ICANCERRESEARCH57. 2104-2108. June1. 9971 Advances in Brief Ornithine Decarboxylase Overexpression Leads to Increased Epithelial Tumor Invasiveness' Mary K. Smith, Mary A. Goral, Jane H. Wright, Lynn M. Matrisian, Rebecca J. Morris, Andres J. P. Klein-Szanto, and Susan K. Gi1mour@ Lankenau Medical Research Center, Wvnnewood, Pennsvli'ania 19096 [M. K. S., M. A. G., R. J. M., S. K. G.J; Department of Cell Biology. Vanderbilt University, Nashville, Tennessee 37232 If. W.. L M. Mi: and Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [A. J. P. K-S.] Abstract Ornithine decarboxylase (ODC) overexpression cooperates with genetic lesions such as an activated c@rashta to enhance epithelial tumorigenesis. To assess the invasiveness of ODC-overexpressing cells, two noninvasive epi dermal cell lines, nontumorigenic BK-i cells, and the papilloma-derived cell line SP-i were infected with a replication-defective retrovirus that overexpresses ODC, inoculated into deepithelialized rat tracheas, and transplanted into athymic nude mice. After 5 weeks, ODC-overexpressing BK-i cells remainedlocalizedon the luminal surface of the tracheal xenotransplants, whereas the ODC-overexpressing SP-i celis were ex tremely invasive, with the whole tracheal wall penetrated. This invasive ness of ODC-overexpressing SP-i cells was accompanied by elevated proteinase expression, including increased urokinase plasminogen activa tor activity in ODC-overexpressingcells and elevatedstromelysin-i mRNA expression in the stromal cells of invaded tracheal transplants. Introduction ODC3 is a key regulatory enzyme in the biosynthesis of poly amines, which are essential for all cell growth and differentiation (1, 2). Although ODC is rapidly induced by a variety of mitogens, tumor promoters, and hormones, ODC expression is tightly regulated in normal cells. The regulation of ODC is altered in tumor cells, yielding constitutively high levels of ODC expression (2—5).As a result, polyamine biosynthesis and intracellular polyamine levels increase (6). Accumulating evidence indicates that ODC overexpression plays an important role in tumorigenesis. Overexpression of ODC causes the transformation of NIH 3T3 cells with tumor formation in nude mice (7—10).Whereas constitutive overexpression of ODC alone is not sufficient to induce tumors in normal diploid epithelial or fibro blast cells (10), ODC overexpression cooperates with other genetic lesions, such as an activated c-rai―, to promote epidermal tumor development (10). Although ODC overexpression plays a causal role in epidermal tumorigenesis, the role of elevated levels of ODC and polyamines in epithelial malignant conversion remains unclear. A major difference in malignant conversion between fibroblasts and epithelial cells is that epithelial tumors must invade through the basement membrane matrices. Conversion to an invasive phenotype involves several events, including the attachment of the tumor cells to the basement membrane via cell surface receptors, the secretion of Received 3/I 3/97: accepted 4/20/97. The costsof publicationof this article weredefrayedin part by the paymentof page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by Grant CA70739 from the National Cancer Institute (to S. K. G.) andin partby GrantCA45293 fromNIH (to R. J. M.) andGrantCA46843 from NIH-National Cancer Institute (to L. M. M.). 2Towhom requests foreprints should beaddressed, atLankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, PA 19096. Phone: (610) 645-8429; Fax: (610) 645-2205; E-mail: sgilmour@voicenet.com. 3 The abbreviations used are: ODC, ornithine decarboxylase; PA, plasminogen acti vator; uPA, urokinase-type PA; DFMO, a-difluoromethylomithine. enzymes that degrade the basement membrane and the underlying stroma, and increased cell motility (11—13). Because a key step in the progression of an epithelial tumor to a malignant phenotype is the acquisition of the ability to degrade its basement membrane and to infiltrate neighboring tissues, we directly tested the effect of elevated levels of ODC on the invasive properties of epidermal cells growing in vivo. Although a variety of in vitro systems have been developed to assess the invasiveness of tumor cells, including the invasion of tumor cells across a reconstituted basement membrane matrix (Matrigel) on a filter (14), we chose to use a tracheal transplant procedure (15, 16) to evaluate the effect of ODC overexpression on the invasive behavior of epidermal cells. This procedure consists of seeding the epidermal cells into deepithelialized rat tracheas transplanted s.c. into nude mice. It offers several advan tages over in vitro procedures for evaluating invasiveness of cells; namely, it is more reliable and more closely simulates the in vivo conditions in which a malignant tumor cell invades neighboring tissues. Using this model, we demonstrated that ODC overexpression plays a causal role in the conversion of a premalignant epithelial cell to one with an invasive phenotype. Materials and Methods CellCulture. Epidermal celllines,including thenontumorigenic BK-l and the premalignantSP-l cells, weregenerouslyprovidedby Dr. StuartYuspa (NIH, Bethesda, MD). SP-l cells were derived from pooled papillomas pro duced on SENCAR mice by initiation with 7,l2-dimethylbenz[a]anthracene and promotion with TPA and are known to possess an activated c-ras'@― mutation (17). In contrast, BK-l cells were derived from a primary culture of newborn keratinocytes and do not have an activated c@ras@Iamutation (18). SP-l cells form papillomaswhen graftedto athymicnude mice; however, no tumors develop from BK-i cells following s.c. injections in nude mice or transplantation in skin grafts. SP-l cells were maintained in low-calcium Eagle's MEM (Whittaker Bioproducts, Inc.) containing 0.05 mr@i calcium and 8% Chelex-treated fetal bovine serum. BK-l cells were grown in SP-l medium but supplemented with 10 ng/ml epidermal growth factor and conditioned medium collected from primary cultures of murine dermal fibroblasts. Replication-defective retroviral vectors pLXSN (19) and pLOSN (10) were used to infect the epidermal cells. The retroviral vector pLXSN contains the neomycin phosphotransferase gene that confers neor. The pLOSN vector contains the neor gene, as well as the mouse ODC cDNA containing an introduced stop codon at position 425, which results in a truncated, more stable ODC protein with full enzymatic activity (20). Cells were infected with pLXSN or pLOSN for 4 h with 4 p.g/ml polybrene. After two days, the cells were selected with G418 (120 p.g/ml) for 3—7 days. Three days after refeeding with serum-containing medium, cells were assayed for ODC enzyme activity by quantifying the production of ‘4C02 from L-['4C]omithine (New England Nuclear) in cell lysates (3). The presence of a truncated ODC protein with a lower molecular weight than endogenous ODC was confirmed in cells infected with the pLOSN virus by immunoblot analysis as reported previously (10). TrachealTransplantProcedure.Tracheal transplants werepreparedas previously described by Terzaghi et a!. (15) and modified by Momiki et a!. (16). Rattracheas(Zivic-MillerLaboratories)weremountedon Teflon tubing 2104 Research. on February 23, 2016. © 1997 American Association for Cancer cancerres.aacrjournals.org Downloaded from