title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, S.U.N.Y. Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: McNairn
authors.forename: David M
authors.initials: DM
authors.lastname: Gilbert
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051738:Origin Recognition Complex; D016196:S Phase; D013347:Subcellular Fractions; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D002843:Chromatin; D051738:Origin Recognition Complex