The mouse Rad5l gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad5l null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad5Sl/-) pups among 148 neonates examined but a few Rad5-/-embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad5l null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.Genetic recombination leads to new associations of genetic elements. In meiosis, recombination between closely paired homologous chromosomes results in extensive reshuffling of paternal and maternal genes, and the progeny can be better fitted to cope with the environment. Recombination occurring in somatic cells is manifested as sister chromatid exchange and the outcome, by itself, does not alter the cellular genotype.Molecular mechanisms of recombination have been studied extensively in bacteria and lower eukaryotes. The recA gene of Escherichia coli plays an essential role in recombination as well as in DNA repair and induction of SOS functions (1-3). The RecA protein has the potential to promote homologous pairing and strand exchange of DNA in the presence of adenosine 5'-triphosphate (ATP) (2-6). In yeast Saccharomyces cerevisiae, RAD51, RAD52, and RAD54 genes, belonging to the RAD52 epistasis group, were initially identified as those involved in the repair of DNA damage induced by ionizing radiation (7,8), and subsequently were shown to be responsible for mitotic recombination (9-12). Among them the RAD51 gene is a homologue of the E. coli recA gene and plays crucial roles in both mitotic and meiotic recombination as well as in repair of double-strand breaks of Isolation of Targeted ES Cell Clones. The ES cell line CCE was cultured on a feeder cell layer and electroporated, using 5 X 107 cells and 50 ,tg of the linearized targeting vector DNA, as described (25,26). Colonies doubly resistant to G418 (250 ,ug/ml) and ganciclovir (5 ,uM) were selected and expanded on feeder layers in 24-well plates. Homologous recombinants were identified by Southern blot analysis of restriction enzymedigested DNA. DNAs were prepared from cells cultured in the absence of feeder cells and subjected to Southern blot analysis.The DNA (8 jig) was cleaved with BamHI, subjected to agarose gel electrophoresis, blotted onto Hybond N+ membrane (Amersham), and hybridized to probe A. To ensure targeted disruption of the Rad5l gene, the DNA was digested with EcoRV or HindIll, followed by hybridization with ...

The resistance of parathyroid cells to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in uremic hyperparathyroidism is thought to be caused, in part, by a 1,25(OH)2D3 receptor (VDR) deficiency in the parathyroids. However, results of biochemical studies addressing VDR numbers in the parathyroids are controversial. Several studies have found VDR content to be decreased in the parathyroids of uremic patients and animals, while others have found no such decrease in the parathyroids of uremic animals.To clarify the role of VDR, we investigated VDR distribution in surgically-excised parathyroids obtained from chronic dialysis patients by immunohistochemistry. We classified the parathyroids as exhibiting nodular or diffuse hyperplasia. Our studies demonstrated a lower density of VDR in the parathyroids showing nodular hyperplasia than in those showing diffuse hyperplasia. Even in the parathyroids showing diffuse hyperplasia, nodule-forming areas were present; these areas were virtually negative for VDR staining. A significant negative correlation was found between VDR density and the weight of the parathyroids. These findings indicate that the conflicting results of biochemical studies may be caused by the heterogeneous distribution of VDR; the decreased VDR density in parathyroids may contribute to the progression of secondary hyperparathyroidism and to the proliferation of parathyroid cells that is seen in uremia. (J. Clin.

The occurrence of inactivating mutations in SWI/SNF chromatin-remodeling genes in common cancers has attracted a great deal of interest. However, mechanistic strategies to target tumor cells carrying such mutations are yet to be developed. This study proposes a synthetic-lethality therapy for treating cancers deficient in the SWI/ SNF catalytic (ATPase) subunit, BRG1/SMARCA4. The strategy relies upon inhibition of BRM/SMARCA2, another catalytic SWI/SNF subunit with a BRG1-related activity. Immunohistochemical analysis of a cohort of non-smallcell lung carcinomas (NSCLC) indicated that 15.5% (16 of 103) of the cohort, corresponding to preferentially undifferentiated tumors, was deficient in BRG1 expression. All BRG1-deficient cases were negative for alterations in known therapeutic target genes, for example, EGFR and DDR2 gene mutations, ALK gene fusions, or FGFR1 gene amplifications. RNA interference (RNAi)-mediated silencing of BRM suppressed the growth of BRG1-deficient cancer cells relative to BRG1-proficient cancer cells, inducing senescence via activation of p21/CDKN1A. This growth suppression was reversed by transduction of wild-type but not ATPase-deficient BRG1. In support of these in vitro results, a conditional RNAi study conducted in vivo revealed that BRM depletion suppressed the growth of BRG1-deficient tumor xenografts. Our results offer a rationale to develop BRM-ATPase inhibitors as a strategy to treat BRG1/SMARCA4-deficient cancers, including NSCLCs that lack mutations in presently known therapeutic target genes. Cancer Res; 73(17); 5508-18. Ó2013 AACR.