BIOCHEMICAL
Vol. 168, No. 3, 1990 May 16, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 905-911
EPIDERMAL GROWTHFACTOR STIMULATES THE ANCHORAGE-INDEPENKNT GROWTHOF HUMAN SQUAMOUSCELL CARCINOMAS OVEREXPRESSING ITS RECEPTORS Kaechoong
Department Orthopedics, Virology.
Lee, Uami Tanaka’, Koichi Ri kimaru4.
Shigeno. Itsuo Y-to2, Hatanaka5. and Junji
Shuichi Konishi
Ohta3.
of
Nuclear Medicine, ‘Department of Pediatrics, 3Department of Kyoto University School of Medicine, and 5Department of Molecular Institute for Virus Research, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606, Japan ‘Department
4Departnent Faculty Received
Chohei Masakazu
March
19,
of Radiology. Science, Otsu,
Shiga Shiga
University 520-21, Japan
of Oral Surgery, Tokyo Medical of Dentistry, Yushima. Bunkyo-ku.
of
Medical
and Dental University. Tokyo 110. Japan
1990
SUMMARY We examined the effects of epidermal growth factor (EGF) on the anchorage-dependent and -independent growth of four human squamous carcinoma cell lines that overexpress EGF receptors. While EGF inhibited anchorage+kpendent growth, it stimulated anchorage-indepemknt growth of all four cell lines tested The results suggest that the proliferative responses to EGF are characterized by a preference for anchorage-independent, rather than dependent growth, In cells overexpresslng EGF receptors. Moreover, as EGF has been shown to stimulate the in vlvo growth of squamouscarcinoma cells overexpresslng EGF receptors, it Is also suggested that the In vitro EGF responsivenessof these cells in soft agar, but not in monolayer, better correlates with the in vivo EGF responsiveness. 0 1990Academic Press,Inc.
Gverexpression of receptors for EGF has been shown to occur at a high incidence both in primary squamous / epidermoid carcinomas and in established cell lines such as A431 (1-4). Amplification
of EGF receptor gene is also quite common
in squamons carcinoma cell lines that overexpress EGF receptors (5-7). On the other hand, EGF has been shown to inhibit the in vitro
growth of EGF-receptor-
hyperproduclng squamous carcinoma cells (8, 9), although, in general, gene amplification results from some growth advantage. We have previously shown that EGF exerts reciprocal effects on the in vitro growth of A431 human epidermoid carcinoma cells depending on culture conditions; 0006-291x/90 905
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Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
168,
while growth
No.
3, 1990
EGF inhibits
BIOCHEMICAL
anchoragedependent
of A431 (10). To determine
a property
common to cells overexpressing
ous / epidermoid
carcinoma
gene amplification
BIOPHYSICAL
growth,
whether
of EGF on the anchoragedependent
scribed
AND
RESEARCH
it stimulates
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anchorage-independent
the reciprocal
responsiveness
EGF receptors,
we examined
to EGF is the effects
and -independent
growth
of four human squam-
cell lines (A431, Ca9-22,
HSC-2,
and NA) in which
both
have been previously
de-
and overexpression
of EGF receptors
(7 & 9). MATERIALS
AND
MEI-HOIX
Celk A431 and I-BC-2 cell lines were obtained from Japan Cancer Research Resource Bank. The characteristics of the four cell lines used in this s y are %? bindsummarized in Table 1. Overexpression of EGF receptors (more than 1 x 10 ing sites per cell) in these cell lines were confirmed by Scatchard analysis as described previously (11). All cell lines were maintained at 37oC in a humidified 59bC0 atmosphere using Dulbecco’s modified Eag$!s medium (DMEM)-I~%FCS. Growt t Assay: (1)Monolayer growth assay. 1 x 10 cells were plated into the wells of 24-well culture plates using DMEM-lO%FCS and cultured overnight. Medium was then replaced with fresh DMEM-O.5%FCS containing the indicated amounts of EGF and cuItures were maintained for 6 days. Cell growth was estimated by crystal violet staining as described previously (10). In brief, cells were stained and fixed with 0.5% crystal violet / 20% methanol at the termination of incubation and cell growth was determined by measuri ng absorbance at 540 nm after the dye was eluted with 0.5M sodium citrate / 50% ethanol. (S)!Soft-agar colony assay. 2 x lo4 cells were plated in lml of 0.2% agar over an underlayer of lml 0.4% agar / DMEM-lO%FCS and cultured overnight. EGF was then added and the cultures were maintained for 10 days. Cell clusters larger than atic particle counter. 60 urn in diayeter were counted as colonies using an aut (3Boft-agar [ Hlthymidine incorporation assay (12). 2 x 1r cells were plated in lml as in the 0.2% agar over an underlayer of lml 0.4% agar / DMEM-15% FCS colony assay. After overnight incubation, EGF was added and the cultures were rgaintained for the time periods indicated. Cells were treated with 1 pCi of [ Hlthymidine for the last 24 hours of the incubation periods. Agar was melted by boiling for 10 minutes and radioactivity in TCA-precipitable materials was determined using a scintillation counter. RISULTS
EGF inhibited dependently the inhibitory
(Fig.
the anchorage-dependent 1A ) as described
effects Table
A431 HSC-2 k-22
of all four
1.
Characteristics
Origin vulva tongue tongue gingiva
of
cell
of EGF receptors (sites / cell) 3 2 4 1
x 106 x 106 x 106 x 106
cell
lines dose-
(9, 10). The dose-response
of EGF on the anchoragedependent
No. Name
previously
growth
lines
used
growth in
this
Amplification EGF receptor + + + +
curves
were
apparently
study of gene
of
(7)
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168, No. 3, 1990
A. Anchorago-drpmdmt
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
growth
A431
nsc-
360 .,6o.L
2
COB-22
.oooL
v-
025 8. Anchorage-indepondmt
VW
HSC-2
400
a400
V. 0 26
400
6,400
400
6,400
growth
A431
0 25
400
NA
WOO
Y0 25
CaO-22
400
6,400 EGF
0v-oo25
NA
Y025
(PM)
&. Effects of EGF on the aachorage-dependent and -independent growth of human sguamcus / epidermkd carcinoma cell lines. Anchorage-dependent growth was assessed by crystal-violet staining (A; mean and SD, n=4), and anchorageindependent growth by soft-agar colony assay (B; mean and SD, n=2) as described in Materials and Methods.
similar among all of these cell lines. Contrary to the effects on monolayer growth, EGF stimulated soft-agar colony formation of these cell lines in a dose-dependent manner, while few colonies were formed in the absence of EGF (Fig. 1B). The effective
EGF doses that stimulated soft-agar colony formation were the same as
those required to inhibit
anchorage+kpendent growth. The maximum stimulatory
effects of EGF on the soft-agar colony formation were achieved at an EGF concentration of around 499 to 1,600 PM. The patterns of the EGF dose-response curves for soft-agar colony formation were also similar among the four cell lines. ‘Ihe stimulatory effects of EGF on the anchorage-independent growth of these four cell lines were also observed with the soft-agar [3Hlthymidine
incorporation
assay (Fig. 2). Although the higher EGF concentration was rather inhibitory
at day
3 with three cell lines (HSC-2, Ca9-22, and NA), EGF exerted stimulatory effects on [3H]thymidine incorporation from day 4 up to day 7 at most of the dosages tested in all four cell lines !I07
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168,
No.
3, 1990
BIOCHEMICAL
AND
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RESEARCH
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A431
6,000,
3
20,000
4
5
6
7
‘r
10,000
0 0
I
I
.
,
I
I
.
n
,
3
4
5
6
7
3
4
5
6
7
Days
in
culture
Fiq. 2. Time course of the effects of I33 on C3Hlthymidine incorporation by human squamous / epidenmid carcinoma cell lines, Cells were cultured in a bi-layer of soft-agar in the presence of 0 (01, 100 (O), 400 (A), and 1,600 (El) pM EGF and labeled with 1 pCi r3Hlthymidine for 24 hours.
DISCU!SSlON Evidence has been accumulated that the overexpression of EGF receptors provides cells with some growth advantage. First, overexpression of the human EGF receptor confers EGF-dependent transformed phenotypes including EGF-depemknt soft-agar growth to NlH 3T3 mouse fibroblasts transfected with expression vectors containing human EGF receptor
cDNA
(13, 14). Second, administration
promotes the tumor growth of EGF-receptor cells implanted into athymic
hyperproducing squamous carcinoma
mice (15,16). Third, 908
of EGF
the in vivo
growth of clonal
Vol.
168, No. 3, 1990
variants of A431
cells
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
is highly correlated with the degree of gene amplification
and expression of EGF receptors (17). Consistent with these reports, we have shown in this study the stimulatory effects of EGF on the anchorage-independent growth of four squamous/ epidermoid carcinoma cell lines that overexpress EGF receptors. The stimulatory
effects
of
EGF on the anchorage-independent growth of these cell lines were observed both with the soft-agar colony assay and with the [3H]thymidine incorporation assay in soft agar. As EGF has been shown to promote the in vivo growth of three of the four cell lines tested in this study (A431, NA, and Ca9-22) (15, 16), the in vlvo responsesto EGF of cells overexpressing EGF receptors may better correlates with their responses in soft agar, but not with those in monolayer cultures, which is similar to the correlation between tumorlgenicity
and anchorage-independence (18,
19). The mechanisms by which EGF exerts reciprocal effects on the growth of squamous carcinoma cells overexpressing EGF receptors between anchorage&pendent and -independent culture conditions remain to be elucidated also transforming growth factor
Not only EGF but
beta (TGF-beta) has been &own to have either
stimulatory or inhibitory effects on the growth of the same target cells depending on whether the culture conditions are anchorage-dependent or -independent. Such culture-condition-linked
bidirectional
effects
of TGF-beta on cellular growth have
been described with normal fibroblasts such as AKRZB and NRK49F (20, 21), and with a transformed cell line, A431 (10). TGF-beta
It should be noted that both EGF and
have been demonstrated to regulate cell-adhesion-associated molecules
(22-26) because it is known that there is tight coupling between cell adhesion and cell proliferation
(27).
These two factors may exert reciprocal effects on cellular
growth depending on the culture conditions, by altering the connection between cell adbesion and cell proliferation. if overexpression of EGF receptors confers a growth advantage to squamous carcinoma cells in response to EGF, it may be possible to use “anti-EGF therapy” against squamous carcinomas, analogous to
the
anti-estrogen
therapy
against
mammary carcinomas. A monoclonal antibody raked against EGF receptors has been 909
Vol. 168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
shown to completely suppress the growth of A431 tumors in athymic
mice (28).
Moreover, a recombinant fusion protein between Pseudomonas toxin and transforming growth factor alpha, another ligand for EGF receptors, has been demonstrated to kill cells expressing EGF receptors, while the protein has little
activity
against
cells with few receptors (29). By examining the levels of EGF receptor expression in biopsied materials, we may be able to identify indicators which could be used in anti-EGF therapy against squamous carcinoma cells
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