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Cancer cell lines are an essential tool of cancer research, but their origin can sometimes be difficult to determine with certainty. In addition to contamination with other cell lines duringin vitro cultivation, errors can arise during the initial resection from the patient because the organ of origin can sometimes be difficult to determine. This is particularly problematic for ovarian cancer, where a substantial fraction of the cases represent metastases from distant nongenital sites(1). Once in culture and without detailed patient data, it can be difficult or impossible to trace the origin of a particular cell population. The difficulty arises from the fact that cells tend to lose their tissue-specific markers during tumorigenesis and in vitro establishment. In the course of our study of b-catenin signaling in ovarian cancer, we discovered several ovarian cancer lines that have constitutively elevated b-catenin-mediated transcription (Furlong MT, Morin PJ: unpublished results). Deregulation of this pathway results in inappropriate activation of T-cell factor-responsive genes and, in colon cancer, is due to mutations either in b-catenin or in the tumor suppressor gene adenomatous polyposis coli (APC) (2,3). Our finding of ovarian cell lines with an activatedb-catenin/T-cell factor pathway is consistent with a recent report (4) that b-catenin is mutated in a subset of ovarian cancers. SW626, a well-studied ovarian cancer cell line, showed high levels ofb-cateninmediated transcriptional activation but contained a wild-typeb-catenin gene, as assessed by direct sequencing. Surprisingly, however, when a cell lysate of SW626 was probed by immunoblotting with an anti-APC antibody, full-length APC was not detected (Fig. 1, A). Instead, SW626 expressed a truncated product of approximately 110 kd. The APC tumor suppressor gene is mutated in the vast majority of all colon cancers (5) but has never been found to be altered in ovarian cancer. Indeed, APC was previously analyzed in 40 sporadic ovarian carcinomas, and no mutations were found(6). To identify the APC mutation present in SW626, part of exon 15 was amplified from genomic DNA by the polymerase chain reaction, and the purified fragment was used as a template for direct sequencing. An insertion of one adenine in a tract of four consecutive adenines was found in the APC gene at nucleotide 2941 (A2941) (Fig. 1, B). The A2941 insertion is predicted to produce a frameshift along with the generation of an in-frame termination codon nine amino acids downstream of the adenine tract. This mutation was not found in the human gene mutation database (7) but is reminiscent of a type of mutation frequently found in colon cancer (8). It is interesting that no wild-type sequence was present, strongly suggesting hemizygosity at the APC locus. This finding is consistent with the observation that no full-length Apc protein was detected by immunoblotting (Fig. 1, A). As a control, the same region of APC was sequenced from ovarian cancer cell line A2780 and was found to be wild type (Fig. 1, B). To rule out possible contamination with a colon cancer cell line in our laboratory or at the American Type Culture Collection (Manassas, VA), an SW626 seed stock was obtained from the American Type Culture Collection, and genomic DNA was prepared directly from the frozen cells without cultivation. APC exon 15 was amplified and sequenced as described above. The A2941 insertion was present, demonstrating that this mutation is not the result of an in vitro culturing contamination. Because the above results suggested that SW626 may be of colonic origin, we decided to investigate this issue further by using immunologic markers often used to differentiate ovarian from colonic primary tumors. SW626 cells were stained with antibodies against cytokeratin (CK) 20, CK7, estrogen receptor/progesterone receptor, CA125, carcinoembryonic antigen, and Calretinin. It has been reported that CK20 and CK7 are the best markers to differentiate colonic from ovarian carcinoma(9,10). Typically, colonic carcinomas are positive for CK20 and negative for CK7; in contrast, ovarian carcinomas are usually positive for CK7 and negative for CK20. The staining pattern of SW626 was completely consistent with a colonic origin because it was positive for CK20

Evidence for the Colonic Origin of Ovarian Cancer Cell Line SW626