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.

In CRC, the methylation status of multiple promoters can be predicted through knowledge of BRAF and, to a lesser extent, KRAS activating mutations, indicating that these mutations are closely associated with different patterns of DNA hypermethylation. These changes may be important events in colorectal tumorigenesis.

SUMMARY Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined. We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells. Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth. Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation. Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function.

Currently available methods for detection of tumors in vivo such as computed tomography and magnetic resonance imaging are not specific for tumors. Here we describe a new approach for visualizing tumors whose fluorescence can be detected using telomerase-specific replication-competent adenovirus expressing green fluorescent protein (GFP) (OBP-401). OBP-401 contains the replication cassette, in which the human telomerase reverse transcriptase (hTERT) promoter drives expression of E1 genes, and the GFP gene for monitoring viral replication. When OBP-401 was intratumorally injected into HT29 tumors orthotopically implanted into the rectum in BALB/c nu/nu mice, para-aortic lymph node metastasis could be visualized at laparotomy under a three-chip color cooled charged-coupled device camera. Our results indicate that OBP-401 causes viral spread into the regional lymphatic area and selectively replicates in neoplastic lesions, resulting in GFP expression in metastatic lymph nodes. This technology is adaptable to detect lymph node metastasis in vivo as a preclinical model of surgical navigation.

We report here a case of torsion of the gallbladder in a 73-year-old woman. The patient was admitted to our hospital with right hypochondralgia. Ultrasonography and computed tomography demonstrated a distended gallbladder, with a multilayered wall, which contained no stones. Since the symptoms did not respond to antibiotics, laparotomy was performed. The gallbladder was found to be twisted around its pedicle and to be gangrenous. Cholecystectomy was performed, and the patient had an uneventful postoperative course. We also reviewed 245 cases reported in the Japanese literature. The clinical features of gallbladder torsion, which include low frequency of fever and jaundice, poor response to antibiotic therapy, and acute onset of abdominal pain, may be helpful in the differential diagnosis from acute cholecystitis. Moreover, a highly suggestive sign of gallbladder torsion observed by ultrasonography or computed tomography is a markedly enlarged "floating" gallbladder with a continuous hypoechoic line indicating edematous change in the wall.