(CANCER RESEARCH 48, 3720-3727, July 1, 1988]

Effects of Estradici on Estrogen Receptor, Progesterone Receptor, and Tyrosinasein Hamster Melanoma Transplanted into Athymic Mice1

M. Helen Hitselberger,2 Rosemary L. Schleicher,3 and Craig W. Beattie4

Department of Pharmacology [M. H. H., R. L. S., C. W. B.] and Division of Surgical Oncology, Department of Surgery [C. W. B.J, University of Illinois College ofMedicine at Chicago; Cook County Hospital; the West Side Veterans Administration Hospital [C. W. BJ; and the Hektoen Institute for Medical Research [C. W. B.J,Chicago, Illinois 60612

ABSTRACT

Nuclear estrogen binding was characterized in IIM-1, a malignant

hamster melanoma cell line transplanted into male and female athymicmice following acute, subchronic, and chronic injection of estradiol.Nuclear binding was saturable, of high affinity ( IO'" M ') and readilysoluble in low salt buffer. Saturation analyses revealed that (3H]estradiol

in excess of 5.0 IIMapparently bound to a second class of lower affinity¡III'1M~'), higher capacity cytosol sites. Enzyme-linked immunoassay

with a specific monoclonal antibody (H222 SPY) directed against thehuman estrogen receptor protein was in excellent agreement (r —0.93)with values obtained using hydroxyapatite to separate bound from freeligand.

Nuclear estrogen receptor content in 11M-1 cells was increased maxi

mally l h after acute s.c. injection of a low dose (0.1 UK}of estradiol. Theincrease in nuclear receptor content was accompanied by an apparentrapid reduction in cytosol binding. Subchronic (3 days) and chronicexposure (35 days) to estradiol also produced a significant, dose-related

increase in tumor nuclear estrogen receptor content.Cytosol binding for progestin was low (

ER IN HAMSTER MELANOMA

mmol), radioinert R5020, and L-[3,5-3H]tyrosine (specific activity, 40-

60 Ci/mmol) were purchased from New England Nuclear Co. (Cambridge, MA). Additional radioinert steroids were purchased from SigmaChemical Co. (St. Louis, MO) as were L-tyrosine, L-dopa, calf thymusDNA, hydroxyapatite, Tris HCl, TES, phenylmethylsulfonylfluoride,A'-ethylmaleimide, EDTA, Triton X-100, sodium azide, sodium molyb-date, and benzamidine. Crystalline bovine serum albumin was obtainedfrom Armour and Co. (Chicago, IL). Activated charcoal was obtainedfrom J. T. Baker Co. (Phillipsburg, NJ) and Dextran T-70 fromPharmacia (Piscataway, NJ). Estradiol pellets (0.1, 0.5 mg) in a timedrelease matrix of cholesterol:methyl cellulose:lactose (vehicle control)were purchased from Innovative Research of America (Rockville, MD).Protein reagents were obtained from Bio-Rad Laboratories (Richmond,CA) as was the Dowex cation exchange resin (SOW x 12, 200-400mesh). Media, antibiotics, fetal bovine serum, and trypsin-EDTA solution were purchased from GIBCO Laboratories (Grand Island, NY).The ER-EIA kit containing a specific monoclonal antibody to human

estrogen receptor (H222 Spy) was a gift from Abbott Laboratories(North Chicago, IL).

Cell Line. HM-1 was subcloned in our laboratory (1) from a spontaneous hamster melanoma (MM1) (25) provided by Dr. J. Fortner(Memorial Sloan-Kettering Institute) and maintained in minimum essential medium with Hanks' salts supplemented with 16% fetal bovineserum and antibiotics. HM-1 cells (5 x 10s) in 0.1 ml minimumessential medium with Hanks' salts were inoculated s.c. in the right

and left flanks of 4- to 5-week-old male or female athymic mice aspreviously described (1).

Athymic Mice. BALB/c derived athymic (nu/nu) mice (NationalCancer Institute, Frederick, MD, courtesy of Dr. C. Reeder) 4-5 weeksof age were used. Mice were maintained as previously described (1, 6).Acutely treated mice were given injections s.c. of estradiol in 6%ethanol/0.9% saline solution (0.1, 2.5, or 10 fig/0.1 ml), progesteronein 20% ethanol/0.9% saline solution (100 Mg/0.1 ml), or vehicle aloneduring log phase growth and killed 1, 2, 6, or 24 h later. Subchronicallytreated mice were given injections s.c. of estradiol (10 ^g) twice/day for3 days with the final injection 12 h prior to sacrifice. Mice were eithersham gonadectomized or bilaterally gonadectomized under aseptic conditions 1 week prior to s.c. implantation of estradiol or vehicle pellets.The pellets were implanted 1 day prior to s.c. inoculation of HM-1cells. Additional pellets were reimplanted 21 days later to maintainelevated plasma levels of estradiol. At necropsy, blood was collected bycardiac puncture and plasma was separated and stored at —20°C.All

flank tumors were carefully excised, trimmed of any necrosis, rinsed inphosphate-buffered saline, blotted dry, and immediately frozen prior to

receptor assay. All statistical procedures were carried out using theBMDP series of programs (BMDP Statistical Software, Los Angeles,CA) on a PDP-11 computer.

Estrogen Receptor Assay. Tumor cytosols were prepared in 10 mMTris-HCl-1.5 mM EDTA-0.5 mM dithiothreitol, pH 7.4 buffer andanalyzed as previously reported (1). Briefly, cytosols (105,000 x g, l h,200 /il) for saturation and Scatchard (26) analysis were incubated at4°Cwith 0.1-10.0 nM [3H]estradiol with or without a 100-fold excess

of diethy 1stilbcstrol for 16 h. Single point assays were done underidentical conditions with a saturating concentration of 13HJestradiol (3HM). Bound and free [3H]estradiol were separated using HAP (27).

Protein was determined by the method of Bradford (28). Nuclei wereseparated using a sucrose pad (29) in 10 mM Tris-HCl-1.5 mM EDTA-0.5 mM dithiothreitol, pH 7.4 buffer at pH 8.O. For Scatchard/satura-tion analyses, nuclei were isolated from tumors carried in intact maleand female mice 1 h postinjection of 2.5 tig estradiol, incubated with0.1-3.5 nM [3H]estradiol with or without a 100-fold excess of diethyl-stilbestrol at 30°Cfor 35 min and bound ligand separated using HAP

(30). DNA was measured by the method of Burton (31) using calfthymus DNA as the standard. When necessary, binding curves werecorrected using the method of Rosenthal (32). ER-EIAs of 10 HM-1cytosols were carried out either on identical cytosols assayed by HAPor samples from the same tumor according to the manufacturers'instructions. Sensitivity of the ER-EIA in our hands is routinely 1-2fmol/mg protein with intra- and interassay coefficients of variation foran internal standard 1.2 and 6.0%, respectively.

Progesterone Receptor Assay. The method used was a modificationofthat of Booms ma et al. (33). Frozen pulverized melanoma specimenswere suspended in 5 volumes of PgRc buffer (0.5 M sucrose-2 mMMgCl2-10 mM Tris-HCl-0.02% NaN3, pH 7.4) and homogenized at 0-4°C(33). All subsequent procedures were carried out at the same

temperature. After centrifugation at 1,300 x g for 10 min to removethe nuclear pellet, the supernatant sol was recentrifuged at 105,000 xg for 1 h and cytosol aliquots (200 ß\)were incubated for 2 h with 3H-labeled R5020 (0.2-5.0 nM) and a 250-fold molar excess of unlabeledcortisol with or without a 250-fold molar excess of unlabeled R5020.Bound and free R5020 were separated by the addition of 300-fil aliquotsof dextran-coated charcoal (0.5% charcoal-0.05% dextran T-70 in 10mM Tris-HCl-1.5 mM EDTA, pH 7.4), incubated for 5 min, centrifugedat 1,800 x g for 10 min, and the supernatant sol counted in 8.0 mlACS scintillation cocktail (Beckman Instruments, Palo Alto, CA).Single-point binding assays were performed under the same conditionsusing a saturating (3.0 nM) concentration of 3H-labeIed R-5020. For

assay of PgR„the pellet was resuspended in assay buffer (same as forPgRc) with 30% glycerol, pH 7.4, at 0-4°C,rehomogenized, recentri

fuged at 1,300 x g for an additional 10 min, and washed again in assaybuffer under the same conditions. The remainder of the assay wascarried out as previously described (33) over a concentration range of0.2-3.0 nM 3H-labeled R5020. A single saturating concentration of 3.0nM 3H-labeled R5020 was used to routinely assay PgR„content.

Ligand Specificity. Cytosol aliquots of HM-1 (105,000 x g supernatant, 200 ^1) were incubated at 0°Cfor 2 h with 5.0 nM 3H-labeled

R5020 in the presence of increasing concentrations of radioinert steroids. Bound and free ligands were separated with dextran-coatedcharcoal (0.5% charcoal-0.05% dextran T-70 in 10 mM Tris-HCl-1.5mM EDTA, pH 7.4). One hundred % specifically bound was calculatedby subtracting the difference in total 3H-labeled R5020 bound from

that bound in the presence of 5 /¿Mradioinert R5020.Tyrosinase Assay. The tyrosinase assay was modified slightly from

the method of Townsend et al. (34). Melanoma specimens identical tothose assayed for receptor were homogenized in three volumes oftyrosinase buffer (236 mM sucrose, 10 mM KC1, 6.67 TES, 3.32 mMTris-base, 2.0 mM phenylmethylsulfonylfluoride, 1.0 mM EDTA, 1.0HIMA'-ethylmaleimide, 1.0 mM benzamidine, and 0.2% Triton X-100,pH 7.4) (34) at 0-4°Cin a glass homogenizer with 20 strokes of a

Teflon pestle and fractionated according to the method of Seiji et al.(35). HM-1 cells (200-^1 aliquots, 400 x g supernatant) were incubatedat 37°Cwith 10 ^1 air-dried aliquots of purified [3H]tyrosine, equivalent

to 5000 dpm, 50 pi of stock L-tyrosine (6 mM L-tyrosine-17.6 mM TES,pH 6.5; 20 ni 6.96 HIML-dopa-33.4 mM TES-0.97 mM Tris, pH 6.0) to

yield final concentrations of 1.1 and 0.52 mM, respectively, whichmaximized tyrosinase activity (34, 36). The remainder of the assay wascarried out essentially according to the procedure of Townsend et al.(34).

Rate constants were obtained by varying the amount of radioinert L-tyrosine from 25 nmol/270 >i\ (0.092 HIM) to 300 nmol/270 n\ (1.1mM). Higher concentrations were not soluble. Since tyrosinase is inhibited if the concentration of L-dopa is high relative to L-tyrosine, theamount of L-dopa was varied to maintain a constant percentage, 46.4%,of L-tyrosine (37). Incubations were carried out at 37°Cfor 3 h and the

Km and enzyme Vm„calculated. The V30owas calculated based uponthe enzyme activity obtained using 300 nmol of L-tyrosine (50 ^1 of 6mM L-tyrosine stock).

Radioimmunoassay. Estradiol was quantitated with an antibody generated against estradiol-6-carboxymethyloxime bovine thyroglobulin(courtesy of Drs. K. Wright and D. C. Collins, Emory University) at afinal antibody dilution of 1:30,000 at which there is negligible (

ER IN HAMSTER MELANOMA

plasma control sample were 13.9 and 10.7%, respectively.Statistical Analysis. Comparisons between groups were made by one

way analysis of variance followed by a Student-Neuman-Keuls a posteriori discrimination test. Single group comparisons were made usingStudent's / test.

RESULTS

Plasma Estradici and Progesterone Levels. One h after maleand female athymic mice received an acute injection of 2.5 /igestradiol, plasma estradiol levels were 33- and 16-fold higherthan control, respectively, and remained 10- to 14-fold higherthan controls 24 h postinjection. The increases were doserelated (Table 1). Plasma estradiol levels in male mice thatreceived subchronic injections of 10.0 /ig estradiol increased 5-fold over vehicle injected controls (16.8 ±4.5 (SE) pg/ml, N =20; 84.5 ±6.9 pg/ml, N = 21; P < 0.01). Plasma estradiollevels in mice given implants of 0.1 and 0.5 mg estradiol pelletswere significantly increased over vehicle controls in intact male(16.8 ±4.5, N = 20; 26.7 ±4.8, 0.1 mg, TV= 5; 92.7 ±8.8, 0.5mg, N = 5), intact female (17.8 ±3.1, N = 7; 104.6 ±28.9, 0.5mg, N = 6), and Ovax female (12.9 ±2.6, N = 8; 90.4 ±12.6,0.1 mg, N = 8; 192.2 ±47.4, 0.5 mg, N = 7) mice. The 0.5-mgpellet increased plasma estradiol significantly in all cases andrepresents the upper limit of normal for the preovulatory increase in estradiol in heterozygous littermates (23). Plasmaprogesterone levels (ng/ml) were increased (P< 0.001) l h afters.c. injection of 100 /ig progesterone (males, 2.1 ±0.2, W = 42;37.3 ±3.7, W = 9; females, 1.9 ±0.3, JV= 18; 36.9 ±5.8, W =

22).Estrogen Receptor. Saturation analyses of HM-1 cytosols

indicated that a [3H]estradiol concentration of ~2.0 nM satu

rated specific (1), high affinity, low capacity sites in tumorsmaintained in mice of either sex (Fig. 1; Table 2). Concentrations of estradiol in excess of 5.0 nM appeared to bind to asecond class of lower affinity higher capacity (>10 fmol/mgprotein) sites (Fig. 1). There was no sex-related difference in

ERc affinity or capacity (Table 2).Specific binding of [3H]estradiol to an HM-1 nuclear fraction

was maximal at 30°Cfor 35 min; ER„levels rapidly decreasedafter 15 min at 37°C(data not shown). High affinity nuclear

sites were saturated at 3.0 nM estradiol with no sex-relateddifference in affinity (ATd)or content (Table 2). Binding of ligandwas apparently specific for estrogen(s) as the regression coefficient for ER-EIA values and those obtained for ERc using HAPto separate bound from free steroid was >0.90 (Fig. 2). Therelatively low capacity of receptor for estradiol did not appear

Table 1 Plasma estradiol concentration of male and female athymic mice withtime after acute injection of estradiol

Type I kd=2.1 x 1(r10M

Type II kd= 7.5 x 10~9M

Estradiolta)0.0"

2.510.0Time

postin-jection

(h)12

624

1Plasma

estradiol concentration(pg/ml)Males16.8±4.5*'c(20)1'

561.3 ±141.1 (3)222.1 ±28.3 (3)28 1.1 ±20.9 (3)236.4 ±18.9(3)

1907.3±291.4/(22)Females20.6

±3.9' (26)

325.3 ±26.5 (3)159.9 + 67.7(3)205.0 + 96.3 (3)218.8 ±10.9(3)

1243.5 ±44.0«(9)

* Mice were given injections s.c. of vehicle (6% ethanol/saline).* Mean + SE.' P < 0.05 versus all estradiol-treated males (ANOVA-SNK).d Numbers in parentheses, number of mice.' P < 0.05 versus all estradiol-treated females (ANOVA-SNK).r I' < 0.05 versus males given injections s.c. of 2.5 *tgestradiol and killed 1

later (ANOVA-SNK).*P< 0.001 versus females given injections s.c. of 2.5 «tgestradioland killed

h later (ANOVA-SNK).

Bound (pm) x 10"

Fig. 1. Scatchard analysis of specific ER binding in HM-1 cytosol. The plotis indicative of two classes of binding sites. The high affinity site saturates at lessthan 3.5 nM [3H]estradiol. A second lower affinity (K¿-7.5 nM) higher capacity(:-!() fmol/mg) site is apparent at [3H]estradiol concentrations >5 nM. B/F,

bound/free; pm, pmol.

Table 2 Affinity (Kj) and maximum binding capacity for ER, and ER, ¡nHM-1tumors

ERcERnSexM

FAd

(nM)0.25±0.07C

0.19 ±0.04B„„

(fmol/mgDNA)27.0

+ 5.926.0 ±3.1SexM FKI

(nM)1.7

±0.21.2 ±0.4B,,»,

(fmol/mgDNA)36.5

+ 5.640.2 + 7.4

* Melanoma from untreated mice. HM-1 cytosol (105,000 x g supernatant,200 fil) was incubated for 18-20 h at 4'C with 0.1-3.5 nM [3H]estradiol with orwithout a 100-fold molar excess of diethylstilbestrol. HAP was used to separatebound from free ligand.

* Melanoma from mice given injections of 10.0 ¿tgof estradiol l h prior tosacrifice. Aliquots (200 *tl) of HM-1 nuclear fraction (1800 x g, sucrose pad-filtered pellet) were incubated for 35 min at 30*C with 0.1-3.5 nM [3H]estradiolwith or without a 100-fold molar excess of diethylstilbestrol. HAP was used toseparate bound from free ligand.

c Mean ±SE; N = 3 for all assays.

o>E

IK

RRA(fm/mg)Fig. 2. Correlation of ER-EIA values with results obtained by HAP assay and

Scatchard analysis on identical tumors. The regression curve obtained was: EIA= 0.49 (Scatchard) + 0.36 fmol (/m)/mg protein; (r = 0.93, N = 11). RRA,radioreceptor assay.

to significantly influence either the accuracy or precision of theER-EIA. Since the ER-EIA buffer contains molybdate ion,significant correlation with the HAP assay also suggests minimal degradation of receptor in HM-1.

ERn in HM-1 carried in male mice was increased relative tocontrols in response to the elevated blood levels of estradiol 1

n h postinjection of 0.1, 2.5, or 10 Mgestradiol. The increase wasi maximal at 0.1 /¿gestradiol (Fig. 3). HRCand ER„content in

HM-1 in mice given injections s.c. of 2.5 ^g estradiol or vehicle

3722

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ER IN HAMSTER MELANOMA

O

0.10 1.00 10.00

Dose Estradici (/¿g)

Fig. 3. ER content of HM-1 from male athymic mice l h after s.c. injectionof estradiol. ERc and ERn values are different (P < 0.01; ANOVA-Student-Newman-Keuls multiple comparison test) from pretreatment levels. Single pointexchange assays were done at 30'C with 3.0 nM [3H]estradiol. Points, three

individual tumors; bars, SE; fin, fmol.

Table 3 Estrogen receptor content of HM-1 xenografts with time after acute s.c.injection of estradiol

Receptor content (fmol/mg DNA)

EstradiolSex0

ER IN HAMSTER MELANOMA

Table 5 Progesterone receptor content ofHM-1 from male and female athymicmice after subchronic and chronic exposure to estradiol

EstradiolSexM

MF

FMTreatmentSubchronic'

EstradiolSubchronic

EstradiolChronic*

EstradiolDose

ER IN HAMSTER MELANOMA

.020

.018

B/F

.012

.010

008

-OO6

004

.002

Table 7 Tyrosinase activity ofHM-l tumors from male and female athymic micewith time after acute, subchronic, or chronic exposure to estradiol

0.5 1.0 1.5 20 2.5 30

3H-RS020 (nM)

1 23456

BOUND (fmoles)

Fig. 6. Scatchard analysis of 3H-labeled RS020 binding to HM-1 nuclearfraction. Incubation was done at 22'C for 4 h with 0.2-3 nM 3H-labeled R5020in the presence and absence of 250-fold molar excess of radioinert RS020 andcortisol. Ki, 0.7 niu; hound,,,.,, 25.0 fmol (/m)/mg DNA; r = 0.96. Inset, saturationanalysis of 3H-labeled R5020 to nuclear fraction. Apparent saturation occurredat -3.0 nM. B/F, bound/free.

Table 6 Affinity (K¿and maximum binding capacity for progesterone receptor inHM-1 cytosol and nuclear fractions

PgRc" PgR„"

Sex (nM)Sound«». Boundmu

(fmol/mgDNA) Sex Kt (nM) (fmol/mg DNA)

MF

0.71 ±0.2'0.54 ±0.2

72.9 ±4.592.9 ±9.1

MF

1.0 ±0.31.5 ±0.2

25.4 ±1.028.6 ±6.7

" Melanoma from mice subchronically primed with estradiol. HM-1 cytosolaliquots (105,000 x g supernatant, 200 M') were incubated for 2 h at ()"(" with0.2-5.0 nM of 3H-labeled R5020 and a 250-fold molar excess of unlabeled cortisolwith or without a 250-fold molar excess of unlabeled R5020. Dextran-coatedcharcoal (0.5% charcoal-0.05% dextran T-70 in 10 mM Tris-HCl-1.5 mM EDTA,pH 7.4) was used to separate bound from free ligand.

b Melanoma from mice subchronically primed with estradiol and given injec

tions s.c. of 100 Mgof progesterone l h prior to sacrifice. HM-1 nuclear fractions(200 ii\) were incubated for 4 h at 22'C with 0.2-3.0 nM 3H-labeled R5020 and a250-fold molar excess of unlabeled cortisol with or without a 250-fold molarexcess of unlabeled R5020. NaOH hydrolysis was used to solubilize the nuclearpellets.

c Mean ±SE; N = 3 for all assays.

served in rat uterus following an acute injection of estriol, whereER„was within control values by 6 h postinjection (43). Theshorter residency time in HM-1 nuclei apparently does not alterthe inhibitory effects of chronic exposure of estradiol on HM-1 growth (1).

Long-term exposure to estrogens can produce a prolongedelevation in ER„in estrogen sensitive tissues (47). Collectively,our data suggest that after subchronic or chronic exposure toestradiol there is a sustained increase in ERn in HM-1 cells, buttotal receptor content does not increase. Although the sustainedincrease in ER„content suggests that stabilization of the estrogen receptor may have occurred in the presence of elevatedlevels of estradiol, the significant decrease in ERt content duringchronic exposure to estradiol also suggests that some degradation of receptor may also be occurring. The ERc in HM-1 cellsappears as a larger species on sucrose density gradients in the

V ,,,, (nmol/h/mgprotein)Estradiol

exposureAcuteSubchronicChronicDose0>g)0.02.50.010.00.0500.00.01

00.0500.0h

afterestradiol012624Males2.5

±0.2°(20)*2.3

±0.2(8)2.7±0.2(11)2.5±0.3(3)2.2±0.4(3)ND

ER IN HAMSTER MELANOMA

depend on prior exposure to estrogen followed by progesteronetreatment. Estrogen priming has been reported to potentiatethe effect of progesterone on melanocytes (64, 65). Carruthers(8) observed that in men progesterone causes melasma, hyper-pigmentation of facial skin regions, but only if estrogen wasadministered simultaneously, and he postulated that progesterone acts on estrogen-primed melanocytes. After estrogen priming has induced the synthesis of progesterone receptor in themelanocyte, progesterone could act synergistically with estrogen to stimulate tyrosinase activity thereby enhancing skinpigmentation.

While the actual mechanism(s) of the inhibitory effect ofestradiol on HM-1 growth (1) remains unknown, present resultssuggest that protein induction or repression in HM-1 cellswould be a likely avenue for investigation.

ACKNOWLEDGMENTS

The authors thank L. Valcourt for performing the estradiol andprogesterone radioimmunoassays and R. Macke for preparation of themanuscript. 32

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1988;48:3720-3727. Cancer Res M. Helen Hitselberger, Rosemary L. Schleicher and Craig W. Beattie into Athymic MiceReceptor, and Tyrosinase in Hamster Melanoma Transplanted Effects of Estradiol on Estrogen Receptor, Progesterone

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