DOWN-REGULATED MELANOMA DIFFERENTIATION ASSOCIATED GENE (MDA-7) EXPRESSION IN HUMAN MELANOMAS Suhendan EKMEKCIOGLU 1 , Julie ELLERHORST 1 , Abner M. MHASHILKAR 2 , Aysegul A. SAHIN 3 , Christine M. READ 1 , Victor G. PRIETO 3 , Sunil CHADA 2 AND Elizabeth A. GRIMM 1 * 1 Department of Molecular and Cellular Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA 2 Introgen Therapeutics, Houston, TX, USA 3 Department of Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, TX, USA The melanoma differentiation associated gene-7 (mda-7) has a potential inhibitory role in melanoma progression, al- though the mechanisms underlying this effect are still un- known. mda-7 mRNA has been found to be present at higher levels in cultured normal melanocytes compared with met- astatic melanoma cell lines. Furthermore, levels of mda-7 message have shown an inverse correlation with melanoma progression in human tumor samples, suggesting that mda-7 may be a novel tumor suppressor gene. We have designed this study to investigate MDA-7 protein expression in differ- ent stages of melanoma progression and to examine its an- tiproliferative effects in vitro. Our data demonstrate that MDA-7 protein can be found in normal melanocytes and early stage melanomas. It is also observed in smooth muscle cells in the skin. However, in keeping with a possible role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated antitumor effects of overexpressed MDA-7 on human melanoma cells in vitro. Our results demonstrate that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes. © 2001 Wiley-Liss, Inc. Key words: melanoma development; tumor suppressor genes; mda-7; gene therapy; adenovirus A feature of malignant tumors that is considered therapeutically exploitable is the loss of cellular differentiation. This has lead to a search for tissue-specific differentiation factors that might be re- introduced into tumors in order to modify their growth and ability to metastasize. In the case of melanoma, several unique genes have been isolated by Jiang et al. 1 from a melanoma cell line induced to differentiate by interferon- and mezerein. One of the most inter- esting and promising of these genes is the melanoma differentia- tion associated gene-7 (mda-7). Since its initial isolation from melanoma cells and subsequent molecular characterization, the mda-7 gene and MDA-7 protein have been studied in numerous other tumor types, some of which have included carcinoma of the lung, breast, prostate and cervix. 2 Experiments using an adenovi- rus/mda-7 vector construct (Ad-mda-7) to infect normal and ma- lignant cells have consistently demonstrated growth-inhibitory ef- fects on various tumor types, but no inhibitory effects on normal cells. 3,4 These findings have prompted the development of the Ad-mda-7 as a potential therapeutic agent, and plans for a phase I clinical trial are under way. Although the initial description of mda-7 occurred in melanoma, most of the clinically relevant research has focused on the more prevalent malignancies. Our laboratory has an ongoing interest in the development of prognostic tools and therapeutic interventions for advanced mela- noma. We have therefore undertaken a study to better define the significance of MDA-7 in this disease. We describe in this paper our data from human tumors and cell lines demonstrating that MDA-7 protein can be found in normal melanocytes, early stage melanomas and smooth muscle cells. However, in keeping with a role as a tumor suppressor, MDA-7 expression is decreased in more advanced melanomas, with nearly undetectable levels in metastatic disease. We also investigated the anti-tumor effects of overexpressed mda-7 on human melanoma cells in vitro. Our results show that Ad-mda-7 induces apoptosis and G2/M cell cycle arrest in melanoma cells, but not in normal human melanocytes. MATERIAL AND METHODS Tumor samples and cell lines The melanoma tumor samples used in this study consist of primary skin and lymph node metastases surgically removed from patients enrolled in institutionally approved trials. Formalin-fixed and paraffin-embedded tissue sections were obtained from the Melanoma and Skin Cancer Core Laboratory, of the University of Texas M.D. Anderson Cancer Center for use in immunohisto- chemical labeling of tumor tissues. Metastatic melanoma cell lines, A375 and A375.S2, were ob- tained from ATCC (American Type Culture Collection, Rockville, MD). The highly metastatic melanoma cell line MeWo was pro- vided by Dr. David Menter (M.D. Anderson Cancer Center, Hous- ton, TX). Primary melanoma cell lines, WM35, WM793, and their more invasive subclones were provided by Dr. Robert Kerbel (Sunnybrook Health Science Center, Toronto, Ontario, Canada). Normal human epidermal melanocytes (NHEM) were obtained from Clonetics (San Diego, CA). Samples of formalin-fixed and paraffin-embedded human benign nevi were retrieved from the Department of Surgical Pathology. Reagents Recombinant MDA-7 protein was expressed in E. coli and purified using a nickel NTA agarose column from Introgen Ther- apeutics (Houston, TX). This recombinant MDA-7 protein was used to generate rabbit polyclonal antibodies, which were purified by affinity chromatography. Preimmune normal rabbit IgG (Vec- tor, Burlingame, CA) was used as a negative control. Antivimentin antibody (BioGenex, San Ramon, CA) was used as a positive control. Replication-deficient human type 5 adenovirus (Ad5) carrying the mda-7 gene was also obtained from Introgen Therapeutics. The mda-7 gene was linked to an internal CMV-IE promoter and followed by SV40 polyadenylation [p(A)]. Ad-Luc and Ad-CMV p(A) (luciferase and empty vector, respectively), were used as control viruses. Gene transfer Cells were plated 1 day prior to infection. Melanoma cells were infected with adenoviral vectors (Ad-mda-7 or Ad-luc) at a mul- Grant sponsor: NCI; grant numbers: RO1 CA64906, P30 CA16672, T32 CA72371, R43 CA86587. *Correspondence to: Department of Molecular and Cellular Oncology, Box 79, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA. Fax: +713-794-4784. E-mail: egrimm@mdanderson.org Received 23 February 2001; Revised 9 April 2001; Accepted 11 May 2001 Published online 25 July 2001; DOI 10.1002/ijc.1437 Int. J. Cancer: 94, 54 –59 (2001) © 2001 Wiley-Liss, Inc. Publication of t he International UnionAgainst Cancer