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Grover C Bagby

Publications and source records attributed to Grover C Bagby.

10 recordsLinked to original sources
PubMed2004

Impaired type I IFN-induced Jak/STAT signaling in FA-C cells and abnormal CD4+ Th cell subsets in Fancc-/- mice.

The Fanconi anemia (FA) group C protein, FANCC, interacts with STAT1 following stimulation with IFN-gamma and is required for proper docking of STAT1 at the IFN-gamma receptor alpha-chain (IFN-gammaRalpha, IFN-gammaR1). Consequently, loss of a functional FANCC results in decreased activation of STAT1 following IFN-gamma stimulation. Because type I IFN receptors influence the function of type II receptors, and vice versa, we conducted experiments designed to determine whether type I IFN-induced activation of other STAT proteins is compromised in FA-C cells and found that activation of STAT 1, 3, and 5 is diminished in type I IFN-stimulated cells bearing Fancc-inactivating mutations. We also determined that the reduced activation of STATs was accompanied by significant reduction of type I IFN-induced tyrosine kinase 2 and Jak1 phosphorylation. Because tyrosine kinase 2 plays a role in differentiation of Th cells, we quantified cytokine secretion from CD4+ cells and in vitro generated CD4+ Th cell subsets from splenocytes of Fancc null mice to that of heterozygous mice and discovered reduced CD4+ IFN-gamma secretion in the Fancc-/- mouse, indicating impaired Th1 differentiation. We suggest that Fancc mutations result in a subtle immunological defect owing to the failure of FANCC to normally support Jak/STAT signaling.

Animals↗
PubMed2004

Hypoxia-induced nucleophosmin protects cell death through inhibition of p53.

Nucleophosmin (NPM) is a multifunctional protein that is overexpressed in actively proliferating cells and cancer cells. Here we report that this proliferation-promoting protein is strongly induced in response to hypoxia in human normal and cancer cells. Up-regulation of NPM is hypoxia-inducible factor-1 (HIF-1)-dependent. The NPM promoter encodes a functional HIF-1-responsive element that can be activated by hypoxia or forced expression of HIF-1alpha. Suppression of NPM expression by small interfering RNA targeting NPM increases hypoxia-induced apoptosis, whereas overexpression of NPM protects against hypoxic cell death of wild-type but not p53-null cells. Moreover, NPM inhibits hypoxia-induced p53 phosphorylation at Ser-15 and interacts with p53 in hypoxic cells. Thus, this study not only demonstrates hypoxia regulation of a proliferation-promoting protein but also suggests that hypoxia-driven cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.

Apoptosis↗
PubMed2004

The Fanconi anemia proteins functionally interact with the protein kinase regulated by RNA (PKR).

Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimer's, Parkinson's, and Huntington's diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-gamma and tumor necrosis factor-alpha. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-gamma and tumor necrosis factor-alpha. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.

Apoptosis↗
PubMed2004

Marrow failure.

New discoveries in cell biology, molecular biology and genetics have unveiled some of the pathophysiological mysteries of some of the bone marrow failure syndromes. Many of these discoveries have revealed why these syndromes show so much clinical overlap and some hold the potential for influencing the development of new therapies. In children and adults with pancytopenia and hypoplastic bone marrows proper differential diagnosis requires that some attention be directed toward defining molecular and cellular pathogenetic mechanisms because, once identified, some of these mechanisms will clearly suggest rational therapeutic approaches, treatment options that should be avoided, or both. In Section I, Drs. Jeffrey Lipton and Grover Bagby review the approach to diagnosis and management of patients with the inherited bone marrow failure syndromes, Fanconi anemia, dyskeratosis congenita, Diamond-Blackfan anemia, and the Shwachman-Diamond syndrome. Extraordinary progress has been made in identifying the genes bearing pathogenetically relevant mutations in these disorders, but slower progress has been made in defining the precise functions of the proteins these genes encode in normal cells, in part because it is increasingly obvious that the proteins are multifunctional. In practice, it is clear that in patients with dyskeratosis congenita and Fanconi anemia, the diagnosis must be considered not only in children but in adults as well. In Section II, Dr. Elaine Sloand outlines a very practical and evidence-based approach to diagnosis and management of acquired hypoplastic states emphasizing overlap between non-clonal and clonal hematopoiesis is such conditions. The pathogenesis of T lymphocyte-mediated marrow failure is presented as a clear-cut rationale for use of immunosuppressive therapy and stem cell transplantation. Practical management of patients with refractory disease with and without evidence of clonal evolution (either paroxysmal nocturnal hemoglobinuria [PNH] or myelodysplasia [MDS]) is presented. In Section III, the challenge of hypoplastic MDS is reviewed by Dr. Charles Schiffer. After reviewing the most up-to-date classification scheme, therapeutic options are reviewed, focusing largely on agents that have most recently shown some promising activity, including DNA demethylating agents, thalidomide and CC5013, arsenic trioxide, and immunosuppressive therapy. Here are also outlined the rationale and the indications for choosing allogeneic bone marrow transplantation, the only therapy with known curative potential.

Anemia, Aplastic↗
PubMed2003

Nucleophosmin interacts with and inhibits the catalytic function of eukaryotic initiation factor 2 kinase PKR.

In normal cells the protein kinase PKR effects apoptosis in response to various extra and intracellular cues and can also function to suppress the neoplastic phenotype. Because most neoplastic cells are resistant to certain apoptotic cues, we reasoned that an early molecular event in carcinogenesis or leukemogenesis might be the inactivation of PKR by expression or activation of intracellular PKR inhibitors. Seeking novel PKR-modulating proteins we report here that nucleophosmin (NPM), a protein frequently overexpressed in a variety of human malignancies, binds to PKR, and inhibits its activation. Co-immunoprecipitation and in vitro binding experiments showed that NPM associated with PKR. Kinase assays demonstrated that recombinant NPM inhibited PKR activation in a dose-dependent manner. In addition, purified recombinant NPM was phosphorylated by activated PKR. Most importantly, overexpression of NPM suppressed PKR activity, enhanced protein synthesis, and inhibited apoptosis. Lymphoblasts from patients with Fanconi anemia (FA) expressed low levels of NPM, which correlated with high ground-state activation of PKR and cellular hypersensitivity to apoptotic cues, but enforced expression of NPM in these mutant cells reduced aberrant apoptotic responses. Inhibition of PKR by NPM may be one mechanism by which neoplastic clones evolve in sporadic malignancies and in neoplastic cells arising in the context of the cancer predisposition syndrome, Fanconi anemia.

Apoptosis↗
PubMed2003

Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia.

Myelodysplastic and leukemic stem cell clones that evolve in children and adults with Fanconi anemia universally bear complex cytogenetic abnormalities. The abnormalities are generally recurring deletions or chromosomal loss and involve precisely the same chromosomes with the same frequency as has been described in marrow cells from patients with secondary acute leukemia induced by alkylating agents. Reasoning that acquired Fanconi anemia protein dysfunction might contribute to cytogenetic instability in secondary acute myelogenous leukemia (AML) cells, we analyzed leukemic cells bearing characteristic complex cytogenetic defects obtained from a 68-year-old man whose lymphoblasts showed no evidence of Fanconi anemia. Unlike the lymphoblasts, this myeloid leukemia cell line (UoC-M1) was hypersensitive to mitomycin-C (MMC) and diepoxybutane (DEB) and exhibited a marked decrease in nuclear FANCA, FANCG, and FANCD2-L. Retroviral transduction of FANCA significantly reduced MMC sensitivity but FANCF, FANCG, and FANCC did not. Overexpression of FANCA restored levels of both FANCA and FANCG, whereas overexpression of FANCG or FANCC did not restore FANCA levels. The molecular mass of cytoplasmic FANCA, FANCG, FANCC, and nuclear FANCD2 were normal. All exons of FANCA and FANCG were sequenced, and no mutations were found. We conclude that perturbations of as yet unidentified factors that govern the binding activity or intracellular localization of FANCA may promote cytogenetic instability and clonal progression in patients with AML who do not have Fanconi anemia.

Aged↗
PubMed2003

Genetic basis of Fanconi anemia.

Fanconi anemia is a rare autosomal recessive disease characterized by bone marrow failure, developmental anomalies, a high incidence of myelodysplasia and acute nonlymphocytic leukemia, and cellular hypersensitivity to cross linking agents. Five of the seven known Fanconi anemia proteins bind together in a complex and influence the function of a sixth, FANCD2, which colocalizes with BRCA1 in nuclear foci after genotoxic stress. Carboxy-terminal truncating mutations of the seventh Fanconi anemia gene, BRCA2, are hypomorphic and lead to FA-D1 and possibly FA-B. Because the Fanconi anemia alleles of BRCA2 fail to bind to Rad51 in response to genotoxic stress and Rad51 therefore fails to localize to nuclear damage foci, many investigators in the field suspect that the Fanconi anemia pathway supports the integrity of the Rad51 and BRCA1 and BRCA2 pathways as they function in homologous recombination repair. Because these abnormalities are common to all somatic cells, it is unlikely that dysfunction of this particular pathway results in tissue-specific apoptosis of hematopoietic cells. Indeed, at least one of the Fanconi anemia proteins, FANCC, exhibits functions in hematopoietic cells in addition to its role in the complex. Because FANCC protects hematopoietic cells from apoptotic cues in ways that do not require an intact heteromeric Fanconi anemia complex, it is reasonable to expect that the other Fanconi anemia gene products will have independent cytoplasmic and nuclear functions, particularly in hematopoietic and germ cells that seem to rely so substantially on an intact portfolio of Fanconi anemia proteins.

Fanconi Anemia↗
PubMed2002

The anti-apoptotic function of Hsp70 in the interferon-inducible double-stranded RNA-dependent protein kinase-mediated death signaling pathway requires the Fanconi anemia protein, FANCC.

Proteins encoded by five of the six known Fanconi anemia (FA) genes form a heteromeric complex that facilitates repair of DNA damage induced by cross-linking agents. A certain number of these proteins, notably FANCC, also function independently to modulate apoptotic signaling, at least in part, by suppressing ground state activation of the pro-apoptotic interferon-inducible double-stranded RNA-dependent protein kinase (PKR). Because certain FANCC mutations interdict its anti-apoptotic function without interfering with the capacity of FANCC to participate functionally in the FA multimeric complex, we suspected that FANCC enhances cell survival independent of its participation in the complex. By investigating this function in both mammalian cells and in yeast, an organism with no FA orthologs, we show that FANCC inhibited the kinase activity of PKR both in vivo and in vitro, and this effect depended upon a physical interaction between FANCC and Hsp70 but not on interactions of FANCC with other Fanconi proteins. Hsp70, FANCC, and PKR form a ternary complex in lymphoblasts and in yeast expressing PKR. We conclude that Hsp70 requires the cooperation of FANCC to suppress PKR activity and support survival of hematopoietic cells and that FANCC does not require the multimeric FA complex to exert this function.

Apoptosis↗
PubMed2002

Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice.

To define the mechanism of cyclosporine (CsA)-induced apoptosis, we investigated the expression of apoptosis-related genes in experimental chronic CsA nephrotoxicity. Mice on a low-salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain, and the expressions of apoptosis-related genes were evaluated by reverse transcription-polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 +/- 41 vs. 3 +/- 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas-ligand mRNA (0.20 vs. 0.02 amol/microgram total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 rho mol/microgram/h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 rho mol/microgram/h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl-2 protein increased significantly in the CsA group (5.3-fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas-ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis-related genes is associated with CsA-induced apoptotic cell death.

Animals↗