The Arf tumor suppressor inhibits cell cycle progression through both p53-dependent and p53-independent mechanisms, including interference with rRNA processing. Using tandem-affinity-tagged p19Arf , we purified Arf-associated proteins from mouse NIH 3T3 fibroblasts undergoing cell cycle arrest. Tagged p19 Arf associated with nucleolar and ribosomal proteins, including nucleophosmin/B23 (NPM), a protein thought to foster the maturation of preribosomal particles. NPM is an abundant protein, only a minor fraction of which binds to p19Arf ; however, a significant proportion of p19 Arf associates with NPM. The interaction between p19 Arf and NPM requires amino acid sequences at the Arf amino terminus, which are also required for Mdm2 binding, as well as the central acidic domain of NPM and an adjacent segment that regulates NPM oligomerization. The interaction between p19Arf and NPM occurs in primary mouse embryonic fibroblasts, including those lacking both Mdm2 and p53. In an NIH 3T3 derivative cell line (MT-Arf) engineered to conditionally express an Arf transgene, induced p19Arf associates with NPM and colocalizes with it in high-molecular-weight complexes (2 to 5 MDa). An NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain oligomerizes with endogenous NPM, inhibits p19Arf from entering into 2-to 5-MDa particles, and overrides the ability of p19Arf to retard rRNA processing.

Germline mutation of the BRCA2 gene carries a high risk of developing breast cancer. To study the function of this gene, we generated a mutation in Brca2 in mice. Unlike other mutations in the Brca2 gene, which are lethal early in embryogenesis when homozygous, some of our homozygous mutant mice survive to adulthood. These animals have a wide range of defects, including small size, improper differentiation of tissues, absence of germ cells and the development of lethal thymic lymphomas. Fibroblasts cultured from BrcaZ-/-embryos have a defect in proliferation that may be mediated by over-expression of p53 and p21Waf1/CIP1. We show that Brca2 is required for efficient DNA repair, and our results suggest that loss of the p53 checkpoint may be essential for tumour progression triggered by mutations in BRCA2.

Unknown mechanisms govern degradation of the p19Arf tumor suppressor, an activator of p53 and inhibitor of ribosomal RNA processing. Kinetic metabolic labeling of cells with [ H]-leucine indicated that p19Arf is a relatively stable protein (half-life ∼6 h) whose degradation depends upon the ubiquitin-proteasome pathway. Although p19Arf binds to the Mdm2 E3 ubiquitin protein ligase to activate p53, neither of these molecules regulates p19Arf turnover. In contrast, the nucleolar protein nucleophosmin/B23, which binds to p19 Arf with high stoichiometry, retards its turnover, and Arf mutants that do not efficiently associate with nucleophosmin/B23 are unstable and functionally impaired. Mouse p19 Arf , although highly basic (22% arginine content), contains only a single lysine residue absent from human p14 ARF , and substitution of arginine for lysine in mouse p19Arf had no effect on its rate of degradation. Mouse p19 Arf (either wild-type or lacking lysine) and human p14 ARF undergo N-terminal polyubiquitination, a process that has not as yet been documented in naturally occurring lysine-less proteins. Re-engineering of the p19Arf N terminus to provide consensus sequences for N-acetylation limited Arf ubiquitination and decelerated its turnover.[Keywords: Arf tumor suppressor; proteasome; ubiquitin; nucleophosmin/B23; p53; Mdm2] Supplemental material is available at http://www.genesdev.org.

Women heterozygous for mutations in the breast-cancer susceptibility genes BRCA1 and BRCA2 have a highly elevated risk of developing breast cancer [1]. BRCA1 and BRCA2 encode large proteins with no sequence similarity to one another. Although involvement in DNA repair and transcription has been suggested, it is still not understood how loss of function of these genes leads to breast cancer [2]. Embryonic fibroblasts (MEFs) derived from mice homozygous for a hypomorphic mutation (Brca2(Tr2014)) within the 3' region of exon 11 in Brca2 [3], or a similar mutation (Brca2(Tr)) [4], proliferate poorly in culture and overexpress the tumour suppressor p53 and the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). These MEFs have intact p53-dependent DNA damage G(1)-S [3] [4] and G(2)-M checkpoints [4], but are impaired in DNA double-strand break repair [3] and develop chromosome aberrations [4]. Here, we report that Brca2(Tr2014/Tr2014) MEFs frequently develop micronuclei. These abnormal DNA-containing bodies were formed through both loss of acentric chromosome fragments and by chromosome missegregation, which resulted in aneuploidy. Absence of Brca2 also led to centrosome amplification, which we found associated with the formation of micronuclei. These data suggest a potential mechanism whereby loss of BRCA2 may, within subclones, drive the loss of cell-cycle regulation genes, enabling proliferation and tumourigenesis.