Targeted disruption of the focal adhesion kinase (FAK) gene in mice is lethal at embryonic day 8.5 (E8.5). Vascular defects in FAK-/- mice result from the inability of FAK-deficient endothelial cells to organize themselves into vascular network. We found that, although fibronectin (FN) levels were similar, its organization was less fibrillar in both FAK-/- endothelial cells and mesoderm of E8.5 FAK-/- embryos, as well as in mouse embryonic fibroblasts isolated from mutant embryos. FAK catalytic activity, proline-rich domains, and location in focal contacts were all required for proper allocation and patterning of FN matrix. Cells lacking FAK in focal adhesions fail to translocate supramolecular complexes of integrin-bound FN and focal adhesion proteins along actin filaments to form mature fibrillar adhesions. Taken together, our data suggest that proper FN allocation and organization are dependent on FAK-mediated remodeling of focal adhesions.

Tumor necrosis factor alpha (TNF-alpha), a potent proinflammatory cytokine, may be involved in the development of chronic obstructive pulmonary disease (COPD). The production of TNF-alpha is elevated in the airways of these patients. A polymorphism at position -308 of the TNF-alpha gene promoter (TNF-alpha-308*1/2) is known to be associated with alteration of TNF-alpha secretion in vitro. In this study we examined the differences in TNF-alpha-308*1/2 allele frequency to investigate the association of this polymorphism with the presence of smoking-related COPD. TNF-alpha-308*1/2 allele frequency in 106 patients (73 men and 33 women) was compared with 110 asymptomatic smoker/ex-smoker control subjects matched for sex and age and population control subjects consisting of 129 blood donors. Genotype was analyzed by the polymerase chain reaction-restriction fragment length polymorphism technique on genomic DNA isolated from peripheral blood lymphocytes. TNF-alpha-308*1/2 allele frequencies were significantly different among the groups: 0.835/0.165 in patients with COPD, 0.918/0.082 in smoker/ex-smoker control subjects, and 0.922/0.078 in population control subjects. These results indicate that TNF-alpha-308*1/2 alleles are significantly associated with the presence of smoking-related COPD.

Hepatitis C virus (HCV) infection is a major problem throughout the world. Combination therapy of interferon (IFN) and ribavirin is the best treatment for eradication at present, but the mechanism is not completely understood. We used the HCV replicon system to investigate this mechanism. The effects of six drugs (UDCA, glycyrrhizin, TJ-9, bezafibrate, ribavirin, and alpha-IFN 2b) on HCV subgenomic RNA (genotype 1b, NS5B 415Y) were examined by reverse transcription polymerase chain reaction, cloning and sequencing. The HCV replication was inhibited by alpha-IFN 2b (7.39-13.2% at 10 U/mL, 3.29-6.12% at 100 U/mL, 1.3-4.86% at 1000 U/mL) and by ribavirin (4.36-13.9% at 100 microg/mL), but not by the other drugs at 24-72 h after treatment. Furthermore, the combination treatment was superior to IFN monotherapy and to ribavirin monotherapy at 72 h post-treatment. Sequence analyses of the double-stranded RNA-activated protein kinase (PKR)-binding domain and flanking regions within the HCV NS5A region revealed that the total numbers of substitutions caused by ribavirin (n = 36) or combination treatment (n = 57) were more than those of IFN alone (n = 5) and controls (n = 6). The HCV replicon system is the most efficient system for HCV replication and is an excellent choice for testing anti-HCV drugs and disinfectants. Our results further suggested that the combination of alpha-IFN 2b and ribavirin might induce mutations, and inhibit HCV RNA synthesis in hepatocytes to a greater extent than ribavirin monotherapy.

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Purpose: Recently, the application of replication-competent viruses has been studied as anticancer agents. Sindbis virus (SIN) is an RNA virus that belongs to the Alphavirus genus in the Togaviridae virus family. The AR339 strain of SIN has not been reported to induce any serious disease to humans. Experimental Design: In this study, we evaluated the feasibility of the replication-competent SIN AR339 strain as an agent for cervical and ovarian cancer therapy. Results: SIN infection was able to induce cytopathic effects and apoptosis in two cervical cancer cells (HeLaS3 and C33A) and three ovarian cancer cells (HOC-1, HAC-2, and OMC-3) but not in normal human keratinocytes in vitro. The analysis of cell viability, virus protein synthesis, and viral growth showed the cancer-specific cytotoxicity and virus growth of SIN. In nude mice, i.t. and i.v. inoculation of SIN resulted in significant regression of established cervical tumors implanted at their backs. Histologic studies revealed that systemic treatment with the single injection of SIN induces necrosis within tumors at a remote site. In the metastasis model of ovarian cancer, suppression of ascites formation was observed in nude mice with i.p. SIN treatment. By using an in vivo green fluorescent protein imaging system, we also showed that systemic treatment with SIN targeted tumors specifically. Conclusions: Our study suggested that SIN AR339 strain has a possibility as a novel agent for human cervical and ovarian cancer therapy.