(CANCER RESEARCH 56. 5490-5498. December I. 19961 ABSTRACT The putative role of the CAPL gene in enhancing the development of human cancer metastasis was examined by transfecting human high expressing osteosarcoma cells with a hammerhead ribozyme directed against the gene transcript. The ability of the ribozyme to cleave target mRNA in intact cells was demonstrated in a 5'-rapid amplification of cDNA ends assay. In transfected cells, a suppression ofthe capacity to give skeletal metastases upon intracardial injection into nude rats was ob served in cell clones with reduced expression of CAPL mRNA and protein, whereas in vitro and in vivo cell proliferation and tumorigenicity were unchanged. The results provide direct evidence that the expression level of the CAPL-encoded protein can determine the metastatic potential of osteosarcoma cells, and they demonstrate an association between reduced gene expression and proliferation-independent inhibition ofthe metastatic capacity ofhuman tumor cells. The effects ofthe specific cleavage of CAPL mRNA indicate that the gene product is involved in key cellular functions associated with the metastatic process and suggest that therapeutic mod ulation of the protein function may represent a novel approach for inhib iting the metastatic spread of cancer cells. INTRODUCTION One of the most challenging tasks in cancer research is to acquire more insight into mechanisms involved in the metastatic process, in which detached cells from the primary tumor can establish overt metastases in distant organs. The process of metastasis involves a cascade of genetic alterations, and expression of the metastatic phe notype depends on a balance between regulatory elements that pro mote or inhibit the process at one or more of the sequential steps. In recent years, several genes thought to be associated with metastasis have been identified (1—3).One of these, mtsl, was cloned from murine mammary carcinoma cells by subtractive colony hybridization and was shown to be specifically expressed in cell variants with high metastatic capacity (4). Transfection of p9Ka, the rat homologue of mis!, into benign rat mammary epithelial cells resulted in increased tumongenicity, proliferation rate and metastasis formation in some of the animals (5). The mis! gene encodes a protein belonging to the SlOO family of Ca2tbinding proteins (4), and it has been suggested that other SlOO proteins, also, may be of relevance for neoplastic progression, tumor igenicity, and/or metastatic potential (6, 7). Although the normal cellular functions of the S 100 proteins are not known, involvement in signal transduction pathways and regulation of cell growth and dif ferentiation have been postulated (8). Recently, Ca2@/Sl00 was sug gested as a physiological activator of a family of giant protein kinases involved in muscle contractions and cytoskeletal structure (9). Inter estingly, it has been proposed that mtsl and its rat homologue may Received 7/ 1/96; accepted I0/3/96. 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. @ This work was supported by the Norwegian Cancer Society, The Jahre Foundation, the National Institutes of Health (CA 50618), Otto and Rakel Bruuns Legacy Gene Shears Ltd.theDanishCancerSocietyandEC. 2 To whom requests for reprints should be addressed. modulate cell motility and influence the invasive and metastatic properties of rodent tumor cells by interacting with components of the cytoskeleton, and filamentous actin (5), myosin (10), and tropomyosin (1 1) have been suggested as target molecules for the mtsI-encoded protein. The human counterpart of mis!, CAPL (also called SIOOA4), is localized to chromosome 1q21 in a cluster together with at least 12 other S100 genes (12, 13). Based on indirect evidence (namely, an observed correlation between enhanced expression of the gene and the presence of the invasive marker urokinase-type plasminogen activa tor) the CAPL-encoded protein was suggested as a marker for breast cancer prognosis (14). We have found the mRNA to be differentially expressed in biopsies from human melanomas, mammary carcinomas, and @ In the two latter types of cancer, a trend toward higher expression in the more advanced lesions were observed.4 However, no direct or experimental evidence for the involvement of CAPL in human tumor metastasis has been provided. To examine this, the effects of specific suppression of CAPL expression was investi gated by transfecting a CAPL-directed hammerhead ribozyme into OHS5 cells that have well defined in viiro and in vivo growth char acteristics with relevance to the metastatic process in humans (15, 16). The observed ribozyme-induced inhibition of the ability to form symptom-giving bone metastasis is highly encouraging and warrants further investigation of the significance of the CAPL gene product in the metastatic process. MATERIALS AND METHODS In Vitro Cell Growth. The OHS cell line was established from a bone tumor biopsy obtained from a patient treated at the Norwegian Radium Hospital and grown as monolayer cultures as described previously (15). Growth curves were constructed, and the cell doubling time was measured from the exponential part of the curves. Cultivation in soft agar was performed (17), and the plating efficiency was defined as the number of colonies formed in percentage of the number viable cells plated (no. of colonies formed/no. of cells plated x 100%). In Vivo Cell Growth. Single-cell suspensions were obtained from subcon fluent monolayer cultures, and I X 106cells were injected s.c. into the flanks of nude mice (18) or intracardially into the lv. of immunodeficient rats (19). The volume of the s.c. growing tumors was calculated according to the formula: 0.5 x length X width2 (20). After lv. injections in rats, the animals were inspected daily and sacrificed at the first sign of paresis or walking impairment, symptoms reflecting metastatic disease in the spine. No metastasis formation in other organs occurs upon lv. injection of the OHS cells. All animals were bred in our own nude rodent facility and kept under specific pathogen-free conditions. Food and water were supplied ad libitum. Housing and all procedures involving animals were performed according to protocols approved by the National Ethical Committee on animal welfare. 3 G. M. Mulandsmo, V. A. Flørenes, T. Mellingsnther, E. Hovig. R. S. Kerbel, and 0. Fodstad.Differentialexpressionpatternsof the 5100genesCACY, CAPLand SICK)L during progression of malignant melanoma, submitted for publication. 4 G. M. Mnlandsmo, V. A. Flørenes, E. Hovig, and 0. Fodstad. unpublished data. 5 The abbreviations used are: OHS, high CAPL expressing human osteosarcoma cell line; rib, ribozyme; lv., left ventricle; RACE, rapid amplification of cDNA ends; RT, reverse transcription; nt, nucleotide(s); pH@3.pH@Apr-l-neo. 5490 Reversal of the in Vivo Metastatic Phenotype of Human Tumor Cells by an Anti CAPL (mtsl) Ribozyme1 Gunhild Marl Ma1andsmo, Eivind Hovig, Martina Skrede, Olav Engebraaten, Vivi Ann Flørenes, Ola Mykiebost, Mariam Grigorian, Eugene Lukanidin, Kevin J. Scanlon, and Oystein Fodstad2 Department of Tumor Biology. the Norwegian Radium Hospital. 0310 Oslo. Norway 1G. M. M., E. H., M. S., 0. E., V. A. F., 0. M.. 0. F.); Department of Molecular Cancer Biology, the Fibiger Institute. 2100 copenhagen. Denmark [M. G.. E. LI: and Berlex Biosciences, Department of Cancer Research, Richmond, California 94804-0999 [K. J. S.) Research. on January 20, 2022. © 1996 American Association for Cancer cancerres.aacrjournals.org Downloaded from