The objective 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-OAc0-AAF), a potent frameshift mutagen, induced conversion of the EBV immortalized lymphocytes into high grade """"""""immunoblastic lymphomas"""""""" on injection into athymic mice, whereas injection of the untreated, original cells, did not. The tumor cells were all of the B cell lineage as determined by the presence of surface immunoglobulines and antigens detected by B cell specific antibodies to B1 and B4 and the absence of the T cell specific markers, 3A1 and LEU-1. The N-OAc-AAF-induced tumor lines displayed abnormal diploid to teraploid karyotypes. The fewest chromosomal rearrangement, excluding tetraploidy, observed in these chemically induced lymphomas involved a deletion in chromosome 6 and additions on both 16 and 4. Neither major rearrangements nor amplifications were found for K-ras, H-ras, N-ras, c-myc, Blym and c-myb in these tumor lines. In order to determine the transforming genetic element(s) in our chemically induced lymphoma cells, we have transfected high molecular weight DNA derived from both the N-OAc-AAF treated CB23 (A23-15) and the original CB23 cell lines into NIH3T3 and have screened the resulting foci for the presence of oncogenes, including EBV fragments. The first round of transfection with DNA isolated from A23-15 cells gave rise to typical multilayered, well-defined foci. No foci were seen following transfection with DNA from the original untreated CB23 cells. Southern blot analysis of genomic DNAs derived from these transfected foci revealed the presence of human DNA as noted by Alu specific repeat sequences, yet none of the above-mentioned oncogenes were found in any of the transfected cells.
