0013.7227/92/1312-0982$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Vol. 131, No. 2 Printed in U.S.A.

Society

TRANSCRIPTIONAL

REGULATION BUTYRATE

IN

OF PROLACTIN MCF-7 HUMAN

RECEPTOR BREAST

GENE CANCER

EXPRESSION CELLS

BY

SODIUM

Christopher J. Ormandy, Anna de Fazio, Paul A. Kelly * and Robert L. Sutherland Cancer Biology Division, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, N.S.W. 2010, AUSTRALIA and *INSERM UnitC 344- Endocrinologie Moltculaire, Facultt de Mtdecine Necker-Enfants Malades, 156, rue de Vaugirard, 75743 Paris CCdex 15, FRANCE. ABSTRACT: The prolactin receptor (PRLR) mediates the diverse effects of prolactin, which in the mammary gland include the development of lobuloalveolar structures and increased tumor cell proliferation. Treatment of mammary carcinoma cells with the differentiating agent sodium butyrate (NaB) is known to reduce PRLR binding activity and PRLR gene expression, however the mechanism which mediates these changes is unknown, prompting this investigation. Using MCF-7 human breast cancer cells, assay of the rate of PRLR gene transcription by the nuclear run-on technique indicated that 3 mM NaB reduced PRLR gene transcription by 50% after 3 h of treatment and that this effect was maintained for at least 24 h. The protein synthesis inhibitor cycloheximide failed to abrogate this effect, which indicated that NaB did not require continuing protein synthesis to reduce the rate of PRLR transcription. Measurement of PRLR mRNA stability, using Northern blot analysis at various times after the inhibition of transcription with actinomycin D, showed that NaB treatment did not alter PRLR mRNA half-life. These results indicate that NaB inhibits PRLR gene expression by a transcriptional mechanism that does not require continuing protein synthesis.

fetal calf serum was from C.S.L.-Novo, Sydney, Australia. Tissue culture flasks were from Corning, Sydney, Australia. All other reagents were of Molecular Biology or AR grade.

INTRODUCTION

The urolactin receutor (PRLR). is a transmembrane glycoprotein ‘with sequence ‘homology to ‘ihe receptors for growth hormone, the interleukins and erythropoietin (reviewed in 1). The PRLR binds and mediates the many effects ascribed to prolactin in vertebrates (reviewed in 2), however the action of prolactin has been best defined in the mammary gland where in rodents and humans urolactin in concert with steroid hormones (estrogen, progestin, glucocorticoid and thyroxine) provides a mitogenic stimulus to the alveolar enithelial cells, which facilitates lobuloalveolar development (reviewed in 3, 4). Prolactin is also mitogenic in mammary tumours and cell lines (reviewed in 5, 6) suggesting a role for the PRLR in the etiology of mammary cancer. Sodium butvrate (NaB). an inhibitor of cell mowth and a differentiation inducing‘ agent (7, 8 and references therein), reduced PRLR binding activity in explants of normal rabbit mammary gland (9), 7,12-dimethylbenzanthracene-induced rat mammary tumours (8) and human breast cancer cells where PRLR mRNA levels were also decreased (10). PRLR levels may therefore fall as a consequence of cellular differentiation, from relatively high levels in proliferating cells to lower levels in nonnroliferatine cells involved in differentiated function. The ;Ilcch;lnisnls”responsible for the NaB-induced reduction in PRI,R levels are however unknown. NaB has previously been shown to exert both post-transcriptional and transcriptional effects on gene expression (11, 12), prompting this investigation of the effect of NaB on these parameters in MCF-7 human breast cancer cells. EXPERIMENTAL

Cell culture: Cell lines were passaged as previously describg>-rl (15) in RPMI-1640 media supplemented with 6 mM L-glutamine, 10 bg/ml human insulin, 20-&ml gentamicin sulphate and 5% fetal calf serum (RPM1 5% FCS). For Northern analysis and assay of transcription rate, 1~10~ exponentially growing cells were plated in 50 ml of RPM1 5% FCS per 150 cm2 flask. NaB, dissolved in water and filter sterilized, was added directly from 0.5 M stock solutions. Northern Analysis: Total cellular mRNA was prepared using the guanidinium isothiocyanate-cesium chloride method and PRLR mRNA levels were measured as ureviouslv described (16) using PRLR cDNA labelled by a[32P]-deoxycytidine triphosphate random primer extension. RNA loading and transfer were checked by probing with a d3*P]-deoxycytidine triphosphate endlabeled oligonucleotide for the 18s ribosomal subunit. As the variation between lanes was small, no corrections for loading were made. Autoradiographs were quantified using a Bio-Rad 620 video-densitometer and analysed with the Bio-Rad 1D Analyst computer programme (version 3.10). Assay of PRLR transcription rate: The rate of transcription of the PRLR gene was measured using approximately 2-3~10~ cells per point by the method of Greenberg and Ziff (17) with modifications as previously described (18). Briefly, intact nuclei isolated at 3 h and 24 h after the addition of NaB or vehicle were allowed to continue transcription for 45 min in the presence of labeled a13*Pluridine triuhosphate, mior to isolation of total RNA and meaiurement of

Transcriptional regulation of prolactin receptor gene expression by sodium butyrate in MCF-7 human breast cancer cells.

The prolactin receptor (PRLR) mediates the diverse effects of prolactin, which in the mammary gland include the development of lobuloalveolar structur...
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