Gary M. Kupfer

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

ABSTRACT 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.

Human T-cell Leukemia Virus-I Tax Oncoprotein Functionally Targets a Subnuclear Complex Involved in Cellular DNA Damage-Response

The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability.

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.

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.

Molecular pathogenesis of Fanconi anemia.

Fanconi anemia (FA) is a rare inherited disorder characterized clinically by aplastic anemia, developmental defects, and a susceptibility to cancer. Eleven complementation groups have been identified (FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, and -L), and the genes responsible for 9 groups (FANCA, B, C, D1, D2, E, F,G, and L) have been cloned.The proteins involved in FA act coordinately in the cellular response to DNA cross-links in a pathway that has been shown to interact physically or functionally with a variety of other proteins involved in DNA repair or cell cycle control, notably BRCA1, Rad51,ATM,ATR, and Nbs1. Considerable advances in the identification and description of proteins involved in FA have been recorded, but the precise biochemical function of the FA pathway remains elusive. As research continues to improve our understanding of FA, insight will be gained into what is a pivotal process in cancer biology.Fanconi anemia (FA) is a rare inherited disorder characterized clinically by aplastic anemia, developmental defects, and a susceptibility to cancer. Eleven complementation groups have been identified (FA-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, and -L), and the genes responsible for 9 groups (FANCA, B, C, D1, D2, E, F, G, and L) have been cloned. The proteins involved in FA act coordinately in the cellular response to DNA cross-links in a pathway that has been shown to interact physically or functionally with a variety of other proteins involved in DNA repair or cell cycle control, notably BRCA1, Rad51,ATM,ATR, and Nbs1. Considerable advances in the identification and description of proteins involved in FA have been recorded, but the precise biochemical function of the FA pathway remains elusive. As research continues to improve our understanding of FA, insight will be gained into what is a pivotal process in cancer biology.

Serum protein expression profiling in pediatric Hodgkin lymphoma: a report from the Children's Oncology Group.

The prognosis for children with Hodgkin lymphoma (HL) treated with a risk adjusted combination of radiation therapy and multi‐drug chemotherapy has markedly improved. There remains a group of patients whose disease either recurs or does not respond to therapy. Protein expression profiling has been used to define protein characteristics of serum from adult patients in order to improve screening for early diagnosis. However, profiling for the purpose of staging and defining prognostic characteristics of childhood diseases is not well studied. The current stage‐based risk assignment of HL cannot predict the patients within a risk group that are destined to recur or do not respond to therapy. Thus, a need exists to develop new methodologies to better stratify the risk classification of pediatric HL.

Fanconi Anemia: A Pathway of Hematopoiesis and Cancer Predisposition

Fanconi anemia (FA) is a rare genetic disease that is a complex, multi-system, and chronic disorder requiring precise diagnosis, careful and judicious treatment delivered as multidisciplinary care across several medical and surgical specialties, and lifetime, close monitoring. While bone marrow failure is ultimately treated with bone marrow transplant, continued monitoring for squamous cell carcinoma and other cancer types is crucial due to the manyfold increased incidence of solid tumors in these patients.