Mechanisms of inactivation of the p16INK4a gene in leiomyosarcoma of soft tissue: decreased p16 expression correlates with promoter methylation and poor prognosis

The p16INK4a tumour suppressor gene, encoding p16 protein, plays a crucial role in regulation of the G1 cell-cycle phase. To investigate the potential role of p16 in soft tissue leiomyosarcoma (LMS), an immunohistochemical analysis was performed of 77 LMSs for p16 expression. Decreased expression of the p16 protein was identified in 25 of 77 LMSs (32%). Decreased expression of p16 correlated significantly with large tumour size (p=0.0038). In a univariate analysis, large tumour size and decreased expression of p16 were statistically significant adverse prognostic factors (p=0.025 and p=0.0021, respectively). In a multivariate analysis including conventional clinicopathological parameters, decreased expression of p16 protein was revealed as the only independent unfavourable prognostic factor (p=0.012). To elucidate the mechanisms of inactivation of the p16INK4a gene, 49 LMSs for which genomic DNA was available were examined; analysis for homozygous deletion, mutation, and promoter hypermethylation was conducted using differential PCR, PCR-SSCP, and methylation-specific PCR, respectively. Promoter hypermethylation was detected in 11 of 49 LMS cases (22%); homozygous deletion was detected in 3 of 49 cases (6%); and mutation was not recognized in any of the cases studied. Eight of 15 cases (53%) with decreased expression of p16 protein revealed methylation of the p16INK4a gene promoter. Promoter hypermethylation correlated closely with decreased expression and poor prognosis (p=0.0014 and p=0.0088, respectively). These results suggest that decreased expression of p16 protein can be considered as an independent reliable prognostic parameter in patients with soft tissue LMS. Furthermore, promoter methylation was more frequent than either homozygous deletion or mutation in this tumour, and promoter methylation was also shown to have a strong association with inactivation of the p16INK4a gene.     

Radiation-induced reactive oxygen species formation prior to oxidative DNA damage in human peripheral T cells

Previously, we demonstrated that human peripheral T lymphocytes revealed early apoptotic changes (annexin V-positive) and late apoptotic changes (propidium iodide-positive), at 13 and 24 h, respectively, after irradiation of 5 Gy. Changes in mitochondrial membrane potential were observed at 10 h after irradiation of 5 Gy. Subsequently, mitochondrial cytochrome c-release was confirmed. In order to elucidate the mechanism which acts prior to the mitochondrial membrane potential changes, we examined in the previous study the radiation dose and the timing of oxidative DNA damage induced in human peripheral T lymphocytes following 10 MV X-ray irradiation. As a result, the production of 8-oxoguanine, i.e., the product of oxidative DNA damage, was clearly identified starting at 10, 6, and 3 h, after 2, 5, and 20 Gy of irradiation, respectively. Therefore, we examined in the present study reactive oxygen species (ROS) formation in T lymphocytes following 5 Gy of irradiation. Using a CCD camera system, we monitored fluorescence in T lymphocytes loaded with the succinimidyl ester of dichlorodihydrofluorescein diacetate (H2DCFDA), which is non-fluorescent until oxidized by ROS. We found that ROS formation occurred immediately after irradiation, continued for several hours, and resulted in oxidative DNA damage. Therefore, the origin of hyper-radiosensitivity of T lymphocytes seemed to be the high production of ROS in the mitochondrial DNA following irradiation.     

Behçet Disease and the HLA System

Seventy-three unrelated patients with Behçet disease together with 33 members of seven families with at least two patients per family were tissue typed for 26 antigens of the HLA system. The patients with the complete type of Behçet disease were found to have HLA-B5 more significanth than healthy individuals. Family studies suggest that the genes closely linked to HLA locus influence the degree of severity of Behçet disease.     

Effect of interferon treatment on serum 2',5'-oligoadenylate synthetase levels in hepatitis C-infected patients

Interferon (IFN) is widely used for patients with hepatitis C. Less than half of treated patients respond to IFN therapy, however, and increased resistance to IFN is particularly observed in genotype 1b patients. Recently, genotype 1b patients with the wild type sequence in the NS5A gene were shown to be resistant to therapy, suggesting that the NS5A protein may be involved to IFN resistance. Thus, we investigated the serum 2',5'-oligoadenylate synthetase (2',5'-OAS) levels before and during IFN treatment. In addition, other biochemical markers and NS5A mutations were also examined in 30 HCV genotype 1b-positive patients. Before IFN treatment, 2',5'-OAS activity in sera was significantly lower in wild type patients than in mutant type patients. All patients were subsequently enrolled in IFN therapy, and 2',5'-OAS activity was elevated both in wild and mutant type patients, irrespective of the number of mutations in NS5A. Logistic regression analysis revealed that clearance of serum HCV RNA was independently related to the pretreatment viral load and NS5A mutations, but not to serum 2',5'-OAS activity. We concluded that the NS5A protein, that is associated with the outcome of IFN therapy, affects the kinetics of IFN-induced molecules, such as 2', 5'-OAS. 2',5'-OAS activity does not, however, seem to be related to long-term virological response to IFN therapy.     

Recruitment of katanin p60 by phosphorylated NDEL1, an LIS1 interacting protein, is essential for mitotic cell division and neuronal migration

LIS1 is mutated in the human neuronal migration defect lissencephaly and along with NDEL1 (formerly NUDEL) participates in the regulation of cytoplasmic dynein function during neuronal development. Targeted disruption of Ndel1 suggested that NDEL1 could have other molecular targets that regulate microtubule organization for proper neuronal migration. To further understanding the molecular mechanism of LIS1 and lissencephaly, we identified the katanin p60 microtubule-severing protein as an additional molecular target of NDEL1. We demonstrate that phosphorylation of NDEL1 by Cdk5 facilitates interaction between NDEL1 and p60, suggesting that P-NDEL1 regulates the distribution of katanin p60. Abnormal accumulation of p60 in nucleus of Ndel1 null mutants supports an essential role of NDEL1 in p60 regulation. Complete loss of NDEL1 or expression of dominant negative mutants of p60 in migrating neurons results in defective migration and elongation of nuclear-centrosomal distance. Our results suggest that NDEL1 is essential for mitotic cell division and neuronal migration not only via regulation of cytoplasmic dynein function but also by modulation of katanin p60 localization and function.     

Nitric oxide induction by pertussis toxin in mouse spleen cells via gamma interferon.

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.