Comparative analysis of pre‐replication complex proteins in transformed and normal cells

Abstract

This study examines the abundance of the major protein constituents of the pre-replication complex (pre-RC), both genome-wide and in association with specific replication origins, namely the lamin B2, c-myc, 20mer1, and 20mer2 origins. Several pre-RC protein components, namely ORC1-6, Cdc6, Cdt1, MCM4, MCM7, as well as additional replication proteins, such as Ku70/86, 14-3-3, Cdc45, and PCNA, were comparatively and quantitatively analyzed in both transformed and normal cells. The results show that these proteins are overexpressed and more abundantly bound to chromatin in the transformed compared to normal cells. Interestingly, the 20mer1, 20mer2, and c-myc origins exhibited a two- to threefold greater origin activity and a two- to threefold greater in vivo association of the pre-RC proteins with these origins in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited both similar levels of activity and in vivo association of these pre-RC proteins in both cell types. Overall, the results indicate that cellular transformation is associated with an overexpression and increased chromatin association of the pre-RC proteins. This study is significant, because it represents the most systematic comprehensive analysis done to date, using multiple replication proteins and different replication origins in both normal and transformed cell lines.

Diminished Origin-Licensing Capacity Specifically Sensitizes Tumor Cells to Replication Stress

“…Consistent with previous findings, we observed that 3 tumor-derived cell lines showed high ORC1 expression compared with nontumor lines (24)(25)(26)(27). In general, higher ORC1 levels were required to maintain viability in the tumor lines (although MRC-5 cells also had a requirement for a higher ORC1 level).…”

“…Most tumor cells suffer high replication stress, due to uncontrolled proliferation and/or enhanced genomic instability. Interestingly, several studies have reported that origin-licensing proteins are overexpressed in tumor-derived cell lines (24)(25)(26)(27). Given this, we reasoned that tumor cells might have a greater demand for origin licensing than nontransformed cells, either to sustain rapid replication and/or to enhance recovery from the increased level of replication stalling/collapse.…”

“…1C; ref. [24][25][26][27]. Although a-ORC1 antibodies are inefficient for immunoblotting, a marked reduction in ORC1 protein could be observed in both lines following siORC1 (Fig.…”

Suppression of the DHX9 Helicase Induces Premature Senescence in Human Diploid Fibroblasts in a p53-dependent Manner

“…Chromatin Immunoprecipitation (ChIP) Assays-Cells cultured in complete media were washed with pre-warmed PBS and treated with 1% formaldehyde for 10 min to cross-link proteins and DNA in vivo (56). They were then washed and scraped into ice-cold PBS and resuspended in lysis buffer (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1% Triton X-100, 2 mM EDTA) supplemented with a complete protease inhibitor tablet (Roche Applied Science).…”

Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy

“…53 Plk1 upregulation is so widespread in malignancies that it has been termed a 'general feature of human cancer.' 54 Although ORC1 and MCM5 display aberrant expression in certain transformed cells, 55 MCM5 is consistently upregulated in cancers and is considered a biomarker. 56,57 Much evidence also links deregulation of centrosome licensing factors with centriole overduplication.…”