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Int. J. Cancer: 67,357-364 (1996)
0 1996 Wiley-L,iss, Inc.
Publication of the International Union Against Cancer
Publication de I‘Union Internationale Contre le Cancer
STEROID-HORMONE RECEPTORS IN CELL LINES AND TUMOR BIOPSIES
OF HUMAN LUNG CANCER
Ulrich KAISER',^, Jurgen HOFMANN~,
Margret SCHILLI~,
Barbel WEGMANN~,
Uwe KLoTzI, Steffen WEDEL’,
Ellen WOLLMER~,
Detlev BRANSCHEID? Adi F. GAZDAR~
and Klaus HAVEMAN”
Arvind K. VIRMANI~,
lPhilipps Universitat Marburg, Department of Internal Medicine, Marburg, Germany; 2Simmons Cancer Center,
Universit)iof Texas Southwestern at Dallas, Dallas, Texas, USA; and 3GrosshansdorfHospital,
Department of Thoracic Surgery, Grosshansdo~Germany.
Female gender is a significant independent favorable prognostic factor in lung cancer. To study the possible role of sex
hormones in lung cancer, the expression of sex-steroid receptors and the glucocorticoid receptor was investigated in 29
lung-cancer cell lines stemming from small-cell lung cancer
(SCLC) and non-small-cell lung cancer (NSCLC) by means of
immunocytochemistry, ligand-binding assays and RNA expression via polymerase chain reaction. In at least 2 methods of
investigation, NSCLC cell lines showed a low expression of
estrogen receptor in 6, progesterone receptor in 13 and
androgen receptor in I2 out of 17 cases examined; sex-steroidreceptor expression was virtually absent in SCLC cell lines. The
glucocorticoid receptor was expressed in all 29 cell lines
studied. Additionally, 52 tumor samples from primary lung
cancer were investigated for their receptor expression by
means of immunohistochernistry. Among patients with primary
lung-cancer sex-steroid-receptor expression in tumor biopsies
was detected most frequently in female patients (in 69% of I6
cases, vs. 42% of 36 tumors from men) and in patients with
adenocarcinoma. Further research will focus on these subgroups. lmmunohistology is a feasible method of studying
steroid-receptorexpression in lung cancer.
o 1996 Wiley-Lhs, Inc.
Analyses of large clinical trials have shown that female
gender is a significant independent favorable prognostic factor
in small-cell lung cancer (Albain et al., 1990; Johnson et al.,
1988). Further multivariate analysis of a German multicenter
study comprising 766 patients revealed significance only for
women under the age of 60 years (Wolf et al., 1991). Two
studies of patients with unresectable non-small-cell lung cancer who were treated with chemotherapy also showed a longer
survival rate for women (O’Connell et al., 1986; Finkelstein et
al., 1986).
To determine the influence and possible therapeutical value
of sex hormones in lung cancer, we decided to study the
expression of their receptors in lung-cancer cell lines and
tumor biopsies, since these are a prerequisite for steroid
action. There are basically 3 methods for the detection of
steroid-hormone receptors: gene expression, binding assays
and immunohistochemistry. The gene expression of receptors
has not been investigated in lung cancer so far. Binding assays
which use radioactive ligands in whole-cell or cytosolic assays
are the standard method for studying receptor expression.
Immunohistochemistry uses monoclonal antibodies and has
been well established for the detection of estrogen receptors
and progesterone receptors in breast cancer. Numerous studies have shown a close correlation between immunocytochemical methods and binding assays in mammary carcinoma
(Charpin et UL, 1986).
The diagnosis of lung cancer is made predominantly on
small bronchoscopic biopsies or cytological specimens. Therefore binding assays for which large amounts of fresh tissue
have to be provided cannot readily be performed. Immunohistochemistry may overcome this problem, since this method can
be applied to small tissue-biopsy slides. Consequently, another
aim of our study was to establish immunocytochemistry on
lung-cancer cell lines as a new and additional method of
studying receptor expression. Both the RNA expression of
steroid receptors and the ligand-binding studies were compared with the results of immunocytochemistry. In addition, 52
samples of primary lung cancer were investigated for receptor
expression by immunohistochemistry.
MATERIAL AND METHODS
Cell lines
The cell lines used in this study have been established and
characterized in the National Cancer Institute (Carney et al.,
1985; Giard et al.. 1973), Dartmouth Medical School (Pettengill et al., 1980), the University of Uppsala (Bergh et al., 1985)
and the University of Marburg (Beppler et al., 1987). They are
listed in Table I. Assays were performed on 12 cell lines of
small-cell lung cancer, 16 cell lines of non-small-cell lung
cancer, and one cell line established from a malignant mesothelioma. As control, we used the breast-carcinoma cell line
MCF-7 (Brooks et al., 1973) and the B-cell line B2077
(obtained from the National Cancer Institute) as cell lines with
known receptor expression.
Primary tissue
Primary tumor tissue specimen were obtained from the
Department of Thoracic Surgery at the Grosshansdorf Hospital, Germany (Dr. Branscheid). All tumor samples were
derived from tumor resections and were stored in liquid
nitrogen at -70°C. Diagnosis was made on representative
slides; for confirmation, cryostat sections used in this study
were stained with hematoxylin and eosin. As control tissues for
immunoreaction, cryostat sections from receptor-positive breast
cancer (estrogen receptor, progesterone receptor), normal
prostate (androgen receptor) and lymph node (glucocorticoid
receptor) were prepared.
Out of 61 tumors, 52 were suitable for evaluation, 36 tumors
from men and 16 tumors from women. Among the 52 cases,
there were 18 squamous-cell carcinomas, 17 adenocarcinomas,
11 large-cell carcinomas, 3 SCLC, 2 carcinoids and 1 bronchoalveolar-cell carcinoma. For distribution of pathological stage
and histological grade, see Table V.
Culture media and buffer systems
Cells were maintained in RPMI-1640 medium (GIBCO,
Grand Island, NY) plus 10% FCS (Boehringer, Mannheim,
Germany). Prior to receptor determination with immunocytochemistry and ligand assays the cells were grown for 3 days in a
4To whom correspondence and reprint requests should be addressed, at Philipps Universitat Marburg, Department of Internal
Medicine. Division of HematologyiOncology, Baldinger Strasse. 35033
Marburg, Germany. Fax: (49) 6421-282700.
Abbreviations: SCLC, small-cell lung cancer; NSCLC, non-small-cell
lung cancer; ER, estrogen receptor; PR, progesterone receptor; AR,
androgen receptor; GR, glucocorticoid receptor; DCM, medium
supplemented with charcoal-treated FCS.
Received: November 27,1995 and in revised form April 18,1996.
358
KAISER E T A L
TABLE I - LUNG-CANCER CELL LINES USED IN THE STUDY
SCLC
SCLC
SCLC
SCLC
SCLC
SCLC
SCLC
SCLC
SCLC
SCLC
SCLC
SCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
NSCLC
Cell line
Source
24H
H60
H69
H82
HI46
H187
N417
H510
H526
H841
H1618
DMS 79
H23
H125
H157
H322
H460
H596
H661
H1155
H1299
A549
EPLC32M1
EPLC272H
U1752
Ul810
LCLC-97TMl
LCLC103H
MSTO-211H
Marburg
NCI
NCI
NCI
NCI
NCI
NCI
NCI
NCI
NCI
NCI
Dartmouth
NCI
NCI
NCI
NCI
NCI
NCI
NCI
NCI
NCI
NCI
Marburg
Marburg
UppsaK
Uppsala
Marburg
Marburi
Marburg
Gender
m
f
m
m
m
m
f
m
m
m
f
n.k.
rn
m
m
m
m
m
rn
m
m
m
m
m
m
m
m
m
m
Histologyiclass
classic
classic
classic
variant
classic
classic
variant
variant
variant
variant
n.d.
n.d.
adenocarcinoma
adenocarcinoma
squamous
broncho-alveolar
large-cell
adenocarcinoma
large-cell
large-cell
I arge-ceI I
adenocarcinoma
squamous-cell
squatnous-cell
squamous-cell
large-cell
large-cell
large-cell
mesothelioma
NCI, National Cancer Institute. m, male; f, female; n.k., not known; n.d., not done.
TABLE I1 - ANTIBODIES USED IN THE STUDY
Antieen
Name
Soecies
Class
Source
estrogen receptor
ERICA
progesterone receptor PGR-1A6
rat monoclonal
IgG Abbott (Chicago, IL)
mouse monoclonal IgG Novocastra (Newcastle,
UIJ)
androgen receptor
AN 1-15
rat monoclonal
IgG Affinity Bioreagents
(Freeport, UK)
androeen receutor
Ab 70
rabbit Dolvclonal
Blauer et aL, 1991
mouse-mdnoclonal IgG Affinity Bioreagents
gluco&rticoidLreceptor BuGR2
glucocorticoid receptor anti-GR 721-727 mouse monoclonal IgG Flach et al., 1992
steroid-depleted medium (DCM) based on phenol-red-free
RPMI-1640 medium with 10% FCS which was freed of steroid
hormones by treatment with dextran-coated charcoal.
For the passaging of the NSCLC cell lines, a solution of
0.02% EDTA in PBS was used to detach the cells from the
culture bottles.
Antibodies
Mono- and polyclonal antibodies against the androgen
receptor, the estrogen receptor, the progesterone receptor and
the glucocorticoid receptor used in this study have all been
established and characterized, and are listed in Table 11.
Immunocytochemistty
For immunologic detection, cytospin slides and cryostat
sections were prepared and fixed in 10% paraformaldehyde for
10 min. Subsequent to incubation with the primary antibody
for 10 min at room temperature, immunodetection was performed by the avidin-biotin-complex method using a biotinylated secondary antibody (Vector, Burlingame, CA). Each
preparation was performed twice. As a negative control,
immunoglobulins of the same Ig class, species and in the same
concentration were employed. Predominantly nuclear expression was detected when using the anti-receptor antibodies.
Cell lines were graded as positive (+) if more than 50% of all
cells exhibited clear nuclear expression. Reactivity in 10 to
50% of cells was regarded as weakly positive [(+)I.
Receptor determination by binding assay
For receptor measurement by ligand binding, whole-cell
assays were used. Receptor values were calculated according
to the method of Scatchard.
NSCLC cell lines and the MCF-7 cell line were seeded in
multiwell dishes (Nunc, Roskilde, Denmark) at a density of 50
x lo3cells per well in 0.5 ml of growth medium. After culturing
for one day, cells were grown for 3 more days in 0.5 ml of
DCM. On the day of the assay, cell numbers were determined
from quadruplicate cultures. The wells were subsequently
washed with fresh DCM and supplied each with 200 pl DCM.
A volume of 25 pI of tritiated ligands in PBS-BSA was added
to achieve final concentrations ranging from 0.1 to 25 nM in
quadruplicate cultures; (6.7-3H(N))-dexamethasone(DuPont
de Nemours, Dreieich, Germany), (6.7-3H(N))-estradio1,
( 17a-methyl-3H)-methyl-trienoloneand ( 1 7 ~ m e t h y l - ~ H ) promegestone were used as ligands. In case of tritiated
methyltrienolone, the assay medium was supplemented with 1
pM triamcinolone acetonide (Sigma, St. Louis, MO) to prevent binding to the glucocorticoid receptor. Thereafter the
probes were adjusted to 250 p1 by adding 25 pl of PBS-BSA
with or without unlabeled ligands. Parallel runs in the pres-
STEROID RECEPTORS IN LUNG CANCER
3.59
ence of an excess (1 pM) of unlabeled ligands (Sigma) allowed Estrogen receptor
the determination of unspecific binding. The wells were
Immunoreaction with the monoclonal antibody (MAb) diincubated at 37°C in 5% C02/95% air. After 2 hr the cellular rected to the estrogen receptor (ER-ICA) resulted in strong
monolayers were washed twice with cold DCM. The cells were nuclear positivity for approximately 80% of MCF-7 cells. B
lysed with 200 p,l 1 M NaOH per well at room temperature lymphoblasts were completely negative. Among the lungovernight. Then 200 p1 1 M HCI and 100 pl 1 M HEPES buffer cancer cell lines, 7 of the 29 cell lines (24%), 2 of the 12 SCLC
were added for neutralization. The radioactivity of the soh- and 5 of the 17 NSCLC cell lines expressed estrogen receptor.
tions was determined in a beta-scintillation counter.
Estrogen-receptor-specific DNA fragments with the exSCLC cell lines which grow in suspension cultures were pected size of 0.4 kb could be detected in MCF-7 (positive
cultivated for 3 days in DC medium and washed. Portions of control) and in 12 of 23 (52%) lung-cancer cell lines. All 10
2 x lo6 cells in 200 p,I of D C medium were pipetted into SCLC cell lines were negative, while 12 of the 13 NSCLC cell
multiwell dishes (Nunc). Incubations with the ligands were lines demonstrated RNA expression (Fig. 2).
performed as described for the NSCLC cell lines. Thereafter
Receptor content of the MCF-7 cell line by ligand binding
5O-pl volumes of a 0.05% Trypan-blue solution was added to was determined as 46000 sites per cell (KD0.1 nM). In 8 of the
facilitate the following manoeuvre. The cell suspensions were 28 cell lines (29%) estrogen receptor was detected. In 2 of the
laid on 2-ml cushions of cold PBS-BSA/20% glycerol in 5-rnl receptor-positive cell lines quantification was not possible; in
tubes. The tubes were then centrifuged at 1000 g for 10 min one case, receptor content was less than 1000 binding sites per
and the liquid contents were discarded. The radioactivity of cell. One of the 12 SCLC cell lines and 7 of the 16 NSCLC cell
the resultant cell pellets was determined as described above.
lines were positive.
Cell lines were graded as positive (+) if specificbinding sites
Comparing the methods employed the data suggest that
could be detected. Quantification was expressed in binding SCLC cell lines do not express the estrogen receptor, whereas
sites per cell. In some cases specific binding sites could be estrogen-receptor-RNA expression is common in NSCLC cell
detected though not quantified, which means that low specific lines. However, protein data as measured by ligand-binding
binding could be demonstrated at all ligand concentrations; assays and immunocytochemistry suggest that less than half of
however, Scatchard plots could not be evaluated. These cases the NSCLC cell lines express detectable amounts of the
and cell lines which had receptor expression of less than 1000 estrogen receptor.
binding sites per cell were interpreted as weakly positive [(+)I.
Progesterone receptor
Preparation of RNA
The MCF-7 cell line showed heterogenous nuclear reaction
For polymerase-chain-reaction (PCR) analysis, cells grow- in the majority of cells with the anti-progesterone-receptor
ing in RPMI-1640 medium plus 10% FCS were collected in the MAb in immunocytochemistry. No immunoreaction was demlogarithmic growth phase. Cells were washed twice in PBS and onstrated in the B-lymphoblast cell line. Nineteen of the 28
were dissolved in 6 M guanidinium thiocyanate/25 nM sodium cell lines tested, 19 (68%) expressed the progesterone recepcitrate/0.5% sodium Iauryl sarcosine. This solution was over- tor, among them 6 of the 11 SCLC cell lines and 13 of the 17
laid on a CsTFA cushion (Pharmacia, Uppsala, Sweden) and NSCLC cell lines.
total RNA was pelleted in a Beckman centrifuge (24 hr, 15”C,
Using PCR, MCF-7 cells were positive (0.6 kb) as were 16 of
125,OOOg). For reverse transcription, PolyA-RNA was isolated the 24 lung-cancer cell lines (66%). In addition, 5 of the 10
from 75 pg total RNA using paramagnetic oligo (dT)25 beads SCLC cell lines examined and 11 of the 14 NSCLC cell lines
(Dynal, Oslo, Norway).
expressed progesterone-receptor RNA.
Ligand-binding studies revealed a receptor content of 6000
PCR
sites/cell (K, 0.3 nM) in MCF-7 cells. Of the 27 lung-cancer
PolyA-RNA isolated from 75 pg total RNA was reverse cell lines, 15 (56%) exhibited receptor expression; in 10 cases
transcribed with Superscript RT (GIBCO-BRL) in 30 p1 among the positive cell lines, quantification could not be
reaction volume. cDNAs were tested by amplification of performed; 2 of the 11SCLC cell lines and 13 of the 16 NSCLC
GAPDH (5’ primer: CGT CTT CAC CAT GGA GA; 3‘ cell lines showed receptor expression.
primer: CGG CCA TCA CGC CAC AGT TT) in the presence
In conclusion, 5 SCLC cell lines showed RNA expression, 6
of [a3’P]-dCTP and were equilibrated to give similar GAPDH cell lines were positive for immunocytochemistry. Overall data
signals. For the amplification of specific cDNAs the following suggest low expression of progesterone receptor in SCLC cell
primer pairs were used:
lines, whereas in 13 of 17 NSCLC cell lines at least 2 methods
demonstrated
receptor expression.
hGR: S’primer: CCA TCC AGC TCC TCA ACA GCA A
3’primer: TCG ACT TTC TTT AAG GCA ACC
Androgen receptor
hAR: S’primer: TGG ACA CGA CAA CAA CCA GC
Immunoreaction with the anti-androgen-receptor MAb was
3 ’primer: CTG GTA GAA GCG TCT TGA GC
strictly nuclear in the MCF-7 line. B-lymphoblast cells were
hPR 5’primer: AAC TCA AGA CCT CAT AAT CC
negative for androgen-receptor expression. Of the twenty-nine
3’primer: CAT TCA GTA TTC 7TG GAT GAG C
lung cancer cell lines, 16 (55%) revealed positivity; 4 of the 12
hER S’primer: CAA GAC ATA CCA AGA GAA GC
SCLC cell lines and 12 of the 17 NSCLC cell lines revealed
3’primer: GTT GCT GTC ACT ACT CAG GC.
nuclear positivity.
Cell lines were graded as positive if a distinct fragment could
RNA expression was demonstrated in the MCF-7 cell line at
be detected. A faint band was evaluated as weakly positive.
0.5 kb. Of the 23, 15 (65%) cell lines were positive, while 4 of
the 10 SCLC cell lines and 11 of the 13 NSCLC cell lines
expressed androgen-receptor RNA (Figs. 3,4).
RESULTS
In the ligand-binding studies, MCF-7 cells had a receptor
Receptor expression in lung-cancer cell lines
content of 14000 sitesicell (K, 0.3 nM). Out of 27 cell lines, 15
The results are summarized in Table 111. Data on receptor (56%) showed receptor expression; in 2 cases quantification
quantification by ligand-binding assays are listed in Table IV. was not possible, in 1 case receptor content was less than 1000
Using all 3 detection methods, receptor expression was deter- binding sites per cell; 3 of the 11 SCLC cell lines and 12 of the
mined as follows (Figs. 1-5).
16 NSCLC cell lines were positive.
360
KAISER E T A L
TABLE 111- STEROID-RECEPTOR CONTENT OF LUNG-CANCER CELL LINES'
Estrogen receptor
Cell line
IC
24H
H60
H69
H82
H146
H187
N417
H510
H526
H84 1
H1618
DMS7V
H23
H125
H157
H322
H460
H5Y6
H661
H1155
H1299
A549
EPLC 32M1
EPLC272H
U1752
U1810
LCLC-97TM1
LCLC103H
MSTO-211H
Progesterone receptor
Ligand
PCR
Cell line
(+)
24H
H60
H69
H82
H146
H187
N417
H510
H526
H841
H1618
DMS79
H23
H125
H157
H322
H460
H596
H661
H1155
H1299
A549
EPLC 32M1
EPLC272H
U1752
U1810
LCLC-V7TM1
LCLC103H
MSTO-211H
-
ti]
-
+
-
-
-
-
-
+
Androgen receptor
Cell line
24H
H60
H69
H82
H146
HI87
N417
H510
H526
H841
H1618
DMS79
H23
H125
H157
H322
H460
H596
H661
H1155
H1299
A549
EPLC 32M1
EPLC272H
U1752
U1810
LCLC-97TM1
LCLC103H
MSTO-211H
IC
Liaand
PCR
-
-
-
+
-
-
++
+
++
t
+
-
(+I
-
111
>+'
Ligand
+
-
PCR
+
+-
(i)
n.d.
+-
+
n.d.
-
+
+
+
+
+
+
n.d.
n.d.
n.d.
+-
-
n.d.
+
(+I
+
+
(+I
+-
+
Glucocort~co~d
receptor
(+)
n.d.
-
(+I
IC
(+I
+
(+)
+
+
+++
n.d.
-
Cell line
-
-
+
n.d.
-
+
+
n.d.
+-
+
+
(+)
n.d.
-
+
n.d.
n.d.
+
+
+
(+I
+
n.d.
(+)
i+)
24H
H60
H69
H82
H146
H187
N417
H5 10
H526
H841
H1618
DMS79
H23
H125
H157
H322
H460
H5Y6
H661
H1155
H1299
A549
EPLC 32M1
EPLC272H
U1752
U1810
LCLC-Y7TM1
LCLC103H
MSO-211H
IC
+
+
n.d.
n.d.
+
+
+
+
+
+
+
+
+
n.d.
+
+
+
+
+
+
+
+
+
n.d.
n.d.
-
n.d.
n.d.
+
Ligand
PCR
+
+
+
+
+
+
+
+
+
+
+
n.d.
+
+
+
+
n.d.
+
+
+
+
+
-
n.d.
(+)
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
n.d.
+
+
n.d.
n.d.
+
+
+
+
+
+
+
+
'As measured by immunocytochemistry (IC), ligand-receptor binding (Ligand) and RNA
expression (PCR) detected via RT-PCR amplification.
Comparing the results, 2 SCLC cell lines were positive for
the androgen receptor with all 3 methods employed. In 2
further cases expression data were incomplete, These results
suggest that androgen-receptor expression in SCLC is uncommon. A few cases, however, may express the androgen receptor. In 12 of the 17 NSCLC cell lines, at least 2 methods
demonstrated receptor expression, although in 4 of these cases
R N A expression was low. Nevertheless, androgen receptor in
NSCLC cell lines is a common event.
Glucocorticoid receptor
In immunocytochemistry, glucocorticoid-receptor expression was high in MCF-7 cells with both MAbs described above.
B-lymphoblast cells showed an intense nuclear reaction pat-
STEROID RECEPTORS IN LUNG CANCER
361
TABLE IV - STEROID-RECEPTORCONTENT OF LUNG-CANCER CELL LINES’
Cell line
24H
H60
H69
H82
H146
H187
N417
H5 10
H526
H841
H1618
DMS 79
H23
HI25
H157
H322
H460
H596
H661
H1155
H1299
‘4549
EPLC 32M1
EPLC272H
U1752
U1810
LCLC-97TM1
LCLC103H
MSTO-211H
ER
Kd
PR
Kd
AR
Kd
(+I
n.d.
-
0.5
1
1.9
0.3
1500
0.3
3500
0.4
12000
10000
18000
111000
1.3
0.7
1.8
41000
200
4000
69000
27000
0.3
2
0.5
0.5
0.3
19000
18000
2.5
0.5
-
3.4
1.5
0.6
4.7
1
0.8
-
(+I
1.6
-
0.4
-
n.d.
-
GR
Kd
3400
1900
2000
1100
5400
5000
n.d.
5.1
1.3
3.2
3.1
5.4
4.7
(A0
2.8
3700
24000
3 1000
36000
211000
42000
85000
186000
86000
20400
42000
314000
141000
147000
157000
47000
139000
66000
63000
18.1
14
3.5
10
7.7
10
7
8.2
2.6
9.8
1.6
9.3
8.2
8.7
7.6
12
7.7
7.3
5.4
-
‘As measured by ligand-receptor binding. Receptor content in binding sites per cell (parts of the
results of the glucocorticoid receptor have been published in Hofmann et al. (1995)).
FIGURE1 - Immunocytochemistry of lung-cancer cell line
NSCLC-EPL32M1 with anti-GR. Scale bar, 10 km.
tern. Among the lung-cancer cell lines, 22 of the 22 lines (95%)
revealed glucocorticoid-receptor expression, while 10 out of 10
SCLC and 11 of the 12 NSCLC cell lines were positive.
In PCR the MCF-7 cell line was positive at 1.15 kb, as were
all 24 (100%) lung-cancer cell lines (Fig. 5 ) .
In the ligand-binding studies, the MCF-7 cell line was
measured as having a receptor content of 19000 sites/cell (K,
1.2 nM). Of 28 cell lines measured, 27 (96%) were receptorpositive; among them quantification was not possible in 1 case;
10 of 11 SCLC cell lines and all 17 NSCLC cell lines were
positive.
All 3 methods demonstrated GR expression in lung-cancer
cell lines, in SCLC and in NSCLC cell lines.
FIGURE2 - cDNA from NSCLC cell lines as indicated were
amplified with specific primers for the human estrogen receptor
(for primer sequences, see “Material and Methods”). The length
of the amplification product is 0.4 kb, as expected. The size marker
(M) consisted of HaeIII fragments of PhiX 174 DNA. The MCF-7
cell line was included as positive control; for negative control,
c~~~ was substituted with water,
Immunohistochemistryin primary lung cancer
Using immunohistochemistry, glucocorticoid receptor was
detected in 49 cases (94%), progesterone receptor in 23 cases
(44%), androgen receptor in 9 cases (17%), and estrogen
receptor in 4 of the 52 tumor samples (8%). When tumors were
grouped according to histology, sex-steroid-receptor expression was most frequent in adenocarcinoma, as indicated in
Table V. Out of 16 cases, 11 (69%) exhibited sex-steroidreceptor expression. Receptor expression was found in 47% of
the 17 squamous-cell carcinomas and 23% of the 13 large-cell
carcinomas.
When tumors were grouped according to the gender of the
patients, there was strikingly higher expression of sex-steroid
receptors in tumors from women than in tumors from men:
KAISER ETAL.
362
TABLE V - STEROID-RECEPTOR EXPRESSION IN PRIMARY LUNG
CANCER DETERMINED BY IMMUNOHISTOCHEMISTRY
FIGURE3-cDNA from NSCLC cell lines as indicated were
amplified with specific primers for the human androgen receptor
(for primer sequences, see “Material and Methods”). The length
of the amplification product is 0.5 kb, as expected. For description
of marker and control, see Figure 2.
FIGURE4-cDNA from SCLC cell lines as indicated were
amplified with specific primers for the human androgen receptor
(for primer sequences, see “Material and Methods”). The length
of the amplification product is 0.5 kb, as expected. For description
of marker and control, see Figure 2.
Gender
Histology
Sl
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
f
squamous-cell
squamous-cell
squamous-cell
squamous-cell
squamous-cell
squamous-cell
squarnous-cell
squamous-cell
squamous-cell
squarnous-cell
squarnous-cell
squamous-cell
squamous-cell
squatnous-cell
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
broncho-alveolar
large-cell
large-cell
large-cell
large-cell
large-cell
large-cell
large-cell
large-cell
large-cell
large-cell
large-cell
SCLC
squamous-cell
squamous-cell
squamous-cell
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
adenocarcinoma
large-cell
large-cell
carcinoid
carcinoid
SCLC
SCLC
IIIA
I
I
I1
I
I
IIIB
IIIA
I1
I1
IIIA
IIIA
IIIA
I
11
IIIB
I
IIIA
IIIB
IIIA
IIIA
IIIA
IIIA
IIIA
I
IV
I1
IIIA
IIIA
I1
IIIB
I1
I1
IIIA
IV
I
IIIB
I1
IIIA
I1
IIIA
IIIA
1
IIIB
IIIA
I
IIIA
IIIA
I
I
I
I
f
f
f
f
f
f
f
f
f
X
2
2
3
2
3
2
2
2
3
I
X
X
X
X
X
X
+
-
+
+
-
-
+
+
+
+
+
+
+
-
-
-
-
+
-
+
-
+
+
-
-
-
+
-
+
+
-
-
-
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
st, staging. Clinical staging of lung cancer was performed
according to the International System for Staging Lung Cancer
(Mountain, 1986). G, cytological grading of tumour cells, 1-111;
X, no grading performed. +, distinct nuclear immuno reaction in
tumour cells; -, no immuno reaction detectable.
FIGURE5 - cDNA from NSCLC cell lines as indicated were
amplified with specific primers for :he human glucocorticoid
receptor (for primer sequences, see Material and Methods”).
The length of the amplification product is 1.15 kb, as expected. For
description of marker and control, see Figure 2.
69% of the 16 tumors from female patients vs. 42% of the 36
tumors from male patients.
DISCUSSION
Differences in survival among men and women with lung
cancer have led to the assumption that sex hormones and their
receptors may play a role in the biology of this tumor.
Bhatavdekar et al. (1994) have shown that serum levels of
various hormones, including FSH, LH and testosterone, in
lung cancer patients differ from the levels in controls. A review
of the literature reveals that in primary lung cancer only
limited data are available on hormone-receptor expression,
which is the prerequisite for hormone action. Binding assays
have been performed to some extent in lung cancer.
The glucocorticoid receptor has been reported to be expressed in a large number of non-small-cell lung cancers.
However there is large variation in reported results, depending
on the binding assay used. Beattie et al. (1985) reported 75% of
NSCLC to be positive for the glucocorticoid receptor, while
Chaudhuri et al. (1982) found that 30% of NSCLC expressed
STEROID RECEPTORS IN LUNG CANCER
363
the glucocorticoid receptor. No standard threshold for recep- than in the cell lines. Loss of protein by storing procedures
tor positivity has been established. Inter-observer differences cannot be completely ruled out. The glucocorticoid receptor is
are also striking for detection of the androgen receptor. While frequently expressed in primary tumors. Among the sexBeattie et al. (1985) found androgen receptors in almost 75% steroid receptors, expression is lowest for estrogen receptor,
of 26 cases, Chaudhuri et al. (1982) and Cagle et al. (1990) while a higher expression rate is seen for progesterone
reported such expression in less than 20% of NSCLC. Data for receptor. Our data confirm the observation that steroidexpression of the estrogen and the progesterone receptors are receptor expression is highest in adenocarcinoma (Beattie et
more conclusive. The estrogen receptor was found in 10 to al., 1985).
30% of NSCLC (Beattie et al., 1995; Chaudhuri et al., 1982;
The clinical impact of these findings remains to be deterCagle et al., 1990; Kobayashi et al., 1982). The progesterone mined. In one sub-group of positive cell lines, receptor
receptor was found in 20 to 25% of NSCLC by means of expression as determined by ligand-binding studies was low.
ligand-binding assays (Beattie et al. 1985; Chaudhuri et al., However, in another sub-group, the glucocorticoid receptor
1982; Cagle et al., 1990).
and the androgen receptor, particularly, were expressed to a
Data on small-cell lung cancer are sparse. At least 0.5 to 1 g considerable level. Hence, these cell lines may represent
of tissue have to be available for binding assays. Since the tumors that are sensitive to hormonal treatment. Only 3 cell
diagnosis of small-cell lung cancer is most frequently made on lines investigated were from female patients; however, data
small bronchoscopic biopsies, binding assays are often not from primary tumors are more conclusive. The better prognosis for women with lung cancer may be related to increased
feasible in this tumor type.
We chose a set of lung-cancer cell lines to study receptor expression of steroid receptors, as seen in tumors from women.
expression using 3 different methods, namely immunocytochem- However, other aspects have to be considered, such as differistry, ligand-binding assays and RNA expression. A survey of ing metabolisation to cytostatic drugs in men and women.
The epidemiologic data and our own findings suggest that
receptor expression on a panel of SCLC and NSCLC cell lines
shows expression of sex-steroid receptors in a sub-group of cell sex hormones may influence lung cancer. Proliferation studies
are currently undertaken to evaluate the influence of estrogen
lines.
Among a number of conclusions, our results indicate strik- and progesterone on lung-cancer cell lines. Depending on
ing differences in sex-hormone-receptor expression between these results, a sub-set of patients may be defined whose
SCLC and NSCLC cell lines. Expression of estrogen, androgen tumors may be susceptible to hormonal influence. Earlier in
and progesterone receptors is rare in SCLC cell lines. In at vitro studies have indicated a growth-inhibitory effect of
least 2 methods of investigation, NSCLC cell lines showed low dexamethasone (Hofmann et al., 1995) and a growth-promotexpression of estrogen receptor in 6 of progesterone receptor ing effect of testosterone in lung-cancer cell lines (Maasberg et
in 13 and of androgen receptor in 12 out of 17 cases examined. nl., 1989). In a pilot study performed at our institute, a
Glucocorticoid-receptor expression is substantial in NSCLC as temporary growth-inhibitory effect of the anti-androgene fluwell as SCLC cell lines. For detection of the glucocorticoid tamide combined with an LHRH-agonist could be detected in
receptor and the sex steroid receptors, immunochemistry patients with extensive small-cell lung cancer.
appears as a feasible method. In a few cases, discrepancies
Adenocarcinomas of the lung and other tumors from female
between results of immunocytochemistry and of PCR were patients preferentially express sex-steroid receptors. Further
detected. Since these were cases in which “borderline” expres- research on the functional activity of sex steroids will focus on
sion was observed, the different threshholds of positivity may these sub-groups.
account for these findings. In a few cases with positive RNA
expression but lack of protein detection receptor expression
ACKNOWLEDGEMENTS
may have been too low to be detected.
Receptor-expression studies in primary tumors using immuWe thank Mrs. B. Safran for excellent laboratory assistance.
nohistochemistry widely confirm the data obtained from cell This work was supported by a grant from the Deutsche
lines. However, expression rates for all receptors are lower Krebshilfe, Bonn, Germany (project W30/93/ka2).
REFERENCES
ALBAIN,K.S., CROWLEY,
J.J., LE BLANC,M. and LIVINGSTON,
R.B.,
Determinants of improved outcome in small-cell lung cancer: an
analysis of the 2,580-patient Southwest Oncology Group data base. J.
din. OncoL, 8, 1563-1574 (1990).
BEATTIE,
C.W., HANSEN,
N.W. and THOMAS,
P.A., Steroid receptors in
human lung cancer. Cancer Res., 45,4206-4214 (1985).
human cell line (MCF-7) from breast carcinoma. J. biol. Chem., 248,
6521-6523 (1973).
CAGLE,P.T., MODY, D.R. and SCHWARTZ,
M.R., Estrogen and
progesterone receptors in bronchogenic carcinoma. Cancer Res., 50,
6632-6635 (1990).
CARNEY,
D.N.. GAZDAR,
A.F., BEPPLER,
G., GUCCION,
J.G., MAFUNBEPPLER,G., JAOUES,G., NEUMANN,
K., AUMULLER,
G., GROPP, GOS,P.J., MOODY,T.W., ZWEIG,M.H. and MINNA,J.D., EstablishK., Establishment, growth properties, and morpho- ment and identification of small-cell-lung-cancer cell lines having
C. and HAVEMANN,
logical characteristics of permanent human small-cell-lung-cancer cell classic and variant features. Cancer Res., 45,2913-2923 (1985).
lines. J. CancerRes. clin. Uncol., 113,3140 (1987).
CHARPIN,
C., MARTIN,P.M., JACQUIMIER,
J., LAVAUT,M.N., POURBERG,J., NILSSON,
K., EKMAN,
R. and GIOVANELLA,
B., Establishment REAU-SCHNEIDER,
N. and TOGA,M., Estrogen-receptor immunocytoand characterization of cell lines from human small-cell and large-cell chemical assay (ER-ICA): computerized image analysis system, immucarcinomas of the lung. Acta path. microbiol. immunol. scand. sect. A., noelectron microscopy, and comparisons with estradiol-binding assays
93,133-147 (1985).
in 115 breast carcinomas. CancerRes., 46,4271~4277s(1986).
BHATAVDEKAR,
J.M., PATEL,D.D., CHIKLIAR,
P.R., MEHTA,R.H., CHAUDHURI,
P.K., THOMAS,P.A., WALKER,M.J., BRIELE,H.A.,
T.P., DAS GUPTA,Y.K. and BEATTIE, C.W., Steroid receptors in human
VORA,H.H., KARELIA,
N.H., GHOSH,N., SHAH,N.G., SUTHAR,
NEEMA,J.P. and BALAR,D.B., Levels of circulating peptide and lung-cancer cytosols. Cancer Leu., 16,327-332 (1982).
steroid hormones in men with lung cancer. Neoplusma, 41. 101-103
FINKELSTEIN,
D.M., ETTINGER,
D.S. and RUCKDESCHEL,
J.C., Long(1994).
term survivors in metastatic non-small-cell lung cancer. A n Eastern
BLAUER,
M., VAALASTI,
A., PAULI,S.L., YLIKOMI,
T., JOENSUU,
T. and Cooperative Oncology Group Study. J. clin. Uncol., 4,702-709 (1986).
TUOHIMAA,
P., Location of androgen receptors in human skin. J.
FLACH,
H., KAISER,
U. and WESTPHAL,
H.M., Monoclonal anti-peptide
Invest. Dermatol., 97,264-268 (1991).
antibodies to the lucocorticoid receptor. J. Steroid Biochem. molec.
BROOKS,
S.C., LOCKE,E.R. and SOULE,H.D., Estrogen receptor in a Biol., 42,467-474 8992).
364
KAISER ETAL.
GIARD,J.D., AARONSON,
S.A., TODARO,
G.J., ARNSTEIN,
P., KERSEY, dent proliferation, and Sa-reductase activity of small-cell lung cancer
J.H., DOSIK,H. and PARKS,
W.P., In vitro cultivation of human tumors: cell lines. IntJ Cancer, 43,685-691 (1989).
establishment of cell lines derived from a series of solid tumors. J. not. MOUNTAIN,
C.F., The new international system for staging lung
Cancerhi., 51,1417-1423 (1973).
cancer. Chest, 89 (Suppl), 2258-233s (1986).
HOFMANN,
J., KAISER,
U., MAASBERG,
M. and HAVEMA”,K., Gluco- O’CONNEL,
J.P., KRIS,M.G., GRALLA,
R.J., GROSHEN,
S., TRUST,A.,
corticoid receptors and growth-inhibitory effects of dexamethasone in FIORE.J.J., KELSEN,D.P., HEELAN,R.T. and GOLBEY,R.B., Frehuman lung-cancer cell lines Europ. J. Cancer, 31A, 2053-2058 (1995). quency and prognostic importance of pretreatment clinical characterJOHNSON,
B.E., STEINBERG,
S.M., PHLEBS,R., EDISON,M., VEACH, istics in patients with advanced non-small-cell lung cancer treated with
S.R. and IHDE, D.C., Female patients with small-cell lung cancer live combination chemotherapy. J. clin. Oncol., 4,1604-1614 (1986).
PETTENGILL.
O.S., SORENSON,
G.D., WURSTER-HILL,
D.H., CURPHEY,
longer than male patients.Amer. J. Med., 85,194-196 (1988).
T.J., NOLL,W.W., CATE, C.C. and MAURER,L.H., Isolation and
KOBAYASHI,
S.. MIZUNO,
T., TOBIOKA,
N.. ICHIMURA,
H., SAMOTO,
T., growth characteristics of continuous cell lines from small-cell carciA., WAKABAYASHI,
S., UMEMURA,
S., FUKUOKA, noma of the lung. Cancer, 45,906-918 (1980).
TANAKA,
H., MASAOKO,
H. and NAGAI,H., Sex-steroid receptors in diverse human tumors. WOLF,M., HOLLE,R., HANS,K., DRINGS,P. and HAVEMANN,
K.,
Gann, 73,439-445 (1982).
Analysis of prognostic factors in 766 patients with small-cell lung
MAASBERG,
M., ROTSCH,M., JAQUES, G., ENDERLE-SCHMIDT,
U., cancer (SCLC): the role of sex as a predictor for survival. Brit. J.
WEHLE,R., HAVEMANN,
K., Androgen receptors, androgen-depen- Cancer, 63,986-992 (1991).
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