Effects of different vertebrate growth factors on ... - Biology of the Cell

Biol Cell (I 996) 86,67-12
f3 Elsevier, Paris
<strong>Effects</strong> <strong>of</strong> <strong>different</strong> <strong>vertebrate</strong> <strong>growth</strong> <strong>factors</strong>
on primary cultures <strong>of</strong> hemocytes
from the gastropod mollusc, HaZiotk tubercukzta
Jean-Marc Lebel, Wilfrid Giard, Pascal Favrel, Eve Boucaud-Camou
L&orato&e de Biobgie et Biotechnobgies Marines, IBBA, IFREMER URM 14,
Universit6 de Caen, Esplanade de la Paix, 14032 Caen cedex, France
(Received 6 January 1996; accepted 12 March 1996)
67
Original article
Summary - A useful experimental system from primary cultures <strong>of</strong> hemocytes from H&Otis tuberculutu has been established. Six days
after initiation <strong>of</strong> the culture, the viability <strong>of</strong> hemocytes remained constant as measured by the M’IT assay. In addition, hemocytes
showed physiological responses as judged by protein and DNA syntheses in response to treatment with <strong>vertebrate</strong> <strong>growth</strong> <strong>factors</strong>. Por-
cine insulin and human epidermal <strong>growth</strong> factor (EGF) stimulated [jH]-leucine and [3H]-thymidine incorporation in hemocytes in a
dose-dependent manner. No additive effect <strong>of</strong> insulin and EGF is observed either for PHI-leucine or for [3H]-thymidine incorporation.
The response <strong>of</strong> primary cultures <strong>of</strong> abalone hemocytes to <strong>vertebrate</strong> <strong>growth</strong> <strong>factors</strong> confirms their <strong>growth</strong> potential in vitro and provides
a suitable model for further studies on regulation <strong>of</strong> the control <strong>of</strong> cellular processes such as cell <strong>growth</strong>, <strong>different</strong>iation and migration in
in<strong>vertebrate</strong> cells.
moliusc / hemocytes / cell culture / <strong>growth</strong> <strong>factors</strong>
Introduction
In <strong>vertebrate</strong>s as well as in insects, primary culture metho-
dology has opened up new approaches for studying cellular
and molecular events occurring during control <strong>of</strong> cell pro-
liferation, cell <strong>different</strong>iation or cell metabolism. Since the
sixties many attempts have been made to establish long-term
primary cultures <strong>of</strong> marine mollwcan cells [35]. However,
despite numerous investigations testing a wide variety <strong>of</strong>
<strong>growth</strong> media, supplements and cultural conditions, very lit-
tle progress has been made [ 191. A major difficulty concerns
the low rate <strong>of</strong> cell proliferation in vitro, even for tissues
with high mitotic potential such as embryonic or larval tis-
sues [24]. The lack <strong>of</strong> appropriate culture media, especially
the absence <strong>of</strong> <strong>growth</strong> promoting substances, is the main
reason <strong>of</strong> fails to obtain continuous cell multiplication in
vitro [22]. Supplementing the basal medium culture with
homologous hemolymph or with extracts from cerebral gan-
glia or genital glands, perhaps a source <strong>of</strong> mitotic promoting
<strong>factors</strong>, has not significantly improved results [7, 10, 191.
Interestingly, the presence <strong>of</strong> fetal calf serum as well as ver-
tebrate <strong>growth</strong> <strong>factors</strong> in the culture medium increased the
viability <strong>of</strong> a wide variety <strong>of</strong> marine in<strong>vertebrate</strong> cells
including marine molluscan cells [1, 7, 241, suggesting that
<strong>vertebrate</strong> <strong>growth</strong>-promoting <strong>factors</strong> may be efficient for
these cells as is the case for cultured insect cells [ 171.
In in<strong>vertebrate</strong>s, the presence <strong>of</strong> substances homologous
to <strong>vertebrate</strong> <strong>growth</strong>-promoting <strong>factors</strong> has been strongly
suggested, as a result <strong>of</strong> immunocytochemical and biochem-
ical investigations. Some <strong>of</strong> these <strong>growth</strong> <strong>factors</strong> belong to
the family <strong>of</strong> the insulin-like peptides (ILPs) [33]. ILPs,
which have been detected in the digestive system and the
brain <strong>of</strong> <strong>different</strong> rnolluscs, present similarities with mam-
malian insulin at both the structural and functional levels [8].
Furthermore, in the pulmonate mollusc Lymnaea stugnuks,
cDNAs encoding a protein with characteristics <strong>of</strong> <strong>vertebrate</strong>
preproinsulins and called molluscan insulin-related peptides
(MIPS), have been identified in <strong>growth</strong>-controlling neurons
suggesting a possible role <strong>of</strong> MIPS in body <strong>growth</strong> [29, 301.
According to these results, the involvement <strong>of</strong> ILPs in body
and shell <strong>growth</strong> and energy metabolism has been estab-
lished in molluscs [28]. Another interesting <strong>growth</strong>-factor
family has also been found in in<strong>vertebrate</strong>s. This family is
composed <strong>of</strong> members analogous to <strong>vertebrate</strong> polypeptidic
<strong>growth</strong> <strong>factors</strong> such as epidem& <strong>growth</strong> factor (EGF). The
presence <strong>of</strong> EGF-like peptides in in<strong>vertebrate</strong>s was first
documented with the identification <strong>of</strong> specific nucleotide
sequences closely homologous to <strong>vertebrate</strong> EGF and/or
EGF-receptor gene in Drosophikz [l& 27, 371, Caenorhub-
ditis [13], <strong>different</strong> sea urchin species [5, 14, 39, 401 and
more recently My?& [ 151. In agreement with this hypothe-
sis, an EGF-like factor has been found in the tissues <strong>of</strong> the
mussel Mytiks edulis [25]. In addition, Cancre et ul [3],
recently demonstrated the presence <strong>of</strong> an EGF-like sub-
stance in hemocytes <strong>of</strong> the crustacean Palaemon serrutus.
In<strong>vertebrate</strong> hemocytes constitute a population with mor-
phological and functional heterogeneity which has been
ascribed to their degree <strong>of</strong> diffe=ntiation [36]. Among their
diverse functions is a major role in the defence mechanisms,
though they are also involved in wound healing and have the
ability to secrete components <strong>of</strong> the extracellular matrix
(EMC) in vitro [32]. Another characteristic <strong>of</strong> these cells
concerns their capacity to divide in viva [6]. Taking into
account these characteristics, it seems likely that hemocytes
could provide a suitable model to investigate the effects <strong>of</strong>
<strong>growth</strong>-promoting <strong>factors</strong> involved in the process <strong>of</strong> prolife-
ration-<strong>different</strong>iation. Up to now, no reports have appeared
concerning the effects <strong>of</strong> <strong>growth</strong>-promoting <strong>factors</strong> on pro-
tein and DNA syntheses in primary cell cultures <strong>of</strong> marine
in<strong>vertebrate</strong>s. The purpose <strong>of</strong> this present study was thus to

establish a suitable primary cell culture <strong>of</strong> hemocytes and
subsequeptIy to investigate the effects <strong>of</strong> <strong>growth</strong> promoting
<strong>factors</strong> such as insulin and EGF on the incorporation <strong>of</strong>
[3H]-leucine and <strong>of</strong> [3H)-thymidine in order to estimate pro-
tein and DNA syntheses in these cultured cells.
Materials and methods
Source and maintenance <strong>of</strong> anitnuis
Adult abalones, Haiiotk tuberculata, 8-12 cm in shell length,
were collected monthly from the West Coast <strong>of</strong> the Cotentin
peninsula (Manche, France) and acclimated to iaboraiory condi-
tions for at least 2 weeks before experimentation. Animals were
Faintained in natural and continuoutiy aerated seawater at sea..
sonal ambient temperature. They were fed daily with a mixed
algal diet but starved 2 days prior to being killed.
Primaty cell cultures
After an incision in the foot, hemolymph was collecte4. (S-10 ml
per animal) using a 5-ml st.eriIe syringe fitted with a Sgauge hypodermic
needle. Hemolymph was transferred to a sterile? tube and
simultane5usly diluted 1:3 in cooled sterile anti-coagulmt m5&
fied Alsever’s solution [2] (115 &- glucose; 27 i&l @iurn iitrate;
11.5 n&l EDTA; 382 mM NaCl). Ijemocytes were rapidly
plated at 0.8 Iti cells per well in &well culture plate3 to which
three volumes <strong>of</strong> sterile artiticiaI seawater were added. Cultures
were maintained at 15°C in a humidified incubator (~COX-free).
After 90 min <strong>of</strong> incubation, cells were washed with Hanks’-199
medium modified by addition <strong>of</strong> 250 mM NaCl; 10 mM KCl; 25
mM MgSOA; 2.S mh4 .CaCJ; 10 mM Hepes: final pH was 7.4 im&
osmolarity 1100 mosmol/l. Then cells were covered with fresh
medium supplemented with antifungal and antibacterial substance5
t 100 pg/ml streptomycin sulfate; 60 wml~penicillin G: -5O-$g/mt
gentamycin &fate; 0.20 .ug/ml amphotericin By and nystatin
(8 pg/ml), L-glutamine (2 mM), concahavalin A (2 mM) and w&c
incubated (CO?-free) at 1 S’C.
MIT reductioti ussay
The M’M red?ction assay is an enzymabc test based on the aetcr.
mination *f the activity <strong>of</strong> mitochtindrial deshydrogenasc
enzymes- This test, developed by Mosmann [23], h&been
adapted to evaluate the viability <strong>of</strong> marine in<strong>vertebrate</strong> cells i??
24i. F5r assays> I?0 ~1 <strong>of</strong> stock MTT solution (5 mg Ml-r/ml
artificial seawater filtered throygh a O-22. pm filter) were addee to
each dish to be tested.. ~Plates were incuiated at i5YJ. Reaction?
were stopped by addition <strong>of</strong> an equal volume <strong>of</strong> isopropanoi COH.
taining 0.04 N HCl. The plates were shaken for 30 min at room
temperature. Absorbances at wavelength <strong>of</strong> 570 nm were meas..
ured with a reference <strong>of</strong> 630 nm (OD 570 nm/ref 630 nm) within
30 tin <strong>of</strong> adding the isopropanol.
Growth <strong>factors</strong> assq
Cells were plated at 0.8 106 cells per well in &well culture
plates. 24 b. after the beginning <strong>of</strong> c.ulture the mediu& was
renewed (l-2 ml p& dish) without loss <strong>of</strong> dells. Then 10 p$
(I PCi) <strong>of</strong> f3H]-thymidine (sp act: 35 Ci/mmol, ICN r?di.ochemi-
cal diluted in culture mechum) or [sH]-leucine (sp act: 12!
Ci/mmol, ICN radiochemical .diluted in culture medium) was
added to each dish. Cultures were perfomed in-absence or ia
presence <strong>of</strong> the <strong>different</strong> <strong>growth</strong> <strong>factors</strong> to be tested for 24 h
Fig
I99 1. One-day-old primary cultures <strong>of</strong> bemocytes. Cells were seeded at 0.8 I@ cells per dish and grown at 1S”C in modified H&:s’
medium in absence (a) or presence (b) <strong>of</strong> conc%navalin A in the medium. In tie presence <strong>of</strong> Con A adherent hemocytes appcAr+.?d
tly as fibroblast-like cells (F) and to a smaller extetit as epitbelial-like (E) cells. Scale bar = 5O~prn.

([3H]-leucine) or 48 h (f3H]-thymidine). Bovine insulin and
human epidermal <strong>growth</strong> factor (EGQ were dissolved in Hanks’-
199 supplemented medium and 10 ~1 <strong>of</strong> stock solutions to be
tested were added to the wells. Incorporation was stopped by
adding an equal volume <strong>of</strong> ice-cold trichloroacetic acid (10%).
After total precipitation (overnight at 4°C) cells were scrapped
and transferred in tubes which were centrifuged at 3000 g for 10
min. The supematant was discarded and the pellets were washed
twice with 10% trichloroacetic acid. Then 500 ~1 <strong>of</strong> KOH (0.3
M) were added and tubes were incubated for 30 min at 60°C in
order to dissolve the pellet. The total fraction was transferred in
scintillation vial and the radioactivity was counted after addition
<strong>of</strong> 4 ml <strong>of</strong> liquid scintillation to each vial. Blank controls were
performed with cells that were precipitated with trichloroacetic
acid prior to the addition <strong>of</strong> r3H]-leucine or [3H]-thymidine.
Stutistical analysis
Significance <strong>of</strong> the difference between mean values was estimated
using the Student’s t-test. Each experiment was repeated at least
three times and for one experiment the means were calculated
from triplicates.
ResUlt.5
Primary cultures qf hemocytes
In absence <strong>of</strong> concanavalin A (Con A) a large part <strong>of</strong> hemo-
cytes remained round like freshly bled cells (fig la). A con-
sistent feature <strong>of</strong> hemocytes cultures grown in Con A-free
medium was a significant increase in non-adherent floating
cells in the culture supematants during experiments (data
not shown). The presence <strong>of</strong> Con A in the medium
(1 pg/ml) enhanced the adherence <strong>of</strong> hemocytes on the
plates. After the initiation <strong>of</strong> cultures, hemocytes adhered
on glass and formed clusters. Rapidly cells migrated out <strong>of</strong>
the aggregates and flattened on the surface <strong>of</strong> dishes.
Hemocytes appeared more and more isolated and then
formed a uniform monolayer (fig lb). Based on their mor-
phological characteristics, two cell categories could be dis-
tinguished. A large majority <strong>of</strong> hemocytes is represented by
the fibroblast-like cells. The remainder is constituted by the
epithelial-like cells (fig la, b).
MT assay and viability <strong>of</strong> hemocytes
Figure 2 represents the validity <strong>of</strong> the MTT assay test for
hemocytes. The absorbance was directly proportional to the
number <strong>of</strong> cells cultured per dish and this linear function
(y = 0.009 x + 0.009, r* = 0.975) included the greater value
tested, 0.9 106 cells per dish. The viability <strong>of</strong> hemocytes
remained constant during the fast 6 days <strong>of</strong> culture (data not
shown).
Efects <strong>of</strong> insulin or EGF on [3H]-leucine incorporation
Insulin and EGF significantly stimulated PHI-leucine incor-
poration in hemocytes in a dose-dependent manner from
10-T M to 10-5 M for insulin (fig 3) and from lO-7 M to
10-6 M for EGF (fig 4). The maximal stimulation induced
an increase <strong>of</strong> 196 k 4% for insulin and 207 & 12% for EGF
with respect to 100% <strong>of</strong> control. The combined effect <strong>of</strong>
both insulin and EGF is illustrated in figure 5. No additive
effect on [3H]-leucine incorporation is observed for insulin
(104 M) and EGF (lO-7 M). For these concentrations the
combined response (227 & 15% relative to the 100% meas-
ured for control) is not significantly higher than the
A test system from H tuberdata 69
OS 10
1 O5 cells / well
Fig 2. Validity <strong>of</strong> the MTT reduction assay. One day after the
beginning <strong>of</strong> cultures the medium was renewed and M’IT test
was performed. Hemocytes were plated at a density from 0.15
106 to 0.9 l@ cells per well in 6-well culture plates and cultured
at WC in modified Hanks’-199 medium. Each data point (OD
570 nrn/ref 630 nm) represents the mean 2 standard deviation <strong>of</strong>
triplicate cultures.
response <strong>of</strong> insulin (104 M) or EGF (lo-7 M) tested alone,
respectively 210 ? 9% and 198 k 10%. [3H]-leucine incor-
poration measured for control represented 1283 *
125 cpm/l@ cells.
<strong>Effects</strong> <strong>of</strong> insulin or EGF on [3H]-thymidine incorporation
Addition <strong>of</strong> insulin and EGF significantly stimulated [3H]-
thymidine incorporation in hemocyte culture (fig 6). How-
ever, no additive effect on the incorporation <strong>of</strong> [YHJ-thymi-
dine is observed when a combined treatment with insulin
(10 -6 M) and EGF (10-T M) is performed. The combined
response (224 k 8% with respect to the 100% measured for
control) is not significantly higher than the response <strong>of</strong> insu-
lin (lo-6 M) or EGF (10-T M) tested alone, respectively 194
* 9% and 213 k 10%. [3H]-thymidine incorporation meas-
ured for control represented 602 ? 15 cpm/l@ cells.
Discussion
The first aim <strong>of</strong> this work was to establish a suitable in vitro
model for studying the cellular metabolism <strong>of</strong> hemocytes.
As previously observed for sea-urchin embryonic cells, the
presence <strong>of</strong> Con A in the medium enhanced hemocyte
adherence on the plates [21]. In our study, cells attached to
the surface <strong>of</strong> the plates after about 1 h <strong>of</strong> incubation and
remained fully spread during the 6-day experiments. Thus
hemocytes may be seeded without cell loss and in a most
reproducible manner resulting in a highly quantifiable

CO -10 -9 -8 -7 -6 .-5
k!@
Fig 3. Dose-response effect <strong>of</strong> insulin on the incorporation <strong>of</strong>
[SHJ-Ieucine in.heinocytes in culture. Ceils were seeded at 0.8 106
cells per dish and grown in absence or presence tif insulin (from
l@-*‘J M to I@5 M) for 24 h. Cells were cultur.%d at WC! in modified
Hanks’-199 medium. Each data point repssents the mean
percentage * standard deviation (with respect to the 100% <strong>of</strong>
incorporation- measured for control) <strong>of</strong> &$&ate cuItures. Such a
typical experiment was repeatedat least three times. Sign&ant
difference from control cells (Co) at P < W301 (**).
system. This first point is an original aspect <strong>of</strong> our model.
Con A, which tiects cell attachment, has b&n also shown
to act on cell proliferation for <strong>different</strong> cell types. Because
aI cell cultures were carried out in the preseti@ <strong>of</strong> Con A,
this possible influence has not been investigated. In addition,
for marine molluscan cells, Domart-Coulon er uZ [7J did not
observe any significant influence <strong>of</strong> Con A on the viability
<strong>of</strong> cell cultures either after 2 or after 6 days <strong>of</strong> incubation for
concentrations corresponding to 1 pg/ml or 25 @@ml. How-
ever, we call not rule out an @feet <strong>of</strong> Con A on thyddine
incorporation .in cultured hemocytes. The viability <strong>of</strong> henio-
cytes cultur& in a medium without <strong>growth</strong> fac@rs remaibe4l
constant during the 6 fast diys <strong>of</strong> culture. In &&ion, the
cells showed physiological responses as jtidg&d by protein
and DNA syntheses in response to treatment witbinsulin or
EGF, confirming their gruwth potential in V&RX
Treatment with porcine in&in stim&ated incorporation
<strong>of</strong> labelled leucine and thymidine in cultured hemocytes,
reflecting to a certain extent an increase in the protein and
DNA syntheses. These effects are significant for about
l @/ml and maximal for 100 fig/ml. EffecEs <strong>of</strong> <strong>vertebrate</strong>
insulin on other cell types from .molluscs have .been reported.
In Htdisomu &ryi, treatment with porcine insulin (0.1 @ml
during 48 h) increased amino acid incorporation.in the man-
tle collar jn vi?ro [28]. Recently, Domwd-Coulon et ul [‘7]
reported that 6 days <strong>of</strong> treatment with bovine insulin (XI
pg/ml) induced a 25% rise in celIulztr viabiliti:in heart cells
from Crussos~rea g&as in vitr.. 1~~expetimen.t~ conducted
with dissociited cells we showed that insuW (50 pg/ml)
co -9 -8 “7 “4
Log M
Fig 4. Dose-response effect <strong>of</strong> EGF on the incorporation <strong>of</strong> [JH]*
leucine in hemocytes in culture. Cells were seeded &t 0.8 W
cells pr dish and grown ig absence or p=sence <strong>of</strong>.EGF (from
IV M to lv-M) for 24 h. Cells were cultured at WC in rqdifie$
Hanks’-l9~~m~d&m. Each data point rqzsents the-&can
percentage * standard deviation (with respect to the. l~OU% <strong>of</strong>
incorporation m&sured for control) <strong>of</strong> triplicate-cultures. &ch a
typical expzriment was rested at least three times: &gnificmt~
difference fr&ti contr<strong>of</strong> ceils (Co) at P < 0.001 (**).
incr?ases incorpQration <strong>of</strong> labelled leucine in mantle cells,
but also in di@tive bells from the molIusc Pec~en-muximy
(Giard et ai, subm@ed). These results dem~strated that ver-
tebrate insulins have biological effects on a large va&ty <strong>of</strong>
in<strong>vertebrate</strong> cell types but the precise functiti <strong>of</strong>this <strong>growth</strong>
factor on these <strong>different</strong> ce& remains to be- elucida&. Usirig
porcine or human insulin antisera, immunoreactive in&in7
like peptide (l&P) was detected in the neurosecretory medio-
dorsal cells and hemolymph <strong>of</strong> the snail, He&om~&q+
[ 16,281, I&Ps appear to arise from &&rent soqces, such -as
the digestive gland or the central wwous system, $zvala et
u2 [28] suggested that lLP from the digestive gland could IX
invalved in sugar met&&sm, whereas ILP from the central
nervous system.could be a shelXgro* f&tor. In the case <strong>of</strong>
hemocytes no eiid&nce is available to date p&&t&g a &s-
tinction betwe& insulin being a .general met&o& &n&a-
tor or having a. more specif?c fumztion.
The other stimulating <strong>growth</strong> factor tested, EGF”:
increased significantly (at a cmcentition <strong>of</strong> 0.6 &ml) the.
.metab& a&@-<strong>of</strong> ct.&red hemocytes. In the same way?
Domart-Coulon et ui [7] showed that treatment with this
poIypeptidic -gFowth factor has a significant posit& effect
on the viability <strong>of</strong> Crossasfrea~ gigtis heart cell cultures+ A
biological Effect <strong>of</strong> EGF on maririe in<strong>vertebrate</strong> cells is in
agreemerit with results <strong>of</strong> Odinstova et ul [25]. Th&e
authors have extracted a substance which may belong t@ the
fatiily <strong>of</strong> EGF&ke <strong>factors</strong> from Myfz’Zus edulzk .‘l%is facto1
showed a mitogqk activity in dissociated mussel m&k
cells as w&tis in mouse fibro.bktsts. A mitogenic factor

co E I E+I
Fig 5. Combined effect <strong>of</strong> insulin and EGF on the incorporation
<strong>of</strong> [sH]-leucine in hemocytes in culture. Cells were seeded at 0.8
106 cells per dish and grown in absence or presence <strong>of</strong> EGF
(10-7 M) or insulin (1V M) alone; or with EGF (10-T M) and
insulin (10-6 M) simultaneously, for 48 h. Cells were cultured at
15T in modilied Hanks’-199 medium. Each data point represents
the mean percentage k standard deviation (with respect to the
100% <strong>of</strong> incorporation measured for control) <strong>of</strong> triplicate cultures.
Such a typical experiment was repeated at least three times. Signi-
ficant difference from control cells (Co) at P < 0.001 (**).
able to regulate the metabolism <strong>of</strong> mantle cells has also
been extracted from cerebral ganglion and hemolymph <strong>of</strong>
the mussel Mytilus eduZis [20, 341. The presence <strong>of</strong> a mito-
genie factor in hemolymph is in agreement with an endo-
crine function <strong>of</strong> these <strong>growth</strong> <strong>factors</strong>. Other possibilities,
however may be postulated. Recently, a protein, mainly
consisting <strong>of</strong> EGF-like repeats, has been detected in the foot
<strong>of</strong> the mussel MytiZus guZZoprovincialis [ 151. Interestingly,
it seems that this protein functions as a matrix protein. In
<strong>vertebrate</strong>s, several extracellular matrix proteins contain
domains with homology to EGF and exhibit <strong>growth</strong> pro-
moting activities including mitogenic activity [ 111. It has
been suggested that diffusible EGF-like peptides may be
released by processing <strong>of</strong> the extracellular domain. These
proteins <strong>of</strong> the ECM may express their <strong>growth</strong> factor-like
function on neighbouring cells by paracrine stimulation.
They may also act on the cells that secrete it by autocrine
stimulation [1 11. Taking into account the fact that hemocy-
tes secrete a lot <strong>of</strong> components <strong>of</strong> the EMC [31], the in vivo
effects <strong>of</strong> some <strong>growth</strong> promoting <strong>factors</strong> on hemocytes
may result from a paracrine as well as autocrine stimula-
tion.
In this study, despite an effect <strong>of</strong> insulin and EGF on the
stimulation <strong>of</strong> DNA synthesis, the induction <strong>of</strong> cell prolife-
ration is not demonstrated. However, the capacity <strong>of</strong> hemo-
cytes to divide in vitro has been demonstrated by Dikke-
boom er aZ [6]. In the same way, division in vitro <strong>of</strong> cells
from larvae <strong>of</strong> the abalone, HuZiotis rufescens was evi-
denced [24]. No additive effects have been evidenced
.4 test system from H tuberdata 71
CO E I E+I
Fig 6. <strong>Effects</strong> <strong>of</strong> EGF and insulin on the incorporation <strong>of</strong> [sH]-
thymidine in hemmytes in culture. Cells went seeded at 0.8 106
cells per dish and grown in absence or presence <strong>of</strong> EGF (10-T M)
or insulin (1tV M) alone; or with EGF (10-T M) and insulin (10-6
M) simultaneously, for 48 h. Cells were cultured at 15°C in modi-
fied Hanks’-199 medium. Each data point represents the mean
percentage i standard deviation (with respect to the 100% <strong>of</strong>
incorporation measured for control) <strong>of</strong> triplicate cultures. Such a
typical experiment was repeated at least three times. Significant
difference from control cells (Co) at P c 0.001 (**).
between the two tested <strong>growth</strong> <strong>factors</strong> either for protein or
DNA syntheses. In <strong>vertebrate</strong>s, it is now well established
that insulin and EGF induce their biological effects through
interactions with their cell surface receptors that contain
intrinsic tyrosine kinase activities [4, 381. Moreover, in
vivo stimulation by insulin and EGF may overlap in term
<strong>of</strong> their intracellular signalling pathways [12, 381. F’rotein
tyrosine kinases signalling pathways have been conserved
throughout evolution [9, 261. In addition, in DrosophiZu a
receptor that shows a dual binding specificity for both
insulin and EGF has been identified [32]. The lack <strong>of</strong> addi-
tive effects observed in this study may result from the
expression <strong>of</strong> a such protein in marine molluscan cells.
In conchtsion, we have established a routine experimental
system <strong>of</strong> primary hemocyte cultures. Hemocytes are target
cells for <strong>vertebrate</strong> <strong>growth</strong> <strong>factors</strong> such as insulin or EGF
which increased hemocyte protein as well as DNA synthe-
ses. This model appears to be particularly useful for investi-
gating the in vitro effects <strong>of</strong> <strong>growth</strong> <strong>factors</strong> on the control <strong>of</strong>
cell <strong>growth</strong>, <strong>different</strong>iation and migration at the cellular
level in in<strong>vertebrate</strong> cells.
Acknowledgments
We are very grateful to Pr<strong>of</strong>essor E Lopez for encouragement and
helpful advice on the manuscript. This study was supported by a
special grant <strong>of</strong> the University <strong>of</strong> Caen (Action specifique
recherche). A doctoral fellowship (W Giard) from the Dielen
society is gratefully acknowledged.

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