Targeted deletion of the gene encoding the La autoantigen (Sjögren's syndrome antigen B) in B cells or the frontal brain causes extensive tissue loss.

La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1(Cre) La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types.

Localization of BRCA1 protein in breast cancer tissue and cell lines with mutations.

BACKGROUND: The breast and ovarian cancer susceptibility gene (BRCA1) encodes a tumor suppressor. The BRCA1 protein is found primarily in cell nuclei and plays an important role in the DNA damage response and transcriptional regulation. Deficiencies in DNA repair capabilities have been associated with higher histopathological grade and worse prognosis in breast cancer. METHODS: In order to investigate the subcellular distribution of BRCA1 in tumor tissue we randomly selected 22 breast carcinomas and tested BRCA1 protein localization in frozen and contiguous formalin-fixed, paraffin embedded (FFPE) tissue, using pressure cooker antigen-retrieval and the MS110 antibody staining. To assess the impact of BRCA1 germline mutations on protein localization, we retrospectively tested 16 of the tumor specimens to determine whether they contained the common Ashkenazi Jewish founder mutations in BRCA1 (185delAG, 5382insC), and BRCA2 (6174delT). We also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC). RESULTS: In FFPE tissue, with MS110 antibody staining, we frequently found reduced BRCA1 nuclear staining in breast tumor tissue compared to normal tissue, and less BRCA1 staining with higher histological grade in the tumors. However, in the frozen sections, BRCA1 antibody staining showed punctate, intra-nuclear granules in varying numbers of tumor, lactating, and normal cells. Two mutation carriers were identified and were confirmed by gene sequencing. We have also compared co-localization of BRCA1 and nucleolin in MCF7 cells (wild type) and a mutant BRCA1 cell line, HCC1937 (5382insC) and found altered sub-nuclear and nucleolar localization patterns consistent with a functional impact of the mutation on protein localization. CONCLUSIONS: The data presented here support a role for BRCA1 in the pathogenesis of sporadic and inherited breast cancers. The use of well-characterized reagents may lead to further insights into the function of BRCA1 and possibly the further development of targeted therapeutics.

BRCA1 protein and nucleolin colocalize in breast carcinoma tissue and cancer cell lines.

The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. Immunoelectron microscopic studies of estrogen-stimulated MCF7 cells demonstrated BRCA1 protein localization in the granular components of the nucleolus. Moreover, immunofluorescence of BRCA1 and nucleolin double-labeling showed colocalization in both nucleoli and nucleoplasmic foci in breast tumor cells and asynchronously growing MCF7 and HeLa cells. Multiparameter analysis of BRCA1 and nucleolin in relation to cell cycle position (DNA content) showed expression during G1-S and persistence of BRCA1 during G2/M. After gamma-irradiation of MCF7 cells, BRCA1 protein dispersed from nucleoli and nucleoplasmic foci to other nucleoplasmic sites, which did not colocalize with nucleolin. Small interfering RNA-mediated knockdown of BRCA1 protein resulted in decreased immunofluorescence staining, which was confirmed by Western blotting. The observed colocalization of BRCA1 and nucleolin raises new possibilities for the nucleoplasm-nucleolus pathways of these proteins and their functional significance.