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.