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Int. J. Cancer: 67,668-675 (1996)
0 1996 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de I’Union Internationale Contre le Cancer
CHARACTERIZATION OF TUMORIGENIC SUB-LINES FROM A POORLY
TUMORIGENIC HUMAN COLON-ADENOCARCINOMA CELL LINE
Teresa LOPEZ-CONEJO,
Nieves OLMO,Javier TURNAY,
Juana NAVARROand Antonia LIZARBE’
Departamento de Bioqiiimica y Biologia Molecular, Facultad de Qiiimrcas, Universidad Complutense, 28040-Madrid, Spain.
The interaction of tumor cells with extracellular-matrix
components is suspected to play an important role in tumorigenesis induction. The tumorigenicity of a poorly tumorigenic
human colon-adenocarcinomacell line (BCS-TC2) was induced
by co-injection with Matrigel. A new cell sub-line, BCS-TC2. I,
was isolated and establishedfrom these tumors. Implantationof
these cells in nude mice in the absence of Matrigel-generated
tumors which allowed the establishment of another tumorigenic cell sub-line, BCS-TC2.2. Matrigel and laminin, but not
collagens, promote the tumorigenicity of BCS-TC2 cells, probably due to specific interactions of a pre-existing minor cell
sub-population with laminin, which facilitate the initial growth
of these cells in vivo. Cytogenetic analysis reveals that both
sub-lines originate from the parental one, but a new marker in
chromosome 9 is observed. These sub-lines present a lower
degree of differentiation, as deduced from the lower CEA
content, 5‘-nucleotidase and alkaline-phosphatase activities.
No variation is observed in the mRNA and protein expression of
the 67-kDa laminin-bindingprotein. However, an increase in PI
integrins and a parallel decrease in p4 integrin were detected.
Thus, the new sub-lines, compared to the parental cells, present
karyotypic and phenotypic differences such as the expression of
a distinctive integrin pattern. This system represents a useful
model for understanding the development and progression of
tumorigenicity in cancer cells.
o I996 Wiley-Liss,Inc.
Data from the American Cancer Society reveal the high
incidence and mortality of colorectal cancer. Lung, prostate,
breast and colorectal cancers are the most common causes of
cancer death. Diagnosis, surgical techniques and therapies
have been greatly improved, but to date most deaths from
colorectal and other cancers are caused by metastasis. Knowledge of the regulatory mechanisms of tumor growth, local
infiltration and metastasis could help to find more effective
therapy to deal with disseminated cancer cells; this is dependent on the availability of relevant in vivo models for this
disease.
The extracellular matrix (ECM) plays an active role during
healing and development as well as in tumor growth and
progression, modifying tumor-cell adhesion, migration and
differentiation (Ruoslahti, 1992; Passaniti et al., 1992; Noel et
al., 1993; Grant et al., 1994). In the carcinogenic process, the
tumor cell must migrate through the surrounding connective
tissue and disrupt basement membranes, a specialized ECM,
allowing the cells to cross these barriers and disseminate
through the organism. Individual ECM components appear to
enhance the malignant phenotype in vitro and promote tumorcell growth in vwo (Passaniti et al., 1992). In fact, the
malignancy of tumor cells has also been associated with
adhesion to specific ECM components (Ruoslahti, 1992; Kim
etal., 1995).
The use of Matrigel, a reconstituted basement membrane
derived from the EHS murine tumor, has greatly improved
understanding of the role of ECM in tumor growth. Matrigel
promotes tumor-cell growth in vitro (Kleinman et al., 1986;
Fridman et al., 1990, 1992; Albini et al.. 1992; Vukicevic et al.,
1992u; Noel et al., 1993) and enhances in vivo tumorigenicity of
transformed cells when co-injected into nude mice (Fridman et
al., 1990, 1992; Albini et ab, 1992; Vukicevic et al., 19926;
Topley rt a/., 1993). The interaction of cell-adhesion receptors
with ECM components can affect the biological behavior of
neoplastic cells by influencing the process of tumor invasion
and metastasis (Castronovo, 1993; Kim et al., 1995). Supporting this notion, a 67-kDa high-affinity laminin-binding protein
has been directly related to the invasive and metastatic
potential of several tumor cell lines (Rao et al., 1989; Castronovo, 1993). It has also been suggested that the modulation of
the levels of specific integrins is associated with these processes. There is increasing evidence that integrin expression is
altered following malignant transformation (Dedhar and Saulnier, 1990; Castronovo, 1993; Kim et al., 1995; Fujita et al.,
1995). However, no uniform pattern of changes with this
process has been described up to date.
BCS-TC2 cells, derived from a primary and poorly differentiated human colon adenocarcinoma, provide an interesting
model for studying the influence of the ECM on the malignant
behavior of epithelial transformed cells. In the present study
we demonstrate that Matrigel modifies the tumorigenicity of
these human transformed cells. BCS-TC2 cells show very low
tumorigenicity after S.C. injection into nude mice, either alone
or in the presence of different types of collagens. However, the
malignant potential is greatly enhanced when the cells are
co-injected with Matrigel or laminin, the major basementmembrane glycoprotein. We show that interaction of Matrigel
with these adenocarcinoma cells selects a sub-set of neoplastic
cells which, after propagation and establishment in culture,
show tumorigenic potential by themselves in the absence of
Matrigel. While the expression of the 67-kDa laminin-binding
protein is apparently not modified, changes in several characteristics and alteration in the expression pattern of different
integrins with affinity to laminin have been detected in these
cells.
MATERIAL AND METHODS
Matrigel, ECM protein preparations and antibodies
The “reconstituted basement membrane” gel, Matrigel,
containing laminin, type-IV collagen, nidogen and heparansulfate proteoglycan, was extracted from the Engelbreth-HolmSwarm (EHS) tumor as described by Kleinman et al. (1986).
Low Matrigel, from which low-molecular-weight components
were partially removed, was prepared according to Vukicevic
et al. (1992b). Nidogen-free laminin, type-I, type-IV and
type-V collagens were prepared as described by Olmo et al.
(1992).
Mouse anti+, chain (Alex 1/4), anti-al chain (TS 217) and
anti-as chain (HP 2/1) monoclonal antibodies (MAbs) were
kindly provided by Dr. Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain). Rat MAb (GoH3) specific for an extracellular epitope on sub-unit, was a generous gift from Dr. A.
Sonnenberg (Blood Transfusion Service, Amsterdam, The
Netherlands). Mouse anti-67-kDa laminin-binding protein
MAb (MLUCS) was a gift of Dr. S. MCnard (Istituto Nazionale
Tumori, Milan, Italy). MAbs against the a2(PlE6). a3 (PlB5)
and pa (3El) integrin sub-units were purchased from Chemicon (Temecula, CA); mouse antibodies to Ki-67 nuclear
antigen (MIB-1) and carcinoembryonic antigen (CEA) were
‘To whom correspondence and reprint requests should be addressed, at fax: +-34-1-3944159.
Received: April 4, 1996 and in revised form May 8, 1996
CHARACTERIZATION OF TUMORICENIC SUB-LINES
from Immunotech (Marseille, France). The murine anticytokeratin (AEI and AE3) and anti-leukocyte common antigen (LCA) antibodies were obtained from Signet (Dedham,
MA).
Tumor cells and culture conditions
BCS-TC2 human colon-adenocarcinoma cells were cultured
in standard conditions; the growth and characteristics of these
cells have been reported (Turnay et al., 1990). Standard
culture medium was Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% FCS, penicillin (50 IU/ml),
streptomycin (SO pgiml) and L-glutamine (300 pg/ml). Cell
harvesting for assays was performed by treatment of nearconfluent cullures with 0.05% (wiv) trypsin, 0.02% (w/v)
EDTA. After centrifugation, cells were allowed to recover the
cell surface for at least 1 hr in supplemented medium, washed
with PBS (50 mM sodium phosphate, p H 7.4, 0.15 M NaCI)
and re-suspended at the required cell density. The cell viability
was measured by the Trypan-blue exclusion test.
669
incubated with the secondary peroxidase-conjugated goat
anti-mouse antibody (Pierce, Rockford, IL). After further
washing, sections were stained using 3,3'-diaminobenzidine
tetrahydrochloride as chromogen, counterstained with hematoxylin, dehydrated and mounted.
Establishment of in vivo-passaged cell lines from BCS- TC2
parental cells
Tumor specimens were dissected aseptically, washed with
PBS and minced into small portions with a surgical blade.
Tumor tissue was dissociated by collagenase treatment (200
units/ml of collagenase Type-I and 270 unitsiml DNase, both
from Sigma, St. Louis, MO) for 4 hr at 37°C. After centrifugation, the resulting material was re-suspended in standard
culture medium and plated onto cell culture flasks. Flattened
cells, mainly exhibiting a polygonal morphology, were obtained, and they grew firmly attached to the culture support
adopting a greatly organized monolayer. These cells (BCSTC2.1), cultured and passaged under standard culture conditions, were characterized and used for a secondary in vivo
Cell-adhesion and (3H]thymidine-incoiporation
studies
passage. The whole procedure was repeated with the tumors
Cell-adhesion assays on different substrates were performed obtained after inoculation of BCS-TC2.1 cells, thus allowing
as described (Turnay et al., 1994). In order to check the effect the establishment of a new cell sub-line (BCS-TC2.2 cells).
of ECM components and serum on cell proliferation, 5 x lo4 Studies with these variant sub-lines were performed with cells
cells were seeded into 96-well plates in the presence of 5% between the 10th and 15th passages. Tumorigenicity assays of
FCS. After 24 hr the serum content was reduced to 0.5%. One the sub-lines were performed preparing the cells as indicated
day later, the medium was replaced by serum-free standard for the parental ones.
culture medium containing [ 3H]thymidine (Amersham, Aylesbury, UK; 35 Ci/mmol; 1 kCi per well) and the ECM factor to FACS
Quantification of cell-surface receptors was performed by
be tested: laminin, collagen type-I or collagen type-IV, each at
a final concentration of 20 pg/ml. The effect of FCS was also flow-cytometry analysis (FACS) using a FACScan (Becton
studied at different concentrations (0, 5 and 10%) in the Dickinson, San Jose, CA) and data were analyzed on a H P 310
culture medium. After 18-hr incubation at 37"C, the medium computer. Briefly, growing cultures were ttypsinized and
was removed and the cells were incubated 10 min at room allowed to recover the cell surface. Tumor cells (loh) were
temperature with 5% tricloroacetic acid, followed by 3 washes stained by incubation with saturating concentrations of the
with ice-cold ethanol. Radioactivity was measured after cell different MAbs, followed by treatment with fluoresceinlysis in 0.1 N NaOH containing 0.1% SDS. Protein content was isothiocyanate(F1TC)-conjugated goat anti-mouse or anti-rat
determined in parallel wells.
IgG (Chemicon). Cells were then rinsed in PBS and resuspended in PBS containing 1 kgiml propidium iodide. In
In vivo tiimor growth studies
order to compare the phenotype of the different cell preparaMale athymic BALBic nude mice (8 weeks old) were tions, all main operative settings were kept constant throughpurchased from IFFA-CREDO (L'Arbresle, France) and out the study.
were housed in a laminar-flow cabinet under specific-pathogenfree conditions. In vivo tumorigenicity of BCS-TC2 cells was Northern -blot hybridization
tested by S.C. inoculation in the lumbar region of 0.2 ml of a
Total cellular R N A was extracted from tumor biopsies or
mixture of tumor cells in DMEM alone or in the presence of cell cultures using the guanidine-thiocyanate method, electrothe studied factor. The mice were surveyed daily to determine phoresed on a 1% formaldehyde-denaturing agarose gel, and
when tumors first became apparent upon visual inspection blotted onto nylon membrane filters. Hybridization was carand, from then on, tumor sizes were measured weekly. Latency ried out at 42°C in 50% formamide buffer with a 32P-laminintime was estimated as the number of days needed to obtain binding protein oligonucleotide probe (complementary to
tumors of 0.2 to 0.3 mm diameter. Tumor size was calculated nucleotides +230 to +259, according to Raoet al., 1989) for 16
by measuring the dimensions of the tumor mass (average of the hr. The stringency wash was performed at 55°C in 2 x SSC
2 right-angle diameters) with a vernier caliper at various times (SSC: 20 mM sodium citrate, pH 7.0, containing 150 mM
after injection. At the end of the experiment, the mice were sodium chloride), 0.1% SDS for 30 min followed by 0.1 x SSC,
killed and the tumors were excised and used in the different 0.1% SDS at room temperature for 15 min. Afterwards, R N A
experiments. For each data point, 6 to 8 mice were given bands were visualized by autoradiography. Hybridization to
injection per group, and each experiment was repeated at least 32P-28S R N A probe was used to test R N A transfer and to
normalize densitometric signals.
3 times.
Immurioliistociiemist~studies
At selected times after S.C. injection of the tumor cells,
animals were killed and autopsied. The tumor at the primary
site of the inoculum was examined, also lungs, kidneys, spleen,
liver and pancreas for gross metastases. Tumor specimens
were excised, fixed in 10% formaldehyde in PBS, and routinely
processed for paraffin embedding. For histological examination, 4-km thick sections were stained with hematoxylin and
eosin. Immunohistochemical staining was performed by incubation of the tumor specimens for 1 hr at 37°C with the primary
antibody (MIB-1, CEA, LCA, AE1 or A E 3 ) at optimal
concentrations. After washing with PBS, the slides were
Other characterizations and procedures
Karyotype studies were performed according to standard
procedures (Turnay et al., 1990). 5'-nucleotidase activity was
measured as described (Olmo et al., 1992). CEA content was
determined in the culture medium and in the cell pellet by
using the commercially available Roche-CEA kit. For this
purpose, 3 x lo6 cells were re-suspended in 15 ml of culture
medium and seeded on 75-cm2 culture flasks. Four days after
plating the medium was changed to another containing only
2% FCS. Seven days after seeding, culture media were
collected and concentrated by lyophilization; cell layers were
rinsed and scraped off with PBS. Alkaline-phosphatase activity
670
LOPEZ-CONEJO ETAL.
was assayed using the Sigma diagnostics kit (procedure 245);
3.5 X lo5 cells were seeded into 60-mm plastic culture plates
and were maintained in continuous culture for 20 days, with
medium changes every 2 days. After washing, the cell monolayers were homogenized, and parallel determinations of alkalinephosphatase activity and protein concentration were performed.
A
2.0
-
0
Control
Matrigel 0.6 mg
Matrigel 1.2 mg
A
E0
Y
1.5 -
RESULTS
The BCS-TC2 cell line was established in culture from a
primary human colon adenocarcinoma. The morphological,
immunological and ultrastructural features of these cells agree
with their epithelial origin (Turnay et al., 1990). They have
poor ability to form colonies (2% of colony-forming efficiency
in semi-solid media and 22% of plating efficiency) thus
showing very low in vitro tumorigenicity. Xenograft studies
indicated low or non-tumorigenic potential in vivo, even when
these cells were injected in large numbers into athymic mice
(Turnay et al., 1990). We have reported that the in vitro
behavior of BCS-TC2 cells is modulated by ECM proteins
(Turnay et al., 1994).
Effect of Matrigel on the tumorigenicity of BCS-TC2 cells
The effect of Matrigel on the ability of BCS-TC2 cells to
grow in vivo was examined by S.C. injection into nude mice of
different numbers of cells mixed with Matrigel. The coinjection of 10h cells with Matrigel resulted in a significant
increase in the incidence, tumor-growth rates and final tumor
sizes compared with cells injected only with medium. The
latency time was also greatly reduced when cells were coinjected with Matrigel (Fig. 1).Tumors grew in all the mice (18
of 18) given a co-injection with Matrigel, whereas only 5 of 42
nude mice given an injection of tumor cells alone produced
viable tumors. When the number of cells was reduced to 2.5 x
lo5, no tumors at all were induced in animals injected with
BCS-TC2 cells alone, while the incidence was maintained
when they were co-injected with Matrigel. Tumor size in
experimental and control groups differed significantly 12
weeks after injection as a result of enhanced growth of tumor
cells in the presence of basement membrane (2.19 2 0.70 vs.
1.12 & 0.21 cm), and the latency period was shorter (5-7 1’s.
35-40 days).
Figure 1 shows the dose-dependent effects of Matrigel on
tumor growth. When 0.6 or 1.2 mg of Matrigel were S.C.
co-injected with BCS-TC2 cells into athymic mice, tumor
growth was significantly higher than in animals receiving an
equal number of cells without Matrigel. Figure 1 also shows
the evolution of the mean tumor diameter as a function of time
after the injection. At 10 weeks, doses as low as 0.6 mg/mouse
cause a 2-fold increase in tumor size. A reduction in the
latency period of the tumors is also observed when the amount
of Matrigel increases.
Histological studies
Cells formed solid tumors surrounded by a limiting membrane when injected S.C.in nude mice. Visual observation of
the tumors showed extensive vascularization. In large tumors,
the central area was replaced by edema, showing necrotic
tissue where blood vessels were not present. No apparent
metastases were detected in the mice bearing S.C.tumors when
different organs were examined. The histological appearance,
morphology and immunohistochemical data of tumors developed in mice in the presence of Matrigel were indistinguishable from those obtained after injection of BCS-TC2 cells in
culture medium alone.
In general, 2 different layers could be observed in the
tumors, an external one, highly cellular and compacted, and an
internal area with less and more disseminated cells. In the
external area of these tumors, the cells are closely associated
cn
1.o
0.5
0.0
2
4
6
8 1 0 1 2
TIME (weeks)
FIGURE
1 - Effect of Matrigel on the in vivo growth of BCS-TC2
cells. The effect of Matrigel on the tumorigenicity of BCS-TC2
cells was examined by S.C.injection into nude mice. Tumor growth
rate was analyzed after injection of 1 x loh BCS-TC2 cells alone
(A)or in the presence of 0.6 mg (0)or 1.2 mg (0)of Matrigel.
Tumor size measurements were performed with a caliper at
designated time points. Studies were done in triplicate with n =
6-8; data represent mean values.
and proliferate actively, as deduced from the positive staining
obtained with anti-67-Ki antibody. They are positively stained
with anti-AEl cytokeratin antibody, but no reaction is detected with anti-AE3 or anti-CEA antibodies, suggesting a
poor degree of differentiation. In contrast, cells from the inner
area of the tumors appear less proliferative (negative staining
with anti-67-Ki antibodies) but apparently more differentiated, as deduced from the positive reaction with anti-CEA and
anti-AE3 antibodies.
Effect of Matrigel components on in vivo tumorigenicity
of BCS-TC2 cells
Type-I collagen gel is not able to support tumor growth
(Table I). This indicates that the physical nature of Matrigel
(gel structure at 37°C) does not appear to be a requirement for
cell growth. Growth factors present in complete Matrigel could
also be at least partially responsible for the mitogenic effects.
However, low-Matrigel, in which the possible content in
growth factors is significantly reduced (Vukicevic et al., 1992b),
is also able to induce tumor growth (Table I, Fig. 20). The
incidence as well as the tumor size is almost the same when
Matrigel or low Matrigel is used; only a small increase in
latency time is observed with the latter (Table I). Latency time
is also dependent on the low Matrigel doses, decreasing from 3
to 2 weeks when the amount used in the inoculation is
increased from 0.2 mg to 0.4 mg.
BCS-TC2 cells were also co-injected S.C.with purified ECM
proteins. W e have observed that when these cells were
co-injected with type-IV or type-V collagen, tumor growth was
not promoted (Table I). However, we have found that the
co-injection of cells with purified laminin significantly increases the growth of BCS-TC2 tumors (Table I, Fig. 2b),
suggesting that this basement-membrane protein has a specific
671
CHARACTERIZATION OF TUMORIGENIC SUB-LINES
role in tumor dcvelopment. Thus, injection of lo6 cells with 0.4
mg of laminin resulted in tumor formation in the 80% of the
mice with a latency time of less than 2 weeks (Table I). Similar
results were obtained using only as little as 0.14 mg of laminin
per injection.
In vitro efect of laminin and collagens on BCS-TC2 cell
brhavror
Wc havc checked the in vitro response of BCS-TC2 cells to
laminin in comparison with type-I and type-IV collagens, both
of them with no effect on in vivo tumorigenicity. Cell attachment is greatly enhanced when the plastic surface is coated
with laminin, but collagens also promote a similar effect (Fig.
3a). The incorporation of [ ’Hlthymidine is activated by laminin in the absence of serum, as observed in Figure 3b.
However, this effect appears not to be directly linked to the in
vivo induction of tumorigenicity by laminin since type-I and
TABLE I - EFFECT OF MATRIGEL AND EXTRACELLULAR-MATRIX
COMPONENTS ON THE IN V N O GROWTH OF BCS-TC2 CELLS
Treatment’
None
Matrigel
Low Matrigel
Type-I collagen gel
Type-IV collagen
Type-V collagen
Laminin
Tumor size
(cm)’
Latency time
(days)
-
-
1.98 t 0.57
1.58 ? 0.61
10
12
-
-
1.19 ? 0.28
10
‘lo6cells were injected S.C. in nude mice in the absence (none)
or in the presence of Matrigel or different extracellular-matrix
components. Doses of 0.4 mg of Matrigel, low Matrigel and
laminin were used, whereas collagens were each used at 2 different
amounts, 1.25 and 2 mg.-?Tumor size was calculated at the end of
the experiment (12 weeks), as described in “Material and
Methods”.
A
type-IV collagens activate the thymidine incorporation almost
twice as effectively as laminin and similarly to 5% FCS.
Isolation and characterization of tumor cellsfrom S.C.
Matrigel-induced t~imors
BCS-TC2.1 cells were isolated from tumors obtained after
S.C. injection of BCS-TC2 cells with Matrigel, and were
established and maintained in culture through successive in
vitro passages. Cell morphology and organization of the culture is similar to that of the parental cell line, showing a
well-spread epitheloid morphology, with some small spindleshaped cells.
When BCS-TC2.1 cells were injected alone into athymic
mice, the obtained tumors were histologically identical to
those obtained when the parental cells were co-injected with
Matrigel. These new tumors were subjected to the same
procedure as the initial ones. A new cell sub-population,
BCS-TC2.2 cells, was obtained in this second in vivo passage
and the cells were also established in culture. Whereas the
parental BCS-TC2 cell line required Matrigel to form tumors,
the new BCS-TC2.1 and BCS-TC2.2 sub-lines were fully
tumorigenic in its absence (Fig. 4). A significant increase in
tumor growth was observed when the tumor size was analyzed
12 weeks after implantation of loh cells in the absence of
Matrigel: values of 1.12 & 0.21,2.37 -t 0.58 and 2.57 & 0.61 cm
were obtained for parental cells and for BCS-TC2.1 and
BCS-TC2.2 cells respectively. Differences were also evident in
tumor latency period, which was reduced from 5 to 6 weeks
(parental cells) to 2 weeks (BCS-TC2.2 cells), and incidence
data, increased from 12% in parental cells to 67% and 100% in
BCS-TC2.1 and BCS-TC2.2 cells respectively.
Some of the characteristics of the parental BCS-TC2 cells
and the 2 in vivo-obtained sub-lines are summarized in Table
11. Chromosome analysis confirms the human origin of the
studied cells. Karyotype studies indicate that the 2 chromosomic abnormalities detected in BCS-TC2 cells (a +der (15)
marker with an inclusion of unknown origin and an altered
B
Low Matrigel 0.2 mg
0
Low Matrigel 0.4 mg
2.0
0
Laminin 0.2 mg
Laminin 0.4 mg
n
E
0
W
1.5
N
v)
PL
0
1.0
E
3
0.5
0.0
-
I
I
I
I
I
I
2
4
6
8
10
12
I
I
I
I
I
I
I
2
4
6
8
10
12
TIME (weeks)
FIGURE
2 -In vivo growth of BCS-TC2 cells in the presence of low Matrigel and laminin. Cells were pre-mixed with 0.2 or 0.4 mg of
(a) low Matrigel or (b) laminin, and injected S.C.into nude mice. Tumor size was measured as described in “Material and Methods”.
Studies were done in triplicate with n = 6-8; data represent mean value.
LOPEZ-CONEJOETA.
672
0.3
A
02.5 x l o 5cells
4.0
1.O x 1o6 cells
T
3.5
0.2
-E
z
BCS-TC2
3.0
BCS-TC2.1
T
0
BCS-TC2.2
T
T
Y
w
2.5
p!
2.0
0
0.1
5I-
1.5
1.o
T
0.5
15
10
5
FIGURE
3 - Effect of lamipin, collagens and serum on BCS-TC2
cell adhesion and [ 3H]thymidine incorporation. (a) BCS-TC2 cell
adhesion to plastic surfaces coated with BSA, laminin and collagens type I and type IV. After 30-min incubation, attached cells
were quantitated by staining with 0.5% crystal violet and analysis
in an ELISA reader. (b) Effect of ECM components and serum on
the incorporation of [3H]thymidine by BCS-TC2 cells. Mitogenic
activity was measured by incubation of the cells in serum-free
media supplemented with the correspondin factor or at different
serum concentrations, in the presence of [!H]thymidine. Studies
were done in triplicate with 4 wells per point; data represent mean
value ? SD.
chromosome 16) were maintained in the studied metaphases
from the sub-lines. However, BCS-TC2.1 and BCS-TC2.2 cells
present a new chromosomic marker, an alteration in chromosome 9. CEA production was also considered in this comparative analysis, and was measured in the culture medium as well
as in t h e cell monolayer. Values detected in both sub-lines are
lower than those obtained for the parental cells. In addition, a
reduction of alkaline-phosphatase and 5'-nucleotidase activities were observed in the sub-lines (Table 11).
Cell adhesion to different sub-strata (BSA, laminin and
collagen types I and IV) is similar in the sub-lines and in the
parental cells. However, ["]thymidine incorporation is greatly
enhanced in the sub-lines in comparison with the parental cells
(2.8- and 12-fold for BCS-TC2.1 and BCS-TC2.2 cells respec-
IN
LT
3
2
FIGURE
4 - Tumorigenicity of BCS-TC2.1 and BCS-TC2.2 sublines. Tumor size, incidence (IN), and latency time (LT) of the in
vivo growth of the parental cells and BCS-TC2.1 and BCS-TC2.2
sub-lines, were determined after S.C.injection of 2.5 X 10' or lo6
cells into nude mice. Other conditions of the experiments are as
stated in Figure 1. Data represent mean value 2 SD.
tively). When the effect of ECM components on the [?H]thymidine uptake of the sub-lines was analyzed, no significant
alterations were observed, indicating in vitro growth independent of the presence of ECM components in the culture
medium for these cells.
Expression of the 67-kDa laminin-bindingprotein mRNA
Northern-blot analysis of total R N A using an specific
oligonucleotide probe shows the expression of the 67-kDa
laminin-binding protein mRNA in tumors induced by BCSTC2 cells alone, in Matrigel-induced tumors and in those
obtained from the BCS-TC2.1 cells. In agreement with other
reports (Rao et al., 1989; Castronovo, 1993). a single mRNA
species of 1.2 kb is detected (Fig. 5 ) . Quantification of the
Northern-blot hybridization was performed by densitometric
analysis. Comparison of the steady-state of this mRNA with
"P-28S rRNA hybridization indicates a similar level of 67-kDalaminin-binding-protein-mRNA expression in the 3 types of
tumors. A similar result was obtained when R N A extracted
from the 3 cell lines was analyzed (Table 11). These results are
in agreement with the cell-surface expression of this protein.
Table I11 shows the FACS analysis of the 3 cell lines using a
MAb specific for the 67-kDa laminin-binding protein. The
percentage of cells expressing this antigen and the mean
fluorescence intensity (MFI) are similar when the 3 cell lines
are considered.
Itiregrin expression on BCS-TC2 cells and sub-lines
The cell-surface expression of integrin chains was examined
by flow-cytometric analysis of the parental cells and in vivoobtained sub-lines. All the integrin chains considered may be
involved in heterodimers, with affinity towards laminin. Table
111 shows the percentage of cells expressing each antigen as
well as the MFI. Data indicate that all 3 cell lines express a
diverse array of integrins on their cell surface. Several observations can be drawn from the comparison of the percentage of
positive cells and the MFI values corresponding to the different antigens and cell lines: (i) PI, aZ,a3and (Y6 integrin chains
CHARACTERIZATION OF TUMORIGENIC SUB-LINES
673
TABLE 11 - CHARACTERISTICS OF BCS-TC2 CELLS AND THE IN VIVO-OBTAINED SUB-LINES
BCS-TC2
BCS-TC2.1
BCS-TCZ.?
KaVotyPe
46XX, +der(l5), 46XX, +der(l5),
46XX, +der(l5),
+der( 16)
+der(l6), +der(9)
+der(16), +der(9)
CEA
cell culture
monolayer
5 ' -nucleotidase3
Alkaline phosphatase4
67-kDa lamininbinding protein5
100%'
loo%*
24 x lo-'
25.7
1
43%
53%
15 x
18.1
38%
55%
12 x 10-5
6.4
1
1
HT-2Y
nd
333%
140%
nd
nd
7
CEA content data are given as percentage values, 100% being the value obtained for BCS-TC2
cells.
activity is expressed as nmol
'0.89 ng CEA/mg protein.-?33.19 ng CEA1 lo6 ~elIs.-~5'-nucleotidase
activity of cells maintained 20 days in culture
AMP hydrolizedil5 min/~ell.-~Alkaline-phosphatase
is expressed as mU/mg protein. Values are the mean of 3 independent experiments with triplicate
samples; SD was always lower than lO%.rData were obtained from the Northern-blot analysis of
total RNA extracted from the different cell lines using a 32P-67-kDalaminin-binding protein probe
normalized to 32P-28SrRNA probe hybridization.-All values are referred to that of BCS-TC2 cells.
The CEA content and expression of the 67-kDa laminin-binding protein are also compared with
HT-29 cells, a more differentiated and tumorigenic cell line.
Antigen
(antihndvl
6i-kDa IMLUCS)
F~GURE
5 - Northern-blot analysis of total RNA using a specific
probe for the 67-kDa laminin-binding protein mRNA. Tumors
obtained when parental cells were injected S.C. in nude mice in
cell-culture medium alone (a), or in the presence of Matrigel (b),
and tumors induced after injection of BCS-TC2.1 cells in culture
medium (c) were used for mRNA expression. Total RNA extracted from the tumor tissues (10 pg) was separated on a
formaldehyde-agarose gel, blotted onto a nylon filter, and hybridized with a 32P-67-kDalaminin-binding protein probe. Densitometric analysis of the 67-kDa laminin-binding protein mRNA signals,
normalized to the hybridization using a 32P-28S rRNA probe,
indicate no significant differences in the expression of this protein.
are expressed by almost 100% of the cells, but an increase in
the MFI is observed from the parental cells to the sub-lines;
(ii) in contrast, a reduction in both parameters for the
expression of p4and aIintegrin chains is detected; and (iii) a4
integrin chain is not expressed by parental cells or by the
sub-lines.
DISCUSSION
Matrigel has been successfully used in in vitro and in vivo
studies contributing to the knowledge of the role of tumor-cell
ECM interactions on the stimulation of tumor growth, promotion of cell differentiation, invasion and metastasis, as well as
the angiogenic process (Fridman et al., 1990,1992; Albini et al.,
1992; Passaniti et al., 1992; Vukicevic et al., 1992u, b; Noel et
al., 1993; Grant et al., 1994; Topley et al., 1993). BCS-TC2 cells,
a human colon-adenocarcinoma cell line, show very low
tumorigenic potential after S.C. injection in nude mice. Here,
BCS-TC2
90 (466)
BCS-TC2.1
BCS-TC2.2
23 (14)
100 228)
100 t318)
8 (10)
100 420)
100 i303)
2 (0)
99 (98)
100 366)
56 !90,
97 (447)
0 0)
98 t139)
100 485)
37 $98,
99 (475)
Cells were pre-incubated with hybridoma-culture supernatants,
followed by washing and labeling with fluorescein-isothiocyanatelabeled goat anti-mouse or goat anti-rat antibodies. Data were
collected in a logarithmic scale and the percentage of positive cells
was obtained by subtracting the background given by the negative
control. MFI values are given in parentheses. a and p, integrin
chains; 67-kDa, 67-kDa laminin-binding protein.
we have observed that these cells greatly increase their
tumorigenicity when co-injected with Matrigel.
Even though specific tumor-cell interactions with Matrigel
are important for the stimulation of tumor-cell proliferation, it
is not yet clear how Matrigel promotes this process. At 37"C,
Matrigel forms a multi-layered basement-membrane-like gel
structure which could facilitate tumor growth by holding the
cells together (Kleinman et al., 1986). However, Matrigel
appears to be not only a passive structural support, since we
have observed that the co-injection of BCS-TC2 cells in a
type-I collagen gel does not result in any improvement in tumor
growth over cells injected with medium only, as reported for other
cell lines (Fridman et al., 1990; Passaniti et al., 1992).
Since Matrigel is rich in growth factors (Vukicevic et nl.,
1992u, b), we have analyzed the influence of a significant
reduction of such agents on tumor growth using low Matrigel.
We have observed that BCS-TC2 tumor-cell growth is also
supported by low Matrigel. In contrast, we have found that the
co-injection of BCS-TC2 cells with type-IV collagen or type-V
collagen does not promote tumor formation. These results
suggest specific roles for other basement-membrane components in tumor growth. In fact, laminin is able to promote
tumor growth in a similar way to Matrigel, even at very low
concentrations. Thus, the induction of tumorigenicity in BCSTC2 cells by Matrigel seems to be triggered mainly by laminin,
even though the influence of other components such as growth
674
LOPEZ-CONEJOE T A L
factors or proteases cannot be excluded and may enhance
laminin effects. The role of the intact laminin molecule as
inducer of in vivo tumor growth has been described in only a
few other cases, such as human colon-adenocarcinoma WiDr
cells and A253 or A549 cells (Topley et al., 1993; Grant et al.,
1994). In most studies, when cells have been co-injected with
laminin, tumor growth was not promoted (Fridman et al.. 1990,
1992; Albini et al., 1992; Vukicevic et al., 1992b).
The mechanisms through which Matrigel or laminin exert
their effects remain to be elucidated. Several possibilities have
been suggested, such as the induction of cell growth in the
initial stages of tumor progression, the promotion of angiogenesis, or the release of host factors that may enhance the
viability of tumor cells (Fridman et al., 1990, 1992; Passaniti et
al., 1992; Vukicevic et al., 199%; Grant et al., 1994). Laminin
increases in vitro proliferation and adhesion of BCS-TC2 cells;
however, this mechanism does not appear to be the only one
involved in the induction of tumorigenicity, since other ECM
molecules such as collagens type I and type IV promote similar
in vitro effects, but do not induce tumorigenesis.
Tumor grafts or cells obtained from Matrigel-induced tumors (A549 cells, SW480 colon-adenocarcinoma cells, SCLC
cells or retinoblastoma cells) show the same characteristics as
the corresponding parental cells, being unable to induce
tumors in the absence of Matrigel (Albini er al., 1992; Topley et
al., 1993). BCS-TC2 cells behave differently, since cells isolated from Matrigel-induced tumors are able to form tumors in
the absence of Matrigel. The characteristics of the new
tumorigenic cell sub-line BCS-TC2.1 are maintained after a
new in vzvo passage (BCS-TC2.2 cells), thus suggesting that a
process of selection and adaptation has taken place in which
the cells acquire a tumorigenic phenotype, as suggested for a
few other tumoral cell lines (Fridman et al., 1992). The
obtained sub-lines keep the specific markers on chromosomes
15 and 16, but they present an additional marker (chromosome
9). They also show higher in vitro [3H]thymidine uptake, which,
in contrast to parental cells, is independent of the presence of
ECM components.
Higher tumorigenicity is normally associated with a lower
degree of differentiation. Our results regarding CEA content,
alkaline-phosphatase and 5'-nucleotidase activities in the parental cells and in the sub-lines could agree with this hypothesis. All these results point to a decrease in the differentiation
degree, even though the Farental cell line is already poorly
differentiated. From our data, it seems that laminin or Matrigel induces selection of a less differentiated, more resistant
and tumorigenic sub-population within the heterogeneous
original cell population.
The process by which ECM molecules initiate the cell
response is known to involve specific cell-surface receptors.
Data from co-injection of BCS-TC2 cells with laminin point
out to a potential importance of this Matrigel component as a
modulator of BCS-TC2 cell function through the interaction
with cellular receptors. Multiple laminin-binding proteins have
been described, including members of the integrin family and
other surface proteins grouped as non-integrin lamininbinding molecules (Castronovo, 1993). A correlation has been
established between cell malignancy and mRNA levels or
protein expression of the high-affinity 67-kDa laminin-binding
protein (Rao et al., 1989; Castronovo, 1993). However, in our
study the enhancement of tumorigenicity is not associated with
an increase of the mRNA levels or of the expression of this
protein in the cell surface. It has been suggested that the
expression of a tumorigenic phenotype may require certain
genetic changes, which may be different from those required
for the development of a metastatic phenotype (Ess et al.,
1995). Thus, the expression of high levels of the 67-kDa
laminin-binding protein may be helpful for the appearance of
an invasive phenotype, facilitating migration and basement-
membrane degradation, but not for the promotion of tumorigenicity.
The role of integrins in the signal transduction of cell ECM
interactions is well known. Changes in the expression of these
receptors have also been described in association with neoplastic transformation (Dedhar and Saulnier, 1990; Castronovo,
1993; Kim er al., 1995; Fujita er al., 1995). The biological
relevance of these changes remains to be elucidated, but a
potential role in the appearance of the tumorigenic or malignant phenotype has been suggested. We have examined
integrin expression in BCS-TC2 cells and the changes that take
place in the tumorigenic sub-lines, focussing mainly on those
integrins with affinity to laminin (a&, and a6P4integrins, and
other integrins such as alp1,a2P1and a3PI,which also possess
affinity with collagens and fibronectin).
We have observed a decrease of P4 integrin expression in the
tumorigenic BCS-TC2.1 and BCS-TC2.2 sublines. The a&
integrin is an essential constituent of hemidesmosomes in
differentiated epithelial cells, contributing to the formation of
tight interactions with the underlaying basement membrane
(Nagle et al., 1995). This integrin appears to be involved in the
endocytotic cycle, and the decrease in its expression could
therefore be related to higher motility of the tumorigenic cells,
as was also suggested for prostate and breast carcinoma cells,
and colorectal tumors (Bretscher, 1992; Natali et al., 1992;
Nagle et al., 1995). The expression of a6P4integrin has also
been described as inversely correlated with the differentiation
degree of epithelial cells (Nagle et al., 1995). O n the other
hand, a decrease of PI integrins in aggressive poorly differentiated tumors has been described as a general process. However,
some of the reported data are still controversial. We have
observed an increase in pI-integrin expression in the tumorigenic BCS-TC2.1 and BCS-TC2.2 sub-lines. Higher levels of
integrins have been reported in other cell types when comparing highly tumorigenic and invasive cells with their nontumorigenic and poorly invasive counterparts (Dedhar and
Saulnier, 1990). Increased expression of PI integrins in colorectal-cancer tissues and in poorly differentiated gastric adenocarcinomas has also been described (Fujita et al., 1995). This
apparent disagreement concerning PI integrin has been discussed by Kim et al. (1995), who argue that the controversy may
be due to different causes, such as the many biological
activities of integrins or the use of different antibodies.
In summary. the use of the nude-mouse-xenograft model in
the presence of Matrigel has allowed us to obtain and establish
in culture, from poorly tumorigenic colon-adenocarcinoma
cells, sub-lines which show tumorigenicity by themselves. Our
results suggest that the interaction of specific ECM components, such as laminin, with a particular cell sub-population
present in the heterogeneous parental cells appears to trigger
specific cellular mechanisms which promote their growth and
allow the selection or survival of this genetically different cell
sub-population. The cell sub-lines present an altered phenotype that affects at least the degree of differentiation and the
expression of ECM receptors which could be result from or be
related to the generation of tumorigenicity. Thus, the system
herein described may prove useful in defining the events that
take place during the progression of tumor cells to a more
malignant status, and could contribute to knowledge of the
function of ECM components in tumorigenesis.
ACKNOWLEDGEMENTS
This work was supported by grant PB92-0552 from the
DGCYT (Spain) and by a grant from the Fundacidn Cientifica
de la A.E.C.C. (Spain). Flow-cytometry analyses were performed at the Flow Cytometry Service of the Complutense
University.
CHARACTERIZATION OF TUMORIGENIC SUB-LINES
675
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