Vol. 90, No. 4, 1979 October 29, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
1348-1354
PRIMARY CULTURES OF RAT HEPATOCYTES SYNTHESIZE FIBRONECTIN + Bruno Voss , Salah Allam+, Jiirgen Rauterberg+, Kurt Ullrich++, ++ Volkmar Gieselmann , Kurt von Figura ++ +
Institute logical
Received
++ Research and Institute of Physioof Miinster, D-4400 Miinster, FRG
of Arteriosclerosis Chemistry, University August
31,
1979
Summary: Fibronectin was detected by indirect immunofluorescence on primary cultures of rat hepatoc tes maintained in the presence or absence of fetal calf serum. I:1X Cl-Fibronectin synthesized in the presence of [I4 Cl-glycine was isolated by immunoprecipitation and visualized by fluorography. Introduction: present
Fibronectin
in an insoluble
and in a soluble see ref.
that
substrates,
a high
form in plasma
such as collagen, that
depends on the
medium. So far,
and other
mediated
adhesion
of rat
presence
of
synthesis
is unclear.
tissue
cells
(for
review
(2-4).
hepatocytes
fibronectin
It
of cells
and glass
of fibronectin
glycoprotein
body fluids
adhesion
plastic
weight
connective
of fibronectin
fibronectin
(5) demonstrated
molecular
form an different
The function
1).
suggested
dishes
is
has been to different Hook et
al.
to culture
in the
by hepatocytes
culture has not
been reported. The present
report
primary
cultures
and its
deposition
describes
of rat
the
synthesis
hepatocytes,
on the
cell
its
of
release
fibronectin into
by
the medium
surface.
Materials and Methods: Female Wistar rats, 200-250 g, were fed with water and Altromin (Lange, Lappe, W.-Germany) ad libitum. Perfusion and preparation of hepatocytes was done according to Seglen (6) with slight modification including isopycnic centrifugation in Percoll (Pharmacia, Uppsala, Sweden) as described (7). The final preparations contained less than 5% non-viable cells as judged by trypan blue exclusion. Cell culture: 2.5 x lo6 Hepatocytes in 5 ml Eagle's minimal essential medium (8) supplemented with 20% fetal calf serum, nonessential amino acids and antibiotics (9) were plated into 60 mm Falcon plastic dishes. After 4-6 h the non-attached cells (20-40%) were sucked off and fresh medium was added. 0006-291X/79/201348-07$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
1348
BIOCHEMICAL
Vol. 90, No. 4, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
In some experiments the fetal calf serum containing medium was replaced 4 h after plating by a chemically defined, serum free medium (IO, modified according to 11). In the presence of the serum free medium the hepatocytes maintained the polygonal shape and association as confluent monolayers for at least five days (Fig. IA and B). The hepatocyte cultures were essentially free of Kupffer cells as demonstrated by the lack of phagocytosis of ink (Giinther Wagner, Hannover, Germany) and of fluorescein isothiocyanate conjugated IgG (Behringwerke, Marburg, Germany). All experiments were done with anti-human fibronectin Antisera: rabbit serum from Behring Werke (Marburg, Germany). The immunofluorescence studies were done in addition with anti-human fibronectin rabbit serum provided by Dr. J. Hijrmann (Max-Planck-Institut fiir Biochemie, Miinchen, Germany) and an antiserum raised in rabbits against fibronectin isolated from human plasma according to Engval and Vaheri (12). The three antisera gave identical results in the immunofluorescence studies and gave rise to a single precipitation line in immunoelectrophoresis. After five washings with phosphate buffered Immunofluorescence: the hepatocytes were incubated for 30 min saline (PBS), pH 7.5 (13) with anti-human fibronectin rabbit serum followed by incubation for 30 min with fluorescein isothiocyanate (FITC) conjugated antirabbit IgG from goat (Behringwerke). Controls were incubated first with rabbit serum and then treated with FITC-conjugated anti-rabbit of intracellular fibronectin the hepatocytes IgG. For detection were dried for 24 h at 37OC and stained as described above. Immunofluorescence was observed with a fluorescence microscope fitted with epi-illumination and a filter system adapted for FITC (Leitz, Wetzlar, Germany, Orthoplan microscope equipped with Orthomat camera). 14 Immunoprecipitation of [ Cl-fibronectin: Hepatocytes were incubated for 24 h with culture medium containing ascorbic acid, and [14C]-glycine, 2.5 uCi/ml, (Amersham-Buchler, speci50 ug/ml, fic activity 118 mCi/mmol). [14Cl-Fibronectin was isolated from culture medium by immunoprecipitation: 100 ul of culture medium was incubated with 0.5 ml of a fibronectin antiserum for 24 h at 4OC. The immunocomplex was precipitated with 100 ~1 of anti-rabbit IgG for 12 h at 4oC. The precipitate was washed three times with PBS containing 1% (w/v) bovine serum albumin, solubilized in 8 M urea with dithioerytrol (50 ug/ml), assayed for radioactivity and subjected to polyacrylamide gel electrophoresis. Controls were processed identically except that antiserum was replaced by rabbit serum. Cell protein was determined according to Kaltwasser et al. (14)
Identification of [I4 Cl-fibronectin by polyacrylamide electroAli uots of medium obtained after incubation in the phoresis: acid were dialysed and presence of [ q4Cl-glycine a nd ascorbic The samples were solubilized in 8 M urea with or freeze dried. without dithioerytrol (50 us/ml) and applied without using a stacking gel to 110 x 110 x 1.5 mm polyacrylamide slab gels. Electrophoresis was carried out in sodium dodecyl sulphate-containing buffers using the system of Laemmli (15). A one-step gradient of acrylamide concentration was used with 12% in the lower and 5% in the upper part. Autofluorography was done as described by Laskey and Mills (16). Fibronectin isolated from human plasma according to Engval and Vaheri (12) served as reference. Results: plating
Immunofluorescence: in fetal
calf
Hepatocytes
serum-containing
1349
examined
4 h after
medium were free
of
fibro-
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 90, No. 4, 1979
nectin
BIOCHEMICAL
as examined
fibronectin
by indirect
appeared
1 D).
covered
in these
medium fibers plating
cultures
fibronectin of
[
Cl-fibronectin:
[14 Cl-glycine
with
immunoprecipitated was dissolved amide gel
under
2 A).
Only
coprecipitated Culture
presence
of
Fluorography
revealed
Fig.
When the 1:
1 F).
for
24 h
medium was
The precipitate
revealed
14
of
(Fig.
were labelled
and subjected
amounts of other
deposition
became visible
antiserum.
from
When cells
washing,
same mobility
3 days old
cultures
to polyacrylamide
SDS with
and non-reduced
fibronectin were grown in
The culture
Fluorography the
3
to polyacryl-
the presence
of
as fibronectin
C-labelled
compounds
fibronectin.
medium of applied
plating.
conditions
with
trace
with
directly
2 B-E).
reducing
material
cells
a fibronectin
electrophoresis.
radiolabelled (Fig.
with
of
culture).
Hepatocytes
3 days after
After
were seen beginning
by extensive the
of
was observed
When hepatocytes
1 C, 2 days-old
underneath
fibers
cultures.
network
fibronectin
were detached
14
a thick
1 E).
of
(Fig.
located
Synthesis
(Fig.
Fine
of fibronectin
cultures
the hepatocytes
one day after
hepatocyte
distribution
On 5 days-old
a serum-free
immunofluorescence.
on one day old
days a more patch-like (Fig.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or without that
gel treatment
the major
14
samples had same mobility cells
were labelled
labelled
as above was
electrophoresis with
in the
dithioerytrol.
C-labelled
band in reduced
as fibronectin
4 h after
(Fig.
plating,
[14C]-
Indirect immunofluorescence localization of cell surface attached fibronectin. (A) Phase contrast micrograph of hepatocytes 4 h after plating. (B) Phase contrast micrograph of hepatocytes 8 h after feeding with serum-free medium. (C) Staining for fibronectin of a culture 2 days after feeding with serum-free medium. (D) Staining for fibronectin of 3 days-old cultures. (E) Staining for fibronectin of 5 days-old cultures. (F) Staining for fibronectin of a culture dish, from
which hepatocytes grown medium had been detached to staining. Magnification: 160-fold for
1351
for 2 days with serum-free by extensive washing prior
A and B,
500-fold
for
C-F.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
55
5% ;
-
12J 12% ,
red.
red
red.
red. Fig.
2:
Autofluorograms of medium obtained after incubation of hepatocytes in the presence of [14C]-glycine. Hepatocytes were grown in the presence of [14C]-glycine for 24 h either 3 days after plating (slot A-E) or 4 h after plating (slot F and G). (A) Immunoprecipitate of the medium after reduction. (5) Non-reduced medium. (C) Reduced medium. (D) Non-reduced medium, film exposure 4 times as long as in (A). (E) Reduced medium, film exposure 4 times as long as
in (B).
(F) Non-reduced medium. (G) Reduced medium. Arrows mark the position standard. red.: Reduced.
fibronectin
appeared
synthesized
14C-labelled
Discussion:
Fibronectin
cytes
as early
staining. likely present
that
secretory could
this
experiments fibronectin
was established products
a human
a minor
plasma
component
products
be detected plating, with
(Fig.
serum-free
of
1352
of newly 2 F and G). hepato-
medium made it
from the calf
its
rat
immunofluorescence
[ 14 Cl-fibronectin
by identification
as fibronectin,by
fibronectin
on cultured
using
originated
in the medium. Synthesis
hepatocytes secretory
to be only
as 24 h after
Control
of
un-
serum by rat
of one of the mobility
in gel
electro-
Vol. 90, No. 4, 1979
phoresis
BIOCHEMICAL
and precipitation
serum.
Surprisingly,
secretory
by anti-human
fibronectin
protein
of
indicate
that
the
creases
markedly
with
culture
The physiological
hepatocytes direved to
serve
of
fibronectin
association
cytes
(18,19).
Thus,
that
freshly
after isolated
collagen
under
This
meinschaft
both the
for
conditions
medium (5).
Collagen
hepatocytes
hepatocytes
(unpublished
surface
Deutsche
of
report is
are hepato-
may play
a
demonstra-
synthesized
do not
by
synthesize
results).
of Miss F. Weber is gratefully by the
absence
and collagen
The present fibronectin
appears
in the
and collagen
whereas
isolated
fibronectin
sinusoidal
lag phase,
Ra 255/3
in-
by hepa-
exogenous
fibronectin
fibronectin
work was supported (Grant
of freshly
culture
hepatocytes, in vitro
release
and spreading
of hepatocytes.
ACKNOWLEDGMENT; The help ledged.
Cl-fibronectin
synthesized
with
an initial
Preliminary
fibronectin
substrate
in vivo
in the anchorage
14
requires
In vivo,
(17).
in close
tes
of
serum in the
as an attachment
[
cultures.
time.
dishes
found
role
of
Attachment
on plastic
from the
rate
rabbit
to be the major
hepatocyte
function
is unknown.
plasma fibronectin
appeared
4 days-old
results
tocytes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acknow-
Forschunqsqe-
and SFB 104).
References: 1.
Vaheri, 515,
A. and Moster,
D.F.
(1978)
Biochim.
Biophys.
Acta
l-26.
Pearlstein, E. (1976) Nature 262, 497-500. 3. Grinell, F., Hays, D.G. and Minter, D. (1977)
2.
110,
Exp.
Cell
Res.
175-190.
4. Pena, S.D.J. and Hughes, R.C. (1978) Nature 276, 80-83. 5. Hook, M., Rubin, K., Oldberg, A. and tibrink, B. (1977) Biochem. Biophys. Res. Comm. 79, 726-733. 6. Seqlen, P-0. (1976) in "Methods in Cell Biology", Vol. XIII (Prescott, D., Ed.) pp 29-83, Academic Press, New York and London. 7. Ullrich, K., Basner, R., Gieselmann, V. and von Figura, K.(1979) Biochem. J. 180, 413-419 8. Eagle, H. (1959) Science 130, 432-437. 9. Cantz, M., Kresse, H., Barton, R.W. and Neufeld, E.F. (1972) Meth. Enzymol. 28, 884-896.
1353
Vol. 90, No. 4, 1979
IO. 11. 12. 13. 14. 15. 16. 17. 18. 19.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Gorham, L.W. and Waymouth, C. (1965) Proc. Sot. Exp. Biol. Med. 119, 287-290. Fratantoni, J.C., Hall, C.W. and Neufeld, E.F. (1969) Proc. Natl. Acad. Sci. )SA 64, 360-366. Dulbecco, R. and Vogt, M. (1954) J. Exp. Med. 99, 167-182. Engvall, E. and Ruoslahti, E. (1977) Int. J. Cancer 10, 1-5. Kaltwasser, F., Wolters, G. and Pieper, J. (1965) Clin. Chim. Acta 15, 347-351. Laemmli, U.K. (1970) Nature 227, 680. Laskey, R.A. and Mills, A.D. (1975) Eur. J. Biochem. 56, 335. Rubin, K., Oldberg, A., Hook, M. and Ubrink, B. (1978) Exp. Cell Res. 117, 165-177. Linder, E., Vaheri, A., Ruoslahti, E. and Wartiovaara, J. (1975) J. Exp. Med. 142, 41-49. Remberger, K., Gay, S. and Fietzek, P.P. (1975) Virchows Arch. A Path. Anat. and Histol. 367, 231-240.
1354
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages
1348-1354
PRIMARY CULTURES OF RAT HEPATOCYTES SYNTHESIZE FIBRONECTIN + Bruno Voss , Salah Allam+, Jiirgen Rauterberg+, Kurt Ullrich++, ++ Volkmar Gieselmann , Kurt von Figura ++ +
Institute logical
Received
++ Research and Institute of Physioof Miinster, D-4400 Miinster, FRG
of Arteriosclerosis Chemistry, University August
31,
1979
Summary: Fibronectin was detected by indirect immunofluorescence on primary cultures of rat hepatoc tes maintained in the presence or absence of fetal calf serum. I:1X Cl-Fibronectin synthesized in the presence of [I4 Cl-glycine was isolated by immunoprecipitation and visualized by fluorography. Introduction: present
Fibronectin
in an insoluble
and in a soluble see ref.
that
substrates,
a high
form in plasma
such as collagen, that
depends on the
medium. So far,
and other
mediated
adhesion
of rat
presence
of
synthesis
is unclear.
tissue
cells
(for
review
(2-4).
hepatocytes
fibronectin
It
of cells
and glass
of fibronectin
glycoprotein
body fluids
adhesion
plastic
weight
connective
of fibronectin
fibronectin
(5) demonstrated
molecular
form an different
The function
1).
suggested
dishes
is
has been to different Hook et
al.
to culture
in the
by hepatocytes
culture has not
been reported. The present
report
primary
cultures
and its
deposition
describes
of rat
the
synthesis
hepatocytes,
on the
cell
its
of
release
fibronectin into
by
the medium
surface.
Materials and Methods: Female Wistar rats, 200-250 g, were fed with water and Altromin (Lange, Lappe, W.-Germany) ad libitum. Perfusion and preparation of hepatocytes was done according to Seglen (6) with slight modification including isopycnic centrifugation in Percoll (Pharmacia, Uppsala, Sweden) as described (7). The final preparations contained less than 5% non-viable cells as judged by trypan blue exclusion. Cell culture: 2.5 x lo6 Hepatocytes in 5 ml Eagle's minimal essential medium (8) supplemented with 20% fetal calf serum, nonessential amino acids and antibiotics (9) were plated into 60 mm Falcon plastic dishes. After 4-6 h the non-attached cells (20-40%) were sucked off and fresh medium was added. 0006-291X/79/201348-07$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
1348
BIOCHEMICAL
Vol. 90, No. 4, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
In some experiments the fetal calf serum containing medium was replaced 4 h after plating by a chemically defined, serum free medium (IO, modified according to 11). In the presence of the serum free medium the hepatocytes maintained the polygonal shape and association as confluent monolayers for at least five days (Fig. IA and B). The hepatocyte cultures were essentially free of Kupffer cells as demonstrated by the lack of phagocytosis of ink (Giinther Wagner, Hannover, Germany) and of fluorescein isothiocyanate conjugated IgG (Behringwerke, Marburg, Germany). All experiments were done with anti-human fibronectin Antisera: rabbit serum from Behring Werke (Marburg, Germany). The immunofluorescence studies were done in addition with anti-human fibronectin rabbit serum provided by Dr. J. Hijrmann (Max-Planck-Institut fiir Biochemie, Miinchen, Germany) and an antiserum raised in rabbits against fibronectin isolated from human plasma according to Engval and Vaheri (12). The three antisera gave identical results in the immunofluorescence studies and gave rise to a single precipitation line in immunoelectrophoresis. After five washings with phosphate buffered Immunofluorescence: the hepatocytes were incubated for 30 min saline (PBS), pH 7.5 (13) with anti-human fibronectin rabbit serum followed by incubation for 30 min with fluorescein isothiocyanate (FITC) conjugated antirabbit IgG from goat (Behringwerke). Controls were incubated first with rabbit serum and then treated with FITC-conjugated anti-rabbit of intracellular fibronectin the hepatocytes IgG. For detection were dried for 24 h at 37OC and stained as described above. Immunofluorescence was observed with a fluorescence microscope fitted with epi-illumination and a filter system adapted for FITC (Leitz, Wetzlar, Germany, Orthoplan microscope equipped with Orthomat camera). 14 Immunoprecipitation of [ Cl-fibronectin: Hepatocytes were incubated for 24 h with culture medium containing ascorbic acid, and [14C]-glycine, 2.5 uCi/ml, (Amersham-Buchler, speci50 ug/ml, fic activity 118 mCi/mmol). [14Cl-Fibronectin was isolated from culture medium by immunoprecipitation: 100 ul of culture medium was incubated with 0.5 ml of a fibronectin antiserum for 24 h at 4OC. The immunocomplex was precipitated with 100 ~1 of anti-rabbit IgG for 12 h at 4oC. The precipitate was washed three times with PBS containing 1% (w/v) bovine serum albumin, solubilized in 8 M urea with dithioerytrol (50 ug/ml), assayed for radioactivity and subjected to polyacrylamide gel electrophoresis. Controls were processed identically except that antiserum was replaced by rabbit serum. Cell protein was determined according to Kaltwasser et al. (14)
Identification of [I4 Cl-fibronectin by polyacrylamide electroAli uots of medium obtained after incubation in the phoresis: acid were dialysed and presence of [ q4Cl-glycine a nd ascorbic The samples were solubilized in 8 M urea with or freeze dried. without dithioerytrol (50 us/ml) and applied without using a stacking gel to 110 x 110 x 1.5 mm polyacrylamide slab gels. Electrophoresis was carried out in sodium dodecyl sulphate-containing buffers using the system of Laemmli (15). A one-step gradient of acrylamide concentration was used with 12% in the lower and 5% in the upper part. Autofluorography was done as described by Laskey and Mills (16). Fibronectin isolated from human plasma according to Engval and Vaheri (12) served as reference. Results: plating
Immunofluorescence: in fetal
calf
Hepatocytes
serum-containing
1349
examined
4 h after
medium were free
of
fibro-
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 90, No. 4, 1979
nectin
BIOCHEMICAL
as examined
fibronectin
by indirect
appeared
1 D).
covered
in these
medium fibers plating
cultures
fibronectin of
[
Cl-fibronectin:
[14 Cl-glycine
with
immunoprecipitated was dissolved amide gel
under
2 A).
Only
coprecipitated Culture
presence
of
Fluorography
revealed
Fig.
When the 1:
1 F).
for
24 h
medium was
The precipitate
revealed
14
of
(Fig.
were labelled
and subjected
amounts of other
deposition
became visible
antiserum.
from
When cells
washing,
same mobility
3 days old
cultures
to polyacrylamide
SDS with
and non-reduced
fibronectin were grown in
The culture
Fluorography the
3
to polyacryl-
the presence
of
as fibronectin
C-labelled
compounds
fibronectin.
medium of applied
plating.
conditions
with
trace
with
directly
2 B-E).
reducing
material
cells
a fibronectin
electrophoresis.
radiolabelled (Fig.
with
of
culture).
Hepatocytes
3 days after
After
were seen beginning
by extensive the
of
was observed
When hepatocytes
1 C, 2 days-old
underneath
fibers
cultures.
network
fibronectin
were detached
14
a thick
1 E).
of
(Fig.
located
Synthesis
(Fig.
Fine
of fibronectin
cultures
the hepatocytes
one day after
hepatocyte
distribution
On 5 days-old
a serum-free
immunofluorescence.
on one day old
days a more patch-like (Fig.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
or without that
gel treatment
the major
14
samples had same mobility cells
were labelled
labelled
as above was
electrophoresis with
in the
dithioerytrol.
C-labelled
band in reduced
as fibronectin
4 h after
(Fig.
plating,
[14C]-
Indirect immunofluorescence localization of cell surface attached fibronectin. (A) Phase contrast micrograph of hepatocytes 4 h after plating. (B) Phase contrast micrograph of hepatocytes 8 h after feeding with serum-free medium. (C) Staining for fibronectin of a culture 2 days after feeding with serum-free medium. (D) Staining for fibronectin of 3 days-old cultures. (E) Staining for fibronectin of 5 days-old cultures. (F) Staining for fibronectin of a culture dish, from
which hepatocytes grown medium had been detached to staining. Magnification: 160-fold for
1351
for 2 days with serum-free by extensive washing prior
A and B,
500-fold
for
C-F.
Vol. 90, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
55
5% ;
-
12J 12% ,
red.
red
red.
red. Fig.
2:
Autofluorograms of medium obtained after incubation of hepatocytes in the presence of [14C]-glycine. Hepatocytes were grown in the presence of [14C]-glycine for 24 h either 3 days after plating (slot A-E) or 4 h after plating (slot F and G). (A) Immunoprecipitate of the medium after reduction. (5) Non-reduced medium. (C) Reduced medium. (D) Non-reduced medium, film exposure 4 times as long as in (A). (E) Reduced medium, film exposure 4 times as long as
in (B).
(F) Non-reduced medium. (G) Reduced medium. Arrows mark the position standard. red.: Reduced.
fibronectin
appeared
synthesized
14C-labelled
Discussion:
Fibronectin
cytes
as early
staining. likely present
that
secretory could
this
experiments fibronectin
was established products
a human
a minor
plasma
component
products
be detected plating, with
(Fig.
serum-free
of
1352
of newly 2 F and G). hepato-
medium made it
from the calf
its
rat
immunofluorescence
[ 14 Cl-fibronectin
by identification
as fibronectin,by
fibronectin
on cultured
using
originated
in the medium. Synthesis
hepatocytes secretory
to be only
as 24 h after
Control
of
un-
serum by rat
of one of the mobility
in gel
electro-
Vol. 90, No. 4, 1979
phoresis
BIOCHEMICAL
and precipitation
serum.
Surprisingly,
secretory
by anti-human
fibronectin
protein
of
indicate
that
the
creases
markedly
with
culture
The physiological
hepatocytes direved to
serve
of
fibronectin
association
cytes
(18,19).
Thus,
that
freshly
after isolated
collagen
under
This
meinschaft
both the
for
conditions
medium (5).
Collagen
hepatocytes
hepatocytes
(unpublished
surface
Deutsche
of
report is
are hepato-
may play
a
demonstra-
synthesized
do not
by
synthesize
results).
of Miss F. Weber is gratefully by the
absence
and collagen
The present fibronectin
appears
in the
and collagen
whereas
isolated
fibronectin
sinusoidal
lag phase,
Ra 255/3
in-
by hepa-
exogenous
fibronectin
fibronectin
work was supported (Grant
of freshly
culture
hepatocytes, in vitro
release
and spreading
of hepatocytes.
ACKNOWLEDGMENT; The help ledged.
Cl-fibronectin
synthesized
with
an initial
Preliminary
fibronectin
substrate
in vivo
in the anchorage
14
requires
In vivo,
(17).
in close
tes
of
serum in the
as an attachment
[
cultures.
time.
dishes
found
role
of
Attachment
on plastic
from the
rate
rabbit
to be the major
hepatocyte
function
is unknown.
plasma fibronectin
appeared
4 days-old
results
tocytes
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
acknow-
Forschunqsqe-
and SFB 104).
References: 1.
Vaheri, 515,
A. and Moster,
D.F.
(1978)
Biochim.
Biophys.
Acta
l-26.
Pearlstein, E. (1976) Nature 262, 497-500. 3. Grinell, F., Hays, D.G. and Minter, D. (1977)
2.
110,
Exp.
Cell
Res.
175-190.
4. Pena, S.D.J. and Hughes, R.C. (1978) Nature 276, 80-83. 5. Hook, M., Rubin, K., Oldberg, A. and tibrink, B. (1977) Biochem. Biophys. Res. Comm. 79, 726-733. 6. Seqlen, P-0. (1976) in "Methods in Cell Biology", Vol. XIII (Prescott, D., Ed.) pp 29-83, Academic Press, New York and London. 7. Ullrich, K., Basner, R., Gieselmann, V. and von Figura, K.(1979) Biochem. J. 180, 413-419 8. Eagle, H. (1959) Science 130, 432-437. 9. Cantz, M., Kresse, H., Barton, R.W. and Neufeld, E.F. (1972) Meth. Enzymol. 28, 884-896.
1353
Vol. 90, No. 4, 1979
IO. 11. 12. 13. 14. 15. 16. 17. 18. 19.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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