(CANCER RESEARCH 47, 3742-3746, July 15, 1987]

A New Glucocorticoid Receptor Detected in Host Rat Liver but not inVarious Hepatomas1

Kenji Hirota, Takako Hirota, Yoshikazu Sanno,2 and Takehiko Tanaka3

Departments of Nutrition and Physiological Chemistry [T. H., Y. S., T. T.) and of Obstetrics and Gynecology [K. H.], Osaka University Medical School,4-3-57 Nakanoshima, Kita-ku, Osaka 530, Japan

ABSTRACT

Previously a new glucocorticoid receptor (Peak C), which eluted with0.12 to 0.14 M NaCl from DEAE-cellulose column, was identified in

addition to another receptor (Peak B), a classic type of glucocorticoidreceptor, which eluted with 0.05 to 0.08 M NaCl. Peak C appeared afterstress or injection of a high dose (20 «tg/100g body weight) of dexameth-

asone into rats. Peak C was also detected in the liver of rats bearingvarious tumors, but it was not found in malignant tumors (Yoshidasarcoma and Yoshida ascites hepatoma AH 130), a less malignantYoshida ascites hepatoma (LY-5), or in minimal deviation-type hepato-

mas (Morris hepatomas 7316A and 7794A). The absence of Peak C inthese tumors coincided with the inability of the glucocorticoid to inducetryptophan oxygenase in these tumors and in the liver of rats during earlypostnatal development. Peak B was consistently observed in varioushepatomas and immature rat liver with capability to induce tyrosineaminotransferase.

Thus Peak C appeared to be a highly differentiated type of glucocorticoid receptor mediating specific hormone actions and to be present inmature liver cells, but not in immature liver or tumor cells, even of theminimal deviation type.

INTRODUCTION

Glucocorticoid has various biological actions, some anabolicand others catabolic, in many target organs. Its effects arethought to be initiated by its binding to a soluble receptorprotein (1-3). However, although there are many reports onthe capacity and affinity of glucocorticoid receptors, there isincreasing recent evidence of discrepancies between the bindingand physiological actions of the hormone (4-9). One possibleexplanation is the existence of multiple forms of glucocorticoidreceptor (10-12). We have been trying to identify differentforms of glucocorticoid receptor to elucidate such various actions of the hormone. Previously we separated at least threetypes of glucocorticoid receptors, named Peaks A, B, and C, byDEAE-cellulose chromatography (13-16). Peak A, whicheluted with 0.02 to 0.03 M NaCl, is a minor peak not foundconsistently in rat liver. It may be similar to Binder IB identifiedby Markovic et al. (17), who suggested that Binder IB mediatesspecific hormone actions, such as stimulation of sodium absorption from the intestine and kidney (18). Alternatively itmay be a proteolytic fragment of Peak B (19). Peak B, whicheluted with 0.05 to 0.08 M NaCl, is a classic type of glucocorticoid receptor, the so-called G protein (1) or Binder II (2),detected in both mature and immature rat liver (13) and mosttarget organs.4 It may mediate many basic and essential actions

of glucocorticoid for cell growth which occur in even immatureliver cells (15), such as TAT5 induction just after birth (20-22).

Received 11/14/86; revised 4/9/87; accepted 4/15/87.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 by a grant from the Ministry of Education, Science, and Culture(59010047, 61010015)of Japan.1 Present address: Department of Home Economics, Mukogawa Women's

College, Hyogo 663, Japan.3To whom requests for reprints should be addressed.4T. Hirota, K. Hirota, Y. Sanno, and T. Tanaka, manuscript in preparation.'The abbreviations used are: TAT, tyrosine aminotransferase (EC 2.6.1.5);

TO, tryptophan oxygenase (EC 1.13.11.11).

Peak C, which eluted with 0.12 to 0.14 M NaCl, was observedin animals only during stress (13) or after administration of ahigh dose of glucocorticoid to adult rats (14), but not onadministration of the hormone to immature animals (15). Repeated treatments with the hormone were necessary to induceTO activity (23) and the appearance of Peak C for maturationof liver cells in young rats (15). Therefore, Peak C mightmediate highly differentiated actions of the hormone, such asTO induction, rather than basic actions. All target organsexamined contained Peak B, but only specific organs4 contained

Peak C.We have identified this new glucocorticoid receptor protein,

Peak C, by its comparison with the classic type of glucocorticoidreceptor, Peak B (13-16). Peak C was significantly differentfrom Peak B in stability (Peak C is more unstable) and immu-nological activity (an antiserum against Peak B did not reactwith Peak C), but it was similar to Peak B in molecular weight(MT 90,000) (16). Then we categorized two biological markersin the actions of the glucocorticoid hormone in rat liver, theinductions of TAT and TO (14). Although both were wellstudied as similar markers of glucocorticoid, Peak B corresponds well with the inducibility of TAT in both mature andimmature rat liver (15). Peak C corresponds well with theinducibility of TO dose and time dependently (14) in mature,but not immature, young rat liver (15).

It has been well documented that, in transplantable hepatomas in rats, marker isozymes specific to liver diminish, andnonhepatic fetal or prototypic isozymes of undifferentiatedtissues appear (24-26). We compared the presence of suchisozyme types of glucocorticoid receptors in various hepatomasand normal liver. The tumors examined varied from the poorlydifferentiated Yoshida ascites hepatoma AH 130 to the minimaldeviation type, Morris 7794A. We proved Peak B in all tumorcells examined, but did not observe either Peak C or TOinduction by glucocorticoid in these tumors. Therefore, wesuggest here that Peak B is the neonatal, or prototype, receptorof undifferentiated tissues and that Peak C is the adult, ordifferentiated, type of receptor of glucocorticoid. This is thefirst paper that presents a comparative analysis of glucocorticoid receptors in normal liver and hepatomas, and that demonstrates a possible existence of different types of glucocorticoid receptors, each of which mediated different hormone actions.

MATERIALS AND METHODSReagents. l,2,4-[3H]Dexamethasone (specific activity, 40 Ci/mmol)

was obtained from Amersham International, Ltd., Buckinghamshire,England. Nonradioactive dexamethasone (9-fluoro-11/3,17,21-trihy-droxy-16-methylpregna-l,4-diene-3,20-dione) was from Sigma Chemical Co., St. Louis, MO. DEAE-cellulose was from Brown Co., Berlin,NH, and Sephadex G-25 was from Pharmacia Fine Chemicals AB,Uppsala, Sweden.

Animals and Tumors. Male Sprague-Dawley rats, female Buffalo rats(Clea Co., Tokyo, Japan), and female Donryu rats (Shizuoka Laboratory Animal Center, Shizuoka, Japan), weighing 140 to 250 g, weremaintained on normal chow diet. Yoshida sarcoma and Yoshida ascites

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NEW GLUCOCORTICOID RECEPTOR IN HOST LIVER AND HEPATOMAS

15

10

ao

CONTROL

10 20 30 40 50 60

FRACTION NUMBER

NaCI(M)

0.3

02

0.1

70 10 20 30 40 SO 60

FRACTION NUMBER

60 70

15

E

aO

NiCl(M)

0.3

0.2

0.1

20 30 40 50 60 70

FRACTION NUMBER FRACTION NUMBERFig. 1. DEAE-cellulose chromatography of liver cytosol labeled with [JH]dexamethasone in rats with and without tumors and with and without hormone treatment.

a, normal rat; A, rat treated with dexamethasone (DEX) 20 h before sacrifice; c, rat bearing Morris hepatoma 7794 A; d, rat bearing Morris hepatoma 7794 A andtreated with cold dexamethasone (100 jig/100 g body weight) 20 h before sacrifice. Liver cytosol fractions from each animal were incubated with [3H]dexamethasonefor 2 h and subjected to DEAE-cellulose chromatography after removing free dexamethasone as described in "Materials and Methods."

Table 1 Serum corticosterone levels in rats bearing various tumorsRats were killed at the times after tumor inoculation described in the text.

Animals

Serumcorticosterone

Normal rats 21.86 ±6.6"(11)*

Rats bearingMorris hepatoma 7794 ALY-5AH 130Yoshida sarcoma77.6

±30.4 (4)c48.0 ±11.5(3)''34.3 ±8.0 (4)c45.0 ±10.3 (4)'

•Mean ±SD.* Numbers in parentheses, number of animals.c P < 0.05 versus normal rats.d P< 0.01 versus normal rats.

hepatoma LY-5 were gifts from Sasaki Institute, Sasaki Foundation,Tokyo, Japan. Yoshida ascites hepatoma AH 130 was a gift from theInstitute for Cancer Research, Osaka University, Osaka, Japan. Morrishepatomas 7316 A and 7794 A were gifts from the National CancerCenter, Tokyo, Japan. Tumor ascites (Yoshida sarcoma, AH 130, LY-5) were transplanted into the abdominal cavity of Donryu rats andtumor cells (Morris hepatomas 7316 A and 7794 A) were transplantedinto the thigh muscle of Buffalo rats.

Preparation of Cytosol for Receptor Assay. Animals were killed bydecapitation. Their livers were perfused in situ with 0.9% saline solutionthrough the portal vein, removed, and homogenized in two volumes ofa solution of 50 IHMKC1, 20 HIMmercaptoethanol, and 20% glycerolin 50 HIMTris-HCl, pH 7.5 (Buffer A). Ascites of Yoshida sarcoma,AH 130, and LY-5 were collected on Days at 5, 7, and 14, respectively,after inoculations. These tumors were washed 3 times with ice-coldphosphate-buffered saline, followed by centrifugation. Fresh cells of

Morris hepatomas 7316 A and 7794 A were carefully collected 1 moafter the inoculation, resuspended in their original volume of Buffer A,and homogenized. All subsequent operations were performed at 0-4*C

on the same day. Liver tissue or tumor cells were homogenized in amotor-driven Potter-Elvehjem homogenizer operated at 1,400 rpm.The homogenate was centrifuged at 100,000 x g for 80 min, and thesupernatant (cytosol) was used after removal of the upper lipoproteinlayer by aspiration.

Glucocorticoid Receptor Binding Assay. The cytosol was incubatedwith 100 11M [3H]dexamethasone with or without a 500-fold excess ofunlabeled steroid at 0"( ' for 2 h. The [3H]dexamethasone-receptor

complex was measured by the dextran-coated charcoal method (27).Column Chromatography. The cytosol labeled with ['Hjdexametha-

sone was promptly applied to a Sephadex G-25 column equilibratedwith buffer of the same composition as the homogenizing buffer but ofpH 8.4 (Buffer B). The unadsorbed fraction was applied to a DEAE-cellulose column (2.5 x 30 cm) equilibrated with Buffer B. The columnwas washed with 2 column volumes of Buffer B, and then proteins wereeluted with a linear gradient of NaCI formed with 140 ml of Buffer Band 140 ml of 0.35 M NaCI in Buffer B. Fractions of 4 ml werecollected, and 1 ml of each fraction was mixed with 6 ml of scintillationfluid (Univer-Gel II; Nakarai Chemicals, Ltd.). Radioactivity was

counted in an Aloka 751 liquid scintillation counter with a meancounting efficiency of 35 to 40% as judged by external standardization.

Enzyme Assay. TO was assayed as described previously (23). Oneunit of TO activity was defined as the amount catalyzing formation of1 /¿molof kynurenine per h at 25"C per g of fresh liver. TAT was

assayed by the method of Granner and Tomkins (28). The cytosol waskept at —20°Covernight, and then its enzyme activity was measured byincubation for 15 min at 37°C.One unit of enzyme activity was defined

as that catalyzing the formation of 1 ¿tmolof p-hydroxy-phenylpyruvate

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NEW GLUCOCORTICOID RECEPTOR IN HOST LIVER AND HEPATOMAS

Table 2 Induction of TO and TAT in Morris hepatoma 7794 Aby dexamethasone

Rats bearing Morris hepatoma 7794 A were treated i.p. with 100 fig/100 gbody weight of dexamethasone and were killed 5 h later. Hepatomas wereremoved, and TO and TAT were assayed as described in "Materials and Methods."

Fig. 2. DEAE-cellulose chromatography of tumor cytosol fractions of tumorslabeled with [3H]dexamethasone (DEX) with or without hormone treatment. Ratsbearing Yoshida sarcoma ( )',V).AH-130, LY-S, and Morris hepatomas 7316 A

and 7794 A with (right) or without (left) treatment with dexamethasone (100 fig/100 g body weight) 20 h previously were sacrificed. The days after inoculation onwhich these tumors were obtained were Day 5 for Yoshida sarcoma, Day 7 forAH 130, Day 14 for LY-5, and Wk 4 for Morris hepatomas. Tumor cells collectedfrom these animals were washed, and the cytosol fractions were incubated with[3H]dexamethasone as described in "Materials and Methods."

per min at 37"C. The effective molar absorbance of the product, p-hydroxybenzaldehyde, was taken as 19,900 M"1cm"1.

Determination of Serum Corticosterone Level. Serum corticosteronewas measured by radioimmunoassay. Sera were obtained at a fixed timeof about 9 a.m.

RESULTS

DEAE-Cellulose Chromatography of Host Liver Cytosol ofRats Bearing Tumors. Buffalo rats with or without treatment ofdexamethasone (100 Mg/100 g body weight) 20 h previouslywere sacrificed, and their liver cytosol was labeled with [3H]-dexamethasone and applied to a DEAE-cellulose column afterremoval of free steroids as described in "Materials and Meth-

Dexamethasone+TO(units/gliver)0.245±0.22" (4)*

0.423±0.17C(3)TAT(units/mg

protein x10')142

±3.51 (3)237 ±16.46' (3)

•Mean ±SD.* Numbers in parentheses, number of animals.' Not significantly different from the TO activity without hormone treatment.'' Significantly different from the TAT activity without hormone treatment (/'

< 0.001).

ods" (Fig. 1, a and b). Macromolecular proteins in untreatedrat liver, which bound specifically to [3H]dexamethasone andeluted with 0.05 to 0.08 M NaCl, were previously named glu-cocorticoid receptor Peak B (13) and corresponded to G proteinor Binder II (Fig. la). Another peak in the flow-through fractionwas free steroid that dissociated from the hormone-receptorcomplex during column chromatography. When rats had beentreated with dexamethasone (100 /¿g/100g body weight) 20 hpreviously, their liver contained less Peak B, and differentmacromolecular proteins specifically bound to [3H]dexametha-

sone, which was eluted with 0.12 to 0.15 M NaCl and has beennamed Peak C (Fig. lò)(13).

In rats bearing Morris hepatoma 7794 A, Peak C was one ofthe predominant receptors in the host liver even without hormone treatment (Fig. le). A similar appearance of Peak C wasobserved in the livers of host with other hepatomas, such asYoshida sarcoma, LY-5, Morris hepatoma 7316 A (data notshown), and AH 130 ( 13). Hormone treatment did not changethe patterns of glucocorticoid receptors in the liver of ratsbearing tumors (Fig. Id). Although Peak C appeared in thecontrol rats only after treatment with hormone, it was consistently found in the liver of rats bearing hepatomas, even withouthormone treatment.

The serum levels of corticosterone in rats bearing varioustumors are shown in Table 1; the mean level was 21.86 ±6.6in intact rats and 77.6 ±30.4 in rats bearing Morris hepatoma7794 A. The serum corticosterone levels in rats with othertumors were also significantly increased. The serum corticosterone levels were low just before the death of hosts (data notshown). Therefore Peak C appeared in host livers concomitantlywith high serum glucocorticoid levels (Fig. 1, b-d). A similarconcomitant increase in the two was also observed in the ratssubjected to various other stresses (13).

Chromatographie Profiles of Receptors in Various TumorsTreated with or without Dexamethasone. Next we examined thereceptors in various tumors by studying the profiles on DEAE-cellulose chromatography of their cytosol fractions (Fig. 2) aswell as host livers (Fig. 1, c and d). Tumors derived fromhepatomas, such as Yoshida asci ics hepatomas AH 130 andLY-5, and Morris hepatomas 7316 A and 7794 A, were examined. Very malignant tumors, such as Yoshida sarcoma, theorigin of which is not clear but may be a hepatoma, were alsoexamined. The differentiation of the tumors examined increasesin the order from Yoshida sarcoma, Yoshida ascites hepatomaAH 130, LY-5, Morris hepatoma 7316 A, to the highly differentiated hepatoma 7794 A. This order was paralleled withmalignancies, or growth rates, of the tumors. Cytosol fromfreshly isolated hepatomas was labeled with [3H]dexamethasoneand subjected to DEAE-cellulose chromatography after removing free steroid as described in "Materials and Methods." Peak

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NEW GLUCOCORTICOID RECEPTOR IN HOST LIVER AND HEPATOMAS

B was the only receptor detected in all the tumors, Yoshidasarcoma, AH 130, LY-5, Morris hepatoma 7316 A, and hepa-toma 7794 A (Fig. 2, left). Next rats bearing these tumors weretreated with dexamethasone (100 Mg/100g body weight) 20 hbefore sacrifice, and then their tumors were excised and examined. As shown in Fig. 2, right, Peak B was predominant inmost tumors. No Peak C was detectable in any tumors, including the minimal deviation-type hepatomas, even after hormonetreatment, but Peak A as well as Peak B was seen after hormonetreatment in some tumors, such as Yoshida sarcoma and Morrishepatomas 7316 A and 7794 A. In contrast, Peak C was foundin the host livers with or without hormone treatment (Fig. 1, cand ¡I).

Inductions of TAT and TO by Dexamethasone in MorrisHepatoma 7794 A. The inductions of TAT and TO as biologicalmarkers of glucocorticoid receptors were examined in Morrishepatoma 7794 A, which is a highly differentiated tumor andlikely to have functions similar to normal hepatocytes. In thistumor, glucocorticoid induced double the basal level of TAT,but did not induce TO (Table 2). In contrast, in normal rat liverglucocorticoid induced 5 times the basal level of TO (14, 23).Similar induction of TAT but not TO by glucocorticoid inneonatal rat liver has been reported (20-22). Glucocorticoidalso did not induce TO in other poorly differentiated hepatomas, such as AH 130, Yoshida sarcoma, and LY-5 (data notshown).

DISCUSSION

We previously identified glucocorticoid receptor species,which we named Peaks A, B, and C, in the liver of rats undervarious biological conditions, such as during stress (13), earlydevelopment (15), and hormonal treatment (14). Peaks A, B,and C all bind displaceably to [3H]dexamethasone, as demonstrated by assay in the presence of 500-fold excess of colddexamethasone (13). Peak B is thought to be a classic type ofglucocorticoid receptor (13). Peak B was found to be the predominant receptor in most organs4 in normal conditions. Peak

C appeared only in such cases after a burst of hormone releasein stress or after a pharmacological dose of the hormone (Fig.1) and was found in specific organs." We have focused attention

on this glucocorticoid receptor, Peak C, which appears anddisappears in response to changes in physiological conditions.Peak C appeared dose and time dependently 15 min to 20 hafter treatment with dexamethasone (more than 20 Mg/100 gbody weight) (14). It also appeared in the nuclear fraction. PeakC binds specifically to glucocorticoid, showing high affinity anda low capacity as a receptor, and it has strict specificity forglucocorticoids, whereas Peak B also binds to other steroids,such as progesterone and aldosterone (16). Peak C was neitheran activated nor a proteolytic form of receptor Peak B (14, 16).

Peak C is not detectable in the liver of rats for the first 14days after birth (15). During this period, some liver enzymes,such as TO, cysteine dioxygenase (29), glucokinase (30), andornithine aminotransferase (31), etc., cannot be induced, butthereafter they appear naturally. Since rat liver cells are notcompletely mature until 14 days after birth, the appearance ofPeak C corresponds well with liver maturation (15). Theseresults suggest that Peak C is a mature or differentiated type ofglucocorticoid receptor.

This conclusion prompted us to study whether Peak C wasdetectable in various types of tumors varying from poorlydifferentiated to well-differentiated types. There have been onlya few reports on the analysis of proteins of glucocorticoid

receptor in tumors (9, 32, 33). Most studies have been on theirpresence, demonstrated by their binding capacities and affinitiesto ligands (4, 34-36). Some studies have shown discrepanciesbetween the glucocorticoid binding capacities and responses tothe hormone (4, 9, 33). In this work, we found that no Peak Cwas detectable chromatographically in any tumors examined(Fig. 2), and that Peak B was a dominant receptor with andwithout hormone treatment in all these tumors. Peak A alsoappeared in Yoshida sarcoma and Morris hepatomas after thehormone treatment. Peak A was rather big in these tumors(Fig. 2), but a minor shoulder in normal liver (Fig. 1). If PeakA is a proteolytic fragment of Peak B, as suggested from thedata of Eisen et al. (19), Peak B in tumors must be easilydegraded by their endogenous protease (9), whereas if it is nota proteolytic product (37), it may mediate some specific hormone action in tumors as Binder IB does in the kidney andintestine (17, 18). Peak A was similar in Chromatographiebehavior to Binder IB from the liver (14) and kidney.4It remainsto be further resolved. We could not find Peak C even in themost highly differentiated hepatoma, 7794 A, which was likelyto have functions similar to those of normal liver. However,Peak C was present in all host livers of rats bearing thesetumors evenwithout hormone treatment (Fig. le). A high serumlevel of corticosterone induced by a burst of endogenous hormone release (Table 1) or hormone treatment induced Peak Cin host livers, but not in their hepatomas. A high level ofglucocorticoid can enter into both hepatomas and hosts owingto its free passing through cell membrane (4, 7, 8, 38).

The TO activity of Morris hepatoma 7794 A was low andwas not induced by the hormone, but TAT activity was inducedby glucocorticoid (Table 2). These data are similar to thosereported for other Morris hepatomas (39, 40). The enzymepattern in hepatoma 7794 A is similar to that of liver in ratssoon after birth, but not in mature rats (15). Thus highlydifferentiated hepatomas have biological functions similar tothose of neonatal liver and, like the latter, have no detectablePeak C.

On the other hand, the isozyme patterns of several enzymesin various rat hepatomas have already been investigated well.Results have shown that prototype isozymes are increased andthat mature liver-type isozymes (differentiated types) are decreased in hepatomas according to the extent of malignancy ofthe tumors (24-26). Fetal liver also contains the prototype ofisozymes, not differentiated types (41).

Based on these observations, we suggest that Peak B is aprototype of glucocorticoid receptor found in most organs andeven in undifferentiated cells, including immature cells andhepatomas. In contrast, Peak C is a differentiated type of thereceptor found in specific organs and in differentiated types ofcells, such as mature liver cells. Therefore, we propose thenames of Isoreceptors B and C for the Peaks B and C, whichare different immunochemically. For further evidence of this,we are now trying to purify Peak C.

ACKNOWLEDGMENTS

We thank Dr. S. Sato of the National Cancer Center, Dr. H. Sato ofthe Sasaki Institute for gifts of various tumors, and Dr. F. Isohashi ofOsaka University for his helpful advice and gift of AH 130. We alsothank H. Tanigawa of Osaka University for skillful assistance in computerizing radioisotope data.

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