Cell. Vol. 25, 355-361, August 1981, Copyright 0 1981 by MIT Three Different Human Tumor Cell Lines Contain Different Oncogenes Mark J. Murray, Ben-Zion Shilo, Chiaho Shih, Deborah Cowing, Ho Wen Hsu* and Robert A. Weinberg Center for Cancer Research and Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Summary We have obtained foci of transformed mouse cells after transfection of human DNA from colon and bladder carcinoma cell lines and a promyelocytic leukemia cell line. These foci can be shown to contain a large number of human DNA sequences by use of highly repetitive human DNA sequence probes. Cell DNA from primary foci can be used in a subsequent cycle of transfection resulting in sec- ondary foci that contain relatively little human DNA. Secondary foci appear to contain only the human sequences proximal to those responsible for the transformed phenotype. A set of characteristic DNA restriction fragments is found in common among secondary foci derived from each tumor cell line DNA. Comparison of the common DNA fragments found in secondary foci derived from three different human tumor cell lines indicates that these three cell lines contain three different transforming genes. introduction The ability to transfer a selectable phenotype to a recipient cell by gene-transfer techniques has made it possible to passage a variety of mammalian genes (Wigler et al., 1978, 1979a, 1980; Graf et al., 1979; Lewis et al., 1980). Recently, this approach has been applied to the study of oncogenic transformation. In- vestigators in our laboratory (Shih et al., 1979, 1981) and others (Cooper et al., 1980; Krontiris and Cooper, 1981) have shown that DNAs from nonvirally trans- formed cells are able to induce transformation of normal mouse fibroblasts. Initial work in this area involved the transfer of the oncogenic phenotype of 3-methylcholanthrene-transformed mouse fibroblasts to NIH/3T3 mouse cells by DNA transfection (Shih et al., 1979). The successful passaging of these traits via gene-transfer procedures suggests the presence, in these cells, of DNA sequences that behave upon transfection like some well studied viral transforming genes (Klein, 1980). Moreover, this work demon- strated the ability of the transforming DNA to act across tissue and species barriers. For example, DNA extracted from a human carcinoma cell line was able * Present address: College of Physicians and Surgeons, Columbia University, New York, New York to transform mouse fibroblasts (Shih et al., 1981; Krontiris and Cooper, 1981). Initial arguments for the authenticity of transformed foci rested on the efficiency of induction of foci in the recipient monolayers. DNAs prepared from these foci also were able to generate foci efficiently in subse- quent cycles of transfection. To provide a biochemical verification for the presence of transformed donor ceil DNA in the recipient cells, we added many copies of a cloned DNA sequence (for example, pBR322 plas- mid DNA) to the donor DNA prior to transfection. The resulting foci could be shown by Southern blotting to contain copies of the cotransfected plasmid DNA (Shilo et al., 1980). This cotransfected plasmid DNA therefore served as a “sequence tag,” signaling the presence of foreign DNAs in the recipient cell. These data showed that these foci belonged to the small fraction (0.1%) of recipient cells in the transfected culture that takes up and stabilizes donor DNA (Per- ucho et al., 1980b). It was concluded therefore that the transformation of these cells depended upon the introduction of exogenous DNA sequences. An alternative biochemical verification was made possible when human tumor cell DNAs were found to be able to induce transformation in recipient mouse cells (Shih et al., 1981; Krontiris and Cooper, 1981). Many human genes are punctuated by arrays of highly repeated sequences scattered throughout the human genome (Jelinek et al., 1980; Schmid and Deininger, 1975; Tashima et al., 1981). The most abundant family of these sequences has been termed the “Alu” family by virtue of an Alu I restriction enzyme site found in a high proportion of these repeated se- quences (Houck et al., 1979). These “Alu” se- quences are 300 base pairs long and are present in as many as 600,000 copies per haploid cell genome (Rinehart et al., 1981). They are found, on average, every few kilobases throughout the genome. These human repetitive sequences can be specifically de- tected in the mouse cell genetic background (Shih et al., 1981). They therefore serve as naturally occurring “sequence tags” that indicate the presence of human DNA in the mouse genetic background. The human repeat DNA sequence blocs provide a powerful analytic tool in addition to the verification of successful gene transfer. They provide a means to characterize the structural outline of the human tumor genes in the mouse recipient cells. Analysis of these structural outlines has allowed us to draw several conclusions concerning these genes. Perhaps the most salient of these conclusions is that different distinct genes appear to be responsible for the onco- genie transformation observed in different types of human tumor cell lines. Results In a previous report, we demonstrated that the DNA of a human bladder carcinoma cell line was able to