[CANCER RESEARCH 49. 5677-5681. October 15. 1989]
Epidermal Growth Factor Receptor Gene Expression, Protein Kinase Activity, and
Terminal Differentiation of Human Malignant Epidermal Cells'
Ivan King and Alan C. Sartorelli
Department of Pharmacology and Developmental
Connecticut 06510
Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven,
ABSTRACT
A number of epidermal growth factor (EGF)-resistant
clones have
been isolated from human epidermoid carcinoma A4 î
I cells (I. King and
A. C. Sartorelli, Biochem. Biophys. Res. Commun., 140: 837, 1986).
These cells had a higher capacity to enter a pathway of terminal differ
entiation, as determined by their ability to form cornified envelopes.
Thus, after 6 days in culture, 10% of parental A431 cells expressed a
differentiated phenotype, while more than 50% of each of the I ( ,1 resistant variants (Ml, A5, and A7) formed cornified envelopes. These
EGF-resistant clones expressed fewer EGF receptors and had a lower
capacity for M.I receptor autophosphorylation than parental A431 cells.
Clone Ml had the highest capacity to form cornified cell envelopes and
expressed about 10% of the EGF receptor autophosphorylation activity
of parental cells. The decrease in the level of EGF receptor autophos
phorylation appeared to be due to a decrease in EGF receptor number
rather than to a lowering of enzyme activity per se. Southern analysis
demonstrated that all of the EGF'-resistant variants contained fewer
copies of the I ( ,1 receptor gene and no apparent gene rearrangement
was detected in these variant cells. A corresponding decrease in EGF
receptor mRNA was also observed in Ml, A5, and A7 cells, with a ratio
of 18:1:10:5 for A431, Ml, AS, and A7 cells, respectively. In addition,
two other malignant epithelial cell lines, SqCC/Yl and lu Du. contained
relatively few copies of EGF receptor genes, had low EGF receptor
kinase activity and showed a relatively high capacity to form cornified
envelopes. These findings suggest that the level of the EGF receptor was
critical to the regulation of the degree of maturation of malignant epider
mal cells.
INTRODUCTION
EGF: is a polypeptide mitogen for a variety of cells that was
originally isolated from mouse submaxillary gland (1). It is of
interest that the growth of the human epidermoid carcinoma
A431 is suppressed by EGF at concentrations that stimulate
the proliferation of other cell lines (2, 3). A431 cells have been
shown to have exceedingly high levels of EGF receptor, express
ing 50-fold more surface receptor than normal fibroblasts (4).
Kawamoto et al. (5) have reported that EGF at picomolar levels
is a mitogen for A431 cells and suggested that A431 cells are
so oversensitive to EGF that it becomes toxic at higher concen
trations.
The EGF receptor is a glycoprotein with 170,000 molecular
weight whose C-terminus bears an intrinsic tyrosine-specific
protein kinase activity. It is highly homologous to \-erbB, an
oncogene isolated from the avian erythroblastosis virus (6). The
binding of EGF to its receptor activates the protein kinase
which phosphorylates a number of cellular proteins. In addition,
other events such as Na* influx, phosphatidylinositol turnover,
and receptor internalization
have been reported to occur as a
Received 11/7/88; revised 5/1/89; accepted 7/20/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported by U. S. P. H. S. Grant CA-02817 from the
National Cancer Institute.
- The abbreviations used are: EGF. epidermal growth factor; DMEM. Dulbecco's modified Eagle's medium; HEPES. 4-(2-hydroxyethyl)-l-pipcrazineethaneMIIdnii. acid: PBS. phosphate buffered saline; SDS. sodium dodecyl sulfate: TGFtt. transforming growth factor-«.
result of the interaction of EGF with its receptor in various cell
types. These actions are considered to be important for some
of the events mediated by EGF.
The EGF receptor has been shown to be overexpressed in
certain carcinomas; thus, for example, several laboratories have
reported 2.5- to 55-fold increases in the number of EGF recep
tors in squamous cell carcinomas compared to normal epider
mal cells (see for example Refs. 7 and 8). Lim and Hauschka
(9) have shown that a marked decrease in the level of EGF
receptor occurs during the terminal differentiation of mouse
myoblasts, and that the decrease in receptor number is not due
to the cessation of cellular proliferation. Nanney et al. (10) have
reported a difference in the number of EGF receptors in epi
dermal layers, decreasing from the stratum basale to the stratum
corneum. This inverse relationship between the number of EGF
receptors and the degree of epidermal cell differentiation sug
gests a physiological role for this receptor in the maturation of
the epidermis. In support of this premise, we (11) and others
(12) have reported that EGF suppresses the terminal differen
tiation of a variety of malignant keratinocytes. In addition,
using epidermal cells that express different amounts of surface
EGF receptor, Boonstra et al. (13) have shown that cells pos
sessing high levels of surface EGF receptor tend to have a
decreased ability to enter a terminal pathway of differentiation.
Consistent with these findings, we have isolated a number of
variants of A431 cells resistant to the cytotoxic action of EGF,
which have decreased levels of EGF receptor and a higher
capacity to form cornified envelopes than parental cells (14).
These variants have been found to be capable of proliferation
in serum-containing medium in the absence of exogenous EGF
for at least 15 passages without significant changes in growth
rate and responsiveness to EGF. The amounts of surface EGF
receptor on these variants have remained unchanged for at least
eight passages under the same conditions. The stability of these
variants in culture beyond this period of time, however, has not
been examined.
To characterize further the relationship between terminal
differentiation and the expression of the EGF receptor and its
protein kinase in various EGF-resistant clones of A431 cells,
we have measured the gene copy number of the EGF receptor
and its mRNA expression in these clones. The amounts of EGF
receptor and the degree of autophosphorylation have also been
assessed.
MATERIALS AND METHODS
Cell Culture. A431 and FaDu cells, obtained from the American
Type Culture Collection (Rockville, MD), and SqCC/Yl cells were
grown in DMEM supplemented with 10% fetal bovine serum (GIBCO;
Grand Island, NY). The development of the EGF-resistant clones of
A431 cells was previously described (14). Cells were routinely main
tained at 37°Cin a humidified atmosphere of 95% air/5% CO2. To
measure the degree of differentiation, cells were seeded at a concentra
tion of 1.8 x IO6cells in 25-cm2 flasks. Fresh medium was added every
3 days. The degree of terminal differentiation was measured by the
ability of cells to form cornified envelopes as described previously (11).
5677
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NEOPLASTIC
KERATINOCVTE
DIFFERENTIATION
Autophosphorylation of the EGF Receptor. Cells were seeded at a
level of 2.0 x IO6cells per 25-cm2 flask for 24 h before incubation with
0.5 mCi/ml of ["Pjorthophosphate (9,000 Ci/mmol; Amersham. Ar
lington Heights, IL) for 4 h in 2.5 ml of phosphate-free DMEM. EGF
(40 ng/ml; Collaborative Research; Bedford. MA) was added and the
incubation was continued for another 45 min. Cells were then washed
with phosphate buffered saline and lysed with RIPA buffer (20 mM
HEPES, pH 7.5; 150 m\i NaCI; 1% Triton X-100; 1.5 mM MgCl2; 40
MMphenylmethylsulfonyl fluoride; and 0.5 trypsin inhibitor unit/ml of
aprotinin). Anti-EGF receptor antibody (ICN; Lisle, IL) was added to
the lysate and incubated for 2 h at 25°C.Anti-mouse IgG-linked agarose
washed with 15 mM NaCI, 1.5 mM sodium citrate, and 0.1% SDS at
65°Cfor 1 h, with two changes of the solution.
was basically the same as described previously (14). Briefly, cells were
incubated with 1.5 ng/ml of [125I]EGF (1-2 x IO5 cpm) with varying
amounts of unlabeled EGF. After incubation at 4°Cfor 4 h, unbound
[125I]EGFwas removed by washing four times with cold binding buffer
(DMEM containing 0.1% bovine serum albumin and 20 mM HEPES,
pH 7.4). Cells were lysed with 0.5 ml of l N NaOH and radioactivity
was determined with an LKB CompuGamma counter.
Isolation and Blotting of RNA. Total RNA was collected from expo
nentially growing cultures by guanidine isothiocyanate solubilization
and CsCl centrifugation (17). Cells were lysed with a solution of 4 M
guanidium isothiocyanate, 5 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, and 0.1 M /i-mercaptoethanol. The mixture was layered onto 2.5
ml of 5.7 M CsCl in O.I M EDTA and centrifuged at 35,000 rpm for
16 h in an SW-40 rotor. The pellet containing the total RNA was
dissolved in distilled water and analyzed on a 1% agarose gel containing
2.2 M formaldehyde (18). RNA was blotted onto GeneScreen (New
England Nuclear Corp.; Boston, MA) by a capillary mechanism using
1.5 M NaCI and 150 m.\i sodium citrate as a convectant.
DNA Extraction and Blotting. The procedure used for DNA extrac
tion was basically that described by Gross-Bellard et al. (19). DNA (IO
Mg)was digested with either £coRIor Hind\\\ (New England Biolabs;
Beverly, MA) using the conditions described by the vendor. The restric
tion fragments were analyzed by electrophoresis on 1% agarose gels
and were then transferred to Zeta-probe (Bio-Rad; Richmond, CA)
using 0.4 N NaOH as the convectant.
Hybridization. A 2.4-kilobase C/al fragment isolated from the EGF
receptor complementary DNA pE7 plasmid was used for hybridization
(20). The probe was labeled by the random primer method described
by Feinberg and Vogelstein (21). The blots were prehybridized with a
solution containing 50% formamide, 10% dextran sulfate (Pharmacia;
Piscataway, NJ), 1 x Denhardt's solution (0.2% polyvinyl pyrrolidine.
0.2% bovine serum albumin, 0.2% Ficoll, 0.05 M Tris-HCI, pH 7.5,
0.1% sodium pyrophosphate, 1.0% SDS. and 1.0 M NaCI). and 0.1 mg/
ml of heat-denatured salmon sperm DNA at 42°Cfor 4 h. The blots
autophosphorylation process was further characterized by elec
trophoresis of membrane proteins followed by precipitation of
the EGF receptor by a monoclonal antibody. The data in Fig.
3 confirm the findings that A431 cells express more EGF
receptor than the EGF-resistant clones and SqCC/Yl and
FaDu, with the ratio of the receptor in A431, Ml, AS, A7,
SqCC/Yl and FaDu cells being 25:1:8:8:3.8:3.6, respectively,
RESULTS
The three EGF-resistant clones derived from A431 cells were
all capable of forming considerably more cornified envelopes in
culture than the parental line, and the degree of differentiation
depended upon the length of the incubation period (Fig. 1).
Thus, although few envelope-competent A431 cells were ob
served after 4 days in culture, 5 to 12% of these cells formed
(Sigma Chemical Co; St. Louis, MO) was then added and the incubation
cornified envelopes after 6 days. In contrast, all of the EGFwas continued for another 45 min. The immune complex was washed
four times with RIPA buffer supplemented with 0.1% SDS, boiled for
resistant clones exhibited an ability to form a comparable
amount of envelopes after only 3 days in culture. Of the EGF2 min in Laemmli sample buffer (15) and subjected to electrophoresis
on a 4-20% SDS-polyacrylamide gradient gel. Proteins were visualized
resistant clones. Ml had the highest capacity to express the
by silver staining, and the gels were dried and exposed to X-ray film
terminally differentiated phenotype, with 55-70% of the cells
(XRP; Kodak: Rochester, NY) at -70°C. Bands corresponding to the
becoming envelope competent after 6 days in culture. In addi
EGF receptor were excised and radioactivities were determined by tion, two other malignant epidermal cell lines, SqCC/Yl and
liquid scintillation spectrometry.
FaDu, produced relatively high amounts of cornified envelopes
EGF Receptor Kinase Assay. Plasma membranes from various epi
(i.e.,
44% and 51%, respectively) under the same conditions
dermal cells were isolated by the methodology of Thorn et al. (16).
after
6
days in culture (Fig. 2).
Phosphorylation was carried out in a solution containing 20 mM
Using
a ligand binding assay, A431 cells were found to exhibit
HEPES (pH 7.4), 4 mM MnCl2, 100 ^M Na,VO4, and 20 Mg of
a greater number of EGF receptors than the EGF-resistant
membrane protein, in the presence and absence of 100 ng/ml of EGF
at 4°C.The reaction was initiated by the addition of 50 ^Ci of [12P]- variants, Ml, A5, and A7, and the SqCC/Yl and FaDu carci
nomas (Table 1). The degree of EGF receptor autophosphoATP (5 Ci/mmol). To terminate the reaction, the mixture was applied
to a piece of Whatman P81 filter paper, which was washed three times
rylation that occurs following the binding of EGF is one of the
with 75 mM phosphoric acid. Radioactivity therein was determined
measures of expression of the EGF receptor. Consistent with
the measurements of receptor number, the degree of phosphowith a liquid scintillation spectrometer.
EGF Receptor Binding Assay. Cells were grown in 24-welI dishes at
rylation of the EGF receptor was greater in A431 cells than in
a concentration of 10*cells/well for 24 h before analysis. The procedure
the other cell lines. Since EGF phosphorylates other cellular
proteins in addition to the EGF receptor even at 0°C,the
used for the measurement of cell surface EGF receptor concentration
Days
in
Culture
Fig. 1. Formation of cornified envelopes by A43I cells and EGF-resistant
variants. The capacity of cells to form envelopes was determined as described in
"Materials and Methods" every day beginning on the second day. Results are the
mean of two separate experiments, each consisting of duplicate samples; varia
tions between experiments were always less than 10%. A431 (•).Ml (*). AS (•),
A7 (A).
were further incubated at the same temperature for another 20 h
following the addition of the radioactive probe. The blots were then
5678
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NEOPLASTIC KERATINOCYTE
DIFFERENTIATION
>
\
ro
S
ü
3
ID N- oO" oo
< < (/> U_
•¿->
40-
—¿
EGFR
Fig. 3. Analysis of the autophosphorylation of the EGF receptor. J2P-Labeled
cell lysates were immunoprecipitated with an anti-EGF receptor antibody fol
lowed by the addition of anti-mouse IgG-linked agarose. Immune complexes were
then analyzed on polyacrylamide gels.
•¿Hind
IH
EcoRl
FaDu
SqCC/Yl
Fig. 2. Formation of cornified envelopes by SqCC/Yl and FaDu cells. Cornified envelopes were determined 3 (D) and 6 (Ü)days after initiation of cultures.
Results are the mean of two separate experiments, each consisting of duplicate
samples.
Table 1 ECF receptor number and associated protein kinase activity of
malignant epidermal cells
Phosphorylation was carried out at 4°Cin a mixture containing 20 ^g of
membrane protein with or without the addition of EGF. EGF-stimulated phosphorylation was calculated from the difference in the degree of phosphorylation
between EGF-treated and untreated samples. Cell surface receptor was measured
by incubating cells with [IJ5I]EGF at 4°Cfor 4 h. The binding parameters were
IO
kb
23.19.46.6~
4.42.32.0-
—¿io
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S
_
-
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determined by Scatchard analysis.
activityEGF
protein*niimhfrCelireceptor
Tyrosine kinase
cpm/^g
10~5°21
lineA431MIA5A7SqCC/YlFaDux
Fig. 4. Analysis of EGF receptor DNA by Southern blotting. Genomic DNA
(10 >jg)was digested with EcoRI or Hind\\l restriction enzymes before analysis
43190
±
39240
±
±0.725680.21.11.00.97
by
agarose gel electrophoresis. DNA was transferred to Zeta-probe, which was
±31310
21210±
±
then hybridized with a radioactive EGF receptor complementary DNA probe.
11190
±35280
±20170 21245
±
19185
±
24257
±
this gene was observed in the various cell lines.
1.0-EGF250 ±13+EGF470
±19Difference22050100907572Relativeactivity1.000.230.450.410.340.33
" Numbers represent the sum of both high and low affinity receptors, as
A431 cells expressed at least three species of EGF receptor
determined by Scatchard analysis. The results are the mean ±standard deviation
RNA (Fig. 5, left). Two of the RNA species (10 and 5.6
of three different sets of experiments.
kilobases) encode for the full length receptor, while the most
* Results represent mean ±standard deviation of three different sets of exper
prominent RNA (2.8 kilobases) codes for the truncated receptor
iments.
as determined by scintillation spectrometry.
It has been reported that cells expressing high levels of EGF
receptor always have an amplification of EGF receptor genes
(22). To determine whether the lower expression of the EGF
receptor by the EGF-resistant variants and other malignant cell
lines relative to A431 cells was due to a decrease in the number
of EGF receptor genes, the DNA from these cells was isolated
and the gene content was measured by Southern analysis (Fig.
4). The gene copy number of the EGF receptor in the Ml, A5,
and A7 clones was greatly reduced in comparison to that of
parental cells. Furthermore, SqCC/Yl and FaDu cells also had
a lower copy number of the EGF receptor gene compared to
A431 cells. Using densitometric scanning, the ratio of the copies
of the EGF receptor gene was found to be 30:2:5:5:1:1 for
A431, Ml, A5, A7, SqCC/Yl, and FaDu cells, respectively. In
addition, the DNA restriction patterns of all of the cell lines
were essentially the same. The chicken ß-actinprobe was em
ployed as a control in these experiments and no difference in
(20). Ml, A5, and A7 cells also expressed these RNA species,
albeit to a lesser extent. In contrast, SqCC/Yl and FaDu cells
did not express the truncated receptor RNA, and the expression
of the 5.6-kilobase RNA was also greatly decreased. The
amounts of the EGF receptor RNA were determined by scan
ning the autoradiogram by densitometry; the ratio of the full
length 10-kilobase EGF receptor RNA in A431, Ml, A5, A7,
SqCC/Yl, and FaDu was estimated to be 18:1:10:5:1:1.5, re
spectively. The same blot was reprobed with chicken /3-actin,
and /3-actin was found to be expressed to a similar extent in all
of the squamous cell lines tested (Fig. 5, right).
DISCUSSION
We have reported the isolation of several EGF-resistant
clones of A431 cells which possessed a greater capacity than
the parental line to terminally differentiate (14). Most of the
variants grow at rates comparable to that of the parental cell
lines, so that changes in the degree of differentiation cannot be
5679
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NEOPLASTIC
8
kb
10-
KERATINOCYTE
EGF receptor kinase activity in A431 cells maintains certain
cellular proteins in the phosphorylated form even in the absence
of exogenous EGF. Phosphorylation of some of these proteins
may be important for the suppression of the terminal differen
tiation of these cells. In contrast, epidermal cells with a rela
tively low level of EGF receptor and its accompanying kinase
activity may require the presence of EGF to maintain these
proteins in the phosphorylated form. This possibility agrees
with our finding that the terminal differentiation of SqCC/Yl
cells is suppressed by EGF, while the degree of differentiation
of A431 cells is exceeding low, even in the absence of EGF.
The importance of the tyrosine-specific kinase activity for the
action of the EGF receptor has recently been shown by Bertics
et al. (23) and Moolenaar et al. (24). Both groups have used
mutants of the EGF receptor deficient in kinase activity to
demonstrate that the phosphorylation step is required for EGFstimulated DNA synthesis.
A431 cells have been shown to secrete TGF-a (25), which
binds to the EGF receptor and is an inhibitor of terminal
differentiation of keratinocytes.1 It is conceivable that the
o
o
ro
Ü
io
<
3
Is- Ocr Qo
< (f> U.
5.6-
2.8-
Fig. 5. Left, analysis of EOF receptor RN A by Northern blotting. Total cellular
RNA was isolated and fractionated on 1.0% agarose gels. RNAs were transferred
to GeneScreen and hybridized with the EGF receptor complementary DNA probe.
Right, the same blot was reprobed with a chicken /3-actin probe.
solely due to differences in the rate of proliferation. These
findings have been extended to two additional malignant epi
dermal cell lines, SqCC/Yl and FaDu, whose growth rates were
not inhibited by EGF (data not shown). Both of these cell lines
had a greater capacity to enter the pathway of terminal differ
entiation than A431 cells. A relationship existed between the
level of surface EGF receptor and the capacity of the cells to
form cornified envelopes, suggesting that the EGF receptor
may play a significant role in the terminal differentiation of
epidermal cells. In addition, a direct relationship was found
with these malignant cell lines between the degree of EGF
receptor gene amplification and the expression of the receptor.
Thus, by DNA restriction analysis, less EGF receptor DNA
was observed in all of the EGF-resistant variants than in paren
tal A431 cells, indicating that less gene amplification was
present in the Ml, A5, and A7 clones. Furthermore, SqCC/Yl
and FaDu cells, which expressed fewer surface EGF receptors
than A431 cells, also had lower quantities of the EGF receptor
gene. No differences in the DNA pattern were found between
these cells when Hindlll, EcoRl, or Sad (data not shown) were
used for the digestion of the DNA, suggesting that gene re
arrangement did not occur during the development of the EGFresistant clones. However, the possibility of other restriction
enzyme-specific polymorphisms exists.
Gene amplification is always expressed at the transcriptional
level. Consistent with the findings by Southern analysis, the
expression of EGF receptor RNA was also decreased in the
EGF-resistant variants and in SqCC/Yl and FaDu cells, rela
tive to the parental A431 carcinoma. Interestingly, the expres
sion of the 2.8-kilobase mRNA was also decreased in the
variants, which suggests that this mRNA may arise from alter
native processing instead of being encoded by a separate gene
(20). A431 is one of the few cell lines described to date that
expresses a truncated form of the EGF receptor. Thus, it is not
surprising that SqCC/Yl and FaDu cells did not express the
RNA coding for the truncated receptor.
The EGF receptor possesses an intrinsic tyrosine-specific
protein kinase activity which is enhanced by the binding of
EGF. We have shown in this report that a correlation exists
between the level of protein kinase activity and the capacity of
epidermal cells to form cornified envelopes. Epidermal cells
that have a large number of EGF receptors will also have a high
basal level of tyrosine-specific kinase activity. A high level of
DIFFERENTIATION
changes in the capacity of the A431 variants to undergo differ
entiation is due to decreases in the secretion of TGF-a. How
ever, it is unlikely that two mutations occurred simultaneously
in all of the variants that decreased both the EGF receptor and
the secretion of TGF-a. The contribution of TGF-« to the
response of these differentiation-prone variants awaits further
studies.
Green et al. (26) have detected EGF receptor in epithelial
cells, overlying the basement membrane of the epidermis, which
have proliferative capacity. In contrast, cells do not show de
tectable EGF receptor when they initiate the differentiation
process and loose their growth potential. Nanney et al. (IO)
have reported a difference in the number of EGF receptors in
various epidermal layers, with decreasing levels of receptor
progressing from basal cells to cornified cells. These findings
indicate that cell surface EGF receptor concentration decreases
during terminal differentiation, either being directly involved
in the maturation process or being a consequence of the differ
entiation. Using a panel of cells expressing different amounts
of EGF receptor, we have demonstrated that a decrease in EGF
receptor expression results in an increased capacity to enter the
differentiation pathway. When basal cells divide, one of the
daughter cells presumably migrates to the suprabasal level (27).
By some unknown mechanism, cells at the suprabasal level,
such as spinosa cells, express less surface receptor and are more
distal to the nutrient and humoral supply. These cells presum
ably do not obtain enough signals generated by the interaction
between EGF and its receptor to maintain their proliferative
activity or to suppress the differentiation process. Such a thesis
is supported by the finding that the differentiation of one of the
EGF-resistant clones, Ml, can be inhibited if exogenous EGF
is added to the culture medium (data not shown). Thus, EGF
and its receptor appear to be important in the decision of
epidermal cells to proliferate or to differentiate. The mechanism
by which suprabasal cells regulate the expression of the EGF
receptor and the biochemical changes that these cells undergo
to initiate the program of terminal differentiation is unknown.
It has been suggested that a loss of the control of terminal
differentiation may be one of the reasons that cells become
tumorigenic (28, 29). The systems described in this report may
well be useful to study the regulation of cellular differentiation
of squamous cell carcinoma.
' Unpublished data.
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NEOPLASTIC KERATINOCVTE
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1989 American Association for Cancer Research.
Epidermal Growth Factor Receptor Gene Expression, Protein
Kinase Activity, and Terminal Differentiation of Human
Malignant Epidermal Cells
Ivan King and Alan C. Sartorelli
Cancer Res 1989;49:5677-5681.
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