 0.6
34.0 ± 1.3**
34.1 ± 1.2**
35.0 ± 1.5**
33.0 ± 1.4**
33.2 ± 1.5**
35.4 ± 2.1**
33.7 ± 1.6**
a
Due to very low percen tage values an d large stan dard deviation s, addition al statistical eval-
uation was om itted. Mean ± stan dard deviation values are given . Sign ifican tly differen t from
con trol: * p < 0.1, ** p < 0.05.
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2
198
Franěk, Eckschlager, Kohout:
th e effects on tran scription an d DNA replication 23. In addition , brassin o-
steroids are capable of in fluen cin g th e electric properties of m em bran es,
6
such as activation of th e m em bran e-boun d proton pum p .
Th e presen t experim en ts were design ed with th e aim to com pare th e ef-
fect of 6 on a con trol “over-fed” culture with th at on a culture carried out
un der a severe n utrition al stress. Rath er surprisin gly, altered values of in tra-
cellular an tibody, altered values of th e m itoch on drial m em bran e poten tial,
an d altered distribution of cell cycle ph ases were foun d even in th e con trol
culture. Th is fin din g leads to a suggestion th at 6 m ay in fluen ce th e ways of
tran scription an d DNA replication irrespective of th e n utrition al status. A
sign ifican t positive effect of 6 on th e growth of th e cultures un der n utri-
tion al stress m an ifested itself in a zon e of con cen tration s en com passin g
two orders of m agn itude, n am ely 10– an d 10
12
–13
m ol l (see Table III). Th e
–1
lack of growth stim ulation at con cen tration s 10– m l l–1 an d h igh er m ay be
explain ed by a com plex action of 6 on several targets with differen t associa-
tion con stan ts. Th e cultures carried out in th e diluted m edium are ch arac-
terized by an extrem ely h igh proportion of cells in th e S ph ase, an d by a
relatively low viability value. Th ese features obviously reflect th e very slow
an d difficult process of th e syn th esis of cell com pon en ts un der an in suffi-
cien t supply of substrates an d en ergy sources.
11
Th e extrem ely low values of active con cen tration s of 6 in dicate th at th e
m ain m ech an ism of action of th is com poun d is an alteration of gen e ex-
pression , followin g th e gen eral sch em e
6
+ receptor → 6-receptor com plex → n uclear DNA → m RNA → protein
(
e.g. en zym e or factor in cell regulation ).
Th e cell param eter alteration s caused by th e presen ce of 6 are in accord
with th e con cept th at brassin osteroids possess an ti-stress activities. As docu-
m en ted by th e in crease in th e m itoch on drial m em bran e poten tial, 6 is able
to support th e activity of m itoch on dria, an organ elle of crucial im portan ce
in th e en ergetic m etabolism . A m ore efficien t con version of substrates in
en ergy available for cellular processes m ay represen t on e of th e ways th e
cells overcom e n utrition al stress.
Th e suspen sion culture of m am m alian cells in vitro is a system defin ed
on a h igh er level th an th e plan t m odels on wh ich th e growth -stim ulatin g
activity of brassin osteroids was revealed. Th e steady-state establish ed in
sem icon tin uous cultures allows us to precisely an alyse th e quan titative rela-
tion sh ips between th e n um ber of cells an d th e n um ber of m olecules of th e
agen t. Our fin din g of sign ifican t alteration s of several culture param eters
Collect. Czech. Chem. Commun. (Vol. 68) (2003)

2
4-Epibrassinolide
2199
at con cen tration s of 6 of th e order of 10–15 m ol l–1 in dicates th at th e com -
poun d m ay be ph ysiologically active even if th e n um ber of m olecules
of th e agen t in th e system is of th e sam e order of m agn itude as th e n um -
ber of viable cells. Th e un usually low value of th e active con cen tration s,
–
1
≈
1 fm ol l , is con ceivable un der th e assum ption th at m ultiple bin din g sites
are in volved in th e in teraction of 6 with th e correspon din g receptor. Th e
fin al ch an ges in th e wh ole cell population obviously follow a cascade of
even ts startin g with an altered gen e expression in a few cells th at h ave been
h it by th e origin al m olecular sign al.
Supported by the Grant Agency of the Czech Republic (grant No. 203/01/0083 to L. K.), by
the grants of the Ministry of Education, Youth and Sports of the Czech Republic (No. 111300005
(
to T. E.), No. 153100008 (to F. F.) and No. OC 844.10 (to F. F.)), and by the Institute research
project Z4 055 905 (to L. K.).
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