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Insulin imprinting of neuroblastoma cells
Cell Biology
International
INSULIN
Kovacs. Department Medicine, Biochemistry,
Reports,
IMPRINTING
P.,lOkano,
Vol.
13, No. 6, June
1989
OF NEUROBLASTOMA
Y.,
INozawa,
Y.
563
CELLS
and
Csaba.G.
of Biology, Semmelweis University Hungary and 'Department Budapest, Gifu University school of Medicine, Gifu, Japan.
of of
INTRODUCTION-
The first encounter of a hormone with the target cell gives rise to hormonal imprinting, giving instructions to the genetically programmed,though still plastic, usually in the perinatal receptors for maturation (4), period. The imprinting by the right molecule in an adequate time and concentration induces the normal development of receptors (5). The presence of molecules able to bind to the receptors however quantitatively or qualitatively disparate, provoke pathological (stronger or weaker) development of thz receptors (7). While a minimal quantity of the given hormone is always present in the cell culture medium and the target cells have encountered the hormone in the living organism (from which it originated), imprinting could be provoked in cultures of cell lines as well (6). In the present experiment, the imprintability of a neuroblastoma cell line by insulin was studied. HETHODS AND MATERIALS.
A cell line of NL-309 neuroblastoma (a neuroblastoma and L cell hybrid clone induced by Sendai virus) (2) was kindly supplied by Dr H.Higashida and was sustained in GDMEM medium for two days. 5% FB and 1% HAT containing monolayer of cells grown in the medium was treated The with insulin (lO-hM, Semilente MC, NOVO, Copenhagen, Denmark) for 1 h, followed by thorough washing with normal media. Control cells were handled similarily After cultivation in normal without insulin treatment. medium for one the cells were fixed in 2% day, formaldehyde (diluted in PBS, pH 7.2) for five min. were then followed by thorough rinsing. The cells incubated with fluorescein isothiocyanate (FITC; BDH, U.K.) - labelled insulin (FITC-protein ratio 0.39, protein content 0.1 mg/ml) for 1 h, rinsed and dried. The fluorescence intensity of plasma and nuclear (1, 9, membranes was determined by measuring the intensity 10) of fluorescence over the nucleus and cytoplasm by an
0309-1651/891060563-5/$03.00/O
0
1989
Academic
Press
Ltd.
564
Cell Biology
international
Reports,
Vol. 13, No. 6, June
of NL-309 hybrid neuroblastoma.The processes are the neurons surrounded Magn.x 600.
Fig.l.The cells cells with long by small cells.
Intensity
Ei
of fkroreacence
cytoplasm
-
% 150
s-pro,01
-’
T”
loo*,--
f WWO”
small
cells
Fig.Z.Effect of hormonal imprinting (insulin pretreatment) on neuroblastoma cells, related to the control is related to the control. as 100%. Significande
big
7989
CeN Biology
International
Reports,
Vol. 13, No. 6, June
1989
665
Olympus MMSP microscope connected with a CanonCanola 321 computer. The experiments were done in triplicate, so that one column of Fig. 2 represents the mean of 90 cells. RESULTS
AND
DISCUSSION.
There were two types of cells in the cultures (Fig. 1): large cells with long processes (neurons) and small round (glia or neuroblast-like) cells The (3). fluorescence of the latter cells was so intense that whole only mean value for the cell could be determiied, instead of measuring the nuclear and plasma membranes separately. As an effect of insulin imprinting, after one day both cell types showed an elevated insulin binding (Fig. 2). The influence of imprinting was stronger on the plasma membrane in the case of neurons. The possibilty of imprinting is restricted to those cells which either bear the receptors for the hormone or can develop them The neurons have insulin (7). receptors (12), so that one can surmise that their malignant form, the neuroblastoma cells, also have insulin receptors. It is also known that the effect of nerve growth factor on neuroblastoma cells is supported the presence of insulin (11). It is possible that by properties of cells promoted the effect of these the insulin in provoking imprinting. The binding-promoting effect of insulin imprinting has been observed in the progeny cell generations, as the cells were observed for periods in excess of 24 h and so cell division had occurred in this time. Interestingly, small cells were also imprintable. The usual intensity of imprinting seems to be remarkable. Using Chang liver cells (the direct target cells for insulin) as in previous experiments (8) the effect of insulin appeared to show a 20-30% elevation of binding (a similar value was observed in the case of cells) compared with a 50% glia or neuroblastoma-like increase on the plasma membrane of neuroblastemic "big cells". Considering the consequential and significant results in repeated experiments, this demonstrates the extraordinary sensitivity to insulin imprinting Of malignant (Fig. 2) neurons in cell cultures.
566 Cell Biology ACKNOWLEDGMENT. This study co-operative Sciences and Science.
International
Reports,
was supported by programme between the the Japanese Society
Vol. 13, No. 6, June
the grant for Hungarian Academy for the Promotion
1989
the of of
REFERENCES Burwen,
S.J.
polypeptide nuclei 159-162.
and Jones, A-L-(1987) hormones and growth target cells. Trends
of
Catterall,
The association factors with Biochem. Sci.
W.A.,and
Nirenberg,m. (1973) Sodium uptake with activation of action potential of cultured neuroblastoma and muscle cells. Acad. Sci. U.S.A. 70: 3759-3763.
associated ionophores Proc. Natl.
Chalazonitis,
A.,
Minna,
J-D.,
and Nirenberg,
M. (1977)
Expression and properties of acetylcholine in several clones of mouse neuroblastoma somatic hybrids. Exp. Cell Res. 105: 269-280.
Csaba,
G.
receptors: and hormonal
Csaba,
of
G.
Csaba,
and
G.
Experientia
G.,
imprinting exposure capacity Hung. 64:
Horvat, binding Biophys.
in 'Tissue E.Bacsy) Publ.
(1986)
imprinting.
Csaba,
The present state hormone receptors.
(1984)
P.Rohlich Budapest.
A.,
receptors XL cell
(1980) Phylogeny and ontogeny of hormone the selection theory of receptor formation imprinting. Biol. Rev. 55: 47-63.
G. (1984)
ontogeny 329-335.
Csaba,
of the 12:
Torok, in to in
57-63. Li. sites Acta.
Receptor 42:
O.,
in the phylogeny Horm. Metab. Res. Culture House
ontogeny 715-718.
Kovaks
P.
and Hung.
RES Acad.
and
(1984)
cell culture I. Impact of insulin on cellular insulin permanent cell lines. Acta
E. and Katsoyannis, for 382:
insulin 609-620.
in
P.G.
(1975)
rat
liver.
and 16: (eds. Sci.
hormonal Hormonal a single binding Physiol. Cellular Biochim.
Cell Biology
International
Reports,
Vol. 13, No. 6, June
1989
Recio-Pinto,
E., Lang, F.F. and Ishii, Insulin and insulin-like growth factor II growth factor binding and the neurite response in cultured human neuroblastoma Natl. Acad. Sci. U.S.A. 81: 2562-2566.
Wajchenberg,
B.L.
and Lerario,
receptor in circulating t0 the red blood cells. disease states (obesity Metab. Res. 20: 133-137. Paper
received
4.2.89
cells, Its and
Revised
567
D.N. (1984) permit nerve formation cells. Proc.
A.C.
(1988) The insulin with special reference characteristics in some diabetes mellitus) Horm.
paper
accepted
18.4.89.
International
INSULIN
Kovacs. Department Medicine, Biochemistry,
Reports,
IMPRINTING
P.,lOkano,
Vol.
13, No. 6, June
1989
OF NEUROBLASTOMA
Y.,
INozawa,
Y.
563
CELLS
and
Csaba.G.
of Biology, Semmelweis University Hungary and 'Department Budapest, Gifu University school of Medicine, Gifu, Japan.
of of
INTRODUCTION-
The first encounter of a hormone with the target cell gives rise to hormonal imprinting, giving instructions to the genetically programmed,though still plastic, usually in the perinatal receptors for maturation (4), period. The imprinting by the right molecule in an adequate time and concentration induces the normal development of receptors (5). The presence of molecules able to bind to the receptors however quantitatively or qualitatively disparate, provoke pathological (stronger or weaker) development of thz receptors (7). While a minimal quantity of the given hormone is always present in the cell culture medium and the target cells have encountered the hormone in the living organism (from which it originated), imprinting could be provoked in cultures of cell lines as well (6). In the present experiment, the imprintability of a neuroblastoma cell line by insulin was studied. HETHODS AND MATERIALS.
A cell line of NL-309 neuroblastoma (a neuroblastoma and L cell hybrid clone induced by Sendai virus) (2) was kindly supplied by Dr H.Higashida and was sustained in GDMEM medium for two days. 5% FB and 1% HAT containing monolayer of cells grown in the medium was treated The with insulin (lO-hM, Semilente MC, NOVO, Copenhagen, Denmark) for 1 h, followed by thorough washing with normal media. Control cells were handled similarily After cultivation in normal without insulin treatment. medium for one the cells were fixed in 2% day, formaldehyde (diluted in PBS, pH 7.2) for five min. were then followed by thorough rinsing. The cells incubated with fluorescein isothiocyanate (FITC; BDH, U.K.) - labelled insulin (FITC-protein ratio 0.39, protein content 0.1 mg/ml) for 1 h, rinsed and dried. The fluorescence intensity of plasma and nuclear (1, 9, membranes was determined by measuring the intensity 10) of fluorescence over the nucleus and cytoplasm by an
0309-1651/891060563-5/$03.00/O
0
1989
Academic
Press
Ltd.
564
Cell Biology
international
Reports,
Vol. 13, No. 6, June
of NL-309 hybrid neuroblastoma.The processes are the neurons surrounded Magn.x 600.
Fig.l.The cells cells with long by small cells.
Intensity
Ei
of fkroreacence
cytoplasm
-
% 150
s-pro,01
-’
T”
loo*,--
f WWO”
small
cells
Fig.Z.Effect of hormonal imprinting (insulin pretreatment) on neuroblastoma cells, related to the control is related to the control. as 100%. Significande
big
7989
CeN Biology
International
Reports,
Vol. 13, No. 6, June
1989
665
Olympus MMSP microscope connected with a CanonCanola 321 computer. The experiments were done in triplicate, so that one column of Fig. 2 represents the mean of 90 cells. RESULTS
AND
DISCUSSION.
There were two types of cells in the cultures (Fig. 1): large cells with long processes (neurons) and small round (glia or neuroblast-like) cells The (3). fluorescence of the latter cells was so intense that whole only mean value for the cell could be determiied, instead of measuring the nuclear and plasma membranes separately. As an effect of insulin imprinting, after one day both cell types showed an elevated insulin binding (Fig. 2). The influence of imprinting was stronger on the plasma membrane in the case of neurons. The possibilty of imprinting is restricted to those cells which either bear the receptors for the hormone or can develop them The neurons have insulin (7). receptors (12), so that one can surmise that their malignant form, the neuroblastoma cells, also have insulin receptors. It is also known that the effect of nerve growth factor on neuroblastoma cells is supported the presence of insulin (11). It is possible that by properties of cells promoted the effect of these the insulin in provoking imprinting. The binding-promoting effect of insulin imprinting has been observed in the progeny cell generations, as the cells were observed for periods in excess of 24 h and so cell division had occurred in this time. Interestingly, small cells were also imprintable. The usual intensity of imprinting seems to be remarkable. Using Chang liver cells (the direct target cells for insulin) as in previous experiments (8) the effect of insulin appeared to show a 20-30% elevation of binding (a similar value was observed in the case of cells) compared with a 50% glia or neuroblastoma-like increase on the plasma membrane of neuroblastemic "big cells". Considering the consequential and significant results in repeated experiments, this demonstrates the extraordinary sensitivity to insulin imprinting Of malignant (Fig. 2) neurons in cell cultures.
566 Cell Biology ACKNOWLEDGMENT. This study co-operative Sciences and Science.
International
Reports,
was supported by programme between the the Japanese Society
Vol. 13, No. 6, June
the grant for Hungarian Academy for the Promotion
1989
the of of
REFERENCES Burwen,
S.J.
polypeptide nuclei 159-162.
and Jones, A-L-(1987) hormones and growth target cells. Trends
of
Catterall,
The association factors with Biochem. Sci.
W.A.,and
Nirenberg,m. (1973) Sodium uptake with activation of action potential of cultured neuroblastoma and muscle cells. Acad. Sci. U.S.A. 70: 3759-3763.
associated ionophores Proc. Natl.
Chalazonitis,
A.,
Minna,
J-D.,
and Nirenberg,
M. (1977)
Expression and properties of acetylcholine in several clones of mouse neuroblastoma somatic hybrids. Exp. Cell Res. 105: 269-280.
Csaba,
G.
receptors: and hormonal
Csaba,
of
G.
Csaba,
and
G.
Experientia
G.,
imprinting exposure capacity Hung. 64:
Horvat, binding Biophys.
in 'Tissue E.Bacsy) Publ.
(1986)
imprinting.
Csaba,
The present state hormone receptors.
(1984)
P.Rohlich Budapest.
A.,
receptors XL cell
(1980) Phylogeny and ontogeny of hormone the selection theory of receptor formation imprinting. Biol. Rev. 55: 47-63.
G. (1984)
ontogeny 329-335.
Csaba,
of the 12:
Torok, in to in
57-63. Li. sites Acta.
Receptor 42:
O.,
in the phylogeny Horm. Metab. Res. Culture House
ontogeny 715-718.
Kovaks
P.
and Hung.
RES Acad.
and
(1984)
cell culture I. Impact of insulin on cellular insulin permanent cell lines. Acta
E. and Katsoyannis, for 382:
insulin 609-620.
in
P.G.
(1975)
rat
liver.
and 16: (eds. Sci.
hormonal Hormonal a single binding Physiol. Cellular Biochim.
Cell Biology
International
Reports,
Vol. 13, No. 6, June
1989
Recio-Pinto,
E., Lang, F.F. and Ishii, Insulin and insulin-like growth factor II growth factor binding and the neurite response in cultured human neuroblastoma Natl. Acad. Sci. U.S.A. 81: 2562-2566.
Wajchenberg,
B.L.
and Lerario,
receptor in circulating t0 the red blood cells. disease states (obesity Metab. Res. 20: 133-137. Paper
received
4.2.89
cells, Its and
Revised
567
D.N. (1984) permit nerve formation cells. Proc.
A.C.
(1988) The insulin with special reference characteristics in some diabetes mellitus) Horm.
paper
accepted
18.4.89.