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1.
J Leukoc Biol ; 90(1): 5-19, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21278234

ABSTRACT

A screen of cell surface markers differentially expressed during peripheral B cell differentiation identified that the CD45RB epitope detected by the mAb MEM-55 was highly expressed on CD27(+) memory B cells and absent on CD27(-) naïve B cells. IgG(+)CD27(-) memory and a previously unacknowledged CD27(-) population in blood also expressed high levels of CD45RB(MEM55). Naïve and memory B cells from tonsils followed the pattern observed in blood, and CD38(high) B cells had a bimodal expression pattern when analyzed using flow cytometry. No CD38(high) GC B cells, however, expressed the CD45RB(MEM55) epitope when assayed using immunohistochemistry. Rather, CD38(high)CD45RB(MEM55high) B cells had a distinct cellular phenotype and were localized outside of GCs. CD45RB epitopes, detected by other antibody clones, were expressed at high levels through B cell differentiation, and no changes in splicing of the CD45RB exon were observed during B cell differentiation. Instead, B cells regulated their expression of the CD45RB(MEM55) epitope through site-specific modifications of an O-linked glycochain. CD4(+) T cells differentially spliced CD45 but did not vary the glycosylation of the CD45RB(MEM55) epitope, and CD8(+) cells modified CD45RB(MEM55) expression in a similar manner as B cells. Monocytes expressed the CD45RB exon but not the CD45RB(MEM55) epitope. As CD45 is a highly expressed tyrosine phosphatase that regulates antigen receptor signaling strength in lymphocytes, we conclude that regulated O-linked glycosylation of CD45RB can be used to follow B cell differentiation and that this regulation may be involved in fine-tuning antigen signaling in the cell.


Subject(s)
B-Lymphocyte Subsets/cytology , B-Lymphocytes/cytology , Cell Differentiation/immunology , Leukocyte Common Antigens/immunology , Leukocyte Common Antigens/metabolism , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Blotting, Western , Cell Separation , Flow Cytometry , Glycosylation , Humans , Immunohistochemistry , Immunologic Memory/immunology , Immunoprecipitation , Lymphocyte Activation/immunology , Signal Transduction
2.
DNA Repair (Amst) ; 3(7): 743-52, 2004 Jul 02.
Article in English | MEDLINE | ID: mdl-15177183

ABSTRACT

The catalytic subunit of polymerase zeta is encoded from the Rev3 gene. The enzyme is conserved through eukaryotic evolution and its main function appears to be translesion synthesis (TLS) over damaged bases that stall DNA replication. In non-vertebrate cells, inactivation of polymerase zeta results in a moderate hypersensitivity to DNA damage but no proliferative defect in the absence of exogenous damage. Mouse embryos that lack Rev3 however have a severe growth defect and are aborted at midgestation. This has suggested that polymerase zeta may be involved in vital processes in mammalian cells. Here we describe the establishment of immortalized mouse fibroblast cell lines that lack a functional Rev3 gene. These were established from homozygously Rev3-targeted mouse embryos that were also heterozygously targeted at the p53 locus, but the cell lines lost the wild type p53 allele during transformation. Cell lines in which the Rev3 gene is targeted on both alleles grow more slowly than control lines and the deficiency is also associated with an increased frequency of cells at the G2/M phase of the cell cycle and augmented apoptosis. Targeted cells are hypersensitive to UV irradiation and cisplatin treatment and arrest at the S or G2/M phase of the cell cycle if exposed to these treatments. Thus, although vital for murine embryonic development, polymerase zeta activity is not essential for continuous proliferation of transformed mammalian cells that lack p53. It does, however, appear to play an important role in allowing mammalian cells to tolerate DNA damage.


Subject(s)
Cisplatin/toxicity , DNA-Directed DNA Polymerase/deficiency , Fibroblasts/drug effects , Fibroblasts/radiation effects , Radiation-Sensitizing Agents/toxicity , Ultraviolet Rays , Animals , Apoptosis/genetics , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Division , Cell Line, Transformed , Cell Size , Cell Survival , DNA Damage/drug effects , DNA Damage/radiation effects , DNA-Directed DNA Polymerase/physiology , Female , Fibroblasts/metabolism , G2 Phase/drug effects , G2 Phase/radiation effects , Mice , Mice, Knockout , Pregnancy , S Phase/drug effects , S Phase/radiation effects , Time Factors , Tumor Suppressor Protein p53/metabolism
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