Overexpression of ORC subunits and increased ORC‐chromatin association in transformed mammalian cells

Abstract

The origin recognition complex (ORC) is a conserved heterohexamer required for the formation of pre-replication (pre-RC) complexes at origins of DNA replication. Many studies of ORC subunits have been carried out in transformed human cell lines but the properties of ORC in primary cells have not been addressed. Here, we compare the expression levels and chromatin-association of ORC subunits in HeLa cells to the primary human cell line, WI38, and a virally transformed derivative of WI38, VA13. ORC subunits 2 and 4 were highly overexpressed in both HeLa and VA13, whereas ORC1 levels were elevated in VA13 but considerably higher in HeLa cells. Cellular extraction revealed that the proportion of ORC2 and ORC4 subunits bound to chromatin was similar in all three cell lines throughout the cell-cycle. In contrast, very little ORC1 was associated with chromatin after extraction of primary WI38 cells, whereas the majority of overexpressed ORC1 in both HeLa and VA13 co-fractionated with chromatin throughout the cell-cycle. Although none of the cell lines displayed significant changes in the levels or chromatin-association of ORC during the cell-cycle, the chromatin-associated fraction of ORC1 displayed an increase in apparent molecular weight during S-phase. Similar experiments comparing immortalized CHO cells to an isogenic virally transformed derivative revealed no changes in levels of ORC subunits but an increase in the proportion of all three ORC subunits associated with chromatin. These results demonstrate a complex influence of cellular immortalization and transformation properties on the expression and regulation of ORC subunits. These results extend the potential link between cancer and deregulation of pre-RC proteins, and underscore the importance of considering the transformation status of cell lines when working with these proteins.

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.…”

Genetic Analysis of Human Orc2 Reveals Specific Domains That Are Required in Vivo for Assembly and Nuclear Localization of the Origin Recognition Complex

“…Therefore, the cellular concentration of ORC in HeLa cells must be in excess of what is needed for cell proliferation. In fact, the relative levels of ORC among human cell lines can vary significantly (30), and the level of chromatin bound ORC in a human colon carcinoma cell line can be reduced 3-fold with only a 30% decrease in the rate of cell proliferation (31). Therefore, binding of ORC-(2-5) to chromatin must require other factors in addition to Orc1.…”

Control of the Cdc6 replication licensing factor in metazoa: The role of nuclear export and the CUL4 ubiquitin ligase

“…The degradation of ORC1 may not be a consistent regulatory feature, as it is observed in some cells but not in others. [18][19][20] The importance of Cdc6 nuclear export is controversial due to conflicting reports on the extent of nuclear export, as described below. Overexpression of a non-degradable Cdt1 produces limited DNA re-replication (significantly less than in fission yeast).…”