Web-based access to mouse models of human cancers: the Mouse Tumor Biology (MTB) Database.

The Mouse Tumor Biology (MTB) Database serves as a curated, integrated resource for information about tumor genetics and pathology in genetically defined strains of mice (i.e., inbred, transgenic and targeted mutation strains). Sources of information for the database include the published scientific literature and direct data submissions by the scientific community. Researchers access MTB using Web-based query forms and can use the database to answer such questions as 'What tumors have been reported in transgenic mice created on a C57BL/6J background?', 'What tumors in mice are associated with mutations in the Trp53 gene?' and 'What pathology images are available for tumors of the mammary gland regardless of genetic background?'. MTB has been available on the Web since 1998 from the Mouse Genome Informatics web site (http://www.informatics.jax.org). We have recently implemented a number of enhancements to MTB including new query options, redesigned query forms and results pages for pathology and genetic data, and the addition of an electronic data submission and annotation tool for pathology data.

Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting that the proteins cooperate in a nuclear function. In this report, we demonstrate that the recently cloned FANCG/XRCC9 protein is required for binding of the FANCA and FANCC proteins. Moreover, the FANCG protein is a component of a nuclear protein complex containing FANCA and FANCC. The amino-terminal region of the FANCA protein is required for FANCG binding, FANCC binding, nuclear localization, and functional activity of the complex. Our results demonstrate that the three cloned FA proteins cooperate in a large multisubunit complex. Disruption of this complex results in the specific cellular and clinical phenotype common to most FA complementation groups.

A patient-derived mutant form of the Fanconi anemia protein, FANCA, is defective in nuclear accumulation.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but the function of the encoded FA proteins remains unknown. We recently demonstrated that the FANCA and FANCC proteins bind and form a nuclear complex. In the current study, we identified a homozygous mutation in the FANCA gene (3329A>C) in an Egyptian FA patient from a consanguineous family. This mutant FANCA allele is predicted to encode a mutant FANCA protein, FANCA(H1110P), in which histidine 1110 is changed to proline. Initially, we characterized the FANCA(H1110P) protein, expressed in an Epstein Barr virus (EBV)-immortalized lymphoblast line derived from the patient. Unlike wild-type FANCA protein expressed in normal lymphoblasts, FANCA(H1110P) was not phosphorylated and failed to bind to FANCC. To test directly the effect of this mutation on FANCA function, we used retroviral-mediated transduction to express either wild-type FANCA or FANCA(H1110P) protein in the FA-A fibroblast line, GM6914. Unlike wild-type FANCA, the mutant protein failed to complement the mitomycin C sensitivity of these cells. In addition, the FANCA(H1110P) protein was defective in nuclear accumulation in the transduced cells. The characteristics of this mutant protein underscore the importance of FANCA phosphorylation, FANCA/FANCC binding, and nuclear accumulation in the function of the FA pathway.

The fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway.

Functional activity of the fanconi anemia protein FAA requires FAC binding and nuclear localization.

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability.

Subtyping analysis of Fanconi anemia by immunoblotting and retroviral gene transfer.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Two of the FA genes (FAA and FAC) have been cloned, and mutations in these genes account for approximately 80% of FA patients. Subtyping of FA patients is an important first step toward identifying candidates for FA gene therapy. In the current study, we analyzed a reference group of 26 FA patients of known subtype. Most of the patients (18/26) were confirmed as either type A or type C by immunoblot analysis with anti-FAA and anti-FAC antisera. In order to resolve the subtype of the remaining patients, we generated retroviral constructs expressing FAA and FAC for transduction of FA cell lines (pMMP-FAA and pMMP-FAC). The pMMP-FAA construct specifically complemented the abnormal phenotype of cell lines from FA-A patients, while pMMP-FAC complemented FA-C cells. In summary, the combination of immunoblot analysis and retroviral-mediated phenotypic correction of FA cells allows a rapid method of FA subtyping.

The Fanconi anaemia proteins, FAA and FAC, interact to form a nuclear complex.

Fanconi anaemia (FA) is an autosomal-recessive disorder characterized by genomic instability, developmental defects, DNA crosslinking agent hypersensitivity and cancer susceptibility. Somatic-cell hybrid studies have revealed five FA complementation groups (A-E; refs 4-6) displaying similar phenotypes, suggesting that FA genes are functionally related. The two cloned FA genes, FAA and FAC, encode proteins that are unrelated to each other or to other proteins in GenBank. In the current study, we demonstrate the FAA and FAC bind each other and form a complex. Protein binding correlates with the functional activity of FAA and FAC, as patient-derived mutant FAC (L554P) fails to bind FAA. Although unbound FAA and FAC localize predominantly to the cytoplasm, the FAA-FAC complex is found in similar abundance in both cytoplasm and nucleus. Our results confirm the interrelatedness of the FA genes in a pathway, suggesting the cooperation of FAA and FAC in a nuclear function.

The Fanconi anemia polypeptide, FAC, binds to the cyclin-dependent kinase, cdc2.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by developmental defects, bone marrow failure, and cancer susceptibility. Cells derived from FA patients are sensitive to crosslinking agents and have a prolonged G2 phase, suggesting a cell cycle abnormality. Although transfection of type-C FA cells with the FAC cDNA corrects these cellular abnormalities, the molecular function of the FAC polypeptide remains unknown. In the current study we show that expression of the FAC polypeptide is regulated during cell cycle progression. In synchronized HeLa cells, FAC protein expression increased during S phase, was maximal at the G2/M transition, and declined during M phase. In addition, the FAC protein coimmunoprecipitated with the cyclin-dependent kinase, cdc2. We next tested various mutant forms of the FAC polypeptide for binding to cdc2. A patient-derived mutant FAC polypeptide, containing a point mutation at L554P, failed to bind to cdc2. The FAC/cdc2 binding interaction therefore correlated with the functional activity of the FAC protein. Moreover, binding of FAC to cdc2 was mediated by the carboxyl-terminal 50 amino acids of FAC in a region of the protein required for FAC function. Taken together, our results suggest that the binding of FAC and cdc2 is required for normal G2/M progression in mammalian cells. Absence of a functional interaction between FAC and cdc2 in FA cells may underlie the cell cycle abnormality and clinical abnormalities of FA.

Molecular biology of Fanconi anemia.

Fanconi anemia (FA) is a rare, autosomal recessive disease characterized by multiple congenital abnormalities, bone marrow failure, and cancer susceptibility. Although traditionally described as a classic clinical syndrome, as more is discovered regarding its basic molecular and cell biology, FA is emerging as a true premalignant syndrome. Two of the genes of the five known complementation groups have been cloned, and work to understand their function is underway. Further understanding of these gene products has lent new ideas concerning modes of novel therapy, including gene therapy. The impact of molecular biology on our understanding of basic biology and the clinical care of FA patients is discussed.

Different pathways mediate virus inducibility of the human IFN-alpha 1 and IFN-beta genes.

Multimerization of GAAANN generates sequences frequent in virus-inducible promoters. We distinguished different types of (GAAANN)4 sequences mediating virus inducibility. Type I (NN = GT, GC, CT, or CC) responds to IFNs and to IRF-1 and causes silencing. Type II (NN = TG) and type III (NN = CG) neither silence nor respond to IRF-1 or IFN. Type III mediates constitutive transcription and binds the constitutive IEFga factor, whereas type II binds the novel "TG protein". IFN-beta and IFN-alpha 1 promoters contain different response elements: The former has a type I-like sequence (PRDI) and an NF-kappa B-binding sequence (PRDII); the latter has a type II-like "TG sequence" and possibly additional elements but does not bind NF-kappa B. Type I, type II, and NF-kappa B elements represent three distinct terminal pathways mediating virus induction.