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.
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.
Vassilev
A P
AP
Institute of Medical Biochemistry, University of Aarhus, Denmark.
Rasmussen
H H
HH
Christensen
E I
EI
Nielsen
S
S
Celis
J E
JE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000235:genetics; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D004261:DNA Replication; D006657:Histones / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D016196:S Phase; D014452:Ubiquitins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; D000941:Antigens; D006657:Histones; D018809:Proliferating Cell Nuclear Antigen; D014452:Ubiquitins; C021645:chromatin conjugate protein A24