Breast Carcinoma Cell Lines Vasoactive Intestinal Polypeptide Receptor Type-1 in Human Retinoic Acid Down-Regulates the Expression of the Updated Version

Four breast carcinoma cell lines (T47D, ZR-75-1, MDA-MB-231, and MCF-7) were tested for regulation of the expression of vasoactive intes tinal polypeptide receptor type-1 (VIP-R1). In all four cell lines, retinole acid (RA) treatment caused a fast and marked decrease in VIP-R1 niRNA level as examined by Northern blots. Cycloheximide pretreatment attenuated the effect from 3to 2-fold, indicating that existing proteins can mediate the decreasing effect of RA, but to attain the maximal effect new protein synthesis might be needed. Transcriptional inhibition with Velinomi odii D showed that RA did not influence the VIP-R1 inKN A half-life, indicating that the decreasing effect of RA on the mRNA level is due to transcriptional inhibition. In agreement with the observations on mRNA level, we found that the VIP receptor number was reduced 3-fold from 88 to 32 fmol/106 cells in T47D cells and from 222 to 73 fmol/10* cells in MDA-MB-231 cells upon RA treatment for 72 h. The promoter and 5'-flanking region of the VIP-RI gene were cloned from a human placenta! cosmid library, and 2.5 kb were sequenced to search for regulatory elements. Our results, therefore, imply that the regulation of VIP-RI gene ex pression by RA could have a role in human mammary tumor biology. INTRODUCTION VIP3 is a pleiotropic neuropeptide with a widespread occurrence in both the central and peripheral nervous systems. VIP has a broad range of biological functions, including a potent growth-related action influencing proliferation, neuronal survival, and differentiation (1-5). Besides these effects. VIP is an important neurotransmitter involved in autonomie nervous control of smooth muscle activity, blood flow, and endocrine and exocrine secretion (6). The actions of VIP are generally associated with an increase of the cAMP level. High levels of VIP are present in milk, and increased VIP gene expression is found in response to suckling in lactating women (7). In the human mammary gland, VIP containing nerve fibers is present around milk ducts in the parenchyma and the nipple (8). This implies that VIP has a physiological role in regulating milk secretion and ejection from the mammary glands. In both normal and transformed mammary cells, the cAMP level increases in response to VIP treat ment (9), and VIP has been found to induce the growth of a murine mammary tumor cell line (10). Furthermore, a VIP receptor antagonist has been found to inhibit breast cancer growth both HIvitro and in vivo (11). Two human receptors for VIP, VIP-RI and VIP-R2, have been cloned, both belonging to a discrete family of seven TM G-protein-coupled receptors (12-14). A high occurrence of VIP-RI Received 4/27/98: accepted 8/26/98. The costs of publication of this article were defrayed in part hy the payment of page charges. This article must therefore he hereby marked iiilveriim-nÃ-t'nÃin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by the Danish Biotechnology Center for Cellular Communication and by a grant from Professor Suad AI-Kassab's foundation. ~ To whom requests for reprints should be addressed, at Department of Clinical Biochemistry. Bispebjerg Hospital, Bispebjerg Bakke 23. Dk-2400 Copenhagen NV. Denmark. Phone: 45-3531-2646; Fax: 45-3531-3955; E-mail: BMA@>biobase.dk. 'The abbreviations use are: VIP. vasoactive intestinal polypeptide: VIP-RI. VIP receptor type-1; ER. estrogen receptor: RA. telinole acid: RAR. RA receptor; ATCC. American Type Culture Collection: CX. Cycloheximide: AD. Aetinomyocin D; TM. transmembrane: AP-1. activator protein 1. gene expression in human breast cancer cell lines and of VIP binding sites in breast carcinomas and metastasis have also been reported (15. 16). A role for VIP on the growth, differentiation, and function in normal and neoplastic breast may, thus, be possible. Several lines of evidence indicate that growth inhibition of breast cancer cells by hormones and vitamins coincides with enhancement of differentiation (17, 18). RA. a metabolite of vitamin A and a potent inducer of differentiation in a large number of cells, has demonstrated promising results in preclinical and clinical trials of cancer prevention and therapy (18-21). The ability of RA to inhibit the growth of breast cancer cell lines has been found to correlate with the status of RARa (22). The biological effect of this compound seems to be mediated through at least two classes of nuclear receptors, RAR and retinoid X receptor, each containing three members (23). RARs and retinoid X receptors are effective transcriptional activators of some genes; how ever, RA is also known to repress transcription of other genes. The inhibitory effect of RA on gene transcription can probably be exerted by various mechanisms, such as competition for overlapping binding sites between receptors and other transcription factors or by compe tition for limiting cofactors (24-27). In light of the growth-inhibiting effect of RA in breast cancer cells, on the one hand, and the growth-promoting effect of VIP on the other, we examined the effects of RA on VIP-RI expression and on VIP binding in a number of breast carcinoma cell lines. In addition, we cloned and sequenced 2.5 kb of the promoter region of the VIP-RI gene to search for regulatory sequences. MATERIALS AND METHODS Cell Culture. The following human breast carcinoma cell lines obtained from ATCC were used: T47D (ATCC HTB 133). MCF-7 (ATCC HTB 22). ZR-75-1 (ATCC CRL-1500). and MDA-MB-231 (ATCC HTB-26). T47D. MCF-7. and MDA-MB-231 cells were cultured in DMEM with 10% FCS. The medium for the T47D cells was supplemented with 8 fig/ml insulin. ZR-75-1 cells were cultured in RPMI with 10% FCS. The cells cultured in DMEM were kept in 10% CO2/90% atmospheric air. whereas the cells cultured in RPMI were kept in 5% CO2/95% atmospheric air. Routinely, the cells, which are adherent, were split and used in the exponential growth phase for the experi ments. Before experiments. T47D. MCF-7. and ZR-75-1 cells were kept in phenol red-free medium for 48 h to avoid the weak estrogenic effect of phenol red (28). The medium was supplemented with 10% activated charcoal-stripped FCS. The ER-negative cell line. MDA-MB-231. was kept in normal medium with 10% activated charcoal-stripped FCS. The serum was stripped to remove steroid hormones. Parallel dishes were then incubated with all-fra/;.v-RA ( 10~5 M). DMSO (0.1%. dissolving agent for RA), CX (5 fig/ml), and AD (2.5 fig/mil for the time period indicated in each experiment. In the time course experiments, the total culture time of all of the plates was the same. RNA Preparation and Analysis. RNA was extracted using Pharmacia's QuickPrep Total RNA Extraction kit. Total RNA (20 fig) of each preparation was run on 1% agarose gels under denaturating conditions and vacuum-blotted to nylon membranes (Hyhond N: Amersham Corp.). The hlots were succes sively hybridized with "P-labeled hVIP-Rl cDNA (kindly donated by Profs. M. Laburthe and A. Couvineau) and oligo probes for 28S rRNA (Calbiochem). The hVIP-Rl cDNA fragment was labeled with 12P using a random primer synthesis kit (Amersham Corp.). and the 28S oligo probe was labeled using a terminal transferase kit (Boehringer Mannheim). The blots were prehybridized in hybridization buffer [0.5 M sodium phosphate (pH 7.2). 7% SDS. I mM 4845 American Association for Cancer Research Copyright © 1998 on February 23, 2013 cancerres.aacrjournals.org Downloaded from