Abstract
To study the possible relationships between origin activities and cellular processes leading to malignancy, we used an isogenic system of human embryo lung fibroblast cells WI38 and a SV40โtransformed variant, WI38 VA13 2RA (WI38(SV40)). We found that the activities of all initiation sites at the cโmyc locus were approximately twoโfold as high in WI38(SV40) cells as in WI38 cells. Thus, higher initiation frequency of origins at certain loci is induced with cell immortalization, one of the steps in the multiโstep process leading to malignancy. We measured the activities of the four cโmyc promoters P0, P1, P2, and P3 with nuclear runon assay in the two cell lines in order to detect potential individual promoter changes that may be also associated with immortalization by SV40 virus. The results show that the activities of the promoters P0, P1, and P3 did not significantly change, but the activity of the major promoter P2 in WI38(SV40) cells was about 7.5โ to 8.0โfold as high as that in WI38 cells. The increased activity of promoter P2, although โผ600 bp downstream of one of the major DNA replication initiation sites, had no preferential influence on the major sites of origin activity. Since the distribution of nascent strand abundance was not significantly altered, binding of transcription factors does not seem to facilitate the assembly of preโreplication complex (preโRC) or otherwise preferentially alter the activities of the DNA replication proteins at this major initiation site. J. Cell. Biochem. 82:522โ534, 2001. ยฉ 2001 WileyโLiss, Inc.