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.
Nucleophosmin (NPM) is frequently overexpressed in leukemias and other tumors. NPM has been reported to suppress oncogene-induced senescence and apoptosis and may represent a therapeutic target for cancer. We fused a NPMderived peptide to the HIV-TAT (TAT-NPM⌬C) and found that the fusion peptide inhibited proliferation and induced apoptotic death of primary fibroblasts and preleukemic stem cells. TAT
IntroductionNucleophosmin (NPM) is a multifunctional protein that plays important roles in the regulation of cell proliferation and apoptosis. 1 The level of NPM is frequently found to be significantly higher in tumor and proliferating cells than in resting cells. [2][3][4][5][6] Normal cells induce genetically encoded programs that prevent deregulated proliferation and thus protect multicellular organisms from cancer progression. Two such programs are apoptosis and senescence that are normally triggered by DNA damage or other stresses. Our recent studies demonstrated that overexpression of NPM suppresses oncogene-induced senescence and apoptosis and accelerated transformation in cells deficient for the Fanconi complementation group C (Fancc) gene and the ataxia telangiectasia mutated (Atm) gene. 7 Therefore, NPM could be a potential therapeutic target for neoplastic diseases.There is increasing interest in employing peptides and proteins as therapeutic compounds for human cancers. For example, several peptides derived from tumor suppressors as well as oncogenes have been developed as antitumorigenic cargoes and proved to be effective in inhibition of tumor cell growth. [8][9][10] Although peptidebased compounds have limitations owing to poor permeability and low selectivity, recent studies have demonstrated that large cargoes, proteins, and peptides can be delivered intracellularly if conjugated to the protein transduction domain (PTD) derived from the HIV-1 TAT protein. 11,12 Indeed, rapid and receptor-independent uptake of TAT-conjugated peptides has been demonstrated to occur in many cell types and animals. [13][14][15] In this study, we generated a NPM-derived peptide fused to TAT (TAT-NPM⌬C) and demonstrated in an inflammation-associated leukemic mouse model that TAT induced rapid and efficient delivery of the peptide into leukemic cells. TAT-NPM⌬C caused growth inhibition and apoptosis of leukemic cells and significantly delayed leukemic development in mice. TAT-NPM⌬C associated with endogenous NPM proteins formed complexes with endogenous NPM and the NF-B subunit p65 and abrogated NF-B transactivation at promoters of several important antiapoptotic and inflammatory genes controlled by NF-B in leukemic cells. Thus, the results suggest that the TAT-delivered NPM mutant may provide a novel therapy for tumors that require NF-B signaling for survival.
Methods
Cloning, expression, and purification of TAT-NPM fusion proteinsThe full-length human NPM (GenBank sequence accession number BC009623) and its N-terminally (NPM⌬N) and C-terminally (NPM⌬C) deleted cDNAs were amplified by polymerase chain reaction (PCR), usi...
The Fanconi anemia (FA) group C protein, FANCC, interacts with STAT1 following stimulation with IFN-γ and is required for proper docking of STAT1 at the IFN-γ receptor α-chain (IFN-γRα, IFN-γR1). Consequently, loss of a functional FANCC results in decreased activation of STAT1 following IFN-γ 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-γ 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.
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