Immortalization of human WI38 cells is associated with differential activation of the c‐myc origins

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 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 WI38 cells. Thus, higher initiation frequency of origins at certain loci is induced with cell immortalization, one of the steps in the multi-step process leading to malignancy. We measured the activities of the four c-myc 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 7.5- to 8.0-fold as high as that in WI38 cells. The increased activity of promoter P2, although approximately 600 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 pre-replication complex (pre-RC) or otherwise preferentially alter the activities of the DNA replication proteins at this major initiation site.

Differentially Active Origins of DNA Replication in Tumor versus Normal Cells

“…25). Although the relative nascent DNA abundance across 12.5 kb of the human c-myc locus had a similar distribution, a 2-fold higher abundance was found in HeLa than in NSF cells at the majority of the initiation sites tested across the c-myc locus (26) 2-fold higher abundance in the transformed [WI38(SV40)] than in the normal (WI38) cells at the majority of sites tested across the c-myc locus, eliminating the possibility that cell type effects were responsible for the observed differences in origin activities in HeLa and NSF cells (27). Overall, these findings suggested that cellular transformation might induce greater frequency of initiation of origins in certain loci.…”

“…2B, lane 3; refs. 26,27); the plasmid DNA was completely phosphorylated and digested by E exonuclease (Fig. 2B, lane 3 versus lane 4), and residual RNA (Fig.…”

“…(Note that this is a representative graph; the copy number of all the primer sets used in this study was measured; see Table 3 To assess the quality of nascent DNA samples, the distribution of nascent DNA from an origin region associated with the lamin B2 locus was quantified. This origin was shown to be active in a number of different proliferating human cells (26,27,41,43,44). Nascent DNA was measured at two reference points, one located at the center of the origin (LB2 primer set = peak activity), the other located f4 kb away (LB2C1 primer set = background activity), using the primer sets described by Ladenburger et al (45).…”

Regulation of Mouse 4-1BB Expression: Multiple Promoter Usages and a Splice Variant

“…The mouse lck proximal promoter was required for tissue-specific expression in the thymus and mtβ was a critical transactivator for the proximal promoter (Reynolds et al, 1990;Yamada et al, 2001). The phenomenon of a differential expression pattern from a single gene by alternative promoters is very common, and is often related to tissuespecific expression of that gene (Christophi et al, 2004;Hai et al, 2001;Jian et al, 2006;Tao et al, 2001;Xiao et al, 2003).…”

A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

“…Indeed, increased activity of DNA replication origins in tumor cells or SV-40 transformed cells have been reported, suggesting that transformation may induce greater frequency of initiation of origins at certain loci. [39][40][41][42] The increased origin activity in tumor/transformed cells compared to normal cells may be influenced by many parameters including concentration and activity of initiation factors, chromatin structure and nuclear organization. 41 Potentially, FA proteins might be involved in these processes to regulate initiation of DNA replication.…”