(CANCER RESEARCH 49, 1247-1253. March 1, 1989]

Effects of Estradici on Prolactin and Growth Hormone Messenger RNAs inCultured Normal and Neoplastic (MtT/W15 and GH3) Rat Pituitary Cells1

Jiangyue Song, Long Jin, and Ricardo V. Lloyd2

Department of Pathology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0054

ABSTRACT

The effects of 17/9-estradiol (E17/3) on prolactin (PRL) cell proliferation and on the expression of PRL and growth hormone (GH) proteinsand mRNAs were analyzed in cultured pituitary cells by immunocyto-chemistry, in situ hybridization, and Northern blot hybridization studies.Three different cell cultures were used: (a) normal pituitary cells; (b)GH3 tumor cell line; and (c) MtT/W 15. a transplantable PRL and (.II-producing pituitary tumor. E170 (10~7 M) caused a significant increase

in PRL cell proliferation in normal pituitary [3.9 ±0.4 versus 7.7 ±0.9%(SEM) of immunostained PRL cells with thymidine incorporation] |/' <

0.01) but produced a significant decrease in PRL cell proliferation inMtT/W15 primary cell cultures (6.7 ±1.0 versus 3.7 ±0.8%) \P< 0.05). PRL mRN A was significantly increased in normal pituitary andin <.ll< tumor cells by El 7/3treatment. There was a significant decreasein PRL mRNA and an increase in GH mRNA expression in culturedMtT/W 15 tumor cells by immunocytochemistry and in situ hybridizationanalyses. The percentage of cells producing both PRL and GH ormammosomatotropic cells analyzed by two different techniques declinedafter one week in culture in normal pituitary cells and in cultured Mil/W15 tumor cells after E170 treatment. These results show that E17/3has a direct stimulatory effect on normal pituitary and Gil, cells and adirect inhibitory effect on MtT/W15 tumor cells with respect to cellproliferation and PRL hormone and mRNA expression.

INTRODUCTION

The effects of estrogens on the development of PRL3 cell

hyperplasia and transplantable tumors in vivo are well known(1-8). Various investigators have shown that estradiol regulateshormone production and PRL mRNA in anterior pituitary cells(9-12). Recent studies have shown a direct effect of estradiolon the proliferation of normal PRL cells in vitro (8). The effectsof estradiol on regulating tumor cell proliferation and prolactinproduction by established tumor cell lines such as the GH3 cellswhich were established by Tashjian et al. (13, 14), have beenanalyzed extensively (15, 16). Estradiol has been found tostimulate PRL production in these cells.

Our recent studies with the transplantable PRL- and GH-producing pituitary tumor, MtT/W15, showed that the growthof this tumor was inhibited by estrogen in vivo. Estradiol-induced growth inhibition was accompanied by a decrease inPRL and an increase in GH production by the tumor cells (17-19). It was not known if this was a direct effect of estradiol onthe regulation of cell growth and hormone expression by thesetumor cells. The GH3 tumor cell line was analyzed along withnormal pituitary and MtT/W15 cells, because various studieshave shown that GH3 and normal pituitary cells respond to

Received 8/3/88; revised 11/29/88; accepted 12/2/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by NIH Grants CA37238 and CA429S1.2To whom requests for reprints should be addressed, at Department of

Pathology, 2G322, Box 0054, 1500 E. Medical Center Drive, Ann Arbor, MI48109.

3The abbreviations used are: PRL, prolactin; GH, growth hormone; MS,

mammosomatotropic; ICC, immunocytochemical staining; ISH, in situ hybridization; rPRL, rat prolactin; rGH, rat growth hormone; SSC, standard saline-citrate; E l Iß,17.¡-estrad i<il: NIDDK, National Institute of Diabetes and Digestiveand Kidney Diseases.

estrogen stimulation in a similar manner. In addition, proliferating fibroblasts were present in cultured normal pituitaryand MtT/W15 cells while fibroblast contamination was notpresent in the GH3 cell line.

In the study presented in this report we examined the effectsof estradiol on PRL and GH expression in normal and neo-plastic cultured rat pituitary cells and showed that estradiolstimulates PRL production in normal pituitary and GH3 cellswhile inhibiting PRL and stimulating GH production in theMtT/W 15 tumor cells directly. Changes in MS cell populationsin vitro under the influence of estradiol were also analyzed,because MS cells may represent a transient cell type withresponses to estrogens that have not been studied in culturedMtT/W 15 tumor cells.

MATERIALS AND METHODS

Animals

Female 60-day-old Wistar-Furth rats (Harían,Indianapolis, IN) wereused for normal pituitaries. MtT/W 15 tumors were implanted into theright flank (1 mm3) of 40-day-old female Wistar-Furth rats. Palpabletumors were obtained 6-8 weeks after implantation and used for cellculture studies. Rats were maintained on 12 h light and 12 h darknessand were allowed to feed ad libitum. All animals were decapitatedbetween 8 and 10 a.m.

Cell Culture

Anterior pituitaries and MtT/W15 tumors were dispersed in 0.25%trypsin in Hank's balanced salt solution (Grand Island Biological

Company, Grand Island, NY) as described previously (19). Approximately 0.5-1.0 x IO6cells was usually obtained from each pituitary or

from 10 mg of tumor tissue. Viability of dissociated cells as tested withtrypan blue usually exceeded 95%. Aliquots of freshly dissociated cellswere prepared for ICC and for ISH. Normal pituitary cells (1 x IO6),MtT/W 15 (1.5 x IO6), and GH3 (0.6 x IO6) cells were plated in 35-

mm dishes coated with extracellular matrix (Accurate, Hicksville, NY)in 2 ml phenol-red free Dulbecco's modified Eagle's medium with

penicillin (100 units/ml), streptomycin (100 ¿ig/ml),Fungizone (0.25Mg/ml), dextran-coated charcoal-treated 15% horse serum, 2.5% dex-tran-coated charcoal-treated fetal bovine serum as reported previously(7, 20) and 5 Mg/ml of insulin (GIBCO) for 24 h at 37°Cin an

atmosphere of 5% COz/95% air. Different cell numbers were usedbecause of differences in growth rate and plating efficiencies of thevarious cell types determined in preliminary experiments. With thereported starting cell numbers the yield of cells for various experimentswas approximately the same for all 3 groups on day 7.

The concentration of estradiol in the charcoal-treated serum wasundetectable by radioimmunoassay. After 24 h, 17/3-estradiol dissolvedin 100% ethanol was directly added at final concentrations of 10~7,10~', and 10~" M. Control dishes received ethanol which was less than

0.1% of the final culture volume of 2 ml of media. The media waschanged every 2 days. After 7 days in culture, the cells were harvestedby washing with 0.02% EDTA followed by a 10-min incubation with0.25% trypsin, counted and used for ICC, ISH, and Northern blotstudies. Cytospin preparations (1 x 10s cells/slide) were made with a

Shandon cytocentrifuge (Shandon Southern Instruments, Sewickly,PA). GH3 tumor cell line was obtained from the American Type CultureCollection (Rockville, MD) and maintained in F-10 complete medium

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in the laboratory. Aliquots of cells were plated on dishes coated withextracellular matrix and analyzed the same way as primary cell cultures.

Thymidine Incorporation

After 7 days in culture, the medium was changed and new mediumand 10 iiC\ [3H]thymidine (specific activity, 15.7 Ci/mmol; New Eng

land Nuclear, Boston, MA) was added for 4 h. The cells were thenwashed and cytocentrifuged on to microscope slides. Cells were im-munostained for PRL and GH; then autoradiography was carried outby dipping in NTB2 emulsion (Eastman Kodak, Rochester, NY) diluted1:1 with distilled water, stored in the dark at 4°C,and developed after

1 week.

Immunocytochemistry

Dispersed cells were attached to poly-L-lysine-coated glass slides bycytocentrifugation and then fixed in 10% phosphate-buffered formalin(pH 7.1) for 24 h. The slides were immunostained as previously reportedusing the avidin-biotin-peroxidase (Vector Kits, Burlingame, CA)method (19). PRL and GH antisera were from the National PituitaryAgency (NIDDK) and were used at 1/1000 and at 1/40,000, respectively. Positive cells with dark brown cytoplasmic precipitate wereenumerated by counting a minimum of 1000 cells/slide. MS cells wereestimated from the difference between the sum of PRL- and GH-positive cells stained separately on different slides and stained togetheron the same slide (19).

Hybridization Studies

Probes. The oligonucleotide probes used were d(GGC TTG CTCCTT GTC TTC AGG) for rPRL, and d(ATC GCT GCG CAT GTTCGC GTC) for rGH (all 5'-3'). The probes were synthesized by thesolid phase 0-cyanoethyl phosphoramidite method (21) on an automated DNA synthesizer (Gene Assembler) by Pharmacia and werepurified by gel electrophoresis on 20% polyacrylamide gels. The oligo-nucleotides were complementary to the region of rPRL 64-70 (22),and rGH 145-151 (23) coding for these amino acid residues.

Labeling. The oligonucleotides were labeled by the 3'-end labeling

method (24-26). Ten pmol of oligonucleotide were incubated with 45uC\ of [35S]dATP (1300-1500 Ci/mmol), 2 mM CoCl2, 0.5 mM dithio-

threitol, and 20 units of terminal deoxynucleotidyl transferase in 100mM potassium cacodylate (pH 7.2) for 45 min at 37°C.The reaction

was stopped by addition of 0.3% xylene cyanol FF, 0.3% bromophenolblue, and 0.3% EDTA (pH 7.0). The reaction products were separatedby electrophoresis on a 12% polyacrylamide gel. The labeled probeswere localized autoradiographically and extracted twice into steriledistilled water with 0.20 mM dithiothreitol for 24 h each time. Theextracted probes were filtered through 0.2-^m Millipore filters and thendried in a Speed Vac (Savant Instruments, Westbury, NY), redissolvedin 0.05 MTris-EDTA buffer (pH 7.8), and used for in situ hybridization.[32P]dATP (5000 Ci/mol) was used to label the oligonucleotide probesby the 3'-end labeling method (24-26) and used for Northern analysis.

For ISH, cells were cytocentrifuged onto 3-aminopropylmethoxysilane-coated glass slide (Aldrich, Milwaukee, WI), fixed in 10% paraformal-dehyde for 20 min, washed in 2x SSC, dehydrated, and kept at -70°C

for up to 1 month before use.In Situ Hybridization. ISH was performed as previously reported

(26). The "S-labeled probes were diluted in hybridization buffer containing 20 mM dithiothreitol and 1 x 10scpm for PRL and GH, applied

to the slides, and incubated at 44V for 18 h in a moist chamber (26).When combined ISH and ICC procedures were done the ICC wascarried out after 2x SSC washings as described previously (26) usingrGH antiserum at 1/40,000 and rPRL antisera at 1/1,000 from theNIDDK with the avidin-biotin-peroxidase system. Autoradiographicdetection of the hybrids was carried out by dipping in Kodak NTB2emulsion diluted 1:1 with distilled water containing 0.3 M ammoniumacetate. Controls for ISH consisted of (a) prior digestion of pituitarytissues with 100 Mg/ml of RNase (Sigma) at 37°Cfor 30 min beforeISH along with positive control slides with rPRL and rGH 35S-labeledoligonucleotide probes, (¿>)competition studies with a 100-fold excessof unlabeled oligonucleotides corresponding to the hormone sequences

of rPRL or rGH to check specificity and cross-hybridization of eachprobe, (c) substituting rat liver for the pituitary tissue in hybridizationstudies, and (d) colocalization of rPRL and rGH proteins and mRNAsin the same cell with a combined ISH-ICC procedure.

Quantitation of ISH. The distribution of silver grains over pituitarycells in one ISH experiment was assessed by taking photographs oflabeled cells in 4 different fields at x60 and magnifying the prints tox 1100. The number of silver grains per cell were evaluated by countingat least 50 cells per treatment. The number of silver grains werenormalized per unit area. Nonspecific hybridization was obtained fromthe number of grains seen with the PRL and GH probes on liver tissuesand on portions of the same slide without cells. This was subtractedfrom the number of silver grains per cell. The enumeration of thepercentage of cells with GH and PRL mRNAs for 4 to 6 differentexperiments was done by counting 1000 cells/treatment group. Thedata were expressed as relative percentages for the primary culturedcells. Relative percentages included only the cells that had immuno-reactive PRL and GH by ICC or cells with GH and PRL mRNAdetermined by ISH (Table 1). This was very important in the [3H]-

thymidine experiments to avoid including fibroblast cells when enumerating the proliferating PRL cells (27). Pituitary epithelial cells stainedpositively with an antibody against neuron-specific enolase, while thefibroblastic cells were negative as reported previously (7). Fibroblastsalso had larger nuclei than pituitary cells as reported previously (28).

Results were expressed as mean ±SEM and the Student t test wasused for analysis of statistical significance.

RNA Isolation. Total RNA was isolated by extraction in guanidiniumisothiocyanate, followed by ultracentrifugation in cesium chloride (29).The yield of total RNA was assessed spectrophotometrically at /íj6o.The purity of the RNA was assessed by A-ua/A-iwwith values ranging

from 1.7 to 2.1. Most RNA samples had an Aiw/Awo ratio around 2.0and samples with a ratio less than 1.7 were not used. Aliquots of theRNA were run on a 1% agarose gel to assess the integrity of the 28Sand 18S bands.

Northern Hybridization. Total cellular RNA was glyoxylated (30),electrophoresed on 1% agarose gels and transferred to nitrocellulosefilters (31). Blots were baked for 60 min at 80°Cin a vacuum oven.Prehybridization was done for 2 h in Denhardt's solution with 0.1%

sodium dodecyl sulfate, 50% deionized formamide, 50 mM sodiumphosphate (pH 7.0), 5x SSC, and 250 fig/ml denatured salmon spermDNA. The filters were then hybridized for 16 h in the same solutioncontaining 10% dextran sulfate and 1 x IO6 cpm/ml of 32P-labeled

probes. Filters were subsequently washed twice for 30 min at roomtemperature and for 1 h at 55°Cin 6x SSC/0.1% sodium dodecyl

sulfate. These filters were air dried and exposed for 1-3 days withKodak X-omat film at -70°C with intensifying screens. Relative

amounts of rPRL mRNA were determined by scanning densitometryof the autoradiograph with an LKB Ultrascan XL from Pharmacia.The results were expressed relative to control samples. The densitom-eter had a linear range of 0-4.0 A.

RESULTS

Cell Culture. Primary cells and the established GH3 cellsattached to the extracellular matrix 4 h after plating. The

Table 1 Percentages of PRL and GH cells in acutely dissociated normal pituitaryand MtT/W15 pituitary cells

NormalpituitaryTechniqueICCGH42.6

±1.4°(53.4 ±1.0)*PRL36.6

±0.7(46.6 ±1.0)MtT/WlSGH12.6

±1.0(17.0 ±1.2)PRL62.3

±0.5(83.0 ±1.2)

ISH 39.7 ±1.4(53.4 ±0.7)

34.5 ±0.6 44.1 ±8.0 62.7 ±0.5(46.6 ±0.7) (40.2 ±4.3) (59.8 ±6.0)

" Mean ±SEM of 4 to 6 experiments for each group.* Numbers in parentheses indicate relative percentages which included only

cells with immunoreactive PRL or GH detected by ICC or cells with PRL andGH mRNA determined by ISH. The absolute percentages of cells in the GH3 cellline before treatment with estradiol (day 0) were 0.7 ±0.2 (GH) and 2.7 ±0.3(PRL) by ICC and 46.8 ±2.1 (GH) and 9.6 ±0.6 (PRL) by ISH.

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cells became flattened after 1 day in culture. Fibroblast cellswere evident after day 3 in the primary cultures of normalpituitary and MtT/W15 cells. Approximately 10% of the cellsfrom these cultures on day 7 were fibroblasts. During enumeration of immunostained cells to estimate the percentage of PRLand GH cells, a relative percentage figure was used whichincluded only the cells expressing PRL and GH protein ormRNA to avoid including fibroblast cells in the cell counts.

Cell Proliferation. After 1 week in culture the PRL cells fromthe normal pituitary showed a significant increase in the percentage of thymidine-labeled cells in the presence of 10~7 M

estradici compared to controls (Fig. 1). The MtT/W15 cellsshowed a significant decrease in the PRL-labeled cells in thepresence of estradiol. Although PRL cells in the estradiol-treated GHj cells were increased these differences were notstatistically significant (Fig. 1) due to the large variation between individual experiments.

ICC. ICC staining of normal pituitary cells showed a significant increase in the relative percentage of PRL cells and asignificant decrease in GH cells in the presence of 10~7M E17/3

(Fig. 2). Cells containing a brown precipitate in the cytoplasmafter the diaminobenzidine reaction were considered positiveby two independent observers. Cultured cells from the MtT/W15 tumors showed the opposite effect with an increase in GHand a decrease in PRL cells. GH3 tumor cells showed a significant increase in the PRL cells after estradiol treatment. Treatment with 10~9M and 10~" M E17/3 did not induce significant

differences compared to the control group.Control sections for ICC staining including absorption stud-

_ 24 -it

oc2

D PRL CONTROLQ PRLE17P

Fig. 1. Thymidine incorporation in cultured pituitary cells (n = 5) for 5different experiments. Cells were cultured for 1 week in the presence or absenceof 1(T7 M 17rf-estradiol (E170). On day 7, the cells were incubated with['Hj-thymidine for 4 h and immunostained for PRL. autoradiography was doneas described in "Materials and Methods." and the replicating PRL cells were

quantitated by counting a minimum of 1000 stained cells. A7. normal pituitary.',/>< 0.01. Bars, SEM.

100 -

90-

80-

70"

SO

SO40-

30-

20-

10-

0

D PRL CONTROL

D PRLE17P•¿�GH CONTROLH GHE17P

MIT/W15 GH3

Fig. 2. Analysis of PRL and GH distribution by immunostaining. The cellswere cultured with or without E17/3 as described in "Materials and Methods."

The results for primary cultured normal pituitary (NI) cells and MtT/W15 tumorcells were expressed as relative percentages of the immunostained PRL and GHcells to avoid fibroblast contamination. The percentage of GH3 cells reflectsabsolute percentage, since fibroblast were not present in this cell line. *,P< 0.05;**. P < 0.01; »•*.P < 0.001. Bars, SEM.

ics with purified rPRL and rGH abolished staining with PRLand GH antisera.

ISH. Oligonucleotide probes for rPRL and rGH resulted inspecific hybridization signals in the pituitary cells in acutelydissociated cells and in cells cultured for 7 days. Pretreatmentof tissues with 100 me/ml of RNase A abolished specific hybridization signals and only a background level of hybridizationwas seen (Fig. 3). Hybridization with PRL and GH probes wasnot seen in liver tissue. Colocalization studies also confirmedthe specificity of the rPRL and rGH probes. The hybridizationsignal with the "S-PRL probe was abolished when the cellswere incubated with a 100-fold excess of an un labeled oligo-nucleotide sequence corresponding to PRL but not with anunlabeled oligonucleotide sequence corresponding to GH before the hybridization procedure. Likewise a 100-fold excess ofunlabeled GH used in the prehybridization reaction abolishedlabeling with the 35S-GH probe, but unlabeled PRL did notabolish labeling with the 35S-GH probes. The cells hybridizing

with the PRL probe from normal pituitary were larger than thecells labeled with the GH probe after 1 week of incubation withE170 (Fig. 4). There were many more GHj cells labeled forPRL and for GH (Fig. 5) than could be detected by ICC (Fig.

.Fig. 3. In situ hybridization with an 35S-labeled oligonucleotide rPRL probe

with acutely dissociated MtT/W15 tumor cells. Many cells show a positivehybridization signal for PRL (A). Pretreatment of the cells with RNase A beforehybridization abolished the positive hybridization signal (B). Bar, SO/mi. Originalmagnification, x 330.

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'.,- .'-" "* "¿•r"-'Map

•¿�-ív>*.,- ^k. •¿� ..•«¿C.-. ";TÕ*•'-.>".*•-.''*•¿�*Hr$pte&:''

Fig. 4. /n M'/Hhybridization with "S-labeled oligonucleotide probes withnormal rat pituitary cells cultured for 7 days in the presence of IO"7 M 17/3-

estradiol. Many of the cells show a strong positive hybridization signal for PRL(A). Some of the smaller cells hybridized with the GH-oligonucleotide probe (B).Bar, 50 tun. Original magnification, x 330.

Fig. 5. In situ hybridization with an '"S labeled oligonucleotide probe show

that most of the cultured (ill> tumor cells express GH mRNA. Bar, 50 urn.Original magnification, x 528.

2). The percentages of cells hybridizing to the PRL and GHoligonucleotide probes in the control dishes on day 7 were 56.2±1.4% and 34.2 ±10%, respectively, for GH3 cells. In contrastthe ICC and ISH enumeration data for normal pituitary werevery concordant (Table 1). The distribution of silver grains over50 cells for each group in one experiment was done by enumerating the number of silver grains and expressing these per cellarea (Fig. 6). Normal pituitary and GH3 cells showed a significant increase in PRL hybridization signal with estradici treatment. CiH.i tumor cells had a significant decrease in GH hybridization signal after estradici treatment. The MtT/W15tumor cells showed a decrease in hybridization signal in PRLcells and an increase in hybridization signal for GH cells withestrogen treatment (Fig. 6).

Analysis of the positive cells by ISH for 4-6 experiments foreach group is shown in Fig. 7. A significant increase in PRLcells and a significant decrease in GH cells was present in boththe normal pituitary and GH3 cells while the MtT/W15 had adecrease in PRL and an increase in GH cell expressing theappropriate mRNAs under the influence of E17/3. E17/3 at 10~9M and 10~" M had an effect similar to that of E17ßat 10~7M,

but the effects were not statistically significant (data not shown).MS. The presence of cells expressing both PRL and GH was

analyzed by ICC and by combined ISH and ICC. MS cells inthe ICC analysis were derived from the difference in cell populations staining positively for GH and PRL separately and thecells staining for both GH and PRL on the same slide. With

D PRLCONTROLQ PRLE17P•¿�GHCONTROLB GHE17P

NI PIT MlT'W15

Fig. 6. Quantitation of in situ hybridization results. The distribution of silvergrains per cell was analyzed in SOcells from each treatment and a control groupand the results were normalized per cell area. One x 10' cpm of "S-GH and "S-PRL were used for normal pituitary (NI) MtT/W15 cells while 1 x 10' cpm forrPRL and rGH were used for GH3 cells. *, P < 0.05; «, P < 0.01; ***, P <0.001. Bars, SEM.

80-1

DD

20-

PRL CONTROLPRLE17PGH CONTROLGHE17P

NI PIT M1T/W15 GH3

Fig. 7. Distribution of cells with positive hybridization signals from 4 to 6experiments. The percentage of positive cells was enumerated for each group.The normal pituitary (A7) and MtT/W15 groups had 1x10' cpm of 35S-labeledPRL and GH for 1SII and the results are expressed as a relative percentage ofPRL- and GH-labeled cells because about 10% of the cells were fibroblastic onday 7 of culture. The GH3 cells had 1 x IO6 cpm of "S-labeled PRL and GH

probes per slide and the results are expressed as the absolute percentage ofpositive GH] cells with PRL or GH probes, because fibroblasts were not presentin this cell line. Bars, SEM. *, P<O.OS: **, /><0.01; "*, /><0.001.

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the ISH-ICC method, ISH was performed with GH probefollowed by ICC with PRL antisera (Fig. 8). The data in Table2 summarize the percentage of MS cells. Both the ICC and theISH-ICC methods revealed MS cells in normal pituitary and inMtT/W15 tumors. The MS cells declined in normal pituitariesafter culturing for 7 days. The decrease in MS cells was greaterin the presence of estradiol for the MtT/W15 tumor cells. Itwas not possible to analyze for MS cells in the GH3 cell linewith these methods, because of the small percentage of immu-noreactive GH cells in GH3. The percentage of GH cells detected by ICC in the GH3 cell was not increased by preincubat-ing cells with colchicine (5 x 10~3 to 5 x 10~6 M colchicine).

The percentage of GH3 cells expressing GH mRNA was 10-fold greater than the percentage expressing GH protein indicating that there were rapid synthesis and release of GH withoutsignificant storage of the hormone product.

Effects of Estrogen on Cell Size. Estrogen treatment resultedin significant increases in the sizes of PRL cells from normalpituitary and from the GH3 tumor cell line, but not from MtT/W15 tumor cells (Table 3). Estrogen treatment did not producesignificant changes in the cell sizes of GH cells in any of the 3groups studied (Table 3).

Northern Hybridization Studies. Northern blots revealed PRLmRNA of approximately 1 kilobase for all three pituitarytissues. Estrogen treatment resulted in a 2.9-fold and 2.1-foldincrease in PRL mRNA in the normal pituitary and GH3 cells,respectively. The MtT/Wl 5 tumor cells had a 2.7-fold decreasein PRL mRNA with estrogen treatment (Fig. 9).

Fig. 8. Combined in situ hybridization for GH mRNA and immunochemicalstaining for PRL hormone to show MS cells in cultured MtT/Wl5 tumor cells.The GH-positive cells had silver grains but no immunochemical dark cytoplasmicreaction (arrowhead). The PRL cells have a dark color from the immunochemicalstaining for PRL but no silver grains. The MS cell has a positive cytoplasmicreaction for PRL immunoreactivity and cytoplasmic silver grains (arrow). Bar, SOfirn. Original magnification, x 528.

DISCUSSION

The effects of estrogens on stimulating pituitary PRL cellproliferation in vivo have been studied by many investigators(1-8). Recent evidence from in vitro studies has also shown thatestrogens have a stimulatory effect on PRL cell numbers aswell as PRL mRNA in the normal rat pituitary (9). Our previousstudies on the transplantable MtT/Wl 5 tumor have shown thatestrogens had an opposite effect in vivo on PRL and GHmRNAs (26) from that seen in normal pituitary cells and theGHj tumor cell line. We have extended our previous analysesto an in vitro system to examine a possible direct effect ofestrogens on the MtT/Wl5 cells during 1 week of culture.Although our in vivo studies with MtT/Wl5 tumors were donefor 3 weeks, after 3 weeks in culture there was an overgrowthof fibroblastic cells, so we performed the current experimentsfor 1 week when the percentage of fibroblast was still relativelylow. These results indicate that estrogens cause a direct inhibition of PRL cell proliferation assessed by thymidine incorporation in immunostained PRL cells while inhibiting the expression of PRL hormone and PRL mRNA in the tumor cells. GHprotein and mRNA production were also stimulated by estrogens in the MtT/Wl5 tumor while PRL biosynthesis wasinhibited. Analysis of primary cultures of normal pituitary cellsand of the established cell line GH3 showed that the in vitroresponse of MtT/Wl 5 cells to estrogens is opposite that of theabove two pituitary tissues. The paradoxical response of anothertransplantable pituitary tumor MtT/F4 which was also inducedby estrogen and in the transplantable 7315a tumor has beenobserved previously in vivo (32, 33).

The results on the direct effect of estrogens on PRL cellproliferation in the normal pituitary agree with the earlierstudies of Lieberman et al. (9) who observed a small relativeincrease in the numbers of PRL cells after 5 days in culture.Our observations are different from the findings of Antakly etal. (28) who did not observe an increase in the numbers of PRLcells in addition to increased PRL secretion in primary culturesof rat pituitary cells. This may be related to the sensitivity ofthe [3H]thymidine technique which has also been used by other

investigators to show an increase in the number of GH cells inthe pituitary after GH-releasing hormone treatment in vitro(27). Although it was not determined in these experimentswhether E17/3 caused increased de novo synthesis or decreaseddegradation of specific mRNAs, most of the recent data wouldsuggest that increase synthesis occurs more commonly afterhormonal stimulation in mammalian pituitary cells (34-36).

ISH studies with oligonucleotide probes showed a directeffect of estrogen on PRL mRNA levels. ISH with oligonucleotide and other probes have been used by various investigatorsrecently to analyze for the expression of gene products in thepituitary and other endocrine cells (37-42). Various controlswere used to assure specificity of the hybridization signals withthe use of oligonucleotide probes in these studies.

Table 2 Effects of 17ß-estradiolon mammosomatotropic cells in cultured normal and neoplaslic rat pituitary cells

Mammosomatotropic cells were analyzed by immunohistochemistry and by combined in situ hybridization and immunocytochemistry with freshly dissociated cellson day 0 and after culturing the cells for 1 week with and without IO M 170-estradiol. MS cells were estimated after ICC staining as described in "Materials andMethods."

Normal pituitary MtT/Wl 5

Day? Day 7

Technique Day 0 Control E170 Day 0 Control E170

ICCCombined ISH-ICC1I.7±

1.3°

2.9 ±0.35.5±1.6

0.8 ±0.35.9±1.7

0.6 ±0.17.8±0.8

19.4 ±6.38.1±0.8

12.0 + 3.45.8±1.18.4±2.5

' P < 0.05, compared to control cultured cells on day 7. Mean ±SEM.

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Table 3 Effects of 170-estradiol on PRL and GH cell area of cultured ratanterior pituitary cells

Cells were cultured with or without IO"7 M 17(3-estradiol for 1 week. After in

situ hybridization with PRL and GH probes, photographs of cultured cells weretaken at x60 and magnified to x 1100. The areas of 20-25 cells from each groupwere measured.

Cell area

GroupNormalMtT/W15GH,E170 PRL436±22°+

557 ±30*432

±20+ 503 ±10233

±12+ 329±12CGH476

±24474 ±26535

±45466 ±30157

±8160 ±8

•¿�Mean ±SEM.* P < 0.01, compared to controls.' P < 0.001, compared to controls.

1 234567

28S —¿�

18S*-

1.0Kb»-

Fig. 9. Northern blot hybridization with "P-labeled PRL oligonucleotide

probe of pituitary cells cultured for 7 days. Normal pituitary, MtT/WlS tumorcells, and GH, tumor cells were cultured with or without IO M estradici.Approximately 20 //u of total RNA per lane for normal pituitary and for Mt'l

W15 and 40 fig of RNA for GH3 cells were used. Lane I, normal pituitary culturedwithout estrogen; Lane 2, normal pituitary culture with 10" M 17/3-estradiol;Lane 3, MtT/W15 tumor cells; Lane 4, MtT/W15 with 10~7 M 17/3-estradiol;Lane 5, GH3 cells; Lane 6, GH3 cells with IO"7 M 17/3-estradiol; Lane 7, rat liver

total RNA. Arrowhead, approximately 1.0 kilobase (Kb) size.

Two techniques, including the use of combined ISH-ICC,revealed MS cells in the normal pituitary and in MtT/W15tumor cells. The data suggest that the combined ISH-ICCmethod might be less sensitive in detecting MS cells than theICC methods or the reverse hemolytic plaque assay techniquesince more MS cells have been detected in the normal ratpituitary with the plaque assay by us and by other investigators(43-45). Both methods used in this study revealed that MScells decrease in number when normal pituitary cells werecultured for 1 week. Although MS cells could not be detectedwith these methods in the (•!1<cell line, recent studies indicatethat these tumors also contain MS cells (43). Although MScells have been observed previously in the normal rat pituitary(44-46), the role of these cells, if any, in tumor development is

unclear. Our recent in vivo studies (19) and the present cellculture analysis would suggest that estrogens and primary cultures of normal pituitary cells lead to a decrease in the numbersof MS cells in the rat pituitary. The presence of MS cells in theMtT/W15 tumors in this report and in the GH3 cells in aprevious report (43) indicates that neoplastic pituitary MS cellsare a dynamic cell population which can be changed by varioushormones.

Our results indicate that estrogens have a direct stimulatoryeffect on some pituitary cells such as normal pituitary and theGH3 cell line and a direct inhibitory effect on other pituitarycells such as the MtT/W15 with respect to cell proliferationand PRL expression. The mechanisms underlying these different regulatory pathways remain to be elucidated.

ACKNOWLEDGMENTS

The authors thank Susan Johnson and Kristina Fields for technicalassistance and the NIDDK and Dr. A. F. Parlow for the rat prolactinand growth hormone antisera.

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1989;49:1247-1253. Cancer Res   Jiangyue Song, Long Jin and Ricardo V. Lloyd 

) Rat Pituitary Cells3and GHMessenger RNAs in Cultured Normal and Neoplastic (MtT/W15 Effects of Estradiol on Prolactin and Growth Hormone

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