The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase

Abstract

The levels and distribution of ubiquitinated histone H2A (uH2A) have been studied in normal and transformed human cells using a monoclonal antibody (mAb E6C5) that reacts specifically with this ubiquitin conjugate as determined by two-dimensional gel western blotting and microsequencing. Immunoblotting experiments demonstrated that the levels of the protein are highly upregulated in SV40-transformed human fibroblasts (WI-38 SV40) and keratinocytes (K14) relative to their normal counterparts, a finding that was further confirmed by indirect immunofluorescence studies of formaldehyde/Triton X-100-treated cells, which showed that about 97% of the transformed cells and 26% of the normal populations reacted with the antibody to yield a fine granular nuclear staining associated with the chromatin. Transformed cells contained in addition clusters of uH2A that were quite abundant and that showed variable size, shape and distribution even within a single cell line. The clusters, which were rare in normal cells, did not colocalize with other known nuclear antigens and may correspond to novel nuclear domains where ubiquitination/deubiquitination takes place. Electron microscopic immunocytochemistry of K14 cells confirmed the existence of the clusters. Double immunofluorescence studies of K14 keratinocytes with proliferating cell nuclear antigen (PCNA)/cyclin antibodies, which react with the nuclei of cells engaged in DNA replication, showed partial colocalization of PCNA/cyclin foci and large uH2A clusters in about 14% of the S-phase cells, and these corresponded mainly to late S-phase cells. Inhibition of DNA replication with hydroxyurea resulted in an overall increase in the intensity of the uH2A staining as well as in a more clear colocalization of uH2A clusters and PCNA/cyclin foci. Taken together, the results support the contention that uH2A plays a role at some stage of DNA replication.