Immortalization of human W138 cells is associated with differential activation of the c-myc origins

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 W138 and a SV40-transformed variant, WI38 VA13 2RA ( WI38(SV40)). We found that the activities of all initiation sites at the c- myc locus were approximately two-fold as high in WI38(SV40) cells as in WI3 8 cells. Thus, higher initiation frequency of origins at certain loci is in duced with cell immortalization, one of the steps in the multi-step process leading to malignancy. We measured the activities of the four c-myc promot ers P0, P1, P2, and P3 with nuclear runon assay in the two cell lines in or der to detect potential individual promoter changes that may be also associ ated with immortalization by SV40 virus. The results show that the activiti es of the promoters Po, P1, and P3 did not significantly change, but the ac tivity of the major promoter P2 in WI38(SV40) cells was about 7.5- to 8.0-f old as high as that in W138 cells. The increased activity of promoter P2, a lthough similar to 600 bp downstream of one of the major DNA replication in itiation sites, had no preferential influence on the major sites of origin activity. Since the distribution of nascent strand abundance was not signif icantly altered, binding of transcription factors does not seem to facilita te the assembly of pre-replication complex (pre-RC) or otherwise preferenti ally after the activities of the DNA replication proteins at this major ini tiation site. (C) 2001 Wiley-Liss, Inc.