CHEMICAL TRANSFORMATION OF HUMAN LYMPHOBLASTOID CELL LINES

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The object of this project is to characterize the nature of chemically induced transition from a benign hyperproliferative to a malignant state in Epstein-Barr virus (EBV) immortalized human lymphocytes. Treatment with N-acetoxy-2-acetyl-aminofluorene (N-OAc-AAF), a potent frameshift mutagen, induced conversion of the EBV immortalized lymphocytes into high grade "immunoblastic lymphomas" upon injection into athymic mice, whereas injection of the untreated, original cells did not. The tumor cells were all of the B-cell lineage. High molecular weight DNA from the chemically transformed EBV immortalized human cord blood lymphocytes was co-transfected with pSV2Neo into NIH 3T3 cells. After 2 weeks selection in 250 micrograms/ml G418, 10 foci were cloned out and expanded. These foci were positive for soft agar growth and gave rise to fibrosarcomas within 2 weeks in athymic mice. The control NIH 3T3 cells were incapable of anchorage-independent growth or tumor formation. Subsequent Southern analysis revealed that the primary transfected 3T3 cells contained approximately 95 kbp of human DNA as indicated by hybridization to the human repeat sequence, Blue 8. Screening of the foci for various oncogenes revealed that the 3' end of c-raf-1 was present in the transfected cells. This constitutes the first report of chemical activation of the c-raf-1 oncogene in human cell lines.