THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc

VOl. 264, No. 1, h u e of January 5, pp. 266-271,1989 Printed in U. S. A.

Transcriptional Regulation of a-Fetoprotein Expression by Dexamethasone in Human Hepatoma Cells*

(Received for publication, July 11,1988)

Hidekazu Nakabayashi, Kazutada WatanabeS, Akira Saitos, Akira Otsurull, Kazuyuki Sawadaishill , and Taiki Tamaoki** From the Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1

The level of a-fetoprotein (AFP) mRNA in HUH-7 human hepatoma cells is elevated by the addition of dexamethasone to the culture medium. To locate the DNA region involved in hormonal regulation of the AFP gene, we constructed recombinant plasmids in which various lengths of the 5”flanking sequence of the human AFP gene were fused to the CAT gene. Various cell lines were transfected with the recombi- nant plasmids, incubated with or without 3 X lo-’ M dexamethasone, and then assayed for chloramphenicol acetyltransferase expression. In hepatoma cells that produce AFP, the dexamethasone treatment resulted in the stimulated chloramphenicol acetyltransferase expression when the transfected plasmids contained 169 base pairs (bp) or longer AFP 5”flanking se- quence. No dexamethasone effect was observed when the 5”flanking sequence was less than 98 bp long. The dexamethasone stimulation was effectively suppressed by the glucocorticoid antagonist RU486, indicating that this effect is mediated by glucocorticoid receptors. The 71-bp region between positions -169 and -98 contains a nucleotide stretch which is similar to the consensus sequence of the glucocorticoid responsive element (GRE). Partial alterations of this sequence resulted in decreased dexamethasone response. The GRE-containing region stimulated heterologous (SV40) promoter activity in response to dexametha- sone treatment in an orientation- and position-inde- pendent manner. The GRE and the upstream AFP en- hancer function independently from each other.

a-Fetoprotein (AFP)’ is a major serum protein during the fetal stage, but it is hardly detectable in adult life (1-3). The AFP expression during development is under the control of several hormones, most notably glucocorticoids (4). It has

* This work was supported by the National Cancer Institute of Canada and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Present address: Dept. of Biochemistry, Metropolitan Institute of Gerontology, Tokyo, Japan.

Present address: National Institute of Agrobiological Resources, Tsukuba Science City, Yatabe, Ibaragi, Japan.

11 Present address: First Dept. of Internal Medicine, Nagasaki University, Nagasaki, Japan.

11 Present address: Dept. of Biotechnology, Kansai New Technol- ogy Research Institute, Torishima, Osaka, Japan.

** Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada. To whom reprint requests should be addressed.

The abbreviations used are: AFP, a-fetoprotein; GRE, glucocor- ticoid responsive element; bp, base pairs; kb, kilobase pairs.

been shown that the administration of dexamethasone to rodent neonates decreases AFP expression at both protein and mRNA levels in the liver (5, 6).

Modulation of AFP gene expression by dexamethasone has also been observed in cultured hepatoma cells. In the case of rat hepatoma cultures, dexamethasone treatment results in either an increase or a decrease in AFP production. For example, dexamethasone suppresses the secretion of AFP and the level of AFP mRNA in the 7777 hepatoma (7-9), whereas increases in the level of both AFP secretion and AFP mRNA concentration have been observed in the McA-RH8994 hep- atoma (10, 11). Increased secretion of AFP is also reported with the H4-II-E-C3-V hepatoma (12), whereas no change in AFP production has been observed in the AH-66 hepatoma (13). In the case of HUH-7 and five other human hepatoma cells, dexamethasone treatment has invariably resulted in increased secretion of AFP (14). The reason why both stim- ulatory and suppressive effects are observed with the rat hepatoma cell lines, whereas only the stimulatory effect was obtained in the human hepatomas, is not known at present. In either case, available evidence shows that dexamethasone regulates the AFP gene primarily at the level of transcription.

Transcriptional regulation by steroid hormones is thought to be mediated by initial binding of the steroid to its receptor followed by specific interaction of the hormone-receptor com- plex with a DNA element, termed “glucocorticoid responsive element” (GRE) (15). In this investigation, we used transient transfection analysis to detect a DNA region upstream of the human AFP gene that mediates positive transcriptional reg- ulation by dexamethasone in human hepatoma cells. We report here the localization and characterization of an active GRE that is present in a 71-bp region in the human AFP gene promoter.

MATERIALS AND METHODS

Cell Cultures-Human hepatoma cell lines, HUH-7 (14, 161, PLC/ PRF/5 (17), and Hep3B (18) were cultured in a chemically defined medium, IS-RPMI (19). HepG2 (18) was subcultured in IS-PRMI containing 1% fetal calf serum for 2 days and then maintained in IS- RPMI without serum. HeLa, BM314 (human colon carcinoma, a gift from Dr. A. Yachi, Sapporo Medical College, Sapporo, Japan), and Ltk- were cultured in RPMI-1640 supplemented with 5% fetal calf serum.

Northern BlotAnulysis-Total RNA was isolated from cell cultures using the guanidinium isothiocyanate procedure (20). Northern blot analysis of AFP mRNA was conducted according to Thomas (21) using 3ZP-labeled AFP cDNA, pHAF-2 (22), as a probe. The amount of hybrids was quantified by densitometric scanning of the autoradi- ograms.

Construction of CAT Fusion Genes-The structures of CAT fusion plasmids used in this work are shown in Fig. 2. They are designated according to the length (kb) of the AFP 5”flanking DNA; for in- stance, pAF0.4-CAT and pAF7.5-CAT contained 0.4 and 7.5 kb of the 5”flanking DNA, respectively. The fusion plasmids containing

266

Hormonal Regulation of the Human a-Fetoprotein Gene 267

0.4-7.5 kb of the human AFP 5'-flanking sequence are described previously (23). To construct the fusion plasmids containing 0.17 kb of the human AFP 5'-flanking sequence (pAFO.l7(Hi)-CAT, pAFO.l7(Bg)-CAT), the ClaI site located 6 bp upstream of the 5' end of the 1-kb AFP flanking sequence in pAF1.O-CAT was converted to a BglII site by the attachment of a synthetic linker. The resultant plasmid is called pAFl.O(Bg)-CAT. This was digested with HindIII to release the 980-bp (positions -951 to +29) AFP 5"flanking se- quence. The remaining plasmid is called pBR(Bg)-CAT. The 980-bp fragment was digested with SstI to release the 198-bp fragment (positions -169 to +29). This was treated with mung bean nuclease, and the HindIII linker was attached and inserted at the HindIII site of pBR(Bg)-CAT to form pAFO.l7(Hi)-CAT.

To construct pAFO.l7(Bg)-CAT, pAFl.O(Bg)-CAT was digested with SstI, followed by mung bean nuclease, and then the BglII linker was attached to it. The DNA was then digested with BgZII to remove the 788-bp BglIIISstI fragment, and the remaining DNA was ligated to form pBRO.l7(Bg)-CAT. To construct pAFO.l(Bg)-CAT, pAFO.l7(Hi)-CAT was digested with S a d , converted to blunt ends by the treatment with the large fragment of DNA polymerase I, and the BglII linker was attached. This DNA was digested with BglII to remove the 77-bp BglIIISauI fragment, and the remaining DNA fragment was ligated.

To construct the AFP/SV40 early promoter/CAT fusion plasmids, the 198-bp DNA from positions -169 to +29 was converted to blunt ends by the large fragment of DNA polymerase I, the BglII linker was attached, and introduced to the BglII site of pSV1'-CAT (23) in normal (pSVAF0.17-CAT.) and reverse (pSVAF0.17[R]-CATa) ori- entations (Fig. 5A). The 198-bp fragment was also inserted to the BamHI site of pSV1'-CAT in the normal orientation to form pSVAF0.17-CATb (Fig. 5A).

Cell Transfection and Chloramphenicol Acetyltransferase Assays- Cells were transfected using the calcium phosphate precipitation method (24) according to Gorman et al. (25) with some modifications as described previously (23). Cells were grown in IS-RPMI and plated at a density of 5-7.5 X lo5 cells/75 cm2 flask 24 h prior to transfection. One hour before transfection, the medium was changed to fresh IS- RPMI supplemented with 10% fetal calf serum. The serum had been treated with charcoal and dextran to remove endogenous steroids (26). Each flask received 20 pg of DNA. Cells were incubated for 4 h at 37 "C, treated with 15% glycerol for 30 s, and rinsed with IS-RPMI medium. The cells were then incubated in IS-RPMI supplemented with or without 3 X M dexamethasone for 48 h and analyzed for chloramphenicol acetyltransferase activity as described previously (23). RU486 was dissolved in dimethyl sulfoxide and diluted with phosphate-buffered saline to make a stock solution of 7.5 X M. This was added to the culture following the glycerol shock at the final concentration of 3 X M.

RESULTS

Dexamethasone Increases AFP mRNA Concentration in HUH-7 Cells-Earlier reports show that dexamethasone treat- ment of HUH-7 cells results in an increase in the AFP con- centration in the medium (14). In this study we analyzed the effect of dexamethasone on the level of AFP mRNA. We incubated HUH-7 cells with 3 x M dexamethasone, ex- tracted RNA at various times, and analyzed AFP mRNA by Northern blot hybridization (Fig. L4). The amount of hybrids was quantified by densitometric tracing and presented as a function of time (Fig. 1B). The AFP mRNA concentration increased significantly within 12 h of incubation with dexa- methasone, reaching a plateau at 24-48 h with an overall increase of about 2.5-fold. In contrast, an increase in the AFP concentration in the medium was observed only after 42 h of dexamethasone treatment (14). Similar delays in the change of secreted protein levels have been reported in other systems (11, 27, 28), which likely reflect the time required for mRNA translation, protein processing, and secretion.

In agreement with a previous report (14), we found that the total number of cells was slightly lower (by about 10%) in dexamethasone-treated cultures than in the control cultures.

Delimitation of Glucocorticoid Responsive Regions Upstream of the AFP Gene-To locate upstream DNA regions that are

A 2 kb-

0 12 24 36 48 60 hours

B h 0

0 10 20 30 40 50 60

TI ME (hours) FIG. 1. Increase in AFP mRNA in HUH-7 cells on incubation

with dexamethasone. HUH-7 cells were incubated in the presence of 3 X M dexamethasone. Total RNA was isolated at indicated times and analyzed for AFP mRNA. A , Northern blot analysis. Hours indicate the duration of dexamethasone treatment. B, autoradiograms shown in A were quantified by densitometric scanning and plotted against the time of incubation with dexamethasone.

- 8 K b - 6 K b - 4 K b - 2 K b + 2 9 5 ' I I l~& I I I cap, 3 '

A 0

-E

pAF3.7-CAT E pAF5.1-CAT E pAF7.5-CAT

1. - - - -4 pAF3.5[A2]-CAT (CATI pAF2.9-CAT (CATI pAFl .E-CAT

pAF1.0-CAT e pAF0.4-CAT pAFO.l7(Hi)-CAT pAFO.l7(Bg)-CAT pAFO.l(Bg)-CAT

FIG. 2. Fusion genes bearing AFP 5"flanking sequences. Numbers on the top indicate the distance from the cap site of the human AFP gene. A and B indicate two AFP enhancer domains (23). Heavy solid lines indicate AFP 5'-flanking DNA. Broken lines indicate a deleted sequence.

involved in the dexamethasone responsiveness, we conducted transfection experiments using recombinant plasmids con- taining the CAT gene to which various sizes of the AFP 5'- flanking DNA were fused. Fig. 2 shows the structure of these fusion genes. They were introduced into HUH-7 cells by the calcium phosphate precipitation method. The transfected cells were incubated with or without 3 X M dexametha- sone for 48 h and then assayed for chloramphenicol acetyl- transferase activity. This concentration of dexamethasone has been shown to induce the highest level of AFP secretion (14). We also found in dose-response analysis that chloramphenicol acetyltransferase was stimulated maximally in the presence

268 Hormonal Regulation of the Human a-Fetoprotein Gene

of 3-6 X M dexamethasone (data not shown). The results of chloramphenicol acetyltransferase assays

showed that the dexamethasone treatment stimulated the chloramphenicol acetyltransferase expression in HUH-7 cells transfected with plasmids containing 0.17-7.5 kb of the AFP 5’-flanking DNA, but not 0.1 kb (Fig. 3, A and B ) . This indicates that sequences critical for dexamethasone respon- siveness reside in the 71-bp region between 98 and 169 bp upstream of the cap site of the AFP gene. pBR-CAT which lacks AFP DNA (23) exhibited no chloramphenicol acetyl- transferase activity either in the presence or absence of dex- amethasone (Fig. 3B).

Although the dexamethasone effect was observed with a wide range of sizes of 5”flanking DNA, the degree of stimu- lation of chloramphenicol acetyltransferase activity by dexa- methasone varied greatly depending on the size of the 5’- flanking sequence. This phenomenon will be analyzed in more detail below.

A

+3-Ac

C 1 -AC

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 - + - + - + - + - + - + ”“WL

7 . 5 5 . 1 3 . 7 3 . 5 A 2 . 9 1 . 8

B

+ ~ - A c

+ l - A c

The Active Region Contains GRE Homologous Sequence- The nucleotide sequence of the human AFP promoter region (29) is shown in Fig. 4A. In the region critical for the dexa- methasone responsive activity (positions -169 to -981, we found a TGTCCT sequence at positions -166 to -161 which

is identical with the consensus GRE hexamer, TGTdCT

(30, 31). The 15-bp sequence, AGAGCTCTGTGTCCT (po- sitions -175 to -161), that includes this hexamer and the adjacent upstream nucleotides, showed 81, 69, and 63% ho- mology to the GRE of rat AFP (32), human metallothionein IIA (33), and growth hormone (34), respectively (Fig. 4B). To assess the importance of the nucleotides other than the hex- amer for GRE activity, we altered six nucleotides at the 5’ end of the putative GRE by replacing them with the BglII or HindIII linker a t position -169. As shown in Fig. 4C, the BglII linker restores five of six original nucleotides, whereas the HindIII linker restores only two, without counting one nucleotide gap in both cases. The sequence modified with the BglII linker was found to retain 85% activity of the original sequence, whereas the sequence modified with the HindIII linker exhibited only 55% activity (Figs. 3A and 4C). These results are consistent with the report that the nucleotides 5’ to the consensus GRE hexamer play a role in dexamethasone responsiveness (35).

The Stimulation of Chloramphenicol Acetyltransferase Expression Is Mediated by Glucocorticoid Receptors-The an- tiglucocorticoid compound RU486 has been shown to strongly interact with the glucocorticoid receptor and to inhibit the action of dexamethasone (36). We examined whether RU486 affects the level of stimulation of chloramphenicol acetyl- transferase activity by dexamethasone. RU486 (3 X hi)

T

-140 -130 -120 -110

& x T A & A A T T A ~ ~ ~ G C ~ A A ~ C ~ ( ~

~ c T A A ~ ~ A ~ T ~ & ~ ~ ~ T A T . ~ ~ ~ A A -100 4 -90

sou I -80 -70

-60 -70 - f O

h A C T A G l T A A C A G 3 X ~ & ~

& m & m d -3‘

-30

-20 -10 tl

B h-AFP -175 A G A G C T C T G T G T C C T-161

r-AFP -175 A G T G G T C T T T G T C C T-161

h-MT -262 G G T A C A C T G T G T C C T-248 h-GH +93 G G C A C A A T G T G T C C T+107 CONCENSUS G G ~9~ c A% N N T G T 8 c T

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 - + - + - + - + - + - + ””” 1 . 0 0.4 0 . 1 7 ( H i ) 0 . 1 7 ( B g ) O . l ( B g ) pBR

FIG. 3. Effect of dexamethasone on chloramphenicol ace- tyltransferase expression directed by various lengths of AFP 5’-flanking DNA. HUH-7 cells were transfected with various fusion genes shown in Fig. 2, incubated with (3 X M) or without dexamethasone for 2 days, and analyzed for chloramphenicol acetyl- transferase activity as described under “Materials and Methods.” Cm, chloramphenicol; I-Ac, 1-acetate chloramphenicol; 3-Ac, 3-acetate chloramphenicol. + and - indicate the presence and absence of dexamethasone, respectively. A, lanes 1 and 2, pAF7.5-CAT; lanes 3 and 4 , pAF5.1-CAT; lunes 5 and 6, pAF3.7-CAT; lanes 7 and 8, pAF3.5[A2]-CAT; lanes 9 and 10, pAF2.9-CAT; and, lanes I 1 and 12, pAF1.8-CAT. B, lanes I and 2, pAF1.0-CAT; lanes 3 and 4 , pAF0.4- CAT, lanes 5 and 6, pAFO.l7(Hi)-CAT; lanes 7 and 8, pAHO.l7(Bg)- CAT; lanes 9 and I O , pAFO.l(Bg)-CAT; lanes 11 and 12, pBR-CAT.

C Dexamethasone

responsiveness

(%)

~ - A F P -175 A G A G c T I C T G T G T c c ~1.161 100 ... .. 85

H d n d I I I linker

FIG. 4. Nucleotide sequence and location of the human AFP GRE. A, nucleotide sequence of the promoter region of the human AFP gene. The sequence is taken from Sakai et al. (29). The cap site is numbered + I . The 15-bp stretches homologous to the GRE con- sensus sequence (30, 31) are underlined. Closed circles indicate the CCAAT pentamer. The TATA sequence is bored. B, comparison of the human AFP GRE with several other GREs. h-AFP, human AFP; r-AFP, rat AFP (32); h-MT, human metallothionein IIA (33); h-GH, human growth hormone (34). C, changes in sequence and activity of the human AFP GRE by the attachment of restriction site linkers. Closed circles indicate matched nucleotides.

Hormonal Regulation of the Human a-Fetoprotein Gene 269

by itself had no effect on chloramphenicol acetyltransferase expression in HUH-7 cells transfected with pAF1.O-CAT, confirming that RU486 lacks agonist activity (Fig. 5) (26, 36, 37). However, the addition of RU486 to HUH-7 cultures simultaneously with dexamethasone suppressed the stimula- tory effect of dexamethasone on chloramphenicol acetyltrans- ferase activity (Fig. 5). These results suggest that the stimu- lation of chloramphenicol acetyltransferase expression by dexamethasone is mediated by the glucocorticoid receptor.

Relationship between GRE and Enhancer-Typical GREs have been shown to be similar to classical enhancer elements,

- 3-Ac

c 1 - A ~

“ C m

D E X : - - + + RU486: - + + -

FIG. 5. Suppression of dexamethasone stimulation of chlor- amphenicol acetyltransferase activity by RU486. Transfection of HUH-7 cells with pAF1.O-CAT and analysis of chloramphenicol acetyltransferase activity were performed as described in the legend of Fig. 3. + and - indicate the presence and absence of dexamethasone (3 X lo-‘ M ) or RU486 (3 X lo-‘ M), respectively. Cm, 1-Ac, and 3- Ac are as described in the legend of Fig. 3. Dex, dexamethasone.

functioning independently of the orientation and location relative to the gene to be acted on (38). To test whether the AFP GRE behaves in a similar manner, we inserted the 198- bp region from positions -169 to +29 of the AFP gene 5’ to the CAT gene in pSVl’-CAT in normal and reverse orienta- tions (Fig. 6A) . In another construct, the 198-bp DNA was inserted 3‘ to the CAT gene in normal orientation (Fig. 6A) . All these CAT plasmids expressed high chloramphenicol ace- tyltransferase activity in response to dexamethasone treat- ment (Fig. 6B). Neither pSV2-CAT (25) nor pSVl’-CAT which lacks the AFP DNA responded to dexamethasone (Fig. 6B).

To examine the relationship between the AFP GRE and the upstream enhancer elements, we analyzed the level of stimulation of chloramphenicol acetyltransferase activity by dexamethasone as a function of the size of the AFP 5’- flanking DNA in the fusion genes (Fig. 7). In the absence of dexamethasone, the chloramphenicol acetyltransferase activ- ity was low (about 1 pmol/h/mg protein) with 5”flanking DNA fragments up to 2.9 kb long (without the enhancer). Dexamethasone treatment increased the chloramphenicol acetyltransferase expression 10- to 15-fold. When the size of the 5”flanking DNA increased to 3.5 and 3.7 kb to contain domain B of the AFP enhancer a higher level of chloram- phenicol acetyltransferase expression (about 8 pmol/h/mg protein) was obtained in the absence of dexamethasone. This activity was further increased 9- to 10-fold in the presence of dexamethasone. When the size of the AFP 5’-flanking DNA increased to 5.1 and 7.5 kb to contain domain A as well as domain B of the AFP enhancer (23), the highest level of chloramphenicol acetyltransferase activity (about 60 pmol/h/ mg protein) was obtained in the absence of dexamethasone, but the dexamethasone treatment resulted in only a 2- to 3- fold increase in chloramphenicol acetyltransferase activity. These results show that the GRE and enhancer elements act

A - 1 6 9 + 2 9

+ 2 9 - - pSVAF0.17-CATa ,-f9 pSVAFO.l7[R]-CATa

Amp‘

- 1 6 9

pSVAFO.l7[R]-cATb - r 2 9

+

B

+ 3 3 - A ~

+ 1-AC

+ Cm

1 2 - + + s v 2

3 4 5 6 7 8 9 1 0 - + - + - + - + ”” 0.17a 0.17[R]a 0.17[R]b SV1’

FIG. 6. Effect of dexamethasone on chloramphenicol acetyltransferase expression from fusion genes containing a 169-bp AFP 5”flanking sequence and the SV40 early promoter. A, construction of AFP/ SV40 promoter/CAT fusion plasmids. The 198-bp SstI/HindIII fragment between -169 and +29 bp of the AFP gene was inserted at the BglII site or the BumHI site of pSV1’-CAT. B, HUH-7 cells were transfected with the CAT fusion plasmids described in A, incubated with (3 X lo-‘ M) or without dexamethasone, and then analyzed for chloramphenicol acetyltransferase activity. Cm, 1-Ac, and 3-Ac are as described in the legend of Fig. 3. + and - indicate the presence and absence of dexamethasone, respectively. Lanes 1 and 2, pSV2-CAT; lanes 3 and 4 , pSVAF0.17-CAT.; lunes 5 and 6, pSVAF0.17[R]-CATa; lunes 7 and 8, pSVAFO.l7[R]-CATb; lunes 9 and 10, pSV1’- CAT

270 Hormonal Regulation of the Human a-Fetoprotein Gene

I I 1 I I I I I 0 1 2 3 4 5 6 7

5"FLANKING SEQUENCE (kb) FIG. 7 . Relationship between dexamethasone stimulation of

chloramphenicol acetyltransferase activity and the length of AFP 5'-flanking DNA. Chloramphenicol acetyltransferase activi- ties in HUH-7 cells transfected with various fusion genes shown in Fig. 2 in the presence (3 X M) and absence of dexamethasone are plotted against the size of AFP 5'-flanking DNA. 0, without dexa- methasone; 0, with dexamethasone.

A B C D E F

L S - A C

' f l - A c

F F F F F F F F S F F S F F S - + - + - + - + - - + - - + -

FIG. 8. Expression of the CAT gene from pAF1.O-CAT in various cell lines with or without dexamethasone. Various cell lines described below were transfected with pAF1.O-CAT or pSV2- CAT in the presence (3 X 1O"j M) and absence of dexamethasone. Cm, 1-Ac, and 3-Ac are as described in the legend of Fig. 3. The amounts of cell extracts and the incubation times used in chloram- phenicol acetyltransferase assays were as follows: A, HepG2: 200 pg of protein, 120 min; B, Hep3B: 150 pg of protein, 60 min; C, PLC/ PRF/5: 200 pg of protein, 60 min; D, BM314: 400 pg of protein, 90 min; E, Ltk-: 200 pg of protein, 135 min; F, HeLa: 1 mg of protein, 230 min. + and - indicate the presence and absence of dexametha- sone, respectively. Lane F , pAF1.O-CAT; lune S, pSV2-CAT.

independently and that the stimulatory effect of dexametha- sone is greater in the absence of the upstream enhancer than its presence.

Dexamethasone Effect Is Cell Type-specific-To examine whether the effect of dexamethasone is dependent on cell type, we transfected pAF1.O-CAT into different cell lines which produce or do not produce AFP. In three human hepatoma cell lines which produce AFP (Hep3B, HepG2, PLC/PRF/5), dexamethasone treatment resulted in 4- to 12- fold enhancement of chloramphenicol acetyltransferase activ- ity (Fig. 8). On the other hand, three cell lines which do not produce AFP (Ltk-, BM314, HeLa) expressed no chloram- phenicol acetyltransferase activity in the presence or absence of dexamethasone.

DISCUSSION

Transient transfection analysis has proved to be of use in defining various cis-acting regulatory regions, including hor- mone responsive elements (28,39,40). In this study, we show that the stimulation of AFP expression by dexamethasone in HUH-7 human hepatoma cells was mediated by the 71-bp region from 98 to 169 bp upstream of the human AFP gene. This stimulation was not due to general effects on cell growth since the total number of cells in the presence of dexameth- asone was either similar to or only slightly lower than the control cultures (14). The dexamethasone stimulation was effectively suppressed by the glucocorticoid antagonist RU486, indicating that the dexamethasone effect is mediated by glucocorticoid receptors. The location of AFP GRE is similar to that of the chicken lysozyme gene (41), the bovine prolactin gene (42), human metallothionein-IIA gene (33), and several viral genes (15). The location of several other GREs has been shown to vary greatly, from 3 kb upstream of the rabbit uteroglobin (43) and rat tyrosin aminotransferase (31) genes to 100 bp downstream of the human growth hor- mone gene (see 33).

The 71-bp region from positions -169 to -98 of the human AFP gene that stimulates chloramphenicol acetyltransferase expression contains TGTCCT at positions -166 to -161. This sequence is identical with the six nucleotides at the 3' end of the GRE consensus sequence. This hexamer is well conserved among known GREs and shown to be essential for mediating dexamethasone effects (30). In addition, several nucleotides immediately upstream of this sequence have been shown to play a role in dexamethasone responsiveness (35). Thus, we observed that a change of four out of six nucleotides in this region (without counting one gap) resulted in a much greater reduction of dexamethasone stimulation than a change of one nucleotide (Fig. 4C). Strahle et al. (35) have shown that a 15-bp sequence with partial symmetry is suffi- cient to confer glucocorticoid inducibility on a heterologous promoter. Our results are compatible with this conclusion and strongly suggest that the sequence, AGAGCTCTGTGTCCT (positions -176 to -161), is in fact an AFP GRE. This conclusion was further supported by the observation that the AFP GRE region was able to activate a heterologous (SV40) promoter in response to dexamethasone in an orientation- and position-independent manner, thus behaving like classi- cal enhancers (15, 38).

Chloramphenicol acetyltransferase expression from the fu- sion genes with or without dexamethasone was seen in AFP- producing cells, but not in non-AFP-producing cells. The lack of dexamethasone response in non-AFP-producing cells is not due to lack of glucocorticoid receptors in these cells. More likely, the AFP promoter functions in a cell-specific manner, and the GRE cannot modulate a nonfunctioning promoter (15).

We have previously identified a typical enhancer element of the human AFP gene between -3.7 and -3.3 kb (domain B), but the maximum transcriptional enhancement is ob- tained together with an upstream region from -4.9 to -3.7 kb (domain A) (23). Analysis of the relationship of these upstream enhancer elements to the GRE showed that the enhancer and the GRE function independently. Thus, dexa- methasone stimulation occurred both in the presence and absence of the enhancer and the effect of the enhancer was seen both in the presence and absence of dexamethasone. However, the magnitude of dexamethasone effect depended on the basal level of expression. Thus, the highest dexameth- asone stimulation (10-fold or more) was obtained in the absence of the enhancer and the lowest stimulation (2- to 3-

Hormonal Regulation of the Human a-Fetoprotein Gene 271

fold) was obtained in the presence of the full complement of enhancer elements. It must be noted that although the mag- nitude of dexamethasone effect was greater in the absence of the enhancer, the overall level of chloramphenicol acetyl- transferase activity was much lower (less than one-tenth) in the absence of the enhancer than in its presence. This is because the effect of the enhancer (approximately 150-fold stimulation) is much greater than that of dexamethasone (approximately 10-fold stimulation). It is tempting to specu- late that the enhancer plays the major role in AFP gene expression during development (44), whereas the GRE mod- ulates AFP gene activity in adult life.

Dexamethasone treatment stimulates AFP synthesis in all human hepatoma cell lines so far examined (14), whereas either positive or negative response has been observed in several rat hepatoma cell lines (see Introduction). Guertin et al. (32) have recently reported that the region between posi- tions -202 and -121 of the rat AFP gene binds to the glucocorticoid receptor and represses chloramphenicol acetyl- transferase expression in the presence of dexamethasone. This region contains a sequence (positions -175 to -161) similar to the human AFP GRE (Fig. 4). This suggests that the same GRE element may mediate both stimulatory and suppressive effects of dexamethasone on AFP expression in rat hepatomas. It is not clear at present why no down regu- lation of the AFP gene by dexamethasone has been observed in human hepatoma cells.

Acknowledgments-We wish to thank Dr. R. Deraedt for the supply of RU486, and Richard Kennedy and Wendy Matsumoto for excellent technical assistance.

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