Glucocorticoid, androgen, and retinoic acid regulation of glutathione S-transferase gene expression in hamster smooth muscle tumor cells

Vol. 184, No. 2, 1992

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April 30, 1992

Pages

GLUCOCORTICOID,

ANDROGEN,

GLUTATHIONE

AND RETINOIC

S-TRANSFERASE

HAMSTER

SMOOTH

MUSCLE

ACID

GENE

REGULATION

EXPRESSION

TUMOR

1108-1113

OF

IN

CELLS

David A. Schwartz’ and James S. Norri~*‘~ ‘Division

of Rheumatology and Immunology, Department of Medicine *Department of Biochemistry and Molecular Biology Medical University of South Carolina 171 Ashley Avenue, Charleston, SC 29425

Received March 24, 1992

A mu class glutathione S-transferase gene (hGSTYBX) is expressed in the DDT, MF-2 hamster smooth muscle tumor cell line. This gene is glucocorticoid responsive, and near maximal induction was found to occur within 24 h. The induced mRNA was very stable with a half-life of more than 48 h. Serum had no effect on either constitutive or glucocorticoid induced hGSTYBX expression. Although dibutyryl CAMP, phenobarbital, and 12-Otetradecanoylphorbol-13-acetate did not alter hGSTYBX expression, testosterone and retinoic acid were each able to increase hGSTYBX expression in a concentration dependent manner. These results demonstrate a unique pattern of responsiveness of the hamster gene compared to the glutathione S-transferase genes of other species. 0 1992 Academic Press, 1°C.

Glutathione

S-transferase (GST, EC 2.5.1.18) is encoded by a family of multi-locus

genes and catalyzes the conjugation Themultiple

of hydrophobic compounds with reduced glutathione

(1).

isoforms of this enzyme can be generally grouped into alpha (basic), mu (neutral),

and pi (acidic) classes (2). Alterations pathophysiological

in GST activity have been correlated with certain

states. For example, lack of mu class GST activity is associated with an

increased susceptibility

to lung cancer (3,4). In contrast, increased GST activity is associatecl

with acquired drug resistance during cancer chemotherapy

(5.6). Thus, delineation

of factors

that can regulate GST activity could lead to improved cancer treatment protocols or reduced frequency of carcinogenesis. We have cloned a glucocorticoid leiomyosarcoma

responsive mu class GST (7) from cultured

cells derived from an androgen and estrogen treated Syrian hamster, and have

found its constitutive expression to be reduced in proliferating 000&291X/92$1.50 Copyright All rights

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

1108

cells as compared to confluent

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cells. We have hypothesized androgen administration proliferate,

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that during steroid induced carcinogenesis in the hamster,

stimulates subpopulations

of cells in susceptible target tissues to

thus reducing GST expression (8). As a result, we further hypothesize that

carcinogenic quinone metabolites

of estrogen (9, lo), which are potential GST substrates (2),

can accumulate and lead to genotoxic events. Therefore, in order to better understand the potential

involvement

of altered expression of this GST during carcinogenesis, we have

continued characterization

of the regulation of its expression.

MATERIALS

AND METHODS

DDT, MF-2 cells (11) were cultured in Dulbecco’s Modified Eagle’s/F-12 medium (Gibco) containing penicillin, streptomycin, and amphotericin B. As noted in the figure legends, cultures in tissue culture-treated plastic dishes (Falcon) were supplemented with 5% bovine calf senm (Defined BCS, Hyclone); cultures in rat tail collagen type I-precoated dishes (Collaborative Research) were grown in the presence or absence of insulin, transferrin, and selenium (ITS, Collaborative Research). Triamcinolone acetonide (TA), phenobarbital (PB), dibutyryl CAMP (dbcAMP), 12-0-tetradecanoylphorbol-13-acetate (TPA), testosterone (T), retinoic acid (RA) (all from Sigma Chemical), or vehicle (ethanol, final concentration was 0.05%) were added as indicated. A emction and Northern blot analysis Total RNA was isolated and used for analysis of Syrian hamster mu class GST (hGSTYBX) and fi actin expression as described (7,121. Relative mRNA levels were quantitated by densitometry of the autoradiograms. RESULTS In DDT, MF-2 cells treated with 1Oe7M triamcinolone levels were increased within 4 h, reached near maximal remained elevated with continued approximately

steroid treatment

acetonide, hGSTYBX

mRNA

levels within 24 h, and thereafter (Fig. I).

Maximal

induction

was

4-fold. Following steroid removal from the cultures, the increased mRNA level

continued for some time (Fig. 2); we estimate that its half-life was greater than 48 h. To examine the potential hGSTYBX

relationship

between DDT, MF-2 cell proliferation

expression, cells were cultured with or without ITS in collagen-coated

and dishes

(collagen is required for cell attachment to the dish in the absence of serum). In the presence of ITS the population

doubling time was approximately

34 h, whereas in the absence of ITS

the doubling time was about 57 h. The results in Fig. 3 show that the cells proliferating two different rates each contained low, yet similar, constitutive levels of hGSTYBX 1109

at the mRNA,

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BIOCHEMICAL

HOURS 0

4

AND BIOPHYSICAL RESEARCH COMMUNICATIONS HOURS AFTER TA REMOVAL

OF TA TREATMENT 8

12

24

48

C

72

hGSTY

TA

24

48

BX

f3 Actin

02

EigJ. Tie courseof triamcinoloneacetonide(TA) induction of hGSTYBX expression. DDT, MF-2 cellswereseededat approximately IO3cells/cm*into untreateddishesin mediumcontaining 5%BCS. After 3 daysof growth, the subconfluentcellsweretreatedwith 1o”M TA. At the indicatedtime points, cellswereharvested,total RNA wasextracted, and 5 pg of total RNA wasusedfor Northern blot analysis. w

Stability of glucocorticoidinducedhGSTYBX mRNA after steroidremoval. Subconfluent DDT, MF-2 cellscultured for 1 day in untreated dishescontaining mediumwith 5% BCS weretreated with vehicle (C) or 10.‘M TA for 24 hours. C and TA treatedcultureswereharvestedat this time; other cultures(all TA treated) wererinsedtwice (5 min eachrinse)by incubation at 37°Cwith freshmediumlacking TA. Incubation in fresh mediumcontinuedfor theindicatedtimes,after whichtotal RNA wasextractedand 10pg was usedfor Northern blot analysis.

A

PHENOBARBITAL c

C

TA

C

TA

-

-

+

+

0.1

(mM) 0.5

4

hGSTYBX ITS

hGSTYBX

I”

B

dbcAMP c

03

p Actin

0 4

0.1

(mM) 0.3

TPA 1

1o-‘O

(M) 1o-9

10-e

hGSTYBX

Eig3, Effect of insulin, transfer& and selenium(ITS) supplementationon constitutive and glucocorticoid inducedhGSTYBX expression. SubconfluentDDT, MF-2 cellsculturedfor 3 daysin collagencoateddishescontaining eithermediumwith no supplements (-) or mediumwith ITS (+) wererefedwith their respective media and either vehicle (C) or 1O”M triamcinoloneacetonide(TA) and cultured for an additional 24 hours. Cells werethen harvestedand 5 tugof total RNA wasanalyzed by Northern blotting. Fig. 4 Lack of effect of phenobarbital (PB), dibutyryl CAMP (dbcAMP), and 12-0tetradecanoylphorbol-13-acetate (TPA) on hGSTYBX expression. Subconfluent DDT, MF-2 cellscultured for 3 daysin untreateddisheswith 5% BCS wererefed with fresh mediumand either vehicle(C) or the indicatedconcentrationsof PB (PanelA), bdcAMP (PanelB), or TPA (PanelB). Cellswereharvested24 hours later, and bromide skning 10 pg of total RNA was subjectedto Northern blot analysis. Ethidium equalamountsof RNA in all gel lanes. showed 1110

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and much less than steroid treated cells. Thus, glucocorticoid was not likely due simply to the reduced cell proliferation addition,

hGSTYBX

induced hGSTYBX

expression

rate effected by the steroid (11). In

expression was equally inducible by the steroid in cells grown in the

presence or absence of ITS. We tested the ability of other agents known to affect GSTlevels in other species to alter hGSTYBX mRNA

expression. Dibutyryl

CAMP, phenobarbital,

and TPA did not affect hGSTYBX

levels (Fig. 4). In contrast, testosterone and retinoic acid each increased hGSTYBX

expression by about 2-fold in a concentration

dependent manner (Fig. 5).

DISCUSSION

Previously, we have shown that glucocorticoid

induced hGSTYBX

MF-2 cells is due in part to a modest increase in its transcription hGSTYBX

mRNA demonstrated

increase in transcription

expression in DDT,

rate (7). The stability of the

in Fig. 2 is therefore consistent with the idea that the small

is sufficient to lead to the appreciable hGSTYBX

mRNA steady state

levels shown in Fig. 1. The hGSTYBX

glucocorticoid

by an as yet unidentified

glucocorticoid

another secondary glucocorticoid

response is a secondary response, presumably mediated induced transcriptional

activator (7). In contrast to

response gene, rat a2u globulin (13). whose glucocorticoiJ

induction requires the presence of serum (14), induction of hGSTYBX occurred in the absence of growth promoting

medium supplements (Fig. 3).

A

TESTOSTERONE c

hGSTY BX

(M)

RETINOIC c

1u9 1o-8 10.’ -w

B

lo-8

ACID 10.’

(M) IO”

;odr

f3 Actin

Fig. 5 Effect of testosteroneand retinoic acid on hGSTYBX expression. SubconfluentDDT, MF-2 cellscultured for 3 daysin collagencoateddisheswith ITS (PanelA) or in untreateddisheswith So/u BCS (PanelB) wererefedwith their respectivemedia

and either vehicle (C) or the indicated concentration of testosterone (Panel A) or retinoic acid (PanelB). After 24 hoursof treatment,cellswereharvestedand5 ug (PanelA) or 10ug (Panel B) of total RNA wasusedfor Northern blot analysis.

1111

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Our data indicate that the mu class GST expressed in hamster leiomyosarcoma regulated differently than other GSTspreviously

described. Forexample,

2 cells in medium devoid ofexogenous growth promoting cell proliferation

cells is

culturing DDT, MF-

factors resulted in a reduced rate of

without increased basal expression ofhGSTYBX

(Fig. 3). Incontrast, serum-

starved mouse NIH 3T3 cells express GST mRNA at higher levels than serum stimulated cells (15). Rat liver alpha, mu, and pi class GST mRNAs with phenobarbital

(16,17,18), whereas hGSTYBX

with up to 4mM phenobarbital

are each inducible by in vivo treatment expression was not altered by treatment

(Fig. 4). A rat pi class GST gene has been shown to contain

two imperfect TPA response elements (TREs) that together act as a transcriptional (19,20). TPA at up to 10.*M had no effect on hGSTYBX 4). We have recently cloned the hGSTYBX it contains two TRE-like

enhancer

expression in DDT, MF-2 cells (Fig.

gene and its 5’ flanking region (21) and although

elements, they are separated by 24 nucleotides.

This spacing may

render them inactive since increasing the distance between the TREs in the rat pi GST gene greatly reduces their activity (20). The hamster hGSTYBX 5A).

gene was modestly responsive to testosterone treatment (Fig.

The response to this hormone

immunologically

resembles the regulation

of a mouse liver GST,

similar to rat liver pi class GST, that has been shown to increase during

puberty in male mice (22). Castration of males greatly reduces GST enzyme levels, and testosterone treatment offemalesincreases their GSTenzyme levels to thosein untreated males. Theseresultssuggest that testosterone may transcriptionally

activate expression of this specific

mouse liver GST. We also found that retinoic acid induced hGSTYBX

expression in a concentration

dependent manner (Fig. 5B), suggesting that this may be a receptor mediated event. Indeed. the 5’ flanking region of the hGSTYBX

gene (21) contains a DNA sequence similar to known

retinoic acid response elements (AGAGTTCTC, a less direct manner, perhaps by regulating

23). Alternatively.

retinoic acid m:ry act ii1

proteins that interact with :I i~elir-loop-il~ll~~

regulatory domain also present in the 5’ tlankin, 0 region of hGSTYBX glucocorticoid hGSTYBX

and known to be

responsive (21). Further experiments may determine whether these or other

gene sequences are involved in the response to retinoic acid. ACKNOWLEDGMENTS

Supported by NIH CA49949, CA52085, and the Health Science Foundation. 1112

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REFERENCES 1. 2. 3. 4. 5. 6. 7.

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19. 20. 21. 22. 23.

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