Oncogenes in human tumor cell line transforming gene from human blac (transfection assays/Alu repetitive sequences)

The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated fromicell lines established from carcinomas and sarcomas of various organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibro- blasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma,. respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited an- chorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phe- notype in additional cycles of transfection. Southern blot analysis of T24-derived transformants showed that a single fragment of human DNA specifically cosegregated with the malignant phe- notype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with A Charon 9A as the cloning vector. The resulting recombinant DNA mole- cule, designated AT24-15A, was shown to contain a 15-kilobase- pair EcoRI insert of human cellular DNA. AT24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of 20,000 focus-forming units per pmol of cloned DNA. Our results indicate that we have molecularly cloned a bi- ologically active oncogene present in T24 human bladder carci- noma cells.