ABSTRACT
The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism that is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provide mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and, thus, contributes to maintenance of genomic stability.
Subject(s)
BRCA1 Protein/metabolism , Cyclin B/metabolism , Proteasome Endopeptidase Complex/metabolism , cdc25 Phosphatases/metabolism , BRCA1 Protein/genetics , Cell Division , Cyclin B/genetics , G2 Phase , Gene Knockdown Techniques , Genomic Instability , Humans , Leupeptins/pharmacology , MCF-7 Cells/drug effects , MCF-7 Cells/metabolism , Proteasome Inhibitors/pharmacology , Protein Interaction Domains and Motifs , RING Finger Domains , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination , cdc25 Phosphatases/geneticsABSTRACT
Wilms' tumor (WT), the most frequent renal solid tumor in children, has been linked to aberrant Wnt signaling. Herein, we demonstrate that different WTs can be grouped according to either sensitivity or resistance to an antibody (Ab) specific to frizzled7 (FZD7), a Wnt receptor. In the FZD7-sensitive WT phenotype, the Ab induced cell death of the FZD7(+) fraction, which in turn depleted primary WT cultures of their clonogenic and sphere-forming cells and decreased in vivo proliferation and survival on xenografting to the chick chorio-allantoic-membrane. In contrast, FZD7-resistant WT in which no cell death was induced showed a different intra-cellular route of the Ab-FZD7 complex compared with sensitive tumors and accumulation of ß-catenin. This coincided with a low sFRP1 and DKK1 (Wnt inhibitors) expression pattern, restored epigenetically with de-methylating agents, and lack of ß-catenin or WTX mutations. The addition of exogenous DKK1 and sFRP1 to the tumor cells enabled the sensitization of FZD7-resistant WT to the FZD7 Ab. Finally, although extremely difficult to achieve because of dynamic cellular localization of FZD7, sorting of FZD7(+) cells from resistant WT, showed them to be highly clonogenic/proliferative, overexpressing WT 'stemness' genes, emphasizing the importance of targeting this fraction. FZD7 Ab therapy alone or in combination with Wnt pathway antagonists may have a significant role in the treatment of WT via targeting of a tumor progenitor population.
Subject(s)
Antineoplastic Agents/pharmacology , Frizzled Receptors/immunology , Kidney Neoplasms/drug therapy , Receptors, G-Protein-Coupled/immunology , Wilms Tumor/drug therapy , Wnt Proteins/metabolism , Adaptor Proteins, Signal Transducing , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/immunology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/pharmacology , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , Intercellular Signaling Peptides and Proteins/biosynthesis , Intercellular Signaling Peptides and Proteins/pharmacology , Kidney Neoplasms/genetics , Kidney Neoplasms/immunology , Kidney Neoplasms/pathology , Mutation , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/pharmacology , Tumor Cells, Cultured , Tumor Suppressor Proteins/genetics , Wilms Tumor/genetics , Wilms Tumor/immunology , Wilms Tumor/pathology , beta Catenin/biosynthesis , beta Catenin/geneticsABSTRACT
Early in mammalian development, the stem cell leukemia (SCL/TAL1) gene and its distinct 3' enhancer (SCL 3'En) specify bipotential progenitor cells that give rise to blood and endothelium, thus termed hemangioblasts. We have previously detected a minor population of SCL (+) cells in the postnatal kidney. Here, we demonstrate that cells expressing the SCL 3'En in the adult kidney are comprised of CD45+CD31- hematopoietic cells, CD45-CD31+ endothelial cells and CD45-CD31- interstitial cells. Creation of bone marrow chimeras of SCL 3'En transgenic mice into wild-type hosts shows that all three types of SCL 3'En-expressing cells in the adult kidney can originate from the bone marrow. Ischemia/reperfusion injury to the adult kidney of SCL 3'En transgenic mice results in the intrarenal elevation of SCL and FLK1 mRNA levels and of cells expressing hem-endothelial progenitor markers (CD45, CD34, c-Kit and FLK1). Furthermore, analysis of SCL 3'En in the ischemic kidneys reveals an increase in the abundance of SCL 3'En-expressing cells, predominantly within the CD45 (+) hematopoietic fraction and to a lesser extent in the CD45 (-) fraction. Our results suggest organ-injury-induced reactivation of bone marrow-derived hemangioblasts and possible local angioblastic progenitors expressing SCL and SCL 3'En.
