ABSTRACT
TCRbeta chain repertoire of peripheral alphabeta T cells is generated through the stepwise assembly and subsequent selection of TCRbeta V region exons during thymocyte development. To evaluate the influence of a two-step recombination process on Vbeta rearrangement and selection, we generated mice with a preassembled Dbeta1Jbeta1.1 complex on the Jbeta1(omega) allele, an endogenous TCRbeta allele that lacks the Dbeta2-Jbeta2 cluster, creating the Jbeta1(DJbeta) allele. As compared with Jbeta1(omega/omega) mice, both Jbeta1(DJbeta/omega) and Jbeta1(DJbeta/DJbeta) mice exhibited grossly normal thymocyte development and TCRbeta allelic exclusion. In addition, Vbeta rearrangements on Jbeta1(DJbeta) and Jbeta1(omega) alleles were similarly regulated by TCRbeta-mediated feedback regulation. However, in-frame VbetaDJbeta rearrangements were present at a higher level on the Jbeta1(DJbeta) alleles of Jbeta1(DJbeta/omega) alphabeta T cell hybridomas, as compared with on the Jbeta1(omega) alleles. This bias was most likely due to both an increased frequency of Vbeta-to-DJbeta rearrangements on Jbeta1(DJbeta) alleles and a preferential selection of cells with in-frame VbetaDJbeta exons assembled on Jbeta1(DJbeta) alleles during the development of Jbeta1(DJbeta/omega) alphabeta T cells. Consistent with the differential selection of in-frame VbetaDJbeta rearrangements on Jbeta1(DJbeta) alleles, the Vbeta repertoire of alphabeta T cells was significantly altered during alphabeta TCR selection in Jbeta1(DJbeta/omega) and Jbeta1(DJbeta/DJbeta) mice, as compared with in Jbeta1(omega/omega) mice. Our data indicate that the diversity of DJbeta complexes assembled during thymocyte development influences TCRbeta chain selection and peripheral Vbeta repertoire.
Subject(s)
Antibody Diversity/genetics , Gene Rearrangement, T-Lymphocyte/immunology , Immunoglobulin Joining Region/genetics , Immunoglobulin Variable Region/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Alleles , Animals , Antibody Diversity/immunology , Base Sequence , Cell Line, Tumor , Cells, Cultured , Gene Targeting , Immunoglobulin Joining Region/biosynthesis , Immunoglobulin Variable Region/biosynthesis , Mice , Mice, Transgenic , Molecular Sequence Data , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Recombination, Genetic , T-Lymphocyte Subsets/cytologyABSTRACT
Deregulation of cyclin D1 occurs in numerous human cancers through mutations, alternative splicing, and gene amplification. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms whereby they contribute to neoplasia are poorly understood. We now provide evidence derived from both mouse models and human cancer-derived cells revealing that nuclear accumulation of catalytically active mutant cyclin D1/CDK4 complexes triggers DNA rereplication, resulting from Cdt1 stabilization, which in turn triggers the DNA damage checkpoint and p53-dependent apoptosis. Loss of p53 through mutations or targeted deletion results in increased genomic instability and neoplastic growth. Collectively, the data presented reveal mechanistic insights into how uncoupling of critical cell cycle regulatory events will perturb DNA replication fidelity, thereby contributing to neoplastic transformation.
Subject(s)
Cell Nucleus/metabolism , Cyclin D1/metabolism , DNA Replication/genetics , S Phase , Tumor Suppressor Protein p53/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , Cullin Proteins/metabolism , Cyclin D1/genetics , DNA/genetics , DNA, Neoplasm/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Hydrolysis , Lipopolysaccharides/pharmacology , Mice , Mice, Transgenic , Mutation , NIH 3T3 Cells , Osteosarcoma/pathology , Spleen/cytology , Spleen/metabolismABSTRACT
The ATM (ataxia-telangiectasia mutated) protein kinase mediates early cellular responses to DNA double-strand breaks (DSBs) generated during metabolic processes or by DNA-damaging agents. ATM deficiency leads to ataxia-telangiectasia, a disease marked by lymphopenia, genomic instability and an increased predisposition to lymphoid malignancies with chromosomal translocations involving lymphocyte antigen receptor loci. ATM activates cell-cycle checkpoints and can induce apoptosis in response to DNA DSBs. However, defects in these pathways of the DNA damage response cannot fully account for the phenotypes of ATM deficiency. Here, we show that ATM also functions directly in the repair of chromosomal DNA DSBs by maintaining DNA ends in repair complexes generated during lymphocyte antigen receptor gene assembly. When coupled with the cell-cycle checkpoint and pro-apoptotic activities of ATM, these findings provide a molecular explanation for the increase in lymphoid tumours with translocations involving antigen receptor loci associated with ataxia-telangiectasia.
