Transcriptional activation of c-myc proto-oncogene by estrogen in human ovarian cancer cells

Moleixlur and Crlldar Endocrinology, Elsevier Science Ireland. Ltd.

99 ( I YY4) 1 1- I9

II

MCE 03122

Transcriptional

activation

of c-myc proto-oncogene cancer cells

by estrogen

in human ovarian

Chin-Hsiang Chien *.a, Fung-Fang Wang ” and Thomas C. Hamilton ’ ‘I Drpuriment

and Institute of Biochemistry, Nutionul Yung-Ming MedIcal College, Shih Pui, Tuiper, Tuwun ” Deprtrtm~nt of Mt&rtl Oncology, Fox Chuse Cunwr Center. Philadelphru 19111, USA (Received

kiy ~uor&

c-myc: Estrogen;

Ovarian

cancer;

Antisense

2 February

c-my

lYY3; accepted

6 September

11221.

lYY3)

oligonucleotide

Summary NIH : OVCAR-3 is a human ovarian cancer cell line that expresses a moderate amount of estrogen receptors, 28 fmol/mg protein. We have found that estrogen at a concentration of lo-’ M induced a 2.3-fold increase in the growth of NIH : OVCAR-3 cells after 48 h stimulation. A 4-fold increase in c-myc mRNA expression at 30 min and a 7.5fold increase at 50 min post-induction with estradiol were observed. Nuclear run-on analysis indicated that c-myc transcripts increased 4-fold within 10 min of estrogen addition. The half-life of c-myc mRNA was 64 min f 5 min and was not affected by estrogen. Antisense oligonucleotide to c-myc specifically inhibited the estrogen stimulated c-myc protein expression as well as the growth of NIH : OVCAR-3 cells. A control ovarian cancer cell line OC-3-VGH that had few estrogen receptors (1 fmol/mg protein) did not respond to estrogen in growth; however, these cells respond to estrogen with a 1.5-fold increase in c-myc mRNA. The stability of c-myc mRNA of these cells was not affected by estrogen. Our results indicate that transcriptional induction of c-myc expression by estrogen plays a critical role in the proliferation of NIH : OVCAR-3 cells.

Introduction Epidemiology and clinical observations have implicated estrogen in the pathogenesis and growth regulation of carcinomas arising from ovary (Hildreth et al., 1981; Young et al., 1985). Experimental ovarian tumors could be induced by diethylstilbestrol (Jabara, 1962; Hoover et al., 1977). However, the relationship between estrogen and cancer initiation, growth regulation of ovarian surface epithelial cells (the normal progenitors from which most ovarian cancers arise) or growth of ovarian cancer cells remains largely unknown. The product of the c-myc proto-oncogene is a nuclear phosphoprotein (Cole, 1986) that contains three structural domains: a leucine zipper domain (Landschulz et al., 1988; Dang et al., 1989), a helix-loop-helix motif (Murre

* Corresponding author. Tel. (02) 8267121; Fax 886-02-8264843. Ahhrrc~iutions; ER, estrogen receptor; PR. progesterone receptor. 20 x SSC = 3 M Na, citrate, pH 7.0; 5 x SSPE = 0.75 M NaCI. 0.05 M NaH,PO,, 0.05 M EDTA, pH 7.4. SSDI 0303-7207(93)E0232-J

et al., 1989) and an adjacent domain rich in basic amino acids (Davis et al., 1990). c-myc has a nuclear targeting signal (Dang et al., 1989; Dan and Lee, 1988) and functions as a potential regulator of gene transcription (Eilers et al., 1991). The expression of the c-myc proto-oncogene is closely correlated with cell proliferation (Persson et al., 1984; Struzinski et al., 1986) and differentiation (Coppola and Cele, 1986; Lachmann and Skoultchi, 1984). A rapid increase in the expression of the c-myc gene has been observed in many quiescent cells after stimulation with platelet derived growth factor (Kelly et al., 19831, fibroblast growth factor, and epidermal growth factor (Kaibuchi et al., 1986; Ran et al., 1986; Murphy et al., 1987). Transient increase in the expression of c-myc gene by estrogen has been observed in a variety of estrogenresponsive target tissues (Murphy et al., 1987; Renpel and Johnston, 1988; Dubik et al., 1987; Hvet-hudson et al., 1989). Despite extensive investigations in a variety of normal and neoplastic cells, the fundamental aspects of the kinetics and mechanisms underlying c-myc ex-

pression modulated by estrogen and the significance of its induction remain undetermined. It is therefore important to understand the role of c-myc expression and the mechanisms of its regulation in the estrogen-treated CCIIS. Two ovarian cancer ccl1 lines, NIH : OVCAR-3 with moderate levels of estrogen receptors, and OC-3-VGH with few estrogen receptors, have been employed to determine the relationship bctwecn receptor levels. cell proliferation, and estrogen inducibility of c-myc. Estrogen was found to increase the transcription of NIH : OVCAR-3 c-myc in the hormone-responsive cells. In OC-3-VGH ovarian cancer cells, estrogen was not a mitogen and although c-myc expression was induced hy estrogen, the effect was small compared to that found in NIH:OVCAR-3 cells. Furthcrmorc, antisensc to c-myc gene was shown to inhibit the estrogen induced growth of NIH : OVCAR-3 cells, suggesting a critical role of c-myc expression in the estrogen-stimulated cell proliferation. Materials

and methods

NIH: OVCAR-3 ovarian cancer cell line was obtained from NIH USA (Hamilton et al., 1983, 1984). OC-3-VGH ovarian cancer cell line (Chao et al., 1987) was obtained from Vertaran General Hospital, Taipei, Taiwan. The histology of two cell lines are characterized as serous adenocarcinoma. Antibodies used for Western blot analysis was a monoclonal antibody against human c-myc, obtained from Oncogene Science. NY. Goat anti-mouse biotinylated antibodies, strcptavidin-alkaline phosphatase, and 5-bromo-4chloro-3-indolylphosphate p-toluidine salt/nitroblue tetrazolium chloride were purchased from Bethesda Research Laboratories, MD. The antisense to c-myc oligonuclcotides sequence were (5’-AAC GTT GAG GGG CAT-.?‘) synthesised by Grow Biotechnology, Taipei, Taiwan. This sequence is 15 base pairs in length and corresponds to the translation-initiation region of exon 2 of the c-nzyc gene. Cell culture NIH : OVCAR-3 cells were cultured in RPM1 1640 medium (Gibco, Grand Island, NY) supplemented with insulin (10 pg/ml), penicillin (100 units/ml), streptomycin (100 pg/ml), 24 mM sodium bicarbonate and heat-inactivated fetal bovine serum (2% v/v>. At the beginning of each experiment, cells were replaced in phenol-red free medium (Berthois et al., 1986) containing 10% charcoal-stripped fetal calf serum (medium C), lo-“’ M tamoxifen (medium T) (Borgma and Scali, 1991), and/or lo-” M RU38 486 (Roussel Uclaf, medium TR) (Horwitz, 1985) for 24 or 48 h, 17 pestradiol (lo-’ M) was then added to release the cells from tamoxifen inhibition of growth.

Extraction of RNA and Northern blot anulysis Total RNA was extracted with guanidinium isothiocyanate and purified by ultracentrifugation through a cushion of cesium chloride (Setzer et al., 1980). The RNA pellet was then dissolved in TES buffer (10 mM Tris-HCI, pH 7.5, 5 mM NaEDTA, 1% SDS) at 25°C extracted with chloroform/n-butanol (4 : 1) and reprecipitated with ethanol. RNA was separated by ethidium-stained minigels and visualized under ultraviolet illumination to assess the integrity of RNA and quantitate the concentration of RNA to be loaded. Twenty pg of total RNA was denatured in 50% (v/v) formamide and fractionated in 1.5% agarosc/formaldehyde gel. RNA gel was transferred to nitrocellulose paper in 20 x SSC over a period of 24 h as described by Thomas (1980). Nitrocellulose filters were prehybridized for 2-12 h at 42°C with the solution containing 0.25 mg/ml sonicated and heat-denatured salmon sperm DNA, 50% (v/v) formamide. The hybridization was carried out for 24-36 h at 42°C in a solution containing 5 x SSPE, 5 X Denhardt’s, 0.1 mg/ml sonicated and heat-denatured salmon sperm DNA, 50% formamide and 4-7 X 10’ cpm “2P-labeled DNA probes (Feinberg and Vogelstein, 1982). Filters were washed twice with 2 x SSC. 0.1% SDS at 42°C for 30 min, twice with 0.5 X SSC, 0.1% SDS at 42°C for 30 min, twice with 0.5 x SSC, 0.1% SDS at room temperature for 15 min, and autoradiographed. Relative intensity of bands on autoradiogram were quantitated by laser densitometer (Biomed. Instrument, USA). c-myc mRNA stability analysis Actinomycin D at a final concentration of 5 pg/ml was added to the estrogen-stimulated cells for O-120 min. At the designated time interval, RNA was isolated, and the expression of c-myc mRNA was analyzed by Northern blot analysis. Nuclear run-on transcription assay Nuclei were isolated (Greenberg and Ziff, 1984) by incubating cells for 5 min on ice in lysis buffer (60 mM KCL, 0.5 M sucrose, 0.5 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 14 mM 2-mercaptoethanol, and 15 mM Hepes, pH 7.5) containing 0.5% Nonidet pluronic 40 detergent. The lysate was layered onto a cushion of 30% sucrose in lysis buffer and centrifuged at 3000 rpm for 10 min. The pellets containing nuclei were resuspended in storage buffer containg 50% glycerol, 5 mM MgCl,, 0.5 mM EDTA, 0.85 mM dithiothreitol, 100 U/ml RNasin, 20 mM Tris, pH 7.9. Freshly prepared nuclei (1 x 10’) were incubated in a 200-300 ~1 reaction mixture containing 200 PCi of [u-12P]UTP (760 Ci/mmol, 20 mCi/ml), 30 mM Tris-HCI at pH 8.0, 5 mM MgCl?, 4 mM MnCl,, 150 mM KCl, 0.05 mM Na EDTA, 1 mM DTT, 10 mM creatine phosphate, 20 units/ml RNasin and 1 mM each of ATP,

CTP, GTP at 30°C for 30 min (M&night and Palmiter, 1979). Labeled RNA was isolated by centrifugation through CsCl cushions, and resuspended in 10 mM Tris-HCI at pH 7.5 and 1 mM EDTA. Plasmids containing exon 2 and 3 of human c-myc gene were linearized with restriction enzyme. DNA was denatured by incubation with 0.2 M NaOH for 30 min at 25°C followed by neutralization with 10 volumes of 6 X SSC. 0.4 pg of denatured insert DNA was immobilized on nitrocellulose filter. The filters were prehybridized for 4 h at 37°C with the 5 x SSC, 5 x Denhardt’s mixture (1 X Denhardt’s mix: 0.1% each of BSA, polyvinylpyrrolidone, and Ficoll), 50 mM sodium phosphate (pH 7.0), 0.2% SDS and 50% formamide. Hybridization was carried out for 48-72 h at 37°C with 3 X SSC, 5 X Denhardt’s mix, 50 mM sodium phosphate (pH 7.0), 0.2% SDS, 50% formamide, 300 pg denaturated, sonicated herring sperm DNA and 2-6 x 10h cpm/ml j2P-labeled RNA. After hybridization, the filters were washed three times for 20 min in 1 X SSC at room temperature and incubated at 37°C in 1 X SSC with 10 mg/ml RNase A for 30 min. The filters were washed again in 1 X SSC and 0.2 X SSC at 50°C for 30 min, respectively. The signals were determined by autoradiography and densitometry. Two or 0.5 pg (Yamanitin was employed in the in vitro run-on assay system to inhibit the transcription reaction. Data were quantified by subtracting background hybridization to pBR322 from all values and corrected for slight variations in quantities in the amounts of [“PIRNA added to filters by normalization relative to the 28s rRNA signal. Rates of c-myc transcription were calculated relative to a constitutively expressed gene Pz-microglobulin by dividing the corrected c-myc value by the corrected /?!,-microglobulin value. Probes used The pMYC 7.4 c-myc probe containing the exon 2 to exon 3 of human c-myc gene (Watt et al., 1983) was used in Southern blot and nuclear run on analysis. A 0.5 kb cDNA probe corresponding to exon 2 of a human c-myc Pst I-PstI fragment was used in Northern analysis (Saito et al., 1983). All the estrogen receptor (Geen et al., 1986), P,-microglobulin (Suggs et al., 1981), pS2 (Jakowlew et al., 1984) and c-myc probes were obtained from ATCC, USA. c-myc protein detection For analyzing the time course of estrogen-stimulated c-myc protein expression, NIH : OVCAR-3 cells were cultured in phenol redfree medium at a density of 5 x 104/cm” in 6-well dishes. The medium was then replaced with T medium. After an additional 48 h, the cells were stimulated with estradiol (lo-’ MI. At various times, cells were lysed in sample-loading buffer with 5% mercaptoethanol. Proteins were separated by

a 10% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose paper for immunoblot analysis. The filters were then incubated at room temperature sequentially with a mouse monoclonal c-r?~~c antibody for 2 h. a goat anti-mouse biotinylated antibody for 90 min. and streptavidin-alkaline phosphatase for SO min, with 3 washes over 40 min in Tris-saline buffer between each step. Alkaline phosphatase activity was detected by incubating the filters with 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt/nitroblue tctrazolium chloride for 30 min. Effect of antisense c-myc DNA on c-myc proteirl expression and cell proliferation For the analysis of the effect of antisense oligonucleotide on c-myc expression, NIH : OVCAR-3 cells were plated in phenol red free RPM1 at a density of 5 X 101/cm2. After 48 h the medium was replaced by T medium, and oligonucleotide was added to a final concentration of 8 FM for 5 days. Estradiol (lO_’ M) was added thereafter without changing the medium. After 20 and 120 min, cells were lysed for c-myc immunoblot analysis. For the analysis of the effect on cell growth by antisense-c-myc, NIH : OVCAR-3 ceils were plated in medium T, at a density of 1 X lO”/cm’ in 96-well plates. After 48 h, antisense-c-myc oligonucleotide was added at a final concentration of 5 PM, together with lo-’ M estradiol. Cell growth was assessed by MTT assay (Alley, 1988). Results Estrogen receptor mRNA expression The expression of estrogen receptor mRNA as analyzed by Northern blot analysis of the two ovarian tumor cell lines is shown in Fig. 1. As can be seen, the expression of the 6.4 kb transcript correlated well with the levels of receptor protein expression in these two cell lines. The estrogen receptor of NIH: OVCAR-3 has been determined to be 28 fmol/mg protein. This is comparable to the number reported previously (Hamilton et al., 1983). About 1 fmol/mg protein of ER was detected in OC-VGH-3 cells. Growth response to estrogen In order to maximize the effects of estradiol, cells were cultured in the presence of lo-“’ M tamoxifen and lo-l2 M RU38 486 for 24 h to off set the effects of endogenous estrogen and progesterone, respectively. The growth of cells was analyzed after the addition of estrogen for 96 h (tamoxifen and RU38 486 were still present during this time). As can be seen in Fig. 2, a slight decrease in cell growth was observed in the presence of lo- “’ M tamoxifen (T), or tamoxifen plus

1

NIH:OVCAR-3

2 C

T

TRE

TE

cmyc

&rnicroglobulin

Fig. I. Northern cancer

OC-3-VGH rated

blot analysis of estrogen receptor

cell lines, 20 pg of total cellular (2) NIH:OVCAR-3

through

ovarian

formaldehyde-agarc,se

lose paper, hybridized

RNA

mRNA

to nitrocellu-

estrogen receptor

autoradiographcd

from (I)

cancer cell lines was sepa-

gel. transferred

with “P-labeled

in ovarian

extracted

cDNA

OC-3-VGH

and

C

for 24 h.

T

TFlE TE

c-myc RU 38 486, suggesting the presence of endogenous estrogen or progesterone in these cells or in the culture medium. The addition of lo-’ M estrogen after preincubation of tamoxifen (TE) caused a 2.3-fold increase in the growth of cells after 48 h, compared to that

lS2-microglobulin Fig. 3. Effect pretreated M RU38

of estrogen

with IO

on c-nz.~ mRNA

I” M tamoxifen

4X6 for 4X h. followed by IOY’

min. RNA

was isolated and Northern

found in T medium. The 486 in the preincubation hibit estrogen mitogenicity. No significant effect of was found in OC-3-VGH Expression

days Fig. 2. Effect

of estrogen

NIH:OVCAR-3. control,

phenol

on the proliferation

Cells were

treated

red free medium

containing

fetal calf serum, with no addition tamoxifen TE:

stimulation.

cells preincubated

tamoxifen

M and 10~‘2

and RU38

M, respectively.

and RU38

with tamoxifen

486 treatment

C:

stripped

for 24 h. and followed

486 for 24 h. followed by estradiol stimulation.

tions of estradiol, lo-”

with tamoxifen

with tamoxifen TRE:

10% charcoal

cancer

media:

of drug. T: cells incubated

cells incubated

cells preincubated

estradiol RU38

only. TR:

of ovarian

with the following

with 4X6. by and

The concentrawere lo-’

M,

The cells were harvested

and

cell number counted on the days indicated. from six experiments.

Results are mean values

expression.

or lo- I” M tamoxifen M estradiol

Cells were and 10

I2

exposure for 30

blot analysis performed.

presence medium

of 10P ” M RU38 (TRE), did not in-

estrogen on cell proliferation under similar conditions.

of c-myc RNA

Southern blot analysis did not show amplification of c-myc gene in both cell lines (data not shown). The expression of the c-myc mRNA was analyzed using exon 2 of c-myc as a probe (Fig. 3). In NIH : OVCAR-3 cells, the 2.3 kb c-myc mRNA increased 4-fold after pretreating cells with tamoxifen followed by 30 min of lo-’ M estrogen exposure (TE) (tamoxifen was present during this time). In the presence of RU38 486 in the pretreating medium (TRE), there was a 3.X-fold increase in c-myc mRNA levels; it appears that RU38 486 did not significantly affect the estrogen induced c-myc mRNA level. The pS2 mRNA (pS2:estrogen response element of MCF-7 cell) expression further confirmed that NIH : OVCAR-3 is responsive to estrogen, the level was elevated 3-fold after estradiol treatment. The expression of the steady state c-myc mRNA in OC-3-VGH increased 15fold after lO_’ M estrogen treatment. Elevation in c-myc expression was not observed when both cell lines were treated with lWx M estrogen (data not shown). The time course of the

Ifi alshort

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0.8

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Fig. 4. (A) Time course of estrogen stimulated c-myc mRNA expression. NIH: OVCAR-3 cells were pretreated with 10~ I” M tamoxtfen for 4X h. IO ’ M estradiol were added for the time designated. At the end of the treatment, RNA was isolated and Northern blot analysis performed. (a) short term estrogen effect (O-2 h) (b) effect of persistent (O-72 h) estrogen exposure. (B) c-rn~~ expression of control NIH : OVCAR-3 cells. Cells were treated as described in panel A except that no estrogen was added, and c-myc mRNA levels determined by Northern blotting. (a) short-term and (b) long-term cell culture.

estrogen induced c-myc mRNA expression is shown in Fig. 4. An 7.5-fold increase in c-myc mRNA in NIH: OVCAR-3 (Fig. 4A) was found at 50 min, the levels declined to the uninduced level after 4-24 h of estrogen exposure. A second increase in mRNA accumulation was found at 48-72 h, indicating biphasic expression of c-myc gene during prolonged estrogen stimulation. c-myc expression remained relatively constant in the absence of estrogen (Fig. 4B). Estradiol increased the transcription of c-myc gene Nuclei were isolated from cells exposed to lo-’ M estradiol for various times, and the in vivo-initiated nascent RNA was allowed to elongate. The results are illustrated in Fig. 5A. A rapid induction in nuclear transcripts at lo-20 min was found [Fig. 5A(a)]. The levels declined to unstimulated levels after 5 h of estradiol exposure. The rate of c-myc nuclear transcription was not affected by estradiol in the period between 5 h and 24 h but increased gradually in the time period between 48 and 72 h [Fig. 5A(b)]. The transcription was inhibited by adding 2 pg/ml of c~amanitin suggesting the participation of RNA polymerase II [Fig. 5A(c)l. No significant stimulations of c-myc transcription in NIH: OVCAR-3 was observed in the estrogen untreated cells (Fig. 5B). The level of c-myc nuclear transcripts in OC-3-VGH cells after exposure to estradiol for various times was also examined. No significant estrogen associated elevation in the level of c-myc nuclear transcripts was detected in these cells [Fig. 5A(d)].

c-myc mRNA stability analysis c-myc mRNA stability was next determined. Cells were exposed to estradiol for 50 min, actinomycin D at a concentration of 5 pg/ml was then added. The degradation of existing c-myc mRNA was monitored at various time intervals for a period of 2 h. The half-life of c-myc mRNA of NIH: OVCAR-3 was estimated to be 64 min f 5 min (Fig. 6) in the presence or absence of estradiol. The half-life of c-myc mRNA of OC-3-VGH with or without estradiol treatment was determined to be 107 + 3 min. The results suggested that estrogen has no effect on the c-myc mRNA stability in both cell lines.

Effect of antisense oligonucleotides on c-myc protein expression and cell growth Estradiol (lo-’ MI stimulated the c-myc protein expression in NIH : OVCAR-3 cells, maximal response was found at approximately 120 min (Fig. 7a, lane 3). This correlated with the peak of transcriptional induction and the accumulation of cytoplasmic c-myc mRNA. In the presence of 8 PM antisense-c-myc oligonucleotide, a significant inhibition on the estrogen stimulated c-myc protein expression was detected at 20 and 120 min (Fig. 7a, lanes 5, 6). When the growth of NIH: OVCAR-3 cells was analyzed by the MTT method in the presence of 5 PM antisense to c-myc DNA and lo-’ M estrogen, it was found that the growth of these cells was blocked completely, as indicated in Fig. 7(b).

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min

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c)

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(W ajshort

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Fig. 5. (A) Effect of estrogen on c-tnyc transcription. (a-c) NIH:OVCAR-3 cells or (d) OC-3-VGH cells were pretreated with IO-” M tamoxifen for 48 h, lo-’ M estradiol were added for the times indicated. At the end of the treatment, nuclei were isolated and nuclear run-on assay of c-myc transcripts performed. (a) short-term estrogen effect (O-3 h). (b) effect of persistent (O-72 h) estrogen exposure. (c) “P-labelled RNA from nuclei stimulated with lo-’ M estradiol for 10 min were incubated in in vitro nuclear run-on assay system in the presence (0.5 fig, 2 pg/ml) or absence of cy-amanitin. (d) short-term estrogen effect (O-60 min) on OC-3-VGH cek. (3) Nuclear run-on assay of c-rrzyc in estrogen untreated NIH: OVCAR-3. Cells were pretreated with lo- “’ M tamoxifen for 48 h. Control experiments without estrogen treatment were performed in (a) short-term (b) long-term period.

17

lo’

I

0

30

90

60 Time

120

(min)

Fig. 6. Stability of c-myc mRNA. NIH:OVCAR-3 cells were cultured in the presence of tamoxifen plus estrogen ( v v) or in v ) alone. Alternatively, OC-3-VGH cells were tamoxifen ( v l)orinT(oin TE to----O) condition for 50 min. Cells were then treated with 5 pg/ml of actinomycin D for the time indicated. RNA was extracted and analyzed by Northern blotting.

Discussion

It has been reported that the proliferation of NIH: OVCAR-3 is not affected by low concentrations (1O-‘2-1O-8 M) o f es t rogen (Nash et al., 1989). In the present investigation, treatment of these cells with lo-’ M estrogen for 48 h after antiestrogen blockade resulted in a 2.3-fold increase in cell growth, suggesting a fully functional estrogen receptor system does exist in NIH: OVCAR-3. It also implies that higher dose (i.e., lo-’ M) of exogenous estrogen can overcome the effect of endogenous progesterone on the conversion of estradiol to estrone (Tseng, 1978) in these cells.

la1

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+E2 20

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240

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c-rnyc induction has been shown to be part of the cascade events associated with the mitogenic response to growth factors (Dean et al., 1986; Blanchard, 1985). For example, the c-myc gene product played a functional role in the growth response to PDGF (Armelin et al., 1984). c-myc nuclear protein may positively regulate the genes involved in the initiation of DNA replication in human HL60 and ML-1 cells (Studzinski et al., 1986). We have found that estrogen increased the c-myc mRNA expression and the proliferation of NIH : OVCAR-3 cells, moreover, inhibition of the estrogen-induced expression of the c-myc protein by an antisense-c-myc oligonucleotide resulted in the arrest of estrogen stimulated cell proliferation. These findings suggest that c-myc protein plays important roles in the mechanism of estrogen-induced cell growth. As a cell cycle regulator (Studzinski et al., 19861, the c-myc protein has been shown to be a target of growth factor (Luscher et al., 1989); it can also act as a transcription factor (Penn et al., 1990, Schweinfest et al., 1988). In this sense, the c-myc protein may play a role in regulating the expression of certain growth related genes in estrogen stimulated cells. It appears from the present findings that the expression of c-myc is a direct effect of estrogen, since results from nuclear run-on indicate that c-myc gene activation is a very early effect of estrogen. In OC-3-VGH cells, estrogen stimulation resulted in a 1.5 fold increase in c-myc mRNA, whereas the cell proliferation was not affected. These data suggest that the cascade of events required for estrogen mediated mitogenicity are not fully coupled in this cell line.

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Fig. 7. Effect of antisense oligonucleotide on c-myc protein expression and cell growth of NIH: OVCAR-3. (a) Lanes l-4, lo-’ M estrogen was added to cells cultured in T medium, time-course studies of estrogen induced c-myc protein expression. Lanes 5-6, 8 PM oligonucleotide was preadded to cells in the T medium for 5 days followed by lo-’ M estrogen stimulation for 2 h. c-myc protein expression was detected by Western blot. Lane 7, egg albumin was substituted for c-myc antibody used in the experiment as described in lane 1. (b) Effects of antisense oligonucleotides on time-course of cell growth. Cells were grown in TE medium with or without 5 PM oligonucleotide. Growth was assessed by MTT assay. C: TE medium without antisense c-myc oligonucleotide. AS: TE medium with antisense c-myc oligonucleotide. Each point was measured from triplicate wells of four independent experiments.

IX

The proliferation of cell is alleviated in the presence of RU38 4X6 tin TRE medium) compared to that cultured in TE medium, this could be attributed to the anti-progesterone action of RU38 486, for it has been reported that RU38 4X6 may impair the steps in PR transactivation functions (El-Ashry et al., 1989, DeMarco et al., 1992). It could be suggested that the decrease in cell growth (in TR medium) was due to the inhibition of PR mediated transactivation gene expression, which may play roles in the growth of ovarian cancer cells. The fact that estrogen stimulated proliferation and c-r)zyc induction of NIH : OVCAR-3 was not fully affected by RU38 4X6 is possibly because that the number and function of ER has not been altered by RU3X 486; it is also because that not a very high concentration of RU38 486 was used in this study; under this circumstance only a small fraction of preexisting or estrogen-induced progesterone receptor molecules were affected. The nuclear run-on experiments indicate that transcription of the c-myc gene is increased by estradiol while mRNA stability was not influenced. Our results are consistent with those previously reported by Dubik and Shiu (IYSS), in which transcriptional regulation of c-myc by estrogen has been demonstrated in MCF-7 breast cancer cells. Our finding that mRNA stability is not influenced by estrogen is, in contrast to data from Santos et al. (1988); they have shown that regulation of estrogen stimulated c-myc expression was primarily post-transcriptional in MCF-7 cells. The stability of RNA was known to be regulated by polyadenylated RNA species (Swartwout and Kinniburgh, 1989), or by A + U rich element RNA binding factor (Brewer, I991 I; apparently it is not altered by estrogen as demonstrated in this study. Computer analysis indicates the existence of estrogcn responsive element-like sequence at the 5’-flanking sequence of human c-myc gene; whether the estrogenresponsive element is present and is mediating the estrogen effect on c-myc gene in NIH : OVCAR-3 cells needs to be investigated further. Acknowledgements This work was supported by a grant (NSC 77-0412BOlO-40) from the National Science Council, Taiwan, Republic of China. We thank Dr. H.T. Ng and Dr. K.C. Chao for kindly providing us with the OC-3-VGH cell line for this study. References Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L., Abbott, D.L., Mayo, J.G., Shoemaker, R.H. and Boyd, M.R. (1988) Cancer Res. 48, 589-681. Armelin. H.A., Armelin, M.C.S., Kelly, K., Stewart, T., Leder, P., Lochran, B.H. and Stiles, CD. (19841 Nature 310, 655-660.

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