Báo cáo Y học: Conditionally immortalized adrenocortical cell lines at undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes pot

Conditionally immortalized adrenocortical cell linesat undifferentiated states exhibit inducible expression of glucocorticoid-synthesizing genes Kuniaki Mukai1, Hideko Nagasawa1,*, Reiko

Conditionally immortalized adrenocortical cell lines

at undifferentiated states exhibit inducible expression

of glucocorticoid-synthesizing genes

Kuniaki Mukai1, Hideko Nagasawa1,*, Reiko Agake-Suzuki1, Fumiko Mitani1, Keiko Totani1,

Nobuaki Yanai2, Masuo Obinata2, Makoto Suematsu1and Yuzuru Ishimura1

1 Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan;

2 Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan

To facilitate studies on di€erentiation of adrenocortical cells

and regulation of steroidogenic genes, we established cell

lines from adrenals of adult transgenic mice harboring a

temperature-sensitive large T-antigen gene of simian virus

40 Adrenal glands of the mice exhibited normal cortical

zonation including a functionally undi€erentiated cell-layer

between the aldosterone-synthesizing zona glomerulosa cells

and the corticosterone-synthesizing zona fasciculata cells At

a permissive temperature (33 °C), established cell lines

AcA201, AcE60 and AcA101 expressed steroidogenic genes

encoding steroidogenic factor-1, cholesterol side-chain

cleavage P450scc, and steroidogenic acute regulatory

pro-tein, which are expressed throughout adrenal cortices and

gonads Genes encoding 3b-hydroxysteroid dehydrogenase

and steroid 21-hydroxylase P450c21, which catalyze the

intermediate steps for syntheses of both aldosterone and

corticosterone, were inducible in the three cell lines in

tem-perature- and/or dibutyryl cAMP-dependent manners

Notably, these cell lines displayed distinct expression pat-terns of the steroid 11b-hydroxylase P45011b gene respon-sible for the zone-speci®c synthesis of corticosterone AcA201 cells expressed the P45011b gene at 33 °C, showing the property of the zona fasciculata cells, while AcE60 cells expressed it upon a shift to a nonpermissive temperature (39 °C) On the other hand, AcA101 expressed the P45011b gene at 39 °C synergistically with exposure to dibutyryl cAMP None of these clones express the zona glomerulosa-speci®c aldosterone synthase P450aldo gene under the con-ditions we tested These results show that AcE60 and AcA101 cells display a pattern of the steroidogenic gene expression similar to that of the undi€erentiated cell-layer and are capable of di€erentiating into the zona fasciculata-like cells in vitro

Keywords: adrenal cortex; steroid hormone; immor-talization; simian virus 40 large T-antigen

Adrenal cortices in mammals are composed of

morpho-logically and functionally differentiated cell zones [1,2]

The outer zone, the zona glomerulosa, synthesizes

aldos-terone, the most potent mineralocorticoid The middle

zone, the zona fasciculata produces corticosterone in

rodents and cortisol in humans and other animals The

inner zone, the zona reticularis secretes adrenal androgens

in humans and in some other animals In rodents,

aldosterone and corticosterone are produced from a

common substrate, deoxycorticosterone

Deoxycorticoster-one is synthesized from cholesterol by a successive action

of cholesterol side-chain cleavage enzyme cytochrome

P450scc (P450scc, or the Cyp11a gene product),

3b-hydroxysteroid dehydrogenase (3bHSD), and 21-hydroxy-lase cytochrome P450c21 (P450c21, the Cyp21a gene product) [1,3] These enzymes are present throughout the adrenal cortex [4±6] On the other hand, two structurally related enzymes, aldosterone synthase cytochrome P450aldo (P450aldo, or the CYP11b-2 gene product) and 11b-hydroxylase cytochrome P45011b (P45011b, or the Cyp11b-1 gene product), convert deoxycorticosterone into aldosterone in the zona glomerulosa and into corticoster-one in the zona fasciculata, respectively [7±9] Thus, the zonal differences in the steroid products are attributable to localization of the two enzymes responsible for the last steps in the steroidogenesis [10]

Correspondence to K Mukai, Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Fax: + 81 3 3358 8138, Tel.: + 81 3 5363 3752, E-mail: mukaik@sc.itc.keio.ac.jp

Abbreviations: StAR, steroid acute regulatory protein; Bt 2 cAMP, dibutyryl cAMP; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 3bHSD, 3b-hydroxyl steroid dehydrogenase and isomerase; P450, cytochrome P450; P450scc, cholesterol side-chain cleavage P450; P450c21, steroid 21-hydrogenase P450; P45011b, steroid 11b-hydroxylase P450; P450aldo, aldosterone synthase P450; PDL, population doubling levels; SF-1, steroidogenic factor-1; SV40, simian virus 40; ts, temperature-sensitive; HBSS, Hank's balanced salt solution.

Enzymes: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.9); 3b-hydroxyl steroid dehydroxylase and isomerase (EC 1.1.1.145);

cytochrome P450 side-chain cleavage (EC 1.14.15.6); cytochrome P450 steroid 21-hydroxylase (EC 1.14.99.10); cytochrome P450 steroid 11b-hydroxylase (EC 1.14.15.4); cytochrome P450 aldosterone synthase (EC 1.14.15.4).

*Present address: Faculty of Engineering, The University of Tokushima, Tokushima, Japan.

(Received 23 July 2001, revised 22 October 2001, accepted 22 October 2001)

Recent studies on regulation of adrenocortical

steroi-dogenesis have focused on mechanisms of cell-speci®c

transcription of genes encoding the steroid hydroxylases

(reviewedin[11])andsteroidacuteregulatoryprotein(StAR)

[12] Among transcription factors that have been found to

regulate the steroidogenic genes, it has been demonstrated

that steroidogenic factor-1 (SF-1, also referred to as Ad4BP)

[13,14] is essential for development of steroidogenic organs

such as adrenal cortex and gonads [15,16] However,

molecular mechanisms for development of the

adrenocor-tical zonation and its maintenance have not been clarified

We previously showed the presence of a functionally

undifferentiated cell layer between the

aldosterone-produc-ing zona glomerulosa cells and the corticosterone-producaldosterone-produc-ing

zona fasciculata cells in rats [17±20] It was

immunohisto-chemically recognized as the region devoid of both

P450aldo and P45011b [17±21] We also showed that the

cell layer is composed of the inner half of the zona

glomerulosa and the transitional zone (also referred to as

zona intermedia) the latter of which has been described by

previous investigators [22±29] We have provided further

evidence that the layer locates in the middle of the region

containing proliferating cells, suggesting the presence of

precursor or progenitor cells that could differentiate into the

glomerulosa and/or fasciculata cells [17,30±33] This view is

consistent with recent observation that adrenocortical cells

radially arranging from the zona glomerulosa to the zona

reticularis share the same clonal origin [34] However, such

processes for development and differentiation of the adrenal

cortices have not fully been investigated because precursor

or progenitor cell lines remain ambiguous

A conditionallyimmortalizing gene such as a

temperature-sensitive (ts) large T-antigen gene of simian virus 40 (SV40)

has been utilized for generation of cell lines [35,36] We have

previously generated transgenic mice [37] carrying a ts SV40

large T-antigen gene tsA58 [38] that is driven by its own

promoter These transgenic mice have been used to establish

various cell lines from different tissues [39,40] In this study,

we have attempted to establish conditionally immortalized

adrenocortical cell lines suitable for in vitro analyses of cell

differentiation by using the transgenic mice [37]

M A T E R I A L S A N D M E T H O D S

Mice and adrenal glands

Adrenal glands used in this study were excised from the

transgenic mice [37] carrying a ts mutant of SV40 large

T-antigen gene tsA58 [38] They were maintained on a

standard diet containing 0.3% (w/w) Na and with water ad

libitum in accordance with the institutional animal care

guidelines of Keio University School of Medicine The

adrenal glands apparently developed no tumor and had

normal histology based on examination with hematoxylin/

eosin staining until at least 10 weeks old

Immunohistochemistry

Immunohistochemical localization of P45011b and

P450aldo was performed on 6-lm sections of fresh-frozen

adrenal glands from the transgenic mice as previously

described [17,41] The antibodies used were raised in rabbits

against rat P45011b and P450aldo [21]

Cell culture Ten adrenal glands from 8-week-old male mice and eight adrenal glands from 10-week-old female mice were used in separate experiments The adrenals were minced and treated with 1.5 mL of Hank's balanced salt solution (HBSS) containing 2 mgámL)1collagenase type V (Sigma, St Louis,

MO, USA), 0.05 mgámL)1 DNase I (Sigma), and

5 mgámL)1bovine serum albumin (Sigma) at 37 °C for 1

h with gentle shaking After pipetting to disperse cells, they were collected by centrifugation, and were resuspended with HBSS The suspension contained 1.0 ´ 106and 7 ´ 105cells from 10 and eight adrenals, respectively The cells were collected by centrifugation and resuspended at 5 ´ 105cells per mL with one of two cell culture media: medium A, a

1 : 1 mixture of Dulbecco's modi®ed Eagle's medium and Ham's F12 medium with 15% heat-inactivated horse serum (Life Technologies, Rockville, MD, USA), 2.5% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA),

200 UámL)1penicillin, and 200 lgámL)1streptomycin (Life Technologies); medium E, RITC80-7 medium ([42]; Kyo-kuto Pharmaceutical Industrial, Tokyo, Japan) with the same additives included in medium A The cell suspension (5 ´ 104cells) was placed on the center of a 9.2-cm2well The dishes had been coated with bovine ®bronectin (Life Technologies) by incubation of multiwell plates overnight at

37 °C with serum-free medium containing 1 lgámL)1

®bronectin After incubation of the cells at 37 °C for 4 h,

2 mL of medium was added gently into each well Gas-phase used was humidi®ed atmosphere containing 5% CO2 The next day, the temperature was shifted to 33 °C and the medium was changed at 3- to 4-day intervals The cells were transferred to ®bronectin-coated plates every week using 0.05% trypsin-0.53 mM EDTA (Life Technologies) At a third transfer, 25±100 cells were plated in ®bronectin-coated 60-mm dishes in the same media Visual inspection of the plates veri®ed the absence of pairs or groups of cells After

4 weeks, colonies (3±4 mm in diameter) were isolated using cloning rings and trypsin-EDTA Each clone was grown successively in 1.8-cm2wells, 9.2-cm2wells, and then larger dishes by subculturing Some cells were used for RNA extraction and others were frozen for subsequent experi-ments

The cell lines obtained at the permissive temperature for the T-antigen were examined for expression of mRNAs for SF-1, P450scc, P45011b, and P450aldo by RT-PCR anal-ysis as described below SF-1 and P450scc mRNAs were used as adrenocortical cell markers The reason that we adopted SF-1 and P450scc as criteria for adrenocortical cells was that they were detected in the adrenogenital

primordi-um and throughout the cortex in adults [43±46] P450aldo and P45011b mRNAs were used as the zonal differentiation markers that were responsible for production of aldosterone and corticosterone, respectively [21] We chose three cell lines AcA101, AcA201 (obtained with medium A), and AcE60 (obtained with medium E) They showed different expression patterns of SF-1, P450scc, and P45011b genes (see Results) Their expression patterns of the steroidogenic genes, morphological appearance, and growth rates were unchanged over population doubling levels (PDL) of 200

To further characterize the cell-lines, cells were treated with porcine corticotropin (23 mUámL)1), human angio-tensin II (50 nM), dibutyryl cAMP (Bt2cAMP) (1 mM), KCl

(medium + 15 mM), BAY K6844 (1 lM), A23187 (1 lM),

ionomycin (1 lM), or 12-O-tetredecanoylphorbol 13 acetate

(TPA; 160 nM) for 24 h or 4 days under the standard culture

media described above These reagents were products of

Sigma During the treatments, the cells were cultured at 33

or 39 °C Total RNA was extracted and analysed as

described below

Northern blot analysis

Total RNA was extracted with a modi®ed single-step

isolation method using Trizol reagent (Life Technologies)

Northern blot analysis was performed as described

previ-ously [47] except that probes were32P-labeled DNA Before

transfer to positively charged nylon membranes (Roche

Diagnostics, Mannheim, Germany) rRNAs were visualized

by ethidium bromide Densitometric analysis of 28S rRNA

bands veri®ed that amounts of RNA loaded were similar

(< ‹ 10%) and that degradation of the RNA

prepara-tions was undetectable under the experimental condiprepara-tions

DNA fragments were labeled with [a-32P]dCTP (3000

Ciámmol)1, Amersham-Pharmacia Biotech, Piscataway,

NJ, USA) and High Prime (Roche Diagnostics) according

to the manufacturer's instructions Hybridization signals

were detected with a Kodak BioMax ®lm with an

intensi-fying screen DNAs used for labeling each contained a

fragment as follows: SV40 large T-antigen gene, 1.7-kb

PvuII±EcoRI fragment of pMT-1ODtsA [48]; SF-1,

AccI±EcoRI fragment corresponding to the 3¢

untransla-ted region of a mouse cDNA [13]; P450scc, a mouse

cDNA fragment corresponding to the rat cDNA

nucleotides 1018±1361 [49]; P45011b, a mouse cDNA

nucleotides 761±950 [8,47]; P450aldo, a mouse

cDNA nucleotides 761±953 [8] The plasmids carrying

SV40 large T-antigen gene and mouse SF-1 were generous

gifts from H Ariga (Hokkaido University) and K L Parker

(University of Texas South-Western Medical Center, TX,

USA), respectively cDNAs clones encoding P450scc,

P45011b, and P450aldo were obtained by PCR with the

primer pairs described below Mouse adrenocortical Y-1

and ®broblast NIH3T3 cells were cultured with Dulbecco's

modi®ed Eagle's medium containing penicillin

(100 IUámL)1), streptomycin sulfate (100 lgámL)1) and

10% heat-inactivated fetal bovine serum at 37 °C under a

humidi®ed atmosphere containing 5% CO2

RT-PCR

Expression of mRNA was analyzed with RT-PCR cDNA

was synthesized from total RNA (2 lg) with an oligo dT18

primer and Moloney murine leukemia virus reverse

transcriptase using a ®rst-strand cDNA synthesis kit

(Amersham-Pharmacia Biotech) according to the

manufac-turer's instructions Aliquots (1 lL) of the reaction solution

were used as template for PCR PCR mixture contained

10 mM Tris/HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2,

0.2 mMeach deoxynucleotide triphosphate, 0.5 lM

deoxy-oligonucleotide primers, and Taq DNA polymerase

(1.25 U, Takara Shuzo, Shiga, Japan) in a total volume of

25 lL Ampli®cation conditions were 45 s at 94 °C, 45 s at

the annealing temperature for each primer pair as described

below, and 2 min at 72 °C for 35 cycles or appropriate cycle

numbers as indicated followed by 7 min at 72 °C The

annealing temperatures for each primer pair were: 56 °C for P450scc, P45011b, P450aldo, and 3bHSD; 69 °C for P450c21; 50 °C for StAR; 54 °C for glyceraldehyde 3-phos-phate dehydrogenase (GAPDH) PCR products (5 lL) were analyzed by agarose gel electorphoresis followed by visualization with ethidium bromide Nucleotide sequences

of primer pairs used for PCR were as follows (numbers in parenthesis were nucleotide positions of the cDNA sequences):

SCC-F, 5¢-GCACACAACTTGAAGGTACAGGAG-3¢ (1018±1041); SCC-R, 5¢-CAGCCAAAGCCCAAGTACC GGAAG-3¢ (1348±1361) [50]

m11b-F,5¢-AAGAAAACTTAGAGTCCTGGGATT-3¢ (761±784); m11b-R, 5¢-GTGTCAGTGCTTCCAGCAAT GAGT-3¢ (927±950) [8]

mAldo-F, 5¢-AAGAACATTTCGATGCCTGGG ATG-3¢ (761±784); mAldo-R, 5¢-GTGTCAACGCTCCC AGCGGTGAGC-3¢ (930±953) [8]

mStAR-F, 5¢-AAGAGCTCAACTGGAGAGCAC-3¢ (170±190); mStAR-R, 5¢-TACTTAGCACTTCGTCCC CGT-3¢ (380±400) [51]

CTG-3¢ (412±436); 3bHSD-R, 5¢-CAAGTGGCTCATAG CCCAGATCTC-3¢ (1160±1137) [50]

m21-F, 5¢-CTTCACGACTGTGTCCAGGACTTG-3¢ (553±576); m21-R, 5¢-CAGCAGAGTGAAGGCCTGCA GCAG-3¢ (1309±1332) [52]

GAPDH-F, 5¢-TGAAGGTCGGTGTGAACGGATT TGGC-3¢ (51±76); GAPDH-R, 5¢-CATGTAGGCCATGA GGTCCACCAC-3¢ (1010±1033) [53]

The forward and reverse primers reside in different exons

of the genes The PCR products were digested with appropriate restriction enzymes to ensure the speci®city of the PCR reactions by comparing of sizes of digests with those expected from published DNA sequences Total RNA from Y-1 cells was used as a positive control for detection of the mRNAs except that adrenal total RNA from C57BL/6 mice was used as a positive control for detection of P450c21 mRNA

To estimate relative amounts of mRNA among the cells cultured under different conditions (33 or 39 °C in the presence or absence of Bt2cAMP), intensities of PCR products stained with ethidium bromide (see below) were determined by densitometric analysis All experiments for the determination were performed within the exponential phase of the ampli®cation reactions to obtain the linear response concerning the initial RNA amounts Each experiment was performed at least twice to assure the reproducibility The intensities were normalized with GAPDH cDNA, and the relative amounts of mRNA were expressed in Table 1 as the values of the mRNA level in Y-1 cells or mouse adrenal glands were taken as 1.0

Analysis of steroids Cells (1±2 ´ 106cells per 21-cm2dish) were cultured at 33 or

39 °C in the presence or absence of 1 mM Bt2cAMP for

4 days Water-soluble cholesterol (20 lM; Sigma) was added at 24 h before removal of the medium Steroids in the medium (2 mL) were extracted with 8 mL of dichlo-romethane The extracts were treated with 2 mL of 0.1M NaOH and then washed with 2 mL of water The resulting extracts were evaporated to dryness and redissolved with

50 lL of methanol, and then 50 lL of water was added An

aliquot (25 lL) of each sample was subjected to HPLC

using a C18 column (4.6 mm ´ 150 mm; Cosmosil

5C18-AR, Nacalai Tesque, Kyoto, Japan) Steroids were

sepa-rated by isocratic elution with 65% methanol in water at

0.8 mLámin)1 and detected at 254 nm For detection of

corticosterone, 55% methanol was used as the eluent

Authentic steroid standards were used for identi®cation of

steroid products by comparing elution times To convert

pregnenolone, which is hardly detectable at 254 nm, into

progesterone, the steroid products were treated with 0.53 U

of cholesterol oxidase (26.8 Uámg)1; Toyo Jozo Co., Ltd,

Shizuoka, Japan) [54] in a reaction mixture of 100 lL

consisting of 20 mMpotassium phosphate buffer, pH 7.4,

and 0.3% (v/v) Tween 20 The reaction mixture was

incubated at 37 °C for 20 min, and extracted with

dichlo-methane The extracts were analyzed by HPLC under the

same conditions

R E S U L T S

Histology of adrenal glands of transgenic mice carrying a temperature-sensitive oncogene Adrenal glands of the transgenic mice harboring SV40 large T-antigen tsA58 gene appeared quite normal in size and shape as compared with those of nontransgenic animals, suggesting that the ts oncogene developed no tumor in the adrenal glands at body temperature As judged by the haematoxylin/eosin staining, zonation of their cortices including the zonae glomerulosa, fasciculata, and reticularis were indistinguishable from those of the normal animals (data not shown) The medullary tissues also appeared to be normal

We then examined imminohistochemically expression of steroidogenic enzymes that occur in a zone-speci®c manner, namely, P45011b and P450aldo As shown in Fig 1,

Table 1 Expression of genes involved in adrenocortical steroidogenesis in cell lines AcA101, AcA201, and AcE60 Based on the results from RT-PCR, relative levels of mRNA were normalized using the results of GAPDH mRNA as described in Materials and methods, and are expressed as the mRNA levels in Y-1 cells or mouse adrenal glands were taken as 1.0 Value 0 indicates that the level was < 0.01 of that of Y-1 cells or mouse adrenal glands ND, not determined Bt 2 cAMP presence or absence is indicated by + and ±, respectively.

mRNA

Adrenal

P450scc 0.04 0.12 0.04 0.30 0.25 0.42 0.71 1.1 0.01 0.06 0.02 0.09 1.0 ND

Fig 1 Adrenocortical zonation of transgenic mice harboring a temperature-sensitive SV40 large T-antigen gene Fresh-frozen sections (6 lm) from adrenal glands of the transgenic mice harboring a temperature-sensitive (ts) SV40 large T-antigen gene were analyzed immunohistochemically with

an antibody speci®c to corticosterone-synthesizing 11b-hydroxylase cytochrome P450 (P45011b) (A) and with an antibody to aldosterone synthase cytochrome P450 (P450aldo) (B) as described in Materials and methods Localization of P45011b is shown with a brown color and that of P450aldo

is shown with a blue color These immunohistochemical results obtained with the transgenic mice were indistinguishable from those with nontransgenic normal mice Sizes, shapes and cytology of the adrenal glands (including medulla) of the transgenic mice also seemed to be normal Note that the thickness (marked with a) where P45011b is absent is larger than the thickness (marked with b) where signals of P450aldo are present, indicating that there is a cell-layer which neither has P45011b nor P450aldo Bar ˆ 50 lm.

P45011b was detected in the entire regions of the zonae

fasculata-reticularis (Fig 1A), while P450aldo was detected

in the outermost cells of the zona glomerulosa (Fig 1B)

Such distribution was indistinguishable from that observed

with nontransgenic animals (data not shown) Thus, the

distribution of the two enzymes was not affected by

introduction of SV40 large T-antigen gene tsA58 These

observations suggest that the transgenic manipulation does

not interfere with development of the adrenal zonation in

the mice

As indicated in Fig 1, the region of P45011b-negative

cells (parenthesis in Fig 1A) was thicker than the region of

P450aldo-positive cells (parenthesis in Fig 1B) Evidently,

there was a cell-layer where neither P45011b nor P450aldo

were detectable, suggesting that the cells in this layer were

unable to produce either corticosterone or aldosterone It

was also noted that P450aldo was only detectable in the

outermost area of the zona glomerulosa under dietary

conditions with normal Na contents Together with our

previous results [17], these results indicate that mice exhibit a

functionally undifferentiated cell layer analogous to that

observed in rats

Establishment of immortalized adrenocortical cell lines

A number of cell lines were derived from primary cells

prepared from whole adrenal glands of the transgenic mice

The permissive temperature (33 °C) for the T-antigen

mutant was used to establish cell lines in which the

oncoprotein was kept active To select cell lines exhibiting

properties of adrenocortical cells, RNAs from the cells were

examined by RT-PCR analysis to detect SF-1 and P450scc

mRNAs The cell lines were further examined for detection

of P45011b and P450aldo mRNAs, the functional markers

for the zone-speci®c differentiation of the cells The results

of RT-PCR analyses indicated that the cell lines were

categorized into three different groups The ®rst group

constituted cell lines expressing SF-1, P450scc, and P45011b

mRNAs but not P450aldo mRNA These cell lines had the

property of the zona fasciculata cells The second group was

composed of a small number of the cell lines that expressed

SF-1 and P450scc mRNAs but not P45011b or P450aldo

mRNAs, showing the gene expression pattern observed in

the undifferentiated cell layer The last cell lines were those

that expressed none of the SF-1, P450scc, P45011b, and

P450aldo mRNAs and were characterized by their

®bro-blast-like appearance There were no cell lines that expressed

P450aldo mRNA regardless of expression of SF-1, P450scc,

or P45011b mRNAs

Among these cell lines, AcA101, AcA201, and AcE60

were chosen for further detailed characterization When

cultured at 33 °C, AcA201 was one of cell lines that

displayed mRNAs for SF-1, P450scc, and P45011b but not

P450aldo mRNA On the other hand, AcA101 and AcE60

were two different cell lines that displayed expression of

SF-1 and P450scc mRNAs but not P45011b and P450aldo

mRNAs at 33 °C; the latter two exhibited distinct

expres-sion patterns of steroidogenic genes that were different to

each other (see below) Their phenotypes and growth rates

of the three cell lines were unchanged over a PDL of 200 at

33 °C Morphologies of these cells cultured at 33 °C are

shown in Fig 2 AcA101 and AcA201 cells displayed

retracted appearances AcE60 cells showed a larger and

¯atter appearance and were less retracted than AcA101 and AcA201 cells The doubling time of these cells was 24±30 h

at 33 °C

Fig 2 Morphology of adrenocortical cell-lines Phase contrast Photomicrographs depict morphologies of adrenocortical cell-lines AcA101 (A), AcA201 (B), and AcE60 (C) which were cultured under the permissive temperature (33 °C) for the ts SV40 large T-antigen The cells were cultured at subcon¯uent stages under the conditions as described in Materials and methods Bar ˆ 50 lm.

Growth of AcA101, AcA201, and AcE60 cells under a

nonpermissive temperature for the T-antigen (39 °C) was

examined upon shifting the temperature from 33 to 39 °C

Their rates of growth were reduced within 2 days after the

start of the temperature shift At one week, AcA101 and

AcA201 cells were mostly detached from the culture dish

surface, and completely lost adhesivity until 4 weeks In

contrast, AcE60 cells were able to attach onto the dishes even

after the disappearance of cell division (data not shown)

The three cell lines cultured at 33 °C were characterized

by Northern blot analysis As shown in Fig 3A, Northern

blotting for SV40 T-antigen mRNA indicated that AcA101 (lane 1), AcA201 (lane 2), and AcE60 (lane 3) cells expressed the transgene mRNA with different signal intensities As expected, Y-1 adrenocortical cells (lane 4) and ®broblast NIH3T3 cells (lane 5) gave no hybridization signal SF-1 mRNA (Fig 3B) was detectable in the established cell lines (lanes 1±3) The electrophoretic mobility of the hybridiza-tion signal was the same as that of Y-1 cells (lane 4) Signal intensities of P450scc mRNA varied markedly among the three cell lines (Fig 3C) The P450scc mRNA level in AcA201 cells was evident, while only a faint signal was detectable in AcA101 cells P450scc mRNA in AcE60 cells was undetectable under the current experimental conditions

As shown in Fig 3B,C, Y-1 cells (lanes 4) produced hybridization signals of SF-1 and P450scc mRNA, whereas NIH3T3 cells (lanes 5) did not, indicating that the hybrid-ization was speci®c As seen in Fig 3D, electrophoretic patterns of ribosomal RNAs indicated that amounts of total RNA subjected to the Northern analyses were equivalent and its degradation was undetectable Although attempting

to detect P45011b and P450aldo mRNAs using the same RNA blot, we could not detect these mRNAs under the current experimental conditions (data not shown) Figure 4 shows the results from RT-PCR analysis with greater sensitivity for the detection of P450scc, P45011b, and P450aldo mRNAs in the three cell lines cultured at

33 °C In addition to AcA101 (Fig 4A, lane 1) and AcA201 (lane 2), AcE60 cells (lane 3) exhibited a detectable level of P450scc mRNA Differences in intensities of the ampli®ed DNA fragments among the three were consistent with the results from Northern blotting (Fig 3C) On the other hand, P45011b mRNA (Fig 4B) was detectable in AcA201 cells (lane 2), but not in AcA101 (lane 1) or in AcE60 (lane 3) cells P450aldo mRNA (Fig 4C) was not detectable in the three cell lines (lanes 1±3), although it was detected in

Y-1 cells (lane 4) Digestion of the ampli®ed DNA fragments with restriction enzymes veri®ed speci®city of PCR (right panels of Fig 4A±C) As judged from the results for GAPDH mRNA, the ef®ciency of RT-PCR was compara-ble among the cell lines (Fig 4D) These results suggest that, under conditions where the T-antigen is active, AcA201 cells have the property of the zona fasciculata cells, while AcA101 and AcE60 cells do not display the zone-speci®c markers of steroidogenesis

Cyclic AMP-dependent alterations in steroidogenic gene expression upon inactivation of the T-antigen

To examine effects of inactivation of the T-antigen on expression of the genes for steroidogenesis, we cultured AcA101, AcA201, and AcE60 cells at 39 °C for up to 4 days and analyzed levels of mRNAs for P450scc, StAR, 3bHSD, P450c21, P45011b, and P450aldo by RT-PCR At the same time, the cells were cultured in the presence of regulators of the steroidogenic gene expression such as Bt2cAMP, ACTH, or angiotensin II, in combination with the temper-ature shift As described below, a simple shift of tempertemper-ature for 4 days affected the mRNA levels of some of these steroidogenic genes Treatments with either corticotropin or angiotgensin II did not alter the mRNA levels signi®cantly

at both 33 and 39 °C under the current experimental conditions (data not shown) On the other hand, treatment with Bt2cAMP for 4 days turned out to alter the mRNA

Fig 3 Northern analysis for expression of SV40 large T-antigen,

steroidogenic factor-1 (SF-1, or Ad4BP), cholesterol side-chain cleavage

cytochrome P450 (P450scc, or Cyp11a) in cell lines AcA101, AcA201,

and AcE60 at a permissive temperature for the ts T-antigen Total RNA

was prepared from AcA101, AcA201, and AcE60 cells which were

cultured at 33 °C with standard media as described in Materials and

methods RNA from mouse adrenocortical Y-1 and ®broblast

NIH3T3 cells, neither of which express the T-antigen gene, were used

as positive and negative controls, respectively, for detection of

adrenocortical cell marker mRNAs Total RNA (10 lg per lane) was

analyzed by Northern blotting with 32 P-labeled cDNA probes

enco-ding (A) SV40 T-antigen (B) SF-1 and (C) P450scc genes as described

in Materials and methods Ribosomal RNAs (D) visualized by

ethi-dium bromide show that amounts of total RNAs were comparable to

each other and that degradation of RNA were undetectable under the

experimental conditions.

levels at both temperatures as described below These results

were summarized in Table 1

P450scc and StAR genes

Figure 5A shows effects of a temperature shift in the

presence or absence of Bt2cAMP on P450scc mRNA levels

in AcA101, AcA201, and AcE60 cells P450scc mRNA

levels upon a simple shift to 39 °C were almost unchanged

in AcA101 (lanes 1 and 3) and AcE60 (lanes 11 and 13) cells,

while in AcA201 cells (lanes 6 and 8), mRNA levels

increased signi®cantly Responsiveness of AcA101 (lanes 2 and 4) and AcE60 (lanes 12 and 14) cells to Bt2cAMP was evident at both 33 and 39 °C, while in AcA201 cells (lanes 7 and 9) responsiveness was much less Synergistic effects of a temperature shift and Bt2cAMP were also shown in these cell lines (lanes 4, 9, and 14)

We next examined the ability of AcA101, AcA201 and AcE60 cells to express StAR mRNA, a key factor for the acute induction of adrenocortical steroidogenesis (Fig 5B)

In the absence of the cAMP analog at 33 °C, the mRNA levels were detectable but with only faint bands in the three

Fig 4 RT-PCR analysis for expression of P450scc, P45011b, and P450aldo genes in the cell lines AcA101, AcA201, and AcE60 cells cultured at

33 °C cDNA was synthesized with oligo dT primer using total RNA (2 lg) from AcA101 (lanes 1 and 5), AcA201 (lanes 2 and 6), AcE60 (lanes 3 and 7), or Y-1 (lanes 4 and 8) cells, and the resulting cDNAs were ampli®ed by PCR using speci®c primer pairs for (A) P450scc, (B) P45011b, (C) P450aldo and (D) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described in Materials and methods Cycle numbers in PCR were 35 in (A), (B), and (C), and 25 in (D) Left, PCR products (5 lL) were analyzed through 1% agarose gels Right, PCR products (5 lL except for lane 7 of panel A where 10 lL was used) were digested with BstXI (A,B) and with SacI (C) The digests were separated on 8% polyacrylamide gels DNA fragments were visualized by ethidium bromide Size marker (lanes M) is HaeIII-digested /X174 DNA Numbers with arrowheads indicate sizes of PCR products or their digests ns (C) indicates a nonspeci®c band Amplifying conditions except for lanes 2 and 4 of (A) were within the exponential phase of the reactions to obtain the linear dose±response concerning the initial RNA amounts.

cell lines (lanes 1, 6, and 11) When AcA101 and AcA201

cells were cultured either in the presence of Bt2cAMP (lanes

2 and 7) or at 39 °C (lanes 3 and 8), the mRNA levels were

increased markedly In contrast, AcE60 cells displayed only

a small induction of the mRNA level in the presence of

Bt2cAMP (lane 12), while showing no notable change upon

a temperature shift (lane 13) Interestingly, however, AcE60

cells exhibited the greatest synergy between the temperature

shift and Bt2cAMP among the three cell lines tested (lanes 4,

9, and 14) On the other hand, the results obtained with

GAPDH primers showed similar intensities of the PCR

products among the groups, indicating that ef®ciencies of

reverse transcription and PCR were comparable to one

another (Fig 5C) These results indicated that P450scc and

StAR genes responsible for the initial steps for synthesis of

both adrenocortical and sex steroid hormones were

expressed constitutively and inducible through a

cAMP-dependent pathway in the established cell lines

3bHSD and P450c21 genes

3bHSD and P450c21 catalyze the reactions in the middle of

the synthetic pathways for both corticosterone and

aldos-terone Figure 6A shows mRNA levels of 3bHSD in the

same sets of RNA preparations used for analysis of P450scc

and StAR mRNA levels in Fig 5 3bHSD mRNA in

AcA101 cells was detected only in the presence of Bt2cAMP

at 39 °C (lanes 1±4) AcA201 cells also expressed 3bHSD

mRNA in a similar manner to that seen in AcA101 cells,

although the level after the treatment was lower (lanes 6±9) Thus, synergies of inactivation of the T-antigen and treatment with the cAMP analog were evident in the induction of 3bHSD mRNA in AcA101 and AcA201 cells The mRNA level in AcE60 cells in the absence of Bt2cAMP

at 33 °C was hardly detected (lane 11), while treatment with the cAMP analog at 33 °C induced the mRNA level (lane 12) A temperature shift to 39 °C did not enhance the 3bHSD mRNA level (lane 13), and the induction by the cAMP analog at 39 °C was smaller than that observed at

33 °C (lane 14) It should be noted that, although being expressed in adrenal cortex and gonads (as are the P450scc and StAR genes), the 3bHSD gene of these cell lines is expressed in a manner (Fig 6A) distinct from those of P450scc and StAR gene expression (Fig 5A,B)

Figure 6B illustrates differences in P450c21 mRNA levels among the cell lines The mRNA level in AcA101 cells in the absence of Bt2cAMP was undetectable at 33 °C (lane 1), whereas treatment with the cAMP analog induced a weak signal at 33 °C (lane 2) A simple shift to 39 °C did not induce the mRNA (lane 3), but the same procedure in the presence of Bt2cAMP increased the level signi®cantly (lane 4) The results obtained with AcA201 cells (lanes 6±9) were similar to those obtained with AcA101 cells except that the induction in AcA201 was weak In AcE60 cells, P450c21 mRNA was detectable upon treatment with Bt2cAMP at

33 °C (lanes 11 and 12) At 39 °C, P450c21 mRNA became undetectable irrespective of the presence of Bt2cAMP (lanes

13 and 14) Thus, AcA101, AcA201 and AcE60 cells were

Fig 5 Expression of P450scc and steroidogenic acute regulatory protein (StAR) genes in AcA101, AcA201, and AcE60 cells upon a temperature shift and/or treatment with dibutyryl cAMP (Bt 2 cAMP) AcA101, AcA201, and AcE60 cells were plated at 33 °C, and allowed to attach to dishes for

24 h, and were further cultured at 33 or 39 °C for 4 days in the absence or presence of 1 m M Bt 2 cAMP Total RNA was prepared from the cells and was subjected to RT-PCR analysis using the primer pairs for (A) P450scc, (B) StAR and (C) GAPDH as described in Materials and methods RNA preparation from Y-1 cells were used as positive controls for P450scc and StAR mRNA PCR products (5 lL) were analyzed through 2% agarose gels followed by visualization with ethidium bromide Cycle numbers in PCR for P450scc (A) were 32 in AcA101, 28 in AcA201, and 35 in AcE60 cells, and those in PCR for StAR (B) and GAPDH (C) were 35 and 25, respectively, for the three cell lines.

able to express the P450c21 gene, the marker expressed

exclusively in the entire regions of adrenal cortices but not in

gonads, indicating that the cell lines retain a feature of

adrenocortical cells

P45011b and P450aldo genes

Figure 7 shows the levels of P45011b mRNA in the absence

or presence of Bt2cAMP at 33 and 39 °C AcA101 cells did

not have a detectable level of P45011b mRNA in the absence

of the cAMP analog at 33 °C (Fig 7, lane 1; Fig 4B,

lane 1) Either treatment with the cAMP analog (Fig 7, lane

2) or a temperature shift (lane 3) did not induce the mRNA

Upon the combined treatment with the cAMP analog and a

temperature shift, however, the cells expressed the P45011b

gene (lane 4) A similar synergistic effect in AcA101 cells was

observed on the levels of 3bHSD mRNA (Fig 6A) The

P45011b mRNA level in AcA201 cells at 33 °C was

detectable in the absence of Bt2cAMP (Fig 7, lane 6;

Fig 4B, lane 2), and was not increased by the Bt2cAMP treatment at 33 °C (lane 7 of Fig 7) A simple shift to 39 °C enhanced the mRNA level (lane 8), and the additive effects

of the cAMP analog appeared to be small, if any (lane 9) AcE60 cells did not exhibit a detectable level of P45011b mRNA either in the absence (lane 11) or presence (lane 12)

of Bt2cAMP at 33 °C Interestingly, AcE60 cells showed the ability to induce the mRNA level upon the temperature shift

to 39 °C (lane 13), although the level after the Bt2cAMP treatment remained almost unchanged at 39 °C (lane 14) These results indicated that the three cell lines are able to express P45011b gene, which is a determinant for synthesis

of corticosterone in the zona fasciculata cells Moreover, these three cell lines responded to distinct stimulatory conditions to express the P45011b gene

As mentioned earlier in this article, P450aldo mRNA was undetectable in AcA101, AcA201, and AcE60 cells at 33 °C cultured in the absence of Bt2cAMP at 33 °C In addition, a temperature shift and/or treatment with Bt2cAMP failed to

Fig 6 Expression of 3b-hydroxysteroid dehydrogenase (3bHSD) and 21-hydroxylase P450 (P450c21) upon a temperature shift and/or treatment with

Bt 2 cAMP PCR was performed with primer pairs for (A) 3bHSD and (B) P450c21 using the cDNAs synthesized in the experiments in Fig 5 Y-1 cells and mouse adrenal glands were used as positive controls for detection of 3bHSD and P450c21 mRNA, respectively PCR products (5 lL) were analyzed through 1% agarose gels followed by visualization with ethidium bromide Cycle numbers in PCR for 3bHSD and P450c21 were 35 for the three cell lines.

Fig 7 Expression of P45011b gene upon a temperature shift and/or treatment with Bt 2 cAMP PCR was performed with the primer pair for P45011b using the cDNAs synthesized in the experiments in Fig 5 Y-1 cells were used as a positive control for detection of P45011b mRNA PCR products (5 lL) were analyzed through 2% gels followed by visualization with ethidium bromide Cycle numbers in PCR were 35 for the three cell lines.

induce P450aldo mRNA (data not shown) Furthermore, to

induce expression of the P450aldo gene, these cells were

cultured in the presence of either of angiotensin II, KCl, or

reagents that could change intracellular concentration of

calcium ion at 33 or 39 °C (see Materials and methods)

These stimuli, however, could not induce detectable levels of

P450aldo mRNA in the cell lines under current

experimen-tal conditions (data not shown)

Steroidogenesis of the cell lines

The ability of the cell lines to produce steroids was examined

by using reversed phase HPLC As shown in Fig 8A±D,

when AcA101 cells were cultured in the presence of

Bt2cAMP for 4 days at 39 °C, progesterone and a small

amount of deoxycorticosterone were detected, while

corti-costerone was undetectable On the other hand, AcA201

cells synthesized only a detectable amount of progesterone

when the cells were stimulated with Bt2cAMP at 39 °C

(Fig 8E) AcE60 cells secreted progesterone in the presence

of Bt2cAMP at both 33 and 39 °C (data not shown) Thus,

the three cell lines showed responsiveness of their

steroido-genesis to activation of protein kinase A Although pregn-enolone is a possible intermediate of the steroidogenesis, it is hardly undetectable in the eluate of HPLC by monitoring at

254 nm To examine amounts of pregnenolone in the culture medium of the three cell lines, the dichloromethane extracts were treated with cholesterol oxidase for conversion into progesterone After treatment, a remarkable peak of progesterone was detected in the culture medium of AcA201 (Fig 8F), suggesting that AcA201 cells secreted large amounts of pregnenolone Similarly, AcA101 and AcE60 cells synthesized signi®cant amounts of pregnenolone when they were cultured in the presence of Bt2cAMP at 39 °C (data not shown) These results were consistent with the results from RT-PCR analysis of mRNA levels of the steroidogenic genes (Figs 5±7)

D I S C U S S I O N

Previous studies on regulatory mechanisms of adrenocor-tical steroidogenesis have often utilized mouse Y-1 [55] and human NCI-H295 [56] cells Y-1 cells have been known to display expression patterns of steroidogenic genes analo-gous to those of the zona fasciculata cells except that the endogenous P450c21 gene is not expressed, and that the P450aldo gene is constitutively expressed though its mRNA level is one-tenth of that of P45011b mRNA [8] On the other hand, NCI-H295 cells have been reported to exhibit the phenotypes of both zona glomerulosa and fasciculata cells simultaneously Although these cell lines have been shown to be useful in vitro cell culture systems, the phenotypes of Y1 and NCI-H295 cells do not precisely correspond to either of the glomerulosa or fasciculata cells Other adrenocortical cell lines were also established by using the wild-type SV40 T-antigen gene [57,58] When compared with the cell lines established previously, our cell lines described in the present study have several distinct features suitable for studies on differentiation of adreno-cortical cells and regulation of the steroidogenic genes First, immortalization is conditional so that activity of the oncogene can be removed Secondly, multiple cell lines with the identical genetic background exhibit different pheno-types in steroidogenic gene expression from one another Finally, and most importantly, established AcA101 and AcE60 cell lines have the ability to convert from an undifferentiated stage into the differentiated one analogous

to zona fasciculata-like cells

Because the ts T-antigen transgene did not affect cytogenesis and zonal differentiation of the adrenocortical cells of the mice, the adrenocortical cells in vivo were likely to

be in a normal pathway of their differentiation It is unknown whether the ts T-antigen is active in the adrenals in vivo However, we have previously noted that amounts of the ts T-antigen protein in other cells, which were obtained

by transfection, were decreased markedly at 37 °C when compared with amounts of wild-type T-antigen at 37 °C It

is presumable that levels of the ts T-antigen protein are very low at body temperature At 33 °C, on the other hand, the capability of the established cell lines to grow over PDL 200, appeared to be a result of T-antigen activation Being consistent with the view, inactivation of the T-antigen by culturing at the nonpermissive temperature reduced their growth rates Thus, the cell lines established under the permissive conditions for the T-antigen could be returned to

Fig 8 Analysis of steroid production Cells were cultured at 33 or

39 °C in the absence or presence of 1 m M Bt 2 cAMP for 4 days A

water-soluble form of cholesterol (20 l M ) was added at 24 h before

removal of the medium Dichloromethane extracts of the incubated

medium were prepared and were analyzed by reversed phase HPLC as

described in Materials and methods AcA101 cells were cultured at

33 °C in the absence (A) or presence (B) of Bt 2 cAMP and at 39 °C in

the absence (C) or presence (D) of Bt 2 cAMP Dichloromethane extract

of the medium of AcA201 cells, which were incubated at 39 °C for

4 days in the presence of 1 m M Bt 2 cAMP (E), was treated with

cho-lesterol oxidase to convert pregnenolone into progesterone (F), and

was analyzed by HPLC Peaks other than those corresponding to

deoxycorticosterone and progesterone are unidenti®ed compounds

which are also extracted from medium without incubation of the cells.

D, deoxycorticosterone P, progesterone.