(CANCER RESEARCH 51. 1738-1740. March 15. 19911
Advances in Brief
Increased Tumorigenicity of Fibronectin Receptor Deficient Chinese Hamster
Ovary Cell Variants
Clara Schreiner, Michael Fisher, Saber Hussein, and R. L. Juliano1
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
Abstract
We have developed a series of variant clona) Chinese hamster ovary
cell sublines which are deficient in the expression of the n- ,i, integrin
fibronectin receptor. When these clonal sublines were injected s.c. into
nude mice, there were marked differences in the rate of tumor growth.
Tumors from clones expressing very low levels of fibronectin receptor
grew most rapidly, clones expressing levels of fibronectin receptor com
parable to wild type cells grew at an intermediate rate, while a clone
expressing elevated levels of fibronectin receptor grew slowly. Cells
recovered from the tumors maintained their original phenotypes in terms
of levels of fibronectin receptor expression. These results suggest that
there is an inverse correlation between the level of expression of the
(n/'/i'i fibronectin receptor and the rate of tumor growth.
matically suppressed the ability of these cells to form tumors
in nude mice. The present study examines the effect of ablation
of the expression of the endogenous «5integrin subunit on
tumorigenicity and is thus quite complementary to the obser
vations of Giancotti and Ruoslahti (15). We have utilized a
series of CHO cell clonal sublines which are deficient in expres
sion of the endogenous CHO as/ß\fibronectin receptor to
demonstrate an inverse correlation between levels of expression
of this fibronectin receptor and the rate of tumor growth in
nude mice.
Materials
and Methods
Introduction
Cell Lines. The isolation of clonal CHO cell sublines deficient in
expression of the «5integrin subunit has been described elsewhere (16).
It has been known for years that many transformed cells Briefly, anti-intcgrin antibodies and flow cytometry were used to enrich
express less cell surface fibronectin than their normal counter
for a population of cells with low levels of «5expression. Subsequently
parts (1,2) and that the reduced levels of fibronectin contribute
the cells were cloned by limiting dilution and clones were screened by
ELISA for stable, low level expression of «5.The phenotypes of the asto the alterations in adhesiveness, motility, and morphology
observed in transformed cells (1, 3). Recent studies have indi
deficient clones obtained in this way have been stable for more than 1
cated that differences in the cell surface display of fibronectin
year in culture.
Flow Cytometry and ELISA. Integrin expression was analyzed by
in normal and malignant cells may relate to alterations in the
using monoclonal antibodies specific for Chinese hamster integrins and
expression or function of cell surface receptors for fibronectin.
commercial fluorescent second antibodies. The 7E2 monoclonal anti
The integrin superfamily of a/ßheterodimeric adhesion recep
tors (4-8) includes at least three members of the ß,subunit
body is directed against the hamster 0, homologue, while the FBI
family which bind fibronectin; these are designated «5//tfi(9, monoclonal antibody is directed against the intact heterodimeric at/ß\
receptor (17). The anti-integrin monoclonal antibodies were used at
10), «j/jtfi(7, 11), and a4/ßt(12). Transformation of rodent
levels sufficient to saturate binding in wild type CHO cells. Flow
cells with ras or tyrosine kinase oncogenes has been reported
cytometric
analysis and sorting was performed as described using an
to reduce expression of the «5subunit but not the «3or ßt
Epics
IV
flow
cytometer (16); using this methodology a change in peak
subunits, and this may explain changes in fibronectin mediated
channel number of 26 corresponds to a doubling in primary antibody
adhesion consequent to transformation (13). However, in con
binding, making it possible to quantitatively compare the relative levels
trast to the case with rodent cells, oncogenic transformation of
of integrin expression in different cell populations. ELISA assays for
human cells seems to affect the rate of biosynthesis and distri
integrin expression were performed with cells plated on to microtiter
bution of the «?/£?!
fibronectin receptor without increasing its wells as described (16).
overall expression (14). Thus it seems clear that malignant
Tumor Formation in Nude Mice. Wild type or «.¡-deficient
CHO
transformation can affect the expression, distribution, and clones were harvested by treatment with trypsin-EDTA (Gibco), resusprobably the functioning of cell surface receptors of the integrin
pended in growth medium, and injected s.c. in a 0.2-ml volume into
class, but the pattern of changes may be complex.
the flanks of BALB/c-/ii//nu mice (HarÃ-anSprague Dawley, Madison,
The reciprocal question of whether integrin expression can WI). Each mouse received cells from two distinct clonal sublines, one
affect the transformed phenotype is just beginning to be ex
in each flank. Tumor size was determined at intervals by measurement
plored. Thus Giancotti and Ruoslahti (15) have used transfecof two right angle diameters with a caliper. At the end of the experiment
tion to express the human «5//J|heterodimer at very high levels the mice were sacrificed and tumors were excised and weighed. CHO
in CHO2 cells, a cell type which possesses its own endogenous
cells were recovered from the tumors by carefully mincing the tumor
as/ßi homologue (9). Although overexpression of xenogeneic
into CHO growth medium («minimal essential medium, 10% fetal calf
serum, antibiotics) and allowing the transformed CHO cells to grow
human «5//8|had only modest effects on the ability of CHO
cells to adhere to or migrate on fibronectin substrata, it dra- out from the untransformed murine host cells over a period of about 2
weeks.
Received 11/28/90: accepted 1/31/91.
RNA Analysis. Total cellular RNA was extracted from wild type or
The costs of publication of this article were defrayed in part by the payment
integrin-deficient clonal sublines and was run on denaturing formaldeof page charges. This article must therefore be hereby marked advertisement in
hyde-agarose gels and transferred to nitrocellulose (18). The RNA was
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by Grant GM26165 to R. L. j. and Grant NRSA F32 CA086I4
probed with 12Prandomly primed double stranded cDNA probes (19),
to C. S.
2 The abbreviations used are: CHO. Chinese hamster ovary; ELISA, enzyme
linked immunosorbent assay; cDNA, complementary DNA.
using a human ßtcDNA (courtesy L. Fitzgerald, Cor Therapeutics,
South San Francisco, CA) or a partial length hamster «5cDNA (16).
1738
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FIBRONECTIN
WT
RECEPTOR DEFICIENT CHO CELL VARIANTS
Table I Tumor formation in nude mice by CHO cells with altered fibronectin
receptor expression
Cells (1 x 10") were inoculated s.c. into athymic BALB/c mice. Tumor size
was determined by calipers after 8- and 11-day intervals and tumor weight was
determined at sacrifice at day 14. The relative levels of a,/ii, fibronectin receptor
expression of the cloned cell lines was determined by flow cytometry. The results
represent means ±SE of 5 tumors for each cell type.
Tumor size*
WT
20
CHO
clone3A1
WT1-23
28S-
ofFnR"130
days39
1002428
3183
±
±41
32216
I40±
±741
9%
' FnR. fibronectin receptor.
1Product of two diameters in mnr.
18S-
BETA
1
ALPHAS
Fig. I. Northern analysis. RNA from a wild type clone (WT) or from clones
expressing approximately 20tt (20) (clone 1-23). or 2% (2) (clone 9). of intact
cell surface «5/^1fibronectin receptor by flow cytometry were probed in a
Northern blot analysis with either /Õ,cDNA or a5 cDNA, as described in
"Materials and Methods."
Fig. 2. Flow cytometric analysis of integrin subunit expression. Clones 3A1,
WT. 1-23. and 9 were labeled with 7E2 monoclonal (anti-/if) or PB1 monoclonal
(anti-intact as/ti\ fibronectin receptor) followed by fluoresceinated rabbit antimouse antibody. A-D. labeling with 7E2: E-H. labeling with FBI; abscissa.
logarithm of fluorescence intensity; ordinate, cell number. O. cell samples treated
with both the primary monoclonal antibody and the fluorescent second antibody:
D, cell samples where the primary antibody was omitted. A. E. clone 3A1; B, F.
clone WT: C G. clone 1-23: D. H. clone 9.
days85
1
(g)0.36 wt
±34
±0.09
188 ±46
0.82 ±0.09
±61341
254
1.09 ±0.37
±77Tumor2.1 5 ±0.66
receptor expression in all clones examined thus far is a defi
ciency in «5message levels leading to reduced synthesis of the
«ssubunit and failure to assemble mature «5//3ireceptor. This
is illustrated in the Northern analysis shown in Fig. 1. The wild
type cells as well as a 20% variant and a 2% variant all produce
similar amounts of a\ message; however, «?message is reduced
in the 20% clone and virtually absent in the 2% clone.
In this study we evaluated the tumorigenicity of four inde
pendent CHO clones. The patterns of cell surface expression
of integrin subunits in these clones are illustrated in Fig 2.
Clone 3A1 was an unexpected by product of our search for
fibronectin receptor deficient CHO cells, since this clone stably
expresses greater than normal levels of this integrin, approxi
mately 130% of wild type levels. Clone WT expresses fibronec
tin receptor at a level close to that found in the unselected wild
type parental population and is thus assigned a value of 100%.
Clone 1-23 expresses 20-25% of WT levels, while clone 9
expresses less than 2% of WT levels of fibronectin receptor.
Each of these clones expresses substantial amounts of ß,
subunit
some of which may be associated with other «subunits and
some of which may be uncomplexed.3
These clones were injected into BALB/c-nw/ww mice and
tumor growth was followed over a period of 2 weeks. As seen
in Table 1, there were major differences in the growth rates of
the four clonal sublines. Clone 3A1 grew poorly, forming small
tumor nodules which did not seem well vascularized. Clones
WT and 1-23 grew at an intermediate rate, with 1-23 slightly
outpacing the WT cells. Clone 9 grew much more rapidly than
the other clones and formed highly vascularized, well demar
cated tumors. The differences in tumor diameter and weight
between clones 3A1 and WT or between clones 9 and WT were
statistically significant. Thus, for tumor weight, the clone 3A1/
WT difference is significant at the 0.01 level, using Student's t
test, while the clone 9/WT difference is significant at the 0.05
level. Although the growth rate of clone 1-23 was consistently
faster than that of clone WT, the differences were not statisti
cally significant. Qualitatively similar results were observed
with these clones in several additional experiments where dif
ferent numbers of cells ranging from 4 x IO4 to 1 x IO7 were
injected into nude mice; i.e., the clone expressing the highest
amount of the a^/ß,fibronectin receptor (3A1) grew most
slowly whereas the clone expressing least receptor (clone 9)
Results and Discussion
grew most rapidly.
The injected cells retained their phenotype in terms of fibro
The fibronectin receptor deficient CHO variants that we have
nectin
receptor expression during the period of tumor growth
isolated fall into two broad categories; one group of clones
in
vivo.
Thus, as shown in Table 2, when expiants of the tumors
express approximately 15-25% of wild type levels of the «s/ßi
were analyzed for the expression of fibronectin receptor by
receptor and we have termed these "20% clones"; the other
ELISA, cells derived from clone 9 tumors expressed low levels
group express less than 2% of wild type levels of receptor and
are termed "2% clones" (16). The basis of reduced fibronectin
3C. Schreiner and R. L. Juliano, manuscript in preparation.
1739
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FIBRONECTIN
RECEPTOR DEFICIENT CHO CELL VARIANTS
Table 2 Fibronectin receptor expression in CHO cells recovered from tumors
Expression of cell surface proteins was evaluated in cells grown out from
tumor explants, as described in "Materials and Methods." An ELISA assay using
anti-CHO cell primary monoclonal antibody and a 0-galactosidase-linked second
antibody was used. Results are means ±SE of triplicate determinations. Absorbances in reagent control wells (no cells) averaged 0.10-0.12. The antibodies used
were PB1 (anti-intact as/ß,fibronectin receptor), 7E2 (ami .;,). and 6C10 (anti
body to a non-integrin cell surface protein on CHO cells) (9, 17).
References
Absorbance in ELISA assay
Tumor clone3A1
from
WT
1-23
9FBI1.30
±0.03
1.15 ±0.04
0.82
0.32 ±0.03
0.32
0.12 ±0.017E21.02
0.34
±0.05
2.24
±0.09
±0.02
1.66
±0.016C102.12
2.37
±0.10
+ 0.14
±0.05
±0.19
of as/ßifibronectin receptor, cells derived from clone WT and
clone 1-23 tumors expressed intermediate levels, and cells from
clone 3A1 tumor expressed the highest level. This analysis was
facilitated by the fact that antibodies PB1 and 7E2 only bind to
hamster integrins (17).
From these results it seems clear that there is an inverse
correlation between the level of a>,/ß\fibronectin receptor
expression and tumorigenicity. A modestly enhanced level of
receptor in 3A1 cells markedly slows their growth in nude mice
as compared to wild type cells. This finding agrees well with
that of Giancotti and Ruoslahti (15), who used very high levels
of transfected xenogeneic fibronectin receptor. Conversely, re
duced levels of fibronectin receptor in clone 1-23 and especially
in clone 9 cells, results in rapidly growing tumors. The mecha
nism whereby the a5//3i receptor regulates tumorigenicity is
unclear at present. Perhaps integrins such as fibronectin recep
tor transduce important growth inhibitory stimuli from the
extracellular matrix. In any case, these observations imply that
integrins are involved not only in the adhesive, motile, and
invasive behavior of tumor cells but in their growth regulation
as well.
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1740
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Increased Tumorigenicity of Fibronectin Receptor Deficient
Chinese Hamster Ovary Cell Variants
Clara Schreiner, Michael Fisher, Saber Hussein, et al.
Cancer Res 1991;51:1738-1740.
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