[CANCER RESEARCH 55, 3158-3164, July 15. 1995] Interferon a-2b and Retinole Acid Combined Treatment Affects Proliferation and Gene Expression of Human Cervical Carcinoma Cells1 Francesca Lancillotti, Valeria Giandomenico, Elisabetta Affabris, Gianna Fiorucci, Giovanna Romeo,2 and Giovanni Battista Rossi Laboratory of Virology, Istituto Superiore di Sanila', Viale Regina Elena 299. 00161 Rome ¡F.L. V. C.. E. A., C. F.. G. R.. G. B. RJ: III Università di Roma, 00154 Rome IE. F.]: ami Istituto di Tecnologie Biomediche, CNR. 00161 Rome ¡C.F., G. R.I, Italy ABSTRACT The in vivo and in vitro antitumor effectiveness of IFNs is well docu mented. Their combination with differentiating agents, such as retinoic acid, has been demonstrated to be a promising therapy for patients with advanced squamous cell cancer of the skin and the cervix. However, the mechanisms that mediate these antitumor responses are not yet known. We studied the epidermoid cell line ME 180 derived from human cervical carcinoma to test its responsiveness to IFN-a-2b (INTRON A) and all- frans-retinoic acid (KA). Both agents have demonstrated ability to inhibit the growth of ME 180 cells in a dose- and time-dependent manner. The antiproliferative effect was further increased by the treatment with II V a-2b and RA combined. In accordance with this result, we found that the combination of the two agents has the effect of increasing the expression of the 2-5A synthetase gene, which is thought to play a key role in antigrowth responses to lENs. At increased levels of 2-5A synthetase mRNA corresponds a significant increase in 2-5A synthetase activity. Although RA per se has no effect on the 2-5A synthetase expression, when it is combined with lFN-a-2b it appears to be able to potentiate the IFN-induced 2-5A synthetase expression. Moreover, the combination of IFN-a-2b and RA produces a similar effect also on the expression of the HLA-A2 gene, which has been shown to be induced in ME 180 cells both by II \-d-2h and RA alone. In view of the possible mechanisms of action of the two agents, it is interesting to note that their combination increases, although transiently, the expression of IRF,, which codes for a transcription factor that regulates II \ gene expression and is thought to be involved in the regulation of IFN-induced effects and in mediating cell death or apoptosis. INTRODUCTION IFNs constitute a heterogeneous family of cytokines that, although generally defined as antiviral proteins, also regulate basic cellular functions including growth, differentiation, and immune reactivity (1-4). They exhibit antiproliferative activity on many normal and transformed cells (5) and have in vivo antitumor effects against a variety of cancers (6, 7), including squamous cell carcinoma (8). Their capacity to affect the differentiation of numerous cell types is also well established (9-11); however, it has been observed that the differentiation-inducing potential of IFNs, as well as their antipro liferative effect, is augmented when they are combined with RA3 (12-15). Because of their strong effect on differentiation, retinoids have been largely utilized to prevent carcinogenesis and to treat various types of cancer (16-18). Nevertheless, since clinical therapeutic results ob tained utilizing monotherapy of either cytokines or retinoids are far Received 7/27/94; accepted 5/10/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by grants from the Associazione Italiana per la Ricerca Sul Cancro. Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica Consiglio Nazionale delle Ricerche and Ministero Università e Ricerca Scientifica e Tecnologica 40%. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: RA, all-rrd/i.v-retinoic acid; ISO. IFN-stimulated gene; ISRE. IFN-stimulated response element; RARE, retinoic acid-responsive element; 1RF,. IFN-regulatory factor 1; RAR. retinoic acid receptor. from being satisfactory, it has been proposed that combination therapy may improve their antitumor effectiveness (19). Synergistic antipro liferative effects have been in fact obtained in in vitro studies utilizing the combination of IFN-a and RA on cell lines derived from human carcinoma of the larinx and the vulva (15). Moreover, the first clinical trials with a combination of IFN-a-2b and 13-c/s-retinoic acid have demonstrated to be very promising for squamous carcinoma of the skin and the cervix (20-25). However, the mechanisms by which interferons and retinoids in duce antitumor effects are still not well clarified, even though their different modes of action have been recently better defined. IFNs exert their biological activities on target cells by inducing a number of effector genes, the so-called ISGs (26-29), the transcriptional activity of which is transiently increased after the binding of IFNs with specific cell surface receptors. The transcriptional stimulation is mediated by preexisting proteins that become activated and function as transcriptional factors which recognize an enhancer element (ISRE) within the regulatory sequence of target genes. One of them, the 2-5A synthetase gene, codes for an enzyme which has been suggested to play a role in the growth inhibitory action of IFNs. It polymerizes ATP into a series of 2',5'-linked oligomers of the series ppp(A2'p)nA (2-5A), which specifically activate a 2-5A-dependent ribonuclease to cleave both ribosomal and messenger RNAs. Its activity has been de scribed in four proteins with different requirements for double-stranded RNA activation and different intracellular locations (30). Retinoid activ ities are instead mediated through ligand-dependent nuclear receptors that transactivate the expression of target genes by binding to specific respon sive elements (RAREs; Refs. 31, 32). Retinoid receptors belong to one of the largest transcription factor family, the regulatory machinery of which appears to involve mechanisms of protein-protein coregulation and pos sible interaction with additional transcription factors (33). With the objective of exploring how the combination of IFNs and retinoids might work in squamous cell carcinomas and which mech anisms might be responsible for its effectiveness, we studied the effect of recombinant IFN-a-2b and all-rrunf-retinoic acid in the cervical carcinoma cell line ME 180. We showed that the combined treatment increases the growth inhibitory effect of the single agents and that the levels of 2-5A synthetase activity are also increased. How RA influ ences the complex expression machinery of an IFN-inducible gene remains to be elucidated. Our study suggests that IRF,, a transcrip tion factor which belongs to the IFN machinery, might be involved because its expression is increased when RA is present. The analysis of the expression of others ISGs has, however, demon strated that RA doesn't have the ability to indistictively potentiate IFN-a-2b action. MATERIALS AND METHODS Cell Culture and Treatments. Human epidermoid carcinoma cell line ME 18(), isolated from an omental metastasis of a rapidly spreading cervical carci noma, was obtained from American Type Culture Collection (Rockville. MD) and maintained in McCoy's 5a medium supplemented with 10% fetal bovine serum, previously inactivated at 56°Cfor 20 min. Cells were grown to approximately 85-90% of confluency in a humidified atmosphere of 5% CO, at 37°C. 3158 on June 3, 2015. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from