Background and aims: Mutations in BRAF have been linked with colorectal cancers (CRC) showing high level microsatellite instability (MSI-H). However, the distribution of BRAF mutations in MSI-H cancers remains to be clarified with respect to precursor lesions and the CpG island methylator phenotype (CIMP). Methods: Forty three hyperplastic polyps (HP), nine mixed polyps (MP), five serrated adenomas (SA), 28 conventional adenomas (AD), 18 hereditary non-polyposis colorectal cancers (HNPCC), and 127 sporadic CRC (46 MSI-H and 81 non-MSI-H) were collected from patients undergoing colectomy for either CRC or hyperplastic polyposis. Twenty five of 57 serrated lesions were derived from four patients with hyperplastic polyposis. HP were further subdivided according to recently documented morphological criteria into 27 classical HP and 16 variant lesions described as ''sessile serrated adenoma'' (SSA). All tumours were screened for BRAF activating mutations. Results: The BRAF mutation was more frequent in SSA (75%) and MP (89%) than in classical HP (19%), SA (20%), and AD (0%) (p,0.0001), and also in sporadic MSI-H cancers (76%) compared with HNPCC (0%) and sporadic non-MSI-H cancers (9%) (p,0.0001). The BRAF mutation was identified more often in CIMPhigh serrated polyps (72%) and CIMP-high CRC (77%) than in CIMP-low (30%) and CIMP-negative (13%) polyps (p = 0.002) as well as CIMP-low (18%) and CIMP-negative (0%) CRC (p,0.0001). Conclusions: The BRAF mutation was frequently seen in SSA and in sporadic MSI-H CRC, both of which were associated with DNA methylation. Sporadic MSI-H cancers may originate in SSA and not adenomas, and BRAF mutation and DNA methylation are early events in this ''serrated'' pathway.

The mammalian target of rapamycin (mTOR) controls multiple cellular functions in response to amino acids and growth factors, in part by regulating the phosphorylation of p70 S6 kinase (p70S6k) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that also binds p70S6k and 4E-BP1 and is essential for TOR signaling in vivo. Herein we demonstrate that raptor binds to p70S6k and 4E-BP1 through their respective TOS (conserved TOR signaling) motifs to be required for amino acid-and mTOR-dependent regulation of these mTOR substrates in vivo. A point mutation of the TOS motif also eliminates all in vitro mTOR-catalyzed 4E-BP1 phosphorylation and abolishes the raptor-dependent component of mTOR-catalyzed p70S6k phosphorylation in vitro. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation in vivo and perhaps for conferring their sensitivity to rapamycin and amino acid sufficiency.The target of rapamycin (TOR) 1 proteins are protein kinases that were first identified in Saccharomyces cerevisiae through mutants that confer resistance to growth inhibition induced by the immunosuppressive macrolide rapamycin (1). In mammalian cells, rapamycin blocks phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (2, 3) and p70 S6 kinase (p70S6k) (4,5) by interfering with the function of mTOR (6, 7) (also known as FRAP, RAFT1, or RAPT). Although mTOR can phosphorylate both these targets directly in vitro (8 -10), the mechanism of mTOR regulation of these phosphorylations in vivo remains incompletely understood (11).The p70S6k is activated through a sequential multisite phosphorylation in response to insulin or mitogens in vivo (11). In addition, nutrients, especially amino acids, have been shown to regulate the phosphorylation of p70S6k and 4E-BP1 and to be necessary for insulin or mitogen regulation (12-17). The activity of p70S6k␣1 in vivo is most closely related to the phosphorylation at Thr-412, situated in a hydrophobic motif C-terminal to the canonical catalytic domain (18,19). The identity of the kinase(s) acting on this site in vivo is uncertain; however, this site can be phosphorylated directly by mTOR in vitro (9, 10). Recently, site-specific mutagenesis was employed to define a five-amino acid sequence called the TOS (TOR signaling) motif as the minimal functionally important region within this p70S6k noncatalytic N-terminal segment (21). As with N-terminal deletion, mutation of a single Phe within the TOS motif to Ala causes marked inhibition of activity of full-length p70S6k and a loss of sensitivity to rapamycin and amino acid withdrawal in the p70S6k-⌬CT104, lacking C-terminal noncatalytic tail, background. In addition, a TOS motif was identified in the 4E-BPs, wherein mutation of 4E-BP1 Phe-114 to Ala inhibits amino acid-and serum-induced 4E-BP1 phosphorylation.Raptor is a recently...

Purpose: Replication-selective tumor-specific viruses present a novel approach for treating neoplastic disease. These vectors are designed to induce virus-mediated lysis of tumor cells after selective viral propagation within the tumor. Telomerase activation is considered to be a critical step in carcinogenesis, and its activity is closely correlated with human telomerase reverse transcriptase (hTERT) expression. We investigated the antitumor effect of the hTERTspecific replication-competent adenovirus on human cancer cells.Experimental Design: We constructed an adenovirus 5 vector [tumor-or telomerase-specific replication-competent adenovirus (TRAD)], in which the hTERT promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, and we examined the selective replication and antitumor effect in human cancer cells in vitro and in vivo.Results: TRAD induced selective E1A and E1B expression in human cancer cells, but not in normal cells such as human fibroblasts. TRAD replicated efficiently and induced marked cell killing in a panel of human cancer cell lines, whereas replication as well as cytotoxicity was highly attenuated in normal human fibroblasts lacking telomerase activity. In nu/nu mice carrying s.c. human lung tumor xenografts, intratumoral injection of TRAD resulted in a significant inhibition of tumor growth. No evidence of TRAD was identified in tissues outside of the tumors, despite the presence of TRAD in the circulation. Moreover, TRAD replication in the distant, noninjected tumors was demonstrated.Conclusions: Our results suggest that the hTERT promoter confers competence for selective replication of TRAD in human cancer cells, an outcome that has important implications for the treatment of human cancers.

Calcineurin inhibitors such as cyclosporine A and FK506 have been used for transplant therapy and treatment of autoimmune diseases. However, the inhibition of calcineurin outside the immune system has a number of side effects, including hyperglycemia. In the search for safer drugs, we developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide provided immunosuppression for fully mismatched islet allografts in mice. In addition, it did not affect insulin secretion, whereas FK506 caused a dose-dependent decrease in insulin secretion. Cell-permeable peptides can thus provide a new strategy for drug development and may eventually be useful clinically.