Document Detail


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.
MedLine Citation:
PMID:  7622605     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
A P Vassilev; H H Rasmussen; E I Christensen; S Nielsen; J E Celis
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of cell science     Volume:  108 ( Pt 3)     ISSN:  0021-9533     ISO Abbreviation:  J. Cell. Sci.     Publication Date:  1995 Mar 
Date Detail:
Created Date:  1995-08-29     Completed Date:  1995-08-29     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0052457     Medline TA:  J Cell Sci     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  1205-15     Citation Subset:  IM    
Affiliation:
Institute of Medical Biochemistry, University of Aarhus, Denmark.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Antibodies, Monoclonal
Antigens / genetics
Cell Line
Cell Line, Transformed
Cell Transformation, Neoplastic
DNA Replication
Histones / genetics,  immunology,  metabolism*
Humans
Microscopy, Fluorescence
Microscopy, Immunoelectron
Molecular Sequence Data
Proliferating Cell Nuclear Antigen / metabolism*
S Phase
Ubiquitins / genetics,  immunology,  metabolism*
Up-Regulation
Chemical
Reg. No./Substance:
0/Antibodies, Monoclonal; 0/Antigens; 0/Histones; 0/Proliferating Cell Nuclear Antigen; 0/Ubiquitins; 0/chromatin conjugate protein A24

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