Steroid Receptors in Human Lung Cancer1 · [CANCER RESEARCH 45, 4206-4214, September 1985] Steroid Receptors in Human Lung Cancer1 Craig W. Seattle, Nancy W. Hansen, and Paul A. Thomas2

[CANCER RESEARCH 45, 4206-4214, September 1985]

Steroid Receptors in Human Lung Cancer1

Craig W. Seattle, Nancy W. Hansen, and Paul A. Thomas2

Division of Surgical Oncology, College of Medicine, University of Illinois at Chicago [C. W. B.Â¡,and Division of Cardio-Thoracic Surgery, Department of Surgery, West

Side Veterans Administration Hospital, Chicago, Illinois 60612 [N. W. H., P. A. T.]

ABSTRACT

We have determined that a significant incidence of specifichigh affinity receptors for androgen (AR), estrogen (ER), andglucocorticoid (GR) is present in normal adult human lung andbronchogenic carcinoma cytosols. In contrast, a limited numberof tumor cytosols bound progesterone. Binding characteristicsfor each class of steroid hormones were similar to those reportedfor other steroid-responsive normal and neoplastic tissues. ERwas evenly distributed between squamous cell and adenocarci-

noma cytosols with a slightly lower affinity, but higher contentthan normal lung. In normal lung, GR resolved into two distinctbinding components based on affinity using a dextran-coated

charcoal assay. AR in squamous cell carcinomas behaved in asimilar manner. This was not observed when hydroxyapatite wasused to separate bound from free ligand. When AR affinity andcontent were stratified on the basis of tumor grade in squamouscell carcinoma, the most undifferentiated tumors had a lower ARcontent and higher affinity. In contrast, there was no differentiation of AR content or affinity based on tumor grade in adenocar-

cinoma where AR also did not resolve into two distinct groupsbased on binding affinity. Although related to tumor grade, ARincidence and content were not related to stage of disease. Inadenocarcinoma, initial results suggest GR affinity and contentwere inversely related to degree of tumor differentiation, whileGR content was slightly lower in poorly differentiated squamouscell carcinomas. GR content in squamous cell carcinoma increased slightly between Stages I and II and declined significantlyin Stage II patients. This was not observed in adenocarcinoma,where GR content appeared to increase with stage of disease.Our results suggest that a significant incidence of specific, highaffinity receptors for estrogen, androgen, and glucocorticoid ispresent in nonsmall cell carcinomas of the lung, which couldprovide a useful starting point for examining whether steroidsinfluence the natural history of selected bronchogenic carcinomas.

INTRODUCTION

Steroids, particularly glucocorticoids and estrogens, regulatemammalian lung differentiation and maturation (1-4). Glucocor

ticoids accelerate the maturation of type II alveolar epithelial cellswhich produce surfactant (5) and induce protein synthesis (6, 7)in rabbit lung. The effects of glucocorticoids in rat and rabbitappear mediated by specific, high affinity, cytoplasmic and nuclear receptors, particularly in pulmonary type II cells (8, 9). Inhumans, glucocorticoids also stimulate fetal surfactant production (10), possibly via specific glucocorticoid receptors on type IIpneumocytes (11).

Estrogens also directly alter fetal lung differentiation and maturation (12). In the presence of maximal stimulatory amounts ofglucocorticoid, low doses of estrone and estradiol increase pulmonary surfactant production, while higher doses are inhibitory(2). Estradiol binds to fetal guinea pig (13) and adult rat lungcytosols (14), sedimenting as a 4S species (15). Human fetal andadult lung also bind estradiol (10, 16, 17), albeit with a slightlylower affinity.

Like estrogen, androgens bind to cytosols from fetal and adultrat lung (14, 18) with no apparent sex difference in androgenbinding.

In addition to promoting surfactant production and cell growth,hormones also strikingly alter the ultrastructural morphology ofhuman lung expiants. Addition of cortisol, prolactin, and insulinin vitro induces the formation of lamellar bodies in type II pneumocytes (3).

These studies suggest that bronchogenic carcinomas arisingfrom hormone-responsive cells could retain, and perhaps magnify, some of the characteristics of the hormone-responsive

state.One potential mechanism for the action of steroids on primary

bronchogenic carcinoma is receptor mediation. Receptors forestrogen (16,19, 20), androgen (16, 20), and glucocorticoid (16,21) are present in histologically diverse primary and metastaticbronchogenic carcinomas. Tumor receptor incidence is variable,however, and may be dependent on either the histogenetic originof the lesion or method of assay. To date, no systematic attempthas been made to characterize steroid binding in normal lungand primary bronchogenic carcinoma cytosols in a large seriesof patients under different assay conditions. The present studyreports that a significant incidence of specific, high affinity receptors for androgen, estrogen, and glucocorticoid is present innormal and bronchogenic carcinoma cytosols.

MATERIALS AND METHODS

Materials. [1,2-3H]DEX3 (20.8 Ci/mmol), [2,4,6,7-3H]estradiol (96 Ci/mmol), [3H]R5020 (promogestone), a synthetic progestin (70-87 Ci/mmol), and [3H]R1881, a synthetic androgen (70-87 Ci/mmol) were

purchased from New England Nuclear (Cambridge, MA) and repunfiedprior to use. Radioinert steroids were purchased from New EnglandNuclear and Sigma Chemical Co. (St. Louis, MO). Trizma base, EDTA,HAP, molybdic acid sodium salt, crystalline bovine serum albumin, -

monothioglycerol, dithiothreitol, glycerol, phospholipase A, DNase,Rnase, and trypsin were also purchased from Sigma. Activated charcoal,ammonium sulfate, and additional reagents were purchased from J. T.Baker Co. (Phillipsburg, NJ), and RNase-free sucrose was from

1Supported by the Veterans Administration Research and Development Service.2To whom requests for reprints should be addressed, at the Division of Cardio-

Thoracic Surgery, Department of Surgery, West Side Veterans AdministrationHospital, 900 S. Damen, Chicago, IL 60612.

Received 1/22/85; revised 5/23/85; accepted 6/5/85.

3The abbreviations used are; DEX, dexamethasone acetate (9-fluoro-11,17-dihydroxy-16-methylpregna-1,4-diene-3,20-dione diacciate); DCC, dextran-coatedcharcoal; R5020,17,21 -dimethyl-19-norpregna-4,9-diene-3-one; R1881,17Â«-meth-yltrienolone; HAP, hydroxyapatite; DTT, dithiothreitol; ER, estrogen receptor; PGR,progesterone receptor; AR, androgen receptor; GR, glucocorticoid receptor; DES,diethylstilbestrol; DHT, dihydrotestosterone.

CANCER RESEARCH VOL. 45 SEPTEMBER 1985

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RECEPTORS IN LUNG CANCER

Schwartz-Mann (Orangeburg, NY). Dextran T-70 was obtained fromPharmacia (Piscataway, NJ). ACS scintillation cocktail was from Amer-sham-Searle Corp. (Arlington Heights, IL).

Methods (Tumor and Normal Lung Specimens). Fifty-five lung can

cer tissue specimens were obtained from 55 adult patients undergoingprimary surgery for lung cancer: 52 were obtained from male patientsand 3 from female patients. Thirty (56%) were squamous cell carcinomas,21 (38%) were adenocarcinomas, and 4 (7%) were anaplastic large celltumors. All tumros were verified histologically and staged using thecriteria established by the American Joint Committee for Cancer Staging(22). Twenty-seven specimens of histologically normal ventilating lung

were recovered from 30 of the patients with squamous cell lung cancer,17 of the 20 patients with adenocarcinoma and all of the patients withlarge cell anaplastic carcinomas.

All specimens were obtained during surgery when a portion of thespecimen was sent for histopathological diagnosis (light and electronmicroscopic examination). The remaining tumor was trimmed of anynecrotic tissue on ice, washed with ice-cold saline, blotted on sterile

gauze, and immediately frozen in liquid nitrogen. Normal lung specimenswere verified histologically free of tumor prior to receptor assay. Totalelapsed time from removal of specimen to freezing was less than 15min. Receptor analysis was either performed immediately or within 1 wkfollowing surgery. Storage during this time was at -80Â°.

Receptor Analysis (DCC). Tumor and normal specimens were initiallyanalyzed for cytosol receptor for ER, PGR, AR, and GR by the standardDCC technique as previously reported (16). Specimens for assay wereweighed while frozen and powdered in a tissue pulverizer (ThermovacIndustries, Copiague, NY) at liquid nitrogen temperatures. All subsequentsteps were performed at 0-4Â°C. The powdered tissue was homogenized

1:6 (w/v) with two 10-s bursts in a P-10 homogenizer (Brinkman Instruments, Westbury, NY) separated by a 30-s cooling period in the followingbuffer systems: estrogen, 10 mw Tris-HCI:5 mw EDTA:1 mw DTT (pH7.4); androgen and glucocorticoid, 20 HIM Tris-HCI:5 rriM EDTA:12 mMÂ«-monothioglycerol:10% (v/v) glycerol with or without 20 mw sodiummolybdate (23) (pH 7.4); progesterone, 10 mw Tris-HCI:5 mw EDTA:10ITIMa-monothioglycerol:10% (v/v) glycerol with or without 20 mM sodium

molybdate (pH 7.4). The homogenate was centrifugea at 105,000 x gÂ«,(Beckman L8-70 centrifuge; Beckman Instruments, Palo Alto, CA) for 50

min, and the supernatant solution was defined as cytosol. All cytosol swere diluted to 2-5 mg of protein per ml (24) with the appropriate buffer

for receptor assay. Cytosols assayed for ER were divided into twoportions, and one half was precipitated with (NH4)2SO4(40% saturation)in an attempt to reduce the amount of nonspecific binding in the DCCassay. The other half was assayed for ER using HAP to separate boundfrom free ligand (25). Concentrations of [3H]estradiol between 0.1 and

5.0 nw (minimum of eight points) were used in each 200-/il cytosol assayin the presence and absence of a 100-fold excess of radioinert DES. AllAR binding assays were carried out at 4Â°Cfor 16 h and incorporated a

100-fold excess of triamcinolone in addition to a 100-fold excess ofradioinert R1881 to reduce the binding of [3H]R1881 to PGR. Bindingwas determined over a range of 0.1-10 nw [3H]R1881 (minimum of eight

points) (26). PGR assays contained a 100-fold excess of cortisol toreduce any potential binding of [3H]R5020 to the glucocorticoid receptor.Binding of [3H]DEX was determined at 4Â°Cfor 18 h over a concentration

range of 0.25-25 nw (minimum of eight points). Radiolabeled cytosolswere counted in 8 ml of ACS cocktail at an efficiency of 36-38% in lowK ' glass vials and corrected to dpm automatically in a Beckman LS-

7500 liquid scintillation counter. Specific binding (content, measured asfmol/mg of cytosol protein) and affinity (measured as Kd) were calculatedfor each sample according to the methods of Scatchard (27) and Hill(28). No changes in binding content or affinty in additional tumor ornormal lung specimens were found when stored at -80Â°C over a 2-3

wk interval.HAP. The incidence of ER, AR, and GR was also determined using

HAP to insure that DCC separation of bound from free ligand wouldaccurately estimate high affinity, low capacity sites in the presence of

substantial lower affinity (100 nM) binding. HAP (DNA grade) was madeup to 60% with appropriate buffer following washing of fines, and 200-

>ilaliquots were added to each incubation tube. The HAP complex wascentifuged at 3000 x g for 15 min, washed 3 times with buffer at 4Â°C,and extracted with 1 ml of absolute alcohol at 30Â°Cfor 15 min (25). The

alcohol was decanted and counted as above.Sucrose Gradients. Frozen tumor specimens were weighed, pulver

ized at liquid nitrogen temperatures, and homogenized at 4Â°C in the

appropriate buffer (1:4, w/v). Following centrifugation for 1 h at 105,000x gm, all the supernatant solutions (cytosols) were removed, and thepellet was discarded. Cytosol aliquots were incubated for 4 h (AR, ER)or 20 h (GR) at 0-4Â°C with 5 nM [3H]estradiol, 10 nM [3H]R1881, or 20nM [3H]DEX with or without a 100-fold excess of radioinert steroid.

Unbound ligand was removed by incubation with a 1-ml DCC pellet for15 min at 4Â°Cand centrigued at 3000 x g for 15 min. A 250-//I aliquot

was layered on top of a 4.7-ml 10-30% sucrose gradient and centrifugea

at 250,000 x gav in a SS50.1 rotor for 16 h. Additional gradients for ARand GR incorporated 0.4 M KCI or 20 mM sodium molybdate. methyl-14C-labeled bovine serum albumin was used as a 4.6S reference marker

according to the method of Martin and Ames (29). Each tube waspunctured, and 135-^1 fractions were collected under a constant pressure

head and counted in 6 ml of ACS cocktail.Specificity and Stability Analysis. Specificity of binding for androgen

and glucocorticoid was determined by incubating competing radioinertligands with 10~9 M [3H]R1881 and 10"8 M [3H]DEX in cytosols from

normal lung for 20 h at 4Â°Cand separating bound from free ligand with

HAP. The degree of competition is expressed as percentage of bound3H-ligand with the binding of 3H-ligand in the absence of competitor taken

as 100%. Concentrations of radioinert competing ligand ranged from10^-10"9 M. Receptor stability in the presence of phospholipase A,

DNase, RNase, trypsin, and (NH4)2SO4(40% saturation) was determinedin the presence and absence of 20 mM molybdate ion. Additional stabilitystudies were carried out at 30Â°C and 37Â°C in the presence of 20 mw

molybdate ion.Statistical Analysis. A value over 3 fmol/mg of cytosol protein was

determined by Scatchard analysis was arbitrarily taken as positive fortumor specimens in our earlier studies (16). No arbitrary cutoff wasestablished for receptor binding to tumor or normal lung cytosols in thecurrent study. The difference in receptor incidence of any particular classof receptor among histological groups was analyzed by Fisher's exact

test (30). Receptor content and affinity among groups were analyzed byeither analysis of variance for multiple group comparisons or simple ttest (30).

RESULTS

Receptor for Estrogen and Progesterone. [3H]Estradiol

bound to approximately 30% of normal lung cytosols assayedwhen HAP was used to separate bound from free ligand (Table1). ER incidence, content, and affinity (Ka) were similar in squamous cell cancer cytosols when assayed by either DCC followingammonium sulfate precipitation, DCC alone (data not shown), orHAP in the presence of 20 mM molybdate ion (Table 1). In spiteof the limited binding capacity, receptor affinity was generallylower than 1 nw. There was no suggestion of multiple bindingsites on Scatchard analysis when either DCC or HAP was usedto separate bound from free ligand or when radioinert DES wasused as a competitive ligand. A similar ER profile was found inadenocarcinoma cytosols assayed by HAP. When present, binding was resistant to phospholipase A, DNase, and RNase butsensitive to trypsin and maximal at 4 h at 4Â°Cin the presenceof 5 nw [3H]estradiol (data not shown). ER prepared from a

squamous cell carcinoma cytosol sedimented at 7-8S on a 10-

30% low salt sucrose gradient (Chart 1), suggesting the binding


4207




Table 1Incidence of ER and PGR in normal lung and bronchogenic carcinoma

PGR affinity and content were estimated over a concentration range of 0.1-10 nM [3H]R5020 at 4Â°Cfor 16-18 h. Bound and free ligand were separated by DCC. ER

assayed by DCC were first precipitated by (NH4)2SO

â€¢-V"?


though apparent affinity was lower. In line with this observation,HAP did increase the ratio of specific to nonspecific binding inthe presence of molybdate and was therefore used to separatebound from free ligand in specificity and saturability studies.There was virtually no contamination of the tissue specimens byblood proteins (as judged by hemoglobin content) due to priorthorough washing in ice-cold saline prior to freezing. GR binding

in four cytosols prepared from histologically normal lung fromnoncancer patients was similar to that from normal lung cytosolsobtained from patients with bronchogenic carcinoma (Kd 4.7 Â±1.9 nM, 76.1 Â±34.5 fmol/mg of protein; range, 9.1-147.3; n =

4), suggesting that there was little, if any, indirect effect on GRin normal lung tissue specimens by the primary carcinoma.

Binding of [3H]DEX to normal lung (Chart 2) and bronchogeniccarcinoma cytosols (data not shown) was specific for glucocor-

ticoids, saturable (Chart 3), and trypsin sensitive. Maximal binding (fmol/mg of protein) was observed at 15 and 30 min at 37Â°and 30Â°C, respectively, with approximately 50% of apparent

receptor binding degraded after 2 h at either temperature. At4Â°C,binding of [3H]DEX did not plateau until approximately 20

h (Chart 3) but was saturable at 20-25 nw (data not shown) and

may represent equilibrium between higher and lower affinity sitesfor glucocorticoid. Following a 20-h incubation with 20 nw [3H]-

DEX, GR sedimented at 9.8S in low salt 10-30% sucrose

gradients, 11.28 in the presence of molybdate (Chart 4A), and4S in the presence of 0.4 M KCI (Chart 40) when molybdate wasomitted from the incubation buffer and gradient. GR was also

100H

90

80

i70C 60O

% 50

Â¡40

30

20

10

1

10-9 10-8 10-7 10-6

MOLAR CONCENTRATION OF COMPETITORChart 2. Specificity of [3H]DEX (20 mM) binding to normal lung cytosols in the

presence of 20 mM MoO4. O, DEX; â€¢,R1881; A, progesterone; A, R5020; x,estradici; G, DHT. HAP separation bound from free ligand.

1 2 3 4 5 6 16 18 20 22 24 26TIME(hr)

Charts. GR stability in the presence of 20 mM MoO4 at 4Â°C. Binding of[3H]DEX was saturable only after 18-20 h at 20-25 HM. HAP separation bound

from free ligand.

present in 17 of 22 (77%) squamous cell carcinoma cytosolsassayed by DCC with a mean binding affinity and content similarto those of normal lung cytosols assayed in the same manner(Table 2). An additional eight specimens assayed with HAP allexpressed a similar binding profile. Ten of 12 (83%) adenocarci-noma cytosols bound [3H]DEX when assayed by DCC, while 8of 8 bound [3H]DEX when assayed with HAP. There was no

significant difference in GR affinity or content between methodsor between control and tumor cytosols (Table 2). GR contentwas significantly higher in adenocarcinoma cytosols with anaffinity for [3H]DEX > 10 nM as determined by DCC when

compared to normal lung cytosols with the same affinity. Thisalso appeared to be the case in four cytosols from large cellanaplastic lesions, although the number of these latter tumorswas small (Table 2).

Receptor for Androgen. R1881 bound with high affinity butrelatively low capacity to 22 of 24 (92%) normal lung cytosolsassayed by DCC (Table 3) with minimal cross-reactivity from

other steroids (Chart 5). A similar specificity profile was developed for squamous cell and adenocarcinoma cytosols (data notshown). Binding of [3H]R1881 was saturable at the temperature

tested, and like GR, it reached maximal concentrations at 15 and30 min at 37Â° and 30Â°C, respectively. Unlike GR, however,binding plateaued at 4 h at 4Â°Cwith 10 HM[3H]R1881 (Chart 6)

and did not resolve into two groups with distinct affinities.Receptor for androgen sedimented at 7S in low salt gradients,11S in the presence of 20 mw molybdate (Chart 7), and shiftedto a lower molecular weight 48 species in 0.4 M KCI. [3H]R1881

also bound to a significant number of squamous cell (60%) andadenocarcinoma cytosols (87%) specifically and with high affinity(Table 3). In contrast to normal lung cytosols, the relative affinityof the putative receptor for androgen in squamous cell carcinomacytosols in the presence of 20 mM molybdate was resolvableinto two distinct groups when assayed by DCC. Seven of 12(58%) of the squamous cell carcinoma cytosols binding [3H]-

R1881 bound the ligand in a nanomolar range, while the remaining five (42%) expressed binding below 1 nM. Mean content was,as in the case of GR, inversely proportional to receptor affinity.


4209



ce

a.

lOOO-

900-

800-

700-

500-

400 -

300-

200-

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BSA 4 6s

BSA 46s

3000 -

2000-

CL0 1000 -

B

BOTTOM 5 K) 15 20 25FRACTION NUMBER

30 35 TOP BOTTOM 5 10 15 20 25FRACTION NUMBER

30 35 TOP

Chart 4. A, sucrose gradient of [3H]DEX binding to normal lung cytosols under low salt conditions. â€¢,[3H]DEX; O, [3H]DEX:20 HIM MoO4; A, [3H]DEX plus 100-fokJexcess unlabeled DEX. B, sucrose gradient of [3H]DEX binding to normal lung cytosol in the presence of 0.4 M KCI. â€¢,[3H]DEX; O, [3H]DEX plus 100-fold excess of

labeled DEX.

Table 3

Incidence and binding characteristics of AR in normal lung parenchyma andbronchogenic carcinoma

HistopathologyNormal

lungDCC

HAPSquamous

cellDCCHAPAdenocarcinoma

DCCHAPLarge

cellDCCN/T823/25

(92)0

14/15(93)12/18(67)

5/12 (42)"

7/12 (58f4/5(80)7/8

(88)7/8(88)2/4

(50)Affinity

Ka(nw)0.4

Â±0.05Â°

0.3 Â±0.061.7

+ 0.50.2 Â±0.04f

2.9 Â±0.90.4 Â±0.20.6

+ 0.20.4 +0.10.3

+ 0.1ARContent

(fmol/mgprotein)6.3

+ 0.8"

2.6 +0.510.1

Â±3.24.2 Â±2.39

14.4 Â±4.71.6 Â±0.23.3

Â±0.91.9 +0.32.6

Â±1.68 N/T, number positive/number assayed." Numbers in parentheses, percentage.c Mean Â±SE.dP


10-9 10-8 10-7 10-6

MOLAR CONCENTRATION OF COMPETITORCharts. Specificity of [3H]R1881 (1 nuÂ«)binding to normal lung cytosol in the

presence of 20 mM MoO4. O, R1881; â€¢,DHT; A, estradici; â€¢,progesterone; Â®,DEX:cortisol. HAP separation of bound from free ligand.

5 6 16TIME (hr)

18 20 22 24 26 28

Charte. Stability of AR in the presence of 10 nu [3H]R1881. At 4Â°Cbinding

was saturable at 4 h and stable for 24 h. Receptor ligand dissociation was rapid at37Â°Cin the presence of 20 mM MoCv HAP separation of bound from free ligand.

10.2 fmol/mg of protein, n = 5) and a sharp decline in Stage IIIlesions (32.8 Â±8.0 fmol/mg of protein, n = 11). When lesions ofStages I and II were grouped, the mean GR content was greaterthan in Stage III lesions (6.1 Â±9.2 fmol/mg of protein versus32.8 Â±8.0, P < 0.05).

This was not observed in adenocarcinomas where there wasan increase in mean GR content with stage of disease (Stage I,53.1 Â±11.8 fmol/mg of protein, n = 6; Stage II, 84.2 Â±9.4 fmol/

1000

900

800

700

O 600

Â£ 500

o 400

300

200

100

[14c]BSA4.6s

BOTTOM 5 10 15 20 25FRACTION NUMBER

30 35 TOP

Chart?. Sucrose gradient profile of [3H]R1881 binding to normal lung cytosols

under low salt conditions. While capacity was limited (Table 3), suppression bindingwas demonstrable at ~7S and 4S in the absence of MoO4 and at 11S in thepresence of 20 mM MoO. (â€¢).A, 13H|R1881 plus 100-fold excess unlabeled R1881.

mg of protein, n = 3; Stage III, 115.8 Â±29.7 fmol/mg of protein,n = 5) in GR-positive lesions. The two AR-negative lesions were

removed from a Stage I and Stage III patient. While the smallnumber in each group prevents this increase from reachingstatistical significance, it suggests that GR content of the primarytumor increases as the disease progresses. There was no relationship between stage of disease and the presence or absenceofGR.

DISCUSSION

The present results suggest that a significant incidence ofspecific receptors for androgen, estrogen, and glucocorticoidwith an affinity for hormone similar to those in other steroid-responsive tissues (8, 11, 13, 14, 17,18, 23, 31-35) is present

in normal adult human lung and bronchogenic carcinoma cytosols. The presence of a single class of high affinity (


Table 4Androgen and glucocorticoid receptor content as a function oÃ-tumor grade

HistopathologySquamous

cellAdenocarcmomaGradel-llIII-IVl-ll

III-IVN/T*1

2/1 7 (71 f4/6(67)3/3

(100)11/13(85)ARAffinity

KÂ«(nM)1.9Â±0.7Â°

0.25 Â±0.060.3

Â±0.050.6 Â±0.2Content

(fmol/mgprotein)9.3

Â±3.51.7Â±0.72.2

Â±0.53.0 Â±0.7N/T19/23

6/73/3

13/13GRAffinity

Ka(nM)6.4

Â±1.15.0Â±2.515.1

Â±3.57.8Â±0.8Content

(fmol/mgprotein)47.1

Â±9.338.9 Â±17.238.1

Â±15.688.6 Â±13.2

* N/T, number positive/number assayed.13Numbers in parentheses,percentage.: Mean Â±SE.

squamous cell cytosols when OCC was used to separate boundfrom free ligand in the absence of molybdate Â¡on(16,20,36) andsuggest the importance of assay conditions when attempting todetermine small amounts of receptor in the presence of significant amounts of nonspecific binding. In this context, Jones et al.(19) have reported an overall ER incidence of 55% in squamouscell and adenocarcinoma cytosols with binding characteristicsclose to the present study using Sephadex G-25 to separate

bound from free ligand. The choice of an arbitrary cutoff incontent as indicative of positive for receptor may also accountfor the reported differences in ER incidence (16,36). The numberof specimens available for assay in the present study was toosmall to draw any correlation between degree of histolÃ³gica!differentiation and ER incidence or content. While a significantincidence of ER in adult lung parenchyma provides initial evidence that estrogens may continue to influence lung function inadults, additional studies will be necessary to better define theincidence and determine the functionality of this estrogen bindingprotein in adult human lung.

PRG incidence, while relatively high in normal lung, declinedsignificantly in tumor cytosols confirming earlier observations(16, 20). There was no correlation between the presence of ERand PGR. These observations coupled with the lack of receptorin tumor cytosols and absence of relationship between putativeprogesterone binding and fetal lung function (12, 37) suggest aminor role for progesterone in lung homeostasis. Reports ofdisplacible [3H]R5020 binding in fetal rabbit lung (18) may reflect

a lower affinity, potentially less specific binding protein or aspecies difference. A larger series of cytosols will have to beassayed to resolve this question.

Androgen(s) clearly exhibit specific, high affinity, low capacitybinding to normal adult human lung cytosols. While the presenceof a cytosol receptor does not necessarily indicate a role forandrogens in fetal lung development (12) and in the function ofadult lung, the high incidence (93%) of AR and similarity to thebinding characteristics reported for fetal and adult rabbit lung(18) and adult rat lung (14) suggest that this is a viable possibility(16). The presence of molybdate appears essential to reproduc-

ibly resolving the low capacity AR sites, particularly in tumorcytosols, as our own initial results (16) and those of others (20)omitting MoQÂ»from the assay system showed a lower incidenceof AR. AR in squamous cell carcinoma was present in twopotentially distinct species based on receptor affinity using theDCC assay. Cytosols assayed at the same protein concentration(1-2 mg/ml) bound [3H]R1881 with an affinity either above or

below 1 nM. This was not observed in a smaller series of cytosolswhen HAP was used to separate bound ligand.

Each Scatchard analysis gave a linear plot further suggestingtwo distinct species are observable when DCC is used to separate bound from free ligand in the presence of MoO4. This is thefirst report of such a phenomenon in tumors of squamous cellorigin, and as yet we have no definitive explanation for it. [3H]-

DHT does, however, bind to normal skin squamous cell cytosolsas two distinct species (38). Alternatively the expression of ARmay be dependent on tumor grade or stage. Although AR affinity(content) did not appear related to tumor grade or stage ofdisease, our series was too small to statistically evaluate properly. It should be reiterated, however, that AR-negative lesionswere either undrfferentiated or poorly differentiated (Grades III-IV). Previous reports on AR in squamous cell lung cytosols showK,,s in a range of 0.1-1.0 nM (16, 20) with normal human lung

less than 1 nM (16), similar to those in the present study. Incontrast, AR affinity in adenocarcinoma cytosols exhibited onlya single class of high affinity sites identical to that reported forAR in adenocarcinoma of the breast (31, 32).

GR was present in 82% of normal lung specimens assayed.Sucrose gradient analysis and specificity studies show the binding protein to be virtually identical with that reported for fetal andadult rabbit, rat, and human lung (4, 6, 8, 11, 35, 39). Ourobservations suggest that glucocorticoids may continue to influence lung function in adult human lung, possibly via receptor-

mediated processes. When present in normal lung, GR wasresolvable into two distinct binding moieties based on Kd whenassayed in the presence of MoO4 using DCC. This could be afunction of the assay system as GR-positive specimens gave a

linear Scatchard analysis with a Ka either above or below 10 nM.Alternatively this may indicate that functionally there is a differential sensitivity to endogenous or exogenous glucocorticoids inadult human lung. An analogous situation appears to be presentin fetal rabbit lung. Pulmonary surfactant synthesis in fetal rabbitlung is sensitive to lower exogenous concentrations (10~10M) ofcortisol than cell growth (10 8 M) (5, 39) and additional proteinsynthesis (10~7 M) (6, 7), while cultured fetal rabbit lung bindsglucocorticoid at roughly 10~8 M (9). Adult rat and rabbit lung

cytosols have also been reported to bind glucocorticoid in ananomolar range as have rabbit alveolar macrophages (8, 34).When the latter are activated by glucocorticoids, there is anincrease in the number of binding sites (34). The slow saturationof GR at 4Â°C,absent in AR and ER binding but present in normal

human fetal and neonatal cytosols (11), also suggests a multi-

component binding system of potential physiological and pharmacological interest. Additional studies are needed to clarify thispoint.

Squamous cell carcinomas also show a significant incidence


4212




of binding. The incidence of binding in squamous cell carcinomais significantly higher than previously reported (16, 21) usingDCC in the absence of molybdate. Binding affinity (nw) wassimilar to that reported for GR in other neoplastic tissues (21,31-33, 40). A single class of high affinity receptors for [3H]DEX

was also present in essentially all primary adenocarcinomas ofthe lung assayed by either DCC or HAP. These results confirmour earlier observations (71%) (16) and suggest that GR contentis greater in adenocarcinoma than normal lung cytosols whengroups with similar binding constants are compared.

Demonstration of high affinity cytosol receptors for androgensand glucocorticoids in squamous cell and adenocarcinomas ofthe lung tempts speculation that these lesions may be manipulated by increasing or decreasing the level of glucocorticoid.Early studies suggest that administration of low doses of cortisone was associated with an apparent decrease in survival inpatients with metastatic lung carcinomas (41). In contrast, dex-

amethasone has been reported to decrease the growth of alveolar cell carcinoma and Lewis lung carcinoma in vitro at lowconcentrations (40, 42) and provide subjective benefit in bron-chogenic carcinoma patients (43).

Tumor grade and stage of disease may also have to be takeninto account in assessing the response of a particular histopath-

ological grouping. We have presented suggestive evidence for adecrease in receptor content with increasing anaplasia and stageof disease in squamous cell carcinoma. However, GR content inadenocarcinoma appears to increase with dedifferentiation andtumor burden in adenocarcinoma.

Administration of testosterone propionate had no apparenteffect on the progress of metastatic lung carcinoma (41) but didapparently relieve bone pain (44). Metabolism of this ester of theendogenous steroid must be taken into account in any estimateof its efficacy. The decrease in AR content coupled with analteration in affinity in the more anaplastic squamous cell carcinomas suggest that only selected lesions in this histopathologicalcategory may respond to changes in circulating androgen.

The usefulness of ER, AR, and GR as an aid in determiningpatient prognosis and the appropriateness of steroid manipulation as future adjunct or palliative agents in bronchogenic carcinomas clearly remain to be determined. While previous reportssuggest little chance of small (oat) cell carcinomas respondingto steroids (16), the similarity in binding characteristics betweenbronchogenic carcinoma and other steroid-responsive tissues

suggest a potential starting point in investigating whether steroids influence the natural history of bronchogenic carcinomas.

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1985;45:4206-4214. Cancer Res Craig W. Beattie, Nancy W. Hansen and Paul A. Thomas Steroid Receptors in Human Lung Cancer

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Steroid Receptors in Human Lung Cancer1 · [CANCER RESEARCH 45, 4206-4214, September 1985] Steroid Receptors in Human Lung Cancer1 Craig W. Seattle, Nancy W. Hansen, and Paul A. Thomas2