Download Laminin, a Noncollagenous Component of

[CANCER RESEARCH 41, 1518-1524,
0008-5472/81
/0041-OOOOS02.00
April 1981]
Laminin, a Noncollagenous Component of Epithelial Basement Membranes
Synthesized by a Rat Yolk Sac Tumor1
Ulla Wewer,2 Reidar Albrechtsen, and Erkki Ruoslahti
The University Institute of Pathological
Anatomy, Frederik V's 11, DK-2100 Copenhagen 0, Denmark [U. W., R. A.], and La Jolla Cancer Research Foundation,
La
Jolla, California [E. R.¡
ABSTRACT
Laminin, a glycoprotein antigenically similar or identical to a
component of epithelial basement membranes, was identified
as a major component of the abundant extracellular matrix
synthesized by an experimentally induced rat yolk sac tumor.
Immunocytochemical staining revealed laminin in cultured tu
mor cells as well as in their extracellular matrix. The presence
of soluble laminin in the culture media of the tumor cells was
demonstrated using metabolic labeling followed by identifica
tion by immunoprecipitation
and sodium dodecyl sulfate:polyacrylamide gel electrophoresis. This revealed two polypeptides
with molecular weights of approximately
200,000
and
400,000. These comigrated with the polypeptides of mouse
laminin isolated previously.
The yolk sac tumor tissue grown in vivo contained laminin in
the tumor cells and in the extracellular material as evidenced
by immunofluorescence
and immunoperoxidase staining. Im
munization with the tumor matrix resulted in an antiserum that
contained antilaminin and antifibronectin and was made spe
cific for laminin by absorption with fibronectin. This antiserum
precipitated laminin polypeptides from culture medium of yolk
sac tumor cells and stained basement membranes in rat tissues
in a manner indistinguishable from antilaminin. The presence
of laminin in rat yolk sac cells, the presumed origin of our yolk
sac tumor, was studied in some detail. Laminin was found to
be present in normal cells of the visceral as well as the parietal
yolk sac layer and in their basement membranes suggesting,
but not proving, that both types of cells have ability to synthe
size laminin.
Production of laminin and the presence of laminin-containing
basement membrane material may be important for the biolog
ical behavior of the yolk sac tumor. This tumor will also be a
useful source of laminin for chemical and biological character
ization of this basement membrane protein.
INTRODUCTION
Basement membranes are believed to be necessary for cell
recognition, adhesion, and other important cell functions (9,
10). Characterization of basement membranes in normal and
pathological states has been hampered by low solubilities of
their constituents and the small quantities of material available
for their examination. However, previous studies have shown
that basement membranes contain collagens, noncollagenous
glycoproteins, and proteoglycans (1, 12-15, 18, 31).
1 Supported
by a grant from the Danish Cancer Society and by Grants CA
27455 and CA 28101 from the National Cancer Institute, Department of Health
and Human Services.
2 To whom requests for reprints should be addressed.
Received September 8, 1980; accepted January 9, 1981.
1518
Recent studies on a mouse embryonal carcinoma-derived
cell line have provided new information about the noncollage
nous basement membrane components. These cells synthesize
an extracellular matrix which primarily consists of 2 glycosylated polypeptides (5, 6). Timpl et al. (35) have isolated similar
polypeptides by extraction of basement membrane material
synthesized by a transplantable mouse tumor, the EHS sar
coma (20). They named the material laminin. Laminin is com
posed of 2 polypeptide chains with molecular weights of about
220,000 and 440,000. The endodermal cell matrix proteins
and laminin have been found to be very similar (29, 35) and
are probably identical. Laminin is distinctly different from an
other noncollagenous component of basement membranes,
fibronectin (for recent reviews on fibronectin, see Refs. 19 and
24), with regard to size, amino acid composition, and immunological reactivity (6, 29, 35). Antisera against laminin react
not only with basement membranes of the cultured cells and
tumor used as source of laminin (6, 35) but also with basement
membranes in normal tissues (6, 8, 17, 23, 29, 35), indicating
that studies on laminin will provide information on basement
membranes of normal as well as tumor tissues. We show here
that laminin is a component of the extracellular matrix of a rat
yolk sac carcinoma cell line that synthesizes large quantities of
basement membrane hyalin in vivo and in vitro. This cell line
may offer a valuable source of laminin and a model for studies
on basement membranes.
MATERIALS
AND METHODS
Yolk Sac Tumor. An experimental yolk sac tumor induced in
a Lewis rat was used.3 A method somewhat similar to that of
Vandeputte ef al. (36) was used, but instead of fetectomy and
tumor virus infection, or fetectomy alone (28, 30, 33), the
tumor was induced by puncturing the embryos through the wall
of the uterus at the tenth day of gestation. Some teratomas and
one yolk sac tumor were obtained in 22 rats. Cell cultures were
established from expiants of the yolk sac tumor, designated
L2, and have been grown in culture for 3 years. The cells were
cultured in MEM4 with Earle's salts supplemented with 10%
fetal bovine serum, glucose (0.5 g/liter), 1% MEM amino acids
solution (50x), 1% MEM vitamins solution (100x) (all from
Grand Island Biological Co., Grand Island, N. Y.), gentamicin
(10 fig/ml) (Schering Corp., Kenilworth, N.J.), and Mycostatin
(5 UE/ml) (E. R. Squibb and Sons, London, England), pH 7.2
to 7.4. Cultures were grown in 25- or 75-sq cm tissue culture
3 U. Wewer and R. Albrechtsen. Experimental teratomas and a yolk sac tumor
induced in Lewis rats by a puncture method, manuscript in preparation.
4 The abbreviations used are: MEM, Eagle's minimal essential medium; PBS,
phosphate-buffered
saline [0.137 M NaCI:0.008 M Na2HPO4:0.015 M KH2PO4
<pH 7.4)]; SDS, sodium dodecyl sulfate; PB. 0.05 M sodium phosphate buffer
(7.4); PAS, periodic acid-Schiff solution.
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Laminin in a Rat Yolk Sac Tumor
flasks (A/S Nunc, Roskilde, Denmark) at 37°in humidified 5%
CO2:95% air. The cells were routinely transferred on a 3- to 4day schedule after detachment by 0.01 % Trypure (Novo Industri A/S. Copenhagen, Denmark). Cultured cells used in this
study were taken from passages 20 to 75. The tumor was also
grown in ascitic form and was serially transplanted every 2 to
3 weeks.
For cytological investigation of the cultures, tumor cells were
grown on coverslips placed in BélicoLeighton tubes (Bélico
Glass, Inc., Vineland, N. J.). For immunofluorescence
and
immunoperoxidase investigótion, cultured cells and pieces of
tumor tissue, including cellular sediment obtained by centrifugation from ascitic fluid, were fixed for 15 min (cultured cells)
or 20 hr (tumor tissue and ascitic cells) in ethanohglacial acetic
acid (99:1, v/v) at +4° (26). The samples were dehydrated in
ethanol and xylene, embedded in paraffin, and serially sec
tioned. Tissue blocks from normal adult rat kidney and from
organs of 16th day rat fetuses including placenta and the yolk
sac membranes were treated and examined in a similar manner.
In some experiments, fixéecultured tumor cells and tissue
sections were pretreated with 1% collagenase (177.2 units/
mg; Worthington Biochemical Corp., Freehold, N. J.) solution
at pH 7.5 and hyaluronidase (5.000 ID/ml; Penetrase; Leo,
Lpvens Kemiske Fabrik, Coaenhagen, Denmark) at pH 8.5 for
1 hr at room temperature. In some cases, frozen sections, air
dried for 1 hr and fixed for ' 5 min in acetone, were also used.
Antisera. Antilaminin was prepared by immunization with
laminin isolated from the matrix of cultured mouse embryonal
carcinoma-derived
endodermal cells. The preparation and
specificity of this antiserum have been described (27, 29). An
antiserum was prepared against hyalin basement membrane
material from the rat yolk sac tumor. Tumor mass with abundant
hyalin (transplant passage 22) was homogenized in PBS con
taining 1% Triton X-100 (Sigma Chemical Co., St. Louis, Mo.),
and the insoluble residue was washed 3 times with the same
solution and used to immunize a rabbit. The rabbit received an
i.m. injection of about 1 mg of the insoluble hyalin material
suspended in 0.5 ml of PBS and mixed with 1 ml of Freund's
complete adjuvant (Grand Island Biological Co.). Similar injec
tions were given 14 and 28 days later and once a month
thereafter. The rabbit was bled after the third and each sub
sequent injection. The resulting antiserum contained antibodies
to fibronectin as evidenced by immunodiffusion tests and bind
ing of 125l-labeled fibronectin in radioimmunoassay. These an
tibodies were removed by absorption with rat fibronectin cou
pled to Sepharose (25). Thu antiserum was also absorbed with
rat serum proteins coupled to Sepharose. The presence of
antibody in the absorbed serum against rat yolk sac tumor
basement membrane was determined by indirect immunofluo
rescence. Antisera against rat fibronectin have been described
(25). Anti-mouse laminin kindly provided by Dr. George Martin,
National Institute of Dental Research, was also used in this
study.
Immunoprecipitation.
Cultures of rat yolk sac tumor cells
were labeled with [35S]me':hionine (5 fiCi/ml; Amersham, Ar
lington Heights, IL.) for 24 ir and added to the culture medium
(Dulbecco's modified MEM without methionine supplemented
with 10% fetal bovine serum). The media were analyzed for the
presence of labeled prote ns reactive with the anti-basement
membrane, antilaminin, ard antifibronectin sera. Ten to 200
/tl of antiserum or normal rabbit serum as a control were added
to 1 ml of medium, and the immune complexes were isolated
after a 2-hr incubation by adding 100 /il of a 50% suspension
of protein A-Sepharose (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) in PBS. Complexes were extracted from protein
A:Sepharose by boiling the washed particles for 2 min in a
solution containing 4% SDS, 6 M urea, and 0.06 M Tris-HCI,
pH 8.8. The extracts were analyzed by polyacrylamide gel
electrophoresis in the presence of SDS and 2-mercaptoethanol
(16). The gels were stained with Coomassie blue, dried, and
subjected to fluorography. Laminin purified from the matrix of
mouse endodermal cells (29) was used as a marker.
Immunological Staining Procedures. Sections used for im
munoperoxidase (34) and immunofluorescent
staining were
washed in PB and then depleted of endogenous peroxidase by
incubation for 30 min in 1% hydrogen peroxide in methanol.
The sections were washed with PB for 10 min and preincubated
at room temperature for 15 min with a 1:10 dilution of normal
swine serum (Dako, Copenhagen, Denmark). Antigens were
demonstrated by a 2-step technique with optimally diluted antibasement membrane (1:10) and antilaminin (1:50) and antifi
bronectin (1:50) sera. The sections were incubated with the
antisera for 1 hr. Fluorescein- or peroxidase-conjugated
swine
anti-rabbit IgG (Dako, Copenhagen, Denmark) was used as a
second layer, diluted to 1:50, and incubated for 60 min. All
incubations were carried out at room temperature. After 3
washes with PB, the coverslips were mounted in Aquamount
(Gurr, London, England) and examined for fluorescence by a
Leitz microscope equipped for epiillumination. The peroxidase
conjugates
were detected with 3-amino-g-ethylcarbazole
(Sigma) followed by a 1-min hematoxylin counterstaining. Dem
onstration of antigens in cultured cells was carried out in a
similar manner. Normal rabbit serum instead of the antisera
was used as a negative control. Additional sections were
stained with hematoxylin:eosin and with PAS after a preceding
treatment with amylase.
RESULTS
Immunocytochemical Staining of Laminin and Fibronectin
in Cultured Yolk Sac Tumor Cells. The cultured yolk sac
tumor cells grew in a monolayer as fusiform cells and as fast
growing small aggregates which detached spontaneously from
the substratum. These aggregates formed small cysts which
contained PAS-positive material. The tumor aggregates often
reattached to the substratum and formed new colonies. The
tumor cells showed nuclear polymorphism and hyperchromasia
with many mitoses. Their cytoplasm was often vacuolated and
contained hyalin droplets which were PAS positive with variable
intensity. Trypsinized tumor cells from cultures transplanted
s.c. into nude mice (107 cells) formed tumors that were histologically similar to the rat transplants. Chromosome analysis
showed aneuploidy. The cultures were screened for Mycoplasma and were found to be negative. The Mycoplasma tests
were kindly performed by Dr. K. Lind, Statens Seruminstitut,
Copenhagen, Denmark.
The 2 antilaminin sera and the antiserum against tumor
basement membrane stained the tumor cells with identical
patterns. The staining was mainly found in the cells located in
the periphery of a colony and in the cells present in the
aggregates. In some cells, the staining was confined to small
granules concentrated around the nuclei (Fig. 1). In other cells,
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1519
U. Weweretal.
positive material was seen surrounding a central negative area,
and in still others, large vacuoles were stained. Extracellular
laminin was seen as fibrillar or granular deposits close to the
cells. Staining for fibronectin revealed only a weak reaction in
the tumor cells (not shown).
Characterization of Labeled Proteins in Cultures of Yolk
Sac Tumor Cells. Antisera to fibronectin and laminin and to
yolk sac tumor basement membrane were used to ¡mmunoprecipitate antigens from cultures of yolk sac tumor cells labeled
with [35S]methionine. SDS:polyacrylamide gel electrophoresis
revealed 2 polypeptides with molecular weights close to
200,000 and 400,000 in the immunoprecipitates obtained with
antilaminin (Fig. 2). These comigrated with the polypeptides of
mouse laminin (29) and were somewhat smaller than the mono
mer and dimer of human plasma fibronectin, respectively. The
anti-basement membrane serum brought down similar polypep
tides but was weaker than the antilaminin serum. No radioactive
bands were detected in the material precipitated with antifibronectin or in the normal rabbit serum control.
Laminin and Fibronectin in Transplantable Yolk Sac Tumor
and in Some Normal Fetal and Adult Tissues. The yolk sac
tumor grew in vivo as peritoneal nodules. Ascitic fluid was
present and contained numerous tumor cell aggregates. The
cell aggregates were similar to those seen in the cell cultures.
A core of hyalin was present in the aggregates and nodules.
The glycoprotein nature of the hyalin material was suggested
by a strong PAS reaction (Fig. 3). Antilaminin and anti-base
ment membrane sera stained the ascitic tumor cells to a vari
able extent (Fig. 4). The material in the hyalin core was nearly
always negative. Pretreatment with collagenase and hyaluronidase did not bring about staining of negative areas. However,
in some areas, positive reaction for laminin was seen as a thin
membrane which separated the tumor cells from the deposit.
Nonorganized tumor cells present in ascites had mainly intracellular laminin (Fig. 4). The intracellular staining tended to
disappear as the tumor cells became part of tumor nodules
with extracellular basement membrane material. In sections of
peritoneal tumor masses, laminin was consistently found
around the tumor cells while the cytoplasm of the cells was less
strongly positive (Fig. 5).
Fibronectin could not be demonstrated immunocytochemically in the tumor cells obtained from ascites or from peritoneal
tumor masses, but it was present in the surrounding loose
connective tissue and in blood vessels (Fig. 6).
Laminin staining in 16th day rat fetuses was strongly positive
in the cells of the visceral yolk sac. Positive reaction was also
found as a linear deposit in the visceral yolk sac membrane as
well as in the mesothelium lining the extraembryonic celom
(not shown). Parietal yolk sac cells stained weakly for laminin
(Fig. 7). Reichert's membrane of the parietal yolk sac endoderm
was consistently negative with the exception of a thin line in
contact with the parietal cells. In the placenta, staining was
confined to the basement membrane of the vessels. The base
ment membranes and the cytoplasm of occasional epithelial
cells of the fetus proper also stained for laminin (not shown).
An identical localization pattern was observed in all these
tissues when antisera to laminin and to yolk sac tumor base
ment membrane were used. The antilaminin and anti-basement
membrane sera also stained basement membranes in adult rat
organs. The staining patterns were similar to what has been
described previously for mouse tissues (6, 29), except in the
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kidney where the staining in the case of rat kidney was largely
confined to the tubular basement membrane (Fig. 8), while
both the tubular and glomerular basement membranes are
positive in the mouse (6, 29).
DISCUSSION
We have identified a component of basement membrane
synthesized by a rat yolk sac tumor as laminin, a recently
described glycoprotein (6, 35). The tumor cells incorporated
[35S]methionine into 2 polypeptides that reacted with antilami
nin and comigrated in SDS:polyacrylamide gel electrophoresis
with the polypeptides with molecular weights of 200,000 and
400,000 of mouse laminin.
Laminin in the mouse is one of the first extracellular matrix
glycoproteins detectable in the embryo during development. It
is found as early as in the 16-cell compacted mórula. At this
stage, laminin is also demonstrable intracellularly, but it dimin
ishes when organoid development has taken place (1 7). The
present ¡mmunocytological findings show a similar shift in the
localization of laminin. Laminin was mainly intracellular in single
tumor cells, but when the tumor cells were growing as a tumor
mass where extracellular laminin was present in the tumor
basement membranes, intracellular laminin was less evident. It
seems that the synthesis of laminin by the tumor cells is
transient and takes place at the time before the basement
membrane is established.
We found immunocytochemically
detectable laminin in the
tumor hyalin, but the staining was variable and occasionally
quite weak. Treatment of the sections with collagenase and
hyaluronidase did not improve the staining. That laminin never
theless is a major component of the basement membrane
material of the yolk sac tumor is suggested by the fact that
immunization with this material gave an antiserum which, after
removal of antibodies to fibronectin, appeared specific for
laminin, as judged from immunoprecipitation and immunofluorescence results. The variable staining for laminin in the tumor
hyalin could be due to masking of laminin by other proteins, or
it could be that the molecular packing in this material is so tight
that it prevents access of antibodies. We also found poor
staining for laminin in the glomeruli of the rat kidney. In Reich
ert's membrane, staining was only present in the layer closest
to the yolk sac cells. This corresponds to the newly synthesized
part of the membrane (18). In the mouse, all of Reichert's
membrane as well as the kidney glomeruli stain intensely for
laminin (6, 8, 29). While we cannot exclude true species
differences in the distribution laminin, it seems more likely that
a penetration problem or masking phenomenon, or both, are
involved. We have found that the tumor hyalin stains strongly
for laminin after a gentle treatment of the sections with trypsin.
It is interesting to note that Pierce et al. (22) almost 20 years
ago prepared antiserum against the basement membrane ma
terial of a mouse teratocarcinoma and that these antibodies
stained basement membranes much the same way as antilam
inin. The antibodies reactive with basement membranes in the
antisera of Pierce ef al. (22) as well as similar antisera de
scribed more recently by others (11, 21) probably consist
largely of antilaminin.
Our rat yolk sac tumor cells also seem to synthesize fibro
nectin but at levels much lower than those of laminin. Fibro
nectin was demonstrable in the tumor cells in the cultures by
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Laminin in a Rat Yolk Sac Tumor
immunocytochemical
stainirg but not by immunoprecipitation
of radiolabeled proteins from culture media. Quantitäten of the
2 proteins in culture media by radioimmunoassay and enzyme
immunoassay has revealed 30 to 100 times more laminin than
fibronectin.5
The precise origin of our tumor is not clear, but its morphol
ogy is similar to that of turners induced by viral infection of the
placental and fetal membranes (32, 36) or by displacement of
the visceral yolk sac outside the uterus after fetectomy (28).
Such tumors have generally been found to product a-fetoprotein (3, 28), a product characteristic of the visceral yolk sac (2,
7). The present tumor also synthesized large quantities of afetoprotein in the first year after it was established.3 The pas
sages used in this work, however, no longer synthesize signif
icant quantities of a-fetoprolein,6 and the tumor in this respect
now resembles the parietal yolk sac endoderm. One explana
tion could be that passaging the tumor selected for the parietaltype cell at the expense of other cells present in the original
tumor, but it is also possible that tumors originating from yolk
sac endoderm have the capacity to show the differentiated
characteristics of both visceral and parietal yolk sac endoderm.
The biological role of laminin is not known, but it has been
suggested that laminin is invalved in the development of kidney
tubuli during embryogenesia (8). Furthermore, cells from re
generating liver attach and spread on surfaces coated with
laminin, and a transient appearance of laminin in the liver
accompanies liver regeneration.7 These results suggest that
laminin may be involved in cellular differentiation and prolifer
ation. The presence of large amounts of laminin thus may
influence the biological behavior of yolk sac tumors.
A number of endodermal cell lines that synthesize laminin in
vitro but are not tumorigenic have been described (4). Timpl ef
al. (35) have described a turror believed to be of a sarcomatous
nature as a source of laminin in the mouse. The present results
show that a similar protein is synthesized by our rat yolk sac
tumor. This tumor offers a source of laminin in a different
species. The availability of laminin should be helpful in the
elucidation of the function of this apparently important base
ment membrane glycoprotein.
ACKNOWLEDGMENTS
We thank Drs. Eva Engvall, Edward Hayman, and Shigeo Sakashita for their
help with several aspects of this stuc y and Dr. Eileen Adamson for her comments
on the manuscript.
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CANCER
RESEARCH
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Fig. 1. Immunofluorescence stain ng in rat yolk sac tumor cells with antilaminin. The cells were cultured for 2 days. Positive reaction is mainly found inside the
cells as granular deposits. Extracellular fibrillar staining is seen in some areas, x 650.
Fig. 2. Polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol. Material precipitated from [35S]methionine-labeled culture media of rat
yolk sac tumor cells by antifibronectin (Õ),normal rabbit serum (2), antilaminin (3), and anti-basement membrane serum (4). Arrows, top of the gel (upper arrow) and
the position of myosin (Vf) standard (VI.W. 200,000).
Fig. 3. PAS staining after amylasu treatment of transplanted yolk sac tumor nodule. Abundant PAS-positive basement membrane material surrounds the tumor
cells, x 180.
Fig. 4. Immunofluorescence staining of tumor aggregates obtained from ascitic fluid with antiserum to yolk sac tumor basement membrane. Note the predominantly
intracytoplasmic staining, x 500.
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Fig. 5. Immunofluorescence
staining with antilaminin of yolk sac tumor nodule. Note intense staining for laminin in the periphery of the hyalin deposits. The
cytoplasm of the tumor cells as well as the connective tissue are negative, x 250.
Fig. 6. Immunofluorescence
staining with antifibronectin of yolk sac tumor nodule similar to that in Fig. 5. The tumor cells and the hyalin are negative. Positive
staining is found in the connective tissue, x 250.
Fig. 7. Immunoperoxidase staining with antilaminin of the parietal layer of the yolk sac endoderm from a 16th day rat fetus. The cells (P£)are positive. A linear
staining separates these cells from the underlying Reichert's membrane (W), the rest of which does not stain. No staining can be found at the transition between the
membrane and the placenta (PL), x 300.
Fig. 8. Immunofluorescence staining for laminin in the cortex of adult rat kidney. The tubular basement membrane is positive, while there is little staining in the
glomeruli. X. beginning of positive staining at the urinary pool, x 250.
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Laminin, a Noncollagenous Component of Epithelial Basement
Membranes Synthesized by a Rat Yolk Sac Tumor
Ulla Wewer, Reidar Albrechtsen and Erkki Ruoslahti
Cancer Res 1981;41:1518-1524.
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