by nocodazole. The proteasome-sensitive ubiquitin conjugates of BRCA1 appear to be distinct from BRCA1 autoubiquitination products and are probably catalyzed by the action of other cellular E3 ligases. Interestingly, co-expression of BARD1 inhibits the formation of these conjugates, suggesting that BARD1 serves to stabilize BRCA1 expression in part by reducing proteasome-sensitive ubiquitination of BRCA1 polypeptides. In summary, these data indicate that the cell cycle-dependent pattern of BRCA1 expression is determined in part by ubiquitin-dependent proteasomal degradation.Germline mutations of the BRCA1 gene are responsible for a substantial proportion of hereditary breast and ovarian cancers (1, 2). In this clinical setting, BRCA1 serves as a tumor suppressor that contributes to tumorigenesis through loss of function. The protein it encodes has been implicated in a number of biological processes, including the cellular response to DNA damage (3, 4). In particular, BRCA1 is required for several checkpoints that control cell cycle progression (5, 6) and inhibit mRNA processing (7, 8) after genotoxic stress, as well as for certain modes of DNA repair such as nucleotide excision repair (9, 10) and homology-directed repair of double-strand DNA breaks (11-13). As a key regulator of the DNA damage response, BRCA1 presumably promotes tumor suppression by preserving genomic stability. However, the molecular mechanisms by which it carries out these functions are not understood and, as a consequence, it is still unclear why inherited mutations of the BRCA1 gene predispose women to breast and ovarian cancer.The BRCA1 polypeptide contains two recognizable amino acid motifs: a RING domain near the N terminus and two tandem copies of the BRCT domain at the C terminus (14). In vivo, BRCA1 exists as a heterodimer with BARD1, a distinct protein that harbors a similar array of RING and BRCT motifs (15). Since the phenotypes of mice null for either Brca1 or Bard1 are essentially indistinguishable, the functions of both proteins are likely to be mediated through the BRCA1/BARD1 heterodimer (16), and indeed BARD1 has already been implicated with BRCA1 in homology-directed repair of chromosomal breaks (17). BRCA1 and BARD1 associate by assembling a stable 4-helix bundle from the ␣ helices that flank their respective RING domains (18), and together they form an enzymatic complex that can catalyze ubiquitin polymerization in vitro (19 -24). This enzymatic activity implies that BRCA1/BARD1 functions as an E3 ligase that promotes ubiquitin modification of specific substrate proteins, and that these are likely to include important effectors of BRCA1-mediated tumor suppression (25,26). Although definitive substrates of BRCA1/BARD1have not yet been identified, autoubiquitination of the BRCA1 subunit is observed during in vitro reactions catalyzed by BRCA1/BARD1 (22). In vitro, BRCA1/BARD1 directs the formation of ubiquitin polymers through an unconventional isopeptide linkage involving lysine residue K6 of ubiquitin (27,28). These K6-link...

Many tumors overexpress members of the inhibitor of apoptosis protein (IAP) family. IAPs contribute to tumor cell apoptosis resistance by the inhibition of caspases, and are degraded by the proteasome to allow further progression of apoptosis. Here we show that tumor cells can alter the specificity of cytosolic proteolysis in order to acquire apoptosis resistance, which promotes formation of rapidly growing tumors. Survival of tumor cells with low proteasomal activity can occur in the presence of high expression of Tri-peptidyl-peptidase II (TPP II), a large subtilisin-like peptidase that complements proteasomal activity. We find that this state leaves tumor cells unable of effectively degrading IAPs, and that cells in this state form rapidly growing tumors in vivo. We also find, in studies of apoptosis resistant cells derived from large in vivo tumors, that these have acquired an altered peptidase activity, with up-regulation of TPP II activity and decreased proteasomal activity. Importantly, we find that growth of subcutaneous tumors is limited by maintenance of the apoptosis resistant phenotype. The apoptosis resistant phenotype was reversed by increased expression of Smac/DIABLO, an antagonist of IAP molecules. Our data suggest a reversible mechanism in regulation of apoptosis resistance that drives tumor progression in vivo. These data are relevant in relation to the multitude of therapy-resistant clinical tumors that have increased levels of IAP molecules.

In this work, NiO thin film was prepared by the sol-gel technique and analysed by thermogravimetry, x-ray diffractometry and x-ray photoelectron spectroscopy. The electrochromic characteristics were studied by ultraviolet spectroscopy. NiO thin film shows electrochromic characteristics. Its colour changes from transparent to brown when a voltage is applied. The transmittance of the film can shift from 90 to 40%. Deterioration of the film caused by colouring and discolouring was not observed for up to 100 cycles.

Checkpoint kinase inhibitors can enhance the cancer killing action of DNA-damaging chemotherapeutic agents by disrupting the S/G 2 cell cycle checkpoints. The in vitro and in vivo effects of the Chk1/2 inhibitor AZD7762 when combined with these agents were examined using neuroblastoma cell lines with known p53/MDM2/p14 ARF genomic status. Four of four p53 mutant lines and three of five MDM2/p14 ARF abnormal lines were defective in G 1 checkpoint, correlating with failure to induce endogenous p21 after treatment with DNA-damaging agents. In cytotoxicity assays, these G 1 checkpoint-defective lines were more resistant to DNA-damaging agents when compared to G 1 checkpoint intact lines, yet becoming more sensitive when AZD7762 was added. Moreover, AZD7762 abrogated DNA damage-induced S/G 2 checkpoint arrest both in vitro and in vivo. In xenograft models, a significant delay in tumor growth accompanied by histological evidence of increased apoptosis was observed, when AZD7762 was added to the DNA-damaging drug gemcitabine. These results suggest a therapeutic potential of combination therapy using checkpoint kinase inhibitor and chemotherapy to reverse or prevent drug resistance in treating neuroblastomas with defective G 1 checkpoints.

Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal ␤-subunits (␤ 1 , ␤ 2 , and ␤ 5 ) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2D b molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways.

Although the BRCA1 tumor suppressor has been implicated in a number of cellular processes, it plays an especially important role in the DNA damage response as a regulator of cell cycle checkpoints and DNA repair pathways. In vivo, BRCA1 exists as a heterodimer with the BARD1 protein, and many of its biological functions are mediated by the BRCA1-BARD1 complex. Here, we show that BARD1 is phosphorylated in a cell cycle-dependent manner and that the hyperphosphorylated forms of BARD1 predominate during M phase. By mobility shift analysis and mass spectrometry, we have identified seven sites of mitotic phosphorylation within BARD1. All sites exist within either an SP or TP sequence, and two sites resemble the consensus motif recognized by cyclin-dependent kinases. To examine the functional consequences of BARD1 phosphorylation, we used a gene targeting knock-in approach to generate isogenic cell lines that express either wild-type or mutant forms of the BARD1 polypeptide. Analysis of these lines in clonogenic survival assays revealed that cells bearing phosphorylation site mutations are hypersensitive to mitomycin C, a genotoxic agent that induces interstrand DNA cross-links. These results implicate BARD1 phosphorylation in the cellular response to DNA damage.Although hereditary breast cancer can often be traced to germ line mutations in the BRCA1 tumor suppressor gene, it is still unclear how BRCA1 lesions promote mammary carcinogenesis (1). The major isoform of BRCA1 is a large polypeptide that contains a RING domain at its N terminus and two tandem copies of the BRCT domain at the C terminus (2). In vivo, BRCA1 exists primarily as a heterodimer with BARD1, a distinct but related protein that also harbors an N-terminal RING motif and two C-terminal BRCT domains (3). Both proteins colocalize within the same nuclear foci of S phase cells (4, 5), and their interaction is mediated by sequences encompassing their respective RING motifs (3). Co-immunoprecipitation analysis indicates that most of the cellular pool of endogenous BRCA1 exists in the form of a heterodimer with BARD1 (6, 7), and structural studies have shown that dimerization occurs through a four-helix bundle formed by ␣-helices flanking the RING motifs of both proteins (8). Because the phenotypes of mice null for either Brca1 or Bard1 are essentially indistinguishable (9), the major functions of both proteins are likely to be mediated by the BRCA1/BARD1 heterodimer. This notion is supported by the fact that BRCA1 and BARD1 together form a potent enzymatic complex that can catalyze ubiquitin polymerization in vitro (10 -17). Recent studies have also shown that BARD1 is required for nuclear retention of BRCA1 (18) and that, together with BRCA1, it modulates mRNA processing during the DNA damage response (19,20) and promotes homology-directed repair of chromosomal breaks (21).It is now apparent that phosphorylation exerts important controls over BRCA1 function. Early studies established that BRCA1 is hyperphosphorylated in several cellular settings. During n...

Background: Novel prognostic biomarkers or therapeutic molecular targets for laryngeal squamous cell carcinoma (LSCC) are an urgent priority. We here sought to identify multiple novel LSCC-associated genes. Methods: Using high-density microarray expression profiling, we identified multiple genes that were significantly altered between human LSCCs and paired normal tissues. Potential oncogenic functions of one such gene, DCUN1D5, were further characterized in vitro. Results: Our results demonstrated that DCUN1D5 was highly expressed in LSCCs. Overexpression of DCUN1D5 in vitro resulted in 2.7-fold increased cellular migration, 67.5% increased invasive capacity, and 2.6-fold increased proliferation. Endogenous DCUN1D5 expression was decreased in a time-dependent manner after genotoxic stress, and silencing of DCUN1D5 by siRNA decreased the number of cells in the S phase by 10.2% and increased apoptosis by 11.7%. Conclusion: Our data suggest that DCUN1D5 in vitro might have vital roles in DNA damage response, but further studies are warranted to assess its significance in vivo.

Cellular responses to ;-irradiation exposure are controlled by phosphatidylinositol 3-kinase-related kinases (PIKK) in the nucleus, and in addition, cytosolic PIKKs may have a role in such responses. Here, we show that the expression of tripeptidyl-peptidase II (TPPII), a high molecular weight cytosolic peptidase, required PIKK signaling and that TPPII was rapidly translocated into the nucleus of ;-irradiated cells. These events were dependent on mammalian target of rapamycin, a cytosolic/mitochondrial PIKK that is activated by ;-irradiation. Lymphoma cells with inhibited expression of TPPII failed to efficiently stabilize p53 and had reduced ability to arrest proliferation in response to ;-irradiation. We observed that TPPII contains a BRCA COOH-terminal-like motif, contained within sequences of several proteins involved in DNA damage signaling pathways, and this motif was important for nuclear translocation of TPPII and stabilization of p53. Novel tripeptide-based inhibitors of TPPII caused complete in vivo tumor regression in mice in response to relatively low doses of ;-irradiation (3-4 Gy/wk). This was observed with established mouse and human tumors of diverse tissue backgrounds, with no tumor regrowth after cancellation of treatment. These TPPII inhibitors had minor effects on tumor growth as single agent and had low cellular toxicity. Our data indicated that TPPII connects signaling by cytosolic/mitochondrial and nuclear PIKK-dependent pathways and that TPPII can be targeted for inhibition of tumor therapy resistance. [Cancer Res 2007; 67(15):7165-74]